**Preface**

In the last several years, issues related to environmental pollution, health, and safety have pushed the global research community to address new challenges in order to find valid solutions able to substitute petroleum-based materials, providing advantages for the environment and humans. In this scenario, bio-polymers have gradually caught on in several application fields in materials science, such as manufacturing, biomedical engineering, the food industry, packaging, cosmetic and pharmaceutical industries, agriculture, the energy sector, green nanotechnology, and recycling by attracting the interest of the industrial world, which is increasingly forced to comply with restrictions for environmental and health protection. This reprint collects 32 scientific works of scholars that contributed their own expertise, passion, and science to expand the boundaries of knowledge by addressing new challenges. The topic of new biopolymer-based composite materials is addressed from several aspects, from the development to characterization and application, in several fields of scientific interest.

> **Raffaella Striani** *Editor*

### *Review* **A Review of Chitosan and Chitosan Nanofiber: Preparation, Characterization, and Its Potential Applications**

**Marwan A. Ibrahim 1,2, Mona H. Alhalafi 3,\*, El-Amir M. Emam 4, Hassan Ibrahim 5,\* and Rehab M. Mosaad 1,2**


**Abstract:** Chitosan is produced by deacetylating the abundant natural chitin polymer. It has been employed in a variety of applications due to its unique solubility as well as its chemical and biological properties. In addition to being biodegradable and biocompatible, it also possesses a lot of reactive amino side groups that allow for chemical modification and the creation of a wide range of useful derivatives. The physical and chemical characteristics of chitosan, as well as how it is used in the food, environmental, and medical industries, have all been covered in a number of academic publications. Chitosan offers a wide range of possibilities in environmentally friendly textile processes because of its superior absorption and biological characteristics. Chitosan has the ability to give textile fibers and fabrics antibacterial, antiviral, anti-odor, and other biological functions. One of the most well-known and frequently used methods to create nanofibers is electrospinning. This technique is adaptable and effective for creating continuous nanofibers. In the field of biomaterials, new materials include nanofibers made of chitosan. Numerous medications, including antibiotics, chemotherapeutic agents, proteins, and analgesics for inflammatory pain, have been successfully loaded onto electro-spun nanofibers, according to recent investigations. Chitosan nanofibers have several exceptional qualities that make them ideal for use in important pharmaceutical applications, such as tissue engineering, drug delivery systems, wound dressing, and enzyme immobilization. The preparation of chitosan nanofibers, followed by a discussion of the biocompatibility and degradation of chitosan nanofibers, followed by a description of how to load the drug into the nanofibers, are the first issues highlighted by this review of chitosan nanofibers in drug delivery applications. The main uses of chitosan nanofibers in drug delivery systems will be discussed last.

**Keywords:** chitosan; nanofiber; preparation; characterization; applications; wound dressing

### **1. Introduction**

Henry Braconot (1780–1855) isolated chitin from mushrooms in 1811. Chitin was the second carbohydrate discovered in 1859 [1]. It was hydrolyzed in an alkaline medium in 1894 to produce a new carbohydrate soluble in dilute acids called chitosan. Chitosan and its oligomers drew a lot of attention because of their unique properties [2–4]. Chitin represents the second natural polysaccharide in nature, after cellulose. Its chemical name is poly *N*-acetamido-2-decoxy-β-D-glucose [5]. Figure 1 shows the chemical structure and some sources of chitin and chitosan [6–9].

**Citation:** Ibrahim, M.A.; Alhalafi, M.H.; Emam, E.-A.M.; Ibrahim, H.; Mosaad, R.M. A Review of Chitosan and Chitosan Nanofiber: Preparation, Characterization, and Its Potential Applications. *Polymers* **2023**,

Academic Editor: Raffaella Striani

*15*, 2820. https://doi.org/10.3390/

Received: 25 April 2023 Revised: 13 June 2023 Accepted: 15 June 2023 Published: 26 June 2023

polym15132820

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

**Figure 1.** Chemical Structure and some sources of chitin and chitosan [5].

#### **2. Structure of Chitin and Chitosan**

Chitosan, a white polymer found in crabs, is a nitrogenous polysaccharide that is rigid inelastic and found both inside and outside the exoskeleton of invertebrates. The primary distinction is the solubility of chitosan in diluted acid (at PH = 7). The ocean, lakes, and seas are the sources of chitosan products [10]. Chitosan has potential applications in various fields, such as biomedical applications and the fabrication of protective clothes [10].

Chitin and chitosan, as natural renewable biopolymers, have unique properties, as they are biocompatible, biodegradable, and non-toxic [11]. Because of the NH2 groups that open the structure of chitosan, it is easier to modify than cellulose. As a result, it can be converted into a variety of chitosan derivatives [12]. The main purpose of this modification is to improve its solubility and chemical reactivity [13,14].

Acetylation degree is the presence of acetyl glucose amine to glucose amine in the chitin structure. Which determines the solubility of chitin and chitosan? Chitosan is more accepted than chitin due to its high degree of solubility in dilute acetic acid [15,16].

The presence of acetyl groups in chitin and chitosan makes their solubility problematic, although M.W.T. represents an important later component for chitin and chitosan properties. There are several methods used to estimate the molecular weight (M.W.T.) of chitin and chitosan, such as light scattering and viscosity [15,16].

Chitin is hydrophobic material insoluble in water and most organic solvents. Its solvent is a mixture of 1,2-diehloroethane and trichloroacetic acid (35:65), fresh saturated solution of lithium thiocyanate, chloroalcohols in conjugation with aqueous solutions of mineral acids and Dimethylacetamide containing 5% lithium chloride [10,17].

Chitin and chitosan are biocompatible, biodegradable, and non-toxic. Because they are amino polysaccharides, they exhibit interesting properties in biology, pharmacology, and physiology and have numerous applications such as wound healing, wound dressing, hemostatic agency, and antimicrobial activity, requiring them to be used as a biomedical material [17].

Figure 2 shows chitosan-based materials with different shapes and sizes: chitosan nanoparticles, chitosan nanofibers fabricated by electrostatic spinning technology, chitosan– pectin hydrogel grid scaffold prepared by 3D printing technology, chitosan core-alginate shell microspheres, chitosan-based fibers fabricated by solvent spinning technology, and 3D-printed chitosan porous structures [18].

**Figure 2.** Chitosan-based materials with different shapes and sizes: (**a**) chitosan nanoparticles; (**b**) chitosan nanofibers fabricated by electrostatic spinning technology; (**c**) chitosan–pectin hydrogel grid scaffold prepared by 3D printing technology; (**d**) chitosan core–alginate shell microspheres (**e**) chitosan-based fibers fabricated by solvent spinning technology; and (**f**) 3D-printed chitosan porous structures. Copyright 2023. Reproduced with permission from Elsevier Science Ltd. [18].

#### **3. Modification of Chitin and Chitosan**

Although chitin and chitosan possess several useful qualities, their application is constrained by their poor solubility, small surface area, and porous makeup. To modify chitin and chitosan physically or chemically, various researchers have tested their theories. The two main benefits of modifying chitin and chitosan are to increase their solubility and improve their ability to absorb metals. The OH at the C3, C6, and NH2 at the C2 groups in chitosan undergo substitution reactions. These changes also improve the capacity of the membrane to swell in water [19–21].

In the molecular structure of chitosan, there are three types of active groups: amino groups, and primary and secondary hydroxyl groups at the C-3 and C-6 positions, which allow for chemical modification of chitosan. C6-OH is a main hydroxyl group with little steric hindrance, whereas C3-OH is a secondary hydroxyl group with a lot. As a result, the main hydroxyl group could freely rotate while the secondary hydroxyl group could not. The amino group is usually more reactive than the primary hydroxyl group, and the primary hydroxyl group is usually more reactive than the secondary hydroxyl group [22–24]. Chitosan

can be chemically modified on the amino, hydroxyl, or both amino and hydroxyl groups to generate N-modified, O-modified, or N, O-modified chitosan derivatives [25] (Figure 3).

**Figure 3.** Chemical modification of chitosan.

The proposed mechanism of chitosan modification can pass through one of the following four mechanisms: (a) free radical-induced conjugation to form a polyphenol chitosan conjugation; (b) carbodiimide chemical mechanism to form Schiff base compounds; (c) functional group conversion strategy, which converts the amino group of chitosan into an azide group, substituted carboxyl group, substituted mercapto group, and where the hydroxyl group can be azidate, aminated, oxidized to an aldehyde or carbonyl group, or further oxidized to a carboxyl group; or (d) conjugation of chitosan with polyphenol via enzymatic assisted coupling reaction, as shown in Figure 4a–d [26].

Chitosan has a low specific surface area (2–30 m2·g−1) [27] and is present in flake, which is unsuitable for many applications, so that it has been modified into beads to increase its value in different fields of application [19–21]. Due to the open structure and pores of chitosan, which has poor mechanical properties, shrinkage, and deformation in dry form and is only soluble in weak acids, numerous modifications have been made to it to improve its properties [28]. Chitosan contains amino and hydroxyl groups, so it is a poly-nucleophilic polymer. Nucleophilic substitution occurs. Protonation in NH2 groups results in the formation of NH3 <sup>+</sup> [29,30], such as N-alkylated chitosan prepared from Schiff base reactions followed by imine reduction by sodium borohydride. Furthermore, positive chitosan charges interacted with polyanion polymers such as alginate, carrageen, and pectin between the COOH and NH3 <sup>+</sup> groups [31]. Chitosan produce new functionalized derivatives via a grafting reaction [32]. The properties of grafted chitosan are determined by the side chains and the cross-liking agent [33]. Chitin threads are prepared to be used in absorbable suture fabrication, dressing, and biodegradable materials for human skin fiber growth [34].

**Figure 4.** *Cont*.

**Figure 4.** Proposed mechanisms (pathway) for chemical modification of chitosan via conjugation (**a**) free radical-induced conjugation to form a polyphenol chitosan conjugation; (**b**) carbodiimide chemical mechanism to form Schiff base compounds; (**c**) functional group conversion strategy, or (**d**) conjugation of chitosan with polyphenol via enzymatic assisted coupling reaction.

Chitosan is a hydrophilic material used to impart hydrophilicity to some other polymer in its composites, such as polyacrylonitrile (PAN) [35]. Hydrophilic polymer nanoparticles were prepared by ionic gelation in mild conditions at room temperature, via two phases; one is for polymers such as chitosan polysaccharide (CS) and polyethylene oxide (PEO), and the other phase contains sodium tripolyphosphate (TPP). Calvo et al. prepared chitosan nanoparticles with high protein loading capacity, which were released within a week [36]. The morphology of these nanoparticles is spherical, as shown in Figure 5.

**Figure 5.** Electron transmission microphotography of: (**a**) CS nanoparticles; (**b**) CS/PEO–PPO nanoparticles (concentration of PEO–PPO in the chitosan solution: 10 mg/mL) prepared by Calvo et al. [36], reproduced with permission from John Wiley and Sons.

Nanoparticles of chitosan by using a variety of agents, the freeze-drying method increases shelf life. Chitosan nanoparticles of two different types were prepared by Alonso et al. in 1999 [37]. The prepared nanoparticles ranged in size from 300 to 400 nm, had a positive surface charge, and were efficient. These nanoparticles are used to address the nasal absorption of insulin. In addition, chitosan nanoparticles were used as a poly load of the anthracycline drug doxorubicin (DOX) [38,39]. In addition, these nanoparticles were used to load dextran sulphate to enhance its drug loading [40]. With cyclosporine serving as the model drug, chitosan nanoparticles (CSNP) were used to enhance the drug at the ocular surface. The size of these nanoparticles was 29 nm [41].

Chitosan-coated PLGA–Lecithin nanoparticles were prepared by the modified double emulsion method; these nanoparticles were used through the oral or nasal administration route. Figure 6 depicts the process of preparing CS–PLGA nanoparticles, and Table 1 summarizes the properties of the prepared nanoparticles [41].

**Figure 6.** Flowchart showing the creation of chitosan–PLGA particles step-by-step.

Chitosan nanoparticles loaded by *Mycobacterium uaceae aerivatiuem* antineoplastic proteoglycans exhibit wide antimicrobial activity, as repeated by Tian and Groves (1999) [42]. The researchers prepared chitosan nanoparticles with particle sizes of 600–700 nm without using organic solvents and discovered that the two reactants affected the absorption and release of bovine, as well as that the initial Nanoparticles of chitosan by using a variety of agents, the freeze-drying method increases shelf life. Chitosan nanoparticles of two different

types were prepared by Alonso et al. in 1999 release was followed by a steady release for 4 h in water [43].


**Table 1.** Particle size zeta potential theoretical loading, and encapsulation efficiency values of CS [41].

Using self-aggregates of chitosan modified by deoxycholic acid, there is a novel and straightforward method of delivering adriamycin [44].

Deoxycholic acid and chitosan are covalently conjugated through an EDC-mediated reaction, resulting in self-aggregating chitosan nanoparticles. The Adriamycin active ingredient was physically trapped in nanoparticles, and the resulting self-aggregates were assessed using a variety of methods, including spectroscopy, which shows that these self-aggregates are spherical and that the drug concentration affects the shape of the particles [44].

These self-aggregates of chitosan modified by deoxycholic acid were used as DNA carriers by Kim et al. in 2001 [45]. Chitosan is used to overcome the side effects of drugs such as their solubility and hydrophobicity so that it can be used as a drug carrier. For example, commercial chitosan can be used to control body weight.

#### **4. Chitosan as Biomaterial**

Chitosan is a semi-crystalline polymer that is partially deacetylated chitin, and the degree of crystallinity is correlated with the degree of deacetylation. Chitin and fully deacetylated chitosan have the highest crystallinity, while intermediate levels of deacetylation have the lowest crystallinity [12]. Chitosan is stable, crystalline, and soluble in aqueous solutions at PH 7, but it is insoluble in weak acids due to the protonation of amino groups. Chitosan solutions could be extruded at higher PH values or in non-solvent baths, e.g., methyl alcohol. Chitosan polymer is used for industrial applications in such forms as fiber or film [46]. Chitosan solution's cationic nature and high charge density make it potentially useful as a biomaterial. Chitosan forms soluble ionic complexes or complexes with water soluble anionic polymers such as alginates and synthetic polymers such as poly (acrylic acid) due to its charge density [21]. When used for local delivery of biologically active poly anions like GAGs and DNA, for example, ionic complexes release heparin to increase the efficacy of growth factor secreted by inflammatory cells [21,47–51].

Chitosan DNA complex is used to facilitate cellular transfection and prevent plasmid degradation by nucleases. Chitosan can become porous through freezing and lyophilization, making it useful for both tissue regeneration and cell transplantation [52]. Figure 7 illustrates how ice crystals form in the freezing process from a solution, grow from the ice crystal phase, and are then removed by lyophilization from a general porous mother.

Pore size and pore orientation affect the mechanical properties of scaffold-based chitosan, with porous chitosan membranes having less elasticity (0.1–0.5 MPa) than nonporous membranes (5–7 MPa).

The maximum strain of the porous membrane of chitosan is greater than that of the non-porous membrane, and the 100% chemical modification of chitosan introduces new biological activity with modified mechanical properties. The chitosan NH2 group is reactive and capable of introducing side group attachment with several reactions that affect primarily crystallinity disruption with lower stiffness and alter soluble derivatives feasible in chemical reactions, such as alkyl derivatives of chitosan, which have lower solubility than chitosan itself and give aggregates miscible for c > 5. In addition, the basic properties of chitosan are hemostatic, cationic, and insoluble at pH 7, which are completely reversed by the solvation process to give ionic, water-soluble derivatives, and anticoagulant properties, so that chitosan can attack with unlimited side groups and be chosen according to specific needed functions, e.g., biological activity or modified properties, etc.

**Figure 7.** SEM micrographs of different hybrid hierarchical structures resulting from freezing hydrogel nanocomposites, with identical CHI and calcium phosphate composition (93.25 and 6.75 wt.%, respectively) at different rates of freezing: (**a**) 0.7 mm/min; (**b**) 2.7 mm/min, and (**c**) 5.7 mm/min. Scale bars are 50 μm. TEM (**d**) micrographs of ACP nanoclusters forming the ACP/CHI hierarchical structure. Ref. [52] Reproduced with permission from MDPI.

#### **5. Applications of Chitosan and Chitosan Derivatives**

Chitosan is a biodegradable, biocompatible, and non-toxic biomaterial; therefore, it has many applications in areas such as medicine, agriculture, food processing, cosmetics, and treatment of water.

#### *5.1. Agricultural Application*

Chitosan can be used safely in agricultural applications because it does not pollute the environment or harm consumers; it is used as a leaf coating, fertilizer, and sea coating [10,53]. The use of chitosan in agricultural fields has increased exponentially, especially for germination improvement, leaf growth, retention of moisture, and fungal and disease reduction [53]. Chitosan boosts photosynthetic efficiency while enhancing plant tolerance to salinity, high temperatures, and drought [54]. Chitosan's hydrophilic properties encourage water absorption while reducing transpiration [55]. To encourage plant development, chitosan can be employed as an additional carbon source in plant synthesis [56]. In comparison to the control group, seedlings treated with Cu-chitosan nanoparticles (NPs) at concentrations of 0.04 percent and 0.12 percent had greater rates of germination, seedling length, root length, and root number [57]. To encourage the mobilization of protein and starch to boost seedling growth, Cu-chitosan NPs can increase protease and -amylase activities [58].

#### *5.2. Wastewater Treatment Applications*

Chitosan contains both OH and NH2 groups as chelating agent groups, which allow it to be used in water treatments from wastes due to the high-power effect of these functional groups to bind with heavy dissolved metals present in wastewater such as Cu, Pb, Hg, and Ur [59]. Furthermore, chitosan can be used to break down food particles, particularly food proteins, as well as remove dyes from wastewater [60].

Cu (II) ion-containing wastewater was examined by Qin et al. [61] using sodium alginate and chitosan as treatment agents. They investigated how various parameters affected this ion removal. According to the findings, chitosan and sodium alginate worked better together than they did separately. Pesticide removal from wastewater was explored by Dwivedi et al. [61] using hydrogel beads made of chitosan and gold nanoparticles. The data obtained indicated that the synthetic sorbent has good pesticide removal capacity.

Heterogeneous catalysis is one technique that heavily relies on adsorption. Purification is one of the earliest documented uses of adsorption. Adsorbents are still used to clarify water [62]. Numerous adsorbents, including activated carbon, low-cost biomass adsorbents, waste sludge, rice husk, sugarcane bagasse, lignite, and chitosan, were used in the adsorption tests. Heavy metal contamination clean-up frequently employs chitosan. In order to study the adsorptions of three metal ions—Cu (II), Zn (II), and Pb (II) ions—in an aqueous solution, cross-linked chitosan was created by the homogeneous reaction of chitosan in an aqueous acetic acid solution with epichlorohydrin [63].

#### *5.3. Food Industry Applications*

Because of the high potential for toxicity of chitosan as a chelating agent and its high functional properties, as previously stated, chitosan is used in a variety of applications in the food industry, such as removing specific elements, particles, and undesirable materials, such as dyes and fats. In addition, it is widely used as a natural, safe preservative in the United States to store food [64,65].

There has been an increase in interest in recent years in studying the potential applications of chitosan as films or coatings in food packaging. This is due to their film-forming, antioxidant, and antibacterial properties, as well as their mechanical and barrier properties, which were studied as films. In order to extend the storability and shelf life of perishable commodities, these experiments sought to develop active packaging based on chitosan, either on its own or in combination with other materials. By combining chitosan with other natural antimicrobial agents, it is also possible to make food products that guarantee food safety against a variety of mutating and pathogenic bacteria [66–68].

An edible coating or thin edible film made of chitosan can be applied to food to act as active food packaging. The edible coating is a thin layer that is added to a food item and is generated by dipping the item in a chitosan solution or spraying, in which case, the film-forming solution is crushed up using an aerosol spray coating. Even though the chitosan film is a thin, prefabricated layer, once it is produced, it can be deposited on the surface of or in between food ingredients [66,67,69]. Studies using chitosan films and coatings on food products are shown in Table 2 [66].


**Table 2.** Chitosan-based films have been used in a wide variety of food products.

#### *5.4. Medical Applications*

Chitosan is applicable in several medical industries, especially periodontal and orthopedic drug delivery, wound healing, and tissue engineering applications [86]. Surgical sutures, contact lenses, eye fluids, artificial skin, artificial blood vessels, bandages, sponges, burn dressings, blood cholesterol vessels, antitumor, antibacterial, antiviral, bane regenerator, antimicrobial, and hemostatic agents are the most well-known examples of these applications [86–91].

Chitosan inhibits tumor cell proliferation, and Liu et al. demonstrated that chitosan induces apoptosis in tumor cells by decreasing Bcl-2 and increasing Caspase-3 expression [92,93]. Carboxymethyl chitosan (CMCS) increases macrophage viability, deeply penetrates the tumor microenvironment, generates cytokines like TNF and IL-1, improves phagocytosis, and increases NO levels. Notably, CM-COS is not significantly toxic to normal liver cells but inhibits the growth of BEL-7402 and sarcoma cells in vivo [94,95]. Additionally, chitosan inhibits the invasion and metastasis of tumor cells. During tumor invasion and metastasis, matrix metalloenzymes (MMP) are involved in the breakdown of extracellular matrix, and MMP-2 can encode an enzyme that breaks down type IV collagen [96].

Chitosan is a great material for creating wound dressings because it can promote wound healing. It exhibits good antibacterial activity due to its alkaline amino groups, which cause the destruction of bacterial cells and protect the wound surface from microbial infection [97]. To improve the antibacterial and coagulation capabilities, Zhang et al. created a nanocomposite hydrogel utilizing zinc oxide (ZnO), chitosan, and aldehydic sodium alginate (SA) [98]. It significantly inhibited the growth of *Escherichia coli* and *Staphylococcus aureus*. I-sexual collagen hydrogels sulphated chitosan-doped by Shen et al. improved macrophage polarization from M1 to M2. The IL-4 and TGF-1 secreted by macrophages were stimulated, which resulted in an increase in collagen synthesis, regeneration epithelialization, and neovascularization [99].

Tissue engineering is an emerging interdisciplinary discipline combining material science, engineering mechanics, and biomedicine. The structure and function of damaged tissues and organs are repaired or replaced by cell transplantation combined with bioactive molecules and 3D scaffolds. Chitosan is similar to mucopolysaccharides of the extracellular matrix and is used as a scaffolding material for cartilage tissue engineering [100].

Kaviani et al. used the cryogel approach to construct nano porous scaffolds from chitosan, collagen, and nanohydroxyapatite, which lowered the rate of biodegradation, increased mechanical characteristics, and enabled cell proliferation and adhesion [58]. Porous scaffolds made from chitosan, gelatin, and silk proteins have higher compressive strength and modulus, whereas incidental chondrocytes can produce seed scaffolds and

stimulate cartilage tissue regeneration [58]. By mixing gelatin, chitosan, and polyvinyl alcohol with nano-hydroxyapatite, Martino et al. created porous composite scaffolds [58]. Table 3 summarizes the biological application of chitosan and its derivatives.

**Table 3.** Biological applications of chitosan and its derivatives.


#### **6. Electro-Spun Nanofibers: Process and Application**

#### *6.1. History of Electrospinning*

In 1897, Rayleigh discovered electrospinning. The early 1700s saw the discovery of electrostatic effects on water behavior and their impact on the dielectric values of liquid excitation. Around the turn of the twentieth century, Cooley and Morton developed electrospinning technology. The rotatory electrode was then incorporated into electrospinning by Cooley. Formhals created yarns using electrospinning in 1930 without the use of a spinneret, and his technique and apparatus granted him a patent for his creation [119–121].

After that, Formhals submitted a patent for a different method of creating electrostatic polymer fibers: composite fibers made of several polymers. In 1969, Taylor conducted research on the composition of the polymer droplet formed at the needle tip by a strong electric field. This proved that the droplet assumed a cone-like shape, with jets emerging from vortices. The "Taylor cone" is the final name given to this cone. Additionally, factors affecting fiber stability, such as the electric field, flow rate, and experimental settings, were examined [122,123].

Compared to conventional spinning, electrospinning produces fibers at a much slower rate. Electrospinning produces yarn at a rate of 30 m per minute, compared to conventional spinning's 200–1500 m per minute [119,120,124]. So, before 1990, melt spinning was the preferred method for creating fibers from natural and synthetic polymers, and only a small number of businesses were interested in electrospinning for fiber production. Nanometerscale fibers cannot be produced through melt spinning [120,124].

For the purpose of removing harmful solvents and for use in tissue engineering applications, Dalton et al. applied an electro spun nanofiber web to tissue cells [125]. The use of electro-spun nanofibers today spans a wide range of potential uses [119,124,126]. Surface nanostructures may produce extraordinary phenomena, such as the lotus effect (self-cleaning effect). Since proteins, viruses, and bacteria all have dimensions in this range, the nanoscale is particularly important for biological systems. Electro spun fibers display a strikingly broad range of sizes when compared to the diameters of these things. Figure 8 depicts the dimensions of bacterial cells, proteins, viruses, and nanofibers [119,124,126,127].

**Figure 8.** Position of nanofibers between protein, bacteria, and viruses [126]. reproduced with permission from John Wiley and Sons.

#### *6.2. Electrospinning Process*

A high electric field (kV) is used to create micro or nanofibers from polymer solutions while maintaining ambient temperature and pressure. There are two primary setups for electrospinning devices: vertical and horizontal [119,124,128].

The major components of the electrospinning device are the power supply (high voltage), the syringe (spinneret), and the collector (electrode) [119,123,129].

High voltage creates electric charges on the surface of the polymer solution during the electrospinning process, and these charges build up on the surface. These charges possess repelling forces in a critical electric field that can dissipate the surface tension of the solution and the unstable charges. A Taylor cone-tip jet ejects a solvent-causing jet [119,123,124,130].

A stable jet created at the spinneret needle then changes into an unstable jet to create electro-spun fibers using a straightforward process. When the applied electric field reaches a critical value, jets shoot out of the cone tip, and the tensile force is transferred to the polymer, creating bending instability in that polymer. After that, a jet moves from the cone's apex to a collector with opposing charges, which has the power to draw charged fibers to it. Jet travel causes the solvent to evaporate, leaving the dry fiber on the collector [119,122–124,131,132].

A typical electrospinning device can be created using a power supply with high voltage power, a syringe, and a collector electrode alone, as demonstrated in the experimental part. Polymeric materials can be electro spun to create continuous nanofibers, and there are a number of variables that affect the nanofibers' properties. These variables are either processing variables (electric field) or polymer properties (concentration, viscosity, surface tension, and conductivity) (extrusion rate: distance from needle tip to collector) [119,123,124,129,133].

The definition of the electrospinning technique is the use of a strong electric field to spin polymer solutions into micro- to nanofibers at room temperature and atmospheric pressure (kV). Electrospinning devices can be set up in either a vertical or horizontal orientation [119,124,134–136], (Figure 9).

**Figure 9.** A diagram of electrospinning equipment with a syringe injecting polymer solution into an electric field generated by a high voltage power source between the spinneret and the grounded collector; a high voltage is applied. (**a**) A typical vertical arrangement of an electrospinning apparatus. (**b**) A typical horizontal setup of an electrospinning apparatus [119].

The three primary components of an electrospinning device are the power supply (high voltage), the syringe (spinneret), and the collector (electrode) [119,123,137,138]. When a polymer solution is subjected to a high voltage, electric charges accumulate on its surface. These charges repel one another so strongly that they can overcome the surface tension of the solution and form a Taylor cone in a critical electric field. When the electric field stretches the Taylor cone tip further, a charged jet is ejected. The jet eventually transforms into solid fibers due to solvent evaporation [123,139–141].

Using a simple process, a stable jet at the spinneret needle is converted to an unstable jet to generate electro-spun fibers. When the applied electric field reaches a critical magnitude, the surface tension is overcome by the charge repulsion force, and jets erupt from the cone tip, transferring the tensile force to the polymer and causing it to bend. The

charged fibers are then attracted by jets, with opposing charges moving from the cone apex to the collector. As the solvent evaporates through the jet, it leaves dry fiber on the collector [119,124,131,139,141].

A high-voltage power source, a syringe, and a collector electrode may be used to construct a standard electrospinning apparatus that can be configured vertically or horizontally, as illustrated in Figure 7. Continuous nanofibers may be generated by electrospinning polymeric materials, and the quality of electro-spun nanofibers is determined by some criteria. Polymer physical properties (concentration, viscosity, surface tension, and conductivity) or processing factors (electric field, flow rate, needle tip to collector distance) are examples [119,124,139,141].

Prior to electrospinning, most polymers are dissolved in a range of solvents; when fully dissolved, they create a polymer solution. The polymer solution is then poured into the capillary tube in preparation for electrospinning. However, because some polymers emit unpleasant or even dangerous odors, the procedures should be done in well-ventilated areas [142]. In the electrospinning process, a polymer solution at the capillary tube's end is exposed to an electric field, which causes an electric charge to form on the liquid's surface. The repelling electrical forces outweigh the surface tension forces when the applied electric field is strong enough. The solvent evaporates, and a polymer is formed when a charged jet of the solution is eventually released from the Taylor cone's tip. The jet is unstable and whips quickly in the area between the capillary tip and collector. Just past the spinneret's tip, where the jet is stable, instability sets in. Consequently, the electrospinning technology makes the process of making a fiber simpler [143].

Solution parameters, process parameters, and environmental or "ambient" parameters are the three main groups into which factors that have an impact on the electrospinning process are divided [119,144]. These factors are gathered in order to produce smooth fibers without beads, so a thorough understanding of these factors is required in order to obtain electro-spun nanofibers that are bead-free.

#### *6.3. Application of Electro-Spun Nanofibers*

Nanofiber membranes are created using electrospinning and used in a variety of applications, including biomedicine, security, clothing, and nano-sensors. Nanofibers are used in the biomedical field to focus on tissue engineering, wound dressing, drug delivery systems, and enzyme immobilization because they resemble the majority of organs and tissues, including skin, collagen, cartilage, and bone [145].

Electro-spun nanofibers are unique in that they have a consistent morphology, a high surface area to volume ratio, and inter- and inner porosity. These characteristics make them promising as scaffold biomaterials [146,147]. Additionally, nanofibers improve protein absorption, cell growth, cell differentiation, and cell adhesion [148,149].

Due to their pores, nanofibers are also used in filtration as micro and nano filters based on membrane design and construction, allowing liquids and small particles to pass while arresting larger particle sizes (contaminants), similar to how paper coffee filters prevent undissolved particles from passing through their pores while allowing dissolved ones to do so [150–153].

Affinity membranes, which have numerous uses in the biomedical and environmental fields, were also developed to select immobilized targets and remove contaminant targets [146,147,152–154]. Although electro-spun nanofibers have many uses, including those for tissue engineering, drug delivery, enzyme immobilization, wound dressing, antibacterial properties, filtration, desalination, and protective clothing, the focus of this article will be on biomedical uses.

#### 6.3.1. Tissue Engineering Applications

Electro-spun nanofibers are used in tissue engineering scaffold construction [155]. The use of biodegradable and biocompatible nanofibers to provide target tissues has increased daily [156]. Due to the similarities between these fibers and the natural extracellular matrix, these fiber scaffolds had an impact on both cell-to-cell and cell-to-matrix communication and produced excellent growth factors [157].

#### 6.3.2. Drug Delivery Applications

Based on the observation that the drug's rate of dissolution increased as the surface area of both the drug and the carrier increased, nanofibers were used to coat the drug and deliver it to the target site [158,159]. Anticancer medications, antibiotics, proteins, ribonucleic acid (RNA), and deoxyribonucleic acid are all delivered by electro-spun nanofibers (DNA) [160].

Recently, Yang et al. created a composite scaffold using nanofibers made of gelatin and polyvinyl alcohol (PVA) to transport raspberry ketone [161,162]. Additionally, to transport the growth factor calcium hydroxyapatite, Haider et al. prepared PLGA nanofibers [146,147].

#### 6.3.3. Enzymes Immobilization Applications

Immobilization of enzymes onto insoluble material is essential to improve durability and maintain the enzyme properties such as bioprocessing and long duration controls [163]. The immunized material essentials are biocompatible, durable and hydrophobic or hydrophilic [164].

Recently, electro-spun nanofiber prepared from the dual electrospinning process increased the enzyme immobilization [165]. Yet, there are some limitations that hinder this technique, and enzymes encapsulation and the enzyme immobilized on the fibers surface are limited [166], so that some chemical modification of the surface needs to overcome these limitations [167,168].

#### 6.3.4. Wound Dressing Applications

Inhibiting microorganisms, removing exudate, and protecting the wound site are all important functions of wound dressing. In addition to having antimicrobial properties, wound dressings provide a pleasant, moist environment to speed up the healing process [169–171]. Consequently, electro-spun wound dressings have more benefits than those made using traditional techniques [172]. These benefits include fiber pores, a large surface area, and the ability to stimulate fibroblast cells, making it suitable for use in cosmetic masks for skin cleansing and healing [142,173]. The electro spun nanofiber matrix incorporates various skin-treatment components [174].

Due to its capacity to create cationic clusters that can bind with anions on red blood cells, chitosan nanofibers have amazing hemostatic capabilities that can speed up platelet and red blood cell aggregation and ultimately reduce blood loss [175]. Additionally, this technique is successful even in individuals with coagulation abnormalities and is independent of the patient's own clotting system [176]. By electrospinning, Ren and colleagues [177] created a medicinal dressing made of a composite of silk fibroin, chitosan, and halloysite nanotubes. Aluminum silicate-based halloysite nanotubes have a hollow tubular structure, can efficiently bind antibacterial medications, and can provide delayed, sustained drug release [177]. The findings showed that using halloysite nanotubes caused the loaded-drug release time to increase by about 8 days. Additionally, the electro-spun chitosan composite membrane demonstrated a better blood coagulation rate, improved tensile property, and antibacterial activity, all of which support its potential value as a medical dressing.

An anti-fibrinolytic medication known as tranexamic acid (TXA) is frequently used during trauma surgery and has been found to improve wound healing [178]. For hemorrhage control applications, Sasmal and colleagues [178] created TXA-loaded chitosan/PVA electro-spun nanofibers. The findings support the role of chitosan in hemostasis by demonstrating that the total blood clotting time of pure chitosan/PVA nanofibrous membranes decreased from 21,010 s to 1676 s as the amount of chitosan increased. Additionally, clotting time and plasma recalcification time were dramatically shortened after TXA was added to chitosan nanofibers, demonstrating the enormous potential of TXA-loaded chitosan

nanofibers for managing civil and military hemostasis. Additionally, by fabricating chitosan within a hydrogel carrier template produced from cyclodextrin through proton exchange and complexation, Leonhardt and colleagues [179] observed the development of nanoscale features in chitosan mats. With nanofiber diameters of 9.23.7 nm and a macroscopic shape resembling a honeycomb, the assembled chitosan was highly entangled. When compared to commercially available absorbable hemostatic dressings, the chitosan-based composite hydrogels result in much less blood loss and faster timeframes for hemostasis.

#### 6.3.5. Antibacterial Applications

Many antibacterial hybrid electro-spun nanofiber scaffolds, including polyacrylonitrile/silver PAN and Ag nanofiber scaffolds, prepared by various research groups, have antibacterial activity against both gram-positive and gram-negative bacteria. Therefore, a wide variety of antimicrobial amidoxime was immobilized using PAN nanofibers. Amidoxime's antibacterial action is caused by its binding to the Mg2+ and Ca2+ ions, which upset the balance of the bacteria and result in bacterial death [180].

#### *6.4. Electrospinning of Chitosan*

Template synthesis, drawing, phase separation, electrospinning, self-assembly, and other techniques have all been used to create nanofibers [181]. For the creation of micro- and nanofibers, electrospinning is one of these techniques that is particularly adaptable [182]. With their high surface area to volume ratio, oxygen permeable porosity, and variety of pore sizes, electro-spun nanofibers act as a wound dressing material by promoting fibroblast growth [183]. Chitosan has a low electro spinnability and needs a strong applied electric field to work properly because of its polycationic nature, which is brought on by the amino groups on its backbone [184,185]. A strong electric field is necessary because the polycationic nature of chitosan results in very viscous solutions with high surface tension. Additionally, the chitosan's strong hydrogen bond network minimizes molecule exposure to the applied voltage [184,185].

Chitosan, like the majority of other polysaccharides, needs a strong acidic environment to dissolve properly, but this can be dangerous and prevent the use of chitosan fibers in some circumstances. Furthermore, when it comes to the creation of pure chitosan fibers, chitosan with a low molecular weight and low biological activity frequently produces better results. This has a negative impact on the biological functioning of the electrospun chitosan [184–186]. As in all electrospinning techniques, changing parameters cause morphological changes in the spun fibers. The correlations between parameters and morphology are generally as follows: The diameter and length of the end fiber are decreased as the applied voltage is increased. Low chitosan concentrations can lead to fiber formation and breakdown, which increases fiber diameter and leads to morphological flaws [184,187].

Chitosan's electro spinnability may be improved by using co-spinning polymers, either natural or synthetic. Frequently, chitosan is employed in the following fields: collagen, zein, silk fibroin, PEO, PVA, PLA, and zein [185,188]. Another method for resolving the issues with electrospinning chitosan is to chemically modify it to produce derivatives that are suitable for electrospinning, as with what was done with cellulose. More often than not, these derivatives have improved solubility and electro spinnability. Chitosan derivatives that have been investigated for this purpose include quarternized chitosan, hexanoyl chitosan, N-carboxyethyl chitosan, and others [189].

Chitosan is soluble in diluted aqueous formic, acetic, and lactic acids but insoluble in most mineral acidic media, alkalis, and water solutions. Chitosan dissolves when a small amount of acid is added to mixtures of water, ethanol, methanol, and acetone. Chitosan is a positively charged polyelectrolyte with a pH range of 2–6, which results in its greater solubility when compared to chitin. This characteristic makes chitosan solutions extremely viscous, making electrospinning difficult [190,191]. In addition, the three-dimensional network created by the potent hydrogen bonds prevents the polymeric chains from moving when they are exposed to an electrical field [192,193].

The unique properties of the polymer in solution, such as its polycationic nature, high molecular weight, and broad molecular weight dispersion, make chitosan electrospinning a challenging process. The inner tip–collector gap, the electric field voltage, the molecular weight, and the input velocity are just a few of the variables that have an impact on the quality of the electrospinning process and the finished product. When the electrostatic force in a solution is greater than the solution's surface tension, the electrospinning process starts. When an electric field is applied, the surface of the polymer solution charges up. A strong electrical charge encourages jet extension and increases the volume of solution the needle can draw. On the other hand, a higher voltage leads to a longer stretch of the solution. This has a significant impact on electro-spun fiber morphology, frequently reducing their diameter, and raising the possibility of bead formation [190,194].

The feed rate, which regulates the number of solutions available, is another crucial component. A solution's jet velocity and transfer rate are also impacted by its feed rate. For the evaporation of the solvent and the production of solid nanofibers, lower feed rates are preferred [119]. The solution must be removed from the tip at a rate that is substantially higher than the feed rate. Low feeding rates may prevent electrospinning, and high feeding rates may cause beaded large diameter fibers because sufficient solvent evaporation time must pass before the collector is reached [195].

SEM images of Figures 10 and 11 show the effect of tip–collector distance and electrical field voltage, respectively, on the structure of electro-spun nanofibers. As shown in these figures, increasing gap distance and voltage not only decreases and refines nanofibers diameters, but also improves the quality of electro-spun nanofibers.

**Figure 10.** SEM images of 5 wt.% chitosan hydrolyzed 48 h nanofibers in aqueous acetic acid 90%, tip needle–collector distance: 14 cm (**a**), 15 cm (**b**), 16 cm (**c**). [119] Copyright 2023. Reproduced with permission from Elsevier Science Ltd.

Another factor that affects the sizes and shapes of the nanofibers is the separation between the tip and the collector. In order to give the fibers enough time to dry before reaching the collector, a minimum distance is necessary; otherwise, beads have been observed when distances are either too close or too far apart [119]. This variable directly affects the strength of the electric field and the duration of the jet's flight. The reduced tip–collector distance has a nearly identical effect on the circuit as increased voltage [196].

Additionally, rheological and electrical properties like viscosity, surface tension, conductivity, and dielectric strength are significantly influenced by molecular weight. High molecular weight nanofiber solutions produce fibers with a larger average diameter while too low a molecular weight solution tends to produce beads instead of fibers [197]. Polymer molecular weight, which is important to the electrospinning process, reveals how many polymer chains condense in a solution. It has been suggested that if there are sufficient interactions between molecules to compensate for chain entanglements, high molecular

weights may not always be required for the electrospinning process. High molecular weight solutions typically result in the production of very long fibers. When a low molecular weight solution is employed, beads rather than fibers are produced [198].

**Figure 11.** SEM images of 5 wt.% chitosan hydrolyzed 48 h nanofibers in aqueous acetic acid 90%, electric field voltage: 14 kV (**a**), 16 kV (**b**), 17 kV (**c**). [196] Copyright 2023. Reproduced with permission from Elsevier Science Ltd.

#### *6.5. Applications of Chitosan Nanofibers*

#### 6.5.1. Chitosan Nanofibers in Tissue Engineering

Tissue-engineered scaffolds should be biocompatible and capable of mimicking the extracellular matrix to create a microenvironment conducive to cell growth. Natural bone extracellular matrix, for example, has a complex microstructure of multi-layered collagen fibers and calcium deposits with fiber sizes in the nano meter range [199].

Nanofibrous scaffolds, as nanostructured scaffolds, have the distinct characteristics of large surface area, high porosity, and mechanical strength, resulting in extraordinary biological properties such as mimicking the nanoscale properties of the extracellular matrix, promoting cell adhesion and migration, transporting nutrients, and discharging waste [200].

Among these properties are the functional groups of nanofibrous polymers, which can coordinate with other components to promote cell adhesion, proliferation, differentiation, and, eventually, tissue regeneration [201]. Nanofibrous scaffolds may be a viable solution for the synthesis of extracellular matrix substitutes with the required biological functions [201].

Chitosans are polysaccharide polymers that are similar to extracellular matrix components, so they can be metabolized and their degradation products can be stored as proteoglycans in vivo [202]. Because of their excellent biological properties, chitosan nanofibers are popular in tissue engineering research [202].

Nanofibrous scaffolds based on chitosan are widely used in a variety of applications, including nerve, bone, and cartilage engineering, cardiac and vascular tissue engineering, tendon, ligament, and skeletal muscle regeneration, and wound healing (Table 4). Because of their chemical properties, chitin and chitosan can be dissolved in solvents such as acetic acid, trifluoroacetic acid, formic acid, succinic acid, and 1,1,1,3,3,3-Hexafluoroisopropanol (HFIP) [203].

Electrospinning, self-polymerization, and thermally induced phase separation can all be used to create chitin/chitosan nanofibrous scaffolds [204]. Furthermore, chitin/chitosan nanofibers derived from polymer blending can alter the biological and mechanical properties of composite scaffolds [205]. To meet the requirements of tissue engineering, these composite nanofibrous scaffolds can mimic the nanoscale structure and porosity of the extracellular matrix [206,207].


**Table 4.** Tissue engineering applications of chitosan nanofibers.

#### 6.5.2. Chitosan Nanofibers in Enzyme Immobilization

Nanofibers, in contrast to other nanomaterials, have recently attracted attention for enzyme immobilization not only due to their higher surface-area-to-volume ratio and greater enzyme-loading capacity, but also due to their higher chemical and mechanical protection, which is crucial to ensure physical resistance for the support. The disadvantages of nanoparticles, such as the behavior of aggregation, which affects the stability and activity of enzymes, are not present in nanofibers. They also do not need an additional procedure (such as centrifugation or membranes) to separate nano capsules. The gathered nanofibers form a macrostructure of nanofibers that is simple to separate and repurpose [217–220]. Therefore, chitosan has been used as a support for enzyme immobilization because of its benefits [221–224]. In fact, it is important to note that chitosan-based nanofibers should be further investigated as a support for enzyme immobilization due to the characteristics of chitosan combined with the benefits of nanofibrous structures. The use of chitosan-based nanofibers in enzyme immobilization is summarized in Table 5.


**Table 5.** Chitosan–based nanofibers in enzyme immobilization.

#### 6.5.3. Chitosan Nanofibers in Cancer Treatment

There are numerous anticancer medications, including Doxorubicin, Paclitaxel, Berberine, Methotrexate, Adriamycin, Curcumin, Vincristine, Mercaptopurine, Indomethacin, Ibuprofen, Ketoprofen, Dexamethasone, Tetracycline, Gemifloxacin, Tetanus Toxoid, and Folic acid. The quantity of these anticancer medications is loaded onto the carriers of fibers during in vitro and in vivo cancer treatment. The polycationic nature of chitosan makes it a good candidate in this field among other fiber carriers. Table 6 lists several anticancer drug-loaded chitosan fiber systems along with information on how they affected cancer tissues in vitro.


**Table 6.** The chitosan nanofibers as anticancer drug delivery in vitro conditions.

#### 6.5.4. Chitosan Nanofibers in Food Technology

Chitosan's polycationic nature, biodegradability, nontoxicity, antimicrobial, chelating, mucoadhesive, and gelling properties set it apart. All these properties, combined with the high surface area: volume ratio of nanofibrous structures, make chitosan-based nanofibers suitable for a wide range of applications in food technology that have yet to be fully explored. As a result, the following sessions highlight some plausible studies in this field.

Food waste poses a problem for the food industry. According to the Food and Agriculture Organization of the United Nations [249], one-third of the world's food is wasted, causing economic and environmental problems. Indeed, food packaging has emerged as a viable option for improving the quality and safety of food products by increasing their biological, physical, and chemical stability. It is necessary to avoid chemical contaminants, oxygen, microorganisms, light, and moisture to achieve these properties. As a result, active packaging with antimicrobial properties has received a lot of attention [250–252].

Nanofibers made of chitosan have enormous potential for use in active food packaging. It has been widely reported that chitosan is effective against bacteria, viruses, and fungi [253–257]. In addition, chitosan, a renewable and biodegradable polymer, presents an intriguing real alternative to petroleum-based polymers in the development of green packaging materials.

Arkoun et al. [258] prepared chitosan/poly(ethylene oxide) (PEO) and tested its antibacterial activity against pathogenic microorganisms such as *E. coli*, *Salmonella enterica serves Typhimurium*, *Staphylococcus aureus*, and *Listeria innocuous*.

The nanofibers had an irreversible antibacterial effect, resulting in a bactericidal rather than bacteriostatic mechanism, according to the authors. Furthermore, bacterial growth was reduced at pH 5.8, which is lower than the pKa of amine groups on chitosan, and as a result, the authors proposed that the nanofibers could be applied to foods such as yoghurt, milk, cheese, meat, and fish, where lactic acid is liberated during the storage period.

Cui et al. [259] formed chitosan/poly(ethylene oxide) loaded with tea tree oil and tested it against *Salmonella enteric* subsp. *enteric serovar Enteritidis* and *Salmonella typhimurium*, two food pathogenic microorganisms. When the concentration of tea tree liposome was increased to 50%, the tensile strength increased by around 350%. Tea tree liposomes improved the antibacterial effect of the chitosan/poly (ethylene oxide) nanofiber as well. Furthermore, the chitosan/poly (ethylene oxide) loaded with liposomes tea tree demonstrated a four-day stable antibiofilm activity in chicken meat samples.

To prevent microbial spoilage of fish fillets, Ceylan et al. [260] created electro spun chitosan/thymol/liquid smoke nanofibers. The electro spun chitosan/thymol/liquid smoke nanofibers effectively reduced nearly 60% of total mesophilic bacteria. Furthermore, the authors stated that the nanofibers were thermostable until 150 ◦C, which is within the temperature range used in traditional food preservation methods.

The field of food packaging is a developing one that will most likely expand in the coming years. There are still numerous opportunities to investigate the role of chitosan-based nanofibers in other pathogenic microorganisms and different food types. Furthermore, the effect of other biopolymers and bioactive compounds on food packaging properties, such as mechanical properties and water vapor permeability, can be evaluated. Chitosanbased nanofibers could also be used to control food quality by monitoring external and internal conditions.

The majority of bioactive substances, volatile molecules, antioxidants, and flavors are unstable or even degradable [261].

Therefore, in addition to increasing bioavailability, chitosan-based nanofibers could improve the stability of functional food compounds. Chitosan's mucoadhesive property can be used to deliver bioactive molecules to the body in a manner similar to how drugs are delivered; the mucoadhesion of the functional compounds increases their absorption through the gastrointestinal tract [262,263]. These qualities have increased interest in using chitosan-based nanofibers as a drug delivery vehicle in addition to their non-toxic and biocompatibility benefits [264,265]. Although some research has been done using various nanomaterials as food carriers [266,267], chitosan-based nanofibers have not yet been sufficiently investigated in this regard.

As a delivery vehicle for curcumin, Shekarforoush et al. [268] created electro spun chitosan/xanthan gum nanofiber. The authors discovered that pH 2.2 had a lower release of curcumin from nanofibers than other studied pH values at 6.5 and 7.4. According to one theory, there were stronger electrostatic interactions between chitosan and xanthan at pH levels below the pKa of the amine groups on chitosan, such as pH 2.2, which decreased the swelling behavior of the nanofiber and, as a result, the diffusion of curcumin. As a result, curcumin was able to be transported using the electro spun chitosan/xanthan gum, which improved its stability and bioavailability.

Biocatalysts called enzymes are essential in the food industry. However, due to their poor operational stability, short shelf life, and challenging recovery and reuse, their green chemistry and substrate specificity are compromised [269,270]. These issues might be solved by the enzyme immobilization on chitosan-based nanofibers, though. Due to their high pore-interconnectivity, which improves mass transfer between the substrate and the enzyme and, as a result, the enzyme activity, they stand out as promising supports.

Chitosan's functional groups can be used to functionalize surfaces, which can then be used to adsorb controlled-release enzymes. Lactose is hydrolyzed using chitosan/poly (vinyl alcohol) nanofibers. According to the authors, at 50 ◦C, the immobilized enzyme was more thermostable than the free enzyme. Additionally, after 28 days of storage at 4 ◦C and 25 ◦C, the immobilized -D-galactosidase retained 77% and 42%, respectively, of its activity. As a result, the thermal and storage stability was increased by immobilizing -D-galactosidase in chitosan/poly (vinyl alcohol) nanofibers.

Nanofibers made of chitosan have been developed for the immobilization of enzymes. Indeed, they offer promising resources for the creation of hypoallergenic foods as well as a number of dairy products (baking, jam, jellies, wine, beer and juices). The application of chitosan-based nanofibers on the immobilization of enzymes in food processing, including amylase, trypsin, pectinase, protease, tyrosinase, lipase, pectin lyase, pectin, laccase, among many others, should therefore receive significant attention.

#### **7. Limitations and Future Perspectives for Chitosan Application**

The use of chitosan has several restrictions in addition to its many benefits. Chitosan's low solubility at a neutral pH is its most significant drawback. Numerous chemical and physical procedures have been employed to increase its solubility in order to get around this drawback. Chitosan has three types of functional groups: an amino group at C-2, a primary hydroxyl group at C-6, and a secondary hydroxyl group at C-3. Recent research has attempted to modify the three reactive functional groups in order to improve antimicrobial properties as well as solubility. For example, adding CH3 to chitosan increased its solubility and allowed it to be used in a wider pH range. The addition of disaccharides and Nalkylation increased its solubility and antimicrobial activity against *E. coli* across a wider pH range.

These findings collectively appear to suggest that chitosan can be modified in a variety of ways to increase its solubility and antimicrobial activity. Chitosan and its derivatives have received a lot of attention in recent decades due to their numerous applications in various fields. Several studies have shown that chitosan's antimicrobial properties are affected by a variety of factors, including pH, temperature, Mw, metal chelation, and microorganism type. In vivo studies have also shown that chitosan and its derivatives can be used to treat microbial infections with no side effects. More research is needed, however, to determine the optimal chitosan conditions.

Chitosan's nontoxic, biocompatible, biodegradable, and antimicrobial properties indicate that this compound and its derivatives have a wide range of applications, which have been discussed in this review. In the future, chitosan may be used as an alternative to synthetic bactericides for crops because it has already been shown to be effective against the treatment of bacterial infections in animals. In the food industry, it can be used in food packaging materials to extend the shelf-life of food products, as well as in dressings that treat wounds in the pharmaceutical industry.

However, more research is required to determine its mode of action. A more standardized and comprehensive description of procedures, on the other hand, is required to match the results of different investigators. More research is needed to understand the molecular events that underpin chitosan's antimicrobial action. Finally, more research should be focused on improving chitosan's antimicrobial activity while maintaining its low toxicity and biodegradability.

#### **8. Conclusions**

Chitosan-based polymers can be used in a variety of biological applications. Chitosan is a biomaterial that is both biocompatible and biodegradable. Chemistry increases the utility of chitosan. Chitosan and its derivatives are employed as nano-delivery systems in biotechnology. Electrospinning includes preparation, fiber configuration, material selection, intended applications, and the spinning method. Foundation polymers based on chitosan are anticipated to be used in biological applications more frequently.

The development of biodiversity materials with applications in biomedicine (tissue engineering, wound treatment, drug delivery), as well as environmental protection (air and water filters), is a promising field. Because of the difficulties, the most common method for electrospinning clean chitosan fibers is blending with a co-spinning agent, which has the advantages of easier electrospinning and complementary qualities for specific applications. The result is that chitosan has been combined with a wide range of synthetic polymers, such as poly (ethylene oxide), polyvinyl alcohol, poly (lactic acid), polycaprolactone, polyurethanes, polyamides, polyacrylates, polyethylene terephthalate, polyacrylonitrile, polyaniline, and natural polyproteins (collagen, gelatin, silk fibroin, sericin), as well as polyanionic polysaccharides (hybrid). The systematic analysis of the properties of these blend nanofibers revealed the advantages and disadvantages of each method, the main rules that should be followed when aiming for a specific morphology, and the impact of the co-spinning agents on the fiber properties, which further directs their use. All this foundational knowledge in chitosan electrospinning is useful for the design of materials for real-world applications, which appear to be focused on the fabrication of complex chitosanbased nanofiber blends or composites in order to meet the need for multifunctionality.

**Author Contributions:** Conceptualization, all authors; methodology, all authors; software, all authors; validation, all authors; formal analysis, all authors; investigation, all authors; resources, all authors; data curation, all authors; writing—original draft preparation, all authors; writing—review and editing, all authors; visualization, all authors; supervision, all authors; project administration, all authors; funding acquisition, all authors. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data is contained within the article.

**Acknowledgments:** The authors extend their appreciation to the Deanship of scientific research Majmaah university, Saudi Arabia, for funding this research work through the Project No: R-2023-495.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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## *Review* **An Overview on Wood Waste Valorization as Biopolymers and Biocomposites: Definition, Classification, Production, Properties and Applications**

**Francesca Ferrari, Raffaella Striani \*, Daniela Fico, Mohammad Mahbubul Alam, Antonio Greco and Carola Esposito Corcione**

> Department of Engineering for Innovation, University of Salento, Via Arnesano, 73100 Lecce, Italy **\*** Correspondence: raffaella.striani@unisalento.it

**Abstract:** Bio-based polymers, obtained from natural biomass, are nowadays considered good candidates for the replacement of traditional fossil-derived plastics. The need for substituting traditional synthetic plastics is mainly driven by many concerns about their detrimental effects on the environment and human health. The most innovative way to produce bioplastics involves the use of raw materials derived from wastes. Raw materials are of vital importance for human and animal health and due to their economic and environmental benefits. Among these, wood waste is gaining popularity as an innovative raw material for biopolymer manufacturing. On the other hand, the use of wastes as a source to produce biopolymers and biocomposites is still under development and the processing methods are currently being studied in order to reach a high reproducibility and thus increase the yield of production. This study therefore aimed to cover the current developments in the classification, manufacturing, performances and fields of application of bio-based polymers, especially focusing on wood waste sources. The work was carried out using both a descriptive and an analytical methodology: first, a description of the state of art as it exists at present was reported, then the available information was analyzed to make a critical evaluation of the results. A second way to employ wood scraps involves their use as bio-reinforcements for composites; therefore, the increase in the mechanical response obtained by the addition of wood waste in different bio-based matrices was explored in this work. Results showed an increase in Young's modulus up to 9 GPa for wood-reinforced PLA and up to 6 GPa for wood-reinforced PHA.

**Keywords:** biopolymers; biocomposites; renewable sources; wood waste; waste valorization

#### **1. Introduction**

Nowadays, the manufacturing of bio-based polymers is characterized by a strong development. Because of the present rapid expansion in the manufacture of these polymers, the use of plastic items created from them is also expanding, according to the European Environment Agency (EEA) [1]. However, they even correspond to a very modest portion of the market, since they are around one percent of the more than 368 million tons of plastic produced yearly. The total amount of biopolymers is going to increase up to 2.11 million tons [2]. The main challenge in the next few years will be to significantly reduce production costs. Economies of scale are vital for competitive pricing. However, because bio-based supply chains are often extensive, scaling them up is difficult, especially since many of the essential technologies have not been validated [3,4].

Today, growing attention is paid to the manufacturing of bio-based materials, starting from scraps. The circular economy has, in fact, grown in importance in academic study over the previous decade. A key development goal of the circular economy is the reuse of different kinds of wastes, particularly wastes from industrial operations [5]. The increase in population and the usage of polymers for disposable items and packaging generate uncontrolled waste, posing severe management and disposal issues. The unrestricted

**Citation:** Ferrari, F.; Striani, R.; Fico, D.; Alam, M.M.; Greco, A.; Esposito Corcione, C. An Overview on Wood Waste Valorization as Biopolymers and Biocomposites: Definition, Classification, Production, Properties and Applications. *Polymers* **2022**, *14*, 5519. https://doi.org/10.3390/ polym14245519

Academic Editor: Antonios N. Papadopoulos

Received: 3 December 2022 Accepted: 13 December 2022 Published: 16 December 2022

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

waste stream from many sources poses a major challenge to waste management [6]. Several researchers have been studying the potential and valorization of the organic component in a circular economy scenario. The valorization technique has numerous benefits over conventional organic fraction collecting and treatment technologies. These treatments use the organic portion as an energy source by burning or composting it [7,8].

The organic fraction of municipal scraps includes a huge quantity of carbohydrate and wood derivatives [9]. In today's world, the increase in population and the continuous usage of throwaway materials for lots of applications causes significant problems in waste management [2].

Among the different kinds of organic wastes, agro-industrial and forestry wastes present an unsustainable environmental and economic scenario [10]. Therefore, the valorization of this class of wastes represents one of the main issues in terms of disposal management. Many studies are currently being carried out to develop new procedures to produce biopolymers and biocomposites, starting with agro-industrial wastes. Due to the innovation of the topic, a summary of the last developments in the production of biopolymers and biocomposites can be useful to help carry out future research.

This work reports the current developments in the classification, production, properties and application of biopolymers, particularly those obtained from the valorization of wood wastes. Among the different application fields, the possibility to use wood scraps as a reinforcement for the production of biocomposites is also reported and the improvements in the performances obtained by the use of wood waste fillers are analyzed.

#### **2. General Definition of Biopolymers and Biocomposites**

#### *2.1. Biopolymers: Difference between Biodegradable and Bio-Based*

A polymer can be defined as a bioplastic when the material exhibits biodegradability, or it comes from bio-based or raw materials, or both [11,12]. Other researchers define a bioplastic as a material that can be decomposed into CO2, H2O and non-organic particles or biomass, generally thanks to the enzymatic decomposition carried out by microorganisms. Nevertheless, a bio-based polymer could be not biodegradable and vice versa. A bio-based polymer is derived from natural sources obtained from biomass, which can be partially or totally renewable. There are three basic methods for creating bio-based polymers. To achieve the performance criteria, one option is to partially change polymers derived from green sources, for example, cellulose, and lipids, using extraction, separation and filtration [13].

Depending on the type of synthesis and source, biopolymers can be classified into three groups (Table 1) [14]. Natural sources, such as carbohydrates and proteins, or monomers, such as lactic acid, can be the raw materials for biopolymers. Furthermore, other biopolymers such as polyhydroxyalkanoate (PHA) can be obtained from microorganisms [15]. Commercially accessible biopolymers are divided into the categories which follow: polyhydroxyalkanoates (PHAs/PHBs), polylactic acid (PLA) polyamides, polyols, bio-PET, butyl rubber and cellulose acetate; these are some of the materials used [16]. For practical usage in plastics or as water-soluble polymers, polysaccharides are mostly confined to starch and cellulose derivatives. Both of these compounds are made up of D-glycopyranoside-repeating units, which result in molecular weights in the thousands [14].


**Table 1.** A brief definition and classification of bio-based polymers.

#### 2.1.1. Biodegradable Polymers

Biodegradability is an important property of biopolymers, which does not refer to the raw materials used for the production. A biodegradable polymer could, in fact, be derived from fossil sources. Biodegradation is a biological process that occurs during composting, which involves the release of carbon dioxide, water, non-organic particles and biomass. In order to be defined as biodegradable, the biopolymer should degrade with a similar rate of certified compostable materials, without leaving hazardous residue [25]. If the plastic is able to decompose, but it doesn't follow the established standards, even if it is biodegradable, it must be classified as non-compostable. Finally, degradable materials should also not be derived from natural sources; for example, oxo-biodegradation is a phenomenon that occurs with some polyolefins through an oxidative process, which implies the breakage of the plastics into small pieces making them easier to biodegrade. Nevertheless, oxo-degradation is not currently classified as biodegradable or compostable, as its decomposition does not occur following established standards [26].

Table 2 shows the biodegradability of the most-used bioplastics in different fields. Biopolymer degradation depends on the physical and chemical structure of the biomaterial [27]. Furthermore, pH, temperature, moisture and oxygen must be considered.

**Table 2.** Definition of compostability and biodegradability of some bioplastics in different environments.



**Table 2.** *Cont.*

#### 2.1.2. Bio-Based Polymers

The greatest part of bioplastics currently available on the market are obtained from biomasses of the first generation, for example, corn, potatoes, sugar cane, palm oil and straw. All these sources have a high amount of carbohydrates and can be eaten by people and animals. Feedstocks of the first generation have a high efficiency for the production of biopolymers, since they need less land to grow and have a high yield of production compared to the other feedstocks. The technical maturity of these feedstocks is then very high [25], although the subtraction of sources to the food chain poses important issues.

The second generation of feedstocks is related to those raw materials which cannot be eaten by animals, such as non-edible harvests (e.g., cellulose) or derivatives of raw materials of the first generation, for example, sugarcane bagasse. Although second-generation feedstocks are commercially available, the use is not so widespread due to a relatively high cost.

Finally, the third-generation feedstock, obtained from food scraps, algae biomass and industrial or municipal waste, is the most innovative and can solve the problems related to the consumption of sources from the food chain. Several studies are underway in order to develop new biopolymers from food wastes [31].

Figure 1 shows a summary of the three generations of feedstocks.

This review is focused on a special class of the third-generation biopolymers, i.e., those obtained from the reuse and valorization of wood-based waste. The following paragraphs, hence, refer to the description of the main production methods, properties and applications of biopolymers from wood sources, with a special focus on wood scraps, such as agricultural waste.

#### *2.2. Biocomposites*

The tendency to substitute the petroleum-based polymers with biopolymers has also made its way into the field of composite materials. In the last decade, it has become a possibility to combine biopolymers with natural fillers in developing new materials, i.e., biocomposites. They have attracted the interest of the research community all over the world, as well as the industrial sector, for a wild spectrum of applications that these new materials can offer, such as aerospace parts, automotive components, consumer goods, sporting goods and their use in the marine and oil industries [32]. Biocomposites are defined as composite materials composed of biodegradable natural fibers used as reinforcement and biodegradable (or non-biodegradable) polymers in a matrix. Starch, cellulose, soya, polylactic acid and polyhydroxyalkanoates are the most commonly available biopolymers [33]. Natural fibers are largely divided into two categories: plant-based and animal-based. In general, plant-based fibers are lignocellulosic in nature, and they are composed of cellulose, hemicellulose and lignin; animal-based fibers consist of proteins, for example, silk and wool [34]. In fact, the great advantage offered by biocomposites with respect to traditional composites is related to low energy and low CO2 emission during their processing, biodegradability, renewability, low specific weight, higher specific

strength, and stiffness, high electrical resistance, low cost and good thermal and acoustic insulating properties [35]. Despite some drawbacks that affect biocomposites, mostly due to their high sensitivity to moisture, low durability and low adhesion between matrix and fiber [36], natural fillers play an important role in order to develop fully biodegradable green composites as a possible solution for contemporary environmental difficulties.

**Figure 1.** An overview of the classification of biopolymers.

#### **3. Production of the Main Biopolymers from Bio-Based Sources**

*3.1. Cellulose Traditional Sources*

Cellulose is constituted of anhydroglucose units linked by a *β*-(1,4) glycosidic bond. The repeating unit of cellulose is the glucose dimer known as cellobiose. In the condensation process, glycosidic oxygen bridges the sugar rings which are formed and the cellulose chains reach a degree of polymerization (DP) around 15,000 in native cellulose

cotton and 10,000 glucose units in wood cellulose. Each monomer has three hydroxyl groups that allow the creation of hydrogen bonds by influencing the crystalline packing and, consequentially, the cellulosic physical characteristics. The van der Waals and intermolecular hydrogen interactions allow numerous cellulose chains to stack in parallel and self-assemble into microfibrils, constituted of crystalline sections in which the cellulose chains are organized in a highly ordered form, and amorphous regions, which are less ordered than the former [37,38]. For such a reason, cellulose is a semi-crystalline substance whose crystallinity is determined by its source, extraction technique and treatments. The degree of crystallinity of wood-based and plant-based cellulose is typically 40–60% [39].

Cellulose is a nearly limitless polymeric raw material since it is the most abundant component in most plants. The availability, renewability and biodegradability of this material, as well as its low cost, are the major benefits, but it has two major drawbacks when compared to synthetic polymers: high hygroscopicity, owing to hydroxyl groups, and limited processing due to rapid disintegration [40]. Cellulose can be extracted from several natural sources, such as wood (lignocellulosic biomass), agricultural scraps, cotton, flax, hemp, sisal and especially vegetable byproducts [40]. Wood pulp is the most widespread raw material for cellulose processing, especially for paper and cardboard manufacture [31,41–43].

It is well known that cellulose, which is extracted from wood such as spruce, pine and many other trees, is being used to produce regenerated fibers such as viscose, lyocell, modal, cellulose acetate and cellulose triacetate. Cellulose esters and ethers are the principal industrially used cellulose derivatives, with the former being used in molding, extrusion and films, and the latter in a varied range of application fields (building materials, food, personal care products, paints and pharmaceuticals). However, until solvent methods for dissolving cellulose became available, the processability and application of this type of cellulose in biodegradable plastic films were limited. In order to modify the mechanical and chemical properties of cellulose, plasticization and blending with other polymers are used [44]. There have been a number of recent developments in the field of polymeric thermoplastic film and functional polymeric materials such as composite and composite films [45]. Cellulose-based biocomposite systems use cellulose as a reinforcement and/or matrix (host material) [45]. Cellulose fibers and derivatives are currently being used to make biopolymeric materials such as fillers and polymer matrices in biopolymer composites. Recent biocomposite research has enabled the replacement of petroleum-based polymers (PE and PP) with naturally generated biopolymers, such as cellulose and starch, and glass fibers with cellulose fibers.

Cellulose could also be used in wastewater treatment because it is naturally hydrophilic, it has been employed as an antifouling hydrophilic coating on membranes in order to increase the flow of the membranes and also the adsorption capability of cellulosebased functional materials is excellent for water treatment applications. It has been found that cellulose has a high adsorption capacity for pollutants after being subjected to appropriate chemical alteration on its surface, with the goal being the absorption of molecules with basic groups, particularly those containing significant concentrations of nitrogen, sulfur and oxygen [45]. In fact, as reported by Li et al. [46], cellulose, as well as other biopolymers such as lignocellulose, chitosan, chitin and lignin, shows a good absorption capability of heavy metal ions from aqueous solutions. Li et al. [46] collected the main studies related to cellulose properties, in particular, the capability of a cellulose-based copolymer to adsorb chromium (VI) and convert it in Cr (III) by means of the ultrasonication method [47]; the capability of cellulose aerogels to encapsulate iron oxides [48] and the employment of carboxylated cellulose derivatives for absorbing Co2+, Cu2+ and Ni2+ in aqueous solutions [49]. Dassanayake et al. [50] reported the use of cellulose-based materials and derivative (chitin and chitosan)-based materials in specific applications in which their absorption properties are employed, such as the removal of organic dyes and heavy metals, oil and solvent spillage cleanup and CO2 adsorption. Furthermore, cellulose-based functional materials are widely employed in biomedical fields such as drug delivery systems [51–53]; in cancer

therapy [54]; in bone regeneration [55,56] and in tissue engineering [57]. Demitri et al. [58], in fact, developed an innovative method for producing cellulose-based (CMCNa) foams, demonstrating an excellent biocompatibility profile with a good cell proliferation rate. When cellulose is linked with conductive polymers, it can form nanocomposites with high conductivity [59,60]. Shahbazi et al. [61] demonstrated that CMC modified by both photo and chemical cross-linking can improve surface hydrophobicity, the water barrier and mechanical properties of food packaging materials.

The applications of cellulose in the field of sensing material was also studied, including employing cellulose as a membrane for inkjet printing [62]; as CNT–cellulose composites on ammonia sensors [63]; as hybrid cellulose hydrogel used in release systems [64]; and as active mesoporous cellulosic materials with potential applications in optics, tissue engineering, chiral separation, functional membranes and biosensing [65]. Li et al. [46] also reported the employment of cellulose nanocrystal (CNC)–polymer nanocomposites as reinforcing material [66] or RGO–cellulose composites employed in storage energy filed as supercapacitors [67]. Furthermore, in the field of solar cells, cellulose-based composites were found to have a collocation, as studied by Bisconti et al. [68], that realized semitransparent perovskite–polymer composites by employing hydroxyethyl cellulose and obtaining advantages in terms of ease of processing; improvement of visible transmittance; and enhancement of thermal stability, by preserving the photovoltaic performances of semi-transparent perovskite solar cells.

#### *3.2. Lignin Traditional Sources*

After cellulose, lignin is the second most predominant sustainable bioresource and it is found in abundance in wood, which is the world's primary supply. It is considered as a waste product in a number of industrial processes [69–71]. Because lignin is found in biomass combined with cellulose and hemicellulose, it serves as a restrictive issue in the bioconversion of wood, which is now under investigation [72,73]. Lignin is a naturally occurring component of wood and plant cell walls. Its polyphenolic chemical structure has been studied for industrial applications. Various delignification chemical procedures can extract lignin from wood, which has a structure and qualities unique to each plant species. In recent years, however, the chemical industry has concentrated on using lignin as a feasible renewable source for the production of innovative and ecological biomaterials. Its organization is complicated and hard to describe, making it difficult to blend into polymers, fibers and other materials [74]. Its hydrophilicity, polyanionic structure and nontoxicity make it an ideal choice for modifying membrane bulk and surface properties. Various lignin derivatives have been studied extensively for bulk modification of polymeric membranes. A preliminary material, such as the wood from which the pulp is made, is normally required for the extraction of lignin from several types of biomass. Lignin yield is influenced by numerous factors, including extraction process, reaction time, medium and temperature. The extraction of lignin from cellulosic pulp can be conducted via enzymatic, chemical and physical techniques. Acid hydrolysis, the kraft process, the lignosulphonate process and organo solvolysis are just a few of the chemical processes used to extract lignin. Acid hydrolysis is a process in which the lignin in wood pulp is dissolved using a mixture of argon and concentrated hydrochloric acid (HCl) [75]. The lignosulphonate procedure includes heating wood with sodium sulfite (aqueous) in acidic conditions to extract lignin. Functions include surfactants, additives, dispersants and flocculants [76].

Lignin is another cheap, renewable, biodegradable plastic preference, which improves matrix polymer compatibility and UV stability. Functional groups in matrix and lignin interact to create positive compatibility with both natural and manufactured polymers. The reinforcing activity and thermal stability of lignin have resulted in a high modulus value. Additionally, thermal insulation ratings are a bit higher than those of other materials [77].

Lignin has been extensively explored for its possible use as a sustainable alternative to petroleum derivatives and chemicals. Its polyphenolic structure makes it suitable for usage in phenolic monomers, polyurethane foams, polyolefins, adhesive resins, packaging materials, unsaturated polyester, epoxy resins and material filler. Lignin can be chemically modified or incorporated into a matrix to provide it with new qualities. Banu et al. [78] selected several pretreatment methods (mechanical, chemical, biological, physical and physiochemical) able to extract lignin from diverse kinds of lignocellulosic biomass and reported the influence on medium and short chains of PHA yield for producing packaging materials, such as films, coatings, bags and bottles [78]. They also reported case studies concerning an increase in the production of PHA using genetically modified and engineered bacteria grown in lignin substrate. Furthermore, Banu et al. reported several synthesis processes aimed at producing lignin nanoparticles and their application in biocide systems [79], drug storage and delivery [80] and coatings [81].

Lignin conversion to high quality products is critical to a biorefinery's economic success. These studies have established catalytic pathways for the production of aromatic chemical reagents and bio-based compounds, epoxy resins, carbon fibers, phenolic adhesive resins, hydrogels, 3D-printed biocomposite and polyurethane foams [74]. To make polymer matrix composites, the amorphous polyphenolic macromolecule Lignin is utilized as a filler. The composite's characteristics are enhanced by the inclusion of lignin. Antioxidant properties of lignin make it a good stabilizer for polymers. Because char inhibits the combustion and the heat release rate of polymeric materials, lignin can produce a significant quantity of carbonize residue when heated at a high temperature in an inert atmosphere. This property is fundamental to flame-retardant additives. Lignin can also influence the structure of thermoplastic polymers by acting as a nucleating agent during the crystallization process [82]. A potential alternative to inorganic fillers is represented by lignin-based nanoparticles due to phenolic groups and their UV resistance and antioxidant properties. Furthermore, it was demonstrated that lignin-based nanoparticles are able to improve the mechanical and physical properties of the final nanocomposite. Banu et al. [78] reported the main lignin nanoparticle synthesis processes. Thanks to the several reaction sites of lignin (hydroxyl, carboxylic acid, phenolic clusters) it is possible to cross-link it with the polymeric monomers (polyesters and polyurethanes; phenol–formaldehyde resins). For such a reason, the development of different lignin-based biopolymers is possible. What has been studied, in fact, is how a lignin presence minimizes the biodegradation of polyhydroxyalkanoates (PHA), increasing the resistance towards the microbial activity [83]; how lignin enhances the biodegradability of polyester, increasing the photoreactivity and the glass transition temperature [84]; how lignin is blended to polylactic acid (PLA) in order to obtain higher flame resistance [85]; and how lignin can substitute 30–50% of petroleum-derived polyols for the synthesis process of polyurethane, etc. [86,87].

Figure 2 reports a schematic overview of the main bio-based polymers from traditional wood sources.

#### *3.3. Cellulose and Lignin Biopolymers from Wood Waste*

One of the most promising challenges is the possibility of exploiting biomasses from wood as biosources of hemicellulose, cellulose and lignin. Lignocellulosic biomass represents, in fact, the highest amount of unused global biomass [88] and it is mostly composed of dry matter with the addition of oils, minerals and other components, which account for less than 10% [89]. Biomasses from wood wastes can be obtained by using different raw materials, such as forest and crop scraps, municipal solid waste, wood and paper wastes [89,90], which influence the quantity of each constituent of the biomass. Lignocellulosic material shows different amounts of each component, in terms of chemical composition, since they are usually altered by the environment [91]. In particular, based on the amount of biomass, woods can be classified into hardwoods and softwoods which contain, respectively, higher (78.8%) and lower (70.3%) amounts of cellulose and emicellulose and, reversely, there is lower lignin content in hardwoods (21.7%) than in softwoods (29.2%) [89]. Since the cellulose, lignin and hemicellulose amount depends on the kind of wood biomass, a suitable material should be chosen for the fermentation.

**Figure 2.** Sources and purposes of biopolymers from traditional wood sources.

Plant biomass can, therefore, be used to produce high-performance functionalized polymers. Large-scale lignocellulosic biomass production will provide plentiful renewable feedstock for biomaterials with physical and chemical performances that are equal to or higher than those of petroleum-based mixtures, including lignin, cellulose and hemicellulosic polysaccharides [92].

Agro-waste recycling by composting and fertilizer manufacturing boosts global carbon emissions, according to data collected in Italy. Wastes from olives (OWC) and anaerobic digester-based compost (ADC), respectively, yielded 64 and 67 kg of CO2 equivalent per milligram. Furthermore, each milligram of compost produced by re-composting and co-composting, ranging from 8 to 31 kg of CO2, was released [93].

Particular attention was given to agro-waste cellulose for producing biopolymers in a critical review by Motaung et al. [94]. As discussed by the authors, even if several studies on the chemical modification of cellulose fibers were well known, very few discuss agricultural cellulose waste fibers. Sundarraj et al. [95] focused their study on the cellulose derived from agro-industrial residues as effective reinforcement for the building construction material industry. Lately, Urbina et al. [96] collected in their review the main case studies about the production of bacteria cellulose by employing agro-wastes (residues of agricultural products), focusing on the applications of this kind of biopolymer for environmental applications, optoelectronic and conductive devices, food ingredients and packaging, biomedicine and 3D-printing technology. El Achaby et al. [97] studied the employment of red algae waste as a natural resource for producing superior cellulose nanocrystals and their capability to act as strengthening filler.

The extraction of lignocellulose from waste materials represents a great environmental advantage since it avoids the problem that agro-waste can become a source of contamination.

Lignin can also be derived from wood waste. In fact, as demonstrated by Zikeli et al. [98], a lignin fraction was isolated from the wood wastes of a wood house producer for the production of lignin nanoparticles and then used for wood surface treatment. The developed coatings showed significant results after an artificial weathering test. Nevertheless, the extraction of lignin from wood waste is an open research question due to the difficulty of processing and the consideration of lignin as a waste material, i.e., an undesirable component in the manufacture of ethanol and paper, as reported by Parvathy et al. [99]. Thanks to its high thermal stability, biodegradability, antioxidant property, cross-linked

structure and UV absorption characteristics, lignin could be effectively employed in several applications to produce valuable materials.

Figure 3 reports a flow production of bio-based polymers from wood waste.

**Figure 3.** Flow production of bio-based polymers from wood waste.

#### *3.4. PHAs' Traditional Sources*

Microbial manufacturing techniques are used to produce polyhydroxyalkanoates (PHAs). PHAs are a class of aliphatic polyester which are naturally obtained in a sugarbased media by bacteria and operate as carbon and energy storage materials. They were the first biodegradable polyesters used in the plastics industry. Aliphatic polyesters are the easiest synthetic polymers to biodegrade [14].

Synthesis of PHAs requires the use of different organisms, especially plants and bacteria, often employed for a large-scale production [100]. On the other hand, plants allow the producing of only small amounts (<10% (*w*/*w*) of dry weight) of PHA, since higher quantities of storage PHAs inside the plants lead to negative effects on the plants' growth [101]. Therefore, synthesis of PHA is actually carried out by bacteria, which naturally accumulate more than 90% *w*/*w* of PHAs in order to store carbon and energy during the metabolism of nutrients [102]. In particular, the accumulation of PHAs only occurs if bacteria grow with a reduction in oxygen, nitrogen and phosphorous, and an increase in carbon sources [103].

Once the soluble nutrients and intermediates are converted into insoluble PHA polymers, PHAs are stored in intracellular granules inside the cell. In this way, the osmotic state of the cell is preserved, which in turn means a secure storage of the nutrients without any losses through the cell membrane [104].

After the production, the PHA pellets are coated with a layer of proteins and phospholipids. This layer, mainly composed of a particular class of proteins, the phasins, changes both the size and the amount of PHA pellets [105].

Among PHAs, PHB is the first and commonly used, obtained by Alcaligens Eutrophorus bacteria through the conversion of acetyl-CoA in the following three steps [106], reported in Figure 4:


**Figure 4.** Synthesis pathway of PHB.

PHB is characterized by high hydrophobicity and can be produced at low temperatures. However, PHB is thermally instable when heated close to the melting of the material. The polymer exhibits essential thermoplasticity and degradability qualities in decomposition and other settings including sea water and, as a result, it has gained a lot of industrial attention [107–109]. PHB, a commercially accessible biopolymer, is one of the most attractive members of the polyhydroxyalkanoates family for the packaging of food. PHB is a polymer which should be modified using standard industrial polymer processing facilities. It also has strong mechanical qualities, such as strength and stiffness, that are equivalent to or better than some of the products (such as PP), as well as good barrier properties (similar to PET). PHB degrades in decomposition situations and in other conditions, such as in seawater [109]. Even though PHB is a good choice for green applications such as packaging, it has significant flaws that prevent it from being widely used in the packaging industry. Owing to room temperature crystallization and physiological ageing phenomena, PHB has a relatively high instability which increases over time. PHB also has a small processing range which makes it difficult to treat in some typical packaging applications, such as heat treating [110]. Another major impediment to its application in the packaging industry is its expensive cost, which continues to surpass EUR 5/kg. In this regard, the inclusion of a long-lasting, low-cost or hard filler might help mitigate the raw price rise by (a) minimizing the overall packaging cost and/or (b) decreasing the thicknesses required in standard packaging [110].

#### *3.5. PHA from Wood Wastes*

A particular problem that limits the use of PHAs is related to its cost of production, starting from the raw materials to the recovery process [111]. In particular, the price of PHA raw materials is strongly attributable to the carbon contribution; therefore, many efforts are currently made in order to replace traditional sources with derivatives of wood scraps [112]. A potential way to replace traditional sources was studied by Kumar [113], who used wood hydrolysates obtained using enzymatic hydrolysis of hemicellulose and

cellulose fermentable sugars. To use substrates of wood hydrolysates, the appropriate bacteria must be selected. In his study, Kumar [113] tested various colonies of different morphologies of bacteria for the production of PHA, using biomass substrates obtained from wood wastes. Results obtained allowed the production of PHA from paper paste and tannery effluent water samples with both using gram-positive and gram-negative bacteria, even if gram-negative bacteria were predominant.

PHA properties are mostly related to the length of the polymer chain. Long-chain (PHAs with C ≥ 15), medium-chain (PHAs with 6 ≤ C ≤ 14) and short-chain (PHAs with C ≤ 5) polymers are the three types of PHAs, considering the amount of carbons in the monomer units [114]. Short-chain polymers cannot be used if a high strength is required, because they are too brittle, high-crystalline and stiff. Medium chains have higher elastic modulus and, therefore, show lower brittleness, higher elongation at break and low-crystalline zones. On the other hand, these PHAs are less suited to high-temperature applications [115,116]. Films, fibers [117,118], foams, food additives, medical implants [119], medication delivery carriers, control release material, medical scaffolds for tissue regeneration [120], biofuels [121] and animal feeds all include PHA. PHA can be converted into chiral hydroxyalkanoic acids (HAs). PHA is a renewable substance that should be approved by the market [122]. The high manufacturing costs of PHA, which are at least three times higher than those of traditional materials, e.g., polypropylene (PP), polyethylene (PE) and related biopolymer polylactic acid, have contributed to their limited success [123]; therefore, as explained in previous paragraphs, alternative sources derived from wastes are currently under investigation, which allow for a strong decrease in the production costs.

PHAs showed a higher barrier and mechanical properties than PLA. Nevertheless, they only account for 1.4 percent of the biopolymer industry, even if their manufacturing is expected to double by 2023. PHA has comparable brittleness constraints to PLA, and its fragility can be reduced by the addition of a specific plasticizer. Though PHA has higher barrier properties than other biomaterials, the disadvantages in manufacturing costs are higher and its recycling process is still under development [109,124]. While PET recycling methods are well known and commonly employed [6], there are few studies of the mechanical and chemical recycling of PHA due to the high production costs and low yields of the recycling process [125]. PHAs can then be manufactured and processed for use in many applications, including packaging, thermoplastic products, protective coatings, nonwoven textiles, resins, sheets and activity enhancers, to name a few. PHAs, in contrast to other biomaterials, have a lot of promise for applications such as packaging because of their superior thermal–mechanical and protective qualities [109].

#### *3.6. PLA's Traditional Sources*

PLA is obtained from lactic acid precursors, which are produced from renewable sources such as sugar feedstock, straw maize, corn and food or agricultural waste products via fermentation [31,126].

Nowadays, fermentation represents the most common way to produce lactic acid, particularly if pure optical isomers are needed.

To obtain lactic acid, three main steps are required:


The main sources of starch which can be used in the first step are as follows: corn (maize), straw, tapioca (cassava), potatoes and other raw materials which, after hydrolysis, are transformed into mono- and disaccharides (Figure 5). The hydrolysis of starch was first carried out by using chemicals, but nowadays enzymatic methods are preferred. On the

other hand, only few bacteria are suitable for fermentation, because maltose results in the key product of enzymatic hydrolysis.

**Figure 5.** PLA from feedstocks to the final product.

Furthermore, sucrose-derived raw materials can be used for the fermentation of lactic acid. Conversely, lactose has a limited use because of the low quantities which can be found in readily available whey, and it also requires a high purification of whey [127,128].

Afterwards, to produce lactic acid, different families of bacteria (LAB) can be used (e.g., *Lactobacillus*, *Streptococcus* and *Pediococcus*), which are characterized by high productivity in very narrow pH ranges [114] and allow the producing not only of lactic acid, but also of other organic acids during fermentation. Therefore, in order to focalize the production to lactic acid, decreasing as much as possible the number of other byproducts, the pH is kept between 5.5 and 6.5, thanks to particular bases such as hydroxides or carbonates.

To select the right raw material for the fermentation, some parameters must be considered, such as the availability, the price and, above all, the purity, which influence the field of application of PLA. Mono- and disaccharide traditionally employed mainly derive from the conversion of different substrates and are:


#### *3.7. PLA from Wood Wastes Sources*

Lactic acid, traditionally used in several fields (chemical, cosmetic, food industries and pharmaceutical), is a hydroxycarboxylic acid characterized by two optical isomers [129–131].

Recently, the use of optically pure lactic acid (l- or d-isomer) was studied as a building block for the polylactic acid (PLA: PLLA and PDLA). As already mentioned, PLA is one of the most eco-friendly biomaterials and can be used as a green alternative to traditional fossil polymers [132,133]. Nevertheless, the traditional ways to produce PLA cause the subtraction of important sources from the food chain; therefore, many efforts are currently made to obtain PLA from scraps, thus producing a biopolymer of the third generation.

An innovative way to optimize PLA mechanical properties, using optically pure lactic acid isomers, involves the mixture of pure PLLA and PDLA, thus obtaining Sc-PLA, a stable stereo-complex with good mechanical properties, higher hydrolysis resistance compared to the use of a single enantiomer [134], a melting point ~50 ◦C higher than the pure materials and higher biodegradability [135].

The pure isomers can be obtained through microbial fermentation [136,137], although there are many issues which hinder the scale-up of the production from laboratory to industries, such as the high cost, the raw materials and the nutrient sources [136,138]. In order to solve these problems, second-generation feedstocks, which involve lignocellulosic biomass from agro-industrial or forest sources, are nowadays studied as inexpensive renewable sources for lactic acid production and the consequent microbial fermentation to produce PLA isomers [139,140] (Figure 6).

**Figure 6.** Synthesis pathway of PLA.

Lignocellulosic feedstocks, although seemingly a promising way to replace traditional sources for PLA production, are characterized by a complex structure; therefore, their conversion in optically pure lactic acid is still a challenge [141]. Furthermore, all the feedstocks must be pretreated in order to remove the lignin, thus allowing the enzymes to access the cellulose. Furthermore, inhibitions of the enzymatic catalysis can occur due to the pretreatment of the material; the inhibition mainly consists of the slowdown of both cellulose hydrolysis of lignocellulosic biomass and of the microbial growth [142].

Another drawback which occurs with the use of lignocellulosic scraps is that they are composed of a heterogeneous mixture of sugars, which cannot be easily used at the same time.

The main bacteria often used to produce lactic acid are lactic acid bacteria (LAB). Nevertheless, their use is still not widespread due to many factors, such as the requirement of specific nutrients, the low resistance to acid and the difficulties with co-utilizing glucose and xylose [143]. Recent improvements in bacterial D-lactic acid involve a genetic modification [144,145]. Some of the recent advanced processes, aiming to increase D-lactic production, include fed-batch fermentation, continuous fermentation with cell recycling and integrated membrane fermentation [146].

The industrial and commercial employment of lignocellulose to produce lactic acid is still an issue. In fact, various processing steps are necessary to convert the lignocellulosic biomass to monomeric sugars that are then fermented to obtain lactic acid. The conventional processes used to produce lactic acid from wood biomass involve four main steps: First, the lignocellulosic raw material needs to be pretreated, to break the structure of the biomass; after pretreatment, enzymatic hydrolysis occurs, thus obtaining fermentative sugars through hydrolytic enzymes. The third step consists of fermentation, which allows the metabolization of sugars to lactic acid, usually via LAB. Finally, in the fourth step, the lactic acid is collected and purified. It is worth highlighting the important role of the pretreatment of lignocellulosic biomass, since the native lignocellulose has a low enzymatic susceptibility due to the association of cellulose and hemicellulose with lignin [147,148]; therefore, the efficiency of the pretreatment plays a key role for the subsequent saccharification via hydrolytic enzymes. Finally, if the pretreatment is too strong, toxic materials can be released with a consequent inhibition of the microbial metabolism and growth [149].

#### **4. Main Properties of Bio-Based Polymers and Biocomposites**

Generally, the most investigated features for newly developed biofilms and biocomposites are morphological, thermal, mechanical, rheological, water absorption and antibacterial properties [40,150–153]. Their characterization involves the use of specific analytical techniques, some of which are discussed in this section and shown in Figure 7. Specifically, microscopy (Scanning Probe Microscopy SPM, Scanning Electron Microscopy SEM and Transmission Electron Microscopy TEM) is utilized for the measurement of morphology and porosity, the picometer is useful for density measurement, X-ray Diffraction (XRD) allows one to determine crystallinity, spectroscopy (UV-Visible and Fourier Transform Infrared Spectroscopy FTIR) is employed for molecular chemical characterization, thermal analysis (Thermogravimetric Analysis TGA, Differential Scanning Calorimetry DSC and Dynamic Mechanical Analysis DMA) is employed for the measurement of thermal properties and the study of thermomechanical degradation, water and oxygen adsorption is considered for the evaluation of barrier properties and Tensile and Flexural Testing, the Charpy impact test and Shore Hardness are utilized for the measurements of mechanical properties, etc. [40,150–155]. Usually, the environmental impact and biodegradability of biopolymers and biocomposites are evaluated according to American Society for Testing and Materials (ASTM) standards [153].

**Figure 7.** Some of the characterization methods commonly used to study the main properties of biopolymers and biocomposites.

The mechanical qualities of biopolymers include strength [15], ductility [156], deformability [157], stress [158] and durability [159]. Mechanical properties are usually detected using a dynamometer and by choosing the geometry, the load cell and the test speed according to the appropriate standard test method. Six replicates for each measurement are usually performed, in order to obtain statistically relevant results. Durability tests are carried out on a climate chamber, following the ageing procedure described in the standard test method. The degradation in the flexural modulus, in the strength of the composites and in the interlaminar strength are evaluated after the ageing.

The intrinsic qualities of a polymer are determined by its structure and/or chemical composition [160]. Density is a fundamental feature of polymers, varying between classes and constituents [160] and often measured using a pycnometer. In comparison to petroleumderived polymers, most biodegradable polymers have higher densities.

Though crystallinity isn't considered a self-reflective feature, it affects many other qualities. High-crystalline biopolymers are more resistant to dissolution than low-crystalline biopolymers. A single biopolymer's crystallization degree affects its melting and glass transition temperatures [160], as well as affects solubility; with greater compactness of the structure, there is a lower chance of dissolution [160]. In order to evaluate the crystallinity of the material, both X-Ray diffraction and Differential Scanning Calorimetry are used.

Scientific studies show that the tensile strength, and thus the crystallinity and solubility of biopolymers, can be increased by inserting different kinds of fillers into the polymer matrix, obtaining biocomposites [161]. For example, both PLA and polyolefin-based composites containing wood fillers have similar mechanical properties [161]. Generally, the addition of wood filler to the polymer matrix has different effects on tensile strength. In fact, the measured values arise from the wood species used as fillers and the amount added, the composites' processing methods and the quality of the PLA [161–163]; an additional cause of the reduced mechanical properties of the polymer composites with wood filler is also due to the poor adhesion at the interface between the filler and polymer matrix [154]. This phenomenon is especially evident from high-magnification images obtained using scanning electron microscopy (SEM) on both biofilms and biocomposites and/or biofilaments for 3D printing. For example, some authors report that the transparent films produced have pores and cracks that facilitate the passage of water vapor and gases [150]. Alternatively, other works highlight in SEM images of composites in general, or composite filaments for 3D printing, an irregular, rough, outer surface with several points of discontinuity due to the addition of wood filler, which is often non-homogeneously distributed in the polymer matrix, with the formation of particle aggregates [154,164]. Furthermore, the presence of voids and poor interfacial adhesion between layers (observable in composite sections and often on 3D prints as defects between layers) are attributed to the different polarity between the biopolymer (which has a non-polar surface) and the wood fillers (which have a polar surface) [154,164]. Instead, SEM results often indicate correct dispersion of the filler in the polymer matrix and an improved hydrophobic nature compared to the neat polymer film [151]. Overall, the results depend on the physical and mechanical properties of the polymers and fillers, concentration of the parts, filler geometry, polarity, compatibility, addition of plasticizers and type [151,152,154,165,166]. However, most studies show that, overall, the stiffness of polymeric composites with wood fillers increases, as does the crystallinity compared to pure polymer, and scientific research has focused on improving structural properties, such as through the use of plasticizers [161]. XRD and DSC analyses are useful in order to evaluate the effect of the addition of a plasticizer to different kinds of biopolymers. As reported by Greco et al. [155], the mechanical properties of PLA can be tailored by the addition of different green plasticizers. The plasticization of PLA can be carried out via the extrusion of the polymer with a specific amount of different bio-based plasticizers (e.g., neat cardanol, epoxidized cardanol acetate (ECA) and poly(ethylene glycol) (PEG 400)). Results evidenced the plasticization effectiveness of the different additives in terms of the reduction in glass transition temperature: compared to neat PLA, which showed a Tg of 60 ◦C, the addition of PEG 400, cardanol and epoxidized cardanol involved a decrease in the Tg to 17.5 ◦C, 16.9 ◦C and 22.3 ◦C, respectively. On the other hand, the addition of the plasticizer can influence the degree of crystallinity of the polymer: faster crystallization and an increase in the degree of crystallinity was found for PLA plasticized by PEG, compared to PLA plasticized by cardanol derivatives. Furthermore, mechanical properties were influenced after plasticization, which involves a decrease in the stiffness of the polymer. In particular, as reported in Table 3, different results were obtained if the plasticized polymer was amorphous or semicrystalline; neat amorphous PLA had a Young modulus of 1740 MPa. When PLA was plasticized with cardanol derivatives and an amorphous structure was detected after plasticization, a good efficiency occurred; however, the thicker crystals formed during crystallization of PLA with PEG and cardanol derivatives led to a high increase in the stiffness of the material.


**Table 3.** Young's modulus (MPa) in relation to the added plasticizer and the initial polymer structure.

Xie et al. [167] studied the influence of plasticizing agents (glycerol and tributyl citrate, TBC) on the stability and water absorption of samples based on poplar wood flour and PLA for 3D printing. Two different weight concentrations were tested for each plasticizer agent, 2% and 4% wt. Good compatibility between filler and polymer, good interfacial adhesion and good mechanical properties were obtained using TBC at 4% wt. [167]. In contrast, Zhang et al. [168] developed high-yield esterified lignocellulose nanofibers (LC-NFs) from lignocellulose (LCs) by swelling with a deep eutectic solvent lactic acid/choline chloride (LA/ChCl DES) (100 ◦C for 3 h), followed by mechanical colloidal grinding. LC-NFs/PLA composites were obtained via direct mixing, and the morphological, structural and mechanical properties were studied. The authors showed a significant improvement in compatibility at the LCNF/PLA interface highlighting the potential of natural woodderived nanofibers for making bio-based composites [168]. However, there is an impact of plasticizers on tensile strength up to a threshold value, because the tensile strength decreases with an increase in the amount of plasticizer, particularly in the case of films produced from starch [169]. Mechanical properties are lower for wood–PHA polymers, and even more so for starch. Young's modulus, tensile strength and stiffness decrease for wood–PHA composites, while strain at break is higher [161,170]. Scientific interest in PHA polymers with wood is growing due precisely to their sustainability and interesting and modular mechanical properties [161,170]. For example, Mehrpouya et al. [171], in their work focused on 3D printing, report some examples where the combination of PHAs and natural wood-derived additives (i.e., fibers, lignin, fibrillated nanocellulose, cellulose nanocrystals, etc.) makes remarkable improvements to the strength and microstructural features of pure PHAs. Wu et al. [172] achieved 20% higher parameters of tensile strength at break (7%) and Young's modulus (65 MPa) than pure PHAs by developing a wood–PHA composite (PHA-g-MA/TPF) with polyhydroxyalkanoate engaged with maleic anhydride (PHA-g-MA) and palm fiber treated with coupling agents (TPF) [172]. In addition, the use of wood–PHA composites also offers the possibility of reducing costs compared to pure PHA, while still using a sustainable material [170,172].

Thermoplastic starch (TPS) is the least-used material for the development of wood plastic composites (WPCs), compared to materials such as PLA and PHAs; it has poor mechanical and water barrier properties, and difficult processability [31,161]. Yet, wood filling to the polymer-based specimen results in improvements in tensile strength at break, elastic modulus and elongation at break, and these changes are even more pronounced than with other biodegradable polyesters, due to greater compatibility between the two materials (starch and wood) due to the hydrogen bonding of OH groups [161,173]. Zeng et al. [174] investigated the consequences of three additives (plasticizer, cross-linking agent and blowing agent) on the mechanical properties of a foamed composite made of starch, wood fibers and polyvinyl alcohol (PVA), using a predictive model. The work shows how using the appropriate amount of plasticizer (glycerin/NaOH) can reduce the crystallinity of starch and increase the compatibility between the different components and the tensile strength of the ultimate material (maximum range of 5.91–6.12 MPa) [174].

Dorigato et al. [175] improved the final mechanical properties of fully biodegradable composites laminated with starch and beech wood by impregnating them with

poly(ethylene glycol) (PEG) and consolidating via hot pressing [175]. Alternatively, Harussani et al. [169] analyzed various quantities of two plasticizers (sorbitol and glycerol) in different concentrations by weight (30%, 45% and 60%) on cornstarch-based composites, obtaining better mechanical performance with the use of 30% sorbitol: increased tensile strength (13.61 MPa) by 46% and unchanged elastic modulus [169].

Unlike PLA and PHAs, research on starch-based bio-based composites has focused more on understanding the increase in mechanical performance due to different types of wood fillers, and not on increasing the adhesion of the interface and material compatibilities through the use of plasticizing agents [161,174]. For example, Curvelo et al. [176] measured a 100% increase in tensile strength and 50% increase in elastic modulus, compared to pure thermoplastic starch, after the addition of Eucalyptus urograndis pulp fibers to the composite in 16% wt. concentration [176].

Until today, the use of polymeric degradable matrices for the production of composites has also sometimes been reduced due to their poor moisture and gas barrier properties and limited stability [161]. Barrier, rheological and thermal properties are in fact also of great importance, and are closely related to the structural and mechanical properties already mentioned. For example, the glass transition temperature (Tg) of different pure materials exhibits great differences (i.e., Starch 31–98 ◦C, PLA 45–60 ◦C, PHA −4–8 ◦C) [161], and the addition of wood fillers makes different changes to this and to the viscosity and processability of wood–bioplastic composites [154,169].

Polymer barrier qualities are strongly linked to their capability to allow the interchange molecules that have small size. The form, orientation and crystalline nature of the diffusing molecule, as well as the degree of polymerization and polymer chains, influence the barrier qualities. No material is totally resistant to ambient gasses, water vapor or to other generic natural compounds. In terms of ability of the biopolymer to permeate water or oxygen molecules, generically it is possible to identify three classes: permeable to both water vapor and oxygen; low ability to permeate water vapor but high barrier against oxygen; or contrastingly, less permeable to oxygen but very permeable to water vapor [161]. Among all bioplastics, starch-based composites usually exhibit higher hydrophobicity and low water resistance [31,161,174]. For this reason, several scientific studies have also been conducted to improve the barrier features of starch-based materials by modifying the structural properties through natural fillers of different origins. Curvelo et al. [176], in their starch-based composites developed by the addition of wood pulp (Eucalyptus urograndis at 16% wt.), also measured a significant improvement in water absorption properties. The new composites were conditioned by maintaining relative humidity values of 43 and 100%, and a temperature of 25 ◦C, and the water absorption values were found to be almost halved, unlike the values of neat starch [176]. Miranda et al. [177] studied changes in the structural and absorption properties of flexible thermoplastic films of corn starch, following the addition of cellulose nanocrystals (CNC) as reinforcement, and measured a significant improvement in the stability and barrier properties of pure starch [173]. Alternatively, Chen et al. [178] investigated the addition of nanoscale cellulose particles of different types (bamboo, cotton linter and sisal) to starch samples, using different content (0–10 wt%), and obtained better water vapor barrier properties with the use of bamboo nanocellulose, among others [178].

PLA and PHA-based composites, on the other hand, have higher intrinsic water resistance properties than starch [161]; however, the water barrier properties of biocomposites, to which a wood filler is added, decrease as the percentage content of wood particles/fibers increases and improvements are needed [178]. Da Silva et al. [178] studied the reaction of Struktol used as an adjuvant on the final features of wood–PHB biocomposites, showing a slight improvement in water absorption properties only for the sample containing 20% of wood filler [178]. Wu et al. [172] showed lower water absorption of the developed wood–PHA composite (PHA-g-MA/TPF) with polyhydroxyalkanoate grafted with maleic anhydride (PHA-g-MA) and palm fiber treated with coupling agents (TPF), compared to the corresponding PHA/PF composite [172]. In addition, Song et al. [179] focused their work

on increasing the water vapor barrier characteristics of PLA films by using nano-cellulose fibers (NCFs) modified by adding hydrophobic molecules onto the NCFs to increase the compatibility between NCFs and PLA during mixing. Paper water vapor transmission rate (WVTR) tests were conducted following different operating parameters and with different weight coatings showed that the coating of NCF/PLA innovative samples reduced the WVTR (up to 34 g/m2/d) [179].

Some important properties of the main types of bio-based wood composites are reported in Table 4.


**Table 4.** Some features of the main types of bio-based wood composites.

#### **5. Market Scenarios and Applications of Biopolymers and Biocomposites**

The global bioplastics market is growing significantly due to an increased focus of manufacturers and users on the use of sustainable products, social and economic factors and the more restrictive legislation implemented in recent years [192]. For example, the United Kingdom (UK) introduced a new tax from April 2022 on plastic packaging (called "Plastic Packaging Tax, PPT"), locally produced or imported, containing less than 30% reused plastic by weight, to increase the use of biopolymers, recycling and sustainable development. Aside from this, the tax in Italy has been postponed to the year 2023 [193]. Overall, estimates report a likely increase in bioplastics production from the current USD 9.2 billion to USD billion by the year 2026 [194]. Among different geographic areas, Asia is the largest production center (producing 46% compared to total bio-based polymers produced worldwide), followed by Europe (26%) and North (17%) and South America (10%) [27,194]. Europe is the geographic area where research activities on bioplastic development are most concentrated, and biopolymers are especially used in food packaging (60%) followed by other industries such as agriculture (13%), consumer goods (9%), coatings and adhesives (9%), textiles (5%), automotive (1%) and all others (3%) [27,192]. Several elements influence the market scenarios of bio-based polymers, and consequently also the application areas: the availability of raw materials and renewable sources, the ease of scalability and the production process, the biodegradability features (i.e., durability, degradation conditions and end-of-life treatment) and costs [27]. By cost it is not only meant that which is associated with the recovery of the raw material, but also that of its processing which must be suitable for market demands so that users prefer biopolymers to traditional polymers of petrochemical origin, which usually possess good mechanical and structural properties and often low costs [192,195]. Cost becomes a significant factor, especially in areas known for the development of single-use plastics for food packaging or low performance. Usually, bioplastics are more expensive than petroleum-based plastics. In addition, bioplastics also often have a higher density, causing further cost increases. However, there are exceptions when prices are compared at the product level; redesign and specific material properties can result in raw material savings, for example, due to the higher stiffness of PLA compared to PS, rigid PLA products can be reduced in thickness or the use of additive techniques, which involve adding natural or recycled fillers to the polymer matrix, can further reduce the initial cost of the biopolymer [12,154,196,197]. For example, in the European market, Nylon 6 (virgin polymer, PA6) costs around EUR 2.5–4.0/kg, while low-density polyethylene (LDPE) costs EUR 1.1–2.7/kg and PET costs around EUR 1.60–5.0/kg [193,198]. The price of petroleum-derived plastics is likely to rise further, due to rising raw material and energy costs in recent months. However, the cost per EUR of bioplastics has decreased significantly in recent years; for example, PLA, which had a cost of EUR 7/kg in the 1990s has dropped to

about EUR 1.6–2.5/kg in 2022 [198]. Rising oil prices have thus made the cost of bio-based plastics similar to that of thermoplastics of synthetic derivation; moreover, recent progress in the development of biopolymers has caused an increase in demand in different industrial sectors, such as food [110,199–201], pharmaceutical and medical [45,202–205], personal care [206,207], cosmetics [206,208] and textile and fashion [12,209,210] (Figure 8).

**Figure 8.** Main application of biopolymers and biocomposites.

#### *5.1. Biopolymers in Food Industry*

Generally, several applications of biopolymers sintered and extracted from animals, plants or marine organisms are known in the literature in the food field. Among these, extracts from plant species, such as cellulose, lignin, polyphenols and essential oils have been extensively used for different aims [211]. For example, they are used for the fabrication of edible films and coatings [212]; these constitute a thin layer of soluble bioactive compounds that is applied to the surface of foods or between layers for different purposes such as extending food shelf life, improving quality or acting as a barrier for oxygen, water and solutes [153,213]. Specifically, these compounds are extracted using conventional or innovative methods for the development of edible films and coatings [214]. Improved mechanical and barrier properties of these films have been achieved through the use of composites (derived by combining multiple biopolymers and layers or by adding fibers and fillers to the biopolymer, such as laponite, montmorillonite, sepiolite, palygorskite, etc.) or biopolymer nanocomposites (i.e., by the addition of nano SiO2-x, nano ZnO, nano oxide and nano TiO2, etc., to the polymer) [214]. Typically, these biopolymer-based coatings, in addition to being biodegradable and nontoxic, may have a natural microbial action; others can serve as a carrier for antioxidant or antimicrobial biopolymers [215]. In fact, better antibacterial, antifungal or antioxidant properties of edible films have been achieved by incorporating active compounds (i.e., antimicrobials, antioxidants, dyes, flavors and nutraceuticals) into filmogenic solutions [216]. For example, organic acids and essential oils (EOs), which have intrinsic antimicrobial and antioxidant properties, have been added to biopolymers derived from cellulose and derivates [217], i.e., thyme, clove, rosemary, oregano, cinnamon and tea oils [211]. Polyphenols (i.e., phenolic acids, flavonoids and proanthocyanidins) are other important active compounds added to polymeric edible films for their antioxidant, aromatic and antimicrobial properties [211]. Scientific studies have shown that nanoma-

terials can also be used to control the release of bioactive agents incorporated into edible packaging, improving its durability [212]. Several reviews have been written in recent years highlighting the progress made by research in the development of new biopolymerbased edible films/coatings [212,214,216]. For example, Das et al. [214] emphasized in their work the use of no thermal techniques (such as cold plasma, ultrasound, UV irradiation, high-pressure homogenization) and the addition of nanomaterials (nanoparticles of silver, zinc oxide, titanium dioxide, montmorillonite) to improve the structural (color, thickness, intermolecular bonds, particle size), water and oxygen barrier properties and mechanical features of edible films. Kumar et al. [212] reported the different natural materials used for either pure or composites and, for each, reported the preparation techniques of edible films/coatings and the antimicrobial, antioxidant, physical and sensory properties, also highlighting any critical issues, recent applications and commercial products. In the last few years, the growth of concern for the environment has led to a preference for the use of agricultural and industrial scraps for the development of edible films/coatings that provide a feasible alternative to the use of plastics or bio-based materials of natural origin or from biomass, in favor of sustainable development and the circular economy [212,216,218,219].

Biopolymers are mainly used in food packaging [110,153,220]. The causes mainly can be attributed to the problems that have emerged from the disposal of traditional petroleum-derived materials and the mandatory regulations now existing and increasingly developed in recent years in food packaging, as well as the significant increase in the cost of petroleum products, due to various reasons [213]. Food packaging is supposed to extend the conservation life of all sorts of foods by storing and protecting them from oxidative and microbiological degradation. Next-generation food packaging must have certain durability and good mechanical and barrier properties, as well as aesthetic functions related to marketing [219]. Among the natural polymers most commonly used for food packaging, cellulose and derivates are usually treated, processed, melted and dried. Chitosan, for example, is an antibacterial biopolymer. The encapsulation of chitosan has an important place in obtaining food and packaging with a long shelf life, as demonstrated in a recent study by Baysal et al. [221]. Other biopolymers used for food packaging are PLA, PHA, PBAT or blends of biopolymers, such as a starch-based blend with PLA, PHB, PHAs, PVAs, PCL, PVOH, etc. [149]. Torres-Giner et al. [222], after a classification of polymers used extensively in food packaging (divided into petroleum materials, and into non-biodegradable and biodegradable products), focused the paper on materials such as PLA, PHAs, PBAT, PBS, bio-based PET, cellulose and derivatives of different origins. Similarly, as for edible films, for food packaging the best mechanical, physical and antimicrobial barrier properties have been achieved through the use of multilayers [223] and by adding active ingredients (usually in the form of a sachet or covering on the packaging material embedded in the surface of the material itself) [211] or even nanometer fillers to biopolymers (nanofillers) [224]. In the former case, traditional multilayer packaging comprising polymeric layers (i.e., PE, PET, HDPE, PP, EVO, EVOH, PA, etc.) and inorganic layers (i.e., Al, SiOx, etc.) [223] is being replaced by biocompostable packaging called "active" packaging, which has a reduced environmental impact. Wang et al. [200] report several advanced examples of biodegradable active multilayer packaging composed of sandwich-like substances. These are based on polymer matrices of different natures (among them, methylcellulose has often been used) and active components, such as polyphenols, potassium sorbate, lysozymes, etc. [200]. The authors in the paper examine different production techniques, focusing on a mathematical pattern for release control of the active component of the packaging [200]. In the second case, nanofillers (classified into nanoplatelets, nanofibers and nanoparticles), having nanometric structures and antibacterial properties, are added to biodegradable and environmentally friendly polymers in so-called "smart" packaging (which allows the freshness of the food to be checked in real time), which has recently been added to "active" packaging (in which the added substances protect the food from UV rays, oxygen and microbes interacting directly with the food) on the market. The addition of nanofillers to green polymers produces a number of benefits, such as reduced risk of spreading pathogens,

improved food quality, reduced material waste and sustainability [224,225]. Among these, the most widely used nanofillers are antibacterial nanoparticles (such as Ag, ZnO, Cu/CuO, TiO2, Fe2O3, Fe3O4 and MgO), mesoporous particles, graphene and carbon dots, added in green polymers [224,225]. However, recent research highlights the importance of prior evaluation of the safety of metal oxide nanoparticle additives (through migration testing), and biocompatibility with polymers to minimize the risk of toxicity [225].

The scientific literature has developed several studies on natural fiber-based biopolymers too. Different lignocellulosic fibers, such as wheat straw, linen fibers, jute, coconut, kenaf and olive pomace, have been investigated for their usefulness as fillers [110]. Even if there is an increasing awareness of environmentally friendly packaging, it is necessary to use bio-based and sustainable packaging solutions. Next-generation packaging (often called 4G) combines all these properties with environmental friendliness, through the use of innovative materials [226]. The commercial and technological potential of industrial by-products for the production of next-generation (active and smart) food packaging to support zero waste activities has been known for some years now. For example, Bhat et al. [227] used lignin derived from oil palm black liquor scrap added to sago palm (*Metroxylon sagu*) films for the development of food packaging. After extraction and solubilization in DMSO, the lignin was added in several percentages (from 1 to 5% *v*/*w*) to the starting solution to form the packaging films. The authors in their work demonstrated an increase in the mechanical properties, resistance to thermal sealing, water vapor permeability, solubility and thermal stability of the films obtained using lignin produced from waste [227]. Sánchez-Safont et al. [110] tested the use of local lignocellulosic scraps (rice husk, almond shell and sea grass) as additives for the development of PHB/fiber composites for use in food packaging. Improved mechanical and permeability properties of the composites were obtained, as well as improved thermoforming ability of the films. Tumwesigye et al. [228] used bitter cassava waste to develop a low-cost food packaging film, turning environmental waste into a sustainable resource. Specifically, two different transparent films were produced and tested by the authors, from intact and decorticated bitter cassava; among these, the former were shown to have the best mechanical qualities and structural and higher thermal stability, while leading to a higher yield with a 16% reduction in waste [228].

#### *5.2. Biopolymers in Pharmacology and Medicine*

Biopolymers and their composites are also used in the pharmacological and medical fields due to their biodegradability, cost-effectiveness, wide availability, processability and especially biocompatibility with human organs and tissues [153,205,229]. For all of these qualities, they are used, for example, as materials for transporting pharmaceutical molecules and substances (such as enzymes, antibiotics and antineoplastic drugs, etc.), in ocular, dental, nasal and other systems [153,229,230]. Biopolymers from different sources and that have pharmaceutically active ingredients (capable of influencing the drug delivery process), are used for the production of Drug Delivery Systems (DDS), usually in the form of microcapsules, microspheres, nanospheres, hydrogels, nanogels and liposomes [153,229]. In this area, polysaccharides have been exploited for years mainly for their properties that can form linkages with proteins and lipids. Among the polysaccharides, cellulose (together with starch) is the most widely used in pharmacology and medicine, in general [203,231]. The use of cellulose (and its derivatives) for the development of DDS has expanded due to its exceptional properties, such as its ability to absorb and retain water, its biocompostability and its structural characteristics that allow the loading of specific molecules [203]; in addition, the possibility of producing nanocellulose from wood pulp, along with the use of advanced technologies (such as 3D printing), has opened up an opportunity for the development of innovative materials for pharmacological applications in recent years [231]. For example, Yu et al. [231] used ethylcellulose and hydroxypropyl methylcellulose for the development of a drug delivery device, prepared automatically using 3D printing, that was capable of providing a linear release profile of acetaminophen. Specifically, hydrophobic ethyl cellulose delays the initial rapid release of the drug, while hydroxypropyl

methylcellulose swells into a gel after contact with the dissolution medium, releasing the drug for an extended period [231]. Biopolymers are also used in the medical field for the production of hydrogels and nanogels, materials suitable for wound healing [203,232,233]. Several hydrogels and biopolymer-based formulations have emerged from the addition of therapeutic and bioactive agents such as antimicrobials, growth factors, antioxidants, antiseptics, etc., that facilitate the skin healing process [203,232,233]. Wound-healing materials use hydrocolloids such as foam, gel or spray [153,203,232]. Many researchers have invented several methods for the development of cellulose, hemicellulose and lignin from agricultural wastes, such as from sugar beet, cashew nuts, sago waste, waste from the cotton ginning industry, etc., using a variety of techniques [234,235]. Among them, Cui et al. [236] tested aqueous-based hydrogels from cellulose derived from industrial durian rind waste, fortified with glycerol to obtain organohydrogels, which proved to be suitable for antimicrobial wound dressing, even under extreme thermal conditions (for example, −30 ◦C). In their work, Amores-Monge et al. [237] investigated the opportunity to build a high-profit market that focuses on the production of products (i.e., cellulose, hemicellulose, lignin and enzymes) with biomedical applications from waste obtained from pineapple (*Bromeliaceae family*); among them, the proteolytic enzyme bromelain was found to have an essential application in skin reconstitution [237].

Properties such as biocompatibility, biodegradation and noncytotoxicity also make biopolymers excellent candidates for use in implantable medical materials and scaffolds [153,238,239]. Implantable medical devices are predominantly used to simulate and replace a human structure that has been damaged, or to support normal body function or control trunk posture [239,240]; scaffolds, on the other hand, are used to facilitate hard and soft tissue regeneration in tissue engineering [153,238,241,242]. Bones, heart, eyes, ears, knees, hips, etc. constitute the anatomical parts that are undergoing integration and replacement with polymeric medical implants the most [153]. Teeth, bones and the cartilage of humans, in contrast, are the human parts most concerned with the application of scaffolds [243,244]. Today, traditional materials such as metals and ceramics have been almost completely replaced by biopolymers, due to the immunological rejection by the body that they can cause; biopolymers, on the other hand, exhibit biocompatibility, good degradability, renewability, anti-toxicity and antibacteriality throughout their life cycle [202,238,239]. For the fabrication of medical devices, the materials usually used are polylactic acid (PLA), polyglycolic acid (PGA), poly(lactic-co-glycolic acid) (PGLA) and polycaprolactone (PCL) [153,243], also known as nanocomposites (nanotubes, nanoparticles and nanofibers) [12]. These polymers have also been combined to produce implants. For example, copolymers of PLA and PGA have often been used in place of their respective homopolymers in orthopedic applications (e.g., for the creation of plates or screws for the treatment of fractures and the filling of bone defects) [153]. Alternatively, PLA and PET have been combined to produce prostheses for vascular surgery [153]. The development of Additive Manufacturing (AM) techniques has also affected the biomedical and tissue engineering fields: 3D printing of tissues, organs and body parts using biopolymer nanocomposites has been made possible by the spread of some easy and low-cost 3D-printing techniques, which also have the advantage of printing complex geometries [12,202,240]. For example, S. Bartlett [245] reports on the 3D printing of a bioresorbable tracheal splint that was successfully implanted in the patient and was produced by combining TC images of the airway with the 3D printer. Gross et al. [246] report that 3D printing has also been used for the reproduction of anatomic parts needed for the preliminary study of surgical procedures, for example, to create a calcified aorta with 3D printing for the study of plaque removal surgery, to optimize the removal of bony outgrowths on a shoulder and for the study of drug delivery into the lungs of a premature infant [246]. The fabrication of scaffolds for bone tissue regeneration requires good biomimetic and bioactive properties; therefore, micro- or nanoparticles, comparable to the natural mineral components of bone, are often added to traditional polymers, such as tricalcium phosphate (TP), hydroxyapatite (HA), calcium phosphate cements (CPC), monetite or brushite [246]. For example, in their work,

Corcione et al. (2017, 2018, 2019) [242,243,247] explored the possibility of using FFF printing to develop an osteogenic bone graft based on hydroxyapatite (HA) and polylactic acid (PLA). PLA, PGA, PCL, etc. have also been used together with other biopolymers, such as hyaluronic acid, cellulose, collagen, gelatin, elastin and fibroin, for the synthesis of tissues such as adipose, ligaments, blood vessels, liver, cartilage, pancreas, spinal cord and bone regeneration [153,244]. For example, collagen was often mixed with PLA, PGA, PCL, etc., to improve wettability and interaction with biological substrates [248,249].

#### *5.3. Biopolymers in Personal Care and Cosmetics*

Biopolymers are also used for the production of personal care products and cosmetics [207,250]. The first group includes sanitary napkins, panty liners, feminine hygiene pads, baby diapers and adult incontinence items, which are aimed at improving peoples' lifestyles [239]. These are products that possess multiple layers, each with specific functions, and are composed of different types of synthetic or natural raw materials [239]. So-called "Superabsorbent polymers" (SAPs) constitute the main absorbent component of the layered structure of a personal care product (such as diapers or sanitary napkins for babies and adults). SAPs are a group of cross-linked hydrophilic polymers that are suitable for absorbing aqueous solutions, such as blood and urine, in a short time while keeping the skin dry and limiting infection or irritation [239]. For example, the most well-known SAPs used in personal care are polysaccharide SAPs (such as cellulose, hemicelluloses, bamboo, etc.), in addition to protein SAPs [250]. The demand for hygiene and personal care products will increase in years to come; conversely, environmental concerns over the use of products that are not fully biodegradable are growing, and there is an increasing need to instigate research activities aimed at producing non-toxic and eco-friendly products. Vivicot (Sanicot s.r.l., Prato, Italy) was the first line of compostable pads marketed in Italy (2011), made of pure organic cotton and certified by Certiquality [251]. Subsequently, the company Intimaluna (Borgo San Giovanni, Lodi, Italy) marketed fully compostable "Ecoluna line" feminine hygiene pads made of mater-bi and 100 percent organic cotton, menstrual cups and other washable feminine products [252]. Also in this area, scientific research is focusing on the recycling of raw materials for the production of new materials. For example, Lacoste et al. [253] produced a bio-based superabsorbent (bio-SAP) polymer for nappies from recycled cellulose. The authors demonstrated that they can recover and reuse waste packaging cellulose through a chlorine-free process, which transforms it into carboxymethylcellulose (CMC) that is cross-linked with citric acid afterwards [249]. In recent years, biopolymers have also been used in cosmetics due to their low cost, durability, versatility and biodegradability, and especially after the discovery of the presence of microplastics in aquatic ecosystems released from facial masks and scrubs, mascaras and lipsticks, shampoo, etc. [254].

Cellulose and derivates, polyhydroxyalkanoates, etc., are used in cosmetics for different purposes: for nanoparticle preparation or fragrance delivery, hair care, skin care and make-up [207]. Generally, biopolymers such as collagen, keratin and chitin are mostly used in this sector. For example, the major application of collagen hydrogels in cosmetics is to act as fillers for wrinkles and correctors of other skin defects [206]. Chitosan, on the other hand, is used more for hair care, and is included in shampoos, hair dyes, styling lotions, hair sprays and gels, or it is included in oral hygiene products, for the purpose of preventing tooth and gum disease [206]. Keratin, on the other hand, is mainly used for hair conditioning [206]. However, these substances are often used in combination with other polymers or biopolymers (such as cellulose and hydroxyethyl cellulose) or collagen hydrogel can be cross-linked with starch dialdehyde, tannic acid, squaric acid, PEG and other substances [206]. Examples of cosmetic actives derived from fish, meat, dairy and agro-industrial waste exist in the literature; products obtained from waste are a viable alternative to the usual plant extracts commonly used in cosmetic formulations, as they are effective, inexpensive and biosustainable [208,255]. In the area of industrialwaste-derived bipolymers, Meyabadi et al. [256] studied the reuse of waste cotton fibers

and their conversion to cellulose powder for various applications, including cosmetics. The authors showed that spherical cellulose nanoparticles (less than 100 nm), produced through enzymatic hydrolysis followed by ultrasonic treatment, do not undergo significant changes in structure and major properties, providing a viable sustainable alternative [256]. In their work, Bongao et al. [257] highlighted the potential of micro- and nanocellulose extracted using conventional methods and synthesized from Pili pulp waste to replace the mineral ingredients used in cosmetics. Innovative research in the field of green chemistry and sustainable production now involves many companies. For example, the company Anomera (Montreal, Canada) has been awarded a 1.7 million grant to carry out in its research labs research into the replacement of environmentally harmful plastic microbeads with biodegradable, environmentally friendly, high-performance ingredients for cosmetics and skin care, made with cellulose derived from wood waste from the paper industry [258].

#### *5.4. Biopolymers in Textile and Fashion*

Biodegradable polymers also support the textile and fashion industry, by reducing raw material processing energy, materials and costs of sourcing, production and disposal [209]. In fact, the textile industry is one of the world's most contaminating sectors, after petroleum; the greatest environmental damage comes from the production, processing and dyeing of the textiles [210]. This sector therefore needs alternative raw materials more than others; biopolymers are a responsible choice. Bio-based textiles, which must contain at least 20% renewable carbon, include natural materials and natural, synthetic or regenerated fibers [209,210]. Natural biopolymers are produced from polysaccharides (i.e., cellulose, lignin, etc.), as well as proteins and lipids of plant or animal origin [30]. In fact, natural fibers also come from plant sources (i.e., hemp, wool, cotton, etc.) [30,209,210]. Of these, cotton (together with silk and wool) is the most widely used in clothing production, as it meets aesthetic and wearability standards [30]. Synthetic and regenerated fibers used in textiles come from bacterial activities (such as polyhydroxyalkanoates, PHA) and from the synthesis of natural raw materials (e.g., polylactides, polyglycols, polycaprolactones, etc.) [30]. In recent years, the need for sustainable production has shifted the textile industry's attention not only to materials such as organic cotton (grown without the use of pesticides, fertilizers or other chemical products), but also to the production of synthetic biodegradable textile fibers, referred to as "biodegradable nonwovens." Among them, the biopolymers that find the most applications used in fiber spinning in the modern biodegradable textile industry are in fact polylactic acid (PLA), butyric acid (PHB), valeric acid (PHV), caprolactone (PCL), etc. [12,209].

Other examples of biodegradable nonwovens include those made of natural cellulosic fiber, cotton (cotton/cellulose or biodegradable cotton/co-polyester), the biodegradable nonwovens mentioned above and laminates (composites in which a layer includes a nonwoven fabric) [30]. In fact, composite materials derived from the addition of natural source fibers (such as hemp, flax, cellulose acetate, jute, pineapple, kenaf and many others) to synthetic biopolymers are often used [30]. For example, Gabry´s et al. [259] transformed viscous, commercial, nonwoven fabrics by adding PLA (in addition to potassium nitrate, KNO3) to impart fertilizing properties to fabrics used in modern agricultural mulching. Many technologies are being developed to manufacture biosynthetic fibers from biomass and waste materials derived from agriculture, forestry and even food [30,201,209,260,261]. Several examples of biodegradable synthetic fibers are already commercially available, such as the biodegradable PLA thermoplastic Ingeo (company NatureWorks LLC, Blair, NE, USA) or Modal biofilters and Tencel/Lyocell products produced from beech and eucalyptus wood pulp (Lenzing Aktiengesellschaft, Lenzing, Austria) [209]. Commercial biodegradable products are often enriched with innovative antimicrobial agents to produce workwear, home wear, sportswear, etc. [209]. Early instances of biosynthetics using novel feedstocks such as algae, fungi, enzymes and bacteria are also available [30]. For example, the use of bacterial cellulose (produced by microorganisms) is growing in the textile sector, compared to the traditional use of plant cellulose, because it is sustainable, is biodegradable, does not pollute and can also be dyed, resulting in an attractive textile surface that meets current market research [210]. In addition, Patti et al. [30] report on several bio-based and sustainable textiles produced by large known companies from microbes, algae and bacteria for the production of jackets, shoes and other garments. The search for new biodegradable materials and the continuing evolution of traditional textile production methods, which usually involve the use of chemicals, have also led to the emergence of 3D-printing techniques [12]. Additive Manufacturing (AM) techniques enable the development of innovative and sustainable models for the textile industry and are being employed recently by major brands to shift the production of shoes, clothing, jewelry and other accessories to environmentally friendly and green materials [12,30]. Companies using 3D-printing techniques and biopolymers such as PLA and softened PLA (along with other materials such as, e.g., Ninjaflex, BendLay, TPE [262,263]), have resulted in nonwoven fabrics with improved morphological and structural properties compared to traditional polymers. For example, Loh et al. [264] developed and studied three different polymer composites for the textile world morphologically and mechanically, using a different combination of PLA, nylon and polyesters, and direct extrusion of the materials.

Table 5 reports the main properties and applications of bio-based polymers and biocomposites.


**Table 5.** Main properties and applications of some bio-based polymers and biocomposites.


**Table 5.** *Cont.*

#### **6. Conclusions**

During the last decade, the production of bioplastics has mainly increased with the intention of decreasing the harmful effects of synthetic polymers on the environment. This study summarizes the current developments in the definition, classification, production, properties and applications of bio-based materials, particularly focusing on wood-waste derivates. The third-generation feedstock, obtained from food scraps, algae biomass and industrial or municipal waste, is the most promising category. It represents an innovative solution to the questions related to the consumption of sources from the food chain, according to the circular economy approach. Among the numerous varieties of organic wastes, agro-industrial and forestry wastes, which are generated in massive quantities each year, represent an unjustifiable environmental and economic scenario. The production of the main classes of biopolymers, starting from wood scraps, was reported in this work. Although lab-scale experiments showed promising ways to produce biopolymers from lignocellulosic wastes, the industrial production is still not sufficiently profitable, due to the high cost of the processes. Nevertheless, thanks to the relevant benefits obtained by the use of wood scraps, several studies regarding the development of genetically modified bacteria for the hydrolytic fermentation are under development, in order to overcome all the issues related to the high cost of the production processes.

Finally, in this work it was reported that wood waste can be used not only as a source for the production of third-generation biopolymers, but it can also be employed as a reinforcement for bio-based matrices, thus obtaining biocomposites with improved mechanical performances, as well as enhanced antibacterial, gas barrier and migration properties. Therefore, following both a descriptive and an analytical methodology, the main properties of bio-based polymers and biocomposites were discussed in this review and a comparison of thermal and mechanical properties of polymer matrices and wood biocomposites was reported.

**Author Contributions:** Conceptualization, C.E.C.; investigation and writing—original draft preparation, F.F., R.S., D.F., M.M.A. and C.E.C.; review, editing and supervision, C.E.C. and A.G. All authors have read and agreed to the published version of the manuscript.

**Funding:** Innovative doctoral scholarship from the Development and Cohesion Fund (FSC)—Extract Plan Research and innovation 2015–2017.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


## *Article* **Antibacterial and Antifungal Fabrication of Natural Lining Leather Using Bio-Synthesized Silver Nanoparticles from** *Piper Betle* **L. Leaf Extract**

**Ngoc-Thang Nguyen 1,\*, Tien-Hieu Vu 1,2 and Van-Huan Bui <sup>1</sup>**


**Abstract:** Leather is often used to make comfortable shoes due to its soft and breathable nature. However, its innate ability to retain moisture, oxygen and nutrients renders it a suitable medium for the adsorption, growth, and survival of potentially pathogenic microorganisms. Consequently, the intimate contact between the foot skin and the leather lining surface in shoes, which are subject to prolonged periods of sweating, may result in the transmission of pathogenic microorganisms and cause discomfort for the wearer. To address such issues, we modified pig leather with silver nanoparticles (AgPBL) that were bio-synthesized from *Piper betle* L. leaf extract as an antimicrobial agent via the padding method. The evidence of AgPBL embedded into the leather matrix, leather surface morphology and element profile of AgPBL-modified leather samples (pLeAg) was investigated using colorimetry, SEM, EDX, AAS and FTIR analyses. The colorimetric data confirmed that the pLeAg samples changed to a more brown color with higher wet pickup and AgPBL concentration, owing to the higher quantity of AgPBL uptake onto the leather surfaces. The antibacterial and antifungal activities of the pLeAg samples were both qualitatively and quantitatively evaluated using AATCC TM90, AATCC TM30 and ISO 16187:2013 test methods, approving a good synergistic antimicrobial efficiency of the modified leather against *Escherichia coli* and *Staphylococcus aureus* bacteria, a yeast *Candida albicans* and a mold *Aspergillus niger*. Additionally, the antimicrobial treatments of pig leather did not negatively impact its physico-mechanical properties, including tear strength, abrasion resistance, flex resistance, water vapour permeability and absorption, water absorption and desorption. These findings affirmed that the AgPBL-modified leather met all the requirements of upper lining according to the standard ISO 20882:2007 for making hygienic shoes.

**Keywords:** silver nanoparticles; green synthesis; *Piper betle* L. leaf; pig lining leather; antibacterial activity; antifungal activity

#### **1. Introduction**

Leather is a natural material obtained through the tanning process of a hide of an animal, bird or reptile. Leather has been extensively employed for making various items, such as footwear, clothing, bags, wallets and other accessories. Owing to its softness, breathability and high moisture-absorbing properties, leather provides comfort to the wearer [1–4]. However, due to its good moisture absorption, sweat-containing proteins may serve as a nutrient source that supports the growth of bacteria and fungi on leather goods, particularly within shoes where the foot skin is in close contact with the lining surface [5–8]. In addition, the collagen fiber network within the leather structure provides suitable conditions of moisture, temperature and oxygen for microorganism growth. Moreover, leather footwear products are commonly not washed during use, leading to the accumulation

**Citation:** Nguyen, N.-T.; Vu, T.-H.; Bui, V.-H. Antibacterial and Antifungal Fabrication of Natural Lining Leather Using Bio-Synthesized Silver Nanoparticles from *Piper Betle* L. Leaf Extract. *Polymers* **2023**, *15*, 2634. https:// doi.org/10.3390/polym15122634

Academic Editor: Raffaella Striani

Received: 11 May 2023 Revised: 3 June 2023 Accepted: 5 June 2023 Published: 9 June 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

and proliferation of microorganisms, resulting in unpleasant odors, discoloration, reduced mechanical strength and skin diseases in wearers [9–11]. The hot and humid climate in Vietnam provides proper conditions for bacterial and fungal growth on leather goods during storage, transportation and use. Hence, the antimicrobial characteristics of leather footwear products are concerns among both consumers and enterprises.

To overcome such issues, antimicrobial finishing of leather footwear products using various antimicrobial agents and treatment methods is often employed [2,3,8–11]. Many antibacterial and antifungal agents have been investigated for their effectiveness in leather treatment, including silver nanoparticles, zinc oxide nanoparticles, polymer compounds containing quaternary ammonium, chitosan and its derivatives [12–16]. These agents work through contact mechanisms and cell membrane disruption of microorganisms [17–19]. Although some chemical antimicrobial agents are used in the tanning process, their main function is to prevent the biodegradation of the leather rather than providing antimicrobial properties [2,12]. Furthermore, the use of some antibacterial and antifungal agents has been limited due to health and environmental concerns [20,21]. Therefore, developing high-performance antimicrobial agents that are effective against broad-spectrum bacterial and mold strains and environmentally friendly for leather material is imperative [20–22].

Thus, the appropriate selection of antimicrobial agents and treatment methods is important for creating durable antimicrobial leather materials that effectively prevent undesirable microbial growth while minimizing negative impacts on the material properties and the environment. To address these concerns, bio-synthesized silver nanoparticles (AgNPs) treated on leather have attracted significant attention from scientists due to their broad antimicrobial activity and durability to microorganisms [17,21,23,24]. The synthesis of green AgNPs involves the utilization of bio-reductants derived from natural resources such as plants, algae and microorganisms. Incorporating AgNPs into the collagen fiber matrix of leather enhances the material's long-lasting antimicrobial effects and exhibits low toxicity towards mammalian cells, making them suitable for producing high-quality leather goods [2,3].

Recently, we reported on a green approach to fabricating silver nanoparticles using *Piper betle* L. leaf extract (PBL) as bio-reductants to reduce Ag+ ions into silver metal, which adheres fully to the principles of green chemistry [25]. The spherical shape and narrow size distribution of the obtained silver nanoparticles (AgPBL) showed good synergistic antibacterial activity against three common bacterial strains, including *Escherichia coli*, *Pseudomonas aeruginosa* and *Staphylococcus aureus*. In this work, we further evaluated the antifungal activity of AgPBL against one mold strain (*Aspergillus niger*) and one yeast strain (*Candida albicans*). We then investigated a simple approach to apply AgPBL onto tanned pig leather utilized for shoe lining (Le) by a padding method. The padding method was selected to apply antimicrobial treatment to the pig leather because it is suitable for use during the wet finishing stage of leather production. The presence and distribution of AgPBL on the pig leather surface were evaluated using various analytical techniques, namely colorimetry, scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX), atomic absorption spectroscopy (AAS) and Fourier-transform infrared spectroscopy (FTIR). The antibacterial and antifungal efficacy of the modified leather was assessed qualitatively and quantitatively using established protocols for antimicrobial testing of textile and leather materials in accordance with AATCC TM90, AATCC TM30 and ISO 16187:2013 against two bacterial strains (*Escherichia coli* and *Staphylococcus aureus*) and two fungal strains (*Aspergillus niger* and *Candida albicans*). To the best of our knowledge, there is no report available on the antibacterial and antifungal treatment of pig leather using bio-synthesized AgPBL for shoe lining application.

#### **2. Materials and Methods**

#### *2.1. Materials*

Analytical grade silver nitrate (Ag(NO)3 99.99%, Aladdin Biochemical Technology Co., Ltd., Shanghai, China) and *Piper betle* L. leaves (PBL, Hai Duong Province, Vietnam) were used for the preparation of the silver nanoparticles (AgPBL) under optimal conditions according to our published research [25]. The samples of pristine pig leather (Le) in the wet-blue tanning form were obtained from Hung Thai Brothers Tannery Co., Ltd., Ho Chi Minh, Vietnam. The leather was then split in our laboratory using a DS818-420L leather splitter (Wenzhou Dashun Machinery Manufacture Co., Ltd., Zhejiang, China) to obtain a uniform thickness of 1 ± 0.1 mm, which is proper for use as shoe lining material. The pig lining leather was further cut into small pieces (100 × 100 mm) and dried in a Mesdan M250-RH conditioning chamber (Brescia, Italy) at 65% RH and 25 ◦C for 24 h before being stored in a plastic bag for further study. In all experiments, double distilled water from an EYELA Still Ace SA-2100E (Tokyo Rikakikai Co., Ltd., Tokyo, Japan) was employed as the solvent. The dedicated medium (SCDLP), Luria-Bertani (LB) agar and Sabouraud dextrose agar (SDA) were supplied by Oxoid (Thermo Fisher Scientific Inc., Waltham, MA, USA). All microbial strains, including two bacterial strains *Escherichia coli* (*E. coli*, ATCC 25922) and *Staphylococcus aureus* (*S. aureus*, ATCC 29213, ATCC, Manassas, VA, USA), a mold strain *Aspergillus niger* (*A. niger*, ATCC 16404) and a yeast strain *Candida albicans* (*C. albicans*, ATCC 10231) were provided from the School of Biotechnology—International University, NTT Hi-Tech Institute—Nguyen Tat Thanh University, Institute of Tropical Biology—Vietnamese Academy of Science and Technology.

#### *2.2. Synthesis and Application of AgPBL to Pig Lining Leather*

We have previously described the bio-synthesis of the silver nanoparticles using *Piper betle* L. leaf extract as bio-reductants [25]. Briefly, dried PBL leaves were boiled with double distilled water at a ratio of 1:40 for 15 min. The mixture was then filtered through Whatman No. 1 filter paper. Subsequently, the resulting PBL filtrate underwent centrifugation at 10,000 rpm for 20 min to eliminate any insoluble residues. Prior to the nanosilver synthesis, the PBL supernatant was further diluted 20 times with double distilled water. For the AgPBL synthesis, 1 mL of 10 mM AgNO3 solution was reduced with 10 mL of the diluted PBL extract and allowed to stand for 4 h at room temperature in the dark to avoid any unnecessary photochemical reactions. The mixtures containing silver nanoparticles were purified by centrifugation at 16,000 rpm, 5 ◦C for 30 min, followed by washing with double distilled water in a UT-106H Ultrasonic Cleaner (Sharp Corporation, Osaka, Japan). The purification process was repeated twice to remove residual reagents, and the AgPBL was collected and re-dispersed in double distilled water to achieve various concentrations for further investigation.

In the next step, the pig lining leather samples were treated with the bio-synthesized AgPBL solutions using the padding method. The leather samples were dipped in AgPBL solutions with various concentrations (160, 80, 40 and 20 μg/mL) for 30 min at a liquor-toleather ratio of 5:1 (*w*/*w*). The wet pickups were set at 70%, 80% and 90%, and the padded samples were then dried at 105 ± 3 ◦C for 3 min using SDL mini-drier 398 laboratory thermofixation (SDL Atlas China, Shenzhen, China). The dipping–padding–drying processes of the leather samples were repeated two times. All processed leather samples were conditioned at 65% RH and 25 ◦C in a Mesdan M250-RH (Mesdan SpA, Brescia, Italy) conditioning chamber for 24 h before storage in plastic bags for microbiological analysis. The processes of synthesizing AgPBL and applying it onto pig lining leather are depicted in Figure 1.

**Figure 1.** Schematic illustration of the processes of AgPBL synthesis and its application onto pig lining leather, namely pLeAg.

#### *2.3. Analytical Methods*

#### 2.3.1. Characterization of the AgPBL

The UV-vis absorption spectrum of the AgPBL was acquired employing a UV-1800 spectrophotometer (Shimadzu, Kyoto, Japan) with a quartz cuvette in the range of 300–700 nm and with a resolution of 1 nm.

The diameters of AgPBL nanoparticles were recorded by transmission electron microscopy (JEOL JEM-1400, JEOL, Tokyo, Japan). A suspension of AgPBL in double distilled water was sonicated for 2 min and dropped onto a Cu-grid for TEM analysis.

#### 2.3.2. Characterization of the Modified Pig Leather

The morphology and chemical content of the control and AgPBL-modified leather samples after platinum sputtering were inspected using SM-6510LV JEOL (JEOL, Tokyo, Japan) scanning electron microscope (SEM) coupled to Oxford EDS Microanalysis System (Oxford Instruments NanoAnalysis, High Wycombe, UK).

A PinAAcle 900T atomic absorption spectrometer (AAS, PerkinElmer, Waltham, MA, USA) was employed to record silver content in the AgPBL-modified leather sample.

A Nicolet 6700 spectrometer (Thermo Scientific, Waltham, MA, USA) was utilized to record the FTIR spectra of the control and modified leather samples within the 4000–500 cm−<sup>1</sup> range.

Ci4200 spectrophotometer (X-rite, Grandville, MI, USA) was employed to report both colorimetric data (L\*, a\* and b\*) and color differences (ΔE\*) of the leather samples before and after treatment with the AgPBL solutions. In the CIELab color space, L\* is lightness from brightest white (100) to darkest black (0); a\* is the color ratio from red (+) to green (−) and b\* is the color ratio from yellow (+) to blue (−). The total color difference was determined using Equation (1):

$$
\Delta \mathbf{E} = \sqrt{\Delta \mathbf{L}^{\*2} + \Delta \mathbf{a}^{\*2} + \Delta \mathbf{b}^{\*2}} \tag{1}
$$

where ΔL\*, Δa\* and Δb\* represent the colorimetric differences in L\*, a\* and b\* values, respectively, of the blank and modified leather samples.

#### 2.3.3. Physico-Mechanical Characterization

The AgPBL-modified leather was evaluated for its physico-mechanical properties, which are essential requirements for shoe lining materials, in accordance with the standard ISO 20882:2007 [26]. These properties include tear strength (ISO 17696), abrasion resistance (ISO 17704), flex resistance (ISO 17694), lining water vapour permeability and absorption (ISO 17699), and lining water absorption and desorption (ISO 22649). The physico-mechanical tests were carried out at the Institute of Footwear Research, Vietnam.

#### *2.4. Antibacterial and Antifungal Activities*

#### 2.4.1. Bio-Synthesized Silver Nanoparticles (AgPBL)

The antifungal activities of the AgPBL against yeast *C. albicans* and mold *A. niger* were studied by well diffusion method and disk diffusion method, respectively, following the Clinical Laboratory Standard Institute guidelines [27]. For the anti-yeast test, a suspension of *C. albicans* strain (0.1 mL, 106 CFU/mL) was spread uniformly on plates containing Sabouraud dextrose agar (SDA). Next, five 6 mm diameter holes were made using a sterile cork borer. Then, 60 μL of AgPBL solutions at various concentrations (100, 50 and 25 μg/mL), a standard antibiotic (Streptomycin, 80 μg/mL) as positive control and double distilled water as negative control were poured into their respective wells. The zone of inhibition (ZOI) produced by *C. albicans* was recorded after 24 h of incubation at 37 ◦C.

For the anti-mold test of *A. niger*, the disk diffusion method was performed using sterile 6 mm paper discs loaded with AgPBL solutions (100, 50 and 25 μg/mL) and double distilled water as the negative control. Aliquots of 0.1 mL of *A. niger* strain (approximately 106 CFU/mL) were spread on SDA agar plates, followed by the placement of the prepared paper discs on their surface. The zone of inhibition against A. niger was measured after 7 and 14 days of growth at 28 ◦C. The results were expressed as the mean ± standard deviation (SD) of three independent tests.

The zone of inhibition (ZOI) was calculated based on Equation (2):

$$\mathbf{W} = (\mathbf{T} - \mathbf{D})/\mathbf{2} \tag{2}$$

where

W is the width of clear zone of inhibition, mm;

T is the total diameter of the test specimen and clear zone, mm;

D is the diameter of the test specimen, mm.

#### 2.4.2. The Modified Pig Leather

The antibacterial and antifungal activities of the control and AgPBL-modified leather samples against *E. coli*, *S. aureus*, *C. albicans* and *A. niger* were investigated qualitatively and quantitatively using established protocols for testing the antimicrobial activity of textile and leather materials, including AATCC TM90, AATCC TM30 and ISO 16187:2013 test methods [28–30].

For qualitative tests (AATCC TM90 and AATCC TM30), the disk diffusion method was used to determine the zone of inhibition. A volume of 0.1 mL of each organism strain (approximately 10<sup>6</sup> CFU/mL) was spread on Luria-Bertani (LB) agar plates for bacteria and SDA agar plates for fungi. Next, the control and AgPBL-modified leather samples were positioned on the surface of agar plates, which were subsequently incubated at 37 ◦C for 24 h for bacteria and *C. albicans*, and at 28 ◦C for 7 and 14 days for *A. niger*. Zones of inhibition around and on the leather samples were visually examined.

For quantitative tests (ISO 16187:2013), the static challenge protocol was performed to determine the percentage reduction of bacteria. Six control samples (pristine leather, Le) and six modified leather samples (pLeAg) at each AgPBL concentration were prepared with dimensions of 25 × 25 × 1 mm and placed in individual sterile glass flasks. To each flask, 1 mL of bacterial suspension with a concentration of 5.0 × 105 CFU/mL was added. At the initial time (zero contact time), three control samples and three modified leather samples were collected and washed out with 20 mL of dedicated medium (SCDLP). The remaining six flasks were incubated for 24 h at 37 ◦C (24 h contact time) and then washed out with 20 mL of the SCDLP medium. All flasks were tightly capped and shaken in an incubator shaker at 120 rpm and 37 ◦C for 30 s. A series of ten-fold dilutions of the bacterial sample solutions were made using NaCl 0.85% aqueous solution, and 100 μL of each diluted bacterial solution was spread over LB agar plates. After incubating the plates

at 37 ◦C for 24 h, the surviving bacteria were enumerated by counting their colonies. The bacterial reduction percentage was measured using Equation (3):

$$R = (C\_t - T\_t) \times 100\% / \text{C}\_t \tag{3}$$

where

R is the bacterial reduction percentage, %;

Ct and Tt are the average number of colonies of three control samples and three test samples after 24 h, respectively, CFU/mL.

#### **3. Results and Discussion**

#### *3.1. Synthesis and Antimicrobial Activity of AgPBL*

In previous research, we optimized the conditions for the bio-synthesis of silver nanoparticles from PBL leaf extract and evaluated their antibacterial activities against three bacterial strains, including *E. coli*, *P. aeruginosa* and *S. aureus* [25]. In the current work, we re-fabricated AgPBL under the optimized conditions and characterized its properties using UV-vis, TEM and antifungal activity analyses. The UV-Vis spectrum in Figure 2a indicated a maximum absorption peak at 442 nm due to the surface plasmon resonance (SPR) of spherical silver nanoparticles [21,22,25]. The TEM image in Figure 2b confirmed well-dispersed, spherical-shaped nanoparticles with a fairly uniform size of about 20 nm. The well-dispersion of AgPBL was attributed to the bio-constituents in the PBL extract, which effectively prevented nanoparticle agglomeration and stabilized them.

**Figure 2.** (**a**) UV-vis spectrum and (**b**) TEM micrographs of AgPBL with different magnification ×30 k and ×200 k (inset).

To assess the antifungal activity of the AgPBL, one mold strain (*A. niger*) and one yeast strain (*C. albicans*) were exposed to various AgPBL concentrations (100, 50 and 25 μg/mL) via the plate diffusion method. As shown in Figure 3a–d, the double distilled water (a negative control) exhibited no antifungal activity in terms of inhibition zone, while the AgPBL and the Streptomycin (Strep) obviously revealed ZOI. It was evident that AgPBL showed promising antifungal activity against both tested fungi. A comparison of the ZOI size for each fungal strain indicated a tendency for decreased antifungal action with decreasing AgPBL concentration, although this tendency was not proportional to the change in AgPBL concentration. For instance, reducing the AgPBL concentration by 50% and 75% resulted in a ZOI reduction for *C. albicans* of 29.9% and 42.5%, respectively. The results observed from Figure 3c,d after 7 and 14 days of the antifungal tests against *A. niger* revealed clearly that mold spores could not grow on the AgPBL-impregnated paper disks at any concentration. However, the paper disk impregnated with 25 μg/mL AgPBL solution did not exhibit a clear inhibition zone, indicating that the antifungal activity of AgPBL against *C. albicans* was better than against *A. niger*. The

antifungal properties exhibited by bio-synthesized AgNPs were consistent with the findings of previous studies [31,32].

**Figure 3.** Antifungal activities of the AgPBL: (**a**) Well diffusion method displaying the anti-*C. albicans* action of AgPBL11 (AgPBL 100 μg/mL), AgPBL12 (AgPBL 50 μg/mL), AgPBL14 (AgPBL 25 μg/mL), Streptomycin (80 μg/mL, positive control) and H2O (negative control); (**b**) Mean zone of inhibition of AgPBL against *C. albicans* (±SD, *n* = 3); Disk diffusion method displaying the anti-*A. niger* action of AgPBL after (**c**) 7 days and (**d**) 14 days of incubation.

#### *3.2. Coloration and Characteristics of the AgPBL-Modified Pig Leather*

The presence of AgPBL on pig lining leather was visually observed through the color change of the sample during the treatment of the leather with bio-synthesized AgPBL solutions using the padding method. Indeed, the color of the leather was changed significantly from bright yellow to light brownish color. To illustrate the effect of the wet pickup and AgPBL concentration on the coloration of the leather surface, the colorimetric data (L\*, a\*, b\*) and color differences (ΔE\*) of the leather samples were evaluated, as shown in Table 1. The color of the blank leather was bright yellow with relatively high L\*, a\* and b\* values of 62.42, 9.77 and 23.86, respectively. To compare with the blank leather, the AgPBL-modified leather samples showed smaller L\*, a\* and b\* values, indicating a browning effect of the AgPBL on the leather samples. The ΔE\* of the modified samples increased with higher wet pickup and AgPBL concentration, owing to the higher quantity of AgPBL uptake onto the leather surfaces.

To get visual evidence of the AgPBL embedded onto the leather, leather surface morphologies and element profile of the blank and modified leather samples were investigated. As shown in Figure 4, the blank and modified leather revealed a distinctive hierarchically suprafibrillar structure of the collagen fiber strands. The micrographs of modified leather samples (Figure 4) revealed the occurrence of nanoparticles loosely attached to the leather surface. In dark-field SEM images, nano metals usually appear as bright spots due to their strong light scattering [22,33]. However, the silver nanoparticles employed in this work were about 20 nm in size and could impregnate deeply into the collagen fiber strands of the leather matrix. As a result, they could be challenging to detect in SEM images, which only provide information on the surface morphology of the sample. Therefore, EDX analytical

technique was performed to validate the presence of nanosilver on the leather sample after treatment.

**Sample Wet Pickup (%) AgPBL (**μ**g/mL) L\* a\* b\* ΔE\* Real Images** Le - - 62.42 9.77 23.86 0 pLeAg11 70 160 57.94 9.13 21.38 2.33 pLeAg12 80 57.97 9.75 21.12 2.41 pLeAg13 90 57.01 9.27 21.38 2.66 pLeAg12 80 160 57.97 9.75 21.12 2.41 pLeAg22 80 59.85 9.29 22.27 1.39 pLeAg32 40 59.94 9.79 23.07 1.15 pLeAg42 20 60.01 9.54 23.74 1.04

**Table 1.** Colorimetric data, color differences and images of the AgPBL-modified leather samples in comparison with the blank pig leather.

The EDX spectrum of the blank leather in Figure 4 revealed no Ag signal, but the occurrence of a Cr signal confirming that the pig leather was chrome-tanned leather. In contrast, the EDX spectra of the AgPBL-modified leather sample via the padding treatment (pLeAg) clearly showed a strong signal of elemental silver at 3 keV which authenticated the existence of AgPBL on the leather surface [9,12]. The EDX spectra of those modified samples confirmed again that the bright points in the SEM images were AgPBL.

To evaluate the amount of AgPBL adhered to the pig leather after padding treatment, the AAS analysis was performed, and the result was presented in Table 2. The data indicates that the total silver content in the padded sample was around 380 mg/kg, whereas the blank leather sample did not contain any silver.

**Table 2.** Total silver content of the leather samples.


In order to determine the possible interaction between AgPBL and the functional groups of collagen proteins on the modified leather, the FTIR analyses of Le and pLeAg samples were carried out, and the spectra were given in Figure 5. The characteristic peaks corresponding to the functional groups of the collagen proteins in the Le sample at 3304.5, 2921.1, 2852.3, 1633.5, 1547.8, 1236.6 and 1030.9 cm−<sup>1</sup> were assigned to –NH, –CH3, =CH2, –C=O, –NH, –C=O (in amide III) and C–N (in amine) groups, respectively [10,13,34]. Compared to the blank leather, the pLeAg spectrum exhibited similarity in their characteristic peaks, except for the stronger peak intensities, suggesting the chemical structure of the leather was mostly unchanged. The collagen proteins of leather contain polar groups on the side chains of their constituent amino acid residues, and as such, the amide and carboxylate functional groups of these residues have a tendency to bind with metal atoms [10,12]. Thus, AgPBL could be

absorbed onto the leather surface through the electrostatic interaction of Ag+ ions with the negative charge of RCOO− or lone-pair electrons of N atoms of amino acids. In addition, the formation of hydrogen bonding between the amide and carboxylate groups of the collagen proteins with the appropriate functional groups of the organic layer existing on the AgPBL surface also contributes to these binding interactions. The shifted peaks of the functional groups in the modified leather samples could be attributed to the interaction of heavy silver atoms with the amino and amide groups of collagen protein molecules, resulting in an increase in peak intensity [12]. The results of coloration, SEM, EDX, AAS and FTIR measurements for both blank and modified leather samples were consistent with each other, providing strong evidence for the incorporation of AgPBL into the leather matrix.

**Figure 4.** The SEM micrographs at different magnifications of ×500, ×3000 and ×5000, and EDX spectra of the blank leather (Le) and the AgPBL-modified leather sample (pLeAg).

**Figure 5.** The FTIR spectra of the Le and pLeAg samples.

#### *3.3. Antibacterial and Antifungal Efficacy of the AgPBL-Modified Pig Leather*

Leather is a naturally hydrophilic material that offers a potential medium for the growth of microorganisms such as bacteria, yeasts and molds. The utilization of silver nanoparticles on the lining of leather could avoid the risk of infection and extend the lifetime of leather products by inhibiting microorganism growth. In this research, we investigated the antibacterial and antifungal efficacy of AgPBL-modified leather against *E. coli*, *S. aureus*, *C. albicans* and *A. niger*. Both qualitative (the disk diffusion method) and quantitative (the static challenge protocol of dynamic contact method) tests were employed to determine the antimicrobial activities of the modified leather.

#### 3.3.1. Antibacterial Efficacy

The AgPBL-modified leather samples obtained via the padding method were evaluated for their antibacterial activities against a gram-negative bacterium *E. coli* and a gram-positive bacterium *S. aureus*. The effect of the wet pickup and AgPBL concentration on the antibacterial efficacy of the modified leather was examined, and results were shown in Figure 6 and Table 3.


**Table 3.** The ZOI of AgPBL-modified leather samples against *E. coli* and *S. aureus* (±SD, *n* = 3).

A quick survey across Figure 6 evidences that all the AgPBL-modified leather samples and the Strep-treated leather exhibited obviously ZOI against *E. coli* and *S. aureus*, whereas the pristine leather (Le) showed no activity at all. These findings demonstrate the antibacterial efficacy of the AgPBL-modified leather in this work. As shown in Figure 6, the ZOI of the modified leather samples did not change significantly with varying wet pickups. The pLeAg12 sample with a wet pickup of 80% exhibited the highest ZOI of 8.7 ± 0.21 and 10.2 ± 0.24 mm against *E. coli* and *S. aureus*, respectively. Accordingly, the wet pickup of 80% was selected for further evaluation of the antibacterial activity.

Table 3 presents the results of the antibacterial tests of the padded leather samples, which showed that higher AgPBL concentrations led to larger inhibition zones. However, the relationship between AgPBL concentration and inhibition zone was not proportional. Indeed, as compared to the pLeAg12 sample, when the AgPBL concentrations were decreased by 50% and 75%, the ZOI of pLeAg22 and pLeAg32 samples against *E. coli* decreased by 0.9% and 42.0%, respectively. Additionally, there was a slight difference in the inhibition zone between leather samples treated with AgPBL concentrations of 160 and 80 μg/mL. Moreover, the antibacterial results in terms of inhibition zone clearly demonstrated that the AgPBL-modified leather samples exhibited higher effectiveness against gram-negative bacteria than gram-positive bacteria, which is in line with previous findings regarding the bactericidal properties of bio-synthesized silver nanoparticles [25,35].

**Figure 6.** The photographs showing zone of inhibition of the Le, pLeAg and Streptomycin (80 μg/mL) treated leather samples against *E. coli* and *S. aureus*, with change in the wet pickup and AgPBL concentration.

The leather samples treated with various AgPBL concentrations at the wet pickup of 80% were subjected to the quantitative test to determine the bacterial reduction percentage. This test was performed at 0 h and 24 h contact times between the sample and the test bacteria inoculate. As shown in Figure 7a, the Petri dishes for 24 h contact time revealed that both tested bacteria grew well on the Petri dishes of blank leather, indicating that Le did not possess bacterial inhibition. Based on the agar's turbidity, the pLeAg12 samples showed the highest antibacterial activity, while the pLeAg42 samples exhibited the lowest.

By counting the number of colonies of tested bacteria on agar dishes, the bacterial reduction percentage of the treated leather samples with different concentrations of AgPBL was plotted in Figure 7b. The results showed that the pLeAg12 samples treated with 160 μg/mL AgPBL exhibited the highest antibacterial efficiency against both *E. coli* and *S. aureus* after 0 h contact time, with bacterial reduction percentages of 47.02% and 68.43%, respectively. Moreover, both achieved a 100% antibacterial rate after a 24 h contact time.

The antibacterial activity of pLeAg22 samples treated with 80 μg/mL AgPBL decreased significantly against *E. coli* after both 0 h and 24 h contact time. In comparison to pLeAg12 samples, the bactericidal rates of pLeAg22 samples against *E. coli* and *S. aureus* decreased by 24.8% and 1.88%, respectively, after 24 h contact time. Similarly, pLeAg42 samples treated with 20 μg/mL AgPBL exhibited a sharp decrease in antibacterial activity against both tested bacteria. Compared to the pLeAg12 samples, the bactericidal rates of the pLeAg42 samples against *E. coli* and *S. aureus* after 24 h contact time declined by 84.93% and 31.13%, respectively. Despite the lack of a zone of inhibition in the AgPBL-modified leather sample at a low concentration (20 μg/mL AgPBL) observed by the qualitative antibacterial method, the sample still possessed a weak antibacterial ability.

Based on the obtained antibacterial results, the quantitative antibacterial efficiency of the modified leather samples is strongly influenced by the initial AgPBL concentration

in the treated solutions. The antibacterial efficiency also depends on the tested bacteria and is not proportional to the change in AgPBL concentration. The findings are consistent with the qualitative antibacterial evaluation of the modified leather samples. The suitable concentration of AgPBL for pig lining leather treatment was 160 μg/mL to achieve 100% antibacterial efficiency after 24 h contact time.

**Figure 7.** (**a**) The photographs of bacterial growth in the nutrient agar plates and (**b**) The bacterial reduction percentage (%R) of the Le and AgPBL-modified leather samples against *E. coli* and *S. aureus*.

3.3.2. Antifungal Efficacy

The AgPBL-modified leather samples were evaluated for their antifungal activities against one yeast strain (*C. albicans*) and one mold strain (*A. niger*) according to AATCC TM30. Figure 8 depicted that the blank leather revealed no antifungal activity, whereas both AgPBLmodified leather samples and the Strep-treated leather exhibited considerable antifungal efficiency against the tested fungi. Figure 8a,b confirmed that a decrease in the treated

AgPBL concentration resulted in a reduction of antifungal efficacy against the *C. albicans* strain, as evidenced by a decrease in the ZOI value by 20.6% and 19.6% when the treated AgPBL concentration was reduced by 50% and 75%, respectively. As shown in Figure 8c, the antifungal tests against *A. niger* after 7 and 14 days revealed that the pLeAg12 sample padded with 160 μg/mL AgPBL solution did not show any mold spore germination or growth. However, the pLeAg22 sample padded with 80 μg/mL AgPBL solution exhibited mold spore growth on its surface after 7 days, which was obviously seen after 14 days of incubation. These findings confirm once again the strong dependence of the antimicrobial effectiveness of the AgPBL-modified samples on the AgPBL concentration in the antimicrobial-treated solution. Consequently, pig leather treated with 160 μg/mL solution at a wet pickup of 80% was selected for further evaluation of its physico-mechanical properties.

**Figure 8.** (**a**) Antifungal activities of the Le, pLeAg and Strep-treated leather samples against *C. albicans* with change in the AgPBL concentration; (**b**) Mean zone of inhibition against *C. albicans* (±SD, *n* = 3); (**c**) Antifungal activities of the Le, pLeAg12 and pLeAg22 samples against *A. niger* after 7 and 14 days of incubation.

#### *3.4. The Physico-Mechanical Properties of the AgPBL-Modified Pig Leather*

The blank leather and AgPBL-modified leather samples were subjected to various tests following standard specifications to evaluate their physico-mechanical properties. The experimental data were compared with the requirements of the shoe lining material according to the standard ISO 20882:2007. As presented in Table 4, the mechanical properties of the AgPBL-modified leather including tear strength, abrasion resistance and flex resistance were nearly identical to those of pristine leather. This is because the antimicrobial treatment process used in this research did not alter the chemical structure of pig leather, and nanosilver adheres to the leather matrix solely through physical bonds. The mechanical data of the modified samples met all the standard requirements for shoe lining, and the tear strength was even over 200% of the standard requirement.

Similarly, the antimicrobial treatment of leather with AgPBL did not affect its water absorption and water desorption. The water absorption of the leather samples before and after treatment was around 90% of the standard requirements for the lining. However, this lining layer is typically combined with a porous layer in the shoe upper structure, resulting in an enhancement of its water absorption capacity. The water desorption of the leather samples was excellent, reaching over 160% of the standard requirement. The water vapour permeability and water vapour absorption of the leather samples were very good, exceeding 150% of the standard requirements for upper lining material. Pore expansion on the modified leather surface after the padding process led to an increase in its water vapour permeability. The leather material swells when it absorbs water and then shrinks during the drying process. However, the collagen fiber strands' shrinkage is greater than that of the pores, leading to pore expansion.

**Table 4.** The physico-mechanical properties of the pristine leather and AgPBL-modified pig leather.


In general, the antimicrobial treatments of pig leather did not negatively impact its physico-mechanical properties. The AgPBL-modified leather met all the requirements according to the standard ISO 20882:2007 for making shoe linings. The padding method is suitable for tanneries, which is carried out at the wet finishing stage (dyeing, oiling, etc.) in a rotary drum, then padded and transferred to the drying finishing stage in a drying chamber.

#### **4. Conclusions**

This research provided a simple approach for the fabrication of highly effective antimicrobial pig leather modified with bio-synthesized silver nanoparticles as an antimicrobial agent via the dipping–padding–drying processes. The effect of the wet pickup and AgPBL concentration on the coloration, antimicrobial activity and physico-mechanical properties of the modified leather were investigated according to standard methods. The experiment data validated that the higher wet pickup and AgPBL concentration led to a browner color and enhanced antimicrobial efficacy of the AgPBL-modified leather against both bacteria (*E. coli* and *S. aureus*) and fungi (a yeast *C. albicans* and a mold *A. niger*). These observed results could be attributed to the increased uptake of AgPBL into the leather matrix, which was verified through SEM, EDX, AAS, and FTIR analyses. However, the antimicrobial treatment of pig leather using the padding method did not have any adverse effect on its physico-mechanical properties, and it met the ISO 20882:2007 standard's requirements for the upper lining. Therefore, based on the efficient antimicrobial and suitable physicomechanical properties, the AgPBL-modified pig leather meets the criteria for making upper lining in hygienic shoe production.

**Author Contributions:** Conceptualization, N.-T.N. and V.-H.B.; methodology, N.-T.N., V.-H.B. and T.-H.V.; formal analysis, T.-H.V.; investigation, N.-T.N., V.-H.B. and T.-H.V.; data curation, N.-T.N. and V.-H.B.; writing—original draft preparation, T.-H.V.; writing—review and editing, N.-T.N. and V.-H.B.; visualization, N.-T.N. and T.-H.V.; supervision, N.-T.N. and V.-H.B.; project administration, N.-T.N.; funding acquisition, N.-T.N. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was funded by the Vietnam Ministry of Education and Training under Grant No. B2022-BKA-23.

**Institutional Review Board Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Acknowledgments:** The authors also would like to thank colleagues from School of Textile—Leather and Fashion, Hanoi University of Science and Technology for their efforts in supporting this work.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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## *Article* **Biochemical and Microstructural Characteristics of Collagen Biopolymer from Unicornfish (***Naso reticulatus* **Randall, 2001) Bone Prepared with Various Acid Types**

**Nurul Syazwanie Fatiroi 1, Abdul Aziz Jaziri 1,2, Rossita Shapawi 3, Ruzaidi Azli Mohd Mokhtar 4, Wan Norhana Md. Noordin <sup>5</sup> and Nurul Huda 6,\***


**Abstract:** Biopolymer-like collagen has great industrial potential in terms of its excellent properties, such as strong biocompatibility, high degradability, and low antigenicity. Collagen derived from fish by-products is preferable as it is safer (free from transmittable diseases) and acceptable to most religious beliefs. This study aimed to characterize the unicornfish (*Naso reticulatus* Randall, 2001) bone collagens prepared with different type of acids, i.e., acetic acid, lactic acid, and citric acid. A higher yield (Y) (*p* < 0.05) was obtained in the citric-acid-soluble collagen (CASC) (Y = 1.36%), followed by the lactic-acid-soluble collagen (LASC) (Y = 1.08%) and acetic-acid-soluble collagen (AASC) (Y = 0.40%). All extracted collagens were classified as type I due to the presence of 2-alpha chains (α1 and α2). Their prominent absorption spectra were located at the wavelengths of 229.83 nm to 231.17 nm. This is similar to wavelengths reported for other fish collagens. The X-ray diffraction (XRD) and infrared (IR) data demonstrated that the triple-helical structure of type I collagens was still preserved after the acid-extraction process. In terms of thermal stability, all samples had similar maximum transition temperatures (*Tmax* = 33.34–33.51 ◦C). A higher relative solubility (RS) of the unicornfish bone collagens was observed at low salt concentration (0–10 g/L) (RS > 80%) and at acidic condition (pH 1.0 to pH 3.0) (RS > 75%). The extracted collagen samples had an irregular and dense flake structure with random coiled filaments. Overall, bones of unicornfish may be used as a substitute source of collagen.

**Keywords:** collagen biopolymer; unicornfish bone; acid extraction; characterization

#### **1. Introduction**

Biopolymer collagen, a fibrillar protein, is one of the main structural components in the connective tissues of mammals and makes up almost 30% of total protein composition [1]. It is characterized by the unique right-handed triple-helical structure, composed of three left-handed polyproline-like helices, each with a (Gly–Xa–Ya) repeating sequence where Xa and Ya are often proline and hydroxyproline [2]. At least 29 types (I–XXIX) of collagens with different structures of polypeptides, amino acid motifs, and molecular characteristics have been studied [3]. Type I collagen has been intensively explored by researchers because of its special traits (i.e., strong biocompatibility, high biodegradability, and lack of antigenicity) [4]. Collagen has great potential in various industrial sectors such as medical, pharmaceutical, nutraceutical, and cosmetic. It has been approved for use in tissue engineering due to its ability to stimulate cellular migration, tissue matrix interaction, and tissue regeneration [5,6]. It is also applicable in developing drug-delivery systems and

**Citation:** Fatiroi, N.S.; Jaziri, A.A.; Shapawi, R.; Mokhtar, R.A.M.; Noordin, W.N.M.; Huda, N. Biochemical and Microstructural Characteristics of Collagen Biopolymer from Unicornfish (*Naso reticulatus* Randall, 2001) Bone Prepared with Various Acid Types. *Polymers* **2023**, *15*, 1054. https:// doi.org/10.3390/polym15041054

Academic Editor: Raffaella Striani

Received: 1 December 2022 Revised: 10 February 2023 Accepted: 13 February 2023 Published: 20 February 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

in treating hypertension, obesity, and diabetes [7]. In addition, it is an important cosmetic ingredient that serves as a natural humectant and moisturizer, preventing aging of the skin [8]. In food manufacturing, collagen is often used as a colloidal stabilizer, emulsifier, and foaming agent [9]. Collagens are mostly derived from the skins and bones of land vertebrates, especially bovine and porcine vertebrates. However, use of these animals raises consumer apprehension due to reported infectious diseases such as bovine spongiform encephalopathy, transmissible spongiform encephalopathy, and foot-and-mouth disease. Another constraint is associated with religious belief. For instance, Muslims and Jews are prohibited to consume or use porcine-derivative products, while cows and beef derivatives are forbidden in Hinduism [10]. To deal with these issues, alternative sources of collagen are necessary.

Over the last decade, fish collagen has gained considerable attention amongst scientists. This is evidenced by the large number of publications related to the extraction of fish collagens. Sources of the collagens include tiger grouper (*Epinephelus fuscoguttatus*) skin [11], bigeye tuna (*Thunnus obesus*) skin, bone, and scale [12], lizardfish (*Saurida tumbil*) skin, bone, and scale [13–15], grass carp (*Ctenopharyngodon idellus*) skin, bone, and scale [16], golden pompano (*Trachinotus blochii*) skin and bone [17], Spanish mackerel (*Scomberomorous niphonius*) skin and bone [18], sturgeon (*Huso huso*) skin [19], puffer fish (*Lagocephalus inermis*) skin [20], red stingray (*Dasytis akajei*) skin [21], Siberian sturgeon (*Acipenser baerii*) cartilage [22], leather jacket (*Odonus niger*) bone [23], tilapia (*Oreochromis mossambicus*) bone [24], grey mullet (*Mugil cephalus*) scale [25], and yellow tuna (*thunnus albacore*) swim bladders [26]. Fish collagens are mostly categorized as type I and their physicochemical properties, including thermal stability, solubility, and triple-helical structures, have also been evaluated. Interestingly, few studies have demonstrated that modification of the extraction process could increase the thermostability of fish collagen [27].

Unicornfish (*Naso reticulatus* Randall, 2001) belongs to the family Acanthuridae. It has a convex head with slight angularity before its eye, no horn on the forehead, and an emarginated caudal fin. This fish mainly lives in tandem with coral reefs, feeds on algae, and has tight skin-like jacket [28]. It is popularly served grilled in restaurants. By-products from *N. reticulatus* are usually discarded after processing, resulting in loss of a valuable biological resource. Therefore, converting the by-products into high-end product such as collagen is highly beneficial. Moreover, there is little information regarding extraction of collagen from this fish. Hence, this work aimed to compare the extractability of collagen from unicornfish bone using different acids (i.e., acetic acid, lactic acid, and citric acid), and determine the physicochemical and structural properties of the collagens.

#### **2. Results and Discussion**

#### *2.1. Yield and Hydroxyproline Content of Acid-Soluble Collagens*

Table 1 shows the yields (%) of acid-soluble collagens derived from unicornfish bone. The highest yield (Y) was recorded in CASC (Y = 1.36%) (*p* < 0.05) compared to AASC (Y = 0.40%) and LASC (Y = 1.08%) suggesting that citric acid may be the most effective acid for extracting collagen from fish bones. This finding was in agreement with the previous report on collagen from lizardfish (*S. tumbil*) bone [13]. The results in the present study were also comparable to those for other acid-soluble fish bone collagens such as bigeye tuna (*T. obesus*) (Y = 0.1%) [12], tilapia (*O. niloticus*) (Y = 0.5%) [24], grass carp (*C. idellus*) (Y = 0.7%) [16], carp (*C. carpio*) (Y = 1.06%) [25], and golden pompano (*T. blochii*) (Y = 1.25%) [17]. Logically different fish species, acids, and extraction procedures might influence the collagen yields [14,15]. There was no significant difference in hydroxyproline (Hyp) content (*p* > 0.05) between AASC and LASC. However, the Hyp content in CASC was significantly lower (*p* < 0.05). The Hyp contents recorded in the present study were lower than those for bigeye tuna (*T. obesus*) (82–87 mg/g) [12], cobia (*Rachycentron canadum*) (84–99 mg/g) [29], and marine eel (*Evencheslys macrura*) (94–98 mg/g) [30] bone collagens. These differences could be attributed to several factors including type of species, size, age, structure, and composition of fish tissue, as well as the extraction process [31]. Collagen

with high Hyp content could help to improve the structural stability of its molecules. Kittiphattanabawon et al. [32] stated that Hyp is a prominent component of amino acids and plays an essential role in stabilizing the triple-helical structure of collagen.

**Table 1.** Yield, Hyp composition, and color parameters of the acid soluble unicornfish (N. reticulatus) bone collagen.


AASC: acetic acid soluble unicornfish (*N. reticulatus*) bone collagen; LASC: lactic acid soluble unicornfish bone collagen; CASC: citric acid soluble unicornfish bone collagen. *L*\* = lightness; *a*\* = redness: green to red; *b*\* = yellowness: blue to yellow; and WI = whiteness index. Different lowercase superscripts in the same column indicate significant difference (*p* < 0.05).

#### *2.2. Color Attributes*

Color attributes of the AASC, LASC, and CASC are presented in Table 1. Sadowska et al. [33] pointed out that a lighter-color collagen is more preferable for developing new products in food, nutraceutical, or medical applications because it does not alter the original color of a product. There were no significant differences (*p* > 0.05) in the lightness (*L*\*) and whiteness (WI) values of all extracted collagens in the present study. However, significantly higher *L*\* values ranging from 79.25 to 82.55 was noted in the unicornfish bone collagens compared to lizardfish (*S. tumbil*) (72.76) [15] and barramundi (*Lates calcalifer*) (44.76–65.41) skin collagens [34]. Addition of hydrogen peroxide (H2O2) could increase the lightness of acid extracted collagens, as reported in lizardfish (*S. tumbil*) bone (88.54) [13] and snakehead fish (*Channa argus*) skin (89.49) [35] collagens so there is potential for further experimentation with different H2O2 concentrations. Moreover, the *a*\* and *b*\* values of LASC were significantly higher (*p* < 0.05) than those of AASC and CASC.

#### *2.3. SDS-PAGE Profile*

An SDS-PAGE image of the unicornfish bone collagens is presented in Figure 1. The electrophoretic pattern of each sample was almost similar, with two alpha (α1 and α2), one beta (β), and one gamma (γ) chains. The molecular weight (MW) of each alpha chain was estimated as 138.0 kDa and 118.3 kDa, respectively. Benjakul et al. [36] suggested that type I collagen was characterized by the presence of two alpha chains (α1 and α2). Based on this, unicornfish bone collagens were also categorized as type I. All acid-soluble collagens assessed in this study were comparable to previous literature on type I collagen fish collagen from seabass (*L. calcarifer*) (α1 = 118 kDa and α2 = 105 kDa) [37], loach (*M. anguillicaudatus*) (α1 = 127 kDa and α2 = 115 kDa) [38], golden pompano (*T. blochii*) (α1 = 120 kDa and α2 = 100 kDa) [17], and Nile tilapia (O. *niloticus*) (α1 = 125 kDa and α2 = 114 kDa) [39]. Other electrophoretic chains found in all extracted collagens (i.e., β = 278.1 kDa and γ = 383.0 kDa), indicate dimer and trimer bands as observed in our previous findings on lizardfish (*S. tumbil*) fish collagens [13–15]. Further analysis with addition of β-ME (reducing) and without β-ME (non-reducing), showed no differences in electrophoretic patterns of AASC, LASC, or CASC, and absence of disulfide bonds, as mentioned in previous literature [13–15].

**Figure 1.** SDS-PAGE image of acid soluble unicornfish bone collagen. M: protein marker; A1 and A2: acetic acid soluble collagen (AASC); B1 and B2: lactic acid soluble collagen (LASC); C1 and C2: citric acid soluble collagen (CASC).

#### *2.4. UV Absorption Spectra*

Figure 2 presents the UV absorption spectra of the AASC, LASC, and CASC. In general, a prominent spectrum of fish collagen was located at the wavelengths of 210 nm to 240 nm [40]. All acid-soluble collagens from the bone of unicornfish were within the maximum spectral ranges proposed by previous works, with no significant differences (*p* > 0.05) among the samples. The highest peak observed in this study was in accordance with other fish collagens, such as Siberian sturgeon (*A. baerii*) [22], red drum (*Sciaenops ocellatus*) [41], lizardfish (*S. tumbil*) [13], and puffer fish (*L. inermis*) [20]. The spectra observed were associated with the functional groups of carboxyl (-COOH), carbonyl (C=O), and amides (CONH2), which belong to the polypeptide chains of fish collagen, as proposed by Jaziri et al. [13]. Other low absorption peaks (300 nm to 250 nm) were also observed in all extracted collagens and were likely related to the aromatic amino acids, such as phenylalanine, tryptophan, and tyrosine. It is therefore assumed that collagens extracted from the bone of unicornfish contain low composition of aromatic amino acids.

**Figure 2.** UV absorption spectra of acid soluble unicornfish (*N. reticulatus*) bone collagen. AASC: acetic acid soluble collagen; LASC: lactic acid- soluble collagen; CASC: citric acid soluble collagen.

#### *2.5. Attenuated Total Reflection–Fourier Transform Infrared Spectroscopy (ATR–FTIR)*

FTIR spectra of AASC, LASC, and CASC are shown in Figure 3. Five significant peaks (Amides: A, B, I, II, and III) were clearly identified in all samples. As described in Table 2, Amide A represents the N-H stretching vibrations with hydrogen bonds which represents the protein molecules and is usually located at 3200–3440 cm–1 region [42], as observed in the present study. For Amide B, it described an asymmetric stretching of CH2 vibrations [21], and the higher wavenumber region of Amide B was observed in the

LASC and CASC samples. Meanwhile, Amide I is often related to the secondary structure of proteins, with wavenumbers ranging from 1600 to 1700 cm–1 [43] and represents the stretching vibration of the backbone carbonyl group (C=O) in polypeptides. The strong bands of Amide I in all acid-soluble collagens in the present study were in accordance with other literature [23]. On the other hand, Amides II and III have been widely used for the identification of triple-helical structure of collagen [44]. Amide II, typically located at the wavenumbers from 1500 cm–1 to 1600 cm–1 [45], which correspond to the N–H bending vibrations combined with the C–N stretching vibrations. The CASC showed lower wavenumbers compared to the AASC and LASC, indicating more H bonds in the CASC. Meanwhile, Amide III reflected the peak combination between C–N stretching and N–H remodeling, resulting in amide linkages which generally occur between 1200 cm–1 and 1350 cm–1 [41]. Similar peaks were also noted in other fish collagens, such as Nile tilapia (*O. niloticus*) [39], lizardfish (*S. tumbil*) [13–15], purple spotted bigeye snapper (*P. tayenus*) [46], sturgeon fish (*H. huso*) [19], barramundi (*L. calcarifer*) [37], and loach (*M. anguilllicaudatus*) [38].

**Figure 3.** IR spectra of acid soluble unicornfish (*N. reticulatus*) bone collagen. AASC: acetic acid soluble collagen; LASC: lactic acid soluble collagen; CASC: citric acid soluble collagen.


**Table 2.** The peak area and the description for the acid soluble unicornfish (*N. reticulatus*) bone collagen.

AASC: acetic acid soluble unicornfish (*N. reticulatus*) bone collagen; LASC: lactic acid soluble unicornfish bone collagen; CASC: citric acid soluble unicornfish bone collagen.

In terms of stability of the triple-helical structures, Benjakul et al. [36] suggested that the triple-helical structure was preserved if the difference in wavenumber between Amides I and II (Δ*v=vI*−*vII*) was less than 100 cm−<sup>1</sup> [47]. Based on this guideline, our results confirmed that the triple-helical structure of all extracted collagens from unicornfish bone were maintained because Δ*v* of the AASC, LASC, and CASC were 95.05 cm–1, 93.19 cm–1, and 76.42 cm–1, respectively. Another approach is through the ratio of the Amide III and

1450 cm−<sup>1</sup> band (AIII/A1450), as proposed by Doyle et al. [48]. After validation, the triplehelical structures of extracted collagens did not change during the extraction process as described from their absorption ratio values (~1.0), suggesting that the use of acetic, citric, and lactic acids during the extraction process could solubilize collagens without damaging the structures.

#### *2.6. Evaluation of X-ray Diffraction (XRD)*

Table 3 presents the diffraction data of the AASC, LASC, and CASC. Generally, two significant peaks were observed. The first peak was sharp, and second peak was broader. The obtained diffraction data were comparable to the triple-helical structure of calf-skin collagen (standard) [15]. Similar diffraction motifs were also noted in tilapia (*O. niloticus*) skin [49], carp fish (*C. carpio*) scale [24], golden pompano (*T. blochii*) skin and bone [27] and lizardfish (*S. tumbil*) skin, scale, and bone [13–15] collagens. In order to predict the minimum value of repeated spacings d (Å), the Bragg formula by Zhang et al. [25]. was used with d(Å) = λ/2sin *θ* (where λ is the X-ray wavelength of 1.54 Å and *θ* is the Bragg diffraction angle). As shown in Table 3, the first peak (d = 1.13–1.14 nm) reflects the distance between the molecular chains of triple-helical structure found in fish collagen, with higher d values being detected in the AASC and LASC samples. Meanwhile, the d value of AASC, LASC, and CASC samples in the second highest peak ranged from 0.33 nm to 0.34 nm, with the lowest observed in the CASC. This peak denotes the distance between skeletons of fish-collagen structure. The diameter (d) of a collagen molecule with a single left-handed helix chain and a triple-helical structure was consistent with the diameter of collagen from the barracuda skin prepared by solubilizing with different acids [13]. Overall, our extracted collagens showed no denaturation in the triple-helical structures and were in their native conformations.


**Table 3.** XRD and DSC analyses of the acid soluble unicornfish (*N. reticulatus*) bone collagen.

AASC: acetic acid soluble unicornfish (*N. reticulatus*) bone collagen; LASC: lactic acid soluble unicornfish bone collagen; CASC: citric acid soluble unicornfish bone collagen.

#### *2.7. Thermostability of Acid-Soluble Collagen*

Thermostability, as determined by *Tmax* value of the AASC, LASC, and CASC, is listed in Table 3. A higher thermostability was demonstrated in the AASC (*Tmax* = 33.51 ◦C), followed by LASC (*Tmax* = 33.39 ◦C) and CASC (*Tmax* = 33.34 ◦C. According to Benjakul et al. [36], thermostability of collagen was related to the presence of amino acids (proline and hydroxyproline), particularly at pyrrolidine rings that are governed by the H bonding via the hydroxyl group of Hyp. In addition, Hyp served as stabilizer of the triple-helical structure through H bonding in coil-coiled alpha chains [50]. Our *Tmax* results (around 33 ◦C) were comparable to other fish bone collagens, such as purple-spotted bigeye (*P. tayenus*) (30.80–31.48 ◦C) [32], Siberian sturgeon (*A. baerii*) skin (28.30 ◦C) [22], grass carp (*C. idellus*) (36 ◦C) [16], and golden pompano (*T. blochii*) skin (38.23 ◦C) [17]. Interestingly, fish from tropical waters showed higher thermostability compared to temperate fish such as Spanish mackerel (*S. niphonius*) (18.02 ◦C) [18]. The delta H value (Δ*H*) was defined as the area located under the thermogram peaks, which reflects the energy required to uncouple the alpha chains of collagen and convert them into random coils. The Δ*H* of the AASC sample was lower than of the LASC and CASC samples, indicating a lower energy used in AASC. The differences in the *Tmax* and Δ*H* values of fish collagen were likely influenced by

many factors, such as the composition of amino acids, extraction process, fish species, and other factors, particularly water temperature and habitat [32].

#### *2.8. Microstructure Profile*

The AASC, LASC, and CASC were scanned under a scanning electron microscope (SEM), and the morphological structures were appraised. As illustrated in Figure 4, the extracted collagens of unicornfish bones showed fibril-forming structures, multi-layered forms, and irregular sheet-like films linked by random-coiled filaments. In addition, the wrinkled and porous structures were also clearly visible at magnification of 500×, indicating the samples were dehydrated during the lyophilization process, as documented by Schuetz et al. [51]. According to Lim et al. [7], fish collagen with fibrillary, interconnectivity, and sheet-like film structures could be a potential source of biomaterials for nutraceutical, pharmaceutical, and biomedical products to be used in wound dressing, skin and bone tissue formation, cell migration, and coating material. The microstructure profiles of Lizardfish (*S. tumbil*) skin, bone, and scale [13–15], miiuy croaker (*M. miiuy*) scale [52], black ruff (*Centrolophus niger*) skin [53], and marine eel (*E. macrura*) skin [30] collagens were in agreement with this recent work.

**Figure 4.** *Cont*.

**Figure 4.** SEM image (magnification 500×) of the acid-soluble unicornfish (*N. reticulatus*) bone collagen. (**A**) acetic acid soluble collagen (AASC); (**B**) lactic acid soluble collagen (LASC); and (**C**) citric acid soluble collagen (CASC).

#### *2.9. Solubility Studies*

Solubility of the AASC, LASC, and CASC samples was evaluated at different sodium chloride (NaCl) concentrations and various pH conditions. Higher solubility (more than 80% of relative solubility (RS)) was observed in all extracted collagens when treated with low NaCl concentration (0–10%). RS of >85% was recorded in the LASC sample (Figure 5A). This might have been due to the effect of the dialysis process in LASC that completely removed the remaining salt after being salted out during the precipitation process. As a result, no salt was detected in the lyophilized collagen. However, at high concentrations of NaCl (30 g/L to 60 g/L), the RS decreased sharply to less than 40% in all extracted collagens. Chen et al. [41] suggested that at high salt concentration, the hydrophobic–hydrophobic interactions in the polypeptide chain were escalated. The competition for water with salt ions was also increased, resulting in protein precipitation. These results were in accordance with collagens isolated from the skin and bone of Spanish mackerel (*S. niphonius*) [18], the skin of lizardfish (*S. tumbil*) [13], the skin and bone of golden pompano (*T. blochii*) [17], and the cartilage of Siberian sturgeon (*A. baerii*) [22].

In the context of pH, the solubility was increased in acidic acid conditions, especially at pH 1.0 and pH 5.0 (Figure 5B). The highest RS value (>90%) was noted in all extracted collagens treated at pH 3.0. In contrast, the solubility decreased at pH 7.0 and alkaline (pH 9.0) conditions. The increase in the hydrophobic–hydrophobic interactions among the collagen molecules might have resulted in the total net charge becoming zero, particularly at the isoelectric point which commonly occurs at slightly acidic and neutral conditions [54]. However, at pH 11.0, the RS were slightly increased to around 25–60%. This could be due to the effect of electrostatic repulsion between collagen molecules and hydration of charged residues at pH values above the isoelectric point (pI) [22]. Chuaychan et al. [22] stated that differences in the solubility of collagen treated with various pH were related to the difference in the molecular properties and conformations of collagen. Our findings were equivalent to those for the collagens extracted from the skin and bone of Spanish mackerel (S. niphonius) [18], skin of lizardfish (*S. tumbil*) [13], skin and bone of golden pompano (*T. blochii*) [17], and cartilage of Siberian sturgeon (*A. baerii*) [22].

**Figure 5.** Relative solubility (RS) of acid soluble collagens from the unicornfish (*N. reticulatus*) bone at (**A**) different NaCl concentrations and (**B**) various pH levels. AASC: acetic acid soluble collagen; LASC: lactic acid soluble collagen; CASC: citric acid soluble collagen.

#### **3. Conclusions**

Unicornfish (*N. reticulatus*) bone collagens extracted with the aid of various organicacid solutions (i.e., acetic, lactic, and citric acids) were evaluated. The highest collagen yield (*p* < 0.05) was recorded for the citric-acid-soluble collagen (CASC) compared to that of acetic-acid-soluble collagen (AASC) and lactic-acid-soluble collagen (LASC). The triple-helical structures of type I extracted collagens were still maintained, indicating no denaturation of all samples during the acid-extraction process as confirmed by FTIR and XRD analysis. Although LASC had a lower collagen yield than CASC, other characteristics (i.e., thermostability, hydroxyproline content, color attributes, and solubility) were found to be preferable in the LASC sample. Thus, lactic-acid-soluble collagen (LASC) could be used as an alternative collagen for further research.

#### **4. Materials and Method**

#### *4.1. Materials*

Fifteen kilograms of fresh unicornfish (*N. reticulatus*) were obtained from a local supplier in Kota Kinabalu, Sabah, Malaysia. Samples were placed in an ice-cooled insulating box (ratio of fish to ice was 1:2 (*w*/*w*)) to maintain their freshness during transportation. Upon arrival, fish were subjected to species identification and weight (521.58 ± 32.27 g) and length (30.52 ± 4.28 cm) measurement. The prepared samples were separated automatically using a mechanical deboner machine (SFD-8 type, Taiwan). The fish bones were subsequently cut into small portions (1.0 × 1.0 cm2) with a stainless-steel knife (Brisscoes, Malaysia), and washed with running tap water. The washed samples were packed in a polyethylene bag and then stored in a freezer (−20 ◦C) for further analyses. Sodium dodecyl sulphate (SDS), acrylamide powder, Coomassie Blue R-250, *N,N,N ,N* -tetramethyl ethylene diamine (TEMED), and Folin–Ciocalteu's phenol reagent acetic acid (glacial) were purchased from Merck (Darmstadt, Germany). Precision Plus Protein Dual Color standards (markers) was purchased from Bio-Rad Laboratories (Hercules, CA, USA). Bovine serum albumin (BSA) and Lowry reagent were supplied from Sigma Chemical Co., (St. Louis, MO, USA). Citric acid and lactic acid solution were supplied from Systerm (Selangor Darul Ehsan, Malaysia) and Bendosen (Selangor, Malaysia), respectively. Other chemicals used in this research were of analytical grade.

#### *4.2. Preparation of Acid-Soluble Collagen*

Extraction of unicornfish bone collagens with different acid solutions was carried out according to Jaziri et al. [13] with slight modification. All steps were strictly performed in a chiller (4 ◦C), and the extraction process is depicted in Figure 6. A total of 100 g fish bones was soaked in ten volumes of 0.1 M sodium hydroxide solution for 6 h (the solution was changed every 3 h) with continuous stirring. The pretreated samples were washed with distilled water to achieve neutral condition (pH 7.0). This step aimed to remove non-collagenous protein and pigmentation. Next, the neutralized samples were demineralized by dissolving in ten volumes of 0.5 M ethylenediaminetetraacetic acid disodium salt solution (pH 7.4) for 24 h with continuous stirring, and the solution was changed every 12 h. The demineralized samples were then washed with cold distilled water for 30 min and the distilled water was replaced every 10 min. After the pre-treatment step, the fish-bone samples were subjected to extraction by adding 15 volumes of 0.5 M organic-acid solutions (i.e., acetic acid, lactic acid, and citric acid) for 72 h. The extracted samples were then filtered through two layers of cheesecloth, and the filtrates were saltedout by dissolving in 2.5 M sodium chloride, followed by 0.05 M Tris (hydroxymethyl) aminomethane hydrochloride at a neutral pH (7.0). After that, the precipitates were centrifuged (Eppendorf Centrifuge 5810R, Hamburg, Germany) at 15,000× *g* for 15 min. The pellets were subsequently dissolved in acid solution (based on each acid used during extraction) at a ratio of 1:1 (*w*/*v*). The solutions were prepared for dialysis by transferring into a cellulose-membrane tubing (average flat width 43 mm, 1.7 in.) (Sigma-Aldrich, St. Louis, MO, USA) and then put in 15 volumes of 0.1 M acid solution for 24 h, followed by a distilled water for 48 h (the distilled water was changed every 24 h). The dialyzed samples were dried using a freeze-dryer machine (Labconco, Kansas City, MO, USA). After freeze drying, all dried samples were stored at a −20 ◦C. The dried collagens were labeled acetic-acid-soluble bone collagen (AASC), lactic-acid-soluble bone collagen (LASC), and citric-acid-soluble bone collagen (CASC).

**Figure 6.** The procedure of acid-soluble unicornfish (*N. reticulatus*) bone collagen. AASC: acetic-acidcollagen; LASC: lactic-acid-soluble collagen; CASC: citric-acid-soluble collagen.

#### *4.3. Analyses*

4.3.1. Yield and Hydroxyproline (Hyp) Measurement

The yields of collagens (AASC, LASC, and CASC) were measured according to the formula developed by Jongjareonrak et al. [54], as noted below:

$$\text{Yield } (\%) = \frac{\text{Weight of dried collagen}}{\text{Initial weight of unicornfish bone}} \times 100\tag{1}$$

The Hyp content (mg/g) of extracted collagens was determined using an established procedure [55]. The lyophilized collagens were subjected to hydrolysis by adding a strong acid solution (6 M of HCl) at high temperature (110 ◦C) for 24 h. Afterwards, the hydrolysates were filtered through a Whatman filter paper No. 4 (Sigma-Aldrich, St. Louis, MO, USA). The filtrates were subsequently adjusted with 2.5 M NaOH solution to achieve pH of 6.5–7.0. Approximately 0.2 mL of hydrolysates was transferred into prepared test tubes and 0.4 mL isopropyl alcohol added. Subsequently, 0.2 mL of oxidant solution was added to the solutions and they were allowed to stand at room temperature for 5 min. After that, a total of 2.3 mL Ehrlich's reagent solution was dropped in and mixed thoroughly. The mixtures were then heated in a water bath (Memmert, Schwabach, Germany) at 60 ◦C for 25 min. After heating, the treated samples were cooled for 10 min at room temperature. The cooled samples were further diluted with an isopropyl alcohol (up to 10 mL). Absorbance against distilled water was determined at a wavelength of 558 nm using a spectrophotometer (Agilent Cary 60, Santa Clara, CA, USA). Hyp standard solution (10 to 70 ppm) was also prepared in this current study.

#### 4.3.2. Color Attributes

Color attributes of AASC, LASC, and CASC samples were determined as described by Ismail et al. [56] using a colorimeter (ColorFlex CX2379, HunterLab, Reston, VA, USA). The attributes examined were *L*\* (lightness), *a*\* (redness: green to red), and *b*\* (yellowness: blue to yellow). The whiteness index (WI) of the extracted collagens was calculated using the following formula [57].

$$\text{WI} = 100 \, - \, \left[ \left( 100 \, - \, L^\* \right)^2 + \left( a^{\*2} \right) + \left( b^{\*2} \right) \right]^{0.5} \tag{2}$$

4.3.3. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)

SDS-PAGE analysis of unicornfish bone collagens was conducted using a Mini-PROTEAN electrophoresis system (Bio-Rad Laboratories, Hercules, CA, USA). We used the established method from Laemmli [58] with minor amendments. Each dried collagen (around 2.5 mg) was dissolved in SDS solution (5%) and mixed thoroughly. The mixture was treated with high thermal condition (85 ◦C) using a water bath (Memmert, Schwabach, Germany) for 1 h. After heating, the mixtures were prepared by centrifuging at 8500× *g* for 5 min to

remove undissolved matter. Around 15 μL of supernatants was transferred into a mini centrifuge tube and subsequently, 15 μL sample buffer in the presence and absence of 10% β-mercaptoethanol (β-ME) was added. After that, the mixtures were reheated at the same temperature for 5 min and then loaded into polyacrylamide gel composed of 4% stacking gel and 7.5% resolving gel. The acrylamide gel was electrophoresed with a constant voltage of 120 volts for 90 min. When electrophoresis ended, the gel was immersed in the staining solution containing 0.1% (*v*/*v*) Coomassie Blue R-250, 30% (*v*/*v*) methanol and 10% (*v*/*v*) acetic acid for approximately 30 min. Next, the stained acrylamide gel was destained with 10% (*v*/*v*) acetic acid and 30% (*v*/*v*) methanol solutions. The electrophoretic bands of AASC, LASC, and CASC were compared to the protein marker (Precision Plus Protein Dual Color Standards).

#### 4.3.4. Ultraviolet–Visible Absorption Spectra

Ultraviolet–visible (UV–vis) absorption spectra of all extracted collagens were determined using a UV–vis spectrophotometer (Agilent Cary 60, Santa Clara, CA, USA). A total of 5 mg of lyophilized collagens was dissolved with acetic acid solution (0.5 M), and well mixed. Next, the mixtures were centrifuged at 8500× *g* for 15 min to separate solubilized and insolubilized matters. The solubilized samples were then placed into a quartz cell (its optical path length was of 10 mm). The spectral wavelengths used in the present study were arranged from 400 nm to 200 nm with a baseline of acetic acid solution [14].

#### 4.3.5. Attenuated Total Reflectance–Fourier Transform Infrared Spectroscopy (ATR–FTIR)

ATR—FTIR data of the AASC, LASC, and CASC were obtained from FTIR spectrometer (Agilent Cary 630, Santa Clara, CA, USA) as described from our previous study [15]. A total of 10 mg dried fish bone collagens was placed onto the crystal cell of FTIR spectrometer. Spectral data were prepared with a resolution of 2 cm–1 throughout a wavenumber range of 4000–1000 cm–1 for 32 scans against a background spectrum found from clean empty cells at ambient temperature. The obtained data were then analyzed using a software of Agilent Microlab.

#### 4.3.6. X-ray Diffraction (XRD) Data

Dried samples (AASC, LASC, and CASC) were scanned using a XRD instrument (Rigaku Smart Lab®, Tokyo, Japan), with copper Kα applied as an x-ray source. The tube voltage and current were set up at 40 kV and 50 mA, respectively. The scanning range in all acid-soluble collagens was prepared by adjusting from 10◦ to 40◦ (2θ) with a speed of 0.06◦ per second. The obtained data were recorded and analyzed by comparing to another previous research. XRD was carried out using the method of Chen et al. [21].

#### 4.3.7. Differential Scanning Calorimetry (DSC)

A DSC machine (Perkin-Elmer, Model DSC7, Norwalk, CA, USA) was used to obtain the thermostability value of the extracted collagens from the unicornfish bones. First, the prepared samples were hydrated by adding deionized water at a ratio of 1:40 (*w*/*v*) for 48 h in a chiller. The hydrates were then weighed from 5 mg to 10 mg into an aluminum pan (Perkin-Elmer, Norwalk, CA, USA), and tightly sealed. The DSC instrument was previously calibrated using an indium as a standard, and the sealed samples were subsequently scanned, ranging from 20 ◦C to 50 ◦C at a rate of 1 ◦C per minute. An empty pan was used as a reference. Thermostability in all samples was determined using the maximum transition temperature (*Tmax*), which was collected from the endothermic peak of thermogram, and the total denaturation enthalpy (Δ*H*) was recorded from the area of thermogram [59].

#### 4.3.8. Scanning Electron Microscopy (SEM)

Morphological evaluation of the AASC, LASC, and CASC samples was carried out using a scanning electron microscopy (Carl Zeiss, model MA 10, Germany). Prior to scanning, all acid-extracted collagens were sputter-coated with the gold for 5 min using a coater device (JEOL JFC-1200, Tokyo Rikakikai Co., Ltd., Tokyo, Japan). Next, all coated samples were imaged with a magnification (500×) [52].

#### 4.3.9. Solubility Study

Solubility of acid-soluble collagen was assessed at different sodium chloride (NaCl) concentrations and pH conditions. The procedure used was as described by Matmaroh et al. [59]. For solubility in NaCl, different NaCl concentrations (0–60 g/L) were used. Approximately 5 mL of solubilized collagens was prepared and transferred into 5 mL of different NaCl concentrations. The mixtures were then stirred continuously using a magnetic stirrer (ST0707V2, Selangor, Malaysia) for 30 min in a chiller. The mixtures were centrifuged (8500× *g*) for 10 min after being dissolved. For pH treatment, the dried collagens were added to 0.5 M of acetic acid solution. Afterwards, the dissolved samples were adjusted with different pH, from pH 1.0 to pH 11.0. The 2.5 N HCl and 2.5 N NaOH solutions were used for pH adjustment. The pH-adjusted samples were stirred for 2 h and subsequently centrifuged at 8500× *g* for 10 min. To determine percentage of relative solubility (% RS), all solubilized collagens (treated by different NaCl or pH) were subjected to protein measurement using the method of Lowry et al. [60], with bovine serum albumin (BSA) used as a standard. The RS (%) of all samples was measured using the following equation:

$$\text{Relative solubility} \left( \% \right) = \frac{\text{Current concentration of protein at current NaCl or pH}}{\text{The highest concentration of protein}} \times 100\tag{3}$$

#### *4.4. Statistical Analysis*

Experiment in this study was carried out in triplicate, and collected data are presented as means with standard deviation. One-way ANOVA was applied, and Duncan's multiple range test was used to compare means with a significant effect signed in *p* < 0.05 under a SPSS Statistics version 28.0 (IBM Corp., Armonk, NY, USA).

**Author Contributions:** Conceptualization, N.H.; methodology, A.A.J. and N.H.; software, N.S.F. and A.A.J.; validation, R.S., R.A.M.M., W.N.M.N. and N.H.; formal analysis, N.S.F..; investigation, N.S.F. and A.A.J.; resources, A.A.J. and N.H.; data curation, R.S., R.A.M.M., W.N.M.N. and N.H.; writing—original draft, N.S.F. and A.A.J.; writing—review and editing, R.S., R.A.M.M., W.N.M.N. and N.H.; visualization, A.A.J.; supervision, N.H.; project administration, N.H.; funding acquisition, N.H. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the Ministry of Higher Education Malaysia (FRGS/1/2019/ STG03/UMS/02/5) and Universiti Malaysia Sabah (UMS) for the payment of APC.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available upon request from the corresponding author.

**Acknowledgments:** The authors are grateful to the Ministry of Higher Education Malaysia for the funds provided through the Fundamental Research Grant Scheme (FRGS) with a grant number FRGS/1/2019/STG03/UMS/02/5 and to Universiti Malaysia Sabah (UMS) for their support in completing this research article.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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## *Review* **Biodegradable Polylactic Acid and Its Composites: Characteristics, Processing, and Sustainable Applications in Sports**

**Yueting Wu 1, Xing Gao 1,\*, Jie Wu 1, Tongxi Zhou 1, Tat Thang Nguyen <sup>2</sup> and Yutong Wang <sup>1</sup>**


**Abstract:** Polylactic acid (PLA) is a biodegradable polyester polymer that is produced from renewable resources, such as corn or other carbohydrate sources. However, its poor toughness limits its commercialization. PLA composites can meet the growing performance needs of various fields, but limited research has focused on their sustainable applications in sports. This paper reviews the latest research on PLA and its composites by describing the characteristics, production, degradation process, and the latest modification methods of PLA. Then, it discusses the inherent advantages of PLA composites and expounds on different biodegradable materials and their relationship with the properties of PLA composites. Finally, the importance and application prospects of PLA composites in the field of sports are emphasized. Although PLA composites mixed with natural biomass materials have not been mass produced, they are expected to be sustainable materials used in various industries because of their simple process, nontoxicity, biodegradability, and low cost.

**Keywords:** PLA; biocomposites; biodegradation; sports equipment manufacturing

#### **1. Introduction**

The continuous advancement of science and technology has increased the global demand for natural resources, leading to frequent problems, such as material shortages and environmental pollution. Rapidly depleting oil reserves, greenhouse gas emissions, and the large-scale use of oil-based products have resulted in a lack of biodegradable products, prompting researchers to explore biodegradable, renewable, and recyclable materials. Polylactic acid (PLA) is a biodegradable bio-based aliphatic polyester that can be extracted from 100% renewable resources, such as corn, potatoes, and sugarcane [1]. Compared with traditional petroleum-based composite materials, PLA has a low density, low cost, good plasticity, and rigidity. PLA possesses excellent workability, making it an ideal choice for 3D printing sports equipment. 3D printing can be used to adjust the density and structure of a material according to specific requirements, allowing for personalized customization and innovative designs based on individual measurements and particular needs. This can achieve an ideal combination of lightweight and high strength to ensure that PLA sports equipment does not impose excessive burdens on athletes, enhances athletic performance, and protects different individuals. Although PLA possesses many characteristics suitable for the fabrication of sports equipment, more research is needed. Figure 1 compares the characteristics of bioplastics and petro plastics, showing that PLA occupies a crucial position in the biopolymer market and plays a vital role in various fields, such as in automotive, aerospace, construction, defense, food packaging, and sports equipment applications [2–5].

**Citation:** Wu, Y.; Gao, X.; Wu, J.; Zhou, T.; Nguyen, T.T.; Wang, Y. Biodegradable Polylactic Acid and Its Composites: Characteristics, Processing, and Sustainable Applications in Sports. *Polymers* **2023**, *15*, 3096. https://doi.org/ 10.3390/polym15143096

Academic Editor: Raffaella Striani

Received: 16 June 2023 Revised: 16 July 2023 Accepted: 17 July 2023 Published: 19 July 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

**Figure 1.** Properties of bioplastics and petro plastics (adapted from Refs. [6,7]).

PLA is an extracted thermoplastic that is suitable for manufacturing composite materials using various methods, such as injection molding, extrusion molding, and compression molding [8]. Increases in the annual supply of PLA (Figure 2) and competitive petroleum costs are key factors driving researchers to develop new PLA-based biocomposites. PLA can biodegrade and bioaccumulate, which helps reduce production and waste disposal costs. PLA can also be treated by landfilling, incineration, or pyrolysis. More than 50% (2.8 kg CO2/kg PLA) of the released CO2 in the PLA life cycle is released during its conversion. By optimizing the conversion process of PLA, there is tremendous potential for PLA to become a low-carbon material [9].

**Figure 2.** Global production capacity of PLA (adapted from Ref. [10]).

This report discusses the latest developments in the research and development of PLA and its composites. It first outlines the basic structure, characteristics, production, degradation process, and latest modification methods of PLA, and it discusses the inherent advantages of selecting PLA composite materials and focuses on the relationship between different biodegradable materials and their performance in the final PLA composite materials. Then, it introduces the application prospects of PLA composite materials in the field of sports. Finally, it discusses the challenges faced by PLA composite materials and competing materials.

#### **2. Overview of PLA**

PLA is an entirely biodegradable polymer hailed as one of the most promising biobased polymers because of its biocompatibility, biodegradability, high mechanical strength, nontoxicity, nonirritation, and processability. PLA can be synthesized by low-energy processes, and it is independent of petroleum resources. Microorganisms can decompose

waste PLA into H2O and CO2. After photosynthesis, CO2 and water are converted back into substances such as starch, which can be used as raw materials to resynthesize PLA, thereby realizing a carbon cycle process [11] that does not pollute the environment.

#### *2.1. PLA Structure*

Lactic acid molecules contain a chiral asymmetric α-carbon atom and exhibit optical activity that can be divided into two configurations: left-handed (L) and right-handed (D). The dehydration of two lactic acid molecules forms three optical isomers of lactide: L-lactide, D-lactide, and meso-lactide. L-lactide is cheaper because it is naturally occurring. The content of D-lactic acid changes the crystallization behavior of PLA, including different crystallization rates, multiple crystal types, different scales, and layer thicknesses. The crystal morphology is closely related to the mechanical properties of the polymer: the larger the PLA crystal, the more defects in the interior and on the surface of the crystal, and the poorer the mechanical properties of the resulting material [12]. Like L-lactide, meso-lactide is a cyclic diester with two chiral carbon atoms that are not optically active. PLA synthesized via lactide ring-opening polymerization (ROP) has three different stereo configurations: left-handed polylactic acid (PLLA), right-handed polylactic acid (PDLA), and racemic polylactic acid (PDLLA) [13]. Figure 3 shows the three stereo configurations of PLA. The properties and applications of these PLA stereo configurations depend on the molecular weight, molecular weight distribution, crystal structure, and melt rheological behavior.

**Figure 3.** Three-dimensional configuration of PLA (adapted from Ref. [13]).

#### *2.2. Properties of PLA*

PLA is a member of the family of aliphatic polyesters and has the essential characteristics of universal polymer materials. PLA has a tensile strength similar to that of polyethylene terephthalate (PET), approximately 54 MPa, while its tensile modulus is 3.4 GPa, which is slightly higher than that of PET [14,15]. The mechanical properties of PLA are greatly affected by its molecular weight (*M*w). When the molecular weight doubles from 50 kDa to 100 kDa, the PLA's tensile strength and elastic modulus also double [16]. The mechanical properties of PLA depend on its semicrystalline structure, amorphous structure, and crystallinity. Semicrystalline PLA shows greater mechanical properties than amorphous PLA. Upon increasing the PLA crystallinity and decreasing the molecular chain mobility, the elongation at the break of the material decreases, while the tensile strength and modulus increase. Table 1 shows the properties of PLA and its different stereo configurations. In addition, the stereochemical structure of lactic acid-based polymers can be controlled by copolymerizing L-lactide, D-lactide, D, L-lactide, and meso-lactide to slow the crystallization rate, which significantly impacts the mechanical properties [17,18].

The environmental degradation process of PLA occurs in two steps: hydrolysis and microbial degradation. PLA first undergoes the hydrolytic cleavage of ester bonds, degrading into PLA oligomers (OLAs) [19]. The hydrolysis of PLA can be catalyzed by acid or alkali and is also affected by temperature and humidity [20,21]. As hydrolysis proceeds, the number of –COOH groups in the system gradually increases, which plays a catalytic role in the cleavage of PLA ester bonds [22]. This makes the degradation of PLA a selfcatalytic process. When the molecular weight of PLA decreases to below 10,000 g/mol, microorganisms can participate in the degradation process of PLA and eventually degrade it into H2O and CO2.



#### *2.3. PLA Production*

Figure 4 shows the synthetic route of PLA. Researchers extract starch from renewable natural resources, such as corn and potatoes, and ferment it to produce PLA. Traditional lactic acid fermentation uses starchy raw materials, and some countries have developed the use of agricultural and sideline products as raw materials for this process. The two main methods for synthesizing PLA are direct condensation and lactide ROP [32].

**Figure 4.** PLA synthetic pathways (adapted from Ref. [32]).

#### 2.3.1. Direct Polycondensation

In the late 1980s, the advancement of direct condensation technology significantly increased the global production of PLA and greatly reduced its costs. Direct condensation involves preparing PLA by dehydrating and condensing lactic acid molecules. The disadvantage of this method is that the reaction system is in a dynamic equilibrium between condensation and depolymerization, and the high viscosity of the system makes it difficult to remove the water by-product. The unremoved water causes the depolymerization reaction to proceed, even under vacuum conditions, making it difficult to extract water and increasing the molecular weight of the PLA. Under a high temperature (>200 ◦C), the PLA will undergo depolymerization, discoloration, and racemization accompanied by a series of side reactions, such as ester exchange, which may form differently sized cyclic products. This results in reduced product properties and poor mechanical properties, which limit their industrial applications. However, the use of direct condensation to produce PLA is a short

and inexpensive method. Chen et al. used a combination of direct condensation and melt polymerization using tetra butyl titanate as a catalyst. They used different vacuum periods, esterification, and condensation reactions. The results showed that this method reduced the system's viscosity, thus helping to remove water and increase the molecular weight [33].

#### 2.3.2. Ring-Opening Polymerization (ROP)

In the early 1990s, Cargill Inc. applied for a patent for a solvent-free process and new distillation technology based on ROP to convert lactic acid into high-molecularweight polymers. This made PLA the second-highest volume bioplastic after starch-based materials. By utilizing specific microbial strains, natural agricultural materials can undergo fermentation to produce lactic acid (LA), which is a precursor for PLA [34,35]. The mature ROP process can make high-molecular-weight and chemically controllable PLA samples with good mechanical properties by controlling lactide's purity and reaction conditions. This is currently the most common method for the industrial production of high-molecularweight PLA. The technical difficulties of ROP production lie in the synthesis and purification of lactide. Lactide ROP, first, generates oligomers via the dehydration–condensation of lactic acid, and then oligomers are cracked into lactide using initiators, and the lactide, finally, undergoes ROP to generate PLA. Only high-purity lactide can be used to synthesize high-molecular-weight PLA with the desirable physical properties. Depending on the initiator used, lactide ROP can be divided into anionic, cationic, or coordination ROP. Among them, cationic ROP uses a smaller amount of catalyst, while anionic ROP has high reactivity and a fast speed [36].

#### *2.4. Modified PLA*

According to Refs. [37–39], the low flexibility, elongation, impact resistance, and heat distortion temperature of PLA results in problems such as low crystallinity, long injection molding cycle, high moisture sensitivity, and low hydrolysis resistance. Researchers have used different modification techniques to improve the performance of PLA, such as copolymers and blending with nanocomposites or other polymers.

#### 2.4.1. Copolymers

PLA is a thermoplastic polymer whose processing temperature is generally between 170 and 230 ◦C. In recent years, researchers have produced self-reinforcing PLA through techniques such as melt extrusion, stretching, and injection molding without the need for additives, which retain the biocompatibility and biodegradability of PLA. This method can also solve the trade-off between the toughness and strength and compatibility of blends. In addition, Cao et al. [40] designed a new modification process. After isothermal crystallization, blow molding was carried out below the melting point of crystalline PLA. A crystal network was formed through stretching and blow-molding to prepare a selfreinforcing PLA film. The elongation at the break of this film increased by approximately 67.50% and 104.83% in the transverse and longitudinal directions, respectively, and the tensile strength increased by approximately 45.4 MPa and 78.0 MPa in the transverse and longitudinal directions. This overcame the trade-off between the toughness and strength.

#### 2.4.2. Blending with Nanocomposites

Chrissafis et al. [41] added 2.5% oxidized multiwalled carbon nanotubes into PLA and found that the thermal stability of the modified PLA material was greater than that of pure PLA, and the thermal conductivity increased by about 60%. The hexagonal mesh structure and stable chemical bonds of oxidized multiwalled carbon nanotubes made them highly durable, with a decomposition temperature above 1000 ◦C. Since oxidized multiwalled carbon nanotubes disperse the heat absorbed by PLA, the modified PLA's thermal conductivity and thermal stability were enhanced. In addition, oxidized multiwalled carbon nanotubes acted as heterogeneous nucleating agents in the PLA matrix. The growth of PLA crystals around the oxidized multiwalled carbon nanotubes shortened the

induction process of PLA nucleation, accelerated the PLA crystallization, and reduced the spherulite size. Seligra et al. [42] grafted modified carbon nanotubes onto PLA, which significantly increased the conductivity of the modified PLA material to 4000 s/m. The added carbon nanotubes formed an electron-conducting network that lowered the percolation threshold, thereby transforming PLA into a conductive polymer. A small amount of carbon nanotubes was sufficient to increase the conductivity without affecting the material's mechanical properties.

#### 2.4.3. Blending with Other Polymers

Researchers can improve the mechanical properties of polymers by changing the structure and composition of copolymers. Adjusting the ratio of lactic acid and other monomers in the copolymer system can produce copolymers with the desired mechanical strength to improve the mechanical properties of PLA. By utilizing the hydroxyl and carboxyl groups on the lactic acid segment, different monomers, such as caprolactone (CL), ethylene oxide (EO), ethylene glycol (EG), and trimethylene carbonate (TMC), can be used to synthesize PLA copolymers with improved mechanical properties, especially toughness. Li et al. [43] prepared alternating and random polyurethane copolymers using PLA and polyethylene glycol (PEG). The alternating polyurethane copolymer had a more controllable structure than the random polyurethane copolymer and, therefore, showed higher crystallinity and mechanical properties. Huang et al. [44] developed an electrochemically controlled switchable copolymer system and used it to quickly synthesize multisegment copolymers of PLA and polycarbonate propylene (PPC) without adding external oxidants or reducing agents. In this way, they exploited the complementary advantages of PPC (toughness) and PLA (mechanical strength).

#### *2.5. PLA Degradation*

PLA is a biopolymer that can also undergo biodegradation under certain conditions without producing environmental pollution [45,46]. Polymer degradation can be divided into heterogeneous and homogeneous degradation, also known as surface and intramolecular polymer degradation, which can occur through three different chemical reactions: (a) main-chain cleavage, (b) side-chain cleavage, and (c) cross-link cleavage. PLA degradation mainly occurs through ester bond cleavage, which splits long polymer chains into shorter oligomers, dimers, or even monomers. Specifically, the ester bonds of PLA are cleaved via chemical hydrolysis, and under the action of salicylic acid, they are split into carboxylic acids and alcohols. These shorter units are small enough to pass through the cell walls of microorganisms, where they serve as substrates for their biochemical processes and are degraded by microbial enzymes. PLA can be composted to produce CO2 and H2O, requiring temperatures near the *T*g (60 ◦C) of the polymer and a high relative humidity [47]. The CO2 emissions are offset by the initial absorption during PLA production. Under such conditions, the degradation time can be as short as 30 days.

Piedmont and Gironi [48] studied the hydrolytic degradation kinetics of PLA at concentrations of 5–50 wt% between temperatures of 140 ◦C and 180 ◦C. The results showed that the reaction kinetics did not depend on the concentration of PLA, and the collected data indicated two different reaction mechanisms. The first mechanism was related to a biphasic reaction (*E*<sup>a</sup> = 53.2 kJ mol<sup>−</sup>1), and the second mechanism was associated with a selfcatalytic effect of increasing carboxylic acid groups during the depolymerization process (*E*<sup>a</sup> = 36.9 kJ mol−1). This effect was previously noted in PLA hydrolysis and lowered the solution pH. The group's further work modeled the hydrolysis of PLA at higher temperatures (170–200 ◦C). The kinetic model described the batch erosion of PLA and subsequent hydrolysis of oligomers, and the model accurately predicted the conversion and concentration of oligomers. Under these conditions, PLA could be completely transformed within 90 min.

#### **3. PLA Composite Materials**

Although PLA has excellent mechanical properties, renewability, biodegradability, and low costs [49], Figure 5 shows PLA composite degradation process, it is also brittle and has low heat resistance [50]. Researchers have explored various reinforcement materials to develop PLA composite materials to overcome these drawbacks [51], such as cellulose, lignin, silk, PBAT, and PHA. Table 2 compares the mechanical properties of different PLA composite materials.


**Table 2.** Comparison of the mechanical properties of different PLA composites.

**Figure 5.** PLA composite degradation process (adapted from Refs. [46,47,57]).

#### *3.1. Natural Fibers*

Natural fibers can be divided into plant and animal fibers according to their sources [58]. Generally, combining natural fibers with PLA significantly improves the tensile strength, flexural strength, elastic modulus, heat distortion temperature, and other properties of PLA composites. This also enhances their impact resistance and dimensional stability [59,60] while reducing costs. Therefore, natural fibers are an ideal choice for preparing PLA composite materials.

#### 3.1.1. Cellulose Nanocrystals

Cellulose nanocrystals (CNCs) are rod-shaped nanoparticles extracted from cellulose through acid hydrolysis. A wide range of sources, including bleached wood pulp, cotton, and hemp fibers, can be used to produce CNCs [61,62]. Because of their high specific surface area, high reactivity, high strength, and low density, CNCs are an attractive reinforcement material.

Since Favier et al. [63] first attempted to use cellulose whiskers to reinforce polymers in 1995, nano cellulose products have been commercialized, which has prompted researchers to develop PLA/CNCs composite materials. Most studies have shown that CNCs can be well dispersed in PLA and act as a heterogeneous nucleating agent that affects the crystallization of PLA [64]. During isothermal or nonisothermal bulk crystallization, the presence of CNCs reduces the activation energy of PLA crystallization and increases

the crystallization rate of PLA. Kamal et al. [65] prepared CNCs/PLA composites by melt blending and found that CNCs acted as a heterogeneous nucleating agent that promoted the formation of PLA crystals, increased the crystallization rate, and improved the crystallinity. Karkhanis et al. [66] used CNCs to prepare packaging film with PLA composites. Compared with a PLA film, the water vapor permeability of the composite film decreased by 40%. The oxygen permeability decreased by 75%, thus significantly improving the barrier properties of the thin film. The presence of numerous hydroxyl groups on the surface of CNCs controlled the degradation performance of the material and enhanced the hydrophilicity of PLA composites. Shuai et al. [67] introduced CNCs into a laser-sintered PLA scaffold and found that CNCs, as a heterogeneous nucleating agent, caused the ordered arrangement of PLLA chains by forming hydrogen bonds between the surface hydroxyl groups of CNCs and PLLA, thereby increasing the crystallization rate and crystallinity. In addition, since the mechanical strength of polymers is closely related to their crystallinity, the addition of 3 wt% CNCs to the PLA scaffold increased its compressive strength, compressive modulus, tensile strength, tensile modulus, and Vickers hardness by 191%, 351%, 34%, 83.5%, and 56%, respectively. Adding hydrophilic CNCs also improved the hydrophilicity and degradation performance of PLLA.

#### 3.1.2. Lignin

Lignin is the most abundant aromatic biomass in nature, accounting for 20–30% of the weight of wood [57,68]. Most natural lignin (approximately 98%) is currently unused as a value-added product and is discarded as industrial waste because its chemical structure in its raw form is fragile and lacks resistance to heat, chemicals, external loads, and other factors. When lignin is mixed with organic polymers, acetylation reactions reduce the strength of the hydrogen bonds in lignin molecules, thereby reducing the size of the structural domains when polymerized lignin is mixed with organic polymers [69]. Interactions between the hydroxyl groups of lignin and the carboxyl groups of PLA underpin the production of PLA/lignin composite materials [70].

Spiridon et al. [71] obtained PLA/lignin biocomposites by melt blending, and a study of the impact of their physicochemical parameters showed that adding different concentrations of lignin increased the Young's modulus and tensile strength of the material. PLA/lignin biocomposites showed excellent mechanical resistance, remained stable during a 30-day degradation process, and maintained their dimensional stability in fluid environments. In addition, lignin did not cause cytotoxicity, demonstrating that PLA/lignin biocomposites have good biocompatibility. Tanase-Opedal et al. [72] studied the 3D printing of PLA/lignin biocomposites. Because of the antioxidant activity of lignin, PLA/lignin biocomposites showed incredibly high antioxidant activity, good extrudability, and excellent flowability, making them a promising renewable substitute for traditional 3D printing materials.

#### 3.1.3. Silk Fiber

Silk fiber is a natural animal protein fiber with a higher crystallinity, toughness, and tensile strength than plant fibers [73]. In addition to having good mechanical properties and biocompatibility, silk fiber is also easier to process. However, its softness may limit its applications in fields that require high hardness and rigidity. Therefore, it is necessary to optimize the properties of silk fiber for specific applications, including by mixing it with other materials such as PLA to produce tough and rigid materials [74] with improved mechanical properties. Silk/PLA composites may also show greater biocompatibility, making them suitable for various sports medicine and bioengineering applications.

Zhao et al. [75] prepared silk/PLA biocomposites by melt blending and found that adding silk fiber improved the dimensional stability. The presence of silk fiber also enhanced the enzymatic degradation of the PLA matrix, thereby controlling its susceptibility to hydrolysis. Cheung et al. [76] studied the mechanical properties and thermal behavior of silk/PLA biocomposites and found that their tensile performance was superior to that of

pure PLA. Therefore, adding silk fiber improved the thermal and physical properties of the composite, making it suitable for use in medical scaffolds.

#### *3.2. PHA*

As a new bio-based polymer material, PHA has diverse structures, various sources, and biodegradability, biocompatibility, optical activity, piezoelectricity, and gas barrier properties. They can be naturally biodegraded into CO2 and H2O and are nontoxic to the soil and air [77,78]. Currently, over 150 different PHA monomers have been discovered and produced by other bacteria and growth conditions of which PHB, PHBV, PHBHHx, and P34HB are the four main types. The discovery of these different PHA monomers has dramatically increased the development of PHA into commercial plastic products [79,80].

Zembouai et al. [81] studied PHBV/PLA blends with different mass ratios and found that PHBV acted as a nucleating agent for PLA, thus improving the crystallization of PLA, and the tensile strength and elongation at the break of PHBV/PLA blends were higher than those of pure PHBV. ePHA is a PHA belonging to the polyhydroxy fatty acid family with the same chemical structure, biodegradability, and renewability. Takagi et al. [82] prepared PLA/PHA blends with different compositions by mixing PLA with PHA and functionalized ePHA containing 30% epoxy groups in the side chains. They found that the Charpy impact strength of the PLA/PHA and PLA/ePHA blends increased with the PHA or ePHA content and was higher than that of pure PLA. Functionalizing ePHA with epoxy side groups enhanced the compatibility of the mix, thereby increasing the tensile strength and Charpy impact strength of the PLA/ePHA mixture. The blending of PHA and PLA improved the properties of PHA and also guaranteed the degradability of the composite material.

#### *3.3. PBAT*

PBAT is a biodegradable material produced on large scales and widely used in packaging materials and biomedical fields. PBAT has good processability and can toughen and modify other polyesters [83], but commercially available PBAT/PLA blends often exhibit macroscale phase separation and show two glass transition temperatures (*Tg*), indicating the poor compatibility of unmodified PBAT/PLA blends. In experimental studies, the preparation of PBAT/PLA blends usually involves melt blending. At high temperatures and sufficient time, ester exchange reactions occur between the two polyesters, thereby improving their compatibility [84]. By increasing the PBAT content within a specific range, the mechanical properties of PLA/PBAT composites, such as impact strength and elongation at break, can be improved [85].

Arruda et al. [86] prepared PLA/PBAT blends using an epoxy-functionalized chain extender and investigated the effect of 0.3% and 0.6% chain extenders on the mechanical properties, thermal properties, and microstructure of PLA/PBAT blends with ratios of 40/60 and 60/40. In the blend containing 40% PLA and no chain extender, the microstructure was significantly affected by the chain extender. PLA exhibited a fibrous dispersed phase, appearing elongated in the film stretching direction. In the mixture containing 60% PLA and no chain extender, PBAT displayed a large, belt-like structure in the middle of the film, with an overall skin-core design. The chain extender increased the crystallization temperature of PLA in both blends with different ratios and reduced the crystallinity of PBAT.

#### *3.4. Methods for Manufacturing PLA-Based Composites*

#### 3.4.1. Microcellular Injection Molding

Microcellular injection molding was first proposed in the 1980s by Nam et al. [87]. The formation of pores in microcellular foams proceeds via four main stages: construction of a polymer/supercritical fluid homogeneous system, bubble nucleation, bubble expansion, and cooling and solidification [88–91]. Microcellular foam injection molding can be used to produce microcellular foam products with micropores, with millions of pores per unit

volume. Compared with nonfoamed substrates, microcellular foam materials exhibit at least a four-fold higher fracture toughness and impact resistance [92].

#### 3.4.2. Extrusion Molding

Extrusion molding can be divided into continuous and intermittent types based on the different pressures used during extrusion. Continuous extrusion applies pressure with the rotation of a screw to uniformly plasticize the material inside the barrel. The material undergoes mixing and heating through the action of the screw during the extrusion process, resulting in good material uniformity [93]. Intermittent extrusion applies pressure to the material through a plunger. While this provides a higher pressure than screw extruders, its ability to generate significant shear action is limited, and its operation is discontinuous, which limits its application range [94].

#### 3.4.3. Compression Molding

Compression molding is a standard processing method for PLA. During compression molding, PLA particles are placed in a heated mold, and pressure is applied to liquefy and flow the material at high temperatures [95]. As the material cools, it resolidifies and shapes the mold. The final product's body, size, and performance can be controlled by adjusting the temperature, pressure, and holding time. Compared with other molding methods, compression molding has lower mold fabrication costs [96].

#### **4. PLA Composites for Sports Applications**

The global production capacity of all biodegradable plastics, including PLA, is expected to increase rapidly to approximately 1.33 million in 2024 [6], with primary applications in the automotive industry, electronic components, and sports equipment. In the automotive industry, 3D printing has had a revolutionary impact by enabling the rapid fabrication of lighter and more complex structures. For instance, in 2014, Local Motors manufactured the first electric car using 3D printing. The automotive industry utilizes 3D printing during the improvement stage to explore various alternative solutions to promote ideal and efficient car design. 3D printing can also reduce material waste and consumption [97]. Because of the ability of 3D printing to create highly integrated three-dimensional multifunctional structures, many researchers have actively explored this emerging technology to fabricate geometrically complex and biocompatible devices and scaffolds. These include biosensors, electrically stimulated tissue-regenerating scaffolds and microelectrodes [98,99]. New technologies for producing high-molecular-weight PLA have expanded their applications in recent years. PLA is becoming a popular substitute for petroleum-based synthetic polymers (PETs, polystyrene (PS), polyethylene (PE), etc.) in various fields, particularly the sports industry [100,101], as shown in Figure 6.

#### *4.1. Sportswear*

PLA fiber is a biodegradable synthetic fiber that is refined and fermented from starch sugar in corn, beets, or wheat. It is a new type of polyester fiber in the textile industry. PLA fiber is 100% compostable and reduces the Earth's carbon dioxide levels throughout its entire life cycle. The cross-section of PLA fiber is generally circular with a smooth surface. Its load–elongation curve is similar to that of wool, while its toughness is lower than that of cotton. PLA fiber has good core absorbency and fast moisture management. Therefore, by blending PLA fiber with cotton, the moisture transmission properties of cotton fabrics can be improved. Guruprasad et al. [102] developed a sports textile by combining cotton and PLA at a ratio of 65:35. Then, they tested the moisture management performance, moisture vapor transmission rate, and thermal performance of the cotton/PLA blended fabric. Experiments showed that the mixture of PLA fiber and cotton provided improved moisture management performance. The liquid transfer rate of cotton/PLA blended fabric was faster than that of 100% cotton fabric. The cotton/PLA composite fabric had a high unidirectional transmission capacity, spreading speed, and bottom absorption rate, giving

it a higher OMMC value and allowing it to transfer sweat to the other side faster. The moisture vapor transmission rate of the cotton/PLA blended fabric was 14% higher than that of the 100% cotton fabric, which helped liquid moisture diffuse quicker, making it an ideal material for sportswear.

**Figure 6.** PLA applications in the sports industry.

#### *4.2. Helmets*

Raykar et al. [103] used PLA plastic to manufacture a bicycle helmet through a combination of fused deposition modeling (FDM) and 3D printing. PLA plastic filaments were used and melted and deposited using layer-by-layer heat extrusion onto the building platform of the 3D model until the entire exterior of the helmet was covered in PLA plastic. After cleaning and trimming, a PLA bicycle sports helmet was produced. Experiments proved that the 3D printed PLA bicycle sports helmet had high safety, good breathability, and lighter weight, thus balancing the safety and comfort of the athlete.

#### *4.3. Protective Sports Gear*

Traditional protective sports gear has a structure consisting of a hard outer shell made of a thermoplastic material and an inner soft foam padding. Currently, there are new "soft shell" technologies for sports protectors based on the use of soft polymer foams typically made of polyurethane or polyacrylate with good cushioning properties. During the manufacturing process of sports protectors, soft polymer foams can be combined with PLA. Soft polymer foams are used as the internal cushioning material. In contrast, PLA can be used as the outer shell material to improve sports knee protectors' lightweight, breathability, and comfort properties, thus achieving better protection results. Yang et al. [104] used tensile materials (PLA and thermoplastic polyurethane (TPU)). They tested them through 3D printing prototyping and compared the results with calculated predictions to evaluate the possibility of using tensile materials in sports protectors. The results showed that the tensile material had a high fracture toughness, high shear modulus, superior specific strength, compressive indentation resistance, strong energy dissipation, and a controllable strain penetration rate, making it suitable for protective sports gear to reduce the risk of injuries.

#### *4.4. Surfboards*

The source of power for a surfboard comes from the movement of waves, and the significant impact force generated by an impact wave can often break a surfboard, mainly when materials such as fiberglass are used in its production. In recent years, researchers have turned their attention to biodegradable materials. Soltani et al. [105] used the finite element method (FEM) and 3D printing to manufacture a surfboard with a uniform honeycomb core structure based on a PLA composite material. They then conducted three-point bending experiments and used accurate finite element tools to simulate surfboards with different core structures. The PLA composite material surfboard passed the three-point bending test, and the overall volume of the surfboard remained unchanged.

#### *4.5. Sports Medicine Tools*

Because of the biocompatibility and biodegradability of PLA when in contact with mammalian bodies, it has been widely used in the biomedical and pharmaceutical fields [106] to manufacture screws, pins, surgical sutures, stents, etc. [107,108]. The unique properties of PLA make it suitable for reinforcing rotator cuff repairs and can help heal tendon tissues in various body parts. PLA and its copolymers are often used in orthopedic surgery to manufacture artificial bones and joints, providing a temporary structure for tissue growth, which eventually decomposes. Koh et al. [109] used PLA-reinforced suture anchors to suture and repair tendons separated from the bone. The tensile strength of PLA is approximately 1200 N, and it can be manufactured to the required size. The experiment showed that adding a PLA scaffold to the bone bridge increased the fixation strength by 1.3 times. The use of PLA scaffolds showed significant advantages when used to fix the rotator cuff.

#### *4.6. 3D Printed Sports Equipment*

Compared with traditional printing materials, PLA produces almost no harmful gases and has a lower shrinkage rate, making it ideal for 3D printing sports equipment. Protective gear, such as mouthguards, helmets, and shin guards [110], can be 3D printed using PLA, providing athletes with customizable, comfortable, and lightweight equipment. Because of its biocompatibility, PLA is the preferred material for 3D printing protective gear, as it can be safely used in contact sports without causing harm to athletes. In addition to protective gear, PLA can be used to 3D print bicycle frames, kayak paddles, and skis [111]. PLA's mechanical properties and biodegradability make it an attractive alternative to durable materials, such as plastics, metals, and other traditional materials for sports applications.

#### *4.7. Limitations of PLA Composites in Sports Applications*

Compared with traditional petroleum-based plastic sports equipment, the green disposal of idle sports equipment meets the requirements of sustainable development. Sports equipment made of PLA composites can be used safely and decomposes after being discarded, which can prevent environmental pollution. In addition, PLA has a lower density, allowing for the production of relatively lightweight sports equipment. Through 3D printing, PLA enables personalized customization, offering more possibilities for the innovative design of sports equipment. However, as a linear thermoplastic polyester, PLA's strength may not meet the requirements of certain sporting equipment in specific environments. For example, because of PLA's high brittleness and low elongation at break [25], sports equipment made from PLA composite materials are more susceptible to rupturing during contact sports. Additionally, prolonged exposure to sunlight can cause a decrease in the molecular weight of PLA composite materials [112], potentially impacting the mechanical performance of outdoor sports equipment.

#### **5. Conclusions**

PLA is a natural, renewable, and low-cost biodegradable material, but its inherently poor toughness limits its broader applications. By adding reinforcement materials to develop PLA composites, it can adapt to the increasing performance requirements of various fields. Compared with most inorganic and synthetic fibers, natural fibers have abundant sources, low prices, complete degradability, low energy consumption, and environmental friendliness. In the future, appropriate additives, modifications to polymerization conditions, and reinforcement techniques will be employed to enhance the strength of PLA and meet specific needs. At the same time, by developing low-cost reinforcement materials and optimizing formulations and processing methods, the manufacturing costs of PLA composites can be reduced. Their performance can be improved to meet various environmentally friendly applications, including sports equipment manufacturing. Currently, the application of PLA composites in the sports field is expanding. Compared with petroleum-based materials, the mechanical properties of PLA composites still need to be improved. However, as biodegradable alternatives to petroleum-based plastics, they still have tremendous potential.

**Author Contributions:** Conceptualization, writing—original draft, Y.W. (Yueting Wu); conceptualization, writing—review and editing, project administration, and funding acquisition, X.G.; investigation and formal analysis, J.W.; investigation and formal analysis, T.Z.; software and visualization, T.T.N.; investigation and formal analysis, Y.W. (Yutong Wang). All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Heilongjiang Natural Science Foundation Joint Guidance Project of China, grant number: LH2022E097.

**Institutional Review Board Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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### *Review* **Bioplastics: Innovation for Green Transition**

**Ana Costa 1, Telma Encarnação 1,2,3, Rafael Tavares 1, Tiago Todo Bom <sup>4</sup> and Artur Mateus 1,\***


**Abstract:** Bioplastics are one of the possible alternative solutions to the polymers of petrochemical origins. Bioplastics have several advantages over traditional plastics in terms of low carbon footprint, energy efficiency, biodegradability and versatility. Although they have numerous benefits and are revolutionizing many application fields, they also have several weaknesses, such as brittleness, high-water absorption, low crystallization ability and low thermal degradation temperature. These drawbacks can be a limiting factor that prevents their use in many applications. Nonetheless, reinforcements and plasticizers can be added to bioplastic production as a way to overcome such limitations. Bioplastics materials are not yet studied in depth, but it is with great optimism that their industrial use and market scenarios are increasing; such growth can be a positive driver for more research in this field. National and international investments in the bioplastics industry can also promote the green transition. International projects, such as EcoPlast and Animpol, aim to study and develop new polymeric materials made from alternative sources. One of their biggest problems is their waste management; there is no separation process yet to recycle the nonbiodegradable bioplastics, and they are considered contaminants when mixed with other polymers. Some materials use additives, and their impact on the microplastics they leave after breaking apart is subject to debate. For this reason, it is important to consider their life cycle analysis and assess their environmental viability. These are materials that can possibly be processed in various ways, including conventional processes used for petrochemical ones. Those include injection moulding and extrusion, as well as digital manufacturing. This and the possibility to use these materials in several applications is one of their greatest strengths. All these aspects will be discussed in this review.

**Keywords:** bioplastics; biopolymers; conventional polymers; biodegradability; renewable resources; LCA

### **1. Introduction**

The use of polymeric materials is widely spread around the world. These materials have significant advantages compared with other, more conventional materials, such as metals and wood, mainly because of their properties and performance.

It is estimated that 99% of these polymeric materials come from fossil fuels. These plastics entail several issues since their primary raw material is a hazard to environment conservation [1].

The durability and degradability of these materials are two contradictory topics. For most applications, it is favourable that the material maintains specific properties throughout time, but it is also desirable to discard them easily after their use. There are some alternative processes usually used to manage this kind of waste: recycling (one of the most sustainable waste management processes but requires a controlled process to have a final product with good properties) and energy recovery (allows the production of energy by burning the waste but ends up producing toxic emissions and greenhouse gases) [2,3]. However, a massive quantity of material ends up in landfills or even abandoned, and some of it reaches

**Citation:** Costa, A.; Encarnação, T.; Tavares, R.; Todo Bom, T.; Mateus, A. Bioplastics: Innovation for Green Transition. *Polymers* **2023**, *15*, 517. https://doi.org/10.3390/polym 15030517

Academic Editor: Raffaella Striani

Received: 7 November 2022 Revised: 16 December 2022 Accepted: 25 December 2022 Published: 18 January 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

the ocean. A long-term study took place on the North Atlantic Sea, where it was observed a seawater sample contained 580,000 pieces of plastic per square kilometre. This waste management has created a crisis, since landfills have a limited capacity, high costs and strict legislation [4].

The remaining percentage of plastics is produced from natural raw materials and are denominated bio-based plastics or bioplastics [1]. The use of bioplastics dates centuries ago. In 1500 BCE, Mesoamerican cultures (Maya, Aztecs) used natural rubber and latex to make containers and waterproof their clothes. However, only in 1862 was the first manmade bioplastic produced (Parkesine, a bioplastic made from cellulose), created by Alexander Parkes. The first company to produce bioplastics was Marlborough Biopolymers in 1983. They produced strips, filaments, chips, panels and powders of bacteria called Biopol. More recently, in 2018, Project Effective was launched with the goal of replacing nylon with bio-nylon, and it created the first bioplastic made from the fruit [5].

Although investigations regarding bioplastics have been done for over a century, their implementation and extensive production is not yet developed. Figure 1 presents a chart of the last few years' production and the forecast for the years to come. In 2019, 1.95 Mt of bioplastic was produced, corresponding to about 0.6% of all plastic production worldwide.

**Figure 1.** Global production capacities of bioplastics 2021–2026. Adapted from European Bioplastics, "Bioplastics Market Development Update 2021". https://docs.european-bioplastics.org/ publications/market\_data/Report\_Bioplastics\_Market\_Data\_2021\_short\_version.pdf (accessed on 29 December 2022) [6].

The small production of these plastics is mainly due to their more expensive manufacturing and generally inferior mechanical properties compared to fossil-based polymers. However, it is necessary to develop these materials to have a sustainable alternative to petrochemical materials [7]. This paper will discuss current scenarios and the inherent production limitations and present the pros and cons of producing and using bioplastics to replace some petrochemical-based polymers. While several reviews on biopolymers have been extensively published [8–11], the contribution of this review is to gather current knowledge in several aspects and present the latest discoveries in this topic. We have selected the most representative biopolymers and composites and present new ones. Several applications are described, and processing techniques are discussed. Moreover, some fundamental approaches to bioplastic waste management are presented. Lastly, we highlight legislation and policies that can contribute to promising future perspectives for innovation for the green transition.

#### **2. Materials**

The European Bioplastics organization classifies bioplastics as "plastics based on renewable resources or as plastics which are biodegradable and/or compostable". When it is possible to decompose a polymer into carbon dioxide (CO2), methane, water, inorganic compounds or biomass through an enzymatic process using microorganisms, the polymer is considered biodegradable. It is possible to compost some of these materials under controlled conditions [12]. Based on this definition, it is possible to organize this classification in a simple graph represented in Figure 2.

**Figure 2.** Classification of bioplastics according to The European Bioplastics Organization. Adapted from European Bioplastics, "What are bioplastics?". https://docs.european-bioplastics.org/ publications/fs/EuBP\_FS\_What\_are\_bioplastics.pdf (accessed on 29 December 2022) [13].

In the bioplastic group, they can be classified under three different classes, as shown in Figure 2: (1) polymers originated from biomass materials, and they can be either modified or not; (2) polymers extracted from natural or genetically modified microorganism production and (3) and polymers produced from renewable raw materials with the involvement of bio-intermediaries. Although only some of these materials are available on a commercial scale, the most used bioplastics are based on cellulosic esters, starch, polyhydroxy butyrate (PHB), polylactic acid (PLA) and polycaprolactone (PCL). Figure 3 shows some of the most used bio-based polymers according to their base of production and raw material [14–16].

Some biopolymers have properties comparable with conventional plastics, such as LDPE (low-density polyethylene), PS (polystyrene) and PET (polyethylene terephthalate); some of these properties are important to predict the behaviour of the material during use and the proper conditions to process it. Some of these characteristics, listed in Table 1, are the glass transition temperature (Tg), melting temperature (Tm), tensile strength, tensile modulus and elongation break [2].

Another characteristic important to consider is the rate of crystallinity of the polymer. It influences a vast quantity of essential properties such as hardness, modulus, tensile strength, stiffness, crease point and the melting point, making it important to pay special attention to this property [2].

**Figure 3.** Types of bioplastics according to their raw material. Adapted from "Innovation and industrial trends in bioplastics", Polymer Reviews vol. 49, no. 2, pp. 65–78, April 2009 [14].

**Table 1.** Comparison of typical biodegradable polymer physical properties with LDPE, PS and PET. [2,17–26].


Another characteristic important to consider is the rate of crystallinity of the polymer. It influences a vast quantity of essential properties such as hardness, modulus, tensile

strength, stiffness, crease point and the melting point, making it important to pay special attention to this property [2].

The values in Table 1 are generic, since it is possible to obtain materials with different properties with different combinations of monomers or through chemical derivatization or introduction of additives such as plasticizers, stabilizers, fillers, processing aid and colourants.

Table 2 summarizes some characteristics that may be important for specific applications listed as guidelines. Some typical applications and the degradability of the discussed bioplastics are also included in Table 2 [27].

**Table 2.** Major bioplastic classes, some properties and average degradation time in different environments [27–29]. (*√***/X:** present/absent).


#### *2.1. Starch*

The synthesis of this bioplastic began in the 1970s and is now produced worldwide by companies such as Futerro, Novamont, Biome and Biotec [26,27]. Starch is one of the common names for carbohydrates, along with sugars, saccharides and polysaccharides. They are formed by photosynthesis when CO2 reacts with water. The chemical symbol is generically represented as Cx(H2O)y, where x and y are numbers between 3 and 12. Starch is a type of polysaccharide obtained from floral sources [28]. The production of polymeric film from starch requires a significant quantity of water or plasticizers (glycerol, sorbitol). They are used widely worldwide as a substitute for PS in several thermal and mechanical applications. There are a lot of different possible sources of starch, but the main ones used are corn, wheat, cassava and potatoes, with 82%, 8%, 5% and 5% starch, respectively. The raw material is prevalent, and the production process allows obtaining large quantities of a biodegradable thermoplastic-like material (TPS) with a fair ease of management. Although starch is not a thermoplastic, starch-based bioplastics melt at high temperatures (91–180 ◦C) and tend to be fluid under shearing. The use of plasticizers helps to achieve this behaviour, making it possible to process the material with injection moulding, extrusion and blow moulding. Plasticizers work embedded between polymer chains, which soften the material and lowers the glass transition temperature by spacing the polymer chains apart. There are several processes involved in the conversion of starch into thermoplastic, such as gelatinization, melting, water diffusion, granule expansion, decomposition and crystallization. The thermoplastic material forms in the presence of heat and shearing forces. The energy absorbed melts the original structure and creates new bonds between the starch and the plasticizer. When the mixture cools down to room temperature and the granules reswell, a new granule structure is formed, and the thermoplastic material is produced. The final material has both amorphous and crystalline regions. Cereplast is a producer of TPS that collects starch from tapioca, corn, wheat and potatoes. Out of 1 t of potatoes, they are capable of gathering 0.18 t of starch, which produces 0.24 t of TPS when adding 0.06 t of plasticizer, a process summarized in Figure 4 [12,30–35].

**Figure 4.** Production flow chart of thermoplastic starch (TPS). Adapted from "Bioplastics: Development, Possibilities and Difficulties", Environmental Research, Engineering and Management, vol. 68, no. 2, July 2014 [36].

Since water has a plasticizer effect on starch, one of the problems of the use of starch is the effect water and humidity have on it, creating, for example, variations in its mechanical properties and low resistance to impact. Some derivates of starch have high permeability to moisture and degrade rapidly in specific applications; solutions used to avoid these problems might make the final material expensive. Throughout time, the material's properties change even when temperature and moisture are controlled, lowering the elongation break and increasing the rigidness [30].

Examples of products made of starch bioplastic are grocery bags and trays (rigid or foamed) used to pack fruits and vegetables. One important material widely used is paper foam, used to pack items where product protection is essential, for example, in egg boxes and the packaging of electronic devices [37,38].

#### *2.2. Cellulose*

This polymer is a biodegradable polysaccharide made from wood pulp or cotton linters. Cellophane film starts by dissolving the raw material in a mixture of sodium hydroxide and carbon disulphide and recast the obtained product into an acid solution (sulfuric acid) [12,32].

Pure cellulose bioplastic is very hard to be produced; it is not possible to make it in an industrial environment with standard processes such as thermoforming or dissolution due to its strong and highly structured intermolecular hydrogen bonding network. For this reason, it is usually produced industrially as cellulose derivatives, such as cellulose esters or ethers, which requires extra time and costly chemical purification steps [39]. Some of these derivates are cellulose nanocrystals (CNC), nano-fibre cellulose (NFC), cellulose acetate butyrate, cellulose acetate and bio-PE, and they are produced by the esterification or etherification of hydroxyl groups. A lot of derivates need additives to produce thermoplastics, and some of them are water-soluble [12,40].

Cellophane is transparent, and it can be pigmented. It is common to use it as candy wrappings, laminates, flower wrapping and pack products ranging from cheese to coffee and chocolate [33].

#### *2.3. Polyhydroxyalkanoates (PHA)*

PHA is a family of biodegradable thermoplastic polymers where more than 160 different monomeric units were identified. The most common one is polyhydroxybutyrate (PHB). PHA is a material regularly used to replace conventional polymers due to their similar chemical and physical properties. This biopolymer is produced by the fermentation process in microbial cells (such as *Cupriavidus necator*, *Bacillus* sp., *Alcaligenes* sp., *Pseudomonas* spp., *Aeromonas hydrophila*, *Rhodopseudomonas palustris*, *Escherichia coli*, *Burkholderia sacchari* and *Halomonas boliviensis*), and the polymeric material is then recovered using solvents (chloroform, methylene chloride or propylene chloride) [8,33]. Corn, whey, wheat and rice bran, starch and starchy wastewaters, effluents from olive and palm oil mills, activated sludge and swine waste are some examples of material sources for the fermentation process. Although the conditions of the fermentation process depend on the demands of the microbes, temperatures of 30 ◦C to 37 ◦C, along with low stirrer speeds (resulting in low dissolved oxygen tension), are the conditions used.

Several national and international programmes foster the development and advancement for a green transition. That can be expressed in several projects' results. In 2010, the "Animpol" project was developed by the European Commission with the goal of developing an efficient process that could convert waste streams from slaughterhouses into improved biodiesel and biodegradable high-value polymeric materials, such as PHA. The Consortium was able to produce 35,000 t per year of PHA from 500,000 yearly t of animal waste on an industrial level, as represented in Figure 5. The introduction of this process in the bioplastic production industry would mean that some of the solvents used in the production of bioplastics would be eliminated, and the slaughterhouse waste could be useful to produce added-value products, while nowadays, this waste material is simply burned [8,32,33,38].

PHB is produced by bacteria, algae and genetically modified plants through enzymatic processes. The process starts with the condensation of two molecules of acetyl-CoA into acetoacetyl-CoA, which is then reduced by acetoacetyl-CoA reductase to produce β-hydroxybutyryl-CoA. PHB is obtained by the polymerization of β-hydroxybutyryl-CoA. To harvest the PHB, it is necessary to destroy the cell, since it is present as cysts within the cytoplasm of the cell [33,39,40].

**Figure 5.** Production flow chart of PHA from waste streams from slaughterhouses. Adapted from "Bioplastics: Development, Possibilities and Difficulties", Environmental Research, Engineering and Management, vol. 68, no. 2, July 2014 [32].

Due to its characteristics, PHA is extensively used in medical fields, especially in tissue engineering. For example, PHA is used in long-term dosage of drugs, medicines, hormones, insecticides and herbicides, as osteosynthetic materials in the stimulation of bone growth owing to their piezoelectric properties in bone plates, surgical sutures and blood vessel replacements. Other products made of this biopolymer are composting bags, food packaging, diapers and fishing nets [33]. Tianan Biopolymer, BASF, Tepha and Biocycle are some examples of companies that produce this type of bioplastics [41].

#### *2.4. Polylactide (PLA)*

PLA was initially used in combination with polyglycolic acid (PGA) under the name Vicryl during the 1970s, but its discovery goes back to 1932 when it was discovered by Carother. At the time, he produced a low molecular weight polylactide by heating lactic acid under vacuum while removing the condensed water. Nowadays, most of the PLA produced is used in packaging (about 70%), although its application in other fields has been increasing, especially in fibres and fabrics. The leading producer of PLA in the world is NatureWorks® (USA), but other companies stand out, such as Ingeo, Toyobo, Dai Nippon Printing Co., Mitsui Chemicals, Shimadzu, NEC, Toyota, Biofront (Japan), PURAC Biomaterials, Hycail, Biofoam (The Netherlands), Galactic (Belgium), Cereplast, FkuR, Biomer, Stanelco, Inventa-Fischer (Germany) and Snamprogetti, Hisun (China) [2,30,42].

Similar to PHA, PLA is one of the bioplastics considered to have significant potential to be widely used as a replacement to several fossil fuel-based polymers, such as LDPE and high-density polyethylene (HDPE), PS and PET [8].

PLA involves different types of sciences to be produced: agriculture, for the growth of the crops; biological, during the fermentation process; and chemical, for polymerization. The PLA monomer is called lactic acid (2-hydroxy propionic acid), and it has two configurations, L(+) and D(−) stereoisomers, produced by the bacterial fermentation of carbohydrates (homofermentative and heterofermentative). Lactic acid is produced using one of two processes, fermentation or chemical synthesis. The first one is usually used industrially, since it does not depend on other processes' by-products, it produces L-lactic acid stereoisomer easier and the manufacturing costs are not as high as the synthesis process. The homofermentative method is also preferable, since it creates less by-products and lactic acid with greater yields, and pure L-lactic acid is used to produce PLA. The production procedure uses *Lactobacillus* genera such as *L. delbrueckii*, *L. amylophilus*, *L. bulgaricus* and *L. leichmanii* under specific conditions (a pH range of 5.4 to 6.4, a temperature range of 38 to 42 ◦C and a low oxygen concentration).

After the production of lactic acid, it is then polymerized into PLA. There are three possible processes to do the polymerization: direct condensation polymerization, direct polycondensation in an azeotropic solution and polymerization through lactide formation. High molecular weight PLA with good mechanical properties is not easily achieved using the direct condensation polymerization method. It involves the esterification of lactic acid with some solvents under progressive vacuum and high temperatures, where water is removed. The second process is a more feasible way to produce PLA with high molecular weight. The azeotropic solution reduces the distillation pressure, and using molecular sieves helps the separation of the PLA and the solvent. Lastly, polymerization through lactide formation is also used in the industrial environment to produce high-weight PLA. Lactide is a cyclic dimer formed by removing water under mild conditions and without solvent. As shown in Figure 6, it is possible to produce 0.42 tons of PLA from 1 ton of corn using processes such as hydrolysis, fermentation, dehydration and polymerization [32,33,43–45].

**Figure 6.** Production flow chart of PLA by synthesizing corn-based starch. Adapted from "Bioplastics: Development, Possibilities and Difficulties", Environmental Research, Engineering and Management, vol. 68, no. 2, July 2014 [32].

PLA is widely used in the food packaging industry for short and long shelf-life products. This bioplastic and its blends are also used to make implants, plates, nails and screws for medical surgery. The application fields are expanding to textile, cosmetic, automobile industries and the household [33].

#### *2.5. Polycaprolactone (PCL)*

PCL is a synthetic polyester that is produced from crude oil. Since it is a biodegradable polymer, it is considered a biopolymer.

PCL is regularly used in tissue engineering and biomedical applications due to its blend compatibility, absorbability, good solubility and low melting point. This is a hydrophobic and semi-crystalline material; its crystallinity depends on its molecular weight (high molecular weight means low crystallinity), which is possible to control using low molecular weight alcohols. This biopolymer is characterized by its ease to manufacture and shaping, tailorable degradation kinetics and mechanical properties, making PCL distinguish itself from other bioplastics.

Initially, when it was studied in the 1930s, this polymer was relatively popular, but soon, its use decreased significantly for a long time due to its weak mechanical properties in comparison to other resorbable polymers such as polylactides and polyglycolide. Recently, this material has regained great interest because of its application in the field of tissue engineering widely developed in the 1990s. It not only possesses superior rheological and viscoelastic properties when compared to other polymers, but its ease of processing also stands out.

Although PCL is a bioplastic biodegraded by specific bacteria and fungi present in outdoor environments, this polymer is not degradable in animal or human bodies due to the lack of those organisms. This particularity makes PCL an excellent material to be used in medicine and tissue engineering fields of study; its degradability is relatively slow when compared to PLA, PGA and other resorbable polymers, making it an excellent material to be used "for long-term degradation applications delivery of encapsulated molecules extending over a period of more than 1 year". The rate at which this drug release is achieved may be manipulated by combining PLC with cellulose propionate, cellulose acetate butyrate, PLA or polylactic acid-co-glycolic acid [46].

#### *2.6. Protein-Based*

Protein is a heteropolymer of amino acids with a fibrous and globular structure arranged by hydrogen, covalent and ionic bonds. Protein-based materials have great mechanical and barrier to gas and aroma properties than lipids and polysaccharides. By incorporating keratin, the material produced has thermal stability, mechanical properties and flame resistance. Due to their abundance, biodegradability, nutritional value and better film development capability, packaging is one of this material's main applications [47]. Other applications include matrices for enzyme immobilization or controlled-release devices and in fields where water absorbency and retention are important, such as water-absorbent materials in healthcare, agriculture and horticulture. With further technological development, packaging technology, natural fibre reinforcements, nanotechnology and innovative product design could be fields to be developed using this type of bioplastic.

To produce products with this material, there are two possible processes, the casting method (or physicochemical method) and the mechanical method (or thermoplastic processing). The first process divides itself into three different steps, starting with using chemical or physical rupturing agents to break the intermolecular bonds that stabilize polymers in their native forms; next, the mobile polymer chains are rearranged and oriented to the intended shape. Finally, the three-dimensional network is stabilized by allowing the formation of new intermolecular bonds and interactions. The second method involves mixing proteins and plasticizers to obtain a dough-like material [31,47]. Techniques that can be used to observe the molecular reorganization and orientation are based on time-resolved small-angle X-ray scattering (SAXS) that allows analysing of the anisotropy. Usually, these techniques are available in research facilities that allow in situ observation. It is possible to, through in situ experiments, observe the molecular rearrangement and reorganization that impose levels of preferential orientations [48]. The time-resolved SAXS allows monitoring the evolution (in time) of changes in the morphological organization.

#### *2.7. Polyamide 11 (PA 11)*

PA 11 or nylon 11 is a nonbiodegradable bioplastic produced from renewable material, such as castor oil. The polymer is obtained by the polymerization of 11-aminoundecanoic acid. Although it is nonbiodegradable, PA11 and its composites can be recycled. Due to its nonbiodegradability, the PA11 has greater longevity than most bioplastics. This characteristic, along with the high melting point (200 ◦C) and mechanical and chemical stability, makes it possible to apply it as reinforcement material in the manufacturing of natural gas piping, water tubing, electrical cables, clips and wires in aerospace and automobile industries, metal coatings, footwear, badminton racket strings and shuttlecocks. Other important characteristics are its good resistance to oil and water, high resistance to ionization radiation, strong resistance to different chemicals, fuels and salt solutions and its resistance to abrasion and cracking. It has low heat resistance and rigidity, low resistance to ultraviolet radiation and weak resistance to acetic acid and phenols, and its electrical properties are highly dependent on the moisture content. However, the market price is relatively higher than other polyamides. One of the main PA11 producers is Arkema [47,49,50].

#### *2.8. Spidroin—Spider Silk*

Spiders can, in a fraction of a second, under ambient conditions and from renewable resources, create a material which mechanical properties outperform any manufactured material: spider silk. It has the potential to be used in a wide variety of fields, for example, for making high-performance textiles and sports goods, durable components for robotics, ropes and reinforcements of composite materials and for applications in medicine (spider silk enhances wound healing and has successfully been used to bridge critical size nerve defects and as fascia replacements in animal models) [39,51,52]. However, it is currently not possible to farm spider silk efficiently on large scales because of spiders' cannibalistic nature, the difficulty of breeding, the low production rate in captivity and the collection of silk from spider webs is very time-consuming and not efficient enough for production. Genetic engineering is the most promising way to produce this material. The plan is to express the spider silk protein (spidroin) into different kinds of hosts, such as bacteria, yeast, plant and in the milk of transgenic mammals [40,51,52]. This technique was widely researched using *Escherichia coli*, a well-established host for the industrial-scale production of proteins. Yet, these materials produced do not have as good mechanical properties as spider silk, so new methods need to be developed. Although spider silk always looks the same to the naked eye, there are several different types of thread; the same spider can produce up to seven different types of silk, each one with different mechanical properties. The strength of these natural threads ranges from 0.02 to 1.7 GPa, and its extensibility varies between 10 and 500%. Additionally, using the same natural fibres, those spun technically show different properties than the natural fibres created by the spider, which shows that this is an important process and is done differently than the technology usually used. Despite a biomimetic spinning process to process the silk not existing, there are other alternatives to use this material since recombinant spider silks can self-assemble into non-natural shapes such as spheres, capsules, films, non-wovens or hydrogels. There are other concerns regarding solubility, storage and assembly of the underlying spider silk proteins [53].

#### **3. Bioplastics Composites**

The synthetic assembly of two or more materials, a matrix binder and selected reinforcing agents, is used for various applications. The goal is to overcome the weaknesses and increase versatility. The most common fibres exercised in recent times are glass fibres, carbon fibres, aramid fibres, natural fibres, nylon and polyester fibres. There is a clear advantage to using natural-based fibres; for example, they are biodegradable, renewable, available in bulk, cheaper and lighter [54–56].

#### *3.1. Coating*

Coating bioplastics is an excellent technique to improve some of the properties of these materials; it is specially used to enhance the barrier properties. By applying a thin layer of other polymers on top of the bioplastic, the tensile strength and elasticity can be improved, as well as increasing oxygen and water vapour permeability and resistance. Some examples of usually used coatings are listed below:


#### *3.2. Nanocomposites*

For a composite to be considered a nanocomposite, it must have at least one of its types of particles with dimensions in the nano range. The composite can be classified as polymer layered crystal nanocomposites (Figure 7a), nanotubes or whiskers (Figure 7b) and isodimensional nanoparticles (Figure 7c), according to the number of dimensions it has: three, two or one, respectively [2].

**Figure 7.** Nanoparticle geometries: (**a**) layered particles (1D), (**b**) acicular or fibrous ones (2D) and (**c**) isodimensional nanoparticles (3D). Adapted from "Block copolymer nanocomposites" [57].

The use of these materials as reinforcement to other polymers depends on the capacity of the matrix (continuous phase) to interact with the fibre (discontinuous phase). There are several ways to mix the phases of the composite; one of them is situ polymerization, which involves the dissolution of the nanoparticles in the monomer solution before polymerization; the addition of the nanoparticles during the extrusion process, a process called melt intercalation; or solvent intercalation (use of a solvent to enhance the affinity between the nanoparticles and the matrix). These processes help change some of the composite properties according to the intention.

Nanoclays are one of the most used fibres as reinforcement of bioplastics. They are usually used as layered particles of 1 μm. Different affinities between the matrix and the nanofibers create different interactions: tactoid, intercalated and exfoliated. The first case occurs when the interaction between the continuous and discontinuous phases is low; this happens because the clay interlayer does not expand within the matrix, so no true nanocomposite is formed. When the affinity is moderate, it is possible for a part of the polymer to penetrate the clay interlayer, since there was some level of expansion of the fibre; this creates an intercalated structure of matrix and fibre. The last situation entails a high affinity, the clay disperses into the polymeric matrix, and the layered structure is lost, forming an exfoliated structure instead [8] (Figure 8).

**Figure 8.** Structures of polymer nanoclay composite.

It is also possible to create composites using bigger particles as reinforcement. The project EcoPlast works with melt intercalation using natural fibres (sawdust, cellulose and cork) and biodegradable polymers as matrices (such as PLA, PBS (polybutylene succinate), starch, cellulose and PCL) in several different combinations. The materials used are represented in Figure 9. They are within different ranges of dimensions and densities, where (a) (less than 0.7 mm) and (b) (between 0.7 and 1.4 mm) are quite homogeneous. At the same time, (c) (between 1.4 and 2.8 mm) has very different particles in shape, size and colouring.

**Figure 9.** Different batches of sawdust used (**a**) (less than 0.7 mm), (**b**) (between 0.7 and 1.4 mm) and (**c**) (between 1.4 and 2.8 mm). From the project EcoPlast.

The dispersion of the fibres is affected by the hydrophobic/hydrophilic character of the polymer and the clay; this can be adapted with chemical modifications such as cationic exchange, ionomers, block copolymers adsorption and organosilane grafting. Since a high surface-to-volume ratio leads to better polymer properties, the exfoliated structure is preferred.

Some of the properties affected are the elongation at break (especially when using PLA film as matrix); barrier properties, explained by the confinement effect (the molecules of the polymeric matrix penetrate the dispersed nanoparticles, creating a denser material and creating a more tortuous path for the water and gas molecules to travel through) and thermal stability [8].

#### *3.3. Cellulose*

Adding cellulose to the bioplastic is another way to influence some properties of the final material. It is possible to have good adhesion between the fibre and the matrix due to the chemical similarity between starch and natural fibres. The main effect this addition has on the composite is the reduction of water vapour permeability due to the fibres' highly crystalline and hydrophobic character, also affecting the Young's modulus, tensile strength and the elongation break [8].

#### **4. Processing**

To achieve the maximum possible benefits of bioplastics, the processing of these materials is well established for each one of them, differing between them according to the characteristics of the specific bioplastic [2].

When processing bioplastics, the technologies used are the same as conventional polymers; it is only necessary to adapt the parameters used for the specific material intended to use [58]. Depending on the material, some processes might not be efficient, sustainable or economically reasonable. That is why it is essential to further research the processability of biopolymers to make their range of applications wider [59]. Table 3 summarizes some of the most common processes where bioplastics might be modified.

**Table 3.** Processing possibilities of typical commercial biopolymers. Adapted from "Poly-Lactic Acid: Production, applications, nanocomposites, and release studies", Comprehensive Reviews in Food Science and Food Safety, vol. 9, no. 5, pp. 552–571, September 2010 [2].


#### *4.1. Injection Moulding and Extrusion*

The possibility of processing bioplastics via injection moulding or extrusion is not very different compared to processing conventional polymers. Each type of bioplastic has its own chemical structure, so each material will have different parameters, the same as happens with conventional plastics. Only a few conditions require some attention; for example, some bioplastics are sensitive to moisture or heat exposure; therefore, it is essential to control the drying process of the bioplastics and the long-time cycles in the case of injection moulding. Thus, it is important to consider these characteristics when choosing materials, equipment, and resources [60].

#### *4.2. Digital Manufacturing*

Several research studies have been developed in the past years with the goal of evaluating the possibility of integrating new bioplastics into production processes usually used to transform conventional polymers. One in specific was conducted by Sneha Gokhale [61]. The objectives were to "Understanding sustainable 3DP (3D printing) in the context of bioplastic filaments; Testing commercial bioplastic filaments for sustainability and material properties; Guiding users in the industry towards green 3DP material and process choices". To achieve this, several polymeric materials were tested in three different phases, evaluating their energy consumption when processed by FDM (Fused Deposition Modelling); comparing their printability and dimensional accuracy and rating their mechanical properties (tensile modulus, ultimate tensile strength and elongation). The materials chosen to test were three Ultimaker standard materials (from the FDM machine used): UM-PLA, UM-TPLA (talc-injected PLA) and UM-CPE (chlorinated polyethylene), and five materials available on the online market: ALGA (PLA + Algae), OMNI (PLA based blend), PLAyPHAb (PLA + PHA from 3DPrintLife), PLAPHA (PLA + PHA from Colorfabb) and BioPETG (bio-polyethylene terephthalate glycol). The obtained results are summarized in Figure 10 (about mechanical properties), Figure 11 (print quality) and Figure 12 (regarding the energy consumption and some material characteristics) [61].

**Figure 10.** Material Guide for Green 3D printing: mechanical properties. Adapted from "3D Printing with Bioplastics", 2020 [61]. Mechanical properties for materials PLAYPHAB and QMNI are estimated to be similar to PLAPHA and UM-TPLA respectively.

**Figure 11.** Material Guide for Green 3D printing: energy consumption and some material characteristics. Adapted from "3D Printing with Bioplastics", 2020 [61]. \* One unit refers to this reference part used for universal comparisons.

PLA-based materials bought scored similarly to the standard Ultimaker PLA for print quality and tensile properties. Although the print quality is likely to improve when building simpler parts. While BIOPETG, the material used to compare with UM-CPE behaved slightly better. During the tests, it was verified that the heating of the build plate of the printer was the parameter that consumed the most energy. With this information, it was possible to say that materials that require lower building plate temperatures consume less energy, making them more eco-friendly. It was possible to conclude that new biomaterials have characteristics similar to conventional materials, making it possible to substitute these with greener materials [62]. This technique is widely used to produce a variety of biomedical devices, such as orthopaedic implants, thanks to the possibility to build manufacturing customized, low-volume and complex implants. Some filaments are already made from biological sources and may utilize waste material from producing beer or coffee grounds as filling. This makes the process eco-friendlier [63].

**Figure 12.** Material Guide for Green 3D printing: print quality. Adapted from "3D Printing with Bioplastics", 2020 [61].

#### *4.3. Electrospinning*

Electrospinning is a relatively inexpensive process used since the 1930s to produce fibres on the micro and nano scales using a high voltage (20 kV) as an electrostatic field on a polymer solution. The polymeric solution becomes electrified and stretches, becoming a thin fibre. Although it has been under development for a long time, it is still in its relative developmental infancy in industrial application.

An electrospun mat is usually made with a carrying polymer, ensuring that the mat is stable and able to incorporate other components. Bioplastics are widely used in biomedical applications since some materials are required to be biodegradable or biocompatible; PLA is one of the most commonly used biopolymers [46,64].

#### **5. Applications**

The applications of bioplastics are several (Figure 13), for example, food packaging (with 48% or 1.15 Mt of the total bioplastics market in 2021), consumer goods (11%), fibres (10%), agriculture (9%), automotive (5%), coating and adhesives (4%), construction (3%), electronics (3%) and other sectors (7%). It is expected that bioplastics will grow and the fields of application expand [6].

#### *5.1. Medical Industry*

Bioplastics are an excellent replacement for conventional polymer, as they may cause less allergies than the chemical-based products usually used. They are a sustainable material for the large amounts of one-time-use products used. Since some bioplastics may be breathable and allow water vapour to permeate and be waterproof at the same time; they can be used as sanitary products such as diaper foils, bed underlay and disposable gloves [33].

**Figure 13.** Global production capacities of bioplastics in 2021 (by market segment). Adapted from European Bioplastics "Global production capacities of bioplastics in 2021 (by market segment)". https://www.european-bioplastics.org/wp-content/uploads/2021/11/Global\_Prod\_ Market\_Segment\_circle\_2021.jpg (accessed on 29 December 2022) [65].

#### *5.2. Food Packaging Industry*

The use of bioplastics in the food packaging industry has been increasing rapidly. The materials used in these types of applications require some specific characteristics to achieve the product's intended shelf-life time and respect the food safety regulations. The mainly used bioplastics are PLA, starch and cellulose-based [33].

#### *5.3. Agriculture*

Some crops require using blankets of biodegradable plastic on part of their fields to increase their product yield. Usually, petroleum-based polymers are used, but bioplastics can achieve the same objective and do not leave residues, unlike conventional polymers. This makes it possible to reduce labour and disposal costs [33].

#### **6. Bioplastic Waste Management**

The waste management process of bioplastics is not as simple as it may seem. Although some of the process's parameters are similar to the ones used for conventional plastics, it is not possible to mix these two types of materials without contaminating either the material itself or the environment. Another big misconception is the idea that there is no harm in dumping bioplastics anywhere with degradation assured. This only applies to some biopolymers, such as those made from seaweed.

The proper way to treat bioplastics is to recycle them mechanically, chemically, or organically, depending on the material's capacity to compost or biodegrade (Figure 14). If a material is compostable, it is possible to obtain enriched compost (a valuable material) under industrial composting conditions. If the material is not compostable, the ideal process to use is chemical or mechanical recycling [31,66,67].

#### *6.1. Mechanical and Chemical Recycling*

Physical or mechanical recycling is already an established technology. The main problem with the mechanical recycling of bioplastics is that it is not possible to obtain a good quality material if more than one type of material is mixed; for example, PLA is a material widely used in packaging, and it is possible to recycle it mechanically; however it is difficult to distinguish it from materials such as PET by mere appearance, and this means that it would be necessary to add additional labelling to enable a correct separation by the consumers, but before that, it is needed to create a separate PLA recycling stream.

**Figure 14.** Waste management process of biopolymers. Adapted from "Bioplastics: A boon or bane?" Renewable and Sustainable Energy Reviews, vol. 147. Elsevier Ltd., 1 September 2021 [31].

In chemical recycling, unlike the mechanical process, the aim is to reuse carbon and biogenic substances obtained from waste material to synthesize new plastic materials throw treatments that can involve hydrolysis/solvolysis, hydrothermal depolymerization and enzymatic depolymerization. Some of the advantages of chemical recycling are related to the simplicity of the overall process, and it does not require sorting or thermomechanical degradation, and it is not a process sensitive to material impurities [31,67].

#### *6.2. Composting*

Some materials are specifically designed to be compostable or organically recyclable; the final product is an enhancer to the soil, providing nitrogen, potassium, phosphorus and organic matter to the soil. This is an aerobic process divided into three stages, mesophilic, thermophilic and maturation. The application of these materials in single-use objects, such as bags, food packaging and cutlery strengthens their industrial utilization. Mechanical processing is necessary to separate missorted materials, reduce particle sizes for better bioavailability and mix different organic substrates for optimal dry matter content and C/Nratio. It is possible to obtain high-value products that can be used as a soil amendment (due to the high capacity to hold water because of its organic matter content), biogas, hydrogen, ethanol and biodiesel.

The composting process at home is very difficult to control, which may result in the formation of methane gas. This process is more variable and less optimized than industrial composting, and the temperature achieved is rarely more than a few degrees Celsius above the ambient temperature [66,67].

#### *6.3. Anaerobic Digestion*

Anaerobic digestion aims to degrade organic wastes to biogas and digestate through four successive phases: hydrolysis, acidogenesis, acetogenesis and methanogenesis. The process can be operated at psychrophilic (18–20 ◦C), mesophilic (35–40 ◦C) and thermophilic (50–60 ◦C) temperature regimes, although the last two conditions are more efficient for the degradation of the bioplastics [67].

#### *6.4. Waste-to-Energy (WTE)*

The waste-to-energy process, also known as incineration, is highly influenced by the capacity of the polymer to degrade in terms of its sustainability, and 56% of the energy comes from the incineration of biogenic organic MSW (municipal solid waste), which means that at least half of the process products does not contribute to an increase of CO2 in the biosphere;

in other words, "incineration of bio-based waste emits CO2 which was recently captured and will be captured again when new bio-based products are produced, whereas incineration of fossil plastics emits CO2 that had been sequestered for millions of years" [66].

#### *6.5. Materials*

#### 6.5.1. Disposal of PHA

The types of microorganisms capable of degrading PHA are numerous, which makes this biopolymer easily compostable under industrial and nonindustrial conditions. Home composting is possible in this case, since the degradation of the polymer may start at 30 ◦C. PHA is known to improve the soil in which it decomposes, since it increases the diversity of microbes present in the soil; in turn, this also increases the composting efficiency, as well as the variety of materials able to be degraded. This means that the separation process of these materials does not need to be so stringent, saving time and money. The waste management process of this material is summarized in Figure 15. Industrial decomposition of PHA takes 124 ± 83 days; under anaerobic digestion, it takes about 31 ± 20 days, while improperly abandoning this type of material on the soil takes 1–2 years.

**Figure 15.** Waste management process of PHA. Adapted from "Bioplastics: A boon or bane?", Renewable and Sustainable Energy Reviews, vol. 147. Elsevier Ltd., 1 September 2021 [31].

PHA is also degradable in aerobic lagoon water treatment systems, which house a wide variety of microorganisms. This process is even more efficient than the one occurring on the soil. It is also possible to degrade these materials in landfills, although a lot slower and not ideal [31,67].

Although it is possible, it is not feasible to recycle PHA since its properties at high temperatures are very unstable and, if not isolated from other materials, it might contaminate the final polymer. Chemical recycling is still a method under study; some materials such as herbicides and plasticizers, among others, can be made from products of the chemical recycling of PHA [31].

#### 6.5.2. Disposal of PLA

Initially, the objective of using PLA on single-use plastic items was to transit from petrochemical plastics to biodegradable bioplastics. However, currently, the disposal process of these materials is not any different from the conventional plastics, which is not the most effective method to use. Some manufacturers, such as NatureWorks, have clearly stated that the materials they produce PLA are to be industrially composted and have even explained how the process works. PLA is commonly used as a 3D printing filament, and there are several machines that convert used filament into a new one, making it possible to recycle these materials more efficiently. The waste management process of this material is summarized in Figure 16 [31].

**Figure 16.** Waste management process of PLA. Adapted from "Bioplastics: A boon or bane?", Renewable and Sustainable Energy Reviews, vol. 147. Elsevier Ltd., 1 September 2021 [31].

It is hard to determine if a material is truly biodegradable; for example, in the case of PLA, the enzymes needed to degrade this biopolymer are not present in the natural environment. This means that to biodegrade PLA efficiently, the environment in which this process occurs must be controlled, especially its temperature and moisture. PLA needs to be at least 60 ◦C and with a lot of moisture to catalyse self-hydrolysis of the material. It is hard to achieve these conditions in home composting, which might lead to the production of inert materials. The conversion of the polymer to lactic acid is aided by the temperature and moisture available; this element is then used as a source of nutrients by several types of microorganisms. Under controlled industrial composting conditions, it takes 84 ± 47 days for the complete degradation of PLA; using anaerobic digestion takes about 423 ± 76 and 116 ± 48 days in mesophilic and thermophilic conditions, respectively, while in soil, 4–5 years are necessary for PLA to disappear.

The end product of the process should be a mix of humus, water and CO2. If not processed, the remaining lactic acid reduces the soil's pH, affecting the development of microorganisms and their activity [31].

#### **7. Degradation Process**

This process may be anaerobic or aerobic, and according to the type of product obtained from the process, biomass or gas and minerals, it can be called complete biodegradation or mineralization, respectively. Both processes are influenced by endogenous (such as molecular weight, crystallinity and flexibility of the molecule) and exogenous (temperature, humidity, pH, availability of oxygen and enzymatic activity) factors, which may directly affect the entire process. The best outcome regarding the degradation of bioplastics is composting, since the final products of the process are a soil-like substance called humus, CO2, water and inorganic compounds, leaving no toxic residues (if no additives were used). However, it is important to consider that this is only achievable if the right conditions are met: precisely controlled temperature, humidity, oxygen, etc. Studies show that composting PLA under natural conditions equals approximately 10–20% efficiency when compared to the process under a controlled environment [68].

Various degradation mechanisms degrade PHA and PLA; those include physical, chemical, oxidative, hydrolytic, enzymatic, microbial, photodegradation and thermodegradation mechanisms and have been studied by many researchers [69]. The different mechanisms in which the polymer degradation may occur are linked to the type of diffusionreaction phenomenon taking place. When the absorption of water into the polymer is slower than the hydrolytic chain scission and the diffusion of the monomers into the surroundings of the plastic, it is said that the degradation is called surface erosion. As represented in Figure 17a, over time, a thinning of the plastic piece occurs without decreasing the internal molecular weight of the polymer.

**Figure 17.** Degradation modes for degradable polymers: surface erosion (**a**), bulk degradation (**b**) and bulk degradation with autocatalysis (**c**). Adapted from [46].

Bulk degradation, on the other hand, happens when water penetrates the entire volume of the polymer, resulting in its molecular weight decrease throughout the piece matrix (Figure 17b). If this process does not occur in equilibrium, the erosion does not occur gradually (water does not penetrate the polymer to hydrolyse the chains, making it impossible for the monomers to diffuse out). In that case, it is possible that internal autocatalysis could take place via the carboxyl and hydroxyl end group by-products. The internal concentration of this autocatalysis may create an acidic gradient, resulting in a nucleus with a faster degradation rate than the polymer's surface. The process evolves with the outer layer with a higher molecular weight than the piece's interior, as shown in Figure 17c. Thus, this polymer is denominated as having a bimodal molecular weight distribution. In time, with the continuous diffusion of the inner polymer through the outer layer, the piece turns into a hollowed-out structure [46].

There is, however, an important notion to consider when referring to the degradation of plastics, which is the possibility of mixing additives with conventional plastics (such as PE, PP (polypropylene), PS, PET or PVC (polyvinylchloride)) to mimic the biodegradation process. These materials are called "oxo-biodegradable" or "oxo-degradable" plastics, and the additives used are usually transition metals such as nickel, iron, manganese and cobalt. Their main function is to make it easier for the polymer to break down into smaller pieces. The idea is to allow microorganisms to process the material and convert it into CO2 and the biomass. This degradation process is called oxo-degradation and is divided into two different stages: the first is related to the fragmentation of the polymer itself, which is an abiotic process where the prooxidant actions create an oxidative degradation of the polymer; in the second stage occurs in the biotic process where microorganisms convert the products of the previous stage into CO2 and the biomass. However, this process is hard to predict concerning the time frame in which it takes place, since it depends on the climate factors such as temperature and intensity of solar radiation.

Oxo-degradable materials, however, are not considered as compostable or recyclable, as defined according to the standards accepted by the industry (ASTM D6400—Standard Specification for Labelling of Plastics Designed to be Aerobically Composted in Municipal or Industrial Facilities; ASTM D6868—Standard Specification for Labelling of End Items that Incorporate Plastics and Polymers as Coatings or Additives with Paper and Other Substrates Designed to be Aerobically Composted in Municipal or Industrial Facilities; EN 13432—Requirements for packaging recoverable through composting and biodegradation—Test scheme and evaluation criteria for the final acceptance of packaging; ISO 17088—Specifications for compostable plastics; ASTM D5338—Standard Test Method for Determining Aerobic Biodegradation of Plastic Materials Under Controlled Composting Conditions, Incorporating Thermophilic Temperatures and ASTM D5929—Standard Test Method for Determining Biodegradability of Materials Exposed to Source-Separated Organic Municipal Solid Waste Mesophilic Composting Conditions by Respirometry); after the first stage of the process, the fragmented materials, although invisible, are still present in the environment as microplastics, and there is no guarantee that the entirety of the material will biodegrade or how long it takes.

In 2019, the European Parliament banned the use of oxo-degradable plastics. Until then, companies such as Pizza Hut, Nescafe, KFC, Tiger Brands, Tesco Barclay and Walmart usually used oxo-degradable materials in their products. On the other hand, countries such as the United States of Emirates, Saudi Arabia, Bahrain and Jordan regularly use oxo-biodegradable plastics. In Saudi Arabia, since April 2017, many single-use plastics have been made of these types of materials, and the intention is to expand this utilization. The available literature is divided regarding the eco-friendliness of these materials, and the companies that produce these polymers strongly defend their biodegradability [32,70–72].

#### **8. Environmental Viability Assessment**

Although bioplastics are known as a green alternative to conventional polymers, some drawbacks are making it a questionable choice. There are some bioplastics that only break down in specific conditions or when treated in municipal composters or digesters. When decomposed in composts, they release methane and CO2 into the atmosphere.

The production of bioplastics is also controversial, because some of them are made from plants which production requires the occupation of land that could be used to plant food. Statistics revealed that 0.7 million ha of agricultural land are used to produce bioplastics (Figure 18). This situation has consequences on food prices and in the economy of some countries [6,39].

**Figure 18.** Land use estimation for bioplastics 2021 and 2026. Adapted from European Bioplastics, "BIOPLASTICS MARKET DEVELOPMENT UPDATE 2021", \* in relation to global agricultural area, \*\* including approx.. 1% fallow land, \*\*\* land-use for bioplastics is part of the 2% material use. https://docs.european-bioplastics.org/publications/market\_data/Report\_Bioplastics\_ Market\_Data\_2021\_short\_version.pdf (accessed on 29 December 2022) [6].

Recently, some studies revealed that, when comparing the production of traditional plastics and bioplastics, the bioplastics production created more pollutants due to the use of pesticides and fertilizers when growing the crops, and bioplastics contribute more to ozone depletion than conventional plastics.

However, recent studies reveal that not only the production of PLA saves two-thirds of energy when compared to traditional plastics production, but also, the disintegration of this material does not increase CO2 in the atmosphere. This is possible, because the plant used as raw material to produce PLA absorbed the same quantity of CO2 as the quantity released during the degradation of this polymer. Plus, the degradation of PLA in landfills emits 70% less greenhouse effect gases than conventional plastics. The use of renewable energies also helps to make the final products greener.

The problem regarding using food sources as raw materials to produce bioplastics is easily avoided by using crop residues as the base production material, such as stems, straws, husks and leaves. By using alternative carbohydrate sources, this problem is averted. It is also possible to use kitchen waste, fish meal wastes and paper sludge as a source of carbohydrates to produce PLA, making it possible to help the waste management in big cities.

To correctly evaluate the environmental impact and viability of bioplastics, it is necessary to assess all the processes from initial production to the final disposal. An LCA (life cycle assessment) is usually used to do this. A cradle-to-grave analysis, for example, helps determine the impact of the use of certain materials and compare them with other ones, making it possible to evaluate the whole life of products from beginning to end and in each stage of utilization. Several scenarios are studied, for example, if it is better to recycle or compost the material, to determine the best possible solution. Recent studies revealed that incineration or landfilling of bioplastics is not a useful option. It was also concluded that using PLA and TPS reduces greenhouse emissions by 50 to 70%. PTT (polytrimethylene terephthalate) and bio-urethanes release 36 and 44% less greenhouse gases, respectively. Studies have shown that the issues observed during the production of bioplastics are still less harmful than the use of conventional plastics, and it is possible to address them verified during the production of bioplastics [2,31,39,73].

There are several factors important to consider when performing an LCA; environmentally, some of the most relevant ones are:

#### *8.1. Abiotic Depletion*

Minerals and fossil fuels are some of the system's inputs that are important to consider, since the extraction of these materials affects the health of humans and the environment. For each extraction of these materials, the abiotic depletion factor is determined. This factor is measured based on kg of antimony (Sb) equivalents per kg of extracted mineral.

#### *8.2. Global Warming*

This is related to the amount of greenhouse gas emissions. Global warming is a hazard that severely affects the ecosystem, human health and material welfare. The absorption of infrared radiation changes the climatic patterns and increases the global average temperatures. This factor is measured based on its kg of CO2 equivalents per kg of emission.

#### *8.3. Human Toxicity*

This category does not include health risks in the work environment; the main concerns are related to toxic substances' effects on the human environment. The purpose is to measure the human toxicity potentials, which may include the fate, exposure and effects of toxic substances for an infinite time. This factor is measured based on its kg of 1,4-DB (1,4-Dichlorobenzene) equivalents per kg of emission.

#### *8.4. Freshwater Aquatic Ecotoxicity*

Similar to human toxicity, the goal is to determine the fate, exposure and effects of toxic substances in the air, water and soil on fresh water. This factor is measured based on its kg of 1,4-DB equivalents per kg of emission.

#### *8.5. Marine Aquatic Ecotoxicology*

The logic used to determine this factor is the same as the human toxicity and the freshwater aquatic ecotoxicity; the characterization factor is the potential of marine aquatic toxicity of each substance emitted into the air, water or/and soil. This factor is measured based on its kg of 1,4-DB equivalents per kg of emission.

#### *8.6. Terrestrial Ecotoxicity*

Once again, the characterization factor is the potential of terrestrial toxicity of each substance emitted into the air, water or/and soil. This factor is measured based on its kg of 1,4-DB equivalents per kg of emission.

#### *8.7. Photochemical Oxidation*

Reactions between NOx (nitrogen oxides) and VOCs (volatile organic compounds) when in contact with UV (ultraviolet) light create photochemical oxidant smog, leading to the formation of ozone in the troposphere. This phenomenon depends on the metrological conditions and the background concentrations of pollutants. This factor is measured based on its kg of C2H4 (Ethylene) equivalents per kg of emission.

#### *8.8. Acidification*

Refers to the increase of potentially toxic elements or the decrease of pH by the deposition of pollutants such as SO2 (sulphur dioxide), NOx, HCl (hydrochloric acid), CO2 and NH3 (ammonia), which may affect soil, groundwater, surface water, organisms, ecosystems and materials. This factor is measured based on its kg of SO2 equivalents per kg of emission.

#### *8.9. Eutrophication*

The deposition of excessive nutrients in a soil or water system, especially phosphates and nitrates, usually leads to excessive algae growth, potentially damaging life forms in the system by affecting the ecosystem equilibrium. This factor is measured based on its kg of PO4 <sup>3</sup><sup>−</sup> (Phosphate) equivalents per kg of emission [68,74,75].

#### **9. Statistics of Bioplastics**

According to European Bioplastics, the production of bioplastics represents only 1% (2.42 Mt) of all plastic production, and the vast majority was produced in Asia, about 50%. Europe is the second continent with a greater capacity to produce bioplastics, as shown in Figure 19. The growth of the last few years is greatly influenced by the incentives of the European Commission to decrease the dependency on fossil fuels and transition to a circular economy [33,75].

**Figure 19.** Global production capacities of bioplastics in 2021 (world map). Adapted from "Global production capacities of bioplastics in 2021 (world map)", https://www.european-bioplastics.org/wpcontent/uploads/2021/11/Global\_Prod\_Capacity2021\_map.jpg (accessed on 29 December 2022) [76].

However, this growth is not yet sufficient to consider it possible to replace the use of petrochemical plastics with biopolymers. A study developed by Janis Brizga et al. (2020) revealed that, to achieve this on packaging application alone, it would be necessary to increase by 8.4 times some of the bioplastics production (Figure 20). Other bioplastics would even need to increase production by 100 times. Although these values are only theoretical and no economic feasibility and resource availability are considered, it is possible to observe that the production of bioplastics is still very far from what it would be necessary to replace petrochemical polymers [77].

**Figure 20.** Current Bioplastic Packaging Production versus Necessary Production Capacity Source. Adapted from "The Unintended Side Effects of Bioplastics: Carbon, Land, and Water Footprints", One Earth, vol. 3, no. 1. Cell Press, pp. 45–53, 24 July 2020 [77].

Another study by F. Klein, A. Emberger-Klein, K. Menrad et al. (2019) evaluated the possible motives influencing the intention to purchase bioplastic products on the German market. The study concluded that green consumer values, attitudes towards bioplastic, product experience and interest in information are crucial factors influencing the community to buy bioplastic products. This means that information and communication are key aspects for people to choose to use bioplastics. To reinforce this, it was mentioned that "the purchase intention for bioplastic products measured for all German citizens is moderate at about 56%. In contrast, about 95% of the consumers with product experience intend to buy bioplastic products". This means that, by increasing the promotion of bioplastics, the community becomes more aware of the importance of using these materials. One good starting point would be to set standards regarding the end-of-life usage of bioplastics, eliminating the confusion of some companies and consumers. Therefore, it is useful to consider the LCA analysis to support the choices made [78].

Numerous analyses can be done regarding the use of bioplastics and the consequences it has on important issues, such as fossil fuel consumption, economics, pollution, energy consumption and health.

#### *9.1. Fossil Fuel Consumption*

Using bioplastics instead of petrochemical ones can reduce fossil fuel consumption, since its production does not depend on them. Although it is not yet possible if all the petrochemical polymers were replaced by bioplastics and considering the energy used for its production as renewable, the consumption of fossil fuels would decrease 4% (3.49 million barrels a day). This value surpasses the daily consumption of every country except the United States, China and Japan. This simplified case reveals that the savings in oil consumption would be significant [79].

#### *9.2. Economics*

Generally, bioplastics are more expensive to produce than petrochemical polymers, but with the development of production techniques and the instability of oil prices, this reality tends to shift. For example, Mirel bioplastic made by Metabolix is about double the price of a petrochemical equivalent. The potential price stability is another benefit bioplastics have, and with the industry's growth, the production prices of bioplastics should decrease [79]. To put it in perspective, Table 4 contains a list of typical market prices for common bioplastics inputs and polymers.

**Table 4.** Bioplastics and raw material prices (2018). Adapted from "Green Bioplastics as Part of a Circular Bioeconomy", Trends in Plant Science, vol. 24, no. 3. Elsevier Ltd., pp. 237–249, 1 March 2019 [23].


#### *9.3. Energy Consumption*

Several examples prove that the energy required to produce petrochemical polymers is higher than bioplastics. The total life cycle of HDPE requires 73.7 MJ kg−<sup>1</sup> of plastic produced, LDPE uses 81.8 MJ kg−<sup>1</sup> and PP 85.9 MJ kg<sup>−</sup>1. On the other hand, PHB requires 44.7 MJ kg−<sup>1</sup> of plastic produced, PLA uses 54.1 MJ kg−<sup>1</sup> and TPS 25.4 MJ kg−1. This represents significant consumption savings, and these values might have even greater differences due to the development of the techniques used to produce bioplastics. PLA energy requirement might reach values as low as 7.4 MJ kg<sup>−</sup>1.

Hypothetically, if all the PP used in the United States were replaced by PHB, PLA or TPS, the annual energy savings would be 363, 280 and 529 PJ, respectively. Considering that it is necessary to use 31,250 metric t of coal to produce 1PJ of energy, the savings would be enormous [79].

#### *9.4. Pollution*

Less pollution is produced by the production of bioplastics when compared with petrochemical polymers, not only by requiring less energy to be produced and emitting less CO2 but also by the possibility of recycling these materials. Bioplastics need to have their own recycling process, but they are not yet produced in enough quantities for that to happen; for this reason, they are considered a contaminant to the process. In perspective, if 0.1% of bioplastic material mixes with PET during its recycling process, the entire batch would become useless. It is important, however, to notice that bioplastics are as easily recyclable as petrochemical ones; they just need to be processed separately [79].

#### *9.5. Health*

So far, bioplastics have not been linked to any type of health problem. However, it is important to consider that the bioplastic industry is still under development, which means that further studies could reveal some issues with the use of these materials. It is essential to consider the use of pesticides, used during crop growth, and plasticizers. The problem with plasticizers is better understood than the bioplastics themselves. Nevertheless, bioplasticizers with low toxicity can be used in bioplastics [79].

#### **10. Advantages and Disadvantages**

Petrochemical-based polymers are a type of material in the process of extinction for several motives, such as the environmental hazard they usually provoke; their waste management difficulties; the risk of toxicity for other materials, animals and plants; the limited oil and gas resources and their increasing prices. However, these disadvantages are balanced by the low cost and high-speed production of pieces made of these materials, their high mechanical performance, good barrier properties and good heat stability.

Based on those disadvantages, the utilization of bioplastics works as an alternative. Bioplastics have a much lower carbon footprint, although if the polymer is biodegradable, the CO2 stored during the formation of the raw material will be released when it degrades, unlike the permanent bioplastics, which can be recycled many times while still storing the CO2 absorbed, but this released CO2 is compensated for by the fact that, during the growth of some types of bioplastics raw materials (plants), they absorbed the same quantity of this gas from the atmosphere. For example, 1 kg of bioplastic resign produces about 0.49 kg of CO2, while petrochemical-based polymers produce 2 to 3 kg of CO2. They make it possible for every country to produce polymeric materials without depending on countries with petroleum reserves. The production of bioplastics also takes less energy than conventional plastics.

However, the overall costs to produce bioplastics are higher than the petroleumbased polymers; nevertheless, it is important to consider the possibility of implementing several cost reduction mechanisms. Currently, bioplastics are considered to contaminate the recycling process of other plastics, but this problem is easily overcome by separating it from conventional plastic from the beginning of the process. The production of bioplastics might also reduce the available resource reserves usually used as food by-products. The composting possibility of some bioplastics might create some confusion. These materials are not compostable in the same way as conventional food waste, as it might be interpreted. The process requires controlled conditions that can only be achieved under an industrial composting site. The production, usage and waste management of bioplastics are not yet under any specific legislation in some countries [2,4,33,80].

#### **11. Regulation**

Currently, bioplastics and biodegradable plastics are identified by the ASTM International Resin Identification Coding System as part of group 7 or 'other' (Figure 21b), an identification system developed by the Society of the Plastics Industry in 1988 and administered by ASTM International since 2008. This means that the polymers included in this category do not have specified characteristics, and their management process is not defined. A solution to this problem would be to globalize a symbol easily identifiable identifying polymers classified as compostable or biodegradable according to proper standards, such as ASTM D6400, ASTM D6868, EN 13432, ISO 17088, ASTM D5338 and ASTM D5929. For example, the European Bioplastics created the Seeding logo (Figure 21a) as a label to identify compostable polymers according to the EN 13432 standard. Usually, this label goes along with one other created by Vinçotte (taken over by TÜV AUSTRIA Group), the "OK compost home", "OK compost industrial", "OK marine biodegradable", "OK soil biodegradable", "OK water biodegradable" and "OK biobased" (Figure 21c), because these should guarantee complete biodegradability in the light of specific requirements [31,39,73].

**Figure 21.** Labels currently used as compost, biobased and biodegradable polymers by European Bioplastics (**a**), ASTM International (**b**) and the Vinçotte/TÜV AUSTRIA Group (**c**) [32,73,81].

Some companies already use bioplastics in their products; for example, Coca-Cola uses bioplastics in its packaging. Recently, Coca-Cola revealed that they are launching their first 100% biobased bottle (excluding the cap and label), made of bio-PX (plant-based paraxylene) converted to bio-PTA (plant-based terephthalic acid) and using a new process, commercially viable developed by Virent. PTA is one of the main components of PET (70%); the remaining 30% is MEG (monoethylene glycol), which was already being produced from sugarcane. Since then, Coca-Cola has allowed non-competitive companies to use the technology and brand in their products, from Heinz Ketchup to the fabric interior in

Ford Fusion hybrid cars. This, however, is done by their own initiative. To incentive the implementation of bioplastics as a substitute for conventional plastics, it is important to encourage companies with certain promotion and support mechanisms, such as regulations and monetary inducement. Financial profit is one of the main reasons a company invests in something. However, this principle is not yet applicable to the industry of bioplastic production. This is due to the fact that consumers are not expected to buy bioplastic goods more expensive than conventional plastics just because they might be more eco-friendly. Cereplast, a TPS producer, estimated that when the oil price reaches around 95 USD per barrel, the company's production costs will be lower than petrochemical plastics, and the demand for bioplastics will increase. According to European Bioplastics, the packaging is still the main application of bioplastics, 48% (1.15 million t) of the total bioplastics market in 2021 (Figure 22) [32,82].

**Figure 22.** Global production capacities of bioplastics 2021 (by market segment) (**A**) and growth of bioplastic production in recent years (**B**). Adapted from European Bioplastics, "Bioplastics market data", https://www.european-bioplastics.org/market/ (accessed on 29 December 2022) [82].

With the objective of creating a simple tool for companies to select the best possible solution when selecting a good bioplastic material for food packaging, the Association of Organic Food Producers in Germany, created an Internet tool called the "Biokunststoff-Tool". This program evaluates areas such as ecology, social acceptability, safety, quality and technology of several bioplastics, which makes it possible to select environmentally and socially responsible production methods and materials.

To make the transition to bioplastics easier, some legal changes should be applied. For example, create a strict separation between biobased and biodegradable plastics using marks and labels as in the previous examples. This facilitates the implementation of waste treatment processes specialized in these materials. However, before that, the labelling information should be spread through consumers and waste management companies [32,83].

#### **12. Conclusions**

Bioplastics are materials with great potential for development. Although it is not yet used industrially on a large scale, the ecological advantages of using this material compared with other plastics are enormous. Less chemical pollution, less energy consumption and less CO2 emissions are some of the major drivers of a transition to a circular economy using bioplastics. On the other hand, its production is costly, and they do not have a proper recycling process. The limitations derived from the mechanical characteristics may be overcome and adapted to the intended application using additives. The disadvantages will be gradually overcome with the development of new technology and further research, over time, regarding these materials. To speed up the process, it is essential to spread the information about bioplastics to companies and consumers to convince the consumers that these materials are an excellent alternative to petrochemical-based polymers. In fact, global production capacities have been increasing and show strong growth trends; as such, new applications can be foreseen when large amounts of biopolymers are available for large-scale productions. However, biopolymers and bioplastics are not exempt from sustainability issues. Recycling these materials is a controversial debate because of their biodegradability-specific conditions, their potential methane emissions with a negative climate impact when discarded in landfills and potential contamination of the petroleumbased recycling stream. Other issues are related to land use due to ethical reasons about the potential competition with food resources. These and other concerns are all essential aspects to debate, at the academic, civil, economic and political levels. Laws, norms and regulations related to the environment have the potential to reduce the impacts on the environment. The LCA analysis can be used as a universal tool to assess those impacts and to support political decisions, therefore paving the way for a green transition.

**Author Contributions:** Conceptualization, A.C., T.E. and A.M.; investigation, A.C., T.E. and A.M.; supervision, A.M. and T.E.; writing, review and editing A.C., T.E. and A.M.; images editing, A.C., R.T. and T.E.; funding acquisition, A.M., T.E. and T.T.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** The authors acknowledge the Fundação para a Ciência e a Tecnologia (FCT) through the projects UIDB/04044/2020 and UIDP/04044/2020, Associate Laboratory ARISE LA/P/0112/2020, and PTDC/BTA-GES/2740/2020\_NABIA. The Coimbra Chemistry Centre (CQC) is supported by the FCT through the projects UIDB/00313/2020 and UIDP/00313/2020. PAMI—ROTEIRO/0328/2013 (No. 022158), EcoPlast, Materiais compósitos eco-sustentáveis para substituição dos plásticos convencionais, ref POCI-01-0247-FEDER-069002, funded by the National Agency of Innovation (ANI). We are grateful for funding from PTScience, which is supported through the programs CENTRO-05- 4740-FSE-001526 and FEDER.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **Nomenclature**


#### **References**


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## *Article* **Carboxymethyl Cellulose/Gelatin Hydrogel Films Loaded with Zinc Oxide Nanoparticles for Sustainable Food Packaging Applications**

**Aqsa Zafar 1, Muhammad Kaleem Khosa 1,\*, Awal Noor 2,\*, Sadaf Qayyum <sup>2</sup> and Muhammad Jawwad Saif <sup>3</sup>**

	- Al-Hassa 31982, Saudi Arabia

**Abstract:** The current research work presented the synthesis of carboxymethyl cellulose–gelatin (CMC/GEL) blend and CMC/GEL/ZnO-Nps hydrogel films which were characterized by FT-IR and XRD, and applied to antibacterial and antioxidant activities for food preservation as well as for biomedical applications. ZnO-Nps were incorporated into the carboxymethyl cellulose (CMC) and gelatin (GEL) film-forming solution by solution casting followed by sonication. Homogenous mixing of ZnO-Nps with CMC/GEL blend improved thermal stability, mechanical properties, and moisture content of the neat CMC/GEL films. Further, a significant improvement was observed in the antibacterial activity and antioxidant properties of CMC/GEL/ZnO films against two food pathogens, *Staphylococcus aureus* and *Escherichia coli.* Overall, CMC/GEL/ZnO films are eco-friendly and can be applied in sustainable food packaging materials.

**Keywords:** hydrogel films; gelatin; antimicrobial activity; food packaging; TGA

### **1. Introduction**

Today, food packaging is a growing technology because of rapid advancements in the fields of biopolymers and materials science. They are being used as new modes of food coatings. Encapsulation of biopolymers matrices, and food packaging materials for functional foods, proteins and polysaccharides have gained a lot of attention recently [1–3]. Active packaging is thought to be the best way to increase the safety and shelf life of food [4]. The presence of antimicrobial agents and antioxidants in active packaging plays a vital role in stopping biological or chemical changes and microbial growth in packaged foods [5]. In addition to antimicrobial agents, antioxidants, essential oils, natural pigments, and plant extracts have also been used in packaging materials [6]. Inorganic materials such as nano-sized metals, metal salts, and metal oxides seem promising for this purpose and are frequently used as antibacterial agents [7,8]. The incorporation of metal nanoparticles (ZnO, TiO2 and silica) into composite films has resulted in nanocomposites that are light in weight, stronger in thermomechanical performance, fire resistant, and less permeable to gases. They also reduce the flow of oxygen inside packaging containers when added to plastic films. To keep food fresh for a very long time, they serve as a barrier against gases and moisture [9]. The many properties of packaging materials are greatly enhanced by nanomaterials, which have a significant impact on the food packaging industry. One of the most significant categories of nanomaterials is zinc oxide nanoparticles to improve thermomechanical and antimicrobial properties [10]. Zinc oxide nanoparticles are now widely available commercially and are easy to be used directly due to their outstanding antibacterial properties, high thermal stability, excellent mechanical properties, and heat resistivity [11–13]. Tetrapod-shaped ZnO nanomaterials have recently been reported to

**Citation:** Zafar, A.; Khosa, M.K.; Noor, A.; Qayyum, S.; Saif, M.J. Carboxymethyl Cellulose/Gelatin Hydrogel Films Loaded with Zinc Oxide Nanoparticles for Sustainable Food Packaging Applications. *Polymers* **2022**, *14*, 5201. https:// doi.org/10.3390/polym14235201

#### Academic Editor: Raffaella Striani

Received: 23 October 2022 Accepted: 25 November 2022 Published: 29 November 2022

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

inhibit herpes simplex virus (HSV) infection. It exhibits adjuvant-like properties, improves viral presentation to dendritic cells, and boosts humoral and cell-mediated immunity. ZnO-Nps have been evaluated for their antiviral properties against HSV-1, HSV-2, and influenza. This is primarily because ZnO nanoparticles have the ability to modulate the immune system [14,15]. As packaging materials and coating agents, natural polymers have numerous other uses. In the pharmaceutical sector, gelatin is commonly employed as an absorbent sheet, a wound dressing, an adhesive, and as an excipient in controlled drug delivery [16]. Several studies have shown that combining gelatin with polysaccharides, including alginate, chitosan, hyaluronic acid and other proteins, can enhance its properties due to their permeability and self-adhesiveness, as well as their capacity to form chemical and physical hydrogel films. They also serve as drug delivery and tissue regeneration support matrices. Cellulose is another biopolymer that is plentiful, renewable, and biodegradable and has been used to prepare biocompatible composite films [17]. Carboxymethyl cellulose (CMC), a cellulose derivative, has been extensively applied in cellular growth, food processing, food packaging, the pharmaceutical industry, and medical fields because of its good hydrophilicity, biocompatibility, and film formability [18,19]. The basic requirements for materials used in packaging are that they have good mechanical and thermal performance, protection against gases, and transparency. Therefore, the aim of the current research was to fabricate carboxymethyl cellulose and gelatin-based films using ZnO nanoparticles as a functional material. After characterization by FT-IR and XRD, CMC/GEL/ZnO composite films were investigated for the water vapor permeability (WVP), moisture contents, thermal and mechanical stability, antimicrobial activity, and free radical inhibition activity.

#### **2. Materials and Methods**

#### *2.1. Chemicals*

Gelatin powder (Food grade; Halal), carboxymethyl cellulose (CMC: pKa =3.5, with medium viscosity; Mw. 90,000), glutaraldehyde (GTA), sodium azide, zinc nitrate hexahydrate (Zn(NO3)2.6H2O), 2,2-diphenyl-1-picrylhydrazyl (DPPH), iron sulphate (FeSO4), 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and ferric chloride (FeCl3) were used as received. All chemicals were procured from Sigma-Aldrich Pakistan.

#### *2.2. Chemical Characterization*

For major functional groups and shifting of absorption bands in synthesized CMC/ GEL/ZnO-nanocomposite films, IR spectrophotometer (Bruker) was used, and spectra were recorded in 4000–400 cm<sup>−</sup>1. TGA was determined, under nitrogen from room temperature to 700 ◦C, using Netzsch and Perkin Elmer TGA-7. Tensile strength and % elongation were determined by DMA Q800 V21.3 Build 96. Surface morphology was examined (FEI-NOVA Nano SEM-450, Hillsboro, OR, USA) by using Chroma meter (Konica Minolta, CR-400, Tokyo, Japan) with white standard colour plate (Lo = 92.15, ao = −0.41, and bo = 4.55) as a background, of surface colour (Hunter L, a, and b-values).

#### *2.3. Green Synthesis of ZnO-Nps*

Green chemistry is extensively used in research and is considered eco-friendly because it uses plant phytochemicals to prepare nanoparticles. Keeping this in mind, ZnO-Nps (<50 nm) were prepared using mint (Mentha longifolia) leaf extract. Fresh mint leaves were cleaned by washing with distilled water and were ground until very fine particle sizes were obtained, and were then air dried. About 10 g of fine powdered mint leaves in distilled water (250 mL) were heated at 70 ◦C. After cooling at room temperature for about 30–40 min, the heavy biomaterial that settled down after centrifugation at 4000 rpm was removed by filtration. The clear solution of mint extract was then stored at 4 ◦C and used for the preparation of nanoparticles. Following that, zinc oxide nanoparticles (50 nm) were prepared using mint leaves extract as an oxidizing agent as per the literature with slight modifications [20]. A mixture of 100 mL zinc nitrate Solution (0.01 M) and 30 mL of mint leave extract was heated at 60 ◦C with constant stirring until the bioreduced ZnO nanoparticles settled down as white precipitates. The resulting, white-colored precipitates were dried at 80 ◦C for approximately six hours before being calcined at 600 ◦C for approximately two hours.

#### *2.4. Fabrication of Carboxymethyl Cellulose-Gelatin-ZnO Composite Films*

To prepare carboxymethyl cellulose-gelatin-ZnO (CMC/GEL/ZnO) hydrogel films, the process of solution casting was employed for CMC/GEL (75:25). Briefly, CMC and GEL solutions (2% *w*/*v*) were mixed at 40 ◦C for 4 h along with 1 mL of glutaraldehyde as a crosslinking agent and 0.02% sodium azide to stop the bacterial growth. The CMC/GEL solutions were then slowly mixed with ZnO nanoparticles (1, 1.5, 2, 2.5 *w*/*w*) by sonication for two hours to obtain a clear solution. Then 10 mL of CMC/GEL/ZnO composite solution was poured into Teflon-made boats and dried at 25 ◦C. The films were peeled then off after drying in an oven at 70 ◦C for about twelve hours [21].

#### *2.5. Moisture Contents and Water Vapor Permeability*

To determine the moisture contents (MC), each CMC/GEL/ZnO nanocomposite film was cut into 2.5 × 2.5 cm square sizes and dried in an oven at 70 ◦C for about twelve hours. The difference in final and initial weight of film was noted as a moisture content. Whereas water vapor permeability (*WVP*) was determined gravimetrically. For this purpose, squareshaped pieces (6 × 6 cm) of the film were mounted on top of water vapor permeability measuring cups horizontally, having water (20 mL) and placed in the oven at 50 ◦C. The weight of the cups was noted at regular intervals of 30 min during twelve hours. [22]. After that *WVP* was calculated as:

$$WVP = \frac{(WVTR \times L)}{\Delta p} \tag{1}$$

Here, *WVTR*: rate of water vapor transmission (g/m2. s), *L*: film thickness (m), Δ*p*: partial water vapor pressure differential across the film.

#### *2.6. Antimicrobial Assay*

Six pathogens were tested using neat CMC, GEL, CMC/GEL, and CMC/GEL/ZnO nanocomposite films. Three of which were gram-positive: *Staphylococcus aureus* (ATCC 6538), *Bacillus subtilis* (ATCC 6633), and *Listeria monocytogenes* (ATCC 19111), and three of which were gram-negative: *Enterobacter aerogenase* (ATCC 13048), *Escherichia coli* (ATCC 15224), and *Bordetella bronchiseptica* (ATCC 4617). Briefly, bacterial strains were cultured for about 24 h in agar-agar nutrient broth at 37 ◦C. 1.5 mL of the broth culture of bacterial strains 104–106 (CFU/mL). In Petri dishes that had been sterilized, strains were added to an agar-agar medium and allowed to set at 45 ◦C. The solutions of CMC, GEL, CMC/GEL, and CMC/GEL/ZnO nanocomposite films in DMSO (10 mg/mL) were then added to each well. Each bacterial strain was prepared in triplicate and was incubated for about 24 h at 37 ◦C. By measuring the size of the inhibition zone, antibacterial activity was calculated (mm). Cefixime (1 mg/mL), a standard antibiotic, was used as a positive control [23,24].

#### *2.7. Antioxidant Assays*

The DPPH method was used to measure activity that scavenges free radicals of neat CMC, neat GEL, CMC/GEL, and CMC/GEL/ZnO nanocomposite films [25]. Stock solutions (5 mg/mL) of neat CMC, GEL, and nanocomposite films were made in DMSO. Serial dilutions of 5, 10, 20, 40, 100, and 200 g/mL were performed. In glass vials, 15 μL of each film solution and solution of DPPH (0.1 mM) were mixed together and diluted to 3 mL with methanol. For about 45 min, the reaction mixture was incubated at 37 ◦C in a dark chamber; this caused the DPPH solution's color to change from deep violet to light yellow. At 517 nm, absorbance was noted by using a spectrophotometer. A standard, butylated hydroxyanisole, was used for each experiment, which was carried out in triplicate (BHA, 5 mg/mL). The decreased absorbance of the mixture indicated higher radical inhibition activity. The % age of scavenging activity was calculated as:

% scavenging activity <sup>=</sup>absorbance of control <sup>−</sup> absorbance of test sample absorbance of control <sup>×</sup> <sup>100</sup>

#### **3. Statistical Analysis**

The obtained data were verified statistically by applying ANOVA using Minitab Software. Each experiment was carried out in triplicate. The statistical significance value was tested at *p* < 0.05.

#### **4. Results and Discussion**

#### *4.1. IR Investigation*

The FT-IR absorption spectra of neat carboxymethyl cellulose, gelatin, CMC/GEL/ glutaraldehyde, and CMC/GEL/ZnO nanocomposites are shown in Figure 1. Peak positions and modes of interaction are presented in Table 1. The crosslinking between CMC/GEL and ZnO-Nps was responsible for the majority of the changes in absorption frequencies that were seen. The bands at 2910, 1600, and 1440 cm−<sup>1</sup> in CMC are due to stretching vibrations of aliphatic C-H, (-COO)asy. and (-COO)sym, respectively (Figure 1a). The absorption bands of glucosidic units (C-O-C) appeared at 1160 and 1070 cm−1. A broad absorption band at 3390 cm−<sup>1</sup> was attributed to the hydrogen bonding of OH groups of absorbed water and secondary alcohols (CMC) [26,27]. Absorption bands at 3390 and 3315 cm−<sup>1</sup> correspond to the hydroxyl and amino groups of gelatin film, respectively (Figure 1b). The absorption bands at 1649 and 1551 cm−<sup>1</sup> are due to the (C=O) and (C-N) absorption bands of amide I and amide II in the gelatin structure. The interaction of CMC's anionic groups with thegelatin's cationic groups was confirmed by the shifting of peaks from 3315, 1450 to 3328, 1460 cm−<sup>1</sup> (Figure 1c). The absorption band of the C-H group has been shifted from 2910 to 2955 cm−<sup>1</sup> in CMC/Gel/GTA (Figure 1d). The reaction of GTA with CMC/GEL was confirmed by an absorption band below 1210 cm−<sup>1</sup> forming a hemiacetal structure. Similarly, homogenous mixing of ZnO-Nps with CMC/GEL film (Figure 1e) was confirmed by an absorption peak at 470 cm−<sup>1</sup> [28].

**Figure 1.** FT-IR Spectra of CMC/Gel/ZnO-Nps hydrogel films: (**a**) CMC, (**b**) Gel, (**c**) CMC/GEL, (**d**) CMC/GEL/GTA, (**e**) CMC/GEL/ZnO.


**Table 1.** Peak positions and vibration modes in CMC/GEL/ZnO nanocomposites hydrogels.

#### *4.2. XRD Analysis*

The homogenous mixing of ZnO nanoparticles in the CMC/GEL film was confirmed by the X-ray diffraction pattern, which also quantified the changes from an amorphous to crystalline structure. The XRD pattern of ZnO-Nps (a), CMC/Gel/ZnO (b), CMC/Gelatin (c), CMC (d), and Gelatin (e) is shown in Figure 2. The sharp peaks of the ZnO nanoparticle (Figure 2a) that correspond with the data in JCPDS No. 36-1451, confirmed the wurtzite crystal structure with a hexagonal phase [29]. The diffraction pattern of CMC/GEL/ZnO composite film revealed a characteristic peak of ZnO at 2θ; 32.130, 34.620, 36.460, 46.930, 53.740, 56.460, and 61.350 with low intensity, which indicated the homogenous mixing of ZnO-Nps in CMC/GEL film (Figure 2b). The CMC has an amorphous structure, while gelatin is partially crystalline with peaks at 2θ = 7.6◦ and 22.31◦, Figure 2d,e. In Figure 2c–e, there is no sharp diffraction peak, and an increased diameter of the interreticular triple helix with increased intensity is observed due to the mixing of CMC and gelatin. The interaction between CMC and gelatin has been confirmed by the absence of X-peaks. This phenomenon shows the reduction of hydrogen bonding between the hydroxyls of gelatin and the cellulose groups of CMC. The cross-linking between CMC and gelatin films by GTA exhibited a negligible triple helix approaching 0.8% are in close agreement with the literature [30].

**Figure 2.** XRD pattern of CMC/Gel/ZnO nanocomposites hydrogels.

#### *4.3. Morphological Studies*

All the prepared CMC/gelatin/ZnO nanocomposites were mechanically flexible and self-standing. SEM was used to examine the surface morphology of CMC, gelatin, and ZnO-nanocomposite films and images are shown in Figure 3. The interaction of zinc oxide nanoparticles with CMC and gelatin controls the networked microstructure. Without ZnO nanoparticles, neat CMC, gelatin biopolymer, and CMC/gelatin blend films showed a uniform surface, demonstrating that the film-forming polymers were mostly amorphous and had little crystallization. While the CMC/gelatin film with zinc oxide-Nps additions had a heterogeneous rough surface, with the zinc nanoparticles being uniformly distributed throughout the films and preventing particle separation. It explained that how metal nanoparticles prevented particle aggregation and created high-viscosity CMC/gelatin films, thus a highly stable CMC/gelatin mixture was used [31].

**Figure 3.** Scanning microscopy images of neat CMC, gelatin, CMC/gletan and CMC/gelatin/ ZnO-Nps.

#### *4.4. Optical Properties*

Optical properties of CMC/GEL-based films loaded with various weight ratios (2.5% *w*/*w*) of ZnO-Nps were studied, and data is shown in Table 2 and in Figure 4. Neat gelatin film had two peaks for typical CMC and gelatin peaks at 230–240 nm and 265–280 nm. The nanocomposite films containing ZnO nanoparticles, on the other hand, revealed an additional peak for the ZnO-nps near 370 nm. The peak intensity increased with increasing ZnO nanoparticle contents in CMC/gelatin matrices from 1% to 2.5% [32].

**Table 2.** Optical data of CMC/gelatin/ZnO composite films loaded with different contents of ZnO-Nps.


± standard deviation. (*p* < 0.05).

**Figure 4.** Optical studies of CMC/gelatin/ZnO-Nps.

The L-values of the CMC, gelatin, and CMC/gelatin films exceeded 91, and the a and b parameters ranged from 0.8 to 0.3 and 5.3 to 6.7, respectively, making the films highly transparent. By incorporating ZnO nanoparticles, L and a values were significantly shifted to the lower end, while b values increased. As a result of these changes, the total colour difference between the films increased significantly (Δ*E*) [33]. The Δ*E* was calculated by:

$$\Delta E = \left( (\Delta L)^2 + (\Delta a)^2 + (\Delta b)^2 \right)^{0.5}$$

where Δ*L,* Δ*a*, and Δ*b* are the differences between value of standard colour plate and composites films. In the presence of UV rays, food ingredients oxidise, destroying nutrients and bioactive compounds. Film transparency and UV protection are essential optical properties for applications in food packaging. For the sake of food safety and quality, oxidation reactions that result in toxic substances, off flavours, discoloration, or rancidity are not allowed [34].

Furthermore, the % transmittance of light was measured at *T280* and *T660* nm. At *T660* nm and *T280* nm, the neat CMC and gelatin films had transmittance values of 87.2 ± 0.50, 90.7 ± 0.15, 58.3 ± 1.25, and 30.4 ± 1.50, respectively. When ZnO-Nps was added to CMC/gelatin blend, the percentage transmittance of films at 280 nm was drastically reduced, falling to just 0.3%, whereas the percentage transmittance values at 660 nm only slightly decreased as ZnO-Nps concentrations were increased [35]. The amount of ZnO-Nps present had a significant impact on the transmittance value of the nanocomposite films.

#### *4.5. Thermal Stability*

The TGA technique was used to study the thermal properties of pure biopolymer films as well as films containing ZnO-Nps, and thermograms are shown in Figure 5, which demonstrate the degradation patterns of neat CMC/GEL films and CMC/GEL/ZnO-Nps. All films showed multiple steps of thermal degradation. The first step of degradation started around 95 to 100 ◦C, with a percentage weight loss ranging from 10 to 20% because of the evaporation of loosely bound gases from films. The second stage of degradation of amino groups in CMC/GEL film was observed at 290–360 ◦C with 84% weight loss. The other loss in weight was observed at 415 and 75 ◦C, which were considered the third and fourth steps of degradation in weight, respectively [33]. Compared to neat CMC/GEL films, the degradation curves of prepared nanocomposites films are shifted towards higher temperatures because biopolymers and ZnO-Nps have a strong interaction and are thoroughly mixed [36].

**Figure 5.** TGA of CMC/Gel/ZnO-nanocomposite hydrogels.

#### *4.6. Mechanical Properties*

Tensile strength, percent elongation at break (EB), and elastic modulus, which are key factors in determining the strength and flexibility of the film, were studied as mechanical properties. The effect of ZnO-Nps on EB, tensile strength, and elastic modulus of CMC/GEL films was determined, and the data is presented in Table 3. The addition of ZnO-Nps to the CMC/GEL films has significantly improved the flexibility (EB) and strengthened the CMC/GEL film. Initially, the CMC/GEL (75:25) film had tensile strength and elongation at break values of 39.25 MPa and 4.41 0.23%, respectively. The incorporation of ZnO nanoparticles has increased the elongation at break, and tensile strength values for the CMC/GEL films to 44.6 MPa and 10%, respectively. The increased tensile strength of CMC/gelatin films may be attributed to the incorporation of the proper amount of nanoparticles as well as the bonding between hdroxy groups of gelatin and the hydrophilic groups of CMC, resulting in the formation of a mechanically stable nanocomposite between CMC, gelatin, and zinc oxide nanoparticles [35].


**Table 3.** Mechanical properties and Physical Data of CMC/GEL/ZnO nanocomposite films.

#### *4.7. Moisture Content and Water Vapor Permeability*

The moisture contents (MC) and water vapour permeability (WVP) of neat CMC, neat GEL, CMC/GEL, and MCM/GE/ZnO- films were determined and the data are

presented in Table 1. CMC/GEL films had slightly lower moisture contents and water vapour permeability, whereas CMC/GEL/ZnO-Nps films had higher values and these values increased with higher zinc oxide nanoparticle concentration. ZnO nanoparticles in the CMC/GEL film matrix make the films porous, increasing water vapour permeability.

#### *4.8. Antibacterial Results*

The antimicrobial screening data of CMC, GEL, and ZnO films are presented in Table 4. As per the literature and experiments, the CMC/GEL film had no antibacterial action against both types of tested microorganisms; however, the composite films incorporating ZnO-Nps had marked antibacterial activity. The antimicrobial activity of composites depends on the nature and type of inorganic fillers and the type of bacterial strains used. On both types of tested bacterial strains, the CMC/GEL hydrogel films had a bacteriostatic effect due to the presence of ZnO-Nps, which delayed the growth of the germs because the structure of their cell walls was different. Generally, gram-negative bacteria have a complex cell wall made of a thin layer of peptidoglycan protected by an extra outer membrane, in contrast to gram-positive pathogens that have a thick cell wall with many layers of peptidoglycan. The size and shape of zinc oxide nanoparticles in CMC/GEL/ZnO composite films mainly increased the antibacterial activity of tested bacterial strains. Though the antibacterial mechanism of ZnO is unknown, it is thought that zinc oxide nanoparticles increased the permeability of the membrane around the bacteria, allowing metal nanoparticles to readily pass through the bacterium's cell wall. Various reactive oxygen species (ROS) such as hydroxyl radicals (OH−), hydrogen peroxide (H2O2), superoxide anions (O2−), and organic hydroperoxides are produced inside the cell during this process. When ROS overcomes the cellular antioxidant defense mechanism, oxidative stress occurs, which is linked to damage of several critical macromolecules that ultimately leads to cell death [34].

**Table 4.** Antibacterial activity of CMC/GEL/ZnO-Nps hydrogel films.


± = SD, standard deviation, (*p* < 0.05 vs. control); 5–10 mm zone of inhibition (Activity present): 11–25 mm zone of inhibition (Moderate activity); 26–40 mm zone of inhibition (Strong activity).

#### *4.9. Antioxidant Activity*

The antioxidant activity of neat CMC, GEL, CMC/GEL and CMC/GEL/ZnO nanocomposite films was evaluated by their DPPH (1,1-diphenyl-2-picrylhydrazyl) free radical scavenging activity. The effect of tested nanocomposite materials on generation of free radical of DPPH or radical scavenging activity is measured at 517 nm by decrease in molar absorptivity of DPPH. The degree of discoloration reveals the antioxidant compounds' scavenging capacity in terms of H-donating capacity. Furthermore, the ability of antioxidant compounds to scavenge free radicals depends on their concentration. The radical-scavenging activity rises with antioxidant compound concentration, and a low *IC50* value (i.e., 50% inhibitory concentration) indicates a high antioxidant activity. The obtained results are shown in Table 5. Synthesized composite films containing ZnO-Nps showed free radical

scavenging activity with IC50 in the range of 39 to 85 μg/mL while their respective matrix polymers (CMC/GEL) showed activity more than 100 μg/mL. On comparison of IC50 values of CMC, GEL and their composite films with different wt ratio of ZnO-Nps, it was observed that such CMC/GEL/ZnO-nanocomposites are more active for DPPH activity than their parent polymers [35,36].


**Table 5.** Antioxidant results of CMC/GEL films loaded with different concentration of ZnO-Nps.

± = SD, standard deviation, (*<sup>p</sup>* < 0.05 vs. control); Antioxidant activity: IC50 0–10 mgmL−1, very strongly active; 10–50 mgmL−1, strongly active; 50–100 mgmL−1, moderately active; 100–250 mgmL−1, weakly active; >250 mgmL−1, inactive.

#### **5. Conclusions**

Owing to the versatile nature of biopolymer composites, ZnO nanoparticles have drawn a lot of attention in the field of food packaging. The purpose of this research was to synthesize zinc oxide nanoparticles (<50 nm) by using plant extract as a reducing agent, and to incorporate this in CMC/gelatin matrices to assess the optical, thermal, mechanical and microbial properties. ZnO-based multifunctional CMC/GEL nanocomposites were prepared. ZnO-Nps affected the bio-functional and physical properties of CMC/gelatin composite films. The FTIR and XRD results confirmed the uniform dispersion of ZnO-Nps in the CMC/GEL, matrix to prepare compatible films. The addition of ZnO-Nps (1–2.5 wt. %) to CMC/GEL improved the thermal stability, mechanical properties, moisture contents, and water vapor permeability. CMC/GEL/ZnO-2.5% composite film showed good thermal stability and mechanical properties. CMC/GEL/ZnO 2.5% composite film showed strong antibacterial activity against foodborne pathogenic bacteria, *E. coli*, and *L. monocytogenes* and had high antioxidant activity. CMC-based composite films, such as CMC/GEL/ZnO 2.5%, showed improved thermo-mechanical properties with higher antioxidant and antibacterial activity as compared to CMC/GEL blend films. On the basis of the obtained results, CMC/GEL/ZnO nanocomposite films can be used to prevent photooxidation, ensure food safety, and increase the shelf life of packaged goods in active food packaging applications.

**Author Contributions:** Conceptualization, M.K.K.; methodology, A.Z.; formal analysis, M.K.K.; writing—original draft preparation, A.Z. and M.K.K.; writing—review and editing, A.N. and S.Q. supervision, M.K.K.; Characterization and discussion, M.J.S.; funding acquisition, writing—review and editing, A.N. and S.Q. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the Deanship of Scientific Research, Vice Presidency for Graduate Studies and Scientific Research, King Faisal University, Saudi Arabia (grant no. Grant1974).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The authors declare that data supporting the findings of this study are available within the article entitled: "Carboxymethyl cellulose/gelatin hydrogel films loaded with Zinc Oxide nanoparticles for sustainable food packaging applications".

**Acknowledgments:** This work was supported by the Deanship of Scientific Research, Vice Presidency for Graduate Studies and Scientific Research, King Faisal University, Saudi Arabia (grant no. Grant 1974).

**Conflicts of Interest:** There is no conflict of interest regarding this publication entitled, Carboxymethyl cellulose/gelatin hydrogel films loaded with Zinc Oxide nanoparticles for sustainable food packaging applications.

#### **References**


### *Article* **Clinical Development and Evaluation of a Multi-Component Dissolving Microneedle Patch for Skin Pigmentation Disorders**

**Chenxin Yan 1,2, Mengzhen Xing 3, Suohui Zhang 1,4,\* and Yunhua Gao 1,2,4,\***


**Abstract:** Excessive melanin deposition in the skin leads to various skin pigmentation diseases, such as chloasma and age spots. The deposition is induced by several factors, including tyrosinase activities and ultraviolet-induced oxidative stress. Herein, we propose a multi-component, multi-pathway drug combination, with glabridin, 3-O-ethyl-L-ascorbic acid, and tranexamic acid employed as, respectively, a tyrosinase inhibitor, an antioxidant, and a melanin transmission inhibitor. Considering the poor skin permeability associated with topical application, dissolving microneedles (MNs) prepared with hyaluronic acid/poly(vinyl alcohol)/poly(vinylpyrrolidone) were developed to load the drug combination. The drug-loaded microneedles (DMNs) presented outstanding skin insertion, dissolution, and drug delivery properties. In vitro experiments confirmed that DMNs loaded with active ingredients had significant antioxidant and inhibitory effects on tyrosinase activity. Furthermore, the production of melanin both in melanoma cells (B16-F10) and in zebrafish was directly reduced after using DMNs. Clinical studies demonstrated the DMNs' safety and showed that they have the ability to effectively reduce chloasma and age spots. This study indicated that a complex DMN based on a multifunctional combination is a valuable depigmentation product worthy of clinical application.

**Keywords:** glabridin; 3-O-Ethyl-L-ascorbic acid; tranexamic acid; microneedles; pigmentation; multifunctional drug

#### **1. Introduction**

The normal deposition of melanin in the basal layer of the skin results in the pigmentation of human skin and hair. Aside from conferring protection against ultraviolet (UV) rays, excessive melanin deposition leads to melasma [1] and age spots [2]. Specifically, melasma occurs because of inducing factors such as UV radiation, hormone levels, and family history [1,3]. However, the exact mechanism of age spot formation is currently unclear, and it is widely believed that UV radiation is the main cause of its formation [4].

Melanin production is governed by the rate-limiting enzyme tyrosinase [5]. UV radiation indirectly stimulates tyrosinase, thus increasing melanin production. Recently, several tyrosinase inhibitors intended for skin whitening have been developed, including hydroquinone [6], kojic acid [7], arbutin [8], and aloesin [9]. However, these agents can cause significant side effects, including skin irritation, inflammation, and potential teratogenicity [10]. Furthermore, UV radiation can induce oxidative stress [11], which leads to the formation of oxidants such as nitric oxide [12] and which indirectly stimulates skin pigmentation. Therefore, certain antioxidants can decrease melanin production, such as glutathione [13], ascorbic acid (ASA) [14], and ferulic acid [15]. Recently, researchers

**Citation:** Yan, C.; Xing, M.; Zhang, S.; Gao, Y. Clinical Development and Evaluation of a Multi-Component Dissolving Microneedle Patch for Skin Pigmentation Disorders. *Polymers* **2023**, *15*, 3296. https:// doi.org/10.3390/polym15153296

Academic Editors: Dimitrios Bikiaris and Raffaella Striani

Received: 27 June 2023 Revised: 24 July 2023 Accepted: 28 July 2023 Published: 4 August 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

have combined tyrosinase inhibitors and antioxidants, to treat pigmented lesions. For instance, Yu et al. [16] showed that combining the tyrosinase inhibitor azelaic acid with the antioxidant taurine significantly improved melanin inhibition compared to using azelaic acid alone.

Tranexamic acid (TXA) (seen in Figure 1a), which is a derivative of lysine, was initially proven to be an anti-fibrinolytic agent [17]. It was discovered that TXA has great whitening effects, and it is commonly used to treat melasma through oral administration, injection, or local application [18,19]. However, the systemic administration of TXA usually leads to adverse effects in patients, such as gastrointestinal discomfort and skin rash [20]. Glabridin (GLA) (seen in Figure 1b), which comes from the flavonoid group of compounds in the root of glycyrrhiza glabra, is an excellent tyrosinase inhibitor. It can effectively reduce the transformation of tyrosine to melanin in melanocytes [21–23]. Additionally, 3-O-Ethyl-Lascorbic acid (EAA) (seen in Figure 1c), a derivative of vitamin C (VC), has better stability and hydrophilic–lipophilic balance than VC [24]. Simultaneously, EAA retains the apparent antioxidant effects of VC. The combined use of three effective components is expected to ameliorate stubborn, hard-to-treat skin hyperpigmentation, lengthy treatment, and high recurrence rates. However, because of the protective barrier function of the stratum corneum, drug absorption through the skin is challenging. Therefore, the key to the drug's intended efficacy lies in achieving efficient transdermal drug delivery.

**Figure 1.** The chemical structural formula of (**a**) TXA; (**b**) GLA; and (**c**) EAA.

Microneedles (MNs) are a recent drug delivery innovation [25]. These needle tips, measuring several hundred micrometers in length, can create micrometer-sized pores on the skin, allowing drugs to bypass the skin barrier and to reach the site of the disease. Their small size ensures that they do not come into contact with blood vessels and nerves, which improves patient compliance and comfort, while reducing usage burden. The use of hyaluronic acid (HA) as a matrix for MNs is preferred, due to its excellent biocompatibility. The hyaluronic acid microneedle delivery system has been successfully applied to transport a variety of drugs, including vaccines [26], exenatide [27], proteins [28], and anesthetics [29]. To fabricate microneedle arrays, a mixture of three polymers with biocompatibility and flexibility was used: Polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP) [30], and hyaluronic acid (HA).

In this study, we employed MNs as drug delivery carriers to combine GLA, TXA, and EAA, aiming to synergistically inhibit melanin production from multiple perspectives. Through performing a series of experiments—including in vitro antioxidant assays, tyrosinase inhibition assays, melanin generation inhibition evaluations, zebrafish assays, and clinical trials—we examined the efficacy of the newly developed drug-loaded microneedles (DMNs). Moreover, the effectiveness of DMNs in decolorization was confirmed by in vitro and ex vivo experiments.

#### **2. Materials and Methods**

#### *2.1. Materials*

Tranexamic acid and Glabridin were purchased from Linkebe Technology Co., Ltd. (Hangzhou, China). 3-O-Ethyl-L-ascorbic acid was procured from Dezhou Anglida Biotechnology Co., Ltd. (Dezhou, China). The hyaluronic acid (HA, Mw: 240,000) was procured from Bloomage Biotech (Beijing, China). Poly(vinylpyrrolidone) (PVP) was bought from Boai Nky Pharma Co., Ltd. (Beijing, China). Poly(vinyl alcohol) (PVA) was purchased from Alpha Hi-Tech Pharm Co., Ltd. (Pingxiang, China). The 2-Phenyl-4,4,5,5 tetramethylimidazoline-1-oxyl-3-oxide (PTIO) was bought from Santa Cruz Biotechnology,

Inc. (Santa Cruz, CA, USA), while 1,1-diphenyl-2-picrylhydrazyl (DPPH) was bought from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China), and L-DOPA from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco's Modified Eagle's Medium (DMEM low glucose), RPMI 1640 medium, fetal bovine serum (FBS), phosphate-buffered saline (pH = 7.0–7.4), penicillin–streptomycin (P/S), and 0.25% trypsin (1×) were procured from Gibco (Rockville, MD, USA). The Cell Counting Kit-8 assay kit was provided by Dojindo (Kumamoto Prefecture, Japan). Non-tissue/cell lysate was ordered from Solarbio Tech Co., Ltd. (Beijing, China), while RIPA buffer was bought from Beyotime Biotechnology (Shanghai, China). Sodium hydroxide and dimethyl sulfoxide were obtained from InnoChem (Beijing, China) and Beijing Chemical Works (Beijing, China), respectively. PBS (50 mM, pH = 6.8) was purchased from Regen Biotechnology Co., Ltd. (Beijing, China).

Sprague Dawley (SD) rats (male, 8 weeks old, 220 ± 20 g) were obtained from SPF Biotech Co., Ltd. (Beijing, China). Procedures for animal studies were approved by the Institutional Animal Care and Utilisation Committee of the Technical Institute of Physics and Chemistry, CAS (approval number, LHDW-23025 and IACUCIPC-23025). The animal experiments followed the Guide for the Care and Use of Laboratory Animals (Eighth Edition, 2011). B16-F10 cells were purchased from Shanghai Enzyme Research Biotechnology Co., Ltd. (Shanghai, China). The wild-type AB strain of zebrafish was obtained from China Zebrafish Resource Center (Wuhan, China), and their breeding and propagation followed the requirements of the international AAALAC certification (certification number: 001458). The animal experimentation procedures were executed according to The Standard Operating Procedures for Evaluating the Whitening Efficacy of Zebrafish.

#### *2.2. Volunteers*

For the safety evaluation of DMNs, we recruited 30 participants, comprising 6 males and 24 females, aged between 20 and 58 years old. To assess the efficacy of DMNs in reducing facial pigmentation, 6 eligible female participants, aged from 18 to 60 years old, with noticeable facial pigmentation were chosen based on a predetermined set of criteria. The recruitment of volunteers was carried out following the medical and ethical principles outlined in the Helsinki Declaration. Before participation, all volunteers had to provide their informed consent to be involved in the study by signing a written consent form. All aspects of the study, including but not limited to conducting the experiment, analyzing data, and compiling the report, adhered to the principles of Good Clinical Practice.

#### *2.3. Preparation of DMNs*

HA, PVA, and PVP were chosen as the primary matrix for MNs fabrication due to their outstanding biocompatibility [31]. When preparing the drug-containing solution, 3% TXA, 0.2% GLA, and 2% EAA were dissolved in deionized water. The pH of the solution was then adjusted to 5.5–6 and filtered through a 220 nm filter. PVA was dissolved in deionized water at 80 °C while HA and PVP were dissolved at room temperature. The three polymers were then mixed to form a homogeneous solution containing 4% PVA, 3% HA, and 1% PVP. After sterilizing the high-molecular-weight mixed solution with high pressure, it was combined with the drug-containing solution to obtain the DMN solution. The DMN solution was then applied onto a polydimethylsiloxane (PDMS) mold using vacuum suction to fill the holes with the solution. Finally, the DMNs were left to dry for more than 5 h in an environment with a relative humidity of not more than 30% (Figure 2).

**Figure 2.** The manufacturing process of DMNs.

#### *2.4. Characterizations of DMNs*

DMNs were fabricated by employing PDMS molds that were designed to have a needle height of 230 μm. We then evaluated the DMNs' morphology and actual height by utilizing fluorescence microscopy (BX51, Olympus, Tokyo, Japan). Additionally, the dissolution characteristics of DMNs were investigated using detached piglet pig skin. DMNs were affixed to a hydrogel backing, and a microneedle applicator (20 N/cm2) was employed to exert pressure for 20 s, followed by a 40 min waiting period before the removal of DMNs. Finally, the needle height of the remaining DMNs was analyzed. According to a previous study [32], Parafilm sealing film was employed to evaluate the penetration performance of DMNs. DMNs were applied with a pressure of 20 N/cm2 for 20 s on an 8-layer sealing film. The DMNs were then removed to observe the puncture holes and maximum puncture depth formed on the sealing film. To further validate the puncture performance of DMNs, a needle injector (20 N/cm2) was used to facilitate the insertion of DMNs into excised porcine skin. Subsequently, the DMNs were removed and stained with a 4 mg/mL trypan blue dye solution for 30 min. Following the staining process, the dye was wiped off, and photographs were taken to document the array of needle punctures. Additionally, DMNs were also applied on the skin of SD rats, and the array formed on the rat's skin was recorded.

#### *2.5. The PTIO Antioxidant Activity of DMNs*

The PTIO antioxidant test was adapted from a study by Li et al. [33]. Firstly, dissolve a single piece of DMN dry film in 1 mL of PBS buffer solution to obtain a 100% active solution. Next, prepare a solution with a concentration of 0.5 mg/mL each for PTIO and ASA. Distribute 1, 5, and 20 μL of the 100% active solution to the wells of a 96-well plate, adding PBS to all wells with less than 20 μL and setting up three replicates for each group. Use PBS as a blank control and ASA as a positive control. Finally, introduce 80 μL of PTIO solution into each well and incubate the plate in an incubator set at a constant temperature of 37 °C for 2 h. Next, complete processing by measuring absorbance at 570 nm using an enzyme-linked immunosorbent assay (ELISA) reader (Epoch2, BioTek). Compute the PTIO• scavenging rate using the formula:

$$\text{PTIO} \bullet \text{scavenging rate} \%= (A\_0 - A) \times 100\% / A\_0 \tag{1}$$

where *A* and *A*<sup>0</sup> represent the absorbance values of the sample group and the control group, respectively.

#### *2.6. DPPH• Scavenging Efficiency of DMNs*

Antioxidant assays were performed according to the procedure described by Kim et al. [34], with appropriate modifications. A 100% DMN water solution was prepared as outlined in Section 2.5 and then diluted into concentrations of 40% and 10% with PBS solutions. Subsequently, 2 mg of DPPH powder was weighed and added to a 5 mL centrifuge tube, followed by the addition of 2 mL anhydrous ethanol to make a 1 mg/mL solution. The solution was sonicated in the dark for 5 min, shaken in the dark for 10 min, and then left undisturbed in the dark for 30 min until it was stable. The solution was further diluted to a concentration of 0.1 mg/mL. Positive control ASA was prepared in water to a concentration of 0.2 mg/mL. A 96-well plate was arranged with 100 μL of the different concentrations of the test solution, the positive control, and the negative control in each well. Each group was replicated three times. Then, 100 μL of DPPH solution was applied to each well. The plate was kept in the dark while it was agitated for 30 min. After this time, the absorbance was measured at 517 nm using an ELISA reader. The scavenging effect of DPPH radicals was measured using the formula:

$$\text{DPPH} \bullet \text{clearance rate } \%= (A\_0 - A) \times 100\% / A\_0 \tag{2}$$

where *A* and *A*<sup>0</sup> represent the absorbance values of the sample group and the control group, respectively.

#### *2.7. Safety on B16-F10 Cells*

First, prepare the DMN extract. Sterilize the dry film with UV light for 24 h, then add a low-sugar DMEM culture medium in a ratio of one sheet per milliliter to obtain 100% extract. Next, prepare four concentration gradients of extract at 50%, 20%, 5%, and 1%. Seed B16-F10 cells in 96-well plates, with each well containing 8000 cells, and incubate for 24 h at 37 °C, 5% CO2, and saturated humidity in a culture box. Then, add 100 μL of different concentrations of DMN extract to each well, with four parallel groups set for each concentration and blank controls established at the same time. After administration, continue incubating for 24 h. After processing, wash the cells with PBS and add 100 μL of 10% Cell Counting Kit-8 (CCK-8) solution to each well, then continue incubation for 50 min. Finally, use an ELISA reader to measure the absorbance at 450 nm, and calculate cell vitality using the following formula:

$$\text{Cell viability }\%= (A\_0/A) \times 100\% \tag{3}$$

where *A* and *A*<sup>0</sup> represent the absorbance values of the sample group and the control group, respectively.

#### *2.8. Effects of Cellular Tyrosinase Activity*

The method developed by No et al. [35] was modified. Firstly, B16-F10 cells were seeded into 96-well plates at a density of 8000 cells/well and cultured for 48 h. The 100% concentration extraction solution was prepared following the method from Section 2.7. Then, it was diluted into three concentration gradients of 20%, 5%, and 1%. The test solution was added after 48 h, and each group was set up with 4 parallel experiments and a blank control. The cells were further incubated for 24 h after drug administration. After treatment, the cells were washed with PBS, and then 40 μL of cell lysis buffer containing 1 mM Phenylmethanesulfonyl fluoride was added to each well. The plate was incubated at 4 °C for 30 min, followed by incubation at 37 °C for 5 min. Subsequently, 100 μL of 1 mg/mL L-DOPA solution was added to each well and incubated in a 37 °C incubator for 2 h. After incubation, the activity of cellular tyrosinase was measured by obtaining the absorbance at 475 nm using an ELISA reader according to the following formula:

$$\text{Tyrosinase activity }\%= (A\_0/A) \times 100\% \tag{4}$$

where *A* and *A*<sup>0</sup> represent the absorbance values of the sample group and the control group, respectively.

#### *2.9. Melanin Measurement*

According to the methods described in a previous study [36], with appropriate modifications, B16-F10 cells were seeded at a density of 200,000 cells per well in a 6-well plate and incubated for 48 h. The extraction method for the solution was performed as described in Section 2.8. Following the incubation period, each well was treated with 2 mL of extraction solution at varying concentrations with a blank control and further incubated for 48 h. The cells were processed by washing with PBS twice and treated with 500 μL of cell lysis solution in each well, followed by incubation at room temperature for two hours for lysis. The melanin component was collected in a 1.5 mL centrifuge tube and centrifuged at a speed of 8000 rpm for 5 min. Then, the supernatant was discarded, and 200 μL of 1 M NaOH solution containing 10% DMSO was added to the samples. The samples were incubated in a 70–80 °C water bath for 4 h to dissolve the melanin. The solution was placed

in a 96-well plate and the absorbance was measured at 405 nm using an ELISA reader. The melanin contents are calculated as follows:

$$\text{Melanin' contents }\%= (A\_0/A) \times 100\% \tag{5}$$

where *A* and *A*<sup>0</sup> represent the absorbance values of the sample group and the control group, respectively.

#### *2.10. Zebrafish Experiment*

Fifteen zebrafish from the wild-type AB strain, exhibiting typical developmental characteristics after six hours of fertilization, were selected and assigned to each well of a six-well plate. Caution should be exercised while removing deionized water from the wells to avoid causing harm to the embryos. Following this, 3 mL of 0.5% DMN solution was rapidly added to each well, and a negative control group (adding 3 mL of deionized water) was established. The plate was wrapped in aluminum foil and incubated in a biochemical incubator at 28.5 °C for 45 h. Subsequently, ten zebrafish were randomly selected from each group for photography and observation. ImageJ software (version 1.53t) was employed to analyze the strength of the melanin signal present in their heads. Whitening efficacy was calculated using the formula below:

$$\text{Whitering effect} \,\%= (\text{S}\_0 - \text{S}) \times 100\% / \text{S}\_0 \tag{6}$$

where *S* and *S*<sup>0</sup> represent the signal intensity of melanin in the head of the zebrafish in the sample group and the control group, respectively.

#### *2.11. Clinical Research in DMNs*

We recruited a total of 30 participants, consisting of 6 males and 24 females, with ages ranging from 20 to 58 years. Each participant applied DMNs for a duration of 24 h. At the conclusion of the application, the DMNs were removed, and the participants' skin conditions were observed at 0.5, 24, and 48 h.

To study the clinical whitening efficacy of DMNs, six subjects were selected, two with facial freckles, three with melasma, and one with age spots. Prior to starting the trial, the 6 participants were required to undergo a standardized cleansing process. Following the cleansing process, the participants were instructed to sit in a room with standard conditions (temperature: 20–24 °C, humidity: 40–60%) for a minimum of 30 min. This was to ensure that the skin could naturally dry. Subsequently, a DMN patch was placed on the area of pigmentation beneath the right eye and left in place for one full night, a minimum of 8 h, before being removed. The DMN patch was applied every 48 h, totaling 28 applications. The use of any other products (such as cosmetics or topical medications) in the treated area was avoide during DMN application. The participants underwent facial imaging using a VISIA (a commercial skin imaging analyzer) before the trial and at weeks 2 (W2), 4 (W4), 6 (W6), and 8 (W8) to obtain pre- and post-treatment comparative data. The measured data comprised the water content of the stratum corneum at the application site, values of trans-epidermal water loss (TEWL), melanin, hematochrome, individual type angle (ITA), and a photograph of the subject's facial area at the site of pigmentation. Tests on the same subject were completed by the same measurer.

#### *2.12. Statistical Analysis*

The quantitative data were analyzed through the IBM SPSS Statistics application, version 23.0, provided by IBM SPSS Inc. located in Chicago, IL, USA. Paired t-tests were used to analyze statistical differences in data that followed a normal distribution, either for within-group comparisons or for comparisons between the experimental and control groups. Significance levels of *p* < 0.05, *p* < 0.01, and *p* < 0.001 are indicated by asterisks: \*, \*\* , and \*\*\*, respectively.

#### **3. Results and Discussion**

#### *3.1. Preparation and Characterization of DMNs*

DMNs were manufactured in a clean room with a classification of ten thousand and packed with an aluminum–plastic foam cover, as illustrated in Figure 3a. A fluorescence microscope (BX51, Olympus, Tokyo, Japan) was used to capture images and observations, revealing that DMNs possess a complete needle tip structure. The needle height of the DMNs measures approximately 224.4 ± 4.4 μm (Figure 3b). In addition, after 40 min of skin (detached pigskin) treatment, the DMNs almost completely dissolved (Figure 3c). This indicates that DMNs have good dissolution ability, which can promote the rapid penetration of drugs into the skin. We conducted the puncture experiment for the DMNs using a sealant film with an average thickness of 126 μm per layer [32]. As illustrated in Figure 4a, the DMNs created a needle hole array on the first and second layers of the sealant film, but not on the third and fourth layers. Moreover, DMNs have the capability to create distinct arrays of needle punctures on both isolated pig skin and SD rat skin as illustrated in Figure 4b,c. These findings suggest that DMNs perform well in puncture tests.

**Figure 3.** (**a**) DMNs prepared in a tens of thousands level clean room and packaged in aluminum and plastic; (**b**) DMN image captured after fabrication using a fluorescence microscope; (**c**) the needle tip that remains after inserting into pig skin and being dissolved in DMNs for 40 min.

**Figure 4.** (**a**) Results of puncturing 1–4 layers of sealing film; the needle hole array obtained by applying DMNs to (**b**) pig skin and (**c**) the SD rat.

The height of the MN tips ranges between 50 and 900 μm, allowing the MN to penetrate the stratum corneum [37]. However, an excessive needle height can cause skin damage. Xing et al. [38] assessed the impact of three various heights of MNs (230 μm, 500 μm, and 700 μm) on penetration depth and skin recovery. Among them, the depth of penetration of the 230 μm MNs into the skin was 85 ± 12 μm, and the skin could recover within 30 min. The stratum corneum has a thickness of approximately 10–20 μm. Consequently, a 230 μm needle can pierce through the stratum corneum, reaching the epidermis or shallow dermis, resulting in swiftly healing small wounds on the skin. As a result, we have selected a 230 μm needle height for the DMNs. Simultaneously, we selected HA, PVP, and PVA as matrix materials for the preparation of MNs. HA can hydrate and quickly absorb moisture, thereby accelerating the dissolution process of the DMNs [39]. The basal part of the DMNs, which is not inserted into the skin, will gradually dissolve and penetrate the skin through the pores created by the DMNs. Additionally, we utilized a PVA/PVP hybrid form to

effectively address the lack of mechanical strength in PVA when used alone. PVA is widely used due to its excellent biocompatibility and ability to form films, yet its inability to provide sufficient mechanical strength can be solved by mixing with PVP [40].

#### *3.2. Antioxidant Results of DMNs*

We conducted two studies to assess the antioxidant capacity of the combination using the DPPH assay [41] and PTIO radical scavenging assay [42]. Results from the PTIO experiment indicate that the 5% DMN extract possesses modest antioxidant activity, with a PTIO• scavenging rate of approximately 30%, while the scavenging rate of the 20% extract surpasses that of the positive control (0.1 mg/mL ASA). Furthermore, the combination's free radical scavenging efficacy positively correlates with the extract concentration (Figure 5a). Additionally, DMNs exhibit superior DPPH• clearing properties, with a 50% clearance rate at a 5% concentration and a higher clearance efficiency at a 20% concentration compared to the positive control (Figure 5b). Oxidative stress reactions arise from reactive oxygen species (ROS) accumulation [43] and may cause acquired melanin pigmentation. This can be countered by activating the cell's antioxidant defense system [44] or by introducing antioxidants [45,46]. Therefore, we introduced the VC derivative EAA to the combination, and the experimental results confirm that the inclusion of EAA enhances the combination's antioxidant effects.

**Figure 5.** (**a**) PTIO• scavenging rate and (**b**) DPPH• scavenging rate of different concentrations of DMNs and positive control group (0.1 mg/mL ASA). Significance levels of *p* < 0.01, and *p* < 0.001 are indicated by asterisks: \*\*, and \*\*\*, respectively.

#### *3.3. Cytotoxicity Results of DMNs*

The objective of this section is to evaluate the potential cytotoxicity of the DMN extract. We exposed the B16-F10 cells to different concentrations of DMN extract solution for 24 h. Then, we performed CCK-8 experiments to analyze their individual cytotoxic activities. The findings indicated that concentrations of 5% and below did not exhibit any cytotoxicity. Similarly, cellular viability at 20% concentration was approximately 75%, indicating non-cytotoxicity. In contrast, the 50% concentration showed higher cytotoxicity, as shown in Figure 6. This experiment provides a basis for our subsequent experiments on anti-melanogenesis and cellular tyrosinase activity.

**Figure 6.** Cell viability of B16-F10 melanoma cells. Cells (8000) were seeded for 24 h, followed by treatment with different concentrations (1%, 5%, 20%, or 50%) of DMN extract for another 24 h. Untreated cells were used as controls.

#### *3.4. Effect of DMNs on The Activity of Tyrosinase in B16-F10 Cells*

Tyrosinase is a crucial enzyme in the melanogenesis process [47]. To enhance the melanin inhibitory effect of the combination, we supplemented the tyrosinase inhibitor GLA. The tyrosinase inhibition effect of the combination was tested with B16-F10 cells. The outcomes demonstrated that at 1% concentration, about 42% of the enzyme activity was inhibited; at 5% concentration, around 74% was inhibited; and the inhibition rate at 20% concentration was over 80% (Figure 7a). It can be observed that the inhibition ability of the combination exhibited a direct correlation with the concentration. As a result, these findings suggest that DMNs have a greater tyrosinase inhibition efficiency.

**Figure 7.** (**a**) Detection of tyrosinase activity in B16-F10 cells using L-DOPA. Cells were treated with various concentrations (1%, 5%, or 20%) of DMNs for 24 h. Controls were untreated. (**b**) Melanin contents of B16-F10 cells. Cells were treated with various concentrations of DMNs for 24 h. Controls were untreated. Significance levels of *p* < 0.01, and *p* < 0.001 are indicated by asterisks: \*\*, and \*\*\*, respectively.

#### *3.5. Effect of DMNs on Melanin Production in B16-F10 Cells*

For better visualization of the DMNs' depigmentation effect, we quantitatively analyzed melanin using B16-F10 cells. As shown in Figure 7b, the DMNs' effect on cellular melanogenesis was dose-dependent, inhibiting at rates of 86.43%, 95.35%, and 95.62% at concentrations of 1%, 5%, and 20% respectively. Previous tyrosinase experiments revealed that the tyrosinase inhibition rate for a 1% DMN extract was approximately 42%, whereas the melanin inhibition rate was 86%. Therefore, the combination of EAA, a potent antioxidant, and TXA, a melanin transfer inhibiting component, work synergistically with GLA to enhance its decolorization ability.

#### *3.6. Effect of DMNs on Melanin Pigmentation of Zebrafish Embryos*

Considering the prolonged duration and intricate procedures associated with guinea pig experiments, we therefore chose zebrafish as an animal model to evaluate the discoloration capacity of DMNs [48,49].

Ten zebrafish were selected randomly for each group to observe the back discoloration. The chromatophores on the backs of treated zebrafish were lighter than those in the control group (Figure 8a,b). Next, we used ImageJ software to evaluate the melanin on the backs of both groups of zebrafish quantitatively, and the decolorization rate in the treatment group rose to 32%, as shown in Figure 9a. The qualitative and quantitative results of the zebrafish experiment preliminarily verified that DMNs have decolorization potential. However, the depigmentation effect of the drug combination on zebrafish is relatively poor compared to its effects on B16-F10 cells. One possible reason is that the GLA in the combination does not exert a depigmentation effect on zebrafish and has a certain teratogenicity on zebrafish embryos [23]. The toxicity of GLA reduces the overall drug concentration of the combination. However, DMNs can achieve a depigmentation rate of 32% even at low administration concentrations and in the absence of GLA's effectiveness, indicating DMNs' whitening potential. In Figure 9b, the red dashed box delineates the region where we employed ImageJ software to quantify the concentration of melanin in the head of the zebrafish.

**Figure 8.** Representative photographs of zebrafish embryos at 51 h post fertilization (hpf). Embryos were treated with (**a**) 0.5% DMNs from 6 to 51 hpf; (**b**) Controls were untreated.

**Figure 9.** (**a**) Whitening efficacy evaluation of 0.5% DMNs and blank control. All groups were quantitatively analyzed for zebrafish head melanin using ImageJ software, and the results are expressed as mean ± SD (n = 10). (**b**) The melanin content is determined by analyzing the region enclosed by the red dashed box. The significance level of *p* < 0.001 is indicated by asterisk \*\*\*.

#### *3.7. The Clinical Trial of DMNs*

Thirty volunteers were recruited to assess the irritability of DMNs. Low-sensitivity adhesive tape was utilized to apply DMNs to the participants' backs for 24 h, followed by removal. The participants' skin reactions were observed at 0.5 h, 24 h, and 48 h, respectively. The results were recorded based on the skin reaction grading standards outlined in the Cosmetic Safety Technical Specifications (2015 version). The results indicated no occurrence of adverse reactions among these 30 volunteers, thus confirming the safety of DMN usage.

Due to its assured safety, we enlisted various volunteers exhibiting visible freckles, melasma, and age spots on their faces for an eight-week efficacy test of DMNs for skin whitening. During the procedure, there were no noticeable changes observed in the moisture content of the stratum corneum and the TEWL of all individuals (Figure 10a,b). This suggests that DMN application does not result in moisture loss in the stratum corneum. The primary component of DMNs, HA, exhibits potent hydrating properties and is extensively utilized in cosmetics to improve skin moisture retention [50], which might be the reason for there being no observed loss of moisture. The skin's melanin and red pigment at the site of drug administration both decreased. Melanin decreased by 10.49% and red pigment decreased by 14.40% (Figure 10c,d). ITA (ITA is used to represent the chromatic individual type angle, with a higher value indicating a brighter skin color) value slightly increased by approximately 5.97% (Figure 10e), signifying that DMNs can reasonably lighten spots and whiten skin. We captured the participants' facial images using VISIA (a commercial skin imaging analyzer) every four weeks. Patients with age spots exhibited a reduction in pigmentation in the brown and red regions (Figure 11a), while patients with melasma showed slight a reduction in the pigment in their faces (Figure 11b), but it was less than that of age spots. Nonetheless, the treatment effect of DMNs on freckles was not significant (Figure 11c). The reason for the different efficacies of DMNs on melasma, age spots, and freckles may be due to the reality that freckles are primarily determined by genetic and congenital factors, whereas melasma and age spots are primarily caused by postnatal factors such as UV exposure and hormone levels [51]. Meanwhile, most drugs are more effective in treating postnatal diseases rather than congenital diseases. In conclusion, clinical trials have validated the safety of DMNs for topical application and have shown that it does not cause any loss of skin moisture. Moreover, DMNs exhibit selective efficacy in treating both age spots and melasma.

**Figure 10.** All subjects were tested at 0 W, 2 W, 4 W, 6 W, and 8 W for (**a**) skin stratum corneum water content, (**b**) TEWL, (**c**) skin melanin, (**d**) skin hematochrome, and (**e**) ITA.

**Figure 11.** Subjects with (**a**) age spots, (**b**) melasma, and (**c**) freckles on the face were taken at 0 W, 4 W, and 8 W using VISIA (a commercial skin imaging analyzer) to capture facial images. The dashed box highlights the area of pigment reduction.

#### **4. Conclusions**

UV radiation triggers oxidative stress, which leads to an increase in intracellular ROS levels in human epidermal melanocytes [52]. Additionally, UV exposure increases the activity of intracellular tyrosinase, accelerating melanin production in the epidermis [53]. In this study, a polymer matrix containing GLA, EAA, and TXA was loaded onto MNs, and the antioxidant effects of DMNs were confirmed. DMNs inhibit intracellular tyrosinase and melanin production, as demonstrated in B16-F10 cells and zebrafish models. DMNs were found safe in clinical trials and selectively lightened chloasma and age spots. Future research will determine the optimal ratios of the three-drug components. In conclusion, the DMNs, which consist of various components and exhibit multiple effects, have the potential for whitening.

**Author Contributions:** Conceptualization, Y.G. and S.Z.; Data curation, C.Y. and M.X.; Supervision, S.Z.; Writing—original draft, C.Y.; Writing—review and editing, C.Y., M.X. and Y.G. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Director's Fund of the Technical Institute of Physics and Chemistry, Chinese Academy of Sciences (grant number: E0AK076M12).

**Institutional Review Board Statement:** The clinical safety test was supported by Suzhou Guochen Bio-tech Co., Ltd., on human subjects. Procedures for animal studies were approved by the Institutional Animal Care and Utilisation Committee of the Technical Institute of Physics and Chemistry, CAS (approval number, LHDW-23025, and IACUCIPC-23025). The animal experiments followed the Guide for the Care and Use of Laboratory Animals (Eighth Edition, 2011). The human efficacy evaluation experiment of DMNs was carried out at the Cosmetics Testing Center under the Medical Biotechnology Research and Development Center at Jinan University in Guangzhou. All clinical studies conducted adhered to the Helsinki Declaration guidelines. However, an ethics review was not carried out at the Jinan University Testing Center due to the small sample size. Additionally, Guochen Bio-tech Co., Ltd., could not provide an ethics number as a result of the confidentiality of their data.

**Data Availability Statement:** Data are available on request due to restrictions, e.g., privacy or ethics; the data presented in this study are available on request from the corresponding author.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **Abbreviations**

MNs, microneedles; DMNs, drug-loaded microneedles; ASA, ascorbic acid; TXA, Tranexamic acid; GLA, Glabridin; EAA, 3-O-Ethyl-L-ascorbic acid; VC, vitamin C; HA, hyaluronic acid; PVA, Poly(vinyl alcohol); PVP, poly(vinylpyrrolidone); PDMS, polydimethylsiloxane; ELISA, enzymelinked immunosorbent assay; CCK-8, Cell Counting Kit-8; TEWL, trans-epidermal water loss; ITA, individual type angle; ROS, reactive oxygen species; PTIO, 2-Phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide; DPPH, 1,1-diphenyl-2-picrylhydrazyl.

#### **References**


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### *Review* **Collagen Derived from Fish Industry Waste: Progresses and Challenges**

**Zahra Rajabimashhadi 1, Nunzia Gallo 1, Luca Salvatore <sup>2</sup> and Francesca Lionetto 1,\***


**Abstract:** Fish collagen garnered significant academic and commercial focus in the last decades featuring prospective applications in a variety of health-related industries, including food, medicine, pharmaceutics, and cosmetics. Due to its distinct advantages over mammalian-based collagen, including the reduced zoonosis transmission risk, the absence of cultural-religious limitations, the costeffectiveness of manufacturing process, and its superior bioavailability, the use of collagen derived from fish wastes (i.e., skin, scales) quickly expanded. Moreover, by-products are low cost and the need to minimize fish industry waste's environmental impact paved the way for the use of discards in the development of collagen-based products with remarkable added value. This review summarizes the recent advances in the valorization of fish industry wastes for the extraction of collagen used in several applications. Issues related to processing and characterization of collagen were presented. Moreover, an overview of the most relevant applications in food industry, nutraceutical, cosmetics, tissue engineering, and food packaging of the last three years was introduced. Lastly, the fishcollagen market and the open technological challenges to a reliable recovery and exploitation of this biopolymer were discussed.

**Keywords:** fish collagen; fish industry waste; collagen extraction; nano collagen; sustainability

#### **1. Introduction**

In order to exploit natural resources as much as possible, a long-term plan titled "Blue Growth" was approved by the European Commission and has been implemented to pay particular attention to fish resources in order to preserve the environment from industrial pollution. The enormous amount of valuable protein that could be extracted [1–5] (about 30–40% of the total weight), is one of the most appealing aspects of seafood by-products. More than 20 million tons of them are produced annually from the fish tissues that are discarded as waste, including fins, heads, skin, and viscera [6–8]. Because of their elevated protein content, absence of disease transmission risks, high bioactivity, and less considerable religious and ethical restrictions, the use of fish by-products as a new source of collagen has drawn increasing attention [9–11].

The importance of both aquaculture and fishing to food security is expanding continuously, particularly in light of the rising global fish production and the United Nations' 2030 program of sustainable development [12]. Approximately 70% of fish and other seafood are processed before being sold, resulting in enormous amounts of solid waste from processes such as beheading, de-shelling, degutting, separating fin and scales, and filleting [13,14]. More than half of the weight of fresh fish becomes by-products of the fish industry. Most of these by-products are buried or burned, causing environmental, health, and economic issues. A minor portion are employed as inexpensive ingredients in animal feeds. Fish waste is a rising problem that requires quick, creative methods and solutions. Numerous initiatives and programs have been performed globally to prevent food waste. In addition to reducing the cost of waste disposal, investing in waste from the fish industry can offer

**Citation:** Rajabimashhadi, Z.; Gallo, N.; Salvatore, L.; Lionetto, F. Collagen Derived from Fish Industry Waste: Progresses and Challenges. *Polymers* **2023**, *15*, 544. https://doi.org/ 10.3390/polym15030544

Academic Editor: George Z. Papageorgiou

Received: 15 November 2022 Revised: 11 January 2023 Accepted: 13 January 2023 Published: 20 January 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

the opportunity to recover other important substances such as oils, proteins, pigments, bioactive peptides, amino acids, collagen, chitin, gelatin, etc. [15–17].

More than two decades ago, research on the extraction of collagen from fish waste started to be conducted. Collagens are one of the most abundant proteins in animals, which are found in the extracellular matrix of connective tissues, including skin, bones, tendons, ligaments, cartilage, intervertebral discs, and blood vessels [18]. Collagens are not only implicated in tissue architecture maintenance and strength, but they also cover regulatory roles (i.e., through mechano-chemical transduction mechanisms) during tissue growth and repair [19,20]. Thanks to their nature, collagens are intrinsically bioactive, biocompatible, and biodegradable [21]. Hence, collagens are valued as the most commonly required and used biomaterials in many fields, including medical, cosmetic, nutraceutical, food and pharmaceutical industries in the forms of injectable solutions, thin substrates, porous sponges, nanofibrous matrices, and micro- and nano-spheres [22–25]. Recent studies revealed many similarities in the molecular structure and biochemical properties between collagen derived from fish and mammalian sources, despite the fact that fish collagen typically has a lower molecular weight and lower denaturation temperature than mammalian collagen [8,12,20,22,24,26–28]. Various extraction techniques for fish collagen have been developed depending on the selected tissue type and fish species. Hence, a considerable collection of literature has been developed on this subject [29–31]. Only in the past five years have researchers concentrated on innovative materials with improved characteristics in addition to developing extraction techniques for mass manufacture.

Collagen nanotechnology has a bright outlook because science in this area is always progressing and will continue to do so in the future. Nano collagen is ordinary collagen that has been sized down to a nanometer scale [32,33]. According to its nano-scale-based technology, which offers a high surface-area-to-volume ratio, an optimal penetration into wound sites and higher cell interaction is enabled. [34]. Moreover, nano collagen has the ability to deliver drugs and to supply a durable microenvironment at wounded sites to promote cellular regrowth and healing [35]. Collagen nanotechnology still presents many shortcomings, including the fact that only a small minority of therapeutic compounds have received commercial approval and that there are still numerous unsolved problems [32]. The complexity of pathophysiological symptoms and the lack of data on its real physiological effects is a further challenge for nanotechnology. Despite these downsides, nanotechnology is still a growing trend, with a huge amount of unrealized potential. This gives rise to the expectation that further research will assist in minimizing these downsides, leading to the creation of secure and efficient nano-based systems. In order to create approved therapeutic agents that take advantage of nanotechnology, additional research and studies must be performed [32]. Indeed, Figure 1 reveals the continuous increasing research interest on collagen, fish collagen, and nano collagen investigation in the last twenty years. In particular, it appears clear that there has been a significant increase in scientific works in the last five years. Nano collagen can be used for a variety of improvements and treatments, such as bone grafting, drug delivery, nerve tissue formation, vascular grafting, articular cartilage regeneration, cosmetics, and wound healing [21,22,36]. It is clear that nano collagen is a progressed type of nanotechnology; thus, further investigation must be attempted to advance this technology with the expectation that, in the future, nano collagen scaffolds will be more widely available [37].

This review aims to provide an overview of recent investigations into fish collagen, with a particular focus on its characteristics, types, and extraction methods, and, finally, on the valuation of fish industry waste for the preparation of biopolymers for various applications areas. Among others, fish-collagen application in the medical, pharmaceuticals, food, and cosmetic sectors are discussed.

**Figure 1.** Increasing research interest in fish collagen (MC) and nano collagen (NC) compared with collagen (C), according to scientific papers analyzed by publication year in the last twenty years up to 2022 (from Scopus database: www.scopus.com, accessed on 15 September 2022).

#### **2. Collagen: Structure and Properties**

Collagens represent about 30% of a mammal's weight [18,38]. Based on the historical order of their discovery, 28 types of collagens—type I through type XXVIII—have been identified and described up to the current day [39]. The oldest collagen identified to date was found in the soft tissue of a fossilized *Tyrannosaurus rex* bone that dates back 68 million years [40,41].

The molecular organization of collagens is highly variable, notwithstanding their general triple-helical structure and the triplet (Gly-X-Y)n repetition, where X and Y can be any amino acid, although proline and hydroxyproline are the most frequent occupants of these locations (Figure 2) [42,43]. Collagen's unit is composed of three α-chains, the amino acid composition of which varies among collagen types. Furthermore, function and distribution in tissues play a role in the diversity of collagen, as well as molecular and supramolecular organization, such as occurrence and length of triple helical domains, packing of the triple helices, etc. [27,44].

**Figure 2.** Exemplary amino acid repetition of the triplet (Gly-X-Y)n characteristic of type I collagen.

The most prevalent and thoroughly studied types of collagens are type I (almost present in all tissues and organs), type II (present in the cartilage, vitreous body, and nucleus pulposus), and type III (present in the skin, blood vessels, lungs, liver, and spleen) [45], which are used in tissue engineering and reconstructive medicine as well as in the pharmaceutical industry as compounds that extend the effects of drugs. Types I, II, and III

collagens, especially type I, are also used as plastics in medicine and cosmetology. Type I collagen represents over 70% of the entire collagen family and makes up more than 90% of the collagen in the human body. It is mainly found in connective tissues such as body joints, cartilages, bones, sclerae, ligaments, tendons, intervertebral discs, corneas, adventitia of blood vessels, skin, and most hollow organs including gastrointestinal and genitourinary tracts [24,39,46]. In contrast, types II, III, and IV collagen are frequently seen. Type II collagen, for instance, is a structurally important part of the hyaline cartilage that lines the adult's articular surfaces in addition to being present in other tissues including the intervertebral disc's nucleus pulposus and the retina, sclera, and lens of the eye. Skin, lungs, intestinal walls, and blood vessel walls all contain type III collagen.

Type I collagen is composed of three polypeptide chains, two identical α1(I) chains and one α1(I) chain, each of which has roughly 1000 amino acid residues [47]. Hydroxylation of proline residues is a typical post-traditional modification of type I collagen that accounts for about 11–14% of amino acid residues and it is commonly used as a marker to detect and quantify collagen in tissues [48,49]. Whereas proline and hydroxyproline are essential for maintaining the triple helical structure under physiological conditions by forming hydrogen bonds that inhibit uncontrolled rotation, glycine is critical for packing the three helices [50,51].

The idea that the type I collagen molecule is made up of a single extended polypeptide chain with all amide bonds was brought forward by Astbury and Bell in 1940 [51]. In 1951, Pauling and Corey provided the correct structures for the α-helix and β-sheet [52]. In that proposal structure, three polypeptide chains were connected in a helical configuration by hydrogen bonds. These hydrogen bonds necessitated the production of two of the three peptide bonds and involved four of the six main chain heteroatoms inside each amino acid triplet [52]. The collagen triple helix structure was reconstructed in 1954 by Ramachandran and Kartha as a right-handed triple helix of three staggered, left-handed helices with one peptide bond and two hydrogen bonds within each triplet [53]. In 1955, this structure was improved by Rich and Crick, North, and Colleagues thanks to which the triple-helical structure that is still used today was unveiled. This structure has helical symmetry and just one crosslinking hydrogen bond per triplet [54]. Changes in the proposed structure of collagen from the beginning and its modification to the final structure accepted by the scientific community are shown in Figure 3.

**Figure 3.** Changes in the proposed structure of type I collagen from the beginning and its modification to the final accepted structure. Adapted from [52]. Reproduced from [51] with permission from springer Nature, 1940. Reproduced from [53] with permission from springer Nature, 1954. Reproduced from [54] with permission from Elsevier, 1955.

As is shown in Figure 4, three polypeptide α-chains form the trimeric molecule that represents the type I collagen unit (length ≈ 300 nm, diameter ≈ 1.5 nm). Three parallel, left-handed polyproline-II helices are arranged in a right-handed bundle [55,56]. Multiple collagen units are assembled into fibrils (length ≈ μm, diameter ≈ 100 nm) and then fibers (length ≈ mmm diameter ≈ 10 μm) with dimensions and orientation that are strictly tissue-dependent [28,57].

**Figure 4.** Type I collagen hierarchical organization.

Thus, type I collagen is a hierarchically organized protein. The primary structure of type I collagen consists of three α helices: two identical α1(I) and one α2(I) helices of approximately 1000 amino acids and a molecular weight of about 130–140 kDa and 110–120 kDa, respectively. The collagen molecule has a triple helical part and two nonhelical parts at both ends (called telopeptides), with a molecular weight of 300–400 kDa, a length of 280 nm, and a width of 1.4 nm [58,59]. The secondary structure consists of each of these chains twisted in the form of a left-handed helix with three amino-acid repetitions in each turn. The tertiary structure, the inflexible structure, is created when the chains are then twisted three times around one another. Finally, in the quaternary structure, collagen molecules assemble into fibrils and then fibers. Because of the intermolecular and intramolecular interactions, this collagen organization is very stable [25,60]. Obviously, the collagen structure's stability is directly dependent on its chemical composition. For instance, the triple helix of collagen grows stronger as the percentage of amino acids is higher, such as proline and hydroxyproline. The pyrrolidine rings are directly responsible for the polypeptide chain's movement reduction [22,61]. Preservation of collagen's structural integrity results in an improvement in physical properties, an increase in thermal stability, and a decrease in the denaturation temperature [62–64].

Theoretical examination of the mechanical characteristics of collagen at several levels, including the main monomer, individual collagen fibrils, and collagen fibers, is possible by studying collagen's structured nature. Studying main monomers and fibrils made from type I collagen has likely provided the most direct measurements of the mechanical properties of collagen. Over the recent decades, researchers have used a variety of biophysical and theoretical methods, and recent developments in the Atomic Force Microscopy (AFM) approach have made it possible to perform more accurate evaluations [65]. According to estimates, the fracture strength of individual collagen triple helices is 11 GPa, which is much higher than that of collagen fibrils, which is 0.5 GPa [66]. This difference makes sense because, whereas the breaking of a fibril does not always entail the breakdown of covalent bonds, the breaking of individual collagen triple helices necessitates the unwinding of the triple helix and ultimately breaking of the covalent bonds [67]. In contrast to dehydrated type I collagen fibrils from mammalian sources, which have a Young's modulus of about 5 GPa according to AFM tests, individual collagen triple helices monomers have a Young's modulus between 6 and 7 GPa. Because collagen fibrils are anisotropic, another crucial measure of a collagen fibril's strength is its shear modulus, which determines stiffness [68].

Furthermore, AFM indicated that the shear modulus of dehydrated fibrils of type I collagen from mammalian sources is between 30 and 35 MPa. These fibrils' shear modulus was drastically decreased by hydration, but was increased by cross-linking. It is important to note that while some cross-linking is beneficial for the mechanical qualities of collagen fibrils, excessive cross-linking causes collagen fibrils to become highly brittle, which is a common sign of aging [69]. Investigation of the mechanical properties of collagen fibrils demonstrated that the length of the individual collagen triple helices monomer has been chosen by nature in a way to maximize the strength of the produced collagen fibril through effective energy dissipation. Simulations indicate that individual collagen triple helices monomers either longer or shorter than the length of a type I collagen triple helix, which is 300 nm, would form collagen fibrils with low mechanical properties [62]. The thermal and structural stability of the collagen triple helix is strongly influenced by the chemical composition of amino acid and its type, which is caused by the type of animal and the living conditions. Indeed, hydroxyproline stabilizes and strengthens the collagen structure [70]. In addition to preserving collagen's structure and enhancing its mechanical properties, hydroxyproline also plays an important role in its thermal stability. The denaturation temperature and denaturation enthalpy of collagen increases due to the presence of the hydroxyl group in hydroxyproline and the bonding with the pyrrolidine ring. The quantity of hydrogen bonds formed between hydroxyproline and pyrrolidine significantly influences the increment in enthalpy of denaturation. Therefore, the triple helix does have greater thermal stability the more water molecules that there are surrounding it [25,71].

One of the most basic roles of collagen in the body is to provide connective tissues with stability, structure, and resistance to stresses [19,20]. Moreover, collagen has the ability to manage a wide range of nonstructural activities, including cell proliferation, migration, differentiation, and communication [60,72].

#### **3. Fish Collagen**

Collagen sources, types, pre-extraction conditions, and process methods are the main parameters that determine extracted product properties, including molecular weight of the peptide chain, amino acid composition, molecular structure, solubility, and functional activity. Although native type I collagen could be extracted from different mammalian sources, the main source of extraction is bovine due to availability and biocompatibility. [73]. There are other alternative sources for extracting type I collagen, among which pig, horse, sheep, and rat can be mentioned [74–77]. It is possible to obtain mammalian collagen from a wide range of tissues, notably skin, bones, tendons, lung tissue, and connective tissues. Due to some restrictions in terms of health, cultural, social, and religious issues that are implied by traditional sources, research has concentrated on the development of a new source of extraction. Various resources from the sea, including vertebrates as well as invertebrates,

have been studied and considered as collagen extraction sources. In particular, several fish species (e.g., *Rachycentron canadum, Esoxlucius, Spotless smooth hound, Sciaenops ocellatus, Sardinella fimbriata, Coryphaena hippurus, Alaska pollock, Takifugu flavidus, Pacu, Labeo rohita, Labeo catla, Tuna, Thunnus obesus, Scomber japonicus, Gadus morhua, Prionace glauca, Cichla ocellaris, Cyprinus carpio, Oreochromis niloticus, etc.*) aquatic reptiles (such as the soft-shelled turtles), sponges, corals, octopuses, squids, starfish, jellyfish, cuttlefish and sea cucumbers, sea anemones, sea urchins, mussels, and shells were considered.

Skin, scales, bones, skull, swimming bladder, and remaining viscera, are by-products of fish that may be used as sources of collagen (Figure 5). Among all fish by-products, skin traditionally has been reported as the best source of fish collagen extraction [12,78–81].

**Figure 5.** By-products of fish as potential sources of collagen extraction.

Fish collagen physicochemical properties were found to be similar to mammalian collagen, but with some advantages such as (1) capability of purification and extraction; (2) aquaculture and accessibility to fishing by-product; (3) lower risk of disease transmission compared to mammalian collagen due to high ontogenetic difference between fish and humans; (4) lack of religious and cultural limitation; (5) slightly different chemical composition; (6) low viscosity; (7) non toxicity; (8) reasonable homeostatic properties; (9) bio-resorbability; (10) more simple extraction method; (11) more adaptable and metabolic compatibility; and (12) minimal inflammatory response (Figure 6) [82–85]. Although fish collagen has several advantages, it suffers from several disadvantages such as low denaturation temperature, low mechanical properties, and high degradation rate [78]. The major drawback of fish collagen compared to mammalian collagen is the lower denaturation temperature, which limits its medical applications [70]. During denaturation, collagen turns into gelatin, where the hydrogen bonds that support the helical structure are partially or completely destroyed, and it loses its structural role and its conformation-related biological activity [42,43]. The second main drawback of fish-derived collagen is its low mechanical resistance which limits its applications. Many efforts have been made to improve its mechanical properties and degradation profiles, including chemical or enzymatic cross-linking [86,87]. The different advantages and disadvantages of fish collagen are shown in Figure 6.

**Figure 6.** Advantages and disadvantages of fish collagen.

#### **4. Collagen Extraction Methods**

Due to the enormous diversity of collagen types, it is challenging to design a standard extraction procedure for collagen from various tissues. However, the collagen extraction process usually consists of around five main steps (Table 1): (i) tissue separation and purification; (ii) tissue size reduction; (iii) non-collagenous components elimination; (vi) collagen extraction through acid and/or enzymatic treatment; (v) and, finally, recovery using salt precipitation. The extraction procedures start with the removal of unneeded portions. Fish by-products are then reduced in size to facilitate the following step which is the removal of non-collagen proteins, lipids, pigments, cell remnants, and minerals. Afterwards, collagen is extracted using an acidic treatment, followed by an optional enzymatic treatment, before being recovered using salt precipitation, dialyzation, and lyophilization. All these steps are performed at about 4◦ C to 10 ◦C, to prevent collagen denaturation [88–90].

**Table 1.** Five steps of collagen extraction process from fish sources [88–90].


The conventional process for collagen extraction, based on acid and/or enzymatic methods, has been improved in recent research. The fish ecosystem, the belonging tissue, and the method of extracting collagen from a fish source have a direct effect on the number of remaining impurities [83,91]. In the following, various sources, methods, advantages, and disadvantages of each method and effective parameters in the extraction of fish collagen have been investigated.

Fish tissues require special treatment before being recovered from fisheries and aquaculture byproducts, including washing with water and sodium chloride to remove impurities and lipids and milling the skin to increase its contact surface with the liquid phase [92]. Following the removal of contaminants and non-collagenous proteins using sodium hydroxide, hydrogen peroxide, calcium hydroxide, or a combination of these, the material is submerged in alkaline solutions, with butyl alcohol (10%) used to remove oily components [93,94].

The real extraction step is based on the solubility of the collagenous molecule taken after the pretreatment. The most common treatments are saline, acid, and/or enzymatic. The saline treatment employs neutral salt, such as sodium chloride and/or guanidine hydrochloride, for precipitation-based extraction, which, among its drawbacks, has a low extraction yield [95]. Once the sodium chloride concentration has been gradually raised through adding NaCl, the collagen is separated. It was shown that the basic salt extraction is ineffective after testing a number of collagen isolation techniques. Additionally, raising the salt content will enhance the ionic power of the resulting solution and boost the solubilization capacity. The ultimate yield is extremely low since, in normal tissues, the amount of neutral salt-soluble collagen is typically insignificant [96,97].

In the acid treatment, several acid types could be employed, such as acetic acid, lactic acid, citric acid, hydrochloric acid, formic acid, sulfuric acid, and tartaric acid [83]. Obviously, this method can solubilize collagen more effectively than basic salt extraction, but it is still only effective on young and uncross-linked collagen [97]. Collagen type I derived from fish skin is often extracted using an acidic treatment with acetic acid, hydrochloric acid, or phosphoric acid. However, this extraction method can be performed using either acids or alkali. These extraction techniques are extremely corrosive and, after neutralization, result in a high salt content. The pH value will influence the electrostatic interaction and structure depending on the acid's concentration. It establishes the ability of animal tissue to be extracted and dissolved [28,98,99].

Enzymatic treatment involves the use of enzymes, such as collagenase, papain, or pepsin [100,101]. Enzymatic hydrolysis has emerged as the best method for collagen extraction from fish because it tends to eliminate the non-helical extremities and increase the solubility of collagen molecules and, thus, increase the extracted material yield [102,103]. The potential for irreversible denaturation of the collagen structure by enzymatic digestion during this procedure could either be a drawback or not, since it could be used for the production of several collagen formulations with different hierarchical organization levels that will have different bioactivity profiles. A more effective collagen extraction method was obtained by integrating both the acidic and enzymatic treatments. The collagen molecule is affected by enzymes, which make it more soluble in an acidic media [61,104].

Figure 7 shows that the denaturation of native collagen results in the formation of randomly coiled α-chains. Thermal treatments above collagen denaturation temperature can be used to obtain them. Proteolytic enzymes are able to hydrolyze the polypeptide chains in shorter polypeptide sequences. The final outcome is typically referred to as hydrolyzed collagen that is made up of short, low-molecular-weight peptides. The kind and level of hydrolysis, as well as the different type of enzyme used in the process, all affect collagen properties and functional activity [105–108].

**Figure 7.** Representative scheme of type I collagen denaturation into low-molecular-weight peptides (red and blue).

The molecule is not altered when collagen is extracted using ultrasonic as a substitute method; instead, this helps the enzymatic process [109,110]. This method can be used to produce higher collagen yields in shorter extraction durations. This approach is more effective than the traditional one because it increases mass transfer by opening the collagen fibrils, permitting acid and/or enzymatic hydrolysis, and subsequently improving the extraction yield [109–111].

Electro dialysis, a quick, effective, and affordable approach, was employed instead of traditional dialysis to boost extraction efficiency and process speed [112]. Isoelectric precipitation is a method frequently used to separate protein biomolecules, which can be used in collagen extraction from fish sources [113,114]. Thermal processing, or treating the protein to high pressure and temperature, constitute further extraction techniques. There is a subcritical water level (SCW) used in thermal processing, which can be found at pressures lower than 22 MPa and temperatures between 100 ◦C and 374 ◦C [97,115]. Figure 8 shows that the yield of collagen obtained varies from 0.05% to 94.4% [8].

**Figure 8.** Yield of collagen obtained from fish sources [8].

Table 2 lists some recent studies on various techniques for collagen extraction from different fish sources. The primary objectives of introducing new methods during the collagen extraction phases are to shorten the extraction process time, energy, and chemicals compared to traditional methods. The efficiency of collagen extraction methods from animal by-products depends on the extraction source, age, and type of animal, as well as the condition of the processed by-products and the technology employed.

**Table 2.** Recent studies on various techniques for fish collagen extraction.



**Table 2.** *Cont.*

#### **5. Collagen Applications**

Given its outstanding biocompatibility and biodegradability, low cytotoxicity, elevated versatility, significant therapeutic loading, affordability, lack of need for a multistep extraction procedure, high digestibility, and ease of absorption and distribution in the human body, fish collagen is even more frequently used in a many industrial areas [129,130]. Besides aforementioned advantages, it has a decreased viscosity in aqueous solution, low allergenicity, transparency, good solubility and dispersibility (i.e., uniform distribution in solution), emulsifying ability, and processability in different kinds of products such as powder, foam, and film [131,132]. Thus, throughout many different industrial sectors, including biomedical, pharmaceutical, food, cosmetic, and leather industries, type I collagen is widely employed, as presented in Figure 9. Some of these applications are mentioned below. For niche but promising applications in energy storage devices, the authors referred to a recent review [133].

**Figure 9.** Application of fish collagen in different industrial fields.

#### *5.1. Food Industry*

In the past, collagen has been used to prepare a variety of goods, including meat products, drinks, soups, and others [123,129]. It aids in enhancing and maintaining their physical, chemical, and sensory qualities. Compared to patties made without fish collagen, those prepared with fish collagen have a higher protein percentage, reduced fat content, comparable sensory acceptance, and better texture. Even in processed foodstuffs including sausages, sausage rolls, ham, hotdogs, and hamburgers, collagen has replaced half-content pork fat leading to enhanced hardness and chewiness, better stability after cooking, and a higher water-holding capacity. Additionally, fish collagen can be added to drinks such as natural fruit juice, to enhance their nutritional and functional qualities due to their greater protein content, bioavailability, moderate viscosity, and excellent water solubility [134–138]. More recently, studies are ongoing on the use of fish (minced fillet) waste in the manufacturing of foodstuffs [139].

#### *5.2. Nutraceuticals*

Collagen plays a crucial role in tissue and organ development, maintenance, and healing. The loss of collagen in the body begins at the end of the second decade of life and reaches 1% per year by the end of the fourth decade. This process continues until the eighth decade, when the body has lost about three quarters of its collagen compared to the youth. Additionally, other factors such as diseases, improper diet, alcoholism, and smoking accelerate this process [140–142].

The largest apparatus in the human body is the integumental system, which is primarily made of proteoglycans, hyaluronic acid and elastic fibers, and collagens (mainly types I, III, V; types IV, VI, VII to a minor extent). Natural aging involves changes in the human

body: the skin deteriorates morphologically, structurally, and functionally; collagen levels decline; and elastin fibers encourage the development of wrinkles. In the dermis, collagen has a double role: i) to serve as a building block for the formation of newly synthetized collagen and elastin fibers; ii) to interact with receptors on the fibroblasts' membrane to promote the synthesis of new collagen, elastin, and hyaluronic acid [143]. Considering that collagen peptides have antioxidant and antibacterial properties and vary in quality depending on the technique of extraction, they can be employed as a component in functional dietary supplements. In view of the fact that collagen oral supplementation reaches the deeper layers of the skin and improves skin physiology and appearance by enhancing hydration, elasticity, firmness, wrinkle reduction, and skin regeneration, oral collagen supplementation has gained popularity in recent years [123,144]. Many studies have concluded that hydrolyzed fish collagen applied as food supplement is able to provide positive effects on skin appearance with enhanced water-holding capacity, moisture absorption, retention, anti-aging, and anti-melanogenic effects [59,145].

Skin condition changes brought on by aging are a crucial concern for preserving the quality of life. As a result, the public is interested in dietary supplementation's ability to treat skin disorders. Naoki Ito postulated that, by elevating the plasma growth hormone, a supplement blend comprising ornithine and fish-derived collagen peptide could enhance skin conditions [146]. In this regard, two groups of volunteers used a supplement or identical placebo for two months. Skin condition, including elasticity and transepidermal water loss, as well as growth hormone levels, was significantly improved in the first group. The combination of amino acids in collagen hydrolysate, known as a safe nutraceutical, stimulated the production of collagen in the extracellular matrix of cartilage and other tissues. Porfírio performed research on the action of collagen hydrolysate in bone and cartilaginous tissue and its therapeutic use against osteoporosis and osteoarthritis, discovering a connection between the maintenance of bone strength and composition, as well as cartilage cell development and proliferation, and the administration of various doses of collagen hydrolysate [147]. This study concluded that hydrolyzed collagen has a protective effect on articular cartilage, and especially helps with symptomatic pain reduction considering the ability to raise bone mineral density [147]. Therefore, it has a good therapeutic effect on osteoporosis and osteoarthritis.

#### *5.3. Cosmetics*

As mentioned in the previous section, the role of collagen in the body is very important because it helps the skin, the largest organ of the human body. The skin protects the organism from external damage, regulates temperature, and performs other body functions. Over the years and in the process of aging, the amount of collagen in the skin decreases and this causes its morphological, structural, and functional deterioration. In fact, the presence of elastin fibers causes lines and wrinkles and shows aging. Controlling skin aging is a challenge in the cosmetic industry, but the use of collagen has been proven to be an alternative solution to reduce the effects of aging. In the studies that have been conducted, fish collagen has shown the capacity to retain water, absorb moisture, and retain it again, which can have anti-aging effects on the skin and can be used as a potential active ingredient in skin-care products [148–151].

#### *5.4. Tissue Engineering and Regenerative Medicine*

Historically, tissue engineering is based on the combination of scaffolds, cells, and signals. The term 'scaffold' is usually referred to as a temporal substitute that should structurally support tissue formation and provide the appropriate environment for cell migration, proliferation, and differentiation, and hence for repairing processes. The prevalence of collagen in human tissues and the important role it plays in the extracellular matrix make it a natural choice for its employment as raw material in the development of implantable devices for tissue engineering and regenerative medicine applications. Common

application areas include bone, vascular tissue, skin, cartilage, corneal tissue, oral mucosa, and dental regeneration [26,73,152].

Numerous studies demonstrated that collagens, especially fish collagens, have intriguing osteoconductive and biomechanical properties and are used more frequently in tissue engineering. Due to its exceptional biocompatibility, collagen has been reported to be employed as a biomaterial in a variety of vascular tissue applications. The bioactivity of collagen has caused this biopolymer to be widely used in skin tissue repair with its healing, antigenic, new-tissue-thickening, and adhesion properties.

One such technique is tissue engineering, which relies on the utilization of autologous chondrocytes and resorbable matrices. Visual acuity depends on a healthy cornea, which is the eye's tough, transparent anterior surface. Damage to the cornea is a significant contributor to the lack of limbal stem cells that results in vision problems. To this goal, a number of treatment modalities are being created to address limbal stem cell insufficiency. The goal of this strategy was to create a biocompatible scaffold for growing limbal stem cells that completely replicate the human amniotic membrane. This was done by using a unique method based on fish collagen. It was discovered that the mechanical and physical forces of fish-scale-derived collagen were adequate for this purpose [153,154]. Collagen was also demonstrated to play a critical role in tooth tissue repair. Indeed, various collagen types retrieved using various procedures have demonstrated their ability to stimulate the regeneration of dental tissue; as a result, they can be employed in biomedical applications to regenerate tooth tissue [155,156].

Because of postoperative problems, including retears at the treated site, large and enormous rotator cuff tears pose a difficulty for surgeons. Since fish byproducts are regarded as a safer collagen source than other animal-derived scaffolds, collagen generated from fish scales has recently attracted more attention. Yamaura et al. [157] assessed the biological effectiveness of Tilapia-scales–derived collagen scaffolds for rotator cuff healing in rat models. In this research, by augmenting the repair site with a Tilapia-scale–derived collagen scaffold, after 6 weeks, an enhanced angiogenesis and fibrocartilage regeneration at the enthesis was observed. Due to osteogenic capacity and the connections between cells and the matrix, extracellular matrix and bioceramics are vital components in bone tissue regeneration. Since scaffolds are typically made up of synthetic polymers and bioceramics, surface modifications with hydrophilic materials, such as proteins, have great prospects for tissue engineering applications. In this study, which was provided by Kim et al. [158], marine atelocollagen was extracted from the bones and skins of *Paralichthys olivaceus*. Then, in vitro and in vivo calvarial implantation of the scaffolds with and without marine atelocollagen was performed to study bone tissue regeneration. The results of mineralization confirmed that scaffolds with marine atelocollagen showed an osteogenic increase from 300% to 1000% in different compositions, compared with pure scaffolds.

#### *5.5. Wound Healing*

The complex process of wound healing is essential for re-establishing the skin's barrier function. Numerous illnesses can halt this process, leaving behind chronic wounds that are extremely expensive to treat. Due to the complicated symptoms brought on by metabolic dysfunction of the wound microenvironment, such wounds fail to heal according to the stages of healing, and the comprehensive treatment of chronic wounds is still recognized as a huge unmet medical need. Consequently, there are three broad categories for wound classification: (i) superficial (involves only the epidermis), (ii) partialthickness (involves epidermis and dermis), (iii) and full-thickness wound (involves also the underlying subcutaneous fat or deeper tissues) [159–163]. The process of wound healing is a physiological process that consists of four main steps: (i) hemostasis, (ii) inflammation, (iii) proliferation, and (iv) remodeling (Figure 10). Therefore, it is vital to choose the right polymers, bioactive chemicals, and wound dressings that can speed up the healing process. There is no one wound dressing that can be used to treat all types of wounds due to their varying etiology. Thus, the development of a smart wound dressing with antibacterial,

anti-inflammatory, and antioxidant capabilities that, most critically, can benefit nearly all types of wounds, is the future challenge [163–166].

**Figure 10.** Schematic of the wound healing steps: (**1**) hemostasis, (**2**) inflammation, (**3**) proliferation, and (**4**) remodeling.

The combination of polymers and bioactive compounds significantly speeds up wound healing. Although the use of natural remedies for wound healing has been extensively studied, only a small number have yet to be commercialized or employed in clinical settings. In order to fully understand the potential of naturally occurring bioactive compounds in skin tissue regeneration, more preclinical studies must be done. Collagen, as a biodegradable organic tissue matrix, is a common option when choosing safe and nontoxic materials because it is one of the most crucial elements in tissue regeneration and wound healing and gives the skin its tensile strength. Collagen also has antimicrobial qualities and can aid in the hemostasis process. Collagen is used in different forms of hydrogel, sponge, and film for wound treatment. The best example of wound dressing devices are hydrogels, three-dimensional networks which can maintain a moist environment at the wound site and promote quicker tissue regeneration [161,164,167].

Several attempts at wound healing using prototypal devices made of fish-derived type I collagen or decellularized fish skin have been made. Hu et al. demonstrated that marine collagen peptides promote wound closure at concentrations of 50 μg.mL−<sup>1</sup> commencing at 12 h after treatment with collagen using an in vitro scratch assay [168]. It was demonstrated that the cell migration that was induced was comparable to migration seen when using 10.0 μg/mL of epidermal growth factor, a factor known to be extremely important in wound healing. In addition, after 11 days, rabbits treated with marine collagen peptides extracted from the skin of Tilapia healed considerably quicker than the control group. Additionally, Yang et al. extracted collagen peptides from Alaska Pollock and showed that giving injured rats collagen peptides orally boosted recovery rates substantially more than those in the control groups [169]. Similarly, Chen et al. extracted collagen from bovine skin collagen nanofibers and marine Tilapia skin and demonstrated that collagentreated rat groups recovered from wounds more quickly than control groups [170]. The study also discovered that collagen's hydroxyproline, which promotes re-epithelization, has a significant influence in the rate of wound healing. In comparison to the control groups, the collagen-treated groups had more fibroblasts, higher vascularization, less inflammation, and more collagen fibers.

#### *5.6. Food Packaging*

Food packaging has the primary function of preserving and protecting food, primarily from oxidative and microbial degeneration, extending the shelf-life of the food by enhanced barrier and mechanical properties [171,172]. Fish collagen has attracted growing interest due to its potential for adding active and intelligent functions to conventional packaging [173,174]. In particular, active packaging can prevent the migration of H2O, O2, CO2, smells, and fats, and can include bioactive compounds such as antioxidants, antimicrobials, and taste to prolong the shelf life of the product [57,175,176]. Active packaging can appear in the form of edible films or coatings. Edible films are first produced by solution casting or compression molding and then applied to food surfaces by coating, wrapping, or spraying, while edible coatings are applied to food by spraying or dipping [177,178].

Films and coatings for food packaging must feature an elevated oxygen barrier and adequate thickness, mechanical properties, and transparency besides microbial stability, non-toxicity, and safety [179–182].

There are some necessary properties of biopolymers for food packaging, such as biodegradability, low water vapor permeability, oxygen barrier, thickness, transparency, edibility, and elasticity [183–186].

The application of fish collagen films is still constrained in the packaging industry due to drawbacks including poor mechanical qualities, low thermal stability, excessive water solubility and a large water vapor permeability. Several studies are in progress to overcome these limitations. For example, to reduce the brittleness, collagen films are usually prepared by using a plasticizer, mainly glycerol in the range 20–30 wt%, a small molecule of low volatility added to decrease attractive intermolecular forces along polymer chains and increase the free volume and chain mobility [187]. Moreover, suitable crosslinking treatments are being studied to improve the thermal stability of fish collagen [188,189]. Other possible solutions could be the blend of collagen with other biopolymers, mainly chitosan [77,190–193], and the addition of active compounds providing functional properties suitable for active packaging [187,194].

Gelatin, extracted from fish collagen by partial hydrolysis followed by thermal treatment, is attracting increasing interest for the development of edible films and coatings with probiotic properties, as recently reported in the literature [195–197]. In order to achieve the properties required for food packaging, several studies report on the physical or chemical modification of fish gelatin with chitosan, starch, soy protein isolate and carboxymethyl cellulose [198–202].

#### **6. Collagen Market**

Collagen and derivates are widely used for various applications, including dietary supplements, anti-aging formulations, soft-tissue growth devices, wound dressings, and food packaging. The achievement of US Food and Drug Administration (FDA) GRAS status (Generally Recognized as Safe) in 1983 for collagen and in 1975 for gelatin [203] boosted collagen's use in several areas of application [204].

The increasing popularity of fish collagen for biomedical, food, cosmetic, nutraceutical, and nutricosmetic application has increased its demand. To this, the global marine collagen market was worth USD 685 million in 2020 [204] and USD 633 million in 2022 [205] and it was estimated to register over 5.3–7.5% of the compound annual growth rate (CAGR) between 2021 and 2029 [203,204] and is expected to reach a market size of USD 1123 million by 2032 [205]. In particular, the fish-collagen market was estimated to be worth USD 320.21 million in 2021 and is predicted to skyrocket to USD 624.12 million by 2029, with a CAGR of 8.7% during the forecast period 2022 to 2029 [206].

The increasing popularity of fish-collagen-based products is principally due to two main factors: (i) aging population, and (ii) environmental issues. The increase of the

mean population age is directly correlated with the increase of age-related diseases (i.e., joint disorders, wrinkles, and wounds) [206,207]. In these circumstances, collagen-based products have been revealed to be effective, quite low-cost, easily accessible, safe, noninvasive, and readily available, and, accordingly, fish-derived-collagen awareness has significantly increased thanks to its additional advantages compared to other collagen types [208]. Therefore, the fish-collagen market for nutraceutical application was valued at over USD 280 million in 2020. Moreover, the rising inclination of consumers towards fat-free and nutritious products has further increased the product demand [204]. Thus, the major factor that is expected to boost the growth of the marine collagen market in the forecast period is a rise in the demand for supplements to control healthcare costs [203]. On the other hand, the environmental problems linked to the disposal of the enormous quantity of by-products of the fishing industry and to the use of plastic have shifted focus toward the search for eco-friendly solutions. In particular, waste recovery technologies were developed to reduce the environmental impact on by-products and to develop new products with added value. Local enterprises profited from this arrangement because fish is more readily available for less money, and the collagen market is booming [207]. Therefore, fish collagen and derivates started to be isolated, studied, and commercialized not only in health-related sectors but also in food packaging.

Fish collagen demand is related to its applications. In North America, it is mainly required for pharmaceutical applications [204]. In Europe and Australia, it its mainly used in the cosmetics industry [204,207]. The boost of fish collagen for cosmetic applications is principally due to the increasing preference for minimally or non-invasive surgical procedures compared to traditional surgical treatments. Additionally, the ease of treatment, the higher safety, and major accessibility have led the European population to prefer topical collagen formulations and food supplements for anti-aging and well-being treatments [209]. In Asia and Latin America, besides age-related issues, the major exploitation of fish collagen as a food supplement has arisen from the fact that, according to the European Nutraceutical Association (ENA), a lack of adequate nutrition accounted for 38.6% of deaths in China, India, and Brazil [204,207,209]. Indeed, the ENA's in-depth investigation highlighted that inadequate nutrition is not related to an economic gap, but to incorrect eating habits [209].

Regarding countries' contributions to the fish-collagen market, in 2012, the CARG of fish-collagen market by region was positive and was projected to reach +18% in North America, +31% in Latin America, +10% in Europe, and +28% in Asia by 2016 [209]. As shown in Figure 11, in 2019 North America (about 29%), Europe (about 30%), and Asia (Asia pacific: 21%, China: 15%) occupy the largest share of the market [209]. Among them, Asia is clearly expected to rule the market with about 36% of the total [207,209]. Actual fish-collagen market distribution by midlands is not available but it is known that North America's contribution remained almost unchanged (31%) and that, in Europe, Germany contributes 23.3% to the total fish-collagen market, while, in Asia, Japan contributes 6.6% and, in Oceania, Australia's contribution is about 2.6% [207].

The cost of fish-collagen is also application-related. The cost for the food industry (as binders, stabilizers, emulsifiers, film-formers, and fat replacers) was reported to be between EUR 8–12/kg, for the nutraceutical industry (for joint diseases) it was about EUR 10–12/kg, and for cosmetic applications it was reported to be about EUR 20–25/kg but could reach also EUR 40/kg [210]. However, the quality of the product obtained from marine life forms (USD 44539/metric ton) costs relatively higher than that from bovine sources (USD 33457/metric ton) [210] due to the complex and cost-intensive process of extracting collagen from marine organisms and by-products of the fishing industry. Moreover, fish waste has been somewhat decreased as a result of changes made to fishing regulations to combat overfishing, which limited the production of fish collagen and related goods. The high cost of fish collagen and the lack of awareness about its benefits among consumers are some of the major challenges faced by manufacturers [203]. These disadvantages allowed bovine collagen to have a leadership position as it holds a great cost advantage in lower-value products (e.g., food) [210].

**Figure 11.** Fish-collagen market segmentation by Continent in 2019 [207].

The major players operating in the marine collagen market are Ajinomoto (Tokyo, Japan), Amicogen Deyan Biotech (Jinseong-myeon, South Korea), Ashland (Wilmington, CA, USA), Athos collagen (Surat, India), BDF Biotech (Girona, Spain), BHN (Tokyo, Japan), Certified Nutraceuticals (Pauma Valley, CA, USA), Cobiosa (Madrid, Spain), ETChem (Suzhou, China), Gelita (Eberbach, Germany), Juncà Gelatines (Girona, Spain), Hangzhou Nutrition Biotechnology (Hangzhou, China), HealthyHey Nutrition (Mumbai, India), Hi-Media Laboratories (Maharashtra, India), Italgel (Cuneo, Italy), Lapi Gelatin (Empoly, Italy), Nippi Incorporated (Burnaby, Canada), Nitta Gelatin (Kokin, India), Norland Products (Jamesburg, NJ, USA), ProPlenish (Armadale, Australia), Rousselot (Gent, Belgium), Seagarden (Husøyvegen, Norway), Tessenderlo Group (Ixelles, Belgium), Weishardt Group (Graulhet, France), among others.

#### **7. Challenges in the Industrial Implementation of Collagen Derived from Fish Waste**

The collagen extraction process is a multistep, time-consuming procedure, which is a disadvantage in the industrial production of it. The issues and related challenges of fish collagen extraction are manifold and are principally linked to the extraction process and to the protein chemical-physical properties.

One of the main troubles is its low extraction yield, a parameter that is both speciesrelated (i.e., taxonomy, age, tissue, and living conditions) and process-related (i.e., time, volumes, instrumentation, sample-volume ratio, types of acid and enzyme used and their concentrations, temperature, pH, ionic strength, and so on [211]). Several attempts were made in order to improve collagen extraction yield. The major steps forward have been made by optimizing solute and solvent concentrations and times in extraction steps 3–5. In particular, the implementation of a discarding phase of non-collagenous components (i.e., step 3 in Table 1) with NaOH 0.05–0.1 M, and an extraction phase (i.e., step 4 in Table 1) with an acetic acid concentration of 0.6 M for 36 h [212] brings a collagen yield increase. Regarding the enzymatic extraction, a pepsin concentration of 1200–1300 U/g is revered as the most effective in increasing collagen yield [98]. However, if, on one hand, the enzymatic extraction is able to significantly increase the yield of collagen, on the other hand, it significantly increases the time of the process and decreases the native conformation degree [213,214]. This consequence may not be industrially advantageous since it can lead to a higher cost of the process and therefore to a higher final cost of the product. For this reason, it is necessary to make a cost/benefit assessment before choosing whether or not to perform the enzymatic extraction. In addition to the 'standard extraction process' steps improvements, some innovative attempts have been made. Several authors demonstrated how the application of ultrasound increased yield and reduced processing time, as well as being greener compared to conventional extraction methods [79,109,213]. Huan et al. developed a novel rapid extrusion-hydro-extraction process for collagen from fish scales at room temperature [215] as an alternative to traditional methods.

Regardless of the process, temperature affects all extraction steps, from the tissue separation to the final collagen precipitation and recovery. Because of fish collagen's low denaturation temperature (<37 ◦C), the need to carry out the entire extraction process at low temperatures (4–10 ◦C), to preserve its native structure and thus its structural properties and bioactivity, makes the procedure expensive. The low denaturation temperature of fish collagen is due to fish's evolutionary adaptation to the characteristics of the aquatic environment in which they live. For this reason, it is not possible to intervene in this aspect. The only thing that can be done is to carefully select the fish species. In particular, the selection of a fish species that lives in a tropical environment—and therefore will have collagen with a physiologically higher denaturation temperature (e.g., 32–36 ◦C in catfish [215], 36–38 ◦C in carp [2,216], 32–37 ◦C in Tilapia [121], and 43 ◦C in lizardfish [217])—compared to a fish species living in cold waters, could be a solution. With this in mind, Pinedo et al. investigated the properties of collagen extracted from a hybrid fish line that, although similar to those of the original strains, was allowed to obtain a more controlled fish growth and, thus, a higher yield [81].

Despite the presence of various issues, it is clear how scientific and industrial research is moving towards the optimization of the extraction process and industrial implementation. In this regard, an advanced pilot plant automation was recently designed to maximize collagen extraction [218]. Therefore, since it is not possible to reduce the time and costs of the extraction process by optimizing it from the point of view of temperature control, a way to increase the denaturation temperature of marine collagen and make it more suitable for a wide range of applications is to induce post-synthesis crosslinking of the products. The increment of fish collagen denaturation temperature is another important issue since it is particularly relevant in some clinical applications. The application of crosslinking treatments also helps in the resolution of other two issues related to fish-collagen use which are the low mechanical properties and the low resistance to degradation, which make it unusable in some applications. Indeed, physical (e.g, UV [219,220], dehydrothermal treatment [219,221], chemical (e.g, methacrylation [222], pullulan [223], carbodiimide [221,224], N-hydroxysuccinimide-activated adipic acid [225]), and enzymatical (e.g., transglutaminase [225]) treatments were performed to enhance collagen properties. Maher et al. made a considerable step forward by successfully printing methacrylated fish collagen and realizing a 3D construct with desired properties, despite the fact that the applicated treatment was not able to increase the resistance to degradation on par with collagen extracted from mammals [222]. Another strategy commonly adopted to improve collagen properties is to blend it with other biomaterials with higher mechanical properties, such as chitosan [77,226–228], poly(lactic acid) [228,229], alginate [230], polyvinyl alcohol [227,231], and cellulose [195,231].

#### **8. Conclusions**

Natural biopolymers have unique biophysical and biochemical properties, including biocompatibility, biodegradability, increased body fluid adsorption capacity, increased gel-forming ability, non-toxic and non-immunogenic capabilities, as well as antifungal, antibacterial, and anticancer activities. One of these biopolymers is collagen, which could be obtained from various sources such as fish, mammalian, and agro-food waste. By turning these wastes into new products with a high functional value, recycling these by-products can assist in decreasing the pollution caused by these sorts of wastes. A potential substitute for bovine collagen is thought to be fish collagen. Fish collagen is cited as an important biomaterial due to its wide range of biological characteristics, including remarkable biocompatibility, high levels of cell adhesion, exceptional biodegradability, and low antigenicity. This review provides a general overview of collagen and its properties, types of sources and extraction methods, and diverse applications in a variety of industries, with a spotlight on fisheries and aquaculture sources.

**Author Contributions:** Conceptualization, F.L. and Z.R.; methodology, Z.R. and F.L.; validation, Z.R., F.L., N.G. and L.S.; data curation, F.L.; writing—original draft preparation, Z.R. and F.L.; writing—review and editing, Z.R., F.L., N.G. and L.S.; supervision, F.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** Z.R. acknowledges Regione Puglia for funding NANOCOLLAGEN-"Development of nanometric collagen from waste from the fish industry" (code 284e667a) in the framework of POC PUGLIA FESR-FSE 2014/2020 RIPARTI project.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data available on request.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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## *Article* **Comparative Study of Flax and Pineapple Leaf Fiber Reinforced Poly(butylene succinate): Effect of Fiber Content on Mechanical Properties**

**Taweechai Amornsakchai 1,2,3,\*, Sorn Duangsuwan 1,2, Karine Mougin <sup>4</sup> and Kheng Lim Goh 5,6**


**Abstract:** In this study, we compare the reinforcing efficiency of pineapple leaf fiber (PALF) and cultivated flax fiber in unidirectional poly(butylene succinate) composites. Flax, known for robust mechanical properties, is contrasted with PALF, a less studied but potentially sustainable alternative. Short fibers (6 mm) were incorporated at 10 and 20% wt. levels. After two-roll mill mixing, uniaxially aligned prepreg sheets were compression molded into composites. At 10 wt.%, PALF and flax exhibited virtually the same stress–strain curve. Interestingly, PALF excelled at 20 wt.%, defying its inherently lower tensile properties compared to flax. PALF/PBS reached 70.7 MPa flexural strength, 2.0 GPa flexural modulus, and 107.3 ◦C heat distortion temperature. Comparable values for flax/PBS were 57.8 MPa, 1.7 GPa, and 103.7 ◦C. X-ray pole figures indicated similar matrix orientations in both composites. An analysis of extracted fibers revealed differences in breakage behavior. This study highlights the potential of PALF as a sustainable reinforcement option. Encouraging the use of PALF in high-performance bio-composites aligns with environmental goals.

**Keywords:** pineapple leaf fiber; flax; poly(butylene succinate); unidirectional composites; microstructure; mechanical properties

#### **1. Introduction**

In recent decades, escalating global concern over climate change has prompted a significant shift towards sustainable and environmentally friendly practices across various industries. One of the pressing issues we face today is the increasing level of carbon dioxide (CO2) in the Earth's atmosphere, contributing to the greenhouse effect and subsequent climate change. As scientists and researchers strive to combat this challenge, exploring alternative materials and manufacturing processes becomes crucial in reducing our reliance on petroleum-based products while simultaneously addressing CO2 emissions using different concepts such as carbon capture utilization and storage (CCUS) using expensive modern technologies [1,2].

Plants, through the process of photosynthesis, have the remarkable ability to convert CO2 into organic compounds, effectively sequestering this greenhouse gas and mitigating its impact on the environment. Leveraging the inherent qualities of natural fibers in composite materials offers a promising avenue to not only decrease dependence on fossil fuel-derived resources, but also actively sequester CO2. By incorporating these natural

**Citation:** Amornsakchai, T.; Duangsuwan, S.; Mougin, K.; Goh, K.L. Comparative Study of Flax and Pineapple Leaf Fiber Reinforced Poly(butylene succinate): Effect of Fiber Content on Mechanical Properties. *Polymers* **2023**, *15*, 3691. https://doi.org/10.3390/polym15183691

Academic Editor: Raffaella Striani

Received: 11 August 2023 Revised: 3 September 2023 Accepted: 4 September 2023 Published: 7 September 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

fibers into polymer matrices, composites can be fabricated with improved mechanical properties [3–5] while simultaneously contributing to carbon capture and reduced environmental impact [6], similar to using wood, but much easier and with lower production cost. Although there are different types of natural fibers with a range of mechanical properties [4,7–10], perhaps it is fair to say that flax and hemp are the most successful commercial examples [11,12].

This study aims to conduct a comparative analysis between two types of natural fibers: flax and pineapple leaf fiber. Flax fiber, derived from the stem of the flax plant (Linum usitatissimum), has long been recognized for its mechanical strength and versatility [13,14]. On the other hand, pineapple leaf fiber, obtained from the waste of pineapple cultivation, has emerged as a potential alternative due to its abundance and renewability [15–19]. Although these two fibers have been studied for a long time, there is still some discrepancy in their reported mechanical properties (cf. Table 1 in [4], Table 2 in [15]). PALF has been used successfully in reinforcing different polymer matrices [20–22] despite the much lower than expected mechanical properties [23] compared to those of flax [24]. So far, there has been no direct comparison in the reinforcement efficiency of the two fibers, and thus this is the main objective of the present work. They are used to reinforce bio-based poly(butylene succinate) (PBS), which provokes an intensive interest among industry and researchers [25,26] and has been employed in automotive applications [27]. Here, we seek to investigate the influence of fiber content on the performance of the resulting composite materials.

Through this comparative study, we aim to contribute to the growing body of research dedicated to sustainable materials and their potential applications. By examining the mechanical properties of flax and pineapple leaf fiber composites, we can gain insights into their suitability for various engineering and manufacturing applications while also addressing the urgent need for carbon sequestration.

#### **2. Materials and Methods**

#### *2.1. Materials*

Poly (butylene succinate) (PBS, BioPBS FZ91PM/FZ91PB), produced from the polymerization of bio-based succinic acid and 1,4-butanediol, was used as the polymer matrix. The material was supplied by PTT MCC Biochem Company Limited (Bangkok, Thailand) and had a density of 1.26 g/cm3 and a melt index of 5 g/10 min (190 ◦C, 2.16 kg). Its reported molecular weight (*M*w) was approximately 170 kDa [28].

Flax fiber (LINTEX, ~6 mm in length) was supplied by Dehondt Composites (Port-Jérôme-sur-Seine, France). According to the Alliance for European Flax-Linen and Hemp, the European flax is dew retted and mechanically scutched [29]. The fiber was supplied already cut to the specified calibrated length intended for composite reinforcement [30]. Pineapple leaf fibers (PALF, ~6 mm in length) were prepared from fresh pineapple leaves using the procedure presented in the literature [17]. Fresh pineapple leaves were collected from Bang Yang District, Phitsanulok Province, Thailand. The leaves were cut across their length into pieces 6 mm long, ground with a stone grinder, and dried to yield the whole ground leaf (WGL). The WGL was further processed by crushing it with a high-speed blender, followed by sieving to achieve the separation between the non-fibrous component and the PALF. The loose particulate non-fibrous component, with a particle size smaller than approximately 1 mm2, was able to pass through the sieve. In contrast, the curly and entangled PALF remained on the sieve, highlighting its distinctive physical properties. For visual reference, photographs of both PALF and flax fibers are presented in Figure 1.

#### *2.2. Composite Prepreg Preparation*

Prior to the melt-mixing process, all materials were dried overnight in a hot air oven at 80 ◦C. The PBS pellets were then heated and melted on a two-roll mill (W100T, Dr. Collin GmbH, Maitenbeth, Germany) for 2 min at a speed of 30 rpm. The front and back roll temperatures were 125 ◦C and 100 ◦C, respectively. Subsequently, a predetermined amount of fiber (10 and 20 wt.% of total weight (PBS + fiber)) was gradually added over a period of 3 min. The mixing speed was then increased to 48 rpm, and the mixing continued for another 10 min to achieve a homogenous molten mixture.

**Figure 1.** Photographs of (**a**) PALF and (**b**) Flax fibers.

The resulting molten mixture was carefully pulled out with slight stretching to maintain the alignment of the fiber parallel to the machine direction. It was then allowed to cool and solidify, forming prepreg, as illustrated in Figure 2. The composites were designated as 10PALF, 20PALF, 10Flax and 20Flax, denoting the respective content of the fiber in the composites.

**Figure 2.** Fiber alignment on a two-roll mill during the uniaxial composite prepreg preparation.

#### *2.3. Compressed Sheet Preparation*

Composite sheets were prepared by stacking ten layers of prepreg between two flat metal sheets and a 3 mm spacer to prevent the excessive flow of the material and the disturbance of the fiber alignment. The stacked prepregs were preheated for 5 min under slight pressure. Then, they were pressed under a pressure of 1500 psi for 5 min, followed by cooling under the same pressure for 5 min. The compression molding was carried out at a temperature of 140 ◦C to destroy the matrix orientation and allow only fiber contribution to be observed [31].

#### *2.4. Characterizations*

#### 2.4.1. Fibers' Chemical Composition

The chemical compositions of PALF and flax fibers were determined according to standard methods [32–35] through a certified local laboratory. Chemical composition is reported in terms of cellulose, holocellulose, acid-soluble lignin and acid-insoluble lignin.

The surface chemical compositions of PALF and flax fibers were observed using Fourier-transform infrared spectroscopy in an attenuated total reflectance mode (ATR-FTIR, Frontier, Perkin Elmer, Waltham, MA, USA). Spectra were recorded with 16 scans over the range of 4000 to 500 cm−<sup>1</sup> with a resolution of 4 cm<sup>−</sup>1.

#### 2.4.2. X-ray Diffraction

X-ray diffraction patterns of the composites were recorded using an X-ray Diffractometer (XRD) (D8 DISCOVER, Bruker AXS GmbH, Karlsruhe, Germany) over the 2θ range between 5◦ and 80◦ with a step size of 0.02◦. The X-ray wavelength was 1.54 Å (Ni-filtered CuKα). Pole figures for different samples were obtained with a cradle sample stage on the same machine. The data were analyzed with DEFFRAC.TEXTURE software (V4.1).

#### 2.4.3. Scanning Electron Microscopy (SEM)

Fibers' shapes and sizes and the fractured surfaces of composites were observed using a scanning electron microscope (JSM-IT500, JEOL, Tokyo, Japan) with an accelerating voltage of 10 kV. Prior to observation, a thin layer of platinum was coated on the samples.

#### 2.4.4. Thermal Properties

The melting and crystallization behavior of the composites were determined with a differential scanning calorimeter (DSC) (Q200-RCS90, TA Instruments, New Castle, DE, USA). The samples were first heated from 25 to 200 ◦C, held for 5 min to completely melt all the crystals, cooled to −70 ◦C and then heated again to 200 ◦C. The heating and cooling rate was 10 ◦C/min under a nitrogen atmosphere. The positions of the melting peak (*T*m), enthalpy of fusion (Δ*H*f) and crystallization peak (*T*c) were determined for each sample using the instrument software. The degree of crystallinity (*X*c) was calculated using Equation (1).

$$X\_{\rm C} = \left(\frac{\Delta H\_{\rm f}}{\Delta H\_{\rm f}^{0} \, (1 - W\_{\rm f})}\right) \times 100\% \tag{1}$$

where Δ*H*<sup>0</sup> <sup>f</sup> is the enthalpy of fusion for 100% crystalline PBS, which is taken as 110.3 J/g [36], and *W*<sup>f</sup> is the weight fraction of fiber in the composites.

In addition, the heat deflection temperature (HDT) was determined with a Gotech testing machine (HV-3000-P3C, Gotech Testing Machines Inc., Taichung City, Taiwan). The specimen sizes were 120 × <sup>13</sup> × 3 mm3. The test was performed following ASTM-D648 under the three-point bending mode with a span of 100 mm under a constant load of 0.455 MPa and a heating rate of 2 ◦C/min. HDT was determined as the temperature at which the specimen bends to 0.25 mm.

#### 2.4.5. Mechanical Properties

Flexural testing: The test was carried out on a universal testing machine (Instron 5569, Instron, High Wycombe, UK) at a crosshead speed of 5 mm/min, 1 kN of load cell and a support span length of 48 mm. The specimens were cut from compressed sheets into strips 12.7 mm wide with a long axis parallel to the machine direction. The average values of flexural strength and secant modulus at 1% stain from 5 specimens were reported.

Impact testing: The test was carried out on a pendulum impact testing machine (HIT5.5P, Zwick/Roell, Ulm, Germany) in Izod configuration. The impact specimens were cut from compressed sheets into strips 60 mm long and 12.7 mm wide. The samples were notched with a Zwick/Roell manual notch cutting machine. The notches were cut across the machine direction. Average values of 5 specimens were reported.

#### **3. Results**

#### *3.1. PALF and Flax Characteristics*

#### 3.1.1. Fiber Composition and Structure

Table 1 displays the chemical composition of PALF and flax fiber. In general, the chemical composition of the two fibers is very similar. PALF has a slightly higher holocellulose content than flax fiber, while flax has about 10% greater cellulose content than PALF. PALF also has about 1.5 times higher acid-soluble lignin content than flax. The greater content of hemicellulose (the difference between holocellulose and cellulose) is reflected in the FTIR spectra shown in Figure 3a, in which the peaks at 1731 cm−<sup>1</sup> and 1244 cm−<sup>1</sup> correspond to the C=O stretching of hemicellulose and lignin and the C–O stretching in lignin, respectively [37,38]. The peak at 2918 cm−<sup>1</sup> corresponds to the C–H stretching of methyl groups (–CH3) in both hemicellulose and cellulose [39].

**Table 1.** Chemical composition of PALF and flax fibers.


**Figure 3.** ATR-FTIR spectra (**a**) and XRD patterns (**b**) of PALF and flax fibers.

The lower hemicellulose content in flax is a result of the enzymatic degradation of the binding material during dew retting [14]. For PALF, only mechanical force was used in the preparation of the fiber. Therefore, not much material was removed.

The crystalline structures of PALF and flax fibers were investigated using X-ray diffraction techniques. The diffraction patterns are shown in Figure 3b. Both fibers exhibited a similar characteristic pattern to cellulose Type I [40]. However, the patterns differed significantly in the resolution and sharpness of the peaks; flax fiber has much sharper peaks than PALF. This indicates that the crystalline structure in flax fiber is more perfect and possibly larger than in PALF. This difference could be the main reason for the higher mechanical performance of flax fiber.

#### 3.1.2. Scanning Electron Microscopy (SEM)

Figure 4 compares the size and shape of the two fibers. PALF had both large bundles and fine elementary fibers. The fibers were not straight, but contained a lot of kinks. On the other hand, flax fibers were rather straight and had both isolated small fibers and bundles of fibers. Flax featured larger fiber bundles than PALF. Additionally, both PALF and flax fibers contained non-fibrous components. The variations in fiber size and shape can be attributed to the specific fiber preparation techniques employed.

**Figure 4.** SEM micrographs of (**a**) PALF and (**b**) flax fibers.

#### 3.1.3. Prepreg Appearance

Figure 5 displays the photographs of PALF/PBS and flax/PBS prepregs. PALF/PBS has a pale color, while flax/PBS is brownish with a much greater number of dark spots of non-fibrous components. Both PALF and flax fibers appear evenly dispersed throughout the prepregs, indicating thorough mixing and alignment, as highlighted by the dark lines within elongated red circles.

**Figure 5.** Optical images of composite prepregs: (**a**) PALF/PBS and (**b**) flax/PBS. Images (**c**) and (**d**) depict magnified views of the regions of interest indicated by squares in images (**a**) and (**b**), respectively. The red elongated circles highlight the alignment of fibers. Machine direction is vertical.

#### *3.2. Mechanical Properties of Composites*

#### 3.2.1. Flexural Properties

Figure 6 displays representative stress–strain curves of PALF/PBS and flax/PBS composites containing different fiber contents, and also that of PBS. With a fiber content of 10 wt.%, the stress at different strains increased over that of PBS throughout the whole range of strain. The composites had roughly similar failure strains to that of PBS. At 10 wt.% content, PALF/PBS and flax/PBS composites exhibited virtually the same behavior.

**Figure 6.** Representative flexural stress–strain curves of PALF/PBS and flax/PBS composites containing different fiber contents.

When the fiber content was increased to 20 wt.%, the stress for the flax/PBS composite increased slightly over that of 10 wt.%, and then the stress gradually decreased and the composite failed at a slightly lower strain than the composite with 10 wt.% fiber. The PALF/PBS composite with 20 wt.% fiber content exhibited a much-improved performance but failed at a much lower strain than the flax/PBS composite.

The average values for the flexural strength and flexural modulus of these composites are shown in Figure 7. The average flexural strength increased from 47 MPa to approximately 54 MPa for both PALF/PBS and flax/PBS composites containing 10 wt.% fiber, and to 70.7 and 57.8 MPa for PALF/PBS and flax/PBS with 20 wt.% fiber, respectively. A similar trend was observed for the flexural modulus. The average flexural modulus increased from 0.90 GPa to 1.25 GPa for both PALF/PBS and flax/PBS composites containing 10 wt.% fiber, and to 2.03 and 1.70 GPa for PALF/PBS and flax/PBS with 20 wt.% fiber, respectively.

The above results clearly indicate a better reinforcement efficiency of PALF over that of flax, despite its inferior mechanical properties. Doubling the flax content from 10 wt.% to 20 wt.% caused the flexural modulus to increase by approximately 10%, but it caused only a marginal change in flexural strength. PALF, on the other hand, caused both the flexural modulus and flexural strength to increase by approximately 62% and 31%, respectively, under a similar change. The reasons for this will be addressed later.

#### 3.2.2. Impact Properties

Figure 8 displays the notched Izod impact strengths of PALF/PBS and flax/PBS composites containing different fiber contents. The introduction of 10 wt.% of fiber to PBS resulted in an impact strength reduction to approximately 70% and 64% of that of PBS for PALF and flax, respectively. With a further increase in fiber content to 20 wt.%, the impact strength dropped even further, to approximately 62% and 54% of that of PBS for PALF and flax, respectively. This indicates that PALF contributes to a smaller reduction in the impact strength of the composite compared to flax fibers. The decrease in impact strength in natural fiber-filled polymers is an anticipated outcome due to the increase in material stiffness and the presence of stress concentrators within [41–43]. A more detailed discussion on the reason for the comparatively smaller reduction in impact strength in the PALF system will follow.

**Figure 7.** Flexural properties of PALF/PBS and flax/PBS composites containing different fiber contents, (**a**) flexural strength and (**b**) flexural modulus at 1% strain. Gray bar represents neat PBS.

**Figure 8.** Impact properties of PALF/PBS and flax/PBS composites containing different fiber contents. Gray bar represents neat PBS.

#### *3.3. Thermal Properties*

#### 3.3.1. DSC

The melting and crystallization behavior of PBS in the composites is shown in Table 2. The presence of both PALF and flax fibers has a negligible effect on the melting temperature (*T*m), degree of crystallinity (*X*c), and crystallization temperature of PBS (*T*c). Thus, it may be stated that both PALF and flax fibers do not influence matrix crystallization, similar to the results observed in other systems [44,45].


**Table 2.** Thermal properties of PBS/PALF and PBS/flax composites.

#### 3.3.2. HDT

Figure 9 displays the heat distortion temperature (HDT) of the composites along with the base PBS. At 10 wt.%, both PALF and flax had a negligible effect on HDT. However, when the fiber content was increased to 20 wt.%, PALF caused a larger increase than flax fiber, being approximately 10 ◦C and 6 ◦C higher than that of the base matrix. This is the consequence of the increase in flexural modulus of the respective materials.

**Figure 9.** HDT of PALF and flax composites containing different fiber contents. Gray bar represents neat PBS.

#### *3.4. Fracture Surfaces*

Figure 10 shows the impact fracture surfaces of PALF/PBS and flax/PBS composites containing different fiber contents. Broken fibers are seen end-on, indicating a good alignment of the fibers along the machine direction (toward the observer). For 10 wt.% fiber, a larger number of fiber bundles can be seen in the PALF/PBS composite compared to the flax/PBS composite. When the fiber content was increased to 20 wt.%, a smaller number of large fiber bundles could be observed, indicating the breaking of large fiber bundles into finer elementary fibers. This phenomenon is likely due to the increase in the viscosity of the mixture (resulting from the higher fiber content), which facilitates higher stress transfer and thus breaks the bundles into finer elementary fibers.

**Figure 10.** Impact fracture surfaces of PALF/PBS and flax/PBS composites containing different fiber contents.

#### **4. Discussion**

The nearly identical curves seen for 10 wt.% PALF/PBS and flax/PBS in Figure 6 signify a remarkable parity in reinforcing efficiency between the two types of fibers, even amidst their distinct mechanical properties. Intriguingly, at a higher fiber content of 20 wt.%, PALF exhibited significantly greater reinforcing efficiency than flax fiber. These findings merit a more in-depth examination.

It is known that for short fiber composites, the mechanical behavior of the composite is determined by several factors, including the mechanical properties of the reinforcing fiber, its orientation, the fiber aspect ratio, the fiber volume fraction, and the nature of the interface between the fiber and the matrix [46]. While we kept most starting parameters of the two types of fibers as close as possible, such as their length, amount, and mixing procedure, the only known parameter that was different was the mechanical properties of the fibers, with flax having much superior values. Surprisingly, this difference in mechanical properties

alone does not fully explain the stark difference in reinforcing efficiency. Therefore, a deeper analysis of the internal structure of the composites, including matrix structure, matrix orientation, and fiber dimension, is required.

#### *4.1. Matrix Orientation via Pole Figures*

The production method for uniaxial prepreg employed in this work could lead to matrix orientation [31]. This had been destroyed during the compression molding by using a high compression molding temperature, as previously described. XRD was used to confirm this. Figure 11a displays the XRD patterns of composite prepregs and sheets. Prepregs display very strong intensity, while the sheets show much less intensity, indicating much relaxation of the polymer matrix. Pole figures for all samples were then determined using the most intense peak, at around 22.7◦, which was associated with the (110) plane of PBS crystalline [47].

**Figure 11.** (**a**) XRD patterns of composite prepregs and sheets; (**b**) X-ray pole figures for (110) plane of PALF/PBS and flax/PBS composite prepregs and sheets compressed at 140 ◦C.

Pole figures for all samples for the (110) plane are shown in Figure 11b. It is clearly evident that the peak intensity of the prepregs is concentrated in the center, indicating a preferred matrix orientation in all prepregs. The presence of a high-intensity region supports the fact that the (110) reflection of the drawn PBS film lies on the equator [48]. However, when the sample was compressed at 140 ◦C, the previous orientation disappeared. These results are consistent with the previous XRD and DSC findings. Notably, with an increased fiber content in the sample compressed at 140 ◦C, a relatively weak molecular orientation can still be observed in the case of PALF/PBS. This suggests that the presence of fibers could slow down the relaxation of the matrix in the vicinity of the fiber, as suggested in the literature [31]. However, it could be assumed that such a marginal orientation of PBS in the PALF/PBS composite would play no role in enhancing the PALF/PBS composite over that of the flax/PBS composite (cf. Figure 7).

#### *4.2. Reinforcing Fiber in the Matrix*

It has been reported that fibers such as jute, flax [49], PALF [50], kenaf [51], poplar wood, radiata pine, and rice husk [52] can break down during incorporation into a polymer matrix, resulting in a lower reinforcing efficiency. To determine whether such a situation had occurred, the fibers were extracted from the composites using hot chloroform. Figure 12 displays optical images (Olympus BX51TRF, Olympus Optical Co. Ltd., Tokyo, Japan) of PALF and flax fibers that were extracted from PALF/PBS and flax/PBS composites with 20 wt.% fiber. It is clear that PALF remained long, while flax broke into very short pieces. In both cases, fine elementary fibers and large bundles can be seen. Thus, it is unquestionable that such fragmented flax fibers would not be able to reinforce the composite effectively. PALF, which remains long, can still effectively reinforce the composite [53]. The longer PALF also gives composites with higher impact strength and HDT (cf. Figures 8 and 9). This observation supports our previous works, where PALF has been shown to outperform short Kevlar in reinforcing rubber matrices [22], and with an appropriate adhesion promoter the effectiveness can be further improved [54].

**Figure 12.** Low magnification optical micrographs of (**a**) PALF and (**b**) flax fibers after solvent extraction from their respective 20 wt.% composite prepregs.

As stated above, all kinds of fiber are prone to breakage during compounding with polymer matrices due to different breakage mechanisms [55] depending on the fiber characteristics, and this includes PALF. It can be easily envisaged that by reducing the stress involved during compounding, either by increasing the temperature or reducing mixing speed, the breakage could be reduced or minimized. Mixing with a two-roll mill involves a much lower shear stress than with an internal mixer or screw extruder. It is clear from the results above that flax fiber still breaks, while PALF does not. The fact that PALF can maintain its length during mixing with thermoplastics and provide a high reinforcement

efficiency certainly encourages its use in this form. Given that the starting length is long enough, and large fiber bundles break into finer elementary fibers during mixing, resulting in a significantly increased aspect ratio, composites with greatly improved properties can be obtained. Moreover, the utilization of PALF offers a promising ecological advantage. Compared to purposely cultivated fibers (such as flax, hemp, and kenaf), PALF exhibits lower carbon emissions and a reduced environmental footprint. Additionally, the use of PALF contributes to sustainable waste management practices by repurposing agricultural waste, making it a more environmentally friendly alternative for composite reinforcement. These ecological benefits further underscore the potential of PALF as a viable and ecoconscious solution in advancing sustainable materials across various industries, especially those that require higher performance or thinner parts.

#### **5. Conclusions**

In this study, we compared PALF with cultivated flax fiber as natural reinforcements in unidirectional PBS matrix composites. PALF showed remarkable potential as a sustainable alternative to flax fiber, well known for its high mechanical properties. PALF's ability to maintain length and integrity during mixing led to significant improvements in the flexural strength and modulus, particularly at 20 wt.% fiber content. Successful PALF dispersion in the matrix, along with fiber bundle disintegration, resulting in higher aspect ratio, further contributed to its superior performance. PALF offers valuable ecological benefits, with a lower carbon footprint and the utilization of agricultural waste. The study highlights PALF's underexplored potential as a sustainable and high-performance natural reinforcement, paving the way for eco-friendly materials in various industries. PALF's effective reinforcement and ecological advantages suggest that it is a promising candidate for developing sustainable and eco-friendly materials.

**Author Contributions:** Conceptualization, T.A. and K.M.; methodology, S.D., T.A. and K.L.G.; validation and data curation, S.D.; writing—original draft preparation, S.D. and T.A.; writing—review and editing, T.A., K.L.G. and K.M.; funding acquisition, T.A. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Mahidol University (Basic Research Fund: fiscal year 2022; Grant no. BRF1-046/2565).

**Institutional Review Board Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** This research project is supported by Mahidol University (Basic Research Fund: fiscal year 2022; Grant no. BRF1-046/2565). We thank Mahidol University Frontier Research Facility (MU-FRF) for instrument support and the MU-FRF scientists, Nawapol Udpuay, Chawalit Takoon and Suwilai Chaveanghong, for their kind assistance in the operations of SEM and XRD.

**Conflicts of Interest:** T.A. declares a conflict of interest related to this research article: As a University Professor at Mahidol University, I have been engaged in developing an innovative method for extracting pineapple leaf fiber. This endeavor led to the establishment of TEAnity Team Co. Ltd., where I hold the position of Chief Technology Officer (CTO). While this research was conducted independently and supported solely by university funding, I acknowledge the potential for a conflict due to my dual roles. I affirm that the research and findings presented in the article remain unbiased and have not been influenced by any financial interests or affiliations with TEAnity Team Co. Ltd. The remaining authors declare no conflicts of interest.

#### **References**


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### *Article* **Durability against Wetting-Drying Cycles of Sustainable Biopolymer-Treated Soil**

**Antonio Soldo <sup>1</sup> and Marta Miletic 2,\***


**Abstract:** The world today is more oriented towards sustainable and environmental-friendly solutions in every field of science, technology, and engineering. Therefore, novel sustainable and eco-friendly approaches for soil improvement have also emerged. One of the effective, promising, and green solutions is the utilization of biopolymers. However, even though the biopolymers proved to be effective in enhancing the soil-mechanical properties, it is still unknown how they behave under real environmental conditions, such as fluctuating temperatures, moisture, plants, microorganisms, to name a few. The main research aim is to investigate the durability of biopolymer-improved soil on the cyclic processes of wetting and drying. Two types of biopolymers (Xanthan Gum and Guar Gum), and two types of soils (clean sand and silty sand) were investigated in this study. The results indicated that some biopolymer-amended specimens kept more than 70% of their original mass during wettingdrying cycles. During the compressive strength analysis, some biopolymer-treated specimens kept up to 45% of their initial strength during seven wetting-drying cycles. Furthermore, this study showed that certain damaged soil-biopolymer bonds could be restored with proper treatment. Repeating the process of wetting and drying can reactivate the bonding properties of biopolymers, which amends the broken bonds in soil. The regenerative property of biopolymers is an important feature that should not be neglected. It gives a clearer picture of the biopolymer utilization and makes it a good option for rapid temporary construction or long-standing construction in the areas with an arid climate.

**Keywords:** biopolymer-treated soil; Xanthan Gum; Guar Gum; soil strength; durability; cyclic wetting-drying

### **1. Introduction**

The expansion of cities often causes the need to construct in an unfavorable environment and on soils with undesirable mechanical characteristics. As a solution, soil's engineering properties can be improved by adding different chemical additives. Currently, cement is one of the most commonly used additives. However, the use of cement raises a series of environmental problems from which the contribution to CO2 concentration on the planet is the most concerning. From the data in 2016, the production of cement contributes approximately 7.4% to the world's CO2 emissions [1]. Furthermore, the use of cement can irreversibly affect the urban environment. Increased urban water runoff, vegetation growth prevention, and heat islands are some of the side effects of using cement as soil stabilizer [2]. Therefore, the need for a sustainable, green, and effective solution for enhancing soil characteristics is continuously increasing.

New bio-inspired solutions for the improvement of mechanical characteristics of soil, such as biopolymer-soil mixtures, are proved to be quite effective [2–6]. A biopolymer is a chain of smaller molecular units extracted from nature-made materials, such as wood, vegetable, algae, and animal shells. To the best of the authors' knowledge, no negative

**Citation:** Soldo, A.; Miletic, M. Durability against Wetting-Drying Cycles of Sustainable Biopolymer-Treated Soil. *Polymers* **2022**, *14*, 4247. https://doi.org/ 10.3390/polym14194247

Academic Editor: Magdalena Czemierska

Received: 14 September 2022 Accepted: 8 October 2022 Published: 10 October 2022

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

effect of biopolymers on the environment has been reported. Throughout recent history, biopolymers were used in the food industry, the cosmetic industry, medicine, and agriculture [7–11]. In previous research, it was found that biopolymers, such as xanthan gum, guar gum, beta-glucan, and chitosan, can improve the strength of soil [5,6,12–15]. In addition, some biopolymers proved effective in reducing the collapsibility of soil [16] and erosion [17–19].

However, the main concern is the durability of biopolymer-amended soils while being exposed to environmental conditions such as wind, moisture, and temperature fluctuations. Kavazanjian et al. [18] investigated the effect of wind on erosion properties of the biopolymer-amended soil. Biopolymer emulsion was sprayed on the surface of the soil, and wind flow was blown over the soil surface. The major finding was that biopolymers could reduce wind-induced detachment of soil particles, but that ultraviolet radiation and heat can diminish their effect.

To date, the research on the durability of biopolymer-amended sand remains limited and insufficiently investigated. Chang et al. [20] explored the properties of biopolymeramended sand against cyclic wetting-drying. They performed a series of unconfined compression tests on gellan gum-improved sand specimens after each wetting and drying cycle. Chen et al. [21] performed a series of direct shear tests on xanthan gum-improved sand. Both of the above-mentioned research studies have found that the strength of biopolymer-amended sand ultimately decreases due to cyclic wetting and drying. Some limitations of each of the mentioned studies are that one type of testing was conducted and the soil (sand) was amended with only one type of biopolymer. Some additional research in the field of cyclic wetting-drying of biopolymer-treated soil is presented in Table 1.


**Table 1.** Previous research related to wetting-drying of biopolymer-treated soil.

The main research aim of our study is to investigate the durability of biopolymerimproved soil on the cyclic processes of wetting and drying. Two soil types were investi-

gated in this study, clean sand and silty sand. Additional testing variables were biopolymer type and concentration. In particular, the soil was treated with two types of biopolymer (Xanthan Gum and Guar Gum) at three different biopolymer concentrations (0.5%, 1%, 2%). Furthermore, plain and biopolymer-amended specimens were tested under two types of water-durability tests. Considering that Xanthan Gum and Guar Gum emerged as biopolymers with high potential for soil stabilization, the investigation of their durability will have a significant impact on their utilization in civil engineering practice.

#### **2. Materials and Methodology**

#### *2.1. Base Soil*

To investigate the effect of the soil type on the biopolymer-amended soil durability, two types of soil were investigated in this study: silty sand (SM), and poorly graded sand (SP). The soils were classified according to the following standards: ASTM D6913-17— Standard Test Methods for Particle-Size Distribution of Soils Using Sieve Analysis [25], and ASTM D4318-17—Standard Test Methods for Liquid Limit, Plastic Limit, and Plasticity Index of Soils [26].

#### 2.1.1. Silty Sand

From the grain size distribution curve (Figure 1), the concentration of fine particles was 39% with a liquid limit of 49, a plastic limit of 29, and an index of plasticity of 20. According to the Unified Soil Classification System (USCS), the soil is classified as silty sand (SM).

**Figure 1.** Grain size distribution of the soils used in this study.

#### 2.1.2. Clean Sand

The sand was characterized by a high percentage of quartz and high uniformity. The coefficient of uniformity and coefficient of curvature were calculated as 1.46, and 0.93, respectively (Figure 1). The percentage of fine particles was below 5%. Therefore, the soil was classified as poorly graded sand (SP), according to USCS.

#### *2.2. Biopolymers*

To study the influence of the biopolymer type on the biopolymer-amended soil durability, two types of biopolymers were used in this study: Xantham Gum and Guar Gum.

#### 2.2.1. Xanthan Gum

Xanthomonas campestris bacterium creates the biopolymer polysaccharide Xanthan Gum (XG) by inducing the fermentation of a medium containing carbohydrate, such as glucose. In other words, XG is a long-chain polysaccharide having d-glucose, d-mannose, and d-glucuronic acid as building blocks in a molecular ratio of 3:3:2 with a high number of trisaccharide side chains [27]. Dissolving XG in hot or warm water creates non-Newtonian solutions with high pseudoplasticity. XG can be found in the cosmetic and food industry, agriculture, and oil drilling industry [9], and it has been researched for civil engineering purposes [5,6,28,29].

#### 2.2.2. Guar Gum

Guar Gum (GG) is a galactomannan polysaccharide extracted from Cyamopsis Tetragonolba, known as guar beans or guar. Chemically, a GG biopolymer mainly consists of a highmolecular-weight polysaccharide galactomannan, which is based on a mannan backbone with galactose side groups. The ratio of the two building blocks in a molecular ratio seems to vary slightly depending on the origin of the seed, but the gum is generally considered to contain approximately one galactose building block for every two mannose building blocks [30]. In addition, GG shares certain similarities with XG. For instance, it can be dissolved in hot and cold water, and in the industry is used for similar purposes as XG. It can be found in cosmetic products, food products, oil, and gas drilling industries [30], and it has been researched in civil engineering [6,31–34].

#### *2.3. Specimen Preparation*

The dry base soils were placed in a metal dish and combined with biopolymer powders until uniformly mixed. The biopolymer concentrations used in this study were 0.5, 1, and 2% with respect to the mass of the plain soil. After carefully mixing the dry components (soil and biopolymers), water was added to the mix by spraying and constant stirring. The targeted water content was 16.5% for silty sand and 12% for the clean sand.

After achieving a uniform mixture, the soil-water-biopolymer mass was placed into molds. Two types of molds (Mold A and Mold B) were used for two different parts of this study.

Mold A was cylindrical with a diameter of 10.2 cm and a height of 11.6 cm. Silty sand was placed into the Mold A in three lifts and it was compacted with a hammer with a weight of 2.5 kg (Proctor hammer). The same type of mold is typically used for compaction efforts for ASTM D559 and ASTM D698. Each lift was compacted by releasing the hammer 25 times from the height of 30.5 cm. After each lift, the surface was scarified to achieve a better bond within the soil sample. Proctor hammer was omitted for the sand material due to its nature. Sand had a low concentration of fine particles that would have hindered the proper compaction if excessive compaction force was applied. Therefore, sand was carefully tapped into Mold A. Additionally, tapping the sand material into the mold kept the density of the biopolymer-treated sand close to its natural density. Specimens prepared in Mold A were used for durability testing during cyclic wetting and drying.

Mold B was cubical, with the inner dimensions of 5 cm. Cubical specimens were used for the testing of the compressive strength changes through the wetting-drying cycles. The specimens made out of silty sand were compacted with a metal rod in four lifts. Each lift was pressed 25 times. Sand specimens were gently tapped into the Mold B due to the aforementioned reasons relating to the nature of sand.

All specimens were air-dried in the laboratory at the temperature of 21 ◦C for five days (cubical specimens, Figure 2a) and seven days (cylindrical specimens, Figure 2b) to increase the biopolymer-soil strength and cure the specimens.

**Figure 2.** Photos of specimens for (**a**) unconfined compression, and (**b**) durability tests.

In addition, specimens made of plain silty sand were prepared in the same manner as the specimens with biopolymer additives. The plain specimens were used for comparison with the biopolymer-treated ones. The samples of the plain sand could not be made because the plain sand used in this research had no cohesion. Therefore, it could not be shaped to the desired dimensions.

#### *2.4. Testing*

#### 2.4.1. Durability

The durability testing during cyclic wetting and drying was performed by the guidance of the ASTM D559—Standard Test Methods for Wetting and Drying Compacted Soil-Cement Mixtures [35]. Since ASTM D559 was originally designated for cemented soil, this study introduced certain modifications to the procedure described in ASTM D559. After compaction and air-drying, we measured the mass of specimens and submerged them in water for one hour. The specimens' mass was measured again after one hour, and specimens were placed in the oven at the temperature of 70 ◦C for 24 h. After 24 h, samples were taken out of the oven, gently stroked by a brush to remove all loose material, and weighed again before submerging them into the water. The same process was repeated ten times, where one hour in water and 24 h in the oven represents one wetting-drying cycle. This procedure was performed on XG-treated sand, XG-treated silty sand, and GG-treated silty sand. The cylindrical sand specimens with GG degraded after one hour in the water. Therefore, they could not be used for the continuation of the experiment. A similar degradation process happened with the cylindrical specimen of the plain silty sand. They degraded after one hour in the water. Thus, the durability testing of plain silty sand could not be continued.

#### 2.4.2. Unconfined Compression Test

The unconfined compression test is a widely used test to determine the compressive strength of cohesive materials. The test was performed on the plain and biopolymeramended silty sand. Moreover, it was performed on the XG-improved sand, whereas untreated sand did not have any cohesion, which was required for this type of test. In addition, sand specimens with GG were not testable for this type of experiment due to their low resistance to water. The unconfined compression test was performed on cubical specimens five days after the preparation and air-drying. Three samples were compressed with an axial strain rate of 1.5%/min, which is in agreement with the ASTM D2166— Standard Test Method for Unconfined Compressive Strength of Cohesive Soil [36]. The remaining specimens were submerged in water at room temperature (21 ◦C) for 20 min. They were subsequently dried in the oven at 70 ◦C for 24 h. This process is referred to as

one wetting-drying cycle for unconfined compression specimens. Cubical samples of plain silty sand were not tested in the unconfined compression test after wetting and drying due to their degradation in water. After each wetting-drying period, three specimens were tested in the unconfined compression test, while the remaining samples were placed back in the water for 20 min. For the specimens with XG, seven cycles of wetting and drying were carried out through seven days. The unconfined compression test was performed after each cycle except for the fourth and sixth. Most of the cubical specimens with GG were heavily damaged after the first 20 min in water. Therefore, the remaining specimens with GG went through two or three cycles of wetting and drying.

Furthermore, the healing potential of the XG biopolymer was also investigated by wetting and re-testing previously loaded specimens after their first unconfined compression test. XG-sand cubes were placed in the water for 20 min after they were broken in the unconfined compression test for the first time. They were subsequently placed back in the oven and re-tested for the unconfined compression. The repeated cycle of wetting and drying was conducted to reactivate XG molecules and investigate their regenerative properties on the treated sand.

#### **3. Results and Discussion**

#### *3.1. Durability*

The durability of the soil was observed through the change of mass through cyclic wetting and drying. The change of mass of the biopolymer-treated soil was calculated for each cycle by the following equation:

$$\text{"\%} \sim \text{loss} = (\text{A/B}) \times 100 \tag{1}$$

where *A* is the mass of the soil after each cycle; *B* is the mass of the dry soil after seven days of curing in the air. Several samples were placed in the oven after the preparation and dried for 24 h at 70 ◦C. Those samples were not tested but they were used for comparison in mass with specimens that were air-dried for 7 days. The differences in mass for the same biopolymer–soil mix were between 2% and 7%. Therefore, we decided that the equation above would be appropriate for the analysis of the durability data. A visual representation of sample degradation through time is shown on biopolymer-treated silty sand in Figure 3.

**Figure 3.** Degradation of silty sand treated with 1% XG (**upper row**) and 1% GG (**bottom row**) due to cyclic wetting and drying.

Figure 4 shows the results of the mass percentages of biopolymer-soil remaining after each cycle. Plain soil samples degraded when submerged under the water for one hour and could not be tested in the designed experiment. The results from Figure 4 indicate that

the presence of biopolymers slowed the degradation process for both types of soil. Figure 4 also indicates that the resistance of the soil to cyclic wetting and drying depends on the type of the soil, type of biopolymer additive, and concentration of biopolymer additive. Different biopolymer types, like different soil types, show different reactions with water, which affects the behavior of the soil–biopolymer mixture when exposed to water.

Interestingly, the silty sand with XG showed the best resistance for cyclic wetting and drying at a concentration of 1% XG (Figure 4a). At that concentration, the specimens kept most of the mass up to the sixth wetting-drying cycle. After that, the loss of mass was more noticeable. The same soil type with 0.5% and 2% XG lost more soil mass which indicates that 1% XG could be the optimal water-resistance concentration for this type of soil. There are two reasons behind this: the binding properties of XG and the absorptive properties of the composite material (silty sand and XG). XG is a glue-like binding agent that bridges soil particles. In the mixture with 0.5% XG, soil particles have a weaker biopolymer bond when compared with mixtures with 1 and 2% XG. Therefore, samples with 0.5% XG lost approximately 65% of their initial mass after the first wetting cycle. The plain specimens of silty sand had the weakest particle bond which is the reason they degraded after the first cycle. On the other hand, a question emerges concerning why samples with the highest XG concentration (2%) did not show the best water resistance. The reason for that is the aforementioned absorptive properties of silty sand and XG. The XG attracts and binds water molecules, which further increases the absorbing potential of already swelling plain soil. In other words, the higher presence of XG caused more trapped water. Therefore, to achieve the same water content after each drying for the specimens with 1 and 2% XG, specimens with 2% XG would require a higher drying temperature or longer drying time. The constant higher presence of water can cause the reduction of negative pore pressures that can lower the apparent cohesion and cause the degradation of the soil mass. That resulted in the faster degradation of specimens with 2% XG. However, the loss of the mass under all biopolymer concentrations was significantly reduced when compared with the loss in mass of the plain soil.

In the case of silty sand amended with GG (Figure 4b), the specimens with higher concentrations have kept more of their soil mass during ten cycles of wetting and drying. When compared with higher concentrations, the samples treated with only 0.5% GG had significantly higher losses of mass between each cycle. Unlike the samples with 1% XG, the samples with 1% GG had a slight gradual loss of mass after each wetting-drying cycle. The samples with 2% GG showed a slight mass increase through the cyclic wetting-drying. That is because of the absorptive properties of silty sand and GG, similar as XG-treated silty sand. The samples with 2% GG lost some amount of soil during wetting-drying, but they also absorbed some water that did not completely evaporate during 24 h in the oven at 70 ◦C. Therefore, due to the higher absorptive properties of GG at 2% than at 0.5 and 1%, a longer period of drying or a higher drying temperature would be more appropriate for silty sand with 2% GG. Both GG and XG demonstrate a water-absorption nature. However, comparing results of silty sand with 2% GG and 2% XG indicates that GG chains release water molecules somewhat easier than XG chains. Even though XG and GG need to absorb water to activate their bonds with soils, releasing water molecules stiffens the soil–biopolymer bond, which gives GG-treated silty sand an edge over the XG-treated silty sand at a concentration of 2%.

For the clean sand specimens amended with XG (Figure 4c), the loss of mass is relatively low throughout the testing for higher concentrations when compared with silty sand. The reason for that is the fact that the sand has a higher porosity and lower water absorption capacity than silty sand. In other words, water absorption happens only due to the presence of XG. Higher porosity makes the water evaporation relatively faster in the sand than in silty sand. The cementitious effect of hardened XG gave the sand relatively good resistivity to water. However, sand with only 0.5% XG went successfully through only six cycles of wetting and drying before it became wholly degraded.

**Figure 4.** Change in mass for (**a**) Silty sand—XG, (**b**) Silty sand—GG, (**c**) Clean sand—XG.

#### *3.2. Unconfined Compression Test*

Figure 5 shows the relationship between the compressive strength and the number of wetting and drying cycles for silty sand (Figure 5a,b) and clean sand (Figure 5c). In all figures, the first point, at cycle zero, represents the compressive strength of specimens tested after five days of air-drying in the laboratory at room temperature. The plain silty sand samples degraded after 20 min in water and could not be tested through cyclic wetting and drying (Figure 5a,b). Plain clean sand samples were not testable because of non-existing cohesion that was needed to fabricate the specimens for this type of testing (Figure 5c). Both types of soil showed fast degradation in water for 0.5% of additives. Therefore, soils

amended with 0.5% of biopolymers could not be used for a detailed comparison with soil amended with higher biopolymer concentrations. The exception was the sand treated with 0.5% XG that showed a slightly higher level of water resistance (Figure 5c).

**Figure 5.** Change in compressive strength for (**a**) Silty sand—XG, (**b**) Silty sand—GG, (**c**) Clean sand—XG.

The change in the compressive strength of biopolymer-improved soil with wettingdrying cycles strongly depends on the biopolymer concentration, biopolymer type, and water content. Lower biopolymer concentration in soil results in a reduced number of biopolymer links and subsequent biopolymer–particle bonding. In other words, soils with a lower biopolymer concentration will have smaller compressive strength. On the other hand, a higher percentage of biopolymer causes greater water absorption. The decrease in the water content and degree of saturation increases the surface tension forces between soil particles, which subsequently increases the soil strength (Figure 6). It is noticeable that the biopolymer bond started to weaken after the first wetting and drying cycle because of constant water absorption and the thinning of the biopolymer links. For the specimens made out of the silty sand mixed with 2% XG, the increased biopolymer concentration resulted in higher water absorption than samples with 1% XG. The higher presence of the trapped water caused a more rapid decrease in the compressive strength because of reduced tension forces and loosened biopolymer links.

**Figure 6.** Interaction of water and soil particles: (**a**) a higher degree of saturation—lower surface tension forces, (**b**) lower degree of saturation—higher surface tension forces.

Figure 5b shows the decrease in the compressive strength for GG-treated silty sand with wetting-drying cycles. The vast majority of the 1% GG-treated cubicles were severely damaged and unusable for the unconfined compression test. Therefore, undischarged specimens went under two cycles of wetting and drying, where the change of their compressive strength was investigated. It is noticeable that after two cycles of wetting and drying, the compressive strength of 1% GG-treated cubicles decreased by 75%. In the case of 1% XG-treated silty sand, the decrease of the compressive strength by 75% would be estimated to happen after the fourth cycle of wetting and drying. The specimens with 2% GG showed better resistivity to water and higher strength through cyclic wetting and drying. The first points, at cycle zero, which represent the specimens after five days of air drying, indicate lower strength for the specimens with 2% GG. This trend was already observed with the samples treated with XG. The reason behind that is that higher concentrations of biopolymer need more air-drying time to completely harden and achieve the maximum strength. The same phenomena happened for the treated sand as well (Figure 5c).

Figure 5c represents the change of the compressive strength of XG-treated sand with wetting-drying cycles. During seven cycles of wetting and drying, sand with 1% XG kept 46% of the initial strength, while the sand with 2% XG kept 75% of the initial strength. However, the biopolymer-amended sand samples did not sustain their shape and strength at the lowest biopolymer concentration. At the concentration of 0.5% XG, they lost 70% of the initial strength after the second cycle and completely degraded during the third cycle.

#### *3.3. Regenerative Properties of Biopolymers*

Xanthan Gum is one of the partially reversible bond-based biopolymers. That means that it can be brought to the previous state by reapplying the processes that initially induced the change of that state. That reversible nature of XG was examined in biopolymer-treated sand samples that were tested under the unconfined compression test. After the third wetting-drying cycle, that was used to investigate the change in the compressive strength. The broken specimens were used to investigate the healing properties of XG. The broken specimens were submerged for 20 min and dried in the oven for 48 h, as described previously. After that, the same specimens were tested again under the unconfined compression test. The same process was repeated one more time. The results of two XG-treated sand specimens are summarized in Figure 7. The repeated process of wetting-drying stiffened the soil-biopolymer bond, and the XG-treated sand specimen regained some level of the initial strength, which is presented in Figure 7. Higher magnitudes of the compressive strength and the level of the regained strength were achieved for the higher concentration of XG. That is not surprising since a higher concentration of XG causes faster and broader linking of XG molecules with each other and with the surrounding sand particles.

**Figure 7.** Compressive strength of regenerated sand XG-treated specimens.

That reversible nature of XG is schematically represented in Figure 8. The sand particles bonded by XG-links (Figure 8a) broke during the unconfined compression test Figure 8b). After the broken specimens of XG-treated sand were put back together and submerged in water, the XG linkages loosened their structure, which allowed them to interact with the nearby sand particles again (Figure 8c) and mend the broken bonds (Figure 8d).

**Figure 8.** Healing cycle of biopolymer-treated sand: (**a**) sand particles bonded by XG-links, (**b**) breakage of the dry XG-links due to the applied mechanical loading, (**c**) XG- linkages loosen their structure in the contact with water which allows them to interact with the nearby sand particles; and (**d**) sand particles bonded again by new XG-links.

#### **4. Conclusions**

Recently, biopolymers XG and GG have been shown to be promising environmentally friendly soil stabilization additives. However, they are prone to environmental influence, especially moisture changes. To best to the authors' knowledge, the previous research studies have not comprehensively investigated the effect of wetting-drying cycles on the strength and mass loss of the different biopolymer-treated soils. Therefore, the main aim of this study is to investigate the effect of wetting-drying cycles on the strength and mass loss of the biopolymer-stabilization. The types of soil used in this study were: silty sand and pure sand. In addition, two types of biopolymers (xanthan gum, and guar gum) and three biopolymer concentrations (0.5%, 1%, 2%) were used as testing variables in this research.

The first experimental study was focused on observing the change of the mass of the plain and biopolymer-treated soil during cyclic wetting and drying. It was shown that XG reduces the loss of mass for both tested soil types, while GG was only effective when mixed with silty sand. For the silty sand, the most effective concentration of XG to reduce the mass loss during the cyclic wetting and drying process was found to be 1%. The highest used concentration of XG (2%) caused higher entrapment of water, which ultimately led to faster loss of mass. On the other hand, the lowest concentration of XG (0.5%) resulted in too weak biopolymer-soil bonds, which degraded faster. That points to an optimum concentration of XG that works the best with a certain type of soil. For the GG-treated silty sand, the loss of mass was more prominent for lower concentrations. For the XG- treated sand, the loss of mass was relatively low for concentrations of 1% and 2%, which can be explained by higher porosity of sand, which makes water evaporation easier in comparison to the silty sand. A low concentration of 0.5% XG caused weak bonding between soil particles that rapidly degraded.

The second experiment investigated the change of the compressive strength of biopolymertreated soil with wetting-drying cycles. XG proved able to reduce the loss of compressive strength in the silty sand and sand, while GG was only mildly effective in the silty sand. However, the increase in the GG concentration reduced strength loss. The concentration of 1% XG was more effective than 2% in reducing the strength loss in the silty sand due to higher water absorption for higher concentrations of XG. The XG-treated sand showed extremely good resistivity to the loss of the compressive strength through cyclic wetting and drying. The higher concentrations of XG resulted in the higher compressive strength of sand. The concentration of 0.5% XG and GG was shown to be mildly or non-effective for the proposed type of testing.

The broken XG-treated sand specimens were re-submerged, dried, and subsequently tested in the unconfined compression test to study the healing properties of XG-treated sand. It was shown that re-wetting and drying could restore some level of the compressive strength of XG-treated sand. The reason behind it is the regenerative nature of XG, which loosens its structure in water and re-attaches to the nearby soil particles. The healed soilbiopolymer bond stiffens while the sample is subsequently dried and mends the cracks in sandy specimens. Sand samples with higher concentrations of XG were shown to regain more of their lost strength.

This research study showed that, even though biopolymers tend to be susceptible to water, certain biopolymer types and concentrations can significantly increase the durability of soil to water. It was also shown that the presence of water could activate the regenerative properties of XG, which accentuates its potential for soil stabilization. This research gives a clearer picture of XG and GG utilization, presenting a good option for rapid temporary construction (e.g., embankments) or long-standing construction in areas with an arid climate. The degradation of XG- and GG-treated soil due to longer exposure to water points to a practical way of disposing of the temporary construction elements that are made of the mentioned materials. Due to the non-hazardous nature of these biopolymers, watering and decomposing the XG- and GG-treated soil should not raise environmental concerns. XG and GG also showed favorable characteristics that can be utilized in dust control, erosion, and subgrade stabilization. However, since the water susceptibility of biopolymers is an important factor for their use in industry, this field of research still requires a significant amount of investigation.

**Author Contributions:** Conceptualization, M.M.; methodology, A.S.; validation, M.M.; formal analysis, M.M., A.S.; writing—original draft preparation, A.S.; writing—review and editing, M.M.; supervision, M.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


### *Article* **Durability, Strength, and Erosion Resistance Assessment of Lignin Biopolymer Treated Soil**

**Pouyan Bagheri 1,\*, Ivan Gratchev 1, Suwon Son <sup>2</sup> and Maksym Rybachuk 3,4**


<sup>3</sup> School of Engineering and Built Environment, Griffith University, 170 Kessels Rd., Nathan, QLD 4111, Australia


**Abstract:** To mitigate the negative environmental effects of the overuse of conventional materials—such as cement—in soil improvement, sustainable engineering techniques need to be applied. The use of biopolymers as an alternative, environmentally friendly solution has received a great deal of attention recently. The application of lignin, a sustainable and ecofriendly biobased adhesive, to enhance soil mechanical properties has been investigated. The changes to engineering properties of lignin-infused soil relative to a lignin addition to soil at 0.5, 1, and 3.0 wt.% (including Atterberg limits, unconfined compression strength, consolidated undrained triaxial characteristics, and mechanical properties under wetting and drying cycles that mimic atmospheric conditions) have been studied. Our findings reveal that the soil's physical and strength characteristics, including unconfined compressive strength and soil cohesion, were improved by adding lignin through the aggregated soil particle process. While the internal friction angle of the soil was slightly decreased, the lignin additive significantly increased soil cohesion; the addition of 3% lignin to the soil doubled the soil's compressive strength and cohesion. Lignin-treated samples experienced less strength loss during wetting and drying cycles. After six repeated wetting and drying cycles, the strength of the 3% lignin-treated sample was twice that of the untreated sample. Soil treated with 3% lignin displayed the highest erosion resistance and minimal soil mass loss of ca. 10% under emulated atmospheric conditions. This study offers useful insights into the utilization of lignin biopolymer in practical engineering applications, such as road stabilization, slope reinforcement, and erosion prevention.

**Keywords:** lignin biopolymer; erosion; soil strength; triaxial test; wetting and drying cycles; silt

### **1. Introduction**

Global climate change has dramatically influenced the environment, leading to irreversible changes in the limited resources we rely on. This has urged fundamental sustainable measures to be taken to reduce the consequences of these effects. A major impact of climate change that is significantly affecting the environment around us is extreme weather events, which results in intense localized rainfall in some geographic areas and drought in others. Intense rainfall events may cause instability in the ground properties, bringing a sudden increase in pore water pressure in soil which incurs a reduction in local soil strength, severe runoff, soil erosion, and eventually landslides and slope failures.

To improve the mechanical properties of soil and soil stability, a range of chemical treatments, including the addition of stabilizers to the soil, are often used. Portland cement has traditionally been the most commonly used additive to enhance soil properties. Although Portland cement has been widely used in different geotechnical engineering practices, its application for soil enhancement has been associated with a negative impact

**Citation:** Bagheri, P.; Gratchev, I.; Son, S.; Rybachuk, M. Durability, Strength, and Erosion Resistance Assessment of Lignin Biopolymer Treated Soil. *Polymers* **2023**, *15*, 1556. https://doi.org/10.3390/ polym15061556

Academic Editors: Raffaella Striani and Antonio Pizzi

Received: 2 January 2023 Revised: 11 March 2023 Accepted: 17 March 2023 Published: 21 March 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

on the environment, alongside the increase of carbon dioxide (CO2) emissions during cement production, as cement industries are believed to be responsible for up to 8% of global CO2 emissions annually [1]. The application of cement for civil and geotechnical engineering purposes is believed to have contributed to several environmental concerns, including the increase in soil pH level [2], cement dust accumulation in soil resulting in soil infertility [3], urban runoff, heat islands, prevention of vegetation growth, and groundwater contamination [4].

Recently, research has been undertaken to use biopolymers, such as organic polymers that occur in abundance in nature and can be extracted from natural resources, as environmentally friendly additives in geotechnical engineering applications [5].

The improvement of soil strength by adding biopolymers, such as xanthan gum, guar gum, beta glucan, chitosan, and lignin, has been attempted before [4–13]. Bagheri et al. [5] examined the effect of xanthan gum on soil strength and confirmed substantial improvement in soil compressive strength within a certain curing time. Through lab studies, Soldo et al. [4] investigated the impact of xanthan gum, beta glucans, guar gum, chitosan, and alginate biopolymers on silty sand soil. They reported significant increases in biopolymer-treated soil strengths over a longer period. The effectiveness of gellan gum biopolymer against soil permeability [14] and dextran for surface erosion [13] were investigated. Ham et al. [13] added a microbial biopolymer, dextran, to the fine silica sand and showed that the biopolymer can increase erosion resistance. Zhang et al. [15] performed the shear-wave velocity test and unconfined compression test to assess the small-strain shear modulus and unconfined compressive strength of lignin-stabilised silty soil. They found that a small-strain shear modulus and the unconfined compressive strength of lignin-treated soil logarithmically increased with curing period.

To increase the effectiveness of biopolymer treatment, in situ influencing factors must be taken into consideration. Although lignin has been shown to boost soil strength [10,15], prior research largely focused on analyzing basic strengthening behavior and confirming viability. In particular, in situ three-dimensional stress conditions have not received significant consideration. In addition, studies that address changes in mechanical strength of lignin-treated soils under saturated conditions have been limited, including those concerned with the erosion resistance of biopolymer-treated soil. Furthermore, the earlier reports mostly evaluated the shear behaviour of biopolymer-treated soils by means of direct shear tests using dried soil samples that may not adequately represent the underground conditions.

Lignin was chosen for use in the current investigation because it has been demonstrated to be one of the most economically advantageous materials among all available biopolymers in geotechnical engineering [16].

This study aims to address the mentioned gaps by performing a thorough study to examine the effect of lignin biopolymer agent on the soil mechanical properties subjected to various conditions. A range of laboratory experiments, including Atterberg limits tests and unconfined compressive strength (UCS) tests for the lignin-treated soil samples were conducted to obtain the engineering performance, soil strength, and plasticity behaviour. To investigate the effect of lignin additive on the soil shear strength and shear parameters and simulate in situ three-dimensional stress conditions, CU triaxial tests were performed. The durability and strength of lignin-treated soil under wetting and drying cycles were examined. The erosion resistance and soil loss of biopolymer-treated specimens exposed to natural atmospheric conditions were also examined. Finally, scanning electron microscopy (SEM) analyses were conducted to evaluate the microstructure mechanism of such treatment approaches.

The outcome of this study provides an enhanced understanding of the engineering behaviour of lignin-treated soil subjected to different conditions and facilitates the use of such sustainable techniques in civil and geotechnical practices.

#### **2. Materials and Methods**

#### *2.1. Materials*

Regarding soil, a low-plasticity silt soil (USCS classification: ML) according to ASTM D2487-17 [17] has been obtained from the Gold Coast area, Australia, with the grain size distribution as shown in Figure 1a obtained following the ASTM D422-63 [18]. The soil sample displayed plastic and liquid limits and a plasticity index of 26.9, 38, and 11.1, respectively, as measured in accordance with ASTM D4318-17 [19]. The specific gravity of the soil was 2.77, according to the ASTM D854-14 [20]. The standard proctor compaction test following ASTM D698-12 [21] was performed to obtain the maximum dry density of (1.72 g/cm3) and optimum moisture content of (21.7%) (Figure 1b).

**Figure 1.** (**a**) Grain size distribution. (**b**) Compaction test result. (**c**) X-ray diffraction (XRD) patterns of soil.

The mineral compositions of the soil were supplied by X-ray diffraction (XRD) analysis (Figure 1c). As seen from the soil XRD patterns, quartz is the main mineral with additional inclusions of kaolinite and calcite.

Regarding biopolymers, the lignin (LIG) was calcium lignosulphonate obtained from Dustex, Australia. The material was a brown viscose liquid with a pH (10% solution) of 5.4 ± 3.0, dry matter of 55.0 ± 1.0%, and a density of 1285 kg/m3. The LIG was a mixture of water (51%) and calcium lignosulfonate (49%).

#### *2.2. Specimen Preparation*

Initially, the soil was oven dried, and then the gravel was removed by crushing and sieving to 2.36 mm. Three concentrations of soil to LIG mixtures at 0.5 wt.%, 1.0 wt.%, and 3.0 wt.% were used in the study.

The wet mixture approach, as described by Ta'negonbadi et al. [10], was used to prepare LIG soil mixture. The LIG liquid was first added to the water to reach the desired moisture content, and then the diluted solution was sprayed and thoroughly mixed with the dry soil to prepare the homogenous blend.

The obtained mixtures were wrapped with double-layer plastic wrap and kept in a controlled temperature room for 24 h to prevent the formation of aggregations and ensure a uniform combination of biopolymer with soil particles. The soil mixtures were placed into a cylindrical metal mold (diameter 50 mm, length 150 mm) and evenly compacted in five layers to prepare the samples. The samples with the same diameter and length of around 110 mm were extruded from the mold following each compaction set. Each sample's dry density was guaranteed to be greater than 95% of the maximum dry soil density.

#### *2.3. Experimental Measurments*

Atterberg limits tests were conducted for the untreated and specified concentrations of LIG-treated soil to evaluate the impact of the biopolymer additive on the soil plasticity.

Regarding UCS tests, the samples were cured in a controlled temperature room for 0, 1, 4, 7, 10, 14, 28, and 35 days. The UCS tests in accordance with [22] were carried out for the cured samples. It is worth noting that three samples for each test were tested to minimize errors. From the outcome of UCS tests, the optimum curing time for treated and untreated samples was chosen for the following experiments.

Triaxial tests were used to examine how saturation conditions affected the strength of soil treated with LIG biopolymer. Consolidated undrained (CU) triaxial tests for the saturated samples were performed at confining pressures of 50, 100, and 200 kPa.

Regarding wetting and drying cycles tests, the durability of biopolymer-treated samples over six wetting/drying cycles was investigated. Polyvinyl chloride (PVC) molds were constructed with a diameter approximately equal to the sample (51 mm) and a higher length of 130 mm, allowing the sample to expand during the wetting cycle. Each wetting and drying cycle was initially started by placing the cured sample into a PVC mold, and then the mold was submerged in water for 24 h. The sample was then dried under room temperature conditions for the given optimum curing time. The UCS test was conducted following each cycle's completion to determine each sample's compressive strength.

Regarding the field experiment, five samples, including two untreated and three treated with the given concentrations of LIG, were exposed to the environment for 30 consecutive days, and soil mass loss of each specimen after exposure to the atmospheric conditions was measured and calculated. Daily temperature, relative humidity, and rainfall were recorded to evaluate the effect of environmental conditions on each sample's integrity.

Regarding SEM analysis, sample morphological examination was performed by using a scanning electron microscopy (SEM) analytical system (TESCAN Mira3, TESCAN Orsay Holdings, Czech Republic) under an acceleration voltage of 5 kV. Magnifications at 2500× and 15,000× were used and reported herein for the untreated, and 3% LIG-treated samples. The samples were platinum sputter coated (ca. 5 nm) immediately before collecting SEM image data.

#### **3. Results and Discussion**

The following provide the results and thorough examinations of the impact of LIG biopolymer on the soil.

#### *3.1. Atterberg Limits*

The results of Atterberg limits tests considering different percentages of LIG are presented in Table 1.


**Table 1.** Results of Atterberg limits tests.

The presence of LIG did not significantly affect PL, while the LIG-treated soil experienced a slight reduction in LL (Table 1). This can be related to the clay particles flocculation [23]. By increasing the content of LIG, the LL slightly decreased while the PL remained almost unchanged. As the soil has some negative charges from clay minerals, LIG may neutralize the negative charges on the surface of soil particles. This brings less thickness to the double electric layer between the soil particles [24]. All LIG-treated samples were placed below the A-Line in the plasticity chart, indicating a slight change in soil plasticity (Figure 2).

**Figure 2.** Plasticity chart for the untreated and LIG-treated soil.

#### *3.2. Unconfined Compressive Strength (UCS)*

A series of UCS tests for the specified percentages of LIG-treated soils were conducted to ascertain the optimum curing time and analyze the compressive strength of the stabilized samples. Figure 3a displays the changes in UCS for the untreated and LIG-treated soils over various curing times.

Increased biopolymer concentration resulted in increased UCS. While the specimens treated with 0.5% and 1% LIG reached their peak strength after 10 days of curing, the 3% LIG-treated specimen reached its maximum strength after 14 days of curing. The

UCS considerably increased within a certain curing time for all LIG concentrations. It demonstrates that additional curing time slightly affects the soil strength. Since moisture content has a significant impact on soil behaviour, the moisture content of each specimen at the end of UCS tests was measured and represented in Figure 3b. A significant drop in water content corresponded to a substantial increase in the soil strength (Figure 3b). When the treated samples dried, the LIG biopolymer acted like a glue leading to a noticeable increase in soil strength.

The curing time corresponding to the highest UCS was considered the optimum and used for the following tests.

#### *3.3. Shear Strength*

CU Triaxial

The effect of LIG biopolymer on the stress-strain curves and shearing-induced pore water pressures for the pure soil, LIG treated specimens are shown in Figure 4.

**Figure 4.** Results of CU triaxial tests, deviatoric stress-axial strain curves for (**a**) 50 kPa confining pressure; (**b**) 100 kPa confining pressure; (**c**) 200 kPa confining pressure; and pore water pressures for (**d**) 50 kPa confining pressure; (**e**) 100 kPa confining pressure; (**f**) 200 kPa confining pressure.

As shown in Figure 4, LIG causes a reduction in the brittleness of soil. Pure soil exhibited brittle behaviour, and peak deviator stress is clearly defined; however, there is no well-outlined peak of shear stress for LIG-treated samples.

The soil treated with 3% LIG experienced slightly higher strength than pure soil, especially at lower confining pressures (Figure 4a). An approximate 10% increase in peak deviator stress was observed once the soil was treated with 3% LIG at 50 kPa confining pressure. This relatively small strength enhancement is attributed to the increase in soil cohesion. In addition, the absolute values of pore water pressures developed upon shearing for soil treated with 3% LIG were marginally higher than the shearing-induced pore water pressures in pure soil (Figure 4d). This implies higher suction upon the shearing stage, which resulted in higher deviatoric stress in 3% LIG treated soil. The higher negative pressure causes an increase in effective stress applied to the soil particles and leads to increased soil shear strength.

To combine the obtained results of shearing of pure soil and LIG-treated soil at various confining pressures, the maximum deviatoric stresses are shown in Figure 5.

**Figure 5.** Results of CU triaxial tests, changes in maximum deviatoric stresses at various confining pressures.

Plotted failure envelope curves were used to derive the soil shear parameters for untreated and LIG-treated soil (Figure 6).

**Figure 6.** Effective shear stress vs. effective normal stress curves.

While soil friction angle reduced to some extent, a significant increase in soil cohesion occurred due to the LIG additive. Typically, the angularity of soil particles, soil gradation, and normal stress affect the soil friction angle. The LIG additive covers soil particles, smoothening the surface of particles, which causes a reduction in soil grain angularity. This results in a reduction in overall friction angle of soil. Table 2 provides the calculated shear parameters.

**Table 2.** Effective friction angle and cohesion of pure soil and LIG-treated soil.


#### *3.4. Wetting and Drying Cycles*

Figure 7 shows the changes in UCS values over six wetting and drying cycles. As the number of cycles increased, the UCS of the untreated soil gradually dropped. Following a dramatic reduction in the second cycle, the soil strength for the 1% LIG-treated soil was slightly decreased by increasing the wetting and drying cycles. For the highest dosage of LIG, as the number of cycles increased, there was a noticeable decline in soil strength. To clarify the effect of wetting/drying cycles and to better understand the rate of strength reduction over each cycle, the reduction factor (*Rf*) defined as Equation (1) and changes in *Rf*, (Δ*Rf*), were computed and summarized in Table 3. We have

$$R\_{f\_i} = \left(\frac{|\mathcal{S}\_i - \mathcal{S}\_0|}{\mathcal{S}\_0}\right) \times 100; \,\Delta R\_f = R\_{f\_{i+1}} - R\_{f\_i \prime} \tag{1}$$

where *Si* is the UCS after each cycle, and *S*<sup>0</sup> is the UCS at cycle 0 (before starting the experiment).

**Figure 7.** Changes of UCS with wetting and drying cycles.


**Table 3.** Values of reduction factor and difference in reduction factor during each wetting/drying cycle.

In all samples, the highest *Rf* occurred during the first cycle. As seen after the second cycle, the Δ*Rf* were relatively low, confirming that further wetting/drying cycles did not significantly affect the soil strength. The LIG-treated samples showed higher soil strength than the pure soil sample despite UCS reduction in all specimens. The reason is that after each wetting cycle, once the sample was exposed to the drying cycle, the LIG reattached soil particles and mended the cracks in samples, leading to stronger grain- bonding. This proves the capability of LIG biopolymer in reduction of soil strength loss in comparison with the untreated soil.

#### *3.5. Exposure to Atmosphere Conditions*

Five samples, including two untreated and three treated with the given concentrations of LIG, were first prepared and placed under atmospheric influences. Daily temperature, relative humidity, and rainfall in the field were recorded (Figure 8). Figure 9 shows the samples exposed to natural atmospheric conditions within 30 days of testing. Even though all samples lost some soil mass during rainfall events, they kept their shapes after two weeks of exposure to the atmospheric conditions. While the untreated samples were damaged and lost almost 22% of their soil masses, the LIG-treated samples kept their shapes. The 3% LIG-treated sample remained nearly intact after 30 days of atmospheric condition exposure. The soil mass loss after 30 days of atmospheric exposure is given in Figure 10. The LIG-treated samples lost less soil mass compared to pure soil samples, confirming the applicability of such treatment technique in improving soil erosion resistance.

**Figure 8.** Recorded daily temperature, relative humidity, and rainfall in the field.

(**a**) (**b**)

**Figure 9.** Samples exposed to the atmospheric conditions at (**a**) day 1, (**b**) day 14, (**c**) day 21, and (**d**) day 28.

#### *3.6. SEM Analysis*

SEM images of untreated and 3% LIG-treated soil are presented in Figure 11. The structure of soil changed due to lignin additive. The structure of pure soil has changed from a clumpy and grained-based form (Figure 11a,b) to a combined and more conjunct configuration (Figure 11c,d).

**Figure 10.** Soil mass loss after exposure to atmospheric conditions.

**Figure 11.** SEM images of the (**a**) pure soil with 2500× magnification; (**b**) untreated soil with 15,000× magnification; (**c**) LIG-treated soil with 2500× magnification; (**d**) LIG treated soil with 15,000× magnification.

The structure of pure soil is uneven, flaky, and disjointed. There are more obvious chunks of soil particles with larger spaces (Figure 11a,b). However, in treated soil, the pores among soil particles are partially filled with LIG, and a clear biopolymer coating of soil particles' surfaces and boundaries are seen. The soil particle aggregations by the LIG coating of the grains verify the soil strength improvement by adding biopolymer (Figure 11c,d).

#### **4. Conclusions**

The major goals of this study were to create an environmentally friendly biopolymerbased soil improvement method and to conduct an experimental investigation into the impact of LIG biopolymer on the mechanical properties and erosion resistance of soil. To achieve these goals, various laboratory and field experiments on three different concentrations of LIG soil mixtures were performed. The following represents a summary of the most significant findings.


**Author Contributions:** Conceptualization, P.B.; methodology, P.B.; validation, P.B.; formal analysis, P.B.; writing—original draft preparation, P.B.; writing—review and editing, I.G., S.S. and M.R.; supervision, I.G. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was supported by Basic Science Research Program through the National Research Foundation of Republic of Korea (NRF) funded by the Ministry of Education (grant number: NRF-2022R1I1A1A01054495).

**Institutional Review Board Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Acknowledgments:** This study was performed with the financial support of the Griffith University Postgraduate Research Scholarship (GUPRS). Dustex Australia Pty Ltd is gratefully acknowledged for providing research samples (i.e., lignin) for this study.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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### *Article* **Effect of Spent Coffee Grounds on the Crystallinity and Viscoelastic Behavior of Polylactic Acid Composites**

**Anne Shayene Campos de Bomfim 1,\*, Daniel Magalhães de Oliveira 1, Kelly Cristina Coelho de Carvalho Benini 1, Maria Odila Hilário Cioffi 1, Herman Jacobus Cornelis Voorwald <sup>1</sup> and Denis Rodrigue <sup>2</sup>**


**Abstract:** This work investigated the addition of spent coffee grounds (SCG) as a valuable resource to produce biocomposites based on polylactic acid (PLA). PLA has a positive biodegradation effect but generates poor proprieties, depending on its molecular structure. The PLA and SCG (0, 10, 20 and 30 wt.%) were mixed via twin-screw extrusion and molded by compression to determine the effect of composition on several properties, including mechanical (impact strength), physical (density and porosity), thermal (crystallinity and transition temperature) and rheological (melt and solid state). The PLA crystallinity was found to increase after processing and filler addition (34–70% in the 1st heating) due to a heterogeneous nucleation effect, leading to composites with lower glass transition temperature (1–3 ◦C) and higher stiffness (~15%). Moreover, the composites had lower density (1.29, 1.24 and 1.16 g/cm3) and toughness (30.2, 26.8 and 19.2 J/m) as the filler content increased, which is associated with the presence of rigid particles and residual extractives from SCG. In the melt state, polymeric chain mobility was enhanced, and composites with a higher filler content became less viscous. Overall, the composite with 20 wt.% SCG provided the most balanced properties being similar to or better than neat PLA but at a lower cost. This composite could be applied not only to replace conventional PLA products, such as packaging and 3D printing, but also to other applications requiring lower density and higher stiffness.

**Keywords:** polylactic acid (PLA); spent coffee grounds (SCG); biocomposites; rheological properties; mechanical properties

#### **1. Introduction**

Polylactic acid (PLA) is a well-known biodegradable thermoplastic polymer and is vastly used for packaging and 3D printing applications. It is polymerized by polycondensation or ring-opening polymerization from the lactic acid monomer. Moreover, lactic acid can be fermented from natural sugars such as glucose or sucrose, including the sugars from spent coffee grounds fermentation [1]. The PLA molecular structure and molar mass directly affect the polymer's properties. In particular, thermal properties, such as melting temperature and glass transition temperature, are strongly related to the molecular structure, as well as the thermo-mechanical history of the polymer [2]. Despite its positive effect on the environment due to its biodegradability, PLA is reported to have low ductility, toughness, resistance to moisture, rate of crystallization, and glass transition temperature [3].

To improve the properties, several efforts have been made to fill PLA with different natural fillers as a disperse phase to modify the polymer chain mobility and consequently

**Citation:** de Bomfim, A.S.C.; de Oliveira, D.M.; Benini, K.C.C.d.C.; Cioffi, M.O.H.; Voorwald, H.J.C.; Rodrigue, D. Effect of Spent Coffee Grounds on the Crystallinity and Viscoelastic Behavior of Polylactic Acid Composites. *Polymers* **2023**, *15*, 2719. https://doi.org/10.3390/ polym15122719

Academic Editors: Raffaella Striani and Swarup Roy

Received: 31 March 2023 Revised: 2 June 2023 Accepted: 8 June 2023 Published: 17 June 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

enhance its properties [3–6]. Most of the PLA have low crystallinity (3–10%), and filler addition was reported to significantly improve PLA crystallinity by reorganizing the polymeric chains and influencing the crystallization rate via heterogeneous nucleation. For example, PLA filled with sisal (10 wt.%) was compared with polypropylene (PP) composites [7]. The results showed that the PLA composite had a 270% increase in crystallinity, while the PP composite was only improved by 12%. Moreover, a recent review concluded that PLA with improved properties could be developed by using different natural fillers, depending on the application requirements, since natural fibers are diversified and abundant [8].

Spent coffee grounds (SCG), as a residue of coffee beverages, is a valuable resource rich in polysaccharides, polyphenols, and proteins being widely studied for different applications, including composite filler [9]. A recent review discussed the valorization of SCG for biopolymers synthesis and as composites filler, highlighting the great versatility of SCG in improving different polymers properties [10]. Other works investigated PLA filled with different SCG concentrations and treatments to compare with other fillers and additives, showing promising results in improving PLA properties and biodegradability [11–14]. Although various techniques have been used to characterize the composites, none of the works focused on the effect of SCG addition on the material's crystallinity and viscoelastic properties. Combined dynamic mechanical analysis (DMA) and rheology, performed respectively in the solid and melt state of PLA filled with SCG, were not found in the current literature.

Moreover, PLA/SCG composites are a recent topic of research. According to the Scopus database, only 21 documents were published on this subject between 2013 and 2023 (May). The data show an increased tendency over the years. This subject is achieving scientific relevance, considering the number of citations peak in 2022 (177) [15].

Thus, the main objective of this work is to investigate the effect of spent coffee grounds content (0, 10, 20, and 30% wt.) on PLA crystallinity, as well as to determine the effect of crystallinity on the thermo-mechanical and viscoelastic properties of the composites produced via extrusion followed by compression molding. Although some previous works discussed the subject of PLA/SCG composites, the novelty of this work focuses on the characterizations being done in both the solid and melt state after SCG addition, as crystallinity and viscoelastic proprieties, which directly affect PLA processing and applications, are important for their applications.

#### **2. Materials and Methods**

#### *2.1. Materials*

Pellets of PLA 2003D were supplied from NatureWorks (Minnetonka, MN, USA), while the spent coffee grounds (SCG) were collected as a post-consumer residue (mixture) from coffee shops on the campus of Laval University. Raw SCG was previously dried in an oven at 60 ◦C for a week to ensure that the moisture was completely removed.

#### *2.2. Composite Preparation*

The materials were first dried in an oven at 60 ◦C for 24 h and then mixed in a twinscrew extruder Leistritz ZSE-27 (Remscheid, Germany) having a screw diameter of 27 mm diameter, a L/D ratio (length/diameter) of 40 and 10 heating zone. The PLA pellets were fed in the first zone of the extruder, while the SCG was fed in the fourth zone via a side stuffer. The composition was determined by controlling the specific flow rate for each component (PLA and SCG) by calculating the weight/time ratio (SCG = 0, 10, 20, and 30 wt.%). The extruder was operated at the same constant mass flow rate of 100 g/min and a screw speed of 60 rpm for each composition. The temperature profile in the extruder was controlled at 175 ◦C (zone 1), 185 ◦C (zones 2–9), and 165 ◦C (last zone). A circular die (2.7 mm in diameter) was used to form the melt into a filament shape. The materials were cooled in a water bath close to the die exit (about 10 cm) and then pelletized using a model 304 pelletizer (Conair, Stanford, CT, USA). Then, the pellets were dried in an oven at 60 ◦C for 48 h before use. Finally, the pellets were molded in an SC7620 automatic

hydraulic press (Carver, Wabash, IN, USA) at 210 ◦C for 7 min with a constant force of 2 tons and compressing pressure in the mold of 0.5 MPa. The mold and plates were cooled with water circulation for about 3 min until getting to 70 ◦C. The plates were produced (210 × <sup>210</sup> × 2.4 mm3) and cut into different specimens for characterization, as described next. The preparation of the composites is illustrated in Figure 1.

**Figure 1.** Preparation of the composites: (1) PLA pellets and raw SCG, (2) the extruder, (3) the composites' extruded pellets, (4) the hydraulic press, and (5) the obtained specimens.

#### *2.3. Characterization*

#### 2.3.1. Differential Scanning Calorimetry (DSC)

Dual scan analysis was carried out on a DSC Q20 (TA Instruments, New Castle, DE, USA) with a sample weight of around 5 mg in a temperature range from 0 to 200 ◦C under constant nitrogen flow (40 mL/min). The heating and cooling rate was 10 ◦C/min. Melting and cold crystallization temperatures (*Tm* and *Tcc*), as well as melting and cold crystallization enthalpies (Δ*Hm* and Δ*Hcc*) and crystallization degree (*Xc*) were determined from the thermograms (heat flow as a function of temperature). The crystallinity degree was calculated as:

$$X\_{\mathcal{L}} = \begin{bmatrix} \frac{\Delta H\_{\mathcal{W}} - \Delta H\_{\mathcal{CC}}}{f \times \Delta H\_{\mathcal{W}100\%}} \end{bmatrix} 100 \tag{1}$$

where Δ*Hm*100% is the enthalpy of a fully crystalline PLA sample (93.7 J/g), while *f* is the weight fraction of PLA (matrix) in the samples [16–18].

#### 2.3.2. Thermo-Gravimetric Analysis (TGA)

TGA was carried out on SII Nanotechnology INC equipment model Exstar 6000—TG/DTA series (Tokyo, Japan) with a sample weight of around 10 mg. The analysis was performed in a platinum pan under a constant nitrogen flow (100 mL/min) in a temperature range from 30 to 600 ◦C and a heating rate of 10 ◦C/min. Thermal degradation temperature (*Tonset*) was determined via DTG curves where an inflection point was observed in the baseline.

#### 2.3.3. Dynamic Mechanical Analysis (DMA)

DMA was carried out on a RSA3 (TA Instruments, New Castle, DE, USA) using rectangular specimens (82.3 × 12.5 × 2.4 mm3) cut from the molded plates. A three-point bending geometry was selected with a span of 40 mm. Two types of characterization were performed after strain sweep verification to stay in the linear viscoelastic range of the materials. First, temperature sweeps were done from 25 to 80 ◦C with a heating rate of 0.5 ◦C/min and a frequency of 1 Hz. Then, frequency sweeps were performed at room temperature (23 ◦C) between 0.01 and 25 Hz with a strain of 0.01%.

#### 2.3.4. Rheology

The shear rheological properties in the melt state were studied using a rotational rheometer (ARES, Rheometric Scientific, TA Instruments, New Castle, DE, USA) with a parallel plate geometry (25 mm in diameter). All the analyses were performed under a nitrogen atmosphere. First, strain sweep tests (0.01 to 100%) were performed at a frequency of 1 Hz to determine the linear viscoelastic zone of the samples. Then, frequency sweeps (0.01 to 40 Hz) were performed at a deformation of 5% using three temperatures: 180, 190, and 200 ◦C.

#### 2.3.5. Impact Test

The Charpy impact strength was performed on a manual impact testing machine Wolfgang Ofist usinga4Jpendulum, type A. Seven rectangular "V" notched specimens (82.3 × 12.5 × 2.4 mm3) of PLA and its composites were tested at room temperature according to ASTM D6110. The "V" notch was produced by a manual milling machine Vigorelli (type 1), using a bi-angular milling cutter.

#### 2.3.6. Density and Porosity

The density of PLA and the composites (*ρr*) was measured by a helium gas pycnometer Ultrapyc 1200e (Quantachrome Instruments, Boynton Beach, FL, USA) at room temperature. The samples were previously dried in an oven at 60 ◦C for 24 h and weighed. To calculate the porosity, the apparent density (*ρa*) of the samples was measured via the Archimedes principle using water and calculated as:

$$\rho\_d = \frac{w\_0 \,\rho\_f}{w\_0 - w\_1} \tag{2}$$

where *ρ<sup>f</sup>* is the density of the fluid (water), while *w*<sup>0</sup> and *w*<sup>1</sup> are the samples' weight in air and immersed in water, respectively.

The porosity of the samples was calculated from the real (*ρr*) and apparent density (*ρa*) as:

$$Porosity = \left(1 - \frac{\rho\_d}{\rho\_r}\right) \times 100\tag{3}$$

#### 2.3.7. Scanning Electron Microscopy

Morphological analysis of the composites was carried out on the fractured surface of samples after impact testing. The specimens were previously dried and fixed on metal support using carbon adhesive tape before being coated (metalized) with gold. The images were taken with a Zeiss EVO LS-15 scanning electron microscopy (Cambridge, UK) combined with EDS/EBDS Oxford INCA Energy 250 system (Oxford Instruments, Oxfordshire, UK) operating at 5 kV.

#### 2.3.8. Statistical Analysis

One-way ANOVA and grouping information using the Tukey method to calculate the statistical significance of impact test results were carried out using the statistical software Minitab 18.1 (Minitab Inc., State College, PA, USA). A significance index of 95% (*p*-value < 0.05) was used.

#### **3. Results and Discussions**

#### *3.1. DSC and TGA*

The DSC curves are presented in Figure 2, and the thermal parameters are summarized in Table 1. The first heating scan eliminates the thermal history from PLA and its composites, providing different values from the second heating scan. The thermal history is directly related to the compression molding conditions, mainly the cooling rate [19]. The cooling scan did not present thermal events, but a crystallization peak appeared in the heating scan, called cold crystallization. According to Table 1, PLA melting temperature (*Tm*) was determined as the endothermic peak at around 150 ◦C, while cold crystallization temperature (*Tcc*) occurred at around 122 ◦C. However, *Tcc* was not observed in the second heating scan (Figure 2b) due to a slow crystallization rate and low crystallinity degree (3.4%). For the composites, SCG addition generated the presence of two *Tm* in both heating scans. These peaks are related to the melting of the original crystals and the newly formed crystals due to the melt-recrystallization process or the presence of a dual lamellae structure formed

by the various heating scans [20]. Moreover, Tcc was well-defined around 112–123 ◦C for the composites in both heating scans. This shows that the polymeric chains were more organized after SCG addition due to a heterogeneous nucleation effect [21]. This observation can be confirmed by the significant improvement (34–70% in the 1st heating) of the composites' crystallinity compared to the neat PLA, especially in the second heating scan (about 1500%) (Table 1). The literature also reported PLA crystallinity increases after SCG addition, but the differences were not as high (93% at 15 wt.% and 66% at 20 wt.%) [11,22]. Although composites usually present higher crystallinity as the filler content increases, there is always a maximum related to chain mobility and spatial hindering. In our case, the maximum (70.3%) was observed for PLA/20SCG. This trend can also be related to particle agglomeration in PLA/30SCG acting as defects.

**Figure 2.** DSC curves for PLA and the composites: (**a**) first heating and (**b**) second heating.


**Table 1.** Thermal parameters obtained via DSC and TGA curves.

Finally, the glass transition temperature (Tg) of neat PLA was found to be around 59 ◦C in the first and second heating scans. This result highlights that the thermal processing of the specimens did not significantly modify the PLA structure (degradation), leading to similar Tg of the matrix and the composites in the first and second heating scans. However, the composites presented slightly lower Tg values (2–3 ◦C difference) than the neat PLA in both scans due to higher polymer chain mobility associated with the presence of rigid particles releasing extractives from SCG. A similar trend of decreasing Tg was reported for PLA composites using treated SCG [22] and PLA with waste paper [23]. Another work used PLA with grapevine biochar (1 and 10 wt.%) and concluded that the filler acted as a nucleating agent by increasing the crystallinity and reducing the Tg values of the composites [24]. However, different behavior was identified for PLA/SCG (1–7 wt.%) 3D printed composites in which the Tg was the same for all samples, mostly influenced by the filler content and the processing conditions [25]. These results show the effect of SCG content, as well as the extrusion/compression steps, on the PLA's crystallinity and chain mobility. Although the presence of rigid fillers should limit polymer chain mobility, SCG

extractives can be released, leading to increased mobility as well as acting as nucleating agents/plasticizers, especially for a higher SCG concentration.

TGA curves are presented in Figure 3, and the thermal parameters are presented in Table 1. The thermal degradation temperature (Tonset) was found to decrease as the filler content decreased (11% for PLA/10SCG and 21% for PLA/30SCG). This behavior is not in agreement with the results reported by previous works, such as PLA filled with SCG [26,27], PLA filled with rice straw hydrochar [28], and polypropylene (PP) filled with SCG [29,30]. Moreover, the Tonset values show that the composites were not degraded in the extrusion/compression processing (T < 210 ◦C). Residual weight at 600 ◦C also increased with increasing filler content, which is related to extractives, mostly polysaccharides [31], and ashes from the natural filler. Some works reported that SCG residual weight was in the range of 5–25% [31,32].

**Figure 3.** TGA curves for PLA and the composites.

#### *3.2. Dynamic Mechanical Analysis (DMA)*

In the solid state, DMA results are presented in terms of storage modulus (E ), loss modulus (E), and tan (δ) (= E/E ) in Figure 4. Figure 4a shows that for E , the values at 30 ◦C were selected for comparison. In this case, PLA/10SCG and PLA/20SCG presented higher values (2.32 and 2.39 GPa, respectively) than PLA/30SCG (1.84 GPa) and PLA (2.07 GPa), indicating that composites with intermediate SCG contents (10–20%) are slightly stiffer and store more energy than neat PLA and 30 wt.% SCG. This trend can be related to the DSC results (Table 1), showing that PLA/20SCG presented the highest crystallinity. Similar storage modulus increases were reported for injection-molded PLA composites filled with SCG and bamboo (30 wt.%) [32] and PLA composites filled with nanocellulose from sugarcane bagasse (1–5 wt.%) [33]. However, recent work reported that PLA/SCG (20 wt.%) with and without lactic acid oligomers had a lower storage modulus compared to neat PLA, which was associated with a plasticization effect caused by the SCG remaining oil [26]. Around 50–60 ◦C, the storage modulus significantly decreases for all samples, which corresponds to the glass transition range where the material significantly losses stiffness. For the loss modulus (E), Figure 4b shows that the maximum energy dissipation can be determined from the maximum on the E curve. By increasing the SCG content, the peak temperature slightly decreased from 61.0 ◦C to 58.2 ◦C. Moreover, two transitions were identified in E curves. The first one (51–55 ◦C) corresponds to a secondary transition related to the motion of localized links (bending and stretching) and the relaxation of lateral groups from the polymeric chain, while the second transition (58–61 ◦C) corresponds to the glass transition temperature (Tg). Finally, the damping factor (tan δ) followed the moduli trends: chain mobility restriction caused by SCG addition directly influenced the damping properties of the polymer [34]. Gonzalez-Lopez et al. (2019) observed a similar trend in PLA composites filled with agave fibers (10–30 wt.%) and stated that lower tan (δ) with increasing filler concentration may be related to better polymer/filler interaction/adhesion [35]. Other studies supported this observation, such as PLA/polyester/kenaf laminated composite and PLA/silk composite [36,37]. As reported by Hassan et al. (2014), the tan δ peak shifts to higher temperatures, combined with a decreased peak intensity, indicating a restriction of molecular mobility due to improved interactions between the filler and matrix [38].

**Figure 4.** DMA curves for PLA and its composites: (**a**) storage modulus as a function of temperature, (**b**) loss modulus as a function of temperature, (**c**) tan (δ) as a function of temperature, and (**d**) Cole–Cole plots.

Cole–Cole plots (E as a function of E ) are presented in Figure 4d. It was noticed that semicircular curves are associated with homogenous systems and well-dispersed fillers in a polymeric matrix [33,39]. According to the curves, PLA/10SCG shows the most semicircular arc, similar to PLA, indicating a homogenous composite and good SCG dispersion. However, higher filler content has a more irregular curve, especially for PLA/30SCG. This indicates a more heterogenous structure with poorly dispersed filler. This is also related to lower crystallinity (Table 1) and lower E (Figure 4b) compared to the other composites.

To complete the analysis, Figure 5 compares the glass transition temperature (Tg) from DSC (1st heating) and DMA. The Tg from DMA was determined from E curves whose value decreased as the filler content increased: 51.8 ◦C for PLA, 51.3 ◦C for PLA/10SCG, 50.6 ◦C for PLA/20 SCG, and 49.7 ◦C for PLA/30SCG. This trend is also observed in the Tg obtained by DSC, emphasizing the effect of filler addition, especially with increasing filler content [40]. The relation between Tg and crystallinity as a function of filler content is also presented in Figure 5d. It was clearly identified that as the crystallinity increased, the Tg from DSC and DMA decreased, supporting the SCG effect observed on the polymeric chains.

**Figure 5.** PLA crystallinity (DSC) and Tg (DSC and DMA) as a function of SCG content.

#### *3.3. Rheology*

The rheological properties in the melt state are reported in Figures 6 and 7 in terms of complex viscosity (\*), storage modulus (G ), loss modulus (G), and damping factor (tan δ = G/G ). Figure 6 is used to determine the effect of temperature, while Figure 7 compares the effect of SCG content.

Figure 6 presents the complex viscosity as a function of frequency for different temperatures. As expected, the viscosity decreases with increasing temperature due to higher chain mobility and less interaction between them (more internal energy). However, the effect of frequency is different with increasing SCG content. For the neat PLA (Figure 6a), typical shear-thinning behavior for the neat polymer is observed with a Newtonian plateau at a low frequency and a power-law (decreasing trend) at a higher frequency. Then, PLA/10SCG and PLA/20SCG mainly have a Newtonian behavior (almost constant viscosity) and a slight shear-thinning behavior at a higher frequency (above 10 Hz). This behavior can be related to several factors, including complex interactions. First, the presence of rigid SCG particles modifies the chain mobility, especially as filler content increases.

The second factor is that higher filler content generates more shear in the extruder, possibly modifying the PLA molecular structure (more polymer degradation). However, this effect should be limited since negligible variations of the thermal properties were observed via DSC (Figure 2) and DMA (Figure 4). There is also a possibility that residual molecules (extractives such as mono/polysaccharides, carbohydrates, lipids, oils, proteins, etc.) are present from the SCG particles themselves (they were only dried and not washed before use) [41], i.e., low molecular weight materials that can act as plasticizers. Actually, this can be associated with the small bump around 155 ◦C that can be seen in TGA curves (Figure 4). As the SCG content increases, their concentration also increases, leading to different effects/trends on the PLA behavior. The effect is more important in the melt state (Figure 6) than in the solid state (Figure 4), and this is why both characterizations are needed to obtain complete information on these samples. Finally, PLA/30SCG presents a highly non-Newtonian behavior which is related to its higher filler content. The disappearance of

the Newtonian behavior (low frequency) has been associated with the presence of rigid particles having interactions between them, i.e., particle–particle contact [42].

**Figure 6.** Complex viscosity as a function of frequency at different temperatures (180, 190, and 200 ◦C) for (**a**) PLA, (**b**) PLA/10SCG, (**c**) PLA/20SCG, and (**d**) PLA/30SCG.

According to Figure 7a, all the composites have a lower viscosity than the neat PLA at 200 ◦C. In fact, viscosity decreased as the filler content increased, which could be related to increased polymeric chain mobility in the melt state [43]. A similar trend was reported for PLA filaments filled with waste paper (5–15 wt.%) [23], PLA filled with biochar (1–7.5 wt.%) [42], and PLA filled with different waxes, such as beeswax, candelilla, carnauba, and cocoa (3–15 wt.%) [44]. A lower viscosity also suggests that the filler acted as a plasticizer from the presence of extractives, as described above [27]. This reduced viscosity would improve the processability of these materials [22].

The storage modulus (G ), loss modulus (G) and loss tangent (tan δ) are presented in Figure 7. All the samples show that G and G increase with frequency due to their viscoelastic nature, but the neat PLA again has higher values. At high frequencies (above 10 Hz), G and G decrease with increasing filler content. In the melt state, PLA is more rigid than the composites. However, as the filler content increases, the composites become less rigid [45]. Nizamuddin et al. (2021) reported on G and G behavior for PLA/rice straw hydrochar composites (5–20 wt.%), in which the values increased with filler content justified by the reduction of chain mobility leading to higher flow resistance [28]. Another work investigated PLA filled with cocoa (3–15 wt.%) and observed that at lower frequencies, G increased with increasing filler content, but at higher frequencies, a reversed trend was observed as G decreased with filler content, which was justified by the plasticizing effect of the natural fillers extractives [44].

**Figure 7.** Viscoelastic properties as a function of frequency (200 ◦C) for PLA and the composites: (**a**) complex viscosity, (**b**) storage modulus, (**c**) loss modulus, and (**d**) tan (δ).

The value of tan (δ) can be compared to unity to determine the viscous/elastic behavior of the materials [46]. According to Figure 7d, all the samples showed increased tan (δ) at a lower frequency, followed by a decrease at a higher frequency. PLA/10SCG and PLA/20SCG behave similarly to PLA at high frequency and then decreasing tan (δ), while PLA/30SCG shows an increasing tan (δ) trend for most of the analysis performed. At lower frequency (0.1 Hz), PLA has the highest tan (δ), indicating a more viscous behavior, while at higher frequency (40 Hz), PLA, PLA/10SCG, and PLA/20SCG present a stiffer behavior and PLA/30SCG a more elastic behavior. This decreasing tan (δ) trend with increasing frequency can be associated with chemical interaction between the polymer and the filler [28]. In this case, PLA/30SCG shows poor polymer-filler interactions, which is consistent with the density/porosity results as described next.

#### *3.4. Impact Strength and Density*

The real and apparent densities of the composites are presented in Figure 8a. It can be seen that the composites have a lower density than PLA, especially the apparent density.

The density decreased with increasing filler content as the polymer fraction was reduced, and natural fibers are known to be low-density materials. The density of SCG was reported to be 0.45 g/cm3 [47]. Such behavior (decreasing composites density) was also identified in PLA composites filled with nettle (10 and 25 wt.%) [48]. Furthermore, it was possible to calculate the porosity of the materials from density values. The results show that the composites have higher porosity than PLA. PLA/10SCG presented the lowest porous content (4.7%), while PLA/20SCG presented the highest value (7.9%), indicating poor dispersion and possible interfacial voids. In addition to the filler addition, the specimens' processing also influenced the porosity of the materials since neat PLA presented 3.4% of porosity. González-López et al. (2019) also reported a decreasing density trend with increasing porosity for PLA/agave composites as the filler content increased (10–30 wt.%), but porosity values were higher than in our work: about 35% for 20 wt.% composite and 60% for 30 wt.% composite [35]. Another work used SCG with high-density polyethylene (HDPE) and found that both density and porosity increased as the filler increased (10–30 wt.%), as 55% of porosity for the 30 wt.% composite [49].

**Figure 8.** Properties of PLA as a function of SCG content: (**a**) density and porosity, and (**b**) impact strength and crystallinity.

PLA has a well-known low toughness. From the PLA 2003D datasheet, a value of 16 J/m is reported [45]. Nevertheless, after extrusion/compression processing, PLA was found to have good impact strength (26.5 J/m). Although PLA/10SCG might have a slightly higher value, the composites present a slightly decreased impact strength with increasing filler content (Figure 8b) [11]. PLA filled with agave fibers (10–30 wt.%) was also reported to show lower impact strength with a reduction of 24% for 10 wt.% composite and 41% for the 30 wt.% composite [35]. In some cases, it was reported that filler addition could improve PLA toughness, but an optimum content of around 20 wt.% was observed [3]. Therefore, adding SCG could improve not only the crystallinity (DSC) and stiffness (DMA) of PLA but also its toughness (PLA/10SCG).

Statistical analysis (ANOVA) of the impact test has been carried out, and the results are shown in Table 2. As the F value (8.40) is greater than the Fcritical value (3.01), the decision is to reject the null hypothesis (H0) (all means are equal) for a significance level of 0.05. In addition, considering that the *p*-value is less than the significance level (α = 0.05), the H0 was rejected since the *p*-value represents the probability against the null hypothesis. This result indicates that the impact strength of all the samples differs significantly. However, to determine whether the mean difference between any pair of groups is statistically significant, grouping information using the Tukey method and a significance level of 95% were carried out, which are presented with the letters a and b in Figure 8b. In these results, group a contains PLA, PLA/10SCG, and PLA/20SCG, while group b contains PLA/30SCG. The differences between the means of the PLA, PLA/10SCG, and PLA/20SCG, which share

the same letter, are not statistically significant. The PLA/30SCG does not share any letters, indicating a significantly lower mean value for impact strength than the other samples.


Note: AG—among groups; WG—within groups; Df—degrees of freedom; SS—sum of squares; MS—mean square; F—F-test for one-way ANOVA; Number of observations = 7; Number of samples = 4.

Comparing toughness and crystallinity results, it is possible to identify that both properties increased for PLA/10SCG compared to PLA. Nevertheless, a different trend was observed for the other composites; PLA/20SCG presented the highest crystallinity leading to similar toughness compared to PLA despite presenting a higher porosity. Moreover, PLA/30SCG presented a high crystallinity but lower toughness compared to PLA, which is related to the filler content and the formation of agglomerates acting as stress concentration points, mainly for PLA/30SCG. Overall, it can be concluded that the final properties represent a balance between complex interactions.

#### *3.5. Scanning Electron Microscopy*

SEM images of the composites are presented in Figures 9 and 10. According to these images, it is possible to identify that PLA/20SCG and PLA/30SCG presented a higher volume and size of voids than PLA/10SCG, which can be seen at lower magnification (Figure 9). These images confirm the relatively high amount of porosity reported in Figure 8 for PLA/20SCG (7.9%) and PLA/30SCG (5.6%).

**Figure 9.** SEM images (magnification of 50×) for (**a**) PLA/10SCG, (**b**) PLA/20SCG, and (**c**) PLA/30SCG.

In addition, small voids were observed all over the matrix for all composites, but they are more evident for PLA/20SCG and PLA/30SCG (Figure 10). It can also be seen that SCG dispersion decreases with increasing filler content as less space is available between the particles leading to more particle–particle contact and agglomeration. Although PLA/30SCG has the highest filler concentration, the particles are very challenging to identify.

It can be concluded that the composites' morphology mostly influenced their thermomechanical and viscoelastic behavior, especially for PLA/20SCG and PLA/30SCG.

**Figure 10.** SEM images with different magnifications (500× and 2000×) for (**a**,**b**) PLA/10SCG, (**c**,**d**) PLA/20SCG, and (**e**,**f**) PLA/30SCG.

#### **4. Conclusions**

PLA and its composites based on spent coffee grounds (SCG) were successfully prepared via twin-screw extrusion and compression molding. For the range of SCG content investigated (0–30 wt.%), a series of characterizations were performed with a focus on crystallinity, thermo-mechanical (solid state), and viscoelastic (melt state) properties.

The results showed that SCG can act as nucleating agents (solid particles) and plasticizers (extractive release). This led to several differences between the neat matrix (PLA) and the composites (effect of SCG content), as well as complex interactions.

For example, the thermal processing significantly increased the PLA crystallinity, which was further improved with filler addition. Although rigid particles limit polymeric

chain mobility, SCG extractives caused a higher crystallinity and lower Tg, which is related to higher polymeric chain mobility. Furthermore, as the crystallinity increased, the density and toughness decreased as the filler content increased, while the composite with the lower filler content (10 wt.%) provided a higher toughness than PLA. However, in the melt state, polymeric chain mobility was enhanced, and composites with a higher filler content became less viscous due to the increased chain mobility. This behavior might also be associated with possible residues/extractives acting as plasticizers in the composites.

Although PLA/20SCG showed a higher porosity (7.9%), it provided the best-balanced performances having similar or even better properties than neat PLA and all the other composites. For example, higher crystallinity (38.6%), higher storage modulus at 30 ◦C (2.38 GPa) and at high frequencies (4.3 GPa at 10 Hz), lower viscosity (19.3 Pa·s at 10 Hz), lower density (1.16 g/cm3) and comparable impact strength (26.8 J/m). Nevertheless, PLA/30SCG presented high crystallinity and low density that could influence, to a greater extent, the biodegradability of the material since it was reported that the presence of a biofiller could accelerate the biodegradation process [11].

This work represents a follow-up on previous studies in our groups and the literature. These materials are interesting because they are fully biobased. In the future, more work will focus on the effect of the SCG morphology (particle geometry and size) and composition (washing, extraction, surface modification, etc.) on the final biocomposites' properties. The effect of processing conditions (temperature, pressure, time, velocity, etc.) and method (injection molding, rotomolding, etc.) should also be compared for commercial and industrial applications. The presence of additives (coupling agents) can also be investigated to improve these results. Finally, it would be interesting to investigate the possibility of recycling and composting these materials.

**Author Contributions:** Conceptualization and methodology, A.S.C.d.B. and D.R.; validation, K.C.C.d.C.B., and H.J.C.V.; formal analysis, D.R.; investigation, A.S.C.d.B. and D.M.d.O.; resources, D.R.; data curation, A.S.C.d.B. and D.M.d.O.; writing—original draft preparation, A.S.C.d.B.; writing—review and editing, D.M.d.O., K.C.C.d.C.B., M.O.H.C., H.J.C.V. and D.R.; visualization, A.S.C.d.B. and D.R.; supervision, D.R. and H.J.C.V.; project administration, D.R.; funding acquisition, A.S.C.d.B. and D.M.d.O. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—Brazil (CAPES), finance code 001, and grant number, 88887.495399/2020-00, and the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), grants 2019/02607-6 and 2020/02361-4.

**Institutional Review Board Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The authors would like to acknowledge Université Laval and São Paulo State University for their administrative and technical support.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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### *Review* **Environmental Properties and Applications of Biodegradable Starch-Based Nanocomposites**

**Ashoka Gamage 1,\*, Punniamoorthy Thiviya 2, Sudhagar Mani 3, Prabaharan Graceraj Ponnusamy 3, Asanga Manamperi 4, Philippe Evon 5, Othmane Merah 5,6,\* and Terrence Madhujith <sup>7</sup>**


**Abstract:** In recent years, the demand for environmental sustainability has caused a great interest in finding novel polymer materials from natural resources that are both biodegradable and eco-friendly. Natural biodegradable polymers can displace the usage of petroleum-based synthetic polymers due to their renewability, low toxicity, low costs, biocompatibility, and biodegradability. The development of novel starch-based bionanocomposites with improved properties has drawn specific attention recently in many applications, including food, agriculture, packaging, environmental remediation, textile, cosmetic, pharmaceutical, and biomedical fields. This paper discusses starch-based nanocomposites, mainly with nanocellulose, chitin nanoparticles, nanoclay, and carbon-based materials, and their applications in the agriculture, packaging, biomedical, and environment fields. This paper also focused on the lifecycle analysis and degradation of various starch-based nanocomposites.

**Keywords:** biodegradability; carbon nanotubes; graphene; life cycle analysis; nanocomposites; packaging; remediation; starch

#### **1. Introduction**

In recent days, nanocomposites have gained much attention over traditional composite materials and are widely used in food, packaging, biomedical applications, electronics, energy storage, optics, the automotive industry, bio-sorbants for environmental remediation, textiles, and many other applications [1,2]. Polymer nanocomposites consist of polymer matrices embedded with nanofillers [3]. Petroleum-based polymers are produced in huge amounts globally. Petroleum-based polymers are non-biodegradable, non-renewable, and produce hazardous substances which can threaten human health and the environment [4]. Furthermore, the depletion of these non-renewable petroleum-based fuels demands alternative resources [5].

Thus, biopolymer-based nanocomposites can be a sustainable alternative for petroleumbased nanocomposites in many applications due to their biodegradability, eco-friendliness, renewability, relatively inexpensive, low toxicity, abundancy, and improved thermal, mechanical, physical, barrier, and functional properties [3,4]. Various natural biopolymers, including starch, cellulose, pectin, lignin, chitin/chitosan, alginates, hyaluronic acid, gelatin, terpenes, gelatin, gluten, and polyhydroxyalkanoates (PHAs) from plants, animals, algae, microorganisms and synthetic biopolymers, including polycaprolactone (PCL),

**Citation:** Gamage, A.; Thiviya, P.; Mani, S.; Ponnusamy, P.G.; Manamperi, A.; Evon, P.; Merah, O.; Madhujith, T. Environmental Properties and Applications of Biodegradable Starch-Based Nanocomposites. *Polymers* **2022**, *14*, 4578. https://doi.org/10.3390/ polym14214578

Academic Editor: Raffaella Striani

Received: 3 October 2022 Accepted: 25 October 2022 Published: 28 October 2022

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

poly(butylene succinate) (PBS), poly(lactic-co-glycolic acids) (PLGA), and polylactic acids (PLA), have been used in nanocomposite materials for various applications [1–3,6–8].

Starch is one of the most abundant natural polymers globally. Starch and its nanocomposites have been extensively studied for their abundance, low cost, ease of processibility, and chemical and physical properties [1,4]. Furthermore, starch can be used in natural or modified form. Native starch has drawbacks, such as poor mechanical properties, high hydrophilicity, and high biodegradability. Thus, researchers are exploring starch modification techniques to improve its properties and develop novel composites [1].

Starch can be modified into nanoparticles and can also undergo various physical (milling, blending with other polymers, extrusion, plasticizers, etc.) and chemical (substitution, graft co-polymerization, cross-linking, oxidation, etherification, esterification, dual modification, etc.) modifications to produce materials with novel properties [9–12].

Starch can be reinforced with starch nanoparticle/starch nanocrystals and nano polymers such as nanoclay (montmorillonites [MMTs], halloysites nanotubes [HNTs]), carbon nanotubes (CNTs), and nanofibers and nanowhiskers (cellulose, chitin) and metal and metal oxides (TiO2 NPs, ZnO NPs, etc.) to achieve desirable properties and produce potential green sustainable nanocomposite materials [4,7,13]. The addition of nanofillers and additives with antioxidant and antimicrobial properties has been shown to improve or minimally affect biodegradation of starch-based nanocomposites [5,14,15]. Lifecycle assessments on starch and starch-based composites ensure their lower environmental impact and sustainable alternative for petrochemical-based polymers [16–18].

This review mainly discusses the starch-based nanocomposites in regard to starch and its nanostructures, various starch-based nanocomposites mainly reinforced with nano polymers, such as nanoclay, carbon-based materials, nanocellulose, and chitin NPs), and their applications, particularly in the fields of agriculture, packaging, biomedicine, and the environment. Moreover, this paper highlights the lifecycle analysis and degradation of various starch-based nanocomposites in order to analyze their environmental impact.

#### **2. Starch**

Starch is a polysaccharide and is renewable, inexpensive, biodegradable, and readily available. Starch contains two polymers (glucans) known as amylose (10–30%) and amylopectin (70–90%). Amylose is a linear chain of D-glucose units linked by the α-(1,4) glycosylic bonds, while amylopectin is a highly branched and high molecular weight chain composed of D-glucose repeating units linked by α-(1,4) glycosylic bonds and α-(1,6) glycosidic bonds. The amylopectin chain contains 10–60 glucose units, and the side chains consist of 15–45 glucose units with about 5% of α-(1,6) branching points [6,7]. Amylose and amylopectin are radially arranged in an alternating concentric (amorphous and semicrystalline) ring in starch granules. Amylopectin is radially arranged in granules and contributes to its crystalline nature (double helices region), and single helices amylose is randomly distributed among amylopectin clusters. Amylose and the branching point of amylopectin form the amorphous region [19–21]. Figure 1 illustrates the structure of the starch granule and the chemical structure of amylopectin and amylose.

Starch is a primary energy source in plants, which is stored in various parts, including the roots, tubers, seeds, and stems [6]. Various plant sources, such as corn, potato, wheat, cassava, rice, corn, barley, rye, millet, peas, mung beans, lentils, arrowroot, sago, sorghum, banana, yam, and many others, are utilized to obtain starch [22–24].

Starches from different sources show variation in their chemical composition (αglucans, moisture, lipids, proteins, and phosphorylated residues), the structure of glucan components (amylose and amylose), and starch granule size and shape due to genetic and environmental factors [25,26].

Starch granules' size and shape can vary with the content, structure, and arrangement of amylose and amylopectin [25]. Starch granules are found in various sizes ranging from 2–150 μm and packed with amylose and amylopectin content. Regular starch granules contain amylose in the range of 15–30% but can be varied in the range of 0–78%. Waxy starch contains lower or no amylose, whereas high-amylose starch consists of more than 50% amylose [7,23]. Table 1 shows the amylose contents of various starch sources.

**Figure 1.** Starch granule structure and the chemical structure of amylopectin and amylose.



Starch-based hydrogel is formed via gelatinization of starch during heating with excess water and followed by three-dimensional network formation by retrogradation [37]. Gelatinization of starch is an irreversible process that occurs through the absorption of water and disruption of the crystalline structure of starch granules by hydrogen bond breakage, swelling, the disintegration of starch granules, leaching of amylose that increases viscosity and solubilization of starch molecules [32,35,37].

Amylose and amylopectin content, amylopectin structure (molar mass or chain length), and starch granule size influence the chemical, physical, optical/transparency, and functional properties (water uptake, swelling, gelatinization, pasting [pasting viscosity and temperature], retrogradation, and susceptibility to enzymatic hydrolysis of starch [7,20,23,36,38].

Amylopectin contributes to water absorption, swelling, and pasting of starch granules, whereas amylose hinders the swelling property in the presence of lipids, thus preventing gelatinization power [32,38]. Furthermore, short-chain amylopectin showed better swelling power than that of long-chain amylopectin, indicating that starch with higher crystallinity reduces the swelling power [38]. Smaller granule size increases hydration, thus increasing the swelling, viscosity, and gelatinization properties [26].

Amylose content is negatively correlated with swelling power, gelatinization temperature, and the enthalpy of gelatinization required to disrupt the crystalline structure [35]. Waxy starch has a higher degree of crystallinity and higher gelatinization temperature than starch with high amylose content [31,35]. Amylose in starch has a high tendency for retrogradation due to its linear structure. However, the retrogradation properties of starch

are mainly determined by the degree of crystallinity and gelatinization temperature than the amylose content [35].

Amylose–amylopectin ratio also influences thermal, mechanical, and barrier properties. Basiak et al. [23] reported that potato starch, containing lower amylose (20%) than that of wheat (25%) and corn (27%) starch, exhibited greater mechanical properties and lower water solubility, water vapor, and oxygen permeability. Other than that, optical properties were influenced by the amylose/amylopectin ratio: the potato (lower amylose) film was transparent, whereas corn and wheat films were opalescent.

However, applications of starch have been limited due to their poor performance, such as through their brittleness, high water sensitivity, poor gas and moisture barrier, susceptibility to retrogradation, high viscosity, and limited solubility [13,39]. Therefore, plasticizers, chemical modifiers, and incorporating nanofillers, such as starch nanoparticles, nanoparticles, nanoclay, nanofibers, and others, have been used to improve the properties of starch [39].

#### **3. Nanomaterials and Nanocomposites**

Nanomaterials are referred to as materials which have at least one of their dimensions less than 100 nm. Based on the definition, a thin film with <100 nm thickness is a nanomaterial as one of the dimensions is nanometric. Likewise, nanomaterials such as nanofibers, nanowires, and nanorods have two dimensions on the nanoscale, whereas quantum dots, nanoparticles, dendrimers, and fullerene have three dimensions in the nanometer range (Figure 2) [40].

**Figure 2.** Examples of various types of nanomaterials based on the number of dimensions in the nanometer range.

Nanomaterials can be classified based on dimensionality (number of dimensions with a length larger than 100 nm), as shown in Figure 3: 0D, 1D, 2D, and 3D. Zero dimension (0D), including spheres, hollow spheres, clusters, quantum dots, and metals, have no dimension of particles larger than 100 nm, i.e., all dimensions in the nanoscale. One-dimensional (1D) nanomaterials, such as nanorods, nanowires, nanofibers, and nanotubes, have one dimension, not in the nanoscale (>100 nm) and the other two are in the nanoscale, whereas two-dimensional (2D) nanomaterials, including thin film, nanocoatings, nanoplates, and nanolayers, have two dimensions, not in nanoscale and another one in nanoscale. Three-dimensional (3D) is the combination of nanocrystals in different directions which have various dimensions above 100 nm. Figure 3 depicts the classification of nanomaterials based on dimensionality [40–42].

Nanomaterials can be synthesized by two approaches: top-down and bottom-up approaches (Figure 4). In the top-down method, the bulk material is restructured into nanomaterials using mechanical grinding/milling, ball milling, polishing, lithography, and other means. While in the bottom-up method, nanomaterials are assembled from atomic range particles/molecules or nanoclusters through the sol–gel method, spinning,

molecular self-assembly, pyrolysis and condensation, vapor phase deposition, and other methods [40,41].

**Figure 3.** Classification of materials based on dimensionality.

**Figure 4.** Nanoparticle synthesis methods: top-down and bottom-up approach.

Composite materials consist of two or more dissimilar materials, which are composed of two major constituents: (1) a matrix as a continuous phase (polymer, ceramic, or metal) and (2) reinforcement materials as an un-continuous phase. Bionanocomposites are composite materials that are composed of biopolymers and particles with at least one dimension in the nanometer range (1–100 nm). Bionanocomposites can also be referred to as green composites or biohybrids, or bioplastics [3,43].

Nanocomposites can be classified into three categories based on the morphology of reinforced nanoparticles: (1) particulate/iso-dimensional (silica, metal NPs, metal oxides), (2) layered (monolayered clays, layered double hydroxides), and (3) elongated (cellulose nanofibrils [CNF], carbon nanotubes [CNTs]) nanoparticles [3,44]. Particulate reinforcements are used to enhance resistance to flammability and reduce permeability and cost, whereas layered reinforcements are used for their superior mechanical behavior [43]. Furthermore, based on the degree of dispersion of particles in the matrix, layered nanocomposites have three subclasses, including intercalated, exfoliated, and flocculated/phase-separated nanocomposites (micro-composites) [3,6,43]. Flocculated/phase-separated nanocomposites are formed without a partition between individual layers due to the particle–particle interactions, polymer chains are intercalated between sheets of layered nanoparticles in intercalated nanocomposites, and exfoliated nanocomposites are formed by partition between individual layers (Figure 5) [43].

**Figure 5.** Classification of the nanocomposites.

#### **4. Starch Nanoparticles (SNPs)**

Starch nanoparticles (SNPs) are mainly synthesized by the methods of hydrolysis (acid or enzymatic), regeneration, and physical treatments (milling, high-pressure homogenization, gamma radiation, and ultra-sonication) [45].

SNPs are mainly used as fillers in a polymer matrix to improve their reinforcing effect and mechanical and barrier properties [13]. Nanoparticles have a large surface area/volume ratio, allowing a great interaction capacity, which makes them potential reinforcement materials [46]. SNPs are non-toxic and can be used to prepare nanocomposite, absorbent, carrier (encapsulation), and emulsion stabilizers for food and non-food applications [45,47,48].

Santana et al. [46] reported the SNP obtained from ultrasound showed a significantly higher yield than SNP synthesized by acid hydrolysis. In addition, incorporating SNPs reduced the water vapor permeability of starch film [46]. Lin et al. [49] prepared debranched starch nanoparticles (DSNPs) by reverse emulsification using debranched waxy corn starch (98% of amylopectin), which showed a higher crystallinity and melting temperature than that of native waxy corn starch. Furthermore, the addition of debranched starch nanoparticles (5 wt.%) into corn starch films improved the tensile strength by 85.9% and decreased water vapor permeability and the oxygen transmission rate by 30.94% and 79.31%, respectively.

In another study, starch NPs prepared by acid hydrolysis containing Ag NPs showed good antibacterial activity against *Staphylococcus aureus*, *Salmonella typhi*, and *Escherichia coli* which has the potential to be used as a coating material for food packaging [50].

#### **5. Starch-Based Nanocomposites**

Native starch or thermoplastic starch (TPS) has poor mechanical properties (fragility/ brittleness), low thermal stability, hydrophilicity, high water vapor permeation, poor resistance to external factors (humidity, tearing, picking, etc.), and a lack of compatibility with hydrophobic polymers [7,12,51]. Therefore, starch is blended with other natural and synthetic polymers or incorporated with various nanomaterials to enhance the physical, mechanical, and barrier properties [7]. Compared with bulk materials, nanoparticles have a surface area/volume ratio and possess unique physical, mechanical, optical, magnetic, electrical, and other properties [42]. Hence, recently, bionanocomposites can be a promising material to enhance mechanical and barrier properties [52]. Starch reinforced with nanofillers, including nanocellulose, chitin nanoparticle, nanoclay, and carbon-based materials, are discussed below.

#### *5.1. Starch/Nanocellulose Composite*

Cellulose is the primary component of the plant cell wall and can be extracted from plants, invertebrates, marine animals, algae, fungi, and bacteria [53]. It is the most abundant natural polymer and is popular for its mechanical properties, reinforcement capabilities, low density, renewability, low toxicity, and biodegradability [54]. Cellulose is the polymer of D-glucose units linked by β-(1,4)-glycosidic bonds, and higher hydroxyl groups (-OH and -CH2-OH) at equatorial positions give higher stability (Figure 6) [55]. Cellulose fibres are formed with strong inter and intramolecular hydrogen bonds and aggregate with highly ordered (crystalline) and disordered regions (amorphous) [56]. Nanocellulose is a nanostructure of cellulose and has drawn much attention over the past years due to its excellent characteristics, including its high aspect ratio (length to diameter), improved mechanical and thermal properties, crystallinity, flexibility, renewability, abundance, biocompatibility, and biodegradability [55,57].

**Figure 6.** Cellulose chemical structure and schematic diagram of the formation of cellulose nanocrystals and cellulose nanofibrils.

Nanocellulose can be produced by top-down and bottom-down processes (Figure 6) [53,54] using various techniques, including enzymatic techniques, chemical hydrolysis, and mechanical treatments, including high-pressure homogenization, grinding, cryo-crushing, micro-fluidization, and high-intensity ultrasonication [46,53,54]. These synthetic techniques and conditions influence the dimensions, composition, and properties of nanocellulose. Nanocellulose can be generated in three forms: (1) cellulose nanofibrils (CNFs) and (2) cellulose nanocrystals (CNCs) from woods and other lignocellulosic materials using a top-down process, and (3) bacterial cellulose (BC) from the biosynthesis of bacteria using a bottom-to-top process. Figure 7 summarizes the three forms of cellulose and synthesis methods [53,55].


**Figure 7.** Types of nanocelluloses.

Nanocellulose is widely used in various applications, such as biomedical engineering, the automotive industry, electronics, food packaging, cosmetics, construction, textiles, wood adhesives, and wastewater treatment applications [53,57].

Othman et al. [58] prepared the corn starch (CS) film reinforced with nanocellulose fiber (NCF) and thymol, a compound extracted from the essential oil of thyme, which has

antioxidant and antimicrobial properties. They reported that adding 1.5% of NCF improved the thermal stability, mechanical, and barrier (water vapor and oxygen) properties of corn starch film. The CS/NCF/thymol composite reported improved thermal stability and flexibility. However, a significant reduction was observed with tensile strength, Young's modulus, and barrier properties [58]. In another study, starch from an unripe plantain bananas reinforced with cellulose nanofibers from banana peels improved the mechanical and water vapor barrier properties [59]. Starch/CNC nanocomposites were reported to improve the tensile strength (2.8 to 17.4 MPa), Young's modulus (112 to 520 MPa), and water barrier properties, as well as reduce the water solubility (26.6 to 18.5%) and contact angle 38.2 to 96.3◦ [60].

#### *5.2. Starch/Chitin Nanoparticles Composites*

Chitin is the second most abundant natural polysaccharide next to cellulose and is found in the shell of crustaceans (crab, lobster, and shrimp), the exoskeleton of arthropods, molluscan shells of squid, mushrooms, the cell wall of algae and fungi (yeast and mold). Chitin is composed of N-acetyl-2-amido-2-deoxy-β-D-glucose (N-acetylglucosamine) units linked with a β-(1,4)-glycosidic bond, in which acetamide groups (−NHCOCH3) consists at the C2 of cellulose monomer. Chitosan is derived from the alkaline deacetylation of chitin (Figure 8). Chitin crystals are found in three forms: α-chitins (which contain antiparallel cellulose chains), β-chitins (parallel cellulose chains), and γ-chitin (among three chains, two of them are in the same direction, and one is in the opposite direction) [61–63].

**Figure 8.** Chemical structure of chitin and schematic diagram of the formation of chitin nanowhiskers.

Chitin nanomaterial can be prepared through top-down and bottom-up approaches. Chitin fibrils consist of amorphous and crystalline regions and thus can be converted into three types of nano-chitins in a top-down approach: nanocrystals (via acid hydrolysis), nanofibers (via mechanical treatments), and nanowhiskers (consecutive acid hydrolysis at a high temperature and mechanical treatments) [39,62].

Nano-chitin has been widely studied for its high aspect ratio, high surface area, good mechanical properties, lightweight/low density, good chemical stability, renewability, non-toxicity, and antibacterial properties, and it is used in biomedicine, packaging, water treatment, green electronics, cosmetics, and many other applications [61,63].

A combination of chitin nanofibers and starch nanoparticles showed higher emulsion stability over a range of pHs and temperatures and can be used as an emulsion stabilizer in various products, such as food, paint, coating, cosmetics, and pharmaceuticals [48].

Chang et al. [64] reported chitin nanoparticles (CNPs) exhibited lower crystallinity than chitin whiskers. At a low level of CNPs, tensile strength, storage modulus, glass transition temperature, and water vapor barrier properties of plasticized potato starch/CNPs nanocomposite due to good interfacial interaction between CNPs' nanofiller and starch matrix.

By adding 5 wt.% chitin nanofibers (CNF) obtained from the fungus *Mucor indicus*, Young's modulus and the tensile strength of TPS were enhanced by 239% and 216%, respectively, and moisture absorption was reduced from 51% to 38%. However, the addition of CNF at a higher level increased moisture absorption and reduced the mechanical properties of TPS [39]. In another study, Heidari et al. [61] reported that CNF/TPS nanocomposite

films were more permeable to water vapor than pure CNF film. CNF at higher levels lowers the dispersion of nanofiller and tends to agglomerate, which leads to poor water vapor barrier and mechanical properties. In addition to that, the presence of excessive NH2 groups at the CNF surface may increase the affinity to water, thereby increasing water absorption [39,61].

#### *5.3. Starch/Nanoclay Nanocomposites*

Clay is a polymer composite of two-dimensional layered mineral silicates. The single layer is formed by the edge-linked octahedral sheet of aluminum or magnesium oxide sandwiched between two tetrahedral silicate sheets. As shown in Figure 6, three types of polymer-based nanocomposites can be obtained based on the polymer and silicate layers. Silicate clay is characterized by important physical properties, such as a cation exchange capacity and specific surface area [65,66]. Polymer/nanoclay composites are used in the automotive industry, aeronautical industry, packaging, flame-resistant materials, biomedical applications, and wastewater treatment [67,68]. Nanoclays can be categorized into several classes: smectite, chlorite, kaolinite, illite, and halloysite [68].

Plate-like montmorillonite (MMT) (smectite), a multilayer-aluminosilicates, has been widely studied as a reinforcing material in polymers due to its excellent cation exchange capacity, swelling behavior, and large surface area [68,69]. MMT also improved the thermal stability, mechanical, optical, and barrier properties, even at their lower concentration [70].

Mohan et al. [15] reported that the incorporation of MMT nanoclay into corn starchbased film resulted in a significant reduction in water absorption (by 22%), moisture uptake (40%), oxygen permeation (30%), and swelling thickness (31%) in comparison to corn starch film. Furthermore, the concentration of MMT nanoclay determines the structure of the nanocomposite. X-ray diffraction (XRD) analysis revealed that the intercalated nanoclay structure forms at a higher concentration (>2%), whereas the exfoliated structure forms at a lower concentration in the polymer matrix [15]. In another study, MMT addition was also shown to improve the tensile strength and biodegradability in cross-linked PLA/maleated TPS nanocomposite [71]. Biodegradable nanocomposites fabricated from cross-linked wheat starch (CLWS)/sodium montmorillonite (Na-MMT)/TiO2 NPs showed an exfoliated structure. Incorporating Na-MMT and TiO2 NPs reduced the water vapor permeability and water solubility of the CLWS film, whereas thermal stability, tensile strength, and Young's modulus were increased. TiO2 NPs showed better UV-blocking properties than Na-MMT [69]. Maize starch/glycerol (20%)/Na-MMT (10%) nanocomposite also showed intercalated structures and improved tensile properties [66].

Iamareerat et al. [72] prepared nanocomposite film with plasticized cassava starch incorporated with sodium-bentonite and cinnamon essential oil. The addition of sodiumbentonite nanoclay (0.5–0.75%) decreased the water vapor permeability in plasticized cassava starch with 2% glycerol film. Further addition of cinnamon essential oil into the CS/glycerol (2%)/sodium-bentonite (0.75%) showed better antibacterial activity and significantly inhibited microbial growth in pork meatballs, despite the increase in water vapor permeability.

Halloysites clay nanotubes (HNTs), aluminosilicate hollow cylinders, have a lower hydroxyl group on the surface than other silicates such as MMT, making them a promising reinforcement material for polymers [68,73]. Furthermore, HNTs exhibit exfoliated structures due to their high aspect ratio [73]. Dang et al. [73] revealed that the addition of modified or unmodified HNTs into the TPS/poly(butylene adipate-co-terephthalate) (PBAT) blend improved the thermal and mechanical properties without loss of ductility of the plasticized wheat starch matrix [74]. Another investigation on PVA/starch/glycerol/HNTs nanocomposites revealed that their hydrophobic nature and biodegradability decreased with the addition of HNTs [75].

#### *5.4. Starch/Carbonaceous Nanocomposites*

Fullerenes, diamonds, carbon nanotubes (CNTs), graphene, and their derivatives are common carbon allotropes used in carbon-based nanocomposites [76].

CNTs found in two forms, single-walled (SWCNT) or multi-walled carbon nanotubes (MWCNT), have been widely studied as reinforcing fillers for TPS nanocomposite films [77]. CNTs have a larger surface area, excellent electrical conductivity, mechanical and thermal properties and they also have a higher volume-to-area ratio compared to that of other nanoparticles and they are widely used in various biomedical applications, environmental pollution control, sensing and detection, the automobile industry, and secondary food packaging. Direct contact food packaging materials are limited by their migration and potential toxicity [76,78,79].

Electrically conductive biocomposite films have gained popularity in various electronic, biomedical, and food packaging applications [22]. Potato starch-based film reinforced with MWCNT and ionic surfactants (sodium cholate, SC; cetyltrimethylammonium bromide, CTAB) decreased the contact angle and showed improved antioxidant properties (30.2 and 12% of scavenging activity, respectively) due to the presence of MWCNT. Surfactant SC showed better dispersibility of MWCNT in a potato starch matrix with improved mechanical properties and crystallinity [22].

Starch plasticized with ionic liquids reduces the retrogradation resulting in increased film stability and it has the potential use in ionically conducting solid polymers. The addition of nanofiller MWCNT at 0.5 wt.% in starch plasticized with ionic liquid, 1-ethyl-3 methylimidazolium acetate ([emim+][Ac−]) significantly increased the tensile strength by 327%, Young's modulus by 2484%, and elongation at break 82% (from 30 to 69%). Moreover, electrical conductivity was increased with MWCNT content (wt.%) and reached a maximum (56.3 S/m) at 5 wt.% MWCNT. MWCNT/starch plasticized with [emim+][Ac−] showed electroconductive properties because of its ionic nature of ionic liquids and the excellent electrical conductivity of MWCNT [77]. A starch–iodine complex matrix reinforced with a small amount of MWCNT (0.055%) reduced the water vapor permeability by 43% [78].

Graphene is a two-dimensional material arranged in a hexagonal lattice. Plasticized starch incorporated with reduced graphene oxide (rGO), a derivative of graphene, exhibited increased conductivity and dielectric properties, which could make it a potential candidate for producing sustainable bio-friendly electronic devices [80].

Investigation of poly(lactic acid) (PLA)/thermoplastic starch (TPS)/graphene nanoplatelets (GNP) blends revealed that the addition of GNP increased the crystallinity of the PLA/TPS blend, and the maximum crystallinity (68.39%) was observed with PLA (70%)/TPS (30%)/GNP (1%). Further increases in GNP resulted in the reduction of compatibility [81].

#### **6. Applications of Biodegradable Starch-Based Nanocomposites**

Biodegradable starch-based nanocomposites have been used in agriculture, packaging, biomedical, environment, and many other fields (Figure 9).

#### *6.1. Agriculture*

In recent years, biodegradable films have been developed for agricultural purposes, particularly for mulching applications, the coverings of a greenhouse, and the controlled/slow release of agrochemicals such as fertilizers and pesticides [82–84].

Agricultural mulches are used to prevent the hindrance caused by the weeds' growth, maintain soil wetness, and regulate soil temperature [85]. Interaction with water (water vapor permeability, contact angle, and water solubility/resistance) and environmental factors (thermal stability) are important parameters in mulch films. Mulch films must have a very low water vapor permeability to maintain the soil moisture by reducing the water loss by evaporation. Since mulch films are exposed to outdoor conditions, improving the thermal stability is therefore essential [83,86].

**Figure 9.** Applications of starch-based nanocomposites.

Pesticides protect the crop from pests, pathogens, weeds, and insects by destroying, attacking, mitigating, or repelling activity, whereas fertilizers are essential in agriculture to increase crop yield. However, in conventional applications, the efficiency of reaching their target sites is relatively low as they are hindered by immobilization, erosion, volatilization, leaching, surface runoff, or scavenging by soil. In addition, water is also an essential factor in crop growth and driving off fertilizers. Therefore, management of nutrient/pesticide active compounds and water loss is essential for crop production. To reduce the loss and improve their utilization efficiency, slow-release fertilizers or controlled-release pesticides with improved water retention and water holding capacity can be formulated by incorporating nanomaterial into biopolymers [82,84,87,88].

Merino et al. [83] investigated the water and light interaction with corn starch-based mulch film. The study revealed that the addition of chitosan/bentonite nanofiller into native and oxidized thermoplastic corn starch improved the water resistance, radiometric, and antibacterial properties without having a significant effect on the water vapor permeation and mechanical properties [83]. In another study, Merino et al. [86] reported that the addition of bentonite/chitosan into both matrixes, native and oxidized thermoplastic corn starch, increased the crystallinity (3.0 and 3.4%) and slightly increased thermal stability in comparison to the addition of natural bentonite.

Superabsorbent hydrogels are widely used in bi-functional (retain and supply water and nutrient over a long period) slow-release fertilizers due to their water retention properties. The addition of natural char nanoparticles (NCNPs) into corn starch-g-poly(AAco-AAm) encapsulated urea provided high biodegradability and improved the soil waterretention capacity along with the slow release of urea [84]. Chitosan (CS)/sago starch (ST)/nano zeolite (NZ) nanocomposite released 64% of phosphorus and 41.93% of urea after 14 days and increased the water retention capacity. Furthermore, CS/ST/NZ nanocomposites showed better growth indexes in *Philodendron* spp. compared to the direct application of urea, suggesting the efficacy of nanocomposites in slow-release fertilizer formulation [88]. Urea encapsulated with starch (10%)/PVA (5%) with crosslinker acrylic acid (2%) and citric acid (2%) showed higher nitrogen-releasing efficiency, 70.10 and 50.74%, respectively, as well as improved growth factors in spinach plants [89]. Modified starch (esterified with dicarboxylic acid chloride)/organobentonite-based composites regulate the effective controlled release of encapsulated pesticide atrazine [90].

#### *6.2. Packaging*

Food packaging protects food from humidity, high/low temperatures, and other physiological factors and aids in food quality monitoring and control in the food supply chain and during storage (gas sensors, electronic nose) [91]. Starch has been used in food packaging applications because of its strong mechanical properties, transparent/translucent appearance, and tasteless and flavorless characteristics [69]. Brittleness and poor water vapor barrier properties limit their applications. Nanoparticle reinforcement can improve the mechanical properties, hydrophobicity, water vapor and oxygen barrier, UV barrier, thermal properties, and other functional properties (antioxidant, antimicrobial, etc.) of starch which makes nanoparticles a potent material for edible film/coating, active and intelligent/smart packaging for protecting or maintaining and monitoring the quality of food materials [91–93].

Organic or inorganic nanofillers have been widely studied for food packaging applications, whereas organic nanofillers include nanoclay (MMTs, HNTs), natural biopolymers (chitosan, cellulose), and natural antimicrobial agents (nisin), and inorganic nanofillers includes metals (Ag, Au, Cu), and metal oxides (ZnO, TiO2, Ag2O, MgO, CuO, SnO2) [44,52,91].

The suitability of a film for packaging materials is mainly assessed by water vapor and oxygen barrier properties and good heat salability [94]. Furthermore, a film with improved mechanical strength and flexibility protects against shock and other physical damage. TiO2 NPs reinforcement in potato starch-based composite films led to a reduction in water solubility, moisture uptake, and water vapor permeability, and an increment of UV barrier properties and tensile strength of the film, showing its potential for food packaging [92]. Na-MMT and TiO2 NPs reduce the hydrophilicity and improve mechanical, water vapor, and UV barrier properties in cross-linked wheat starch, which makes them a suitable material for food packaging [69]. UV barrier packaging film from starch/kefiran/ZnO NPs showed improved tensile strength, Young's modulus, and thermal stability (increased melting temperature), which are beneficial to the packaging system [95]. Starch NPs/Ag NPs showed increased antibacterial activity against *Staphylococcus aureus*, *Salmonella typhi*, and *Escherichia coli* and can be used as an antibacterial food coating material [50]. Linseed polyol increased the contact angle, water absorption capacity, thermal stability, and biodegradation of polyvinyl alcohol/corn starch film. Further addition of Ag NPs showed antimicrobial behavior against *Proteus mirabilis*, *Candida albicans*, *Escherichia coli*, *Enterococcus faecalis*, *Staphylococcus aureus*, *Klebsiella pneumoniae*, among others, which shows the potential applications in antimicrobial packaging [96]. Poly(ethyl methacrylate)-co-starch (PEMA-costarch)/graphene oxide/Ag NPs (2 wt.%) nanocomposite film showed improved thermal stability, chemical resistance, tensile strength, oxygen barrier properties, and antimicrobial properties against *Escherichia coli*, *Pseudomonas aeruginosa*, *Staphylococcus aureus*, and *Bacillus subtilis* [97].

Plasticised corn starch films reinforced with nanocellulose improved the mechanical strength, flexibility, and water vapor and oxygen barrier properties that have a beneficial effect on reducing the oxidation of oil during storage. This film showed good heat salability, which further prevents oxygen and water vapor transmission. Moreover, the storage study ensures that this plasticized corn starch-based nanocomposite can be used as an alternative packaging material for storing edible oils at ambient conditions (27 ± 3 ◦C temperature, 65 ± 5% RH) for more than three months without affecting the oil quality in terms of rancidity, viscosity, and color [94].

Starch from potato, wheat, and corn blended with carboxyl methylcellulose (CMC)/Na-MMT has potential applications in food packaging [98]. Cellulose nanocrystals (CNC) obtained from sugarcane bagasse blending with starch improved mechanical, water resistance, and water barrier properties and decreased surface hydrophilicity (contact angle > 90◦), which makes this starch/CNC nanocomposite a potential food packaging material [60]. Heidari et al. [61] developed edible food packaging using chitin nanofibers (CNF)/ TPS nanocomposite.

TPS/MMT/carvacrol essential oil showed biocidal effects against *Escherichia coli* due to the synergistic antibacterial effect of carvacrol essential oil and MMT suggesting the applications in antimicrobial packaging [99]. Packaging material fabricated with sweet potato starch (SPS)/MMT/thyme essential oil (TEO) was studied by Issa et al. [100]. They reported that the addition of MMT improved the mechanical and water barrier properties of SPS, whereas biodegradability decreased. However, incorporating TEO decreased the tensile strength, elongation, Young's modulus, and water barrier with improved biodegradability in SPS/MMT. The nanocomposite made from cassava starch/glycerol (2%)/Na-bentonite (0.75%)/cinnamon essential oil (2.5%) exhibited antibacterial activity against *Escherichia coli*, *Salmonella typhimurium,* and *Staphylococcus aureus*, and significantly inhibited the microbial growth in pork meatballs stored under ambient and refrigeration conditions [72].

The addition of potassium sorbate, a commonly used preservative, into starch/nanoclay films controlled the migration of sorbate, resulting in the retention of antimicrobial activity for a long period [101]. Chen et al. [102] also developed a controlled-release active film from starch/polyvinyl alcohol (PVA) incorporated with cinnamaldehyde and microfibrillated cellulose (MFC). The addition of MFC was found to improve the tensile strength, crystallinity, hydrophobicity, and antimicrobial activity (against *S. putrefaciens*) with reduced flexibility. The oxygen and water vapor permeability reduced at 1.0 and 2.5% MFC but increased at higher concentrations. In addition, MFC, at 1 and 7.5%, controlled the release of cinnamaldehyde.

Smart packaging materials for monitoring the spoilage of milk packed in a bottle were developed by incorporating pH indicators, including bromocresol green (BG) and methyl orange (MO), into a starch/nanoclay nanocomposite [93]. Further nanometals (TiO2, SnO2, Ag2O, MgO, ZnO, CuO) can be used in gas sensors to monitor food quality [91].

#### *6.3. Biomedical*

Biodegradable polymers, including starch-based bionanocomposites, are widely used as scaffolds for tissue engineering, drug delivery systems/drug carriers, wound dressing, surgical sutures, and implants due to their mechanical properties, biocompatibility, biodegradability, and also the generation of non-toxic, biodegradable products [103–105].

Biopolymers in the repair of healing tissues accelerate treatment processes and eliminate implant removal surgery. Furthermore, implant materials and their biodegradable products must be non-cytotoxic and biocompatible [105]. Incorporating bioactive betatricalcium phosphate (β-TCP) nanoparticles (at 10%) into thermoplastic starch (TPS) drastically improved the mechanical properties and showed excellent biocompatibility with no cytotoxic effect for bone tissue engineering materials [105]. Waghmare et al. [106] fabricated starch-based nanofibrous scaffolds by electrospinning for wound healing applications.

Hydroxyapatite has been used widely in biomedical applications due to its biocompatibility and osteoconductive (cell regeneration process) properties. However, brittleness and lack of flexibility limit the applications. The combination of hydroxyapatite with starch materials can reduce brittleness, and the polar nature of starch encourages a good adhesion between starch and hydroxyapatite. Sadjadi et al. [107] synthesized a nanocomposite from starch/nano-hydroxyapatite, which possesses mechanical and biological properties identical to natural bone.

Abdel-Halim and Al-Deyab [108] reported that Ag NPs/starch/polyacrylamide nanocomposite hydrogel showed antimicrobial activity against fungi (*Aspergillus flavus* and *Candida albicans*) and bacteria (*Staphylococcus aureus* and *Escherichia coli*). PVA/starch incorporated with Ag NPs synthesized from green methods (*Diospyros lotus* fruit extract) has the potential to be used in wound dressing as it shows increased swelling and moisture retention capacity and reduced water vapor transmission that prevents the wound from dehydration and better antimicrobial activity against *Escherichia coli* and *Staphylococcus aureus* [109].

The ternary blend was developed by mixing polylactic acid (PLA)/starch (S)/polyε-caprolactone (PCL) with nano-hydroxyapatite (nHAp) for controlled release of antibacterial triclosan. The incorporation of nHA (3%) improved the hydrolytic hydrophilicity, hydrolytic degradation, antibacterial activity (against *Escherichia coli* and *Staphylococcus aureus*), and drug release of PLA/S/PCL film. An increase in nHA content (1–7%) improved the biodegradation (13–10 months), and the antibacterial triclosan release rate of PLA/S/PCL/nHA film at 37 ◦C in buffer solution was increased (0.12–0.18 μg/mL every day), which is in the range of MIC of triclosan (0.025–1 μg/mL). Furthermore, the degradation and release time of PLA/S/PCL/nHA (3 wt.%) nanocomposite showed similar profiles that ensure continuous drug release during the application [110]. Mallakpour and khodadadzadeh [111] also developed starch/MWCNT modified with glucose (MWCNT-G) nanocomposites for slow release of zolpidem drug delivery. Gao et al. [112] developed spherical core-shell Ag/starch NPs using green synthesis for slow-released nano silver as an antibacterial material which can be used in pharmaceutical and biomedical applications.

Nezami et al. [113] fabricated pH-sensitive magnetic nanocomposite hydrogel using graft copolymerization of itaconic acid (IA) and starch in the presence of magnetic Fe3O4 NPs (St-IA/Fe3O4) for the controlled-release of guaifenesin (GFN) with low cytotoxicity. A nanocomposite with magnetic Fe3O4 NPs at 0.83% significantly enhanced the drug release from 54.1 to 90.4% within 24 h in pH 7.4 [113].

Starch-based-fluorescent organic nanoparticles (FONs) reported high water dispersibility and excellent biocompatibility (cell viability was 99.69% at the concentration of FONs 100 μg/mL after 24 h). Thus, FONs are a promising candidate for biomedical applications that can be potentially used as fluorescence probes and carriers for delivering biologically active components [114].

#### *6.4. Environment*

Extensive agricultural and industrial practices lead to the accumulation of various contaminants, including heavy metals and metalloids (Cr6+, Hg2+, Zn2+, Pb2+, Co2+, Cd2+, Cu2+, etc.), dyes, organic substances (pesticides, herbicides, fertilizers, aliphatic and aromatic hydrocarbons, volatile organic compounds [VOCs], oil spills), pathogenic microbes (virus, bacteria, fungi), and toxic gases (nitrogen oxides, SO2, CO) in water, soil, and air [115].

Starch-based nanocomposites with various nanofillers, including metal (Ag, Au, and Pd NPs), bimetal (Ag/Au), metal oxides (TiO2, ZnO, Fe2O3, MnO2), nanoscale zero-valent iron (nZVI) (Fe0), carbonaceous materials (CNTs [SWCNTs and MWCNTs], graphene, graphene oxide), nanoclays (MMTs, HNTs, bentonite), and polymers (chitin, cellulose nanowhiskers) are used in materials as recyclable and reusable filters, absorbents, reductants, photocatalysts, coagulants and flocculants, disinfectants, and gas sensors to detect or remediate contaminants, such as dyes, heavy metals ions (As, Pb2+, Cr6+, Cu2+, Cd2+, Hg2+, Ni2+, Co2+, etc.), various aromatic derivatives, fertilizers (urea), and other organic pollutants [116–124].

Green synthesis of Ag/Au bimetallic nanocomposite using graft copolymer hydroxyethyl starch-g-poly(acrylamide-co-acrylic acid) reported catalytic activities that involve the reduction of 4-nitrophenol to 4-aminophenol and degradation of azo dyes (congo red, Sudan-1, and methyl orange) by cleavage of −N = N-bond thus can be used in water treatment [122]. Gomes et al. [125] analyzed a starch/cellulose nanowhiskers hydrogel composite and highlighted the outstanding capacity for methylene blue dye removal.

Starch-graft-poly(acrylamide) (PAM)/graphene oxide (GO)/hydroxyapatite NPs (nHAp) nanocomposite was developed as a recyclable adsorbent for efficient removal of malachite green (MG) and other cationic dye from aqueous solution. The introduction of nHAp improved the biocompatibility of the PAM/GO composite, whereas the biodegradability, porosity, water content, and water uptake decreased with increasing nHAp content. Adsorption capacity increased with agitation time, pH, nHAp content, and initial dye concentration, and the optimum conditions were 60 min, pH 10, 5% nHAp, and 100 mg/L. PAM/GO and nHAp at 1–5 wt.% reported excellent porosity (31–11%), degradability (41–11% after 15 days), the maximum adsorption capacity of 297 mg/g, excellent regeneration capacity after five consecutive adsorption-desorption cycles of dye with high removal efficiency (77–86%) [126].

Adsorption is a basic principle of mechanism in targeted drug delivery, controlled release of pharmaceutically active compounds, and treatment of chemical water pollution [11]. The degree of the time dependency of kinetic coefficient (kobs) and the influencing factors (pH, temperature, initial concentration of tetracycline) are important to explore the suitability of materials in adsorption-based applications. Monodispersed starch stabilized magnetite nanoparticle (MSM) showed 70% absorption of antibiotic tetracycline within the

first 5 min and reached 90% after 1 h. The degree of the time dependency of the kinetic coefficient (kobs) had a negative correlation with the initial tetracycline concentration [11].

Chitin nanowhiskers (CNW) are better nano-adsorbents due to their high surface/volume ratio and abundant hydroxyl and acetamide functional groups on the surface [63]. MMT is hydrophilic and has a high specific area [127]. The bean starch/Na-MMT nanocomposite showed high absorption capacity for heavy metals Ni2+ (97.1% at pH 4.5, initial concentration of 100 ppm) and Co2+ (78.03% at pH 6, initial concentration of 140) in comparison to the starch matrix (72 and 74.2%, respectively) [116]. Yang et al. [123] studied the material nZVI loaded on biochar stabilized by starch to remediate Cr6+.

Enzyme immobilization is an emerging technology for environmental remediation which gives many advantages over free enzymes, which include the efficiency and stability of catalytic enzymes and their enhanced recovery and reusability [128]. Further, the immobilized enzyme can be used as biosensors and biocatalysts to degrade dye from textile, leather, coloring, and printing industries [129]. Immobilized peroxidase on polymer/Fe3O4 magnetic NPs has been successfully used to remediate wastewater containing different dyes in the textile industry [128]. Immobilized phenoloxidases other than peroxidase, including laccase and tyrosinase, are also used to degrade dyes and phenolic pollutants, and lipases are used to remediate oily wastewater [130]. Mehde [131] reported that magnetic NPs/tannic acid/starch/cross-linked enzyme aggregates-peroxidase are used to remove different types of dyes, such as methylene blue, Congo red, indigo carmine, and malachite green.

#### *6.5. Other Applications*

Plasticized starch/reduced graphene oxide (rGO) nanocomposites with improved conductivity and dielectric properties can be used in bio-friendly flexible electronic devices [80]. The maize starch/glycerol (20%)/Na-MMT (10%) nanocomposite showed improved tensile properties, which can be used in lightweight architectural constructions [66]. Starch-based nanocomposites can also be used in lithium batteries, fuel cells, dye-sensitized solar cells, and electrically conductive biocomposite film for various other purposes [22,77].

Table 2 summarizes the studies reported on various biodegradable starch-based nanocomposites in regard to their applications and properties.


**Table 2.** Starch-based nanocomposites using various biodegradable polymers in regard to their applications and properties.


#### **Table 2.** *Cont.*


#### **Table 2.** *Cont.*

#### **7. Lifecycle Analysis of Nanocomposites**

With increasing fossil depletion and environmental concerns, sustainable biobased materials have gained increasing interest. For biobased materials to be sustainable, preparation and processing should have limited environmental impacts [132].

The environmental credentials of bionanocomposites are evaluated by assessing their material production, product manufacturing, and product end-of-life. Many tools, including environmental impact analysis (EIA), life cycle analysis (LCA), material flow analysis (MFA), and ecological footprint (EF), are used for analyzing the environmental impacts of materials and manufacturing processes [133]. Life cycle assessment (LCA) is the most widely accepted method to assess environmental impact [134]. LCA is a science-based tool to comparatively analyze the environmental impacts of product systems concerning the extraction of raw materials, manufacturing, the use of final products, and their disposal [133,135,136].

The international organization for standardization (ISO) standardized the LCA via ISO 14040 series [134]. The two most commonly used methods are "*cradle to grave*" and "*cradle to gate*" [134,135]. The "*cradle to gate*" system covers all the steps from raw material extraction and energy to product conversion and delivery at the factory gate, whereas "*cradle to grave*" covers all phases of the lifecycle of a product, i.e., includes all steps of "*cradle to gate*" and usage and disposal phase [134]. LCA can be investigated through several environmental impact categories, such as global warming, ozone depletion, acidification, eutrophication, resource depletion (fossil fuel), ecotoxicity, human toxicity, photo oxidant formation, smog air, etc. [136–138]. Thus it is difficult to compare the results between studies [138]. Furthermore, there are only very few mentions in the literature about the environmental performance of nanomaterials based on LCA methods which also has some limitations, including a lack of life cycle inventory data and characterization factors for NMs' emissions [139,140]. Figure 10 depicts the simplified framework for the LCA of nanocomposite materials.

**Figure 10.** A general framework for the LCA of nanocomposite materials.

This section covers the environmental profile of starch-based nanocomposites in comparison to nonconventional counterparts. The environmental impacts of starch-based composites production with PBS, PLA/PBAT, PHB, PLA, PBS/fiber, and recycled-PLA were greatly varied: non-renewable energy use (NREU) (33–72 MJ/kg, when using virgin starch), eutrophication (1.2–1.9 g P eq./kg), greenhouse gas (GHG) emissions (1.8–3.7 kg CO2 eq./kg) and agricultural land use (0.3–1.3 m2yr/kg) (Table 3). Compared to petrochemical polymers, LDPE and PP, virgin starch-based polymers reduced GHG emissions (up to 80%, except starch/PBS, starch/PLA/PBAT) and NREU (up to 60%) but increased eutrophication potential (up to 400%) and agricultural land use. Furthermore, reclaimed starch from wastewater instead of virgin starch reduced environmental impacts [141].

The microwave-assisted technique can be an environmentally friendly alternative for glucose-reduced and starch-stabilized Ag NPs production [137].

LeCorre et al. [132] compared the sustainability of extraction of nanofillers' starch nanocrystals (SNC) and organically modified nanoclay montmorillonite (OMMT). Though global warming and acidification potential indicators of SNC were higher than those of OMMT, SNC has more positive impacts than OMMT, which contributes to non-renewable energy and mineral depletion.

The choice of starch sources and plasticizers influences the environmental impacts displayed by the production of composites. Corn starch/glycerol exhibited the lowest impact on the ecosystem, human health, and resources [142].

**Table 3.** Environmental impacts of starch polymer and nanofiller compared with LDPE polymer (Functional unit = 1 kg).


a, kg/NOx eq.; b, H+ moles eq.; c, kg PO4 3- eq.; d, g P eq./kg; e, kg benzene eq.; <sup>f</sup> , kg toluene eq.; g, g PM; h, kg 2,4-D eq. PM2.5, particulate matter of size under 2.5 μm; 2,4-D, 2,4-dichlorophenoxyacetic acid used as a herbicide and pesticide.

#### **8. Biodegradation of Starch**

Based on ASTM, biodegradable is defined as 'capable of undergoing decomposition into carbon dioxide, methane, water, inorganic compounds, or biomass in which the predominant mechanism is the enzymatic action of microorganisms that can be measured by standard tests, in a specified period, reflecting available disposal condition' [44]. Biodegradable polymers play a critical role in environmental sustainability as they take part in the natural cycle "from nature to nature" [145]. With regard to biopolymer, to be certified as a

biodegradable material, 90% of its mass should be decomposed in composting conditions within 90 days [146].

The type, nature, concentration, chemical modification, and antimicrobial properties of nanofiller, biodegradation test methods, and parameters, including temperature, moisture, humidity, pH, quantity and type of microorganisms, etc., can influence the biodegradability of nanocomposites [15,145,147].

Starch modification and incorporation of nanomaterials as nanofiller have been shown to alter biodegradability. For example, the biodegradability of starch increased with the addition of MMT at lower concentrations because of increased hydrophilicity that permits the microorganisms to enter into the polymer. In contrast, chemical modification of starch, nanofillers such as TiO2, graphene oxide, etc., reduce the biodegradability of starch-based nanocomposite because of their antioxidant potential [5,14,15,148].

Crosslinked nanocomposite film produced from thermoplastic corn starch crosslinked with oxidized sucrose and reinforced with cellulose nanofibrils from a pineapple leaf was reported to have a 30% weight loss rate after 30 days of burial, much lower than that of thermoplastic starch (80%) [5]. Crosslinking thermoplastic starch is hard to decompose due to the formation of acetal/hemiacetals and reduction of hydrophilicity and water permeability of nanocomposite, which decrease the attraction and permeability of microorganisms into the polymer matrix [5].

The addition of MMT into sweet potato starch (SPS) hindered biodegradability in soil burial tests due to the strong hydrogen bond between the hydroxyl groups of SPS and MMT and decreased water solubility that prevents water diffusion into the film [100]. However, the effect of MMT on biodegradability is concentration dependent. In corn starch-based film, adding MMT nanoclay at a lower concentration (1–3%) delayed the biodegradation rate (22–23 days for complete degradation), which may be attributed to the formation of the exfoliated structure at a lower concentration of MMT, which ensures good interaction between MMT and the polymer matrix. The biodegradability was increased at a higher level (>3%) of MMT due to agglomeration [15].

The cationic starch-based film incorporated with MMT and nanocrystalline cellulose degrade faster than the pure cationic starch film in composting at 58 ◦C, which may be attributed to hydrophilic nanocrystalline cellulose [127]. Thyme essential oil (TEO) and MMT incorporation have also been shown to increase biodegradation in SPS/MMT nanocomposites [100].

Incorporating fibrous TiO2 (0.01 and 0.05 wt.%) in maize starch/PVA composite films improved the tensile strength, water vapor, and UV barrier properties with little effect on biodegradability in soil [146]. The addition of nanoclay fillers delays the biodegradation of corn starch when buried in a microbiological medium of pure *Micrococcus luteus* culture at room temperature for 30 days [15]. Incorporating antimicrobial Ag NPs into starch/PVA composite film reduced its biodegradability [14].

The addition of CaCO3 in starch/polyethylhexylacrylate (PEHA)/PVA composite film improved the tensile strength, thermal stability, chemical resistance, and antimicrobial properties, which can be suitable for packaging. Starch/PEHA/PVA/CaCO3 degraded by 65% after 15 days in activated sludge water [149]. Food packaging material prepared from poly(ethyl methacrylate)-co-starch/graphene oxide/AgNPs showed only a 4.5% biodegradation in active sludge water after 180 days [97].

Poly(lactic acid) (PLA)/thermoplastic cassava starch (TPCS)/graphene nanoplatelets (GRH) nanocomposite film showed a lower biodegradation rate than PLA film in vermiculite (0.11 to 0.06 d<sup>−</sup>1) and compost media (0.09 to 0.08 d−1) [148].

In slow-release fertilizer formulation, the incorporation of natural char nanoparticles (NCNPs) into corn starch-g-poly (acrylic acid-co-acrylamide)/urea composite increased the degradation rate (23.9% after 30 days in soil), which may be attributed to the increment in water absorbance that promotes the soil microorganisms to enter into the polymer matrix [84].

The biodegradability of nanocomposite film polylactic acid/starch/poly-ε-caprolactone/ nano hydroxyapatite (nHAp) was increased with the nHAp content [110]. Hosseinzadeh and Ramin [126] reported that the degradability of starch-graft-poly(acrylamide) (PAM)/graphene oxide (GO) nanocomposite decreased with increasing nHAp addition in buffer solution due to the higher crystallinity, compressive strength, and elastic modulus of nanocomposite film.

In vitro degradation tests performed in a simulated body fluid (SBF) showed that thermoplastic starch (TPS)/beta-tricalcium phosphate (β-TCP) NPs degraded 51% after 28 days, higher than that of TPS (47%) [105]. Table 4 summarizes the recent findings about the biodegradability of different starch-based biopolymers.


**Table 4.** Biodegradability of different starch-based biopolymers.


#### **Table 4.** *Cont.*

#### **9. Conclusions and Future Perspectives**

In summary, starch is a natural polymer with outstanding biocompatible characteristics and can be used as both a matrix and reinforcement material for the development of new bionanocomposites. Starch nanoparticles and other nanofillers, including nanocellulose, chitin NPs, nanoclay (MMT, HNTs, bentonites), carbon nanoparticles (MWCNTs, SWCNTs, graphene, graphene oxides), metal and metal oxides (Ag NPs, TiO2, ZnO, CaCO3, etc.), have been widely used for the creation of new starch-based bionanocomposites and are promising candidates for various industrial applications.

The excellent biocompatibility, complete degradability without toxic residues, low cost, wide availability, and renewability of starch-based nanocomposites would open up many applications in agriculture, packaging, environmental remediation, biomedicine, and many other fields. Some of the reported applications are edible food coating, active and intelligent food packaging, controlled/slow-released pesticides and fertilizers, mulch films, drug carriers (controlled/target specific), wound healing, scaffolds in tissue engineering, absorbents, filters, catalysts, or disinfectants for environmental remediation, electronic devices, lightweight architectural constructions, stabilizers in food and paints such as non-food applications, and many others.

Modification of starch or reinforcement with other materials to form a nanocomposite may alter biodegradability. Therefore, regarding the biodegradability of starch-based nanocomposites is important for them to be claimed as being biodegradable materials. Life cycle assessment of starch-based biocomposite materials for their respective applications provides critical information regarding the environmental and ecological benefits of the materials over fossil-based synthetic polymers for developing sustainable nanocomposites. However, only few studies have focused on life cycle assessment. Therefore, further studies on life cycle assessment of starch-based nanocomposites needs to be investigated. Nanomaterials can also enter the human body through inhalation, contact, and ingestion, which can lead to their accumulation in the human body, Therefore, further investigations on toxicity and risk factor analysis are necessary to find the most suitable starch-based nanocomposite materials.

**Author Contributions:** Writing—original draft, A.G., P.T., S.M., P.G.P., P.E. and T.M. Review and editing, A.G., P.T., S.M., P.G.P., A.M., O.M. and T.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


## *Article* **Fabrication, Characterization, and Microbial Biodegradation of Transparent Nanodehydrated Bioplastic (NDB) Membranes Using Novel Casting, Dehydration, and Peeling Techniques**

**Sherif S. Hindi 1,\* and Mona Othman I. Albureikan <sup>2</sup>**


**Abstract:** NDBs were fabricated from gum Arabic (GA) and polyvinyl alcohol (PVA) in different ratios using novel techniques (casting, dehydration, and peeling). The GA/PVA blends were cast with a novel vibration-free horizontal flow (VFHF) technique, producing membranes free of air bubble defects with a homogenous texture, smooth surface, and constant thickness. The casting process was achieved on a self-electrostatic template (SET) made of poly-(methyl methacrylate), which made peeling the final product membranes easy due to its non-stick behavior. After settling the casting of the membranous, while blind, the sheets were dried using nanometric dehydration under a mild vacuum stream using a novel stratified nano-dehydrator (SND) loaded with P2O5. After drying the NDB, the dry, smooth membranes were peeled easily without scratching defects. The physicochemical properties of the NDBs were investigated using FTIR, XRD, TGA, DTA, and AFM to ensure that the novel techniques did not distort the product quality. The NDBs retained their virgin characteristics, namely, their chemical functional groups (FTIR results), crystallinity index (XRD data), thermal stability (TGA and DTA), and ultrastructural features (surface roughness and permeability), as well as their microbial biodegradation ability. Adding PVA enhanced the membrane's properties except for mass loss, whereby increasing the GA allocation in the NDB blend reduces its mass loss at elevated temperatures. The produced bioplastic membranes showed suitable mechanical properties for food packaging applications and in the pharmaceutical industry for the controlled release of drugs. In comparison to control samples, the separated bacteria and fungi destroyed the bioplastic membranes. *Pseudomonas* spp. and *Bacillus* spp. were the two main strains of isolated bacteria, and *Rhizobus* spp. was the main fungus. The nano-dehydration method gave the best solution for the prompt drying of water-based biopolymers free of manufacturing defects, with simple and easily acquired machinery required for the casting and peeling tasks, in addition to its wonderful biodegradation behavior when buried in wet soil.

**Keywords:** hydrophilic bioplastics; gum Arabic; polyvinyl alcohol; casting of poly-(methyl methacrylate); dehydration; phosphorus pentoxide; peeling; microbial biodegradation

#### **1. Introduction**

Despite petroleum-based polymers' lower density and greater mechanical characteristics, biobased polymers have gained significant interest due to growing environmental concerns about their sustainability and biodegradability [1–6]. Many biopolymers, including poly-lactic acid/poly-lactide, poly–3–hydroxy-butyrate, starch, gelatin, alginate, agar agar, guar gum, and GA, have been used in various industrial applications.

GA is a type of edible and natural exudate gum arising from the mature trunks and branches of various acacia species, especially the *Acacia senegal*, *A. seyal*, *A. nilotica*, and *A. mellifera* (family: Fabaceae) [7]. The GA yield can be enhanced by drought conditions,

**Citation:** Hindi, S.S.; Albureikan, M.O.I. Fabrication, Characterization, and Microbial Biodegradation of Transparent Nanodehydrated Bioplastic (NDB) Membranes Using Novel Casting, Dehydration, and Peeling Techniques. *Polymers* **2023**, *15*, 3303. https:// doi.org/10.3390/polym15153303

Academic Editor: Alberto Romero Garcia

Received: 21 May 2023 Revised: 15 July 2023 Accepted: 29 July 2023 Published: 4 August 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

poor soil fertility, and injured or scratched plants to cover the huge demand for domestic and industrial applications due to its water-soluble and polysaccharide nature [8–10]. Chemically, GA has hydrophilic carbohydrates (arabinogalactans) as well as hydrophobic proteins (the arabinogalactan–protein complex and glycoproteins) that exhibit various functional properties in food additives [11–15]. It is well known that D-galactose, l-arabinose, L-rhamnose, D-glucuronic acid, and 4–O-methyl-d–glucuronic acid make up the highly branched complexes known as arabinogalactan proteins, along with a minor amount of proteins. While hydrophilic arabinogalactan provides steric and electrostatic stability, this hydrophobic polypeptide chain can tie gum to oil droplets in an emulsion [15]. The benefit of the amphiphilic nature of GA is that it prevents coalescence, which can promote film formation and display steric stabilization. Furthermore, GAs have significant effects on various emulsion factors, including opacity, specific gravity, zeta potential, and surface tension [15]. GA is used primarily in the food, medicinal pharmaceutical, and wood technology industries [15–33] and as a stabilizer, emulsifier, and thickening agent [15–18] because of its high hydrophilicity, low fluidic viscosity, good surficial activities, and ability to form a protective film around emulsion droplets [15]. Recently, the use of GA has been extended into the nanotechnology and nanomedicine fields due to its biocompatibility for both in vivo and in vitro applications and its stabilization of nanostructures. GA has been probed for its coating properties and increased biocompatibility of iron oxide magnetic nanoparticles [34,35], gold nanoparticles [36], carbon nanotubes [37], and quantum dot nanocolloids [38].

PVA is a synthetic polymer with a wide range of commercial applications due to its high crystallinity, good mechanical properties, water solubility, and adhesive properties [39–42]. These applications extend to the industrial, medical, pharmaceutical, and food sectors and include lacquers, resins, surgical threads, food packaging materials [26,29,40], paper coating, textile sizing [43], and coating purposes [44], where they are used in order to enhance the mechanical properties due to natural properties such as compatible structure and hydrophilic properties [45]. Furthermore, it is used as a thermoplastic polymer for living tissues because it is harmless and non-toxic, as well as functioning as a cross-linker and nanofiller [46–48]. Furthermore, since PVA has excellent chemical resistance, it is used as a good emulsifier [49]. Practically, PVA is widely blended with other hydrophilic polymers [45], such as GA, to enhance its mechanical properties as well as its biodegradability [42,50–56]. However, the drawbacks that are associated with the manufacturing of bioplastic materials are the drying process for the cast GA membrane due to the high water content of the parent material, thereby making the entire process time- and cost-intensive. Hence, there is a need to develop an apparatus or method that overcomes the above limitations [57]. The presence of a large number of hydroxyl, carboxylic, and carbonyl groups in the GA/PVA blends makes it a chemical reductant and environmentally benign medium [58]. PVA is easily degradable by biological organisms [39,58], and many microorganisms that are able to degrade PVA and GA have been identified [59–62].

Different GA composites were prepared and characterized by several researchers for fabricating films and membranes [63–65], as well as for the anodes of lithium-ion batteries [66]. In addition, Silvestri et al. [67] produced nanofibers from a blend containing GA (10 wt% solution), graphene oxide (GO), and PVA, while Lubambo et al. [68] obtained nanofibers from guar gum. The processing of GA includes impurity removal, kibbling, granulating, grinding, powdering, acid or enzymatic hydrolysis, clarification and discoloration, centrifugation (10,000–16,000 rpm/min), purification of the ceramic membrane, desalination, and spray drying [69–73].

Several researchers examined the mechanical characteristics of bioplastic materials [74–79]. These characteristics play a crucial role in determining the appropriateness of their use in particular applications. Electrospun scaffolds, for instance, should have adequate mechanical characteristics to withstand tension or compression pressures in bone tissue engineering. As assessed by Masti et al. [74], the mechanical properties of their fabricated bioplastic films were assessed by tensile strength (TS) and Young's modulus (Ym), termed modulus of elasticity (MoE). The addition of gum acacia (GA) to the equal quantity of PVA/CS in the blend film shows improved Ts and Ym. The increase in the Ts and Ym of the blend due to the addition of the GA suggested that interfacial strength (adhesion) could be improved [74,76]. The further addition of GA decreased both Ts and Ym [74,75].

Based on scientific investigations performed by Chougale et al. [76] and Ibrahim et al. [78], their FTIR studies confirmed that due to the crosslinking and intermolecular interactions caused by esterification during the heat treatment in the blend films, there is a significant interaction between each component of their bioplastic mix. Additionally, when compared to the transverse direction, the mechanical performance of the membranes demonstrated an increase in the elasticity modulus in the longitudinal direction from 85 MPa to 148 MPa [78].

A particular category of polymers known as biodegradable polymers [80–93] degrade naturally into byproducts such as gases (CO2 and N2), water, biomass, and inorganic salts [57] after serving their intended purpose. The abiotic and biotic components of the biodegradation mechanism coexist naturally in the soil [2,6]. Recently, a lot of biodegradable polymers have been created, and some known microbial enzymes can break them down [5]. Numerous microbial communities of bacteria, fungi, and yeasts, including but not limited to Gram-negative species, such as *Escherichia coli* and *Pseudomonas aeruginosa*, and Gram-positive species, such as *Staphylococcus aureus*, can use bioplastic as a nutrition source throughout the biodegradation process [1,3,4,55]. These include *Rhizobium meliloti* [5], *Bacillus* spp. [58], *Pseudomonas* spp., *Aspergillus* spp., *Rhizorpous* spp., *Fusarium* spp., *Penicillium* spp., *Saccharomyces* as yeast [59], and *Elite Aspergillus* [91]. Under aerobic (composting) or anaerobic (landfill) conditions, several petroleum-based polymers are biologically decomposable [47,94]. By combining synthetic and natural polymers, researchers have been able to improve the processing capacity, physicochemical characteristics, and biodegradability of synthetic polymers [1,4,6,50–53]. For various polymers, including PVA, the rates and environmental factors that influence breakdown might vary [47,60,95–97]. Composting can take place under different conditions, including anaerobic environments, underground soil layers, aqueous media, and even in the presence of oxygen.

The aims of the present work were: (a) to invent a more reliable bioplastic membrane that is suitable for different applications; (b) to overcome the casting, drying, and peeling problems of the hydrophilic bioplastic (GA/PVA) blends; (c) to compare the ordinary GA/PVA bioplastic membranes (ADBs) with those synthesized using the novel methods in the present investigation (NDBs); and (d) to study the biodegradation behaviors of the NDBs when buried in wet soil.

#### **2. Materials and Methods**

The management plan for the production of novel transparent NDBs is illustrated in Figure 1.

#### *2.1. Raw Material*

#### 2.1.1. GA

GA (~Mw: 1.827 × 106 g/mol) was harvested from the trunks and branches of *Acacia seyal* trees (Figure 2(a1)) habituated at Hada Al-Sham (about 120 km apart from Jeddah), Saudi Arabia. As shown in Figure 2A, the naturally hardened sap excreted on a branch of an *Acacia seyal* tree was collected after tapping the woody tissues of the tree and making incisions (60 cm × 5 cm). (Figure 2(a1)) and cured into crude granules (Figure 2(a2)). The solid granules were ground using a mechanical grinding machine (Figure 2(a3)), passed through a standard 60 mesh sieve, and retained particles of 80 mesh size (60/80 mesh) using a vacuum-assisted sieving system (Figure 2(a4)). About 50 g of air-dried uniform GA powder (Figure 1(a5)) was dissolved in one liter of deionized water at ambient temperature (25 ◦C) and heated up to 80 ◦C with continuous stirring until all particles were completely dissolved (Figure 1(a6)). Removing the insoluble components of the resultant solution

was achieved via vacuum filtration (Figure 2(a7)) to obtain the clear GA precursor at a concentration of 5% *wt*/*wt* (Figure 2(a8)).

**Figure 1.** The management plan performed to study the efficiency of the novel manufacturing processes (vibrational casting, nano-dehydration, and self-electrostatic peeling) on the quality of the NDB membranes.

**Figure 2.** Manufacturing of NDBs: (**A**) Harvesting and processing of the GA principle precursor: (**a1**) hardened sap naturally excreted on a branch of an *Acacia seyal* tree; (**a2**) crude GA granules; (**a3**) mechanical grinder; (**a4**) vacuum-assisted sieving system; (**a5**) uniform powder of crude GA; (**a6**) crude solution of well-dissolved GA; (**a7**) vacuum-filtration device; and (**a8**) clear vacuum-filtered solution. (**B**) PVA-modifier precursor: (**b1**) analytical-grade bottle; (**b2**) powder form; (**b3**) crude solution of well-dissolved PVA; (**b4**) vacuum-filtration device; and (**b5**) clear vacuum-filtered solution; (**C**) VFHF device; (**D**) the NDB final product.

#### 2.1.2. PVA

PVA was used as a modifier precursor to synthesize the NDB, along with GA. PVA used in this investigation (Figure 2B) was of ACS reagent quality (Figure 2(b1)), Mw 88,000 Da, and 88% deacetylated. About 50 g of PVA crystals (Figure 2(b2)) were dissolved in one liter of deionized water to obtain a crude solution (Figure 2(b3)) as a result of heating under continuous stirring at 80 ◦C until complete clearance and subsequently vacuumfiltered (Figure 2(b4)) to obtain the clear PVA precursor at a concentration of 5% *wt*/*wt* (Figure 2(b5)).

#### *2.2. Preparation of the Bioplastic Blends*

The practical procedure used for the novel casting of the bioplastic blends is presented in Figure S2. Six different bioplastic blends of GA and PVA in different ratios were prepared by mixing their aqueous solutions (5% *wt*/*wt* each) under continuous and calm stirring until the solution became completely homogenous (see Figure 2). It is crucial to stir slowly and carefully to prevent the addition of too many air bubbles to the solution, which can result in aeration defects in the final membranous product.

#### *2.3. The Casting Platform*

The practical procedure used for casting the bioplastic blends is presented at Figure S1 (see in Supplementary Materials). After mixing known concentrations of GA and PVA to obtain a homogenous clear solution, the blend was casted on a novel platform made up of poly-(methyl methacrylate) abbreviated as PMMA and known to be acrylic panel. We chose this material in the current experiment as an ideal casting platform for polymers, particularly water-based ones. This selection of PMMA was due to its non-stick surface, which can help the bioplastic blend be peeled easily after drying and curing [57]. The PMMA panel was irradiated using UV-waves to activate its electrostatic charges and rising its temperature up to 50 ◦C.

As shown in Table S1 and Figures 2C and S2, the PMMA panel (180 cm in width, 2 m longitudinally, 8 mm in thickness) was fixed on the upper panel of the casting table. Furthermore, adjacent strips of PMMA can be arranged to cover a movable belt that may be used as a casting surface [57]. Before pouring the bioplastic blind onto the PMMA's substrate, vibrational forces were generated by using suitable solenoids to ease spreading the blend in a definite velocity over the worm casting platform. After that, a mild air drying of the molten polymeric membrane was applied in order to thicken its viscosity.

#### *2.4. Casting the Bioplastic Blends*

After obtaining complete homogeneity for the biopolymer blend, the bubble-free ternary blend solution was poured onto a clean acrylic panel. This panel is the surficial layer of the VFHF device (Figures 2C and S3), with a prominent frame where it is necessary to adjust the initial thickness of the bioplastic membrane to determine its final thickness. After that, the cast blend was allowed to dry at ambient temperature using the novel stratified nano-dehydrator (SND) apparatus [57,98,99], as shown in Figure 3.

The novel VFHF device (Figures 2C and S2, see in Supplementary Materials) features both the ease of casting the blend and peeling the membranes with a constant thickness free of air bubbles. For manufacturing a NDB, the blend was poured after adjusting the slope angle of the acrylic ground template (Figures 2C and S2) to a slight angle (about 15◦) in order to accelerate the blended fluid movement. The slow motion of the blend protects its matrix from forcing more bubbles inside it. After the blend reached the opposite side of the template, the slope angle was re-adjusted to zero degrees to ensure exact horizontality in order to obtain identical thicknesses. It is worth mentioning that the thickness of the bioplastic sheets was controlled by two critical actions: (a) pouring a definite quantity of blend solution onto the same template area and (b) accurate adjustment for the viscosities of these blends [57].

In order to obtain a gentle, steady, and efficient flow for the viscous bioplastic blend, each of the four legs of the VFHF was fixed with a vibrating magnetic solenoid (a Kendrion OSR series shaker) that was designed with two excitation coils to generate a harmonious vibrating movement in the blend [57]. The magnetic vibrating system (Figure S3B) consists of a permanent magnet at the bottom, connecting the magnetic body's two halves and two excitation windings. The body to be vibrated, which serves as the armature, closes the magnetic circle through an air gap. A steady pulling force between the magnetic body and armature is produced by the permanent magnet that is integrated into the magnetic body, biasing the system. The alternating electromagnetic field's force effect superimposes itself onto the permanent magnet's force effect when an AC voltage is delivered to the

excitation coil. The fully encapsulated bobbin and coils achieve reliable long-life service and maintenance-free operation. In addition, OSR shaker solenoids are not susceptible to dust or moisture when operational under rough or adverse conditions [57]. It is worth mentioning that a permanent magnetic attachment serves to mount the OSR shaker solenoid freely and that it is detachable from the vibrating surface. Angle mounts permanently fix the OSR shaker solenoid to a vibrating surface. In addition, phase angle controllers were installed separately for the fine adjustment of vibration through alternating or direct current (via an integrated one-way rectifier), and they can be DIN-rail mounted within cabinets with minimal space.

**Figure 3.** The novel nano-dehydration technique: (**A**) the stratified air-dryer apparatus, and (**B**) the non-woven textile of polypropylene: (**b1**) an optical image, and (**b2**) microscope image (40×) according to Hindi and Albureikan [57].

#### *2.5. Drying the NDBs*

The Stratified Nano-Dehydrator (SND)

The most critical problem concerning the manufacturing of bioplastic sheets is their drying process, where it is crucial to obtain high-quality products. This problem arises from the highly hydrophilic nature of bioplastic sheets blended with mixtures of hydrophilic GA and PVA in different ratios.

The SND was invented to accelerate the dehydration process of bioplastic sheets [57]. As shown in Figure 3, it consists of three stratified and perforated acrylic panels (poly- (methyl methacrylate)). These panels were arranged in a vertically alternating pattern, with three sub-layers consisting of dehydrant-loaded fibrous materials. Each sub-layer constitutes two non-woven polypropylene textiles, restricting an intermediate net of loosened Egyptian cotton floss (ECF). All fibrous materials constructing the sub-layer are saturated with a strong dehydrant, such as phosphorus pentoxide (P4O10), the most highly preferred dehydrant reagent, rather than calcium chloride, magnesium sulfate, aluminum oxide, lithium aluminum hydride, metallic sodium, or silica gel. Removing water by P2O5 was found to be more complete, quickly, effectively, and at a faster rate than many other dehydrants.

This cellulosic material was selected for this task due to its high content of alpha cellulose, well-known for its high hydrophilicity, which is essential to attaining good affinity to both moisture molecules as well as dehydrant crystals. The manner of loading dehydrant onto the cellulosic fibers can be summarized as follows: (1) air-drying of cellulosic fibers used as a core skeleton of the SND; (2) preparing dehydrant-saturated solution; (3) soaking cellulosic fibers in the salt-saturated solution via vacuum forces to ensure complete immersion and saturation of the fiber cells and penetration of the salt solution into the cell cavities, as well as the cell wall, through the internal border pits of the cellulosic fibers; (4) discarding excess salt solution and drying the cellulosic fibers by air-drying and finally oven-drying at 80 ◦C ± 5 ◦C for 2 h; and (5) this medium layer was inserted between the first and third layers, which were made of non-woven polypropylene textile. The edges of the outer layers were welded thermally due to the nature of polypropylene as a thermoplastic material. Furthermore, the three-layer textile was reinforced upon stitching using a sewing machine.

#### *2.6. Peeling off the Bioplastic Membranes*

A self-electrostatic charged-template (SECT) made of poly-(methyl methacrylate) was used as a casting platform, which made peeling the final product membranes easy due to its non-stick behavior [57]. It can be used as a table cover termed an acrylic platform (Figure 2C) or as a covering layer (by coating or adhesion) for a movable belt to give production continuity.

The resulting transparent bioplastic sheet (Figure 2D) was peeled slowly and accurately away from the PMMA platform, rolled, and stored under dry circumstances until used.

#### *2.7. Characterization of the Bioplastic Membranes*

The values of the different physical and chemical properties of the bioplastic sheets were calculated as presented in Table 1.

**Table 1.** Arithmetic formulas for calculating different chemical and physical properties of the bioplastic sheets.



**Table 1.** *Cont.*

<sup>1</sup> Crystallinity index (%),2 Mass loss of the bioplastic membrane (g), <sup>3</sup> Temperature range (◦C), <sup>4</sup> Enthalpy change (μV/mg), <sup>5</sup> Tensile strength (MPa), <sup>6</sup> Modulus of elasticity (GPa), <sup>7</sup> Tensile strain, <sup>8</sup> Elongation at failure (%), <sup>9</sup> Slope of the HD-curve.

#### 2.7.1. FTIR

Using a Bruker Tensor 37 FTIR spectrophotometer, the chemical constituents and functional groups of the six bioplastic membranous samples were investigated. The samples were combined with KBr at a ratio of 1:200 *wt*/*wt* and compressed under vacuum to form pellets after being oven-dried at 100 ◦C for 4–5 h. The materials' FTIR spectra were captured between 400 and 500 cm−<sup>1</sup> in transmittance mode.

#### 2.7.2. X-ray Diffraction (XRD)

An XRD 7000 Shimadzu diffractometer (Kyoto, Japan) was used to determine the XRD spectra of the six bioplastic sheets. An anode generator, a copper target, and a wide-angle powder goniometer are all parts of the system. Measurements were performed with the aid of CuK radiation arising at 30 kV and 30 mA. The Kα1 (0.15406 nm) and Kα2 (0.15444 nm) components of the CuK radiation are present in the resulting XRD data.

A single-channel analyzer was used to extract the data resulting from the semiconductor detector. The reception slit was 0.15 mm at the same radius, and the divergence and scatter slits were each 10 m wide. Several droplets of diluted amorphous glue were used to mount dried bioplastic sheets (weighing around 0.5 g) onto a quartz platform. Each sample was scanned in the 2θ◦ range between 5◦ and 40◦. Every experiment was run in reflection mode, with increments of 0.05◦ and a scan speed of 4◦/min [57,98].

The crystallinity index (CI) was computed by dividing the diffractogram area of crystalline cellulose by the entire area of the original diffractogram after smoothing the resulting crystalline peaks from the diffraction intensity profiles. Using Microsoft Excel (USA), the area under the curve was calculated by adding adjacent trapezoids [98–103].

#### 2.7.3. Thermal Analysis

Since DTA typically complements TGA with information on phase transitions [93], the TGA and DTA of each blend were conducted simultaneously. These characterizations were carried out for TGA and DTA for all six bioplastic blends [57,98,99,101] using a Seiko and Star 6300 analyzer, Central Laboratory, Faculty of Science, Alexandria University, Egypt.

Using a heating rate of 20 ◦C/min under a nitrogen atmosphere, heating scans were carried out from 30 ◦C up to the final maximum temperature of 450 ◦C [57,98,101].

Determination of the NDB mass loss estimated from the TGA curves was achieved as illustrated in Table 1.

#### 2.7.4. Surface Topography (ST)

Atomic force microscopy (AFM) was used to examine the surfaces of the six NDBs in order to observe the full 3D membranous surface structures down to the nanometric scale. Four distinct characteristics of the NDB were revealed by AFM [57,98]: surface roughness (SR), nanometric particle size (NPS), pore diameter (PD), and void volume (VV).

#### *2.8. Mechanical Properties of the Bioplastic Membranes*

The stress–strain behavior of the six bioplastic membranes, else ADB NDB, was measured using The Instron universal testing machine, model 1193, Instron Co., Ltd., London, UK, with a 200 N-load cell according to the ASTM D–882 standard test, 1989. The bioplastic membrane samples were rectangular-shaped (2.5 × 10 cm) for each of the ADBs and NDBs membranes. The device with two metallic grips was installed to hold the test sample at both ends. The starting grip separation for all samples was 50 mm, and the upper grip was extended at a constant rate of 50 mm per minute while the lower grip remained stationary. An automatic speed controller was fitted to the electrically powered machine to maintain the upper grip's speed. The ambient temperature was used for all measurements. From the plot of stress–strain curves, the UTS, MoE, and EaB for each film were estimated, as illustrated in Table 1.

The mechanical properties, namely, ultimate tensile strength (UTS) in MPa, modulus of elasticity (MoE) in MPa, and elongation at failure (EaF) as a percentage, were calculated.

#### 2.8.1. Ultimate Tensile Strength (UTS)

The UTS shows the film's maximum allowable tensile stress [74–76]. The UTS property of the bioplastic sample was calculated by dividing the maximum load causing the film failure by the cross-sectional area of the film, as explained in Table 1 [74–76].

#### 2.8.2. Modulus of Elasticity (MoE)

The membrane's MoE is a reliable indicator of its stiffness [74–76]. The MoE was computed by dividing the length of the membrane sample at yield by the stress at yield, as expressed in Table 1.

#### 2.8.3. Elongation at Failure (EaF)

The EaF was calculated by dividing the elongation at failure of the sample by the initial gauge length, as shown in Table 1 [74–76].

#### *2.9. Microbial Biodegradation*

Microbial biodegradation was assessed to investigate the microbes' capacity to break down the buildup of bioplastic in soil. Upon calculating the percentage of biodegradation (weight loss), counting the microbe population isolated from the surfaces of bioplastic sheets, and evaluating the various morphological changes in these surfaces as a result of degradation, it was possible to determine the amount of biodegradation [98,99].

The soil was collected from the Agricultural Research Station (ARS) of King Abdullaziz University's Faculty of Environmental Sciences in Hada Al–Sham and was used to bury the bioplastic samples. The location is situated 240 m above sea level, around 120 km to the northwest of Jeddah (N = 21◦48 3, E = 39◦43 25). The pH of the soil at the site ranged from 7.1 to 7.9, along with low levels of CaCO3, organic matter, and cation exchange capability [104,105].

#### 2.9.1. Sample Preparation and Soil Burial Studies

The various bioplastic sheets were shredded into 2 cm × 2 cm pieces and buried 10 cm deep in 100-g wet soil boxes. Before being buried in the ground, each bioplastic piece was weighed (0.040–0.038 g). By adding sterile water to the soil samples to counteract water evaporation, the moisture content of the soil samples was kept constant [98].

Each sample box had a hole at the bottom for the excess water to drain through. After 30 and 60 days, soil samples were carefully removed, and the weight loss was calculated in order to separate, count, and compare the microbial community, as well as speculate on microbial morphology changes as a result of degradation [98,99,106].

#### 2.9.2. Isolation and Counting of Microbial Communities

About 95 mL of sodium pyrophosphate solution at 0.1% *wt*/*v* was used to suspend one gram of soil collected from the surface of the bioplastic sheets from each sample. The samples were left to stand for 30 min. Then, using the serial dilution method, the supernatant was divided among six tubes, and one milliliter (mL) of each dilution was plated on nutrient agar medium, NA (Oxoid), for the isolation of bacteria, while potato dextrose agar medium, PDA (Oxoid), was used for isolation of fungi.

Finally, in order to determine the colony-forming units (CFUs), plates were incubated at 30 ◦C and pH 7 for 720 h (for bacteria) and at 25 ◦C and pH 5 for 1440 h (for fungi). Based on their cultural and physical characteristics, microorganisms were separated and identified using conventional assays [1,57,98,99].

#### *2.10. Statistical Design and Analysis*

Various properties of the six bioplastic membranes synthesized from the aqueous solutions of GA and PVA were assessed using a randomized complete block design. According to El–Nakhlawy [107], a statistical analysis of the obtained data was carried out using the analysis of variance approach and the least significant difference test (LSD) at 0.05.

#### **3. Results**

### *3.1. Chemical and Physical Properties of the Bioplastic Membranes*

#### 3.1.1. FTIR

FTIR spectroscopy was used to determine the chemical functionality of the bioplastic sheets (the nanodehydrated membranes (NDMs), as illustrated in Figure 4. The spectra of the resulting NDBs exhibited chemical group absorption bands that were typical of the gummy products made from both GA and PVA in different ratios. The absorption bands spanned an area between 500 and 4000 cm−1. Several chemical groups were precisely found at the following wavenumbers: 900–1250, 1426, 1402, 1625.4, 1627.4, 1430, 1436.91, 1437, 1641, 1047, 2800–3000, 2885, 2910.87, 2939, 3000, 3261, 3416, 3000, and 3600 cm−<sup>1</sup> [57,98,99,101,108–111].

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**Figure 4.** FTIR spectra of the six transparent nanodehydrated bioplastic (NDB) membranes over the wavenumber range of 4000 to 500 cm<sup>−</sup>1, fabricated from various gum Arabic (GA)/polyvinyl alcohol (PVA) blends.

#### 3.1.2. XRD

Figure 5 displays the XRD patterns of the six bioplastic membranes. The maximum intensity of the GA-broad diffractogram was obtained at 2θ = 20◦, which confirms the amorphous nature of the gum Arabic [13]. Moreover, a typical peak for pure PVA, a semicrystalline polymer, was visible at 2θ◦ = 19.9◦. With the increase in PVA allocation in the blend, the crystallinity index values increased (Figure 5).

**Figure 5.** XRD diffractogram spectra of the six transparent nanodehydrated bioplastic (NDB) membranes over a wavenumber range of 4000 to 500 cm−1, fabricated from various gum Arabic (GA)/polyvinyl alcohol (PVA) blends, showing the crystallinity index (CI) values.

As demonstrated in Figure 5, the CI values of the NDBs were found to increase from 19.4% (for pure GA) to 54.81% (for pure PVA). Accordingly, it is clear that the increase in CI of the bioplastic blends can be attributed to an increase in the PVA allocation in the blend.

#### 3.1.3. TGA

The TGA results are presented in Figure 6 and in Tables 2 and S2. The mass losses of the six NDBS samples were focused on eight temperature regions, namely, 50–100 ◦C, 100–150 ◦C, 150–200 ◦C, 200–250 ◦C, 250–300 ◦C, 300–350 ◦C, 350–400 ◦C, and 400–450 ◦C (Tables 2 and S2; Figure 6).

**Figure 6.** Thermogravimetric analysis (TGA) thermogram spectra of the six bioplastic membranes (NDBs) in the wavenumber range of 4000 to 500 cm−1, fabricated from various gum Arabic (GA)/polyvinyl alcohol (PVA) blends.


**Table 2.** Mean 1–7 values of mass loss (ML) of the six transparent nanodehydrated bioplastic (NDB) membranes over a wavenumber range of 4000 to 500 cm−1, fabricated from various gum Arabic (GA)/polyvinyl alcohol (PVA) blends over different ratios and temperature (T) zones.

<sup>1</sup> Each value is an average of 3 samples. <sup>2</sup> Based on the original oven-dry weight. <sup>3</sup> Superscript capital letters for comparing blend ratios within the same temperature zone. <sup>4</sup> Subscript small letters for comparing temperature zones within the same blend ratio. <sup>5</sup> Means with the same letter are not significantly different at the 5% level. <sup>6</sup> Initial starting weight of the NDBs sample. <sup>7</sup> Final starting weight of the NDB sample.

The thermal degradation of the samples increased with rising temperatures for all six bioplastic blends, according to a comparison of mass losses between temperature zones (at the same bioplastic blend ratio).

Comparing the mass losses within the temperature zone meant studying the differences between bioplastic blend ratios in the same temperature zone. It is clear from Table 2 and Table S2 and from Figure 6 that at lower temperatures (≤150 ◦C), PVA lost more weight (5.69% and 8.98% for 50–100 ◦C and 100–150 ◦C zones, respectively) than GA (13.68% and 11.11%) in the same temperature zones. On the other hand, at higher temperatures, this trend was reversed, whereby PVA lost more weight (79.01% and 58.8% for the 400–500 ◦C and 450–500 ◦C zones, respectively) than GA (28.6% and 15.6% for the same zones, respectively).

#### 3.1.4. DTA

The DTA analysis findings of the six nanodehydrated NDBs are presented in Figure 7 and Table 3.

**Figure 7.** Thermograms of differential thermal analysis (DTA) of the six nanodehydrated bioplastic (NDB) membranes over a wavenumber range of 4000 to 500 cm−1, fabricated from various gum Arabic (GA)/polyvinyl alcohol (PVA) blends.


**Table 3.** DTA results of the six transparent nanodehydrated bioplastic (NDB) membranes in the wavenumber range of 4000 to 500 cm−1, fabricated from various gum Arabic (GA)/polyvinyl alcohol (PVA) blends: points of reaction, thermogram type, temperature range (TR), and enthalpy change (EC).

Examining Figure 7 and Table 3, the NDB thermograms were found to be divided into two sets representing the bioplastic blends, namely, the single-phase and doublephase thermograms. The single-phase thermogram is composed of one endothermal phase, namely, curve 'b' (GA/PVA of 1:0.25), curve 'e' (GA/PVA of 1:1), and curve 'f' (PVA = 100%). On the other hand, the double-phase thermogram is differentiated into two distinct regions (endothermic and exothermic), namely, curve 'a' (GA = 100%), curve 'c' (GA/PVA of 1:0.5), and curve 'd' (GA/PVA of 1:0.75).

For more information, see Table 3. It is evident that the temperature ranges of each thermogram and the absolute values of the heat change (HC) values for the endotherms (16 Vs/mg–52.4 Vs/mg) were larger than those for the exotherms. Additionally, among the other bioplastic blends, the pure PVA endotherm absorbed the most energy (2119.7 Vs/mg), but GA had the lowest value of heat change (−1017.3 Vs/mg).

#### *3.2. Ultrastructure of the Bioplastic Membrane*

#### 3.2.1. Surface Roughness (SR) and Particle Size (PS)

In order to confirm the similarity between the ultrastructure features of ADBs and NDBs, the RS was investigated via atomic force microscopy (AFM) and is presented in Figure 8 for each of the six bioplastic blends. For clear, the GA/PVA blends' membranes dried by air are presented in Figure 8(a1–f1), while those for the NDBs are found in Figure 8(a2–f2). Since the nanometric PS is known to be intimately related to the SR, its data shown in Table 4 and Figure 9a are useful to shed an excess of light over the SR of the bioplastic membranes.

Paying attention to the AFM's images (Figure 8) revealed to that the RS was increased from the 1st blend ratio (GA = 100 %) until reaching the 6th blend ratio (PVA = 100 %). This finding can be attributed to the higher PS value of the PVA comparing to that for the pure GA as clear when speculating the gradual increasing of the PS values along with the six bioplastic blends. However, this trend was found to be similar for the ADBs and NDBs.

Regarding to the PS' results, see Table 4, statistical comparisons were performed between the membranes (ADEs and NDBs) as well as within the membranes (between the GA/PVA blends, namely, 1/0, 1/0.25, 1/0.5, 1/0.75, 1/1, and 0/1). Comparing membranes, there was no statistical difference between the ADBs and NDBs concerning their particle size (13.57 and 14.77 nm, respectively). On the other hand, comparing the blend ratios within the membrane (Table 4, Figures 8(a1,a2) and 9a) revealed that the GA membrane (GA = 100%) had the lowest PS for each of the means (13.57 nm), with a maximum value (55.44 nm). Furthermore, PVA sheets had the highest PS values (20.34 and 89.75 nm for the mean and maximum values, respectively). In between, increasing the PVA concentration

in the bioplastic blends increased the PS gradually (Table 4 and Figure 8(f1,f2) as well as Figure 9a).

**Figure 8.** AFM images of surface roughness of the bioplastic membranes blended from gum Arabic (GA) and/or polyvinyl alcohol (PVA) precursors in different ratios: (**a1**–**f1**) air-dried membrane (ADB) and (**a2**–**f2**) nanodehydrated bioplastic membrane (NDB).


**Table 4.** Statistical parameters (SPs) of the ultrastructural features of the bioplastic membranes.

<sup>1</sup> Mean of the population members. <sup>2</sup> The number of observations is 1000 individuals. <sup>3</sup> Max. is the maximum value. <sup>4</sup> Min. is the minimum value. <sup>5</sup> SD are standard deviation values present within the parentheses.

**Figure 9.** Ultrastructure features of the air-dried membrane (ADB) and the nano-dehydratedbioplastic membrane (NDB): (**a**) particle size, (**b**) pore diameter, and (**c**) void volume as affected by different allocations of GA and PVA (GA/PVA blends).

#### 3.2.2. Pore Diameter (PD) and Void Volume (VV) of the NDB Membranes

Data produced for PD are presented in Table 4 and Figures 9b, 10 and S4, while Table 4 and Figure 9c represent the VV's results. The same ascending trend was noticed for both PD and VV regarding their influence, with an increase in the PVA allocation in the ADBs as well as the NDBs. The PD of the ADB increased from 0.91 nm to 1.485 nm for the GA/PVA ratios of 1/0 and 0/1, respectively. In addition, the VV of the ADB increased from 83.24 nm3 to 548.95 nm<sup>3</sup> for the GA/PVA ratios of 1/0 and 0/1, respectively.

**Figure 10.** Permeability of the nanodehydrated bioplastic membranes blended from gum Arabic (GA) and polyvinyl alcohol (PVA) precursors in different ratios of GA/PVA: (**a**) 1/0, (**b**) 1:0.25, (**c**) 1:0.5, (**d**) 1:1.75, (**e**) 1:1, and (**f**) 0/1, respectively (AFM images).

In addition, there was no statistical difference between ADBs and NDBs in their PS, PD, and VV; consequently, there is no evidence that the novel procedures used in the bioplastic membrane preparation alter their parent ultrastructure.

#### *3.3. Mechanical Properties of the Bioplastic Membranes*

The results of the mechanical investigation of gum Arabic/poly (vinyl alcohol)/blend films were presented in Figures 11–14. Stress–strain curves of the six bioplastic membranes fabricated from GA and PVA are shown in Table 5 and Figure 11. For more specification, ultimate tensile strength (UTS), modulus of elasticity (MoE), and elongation at failure (EaF) are clear in Figures 12–14, respectively. Concerning Figure 11, it is worth mentioning that the disappearance of air bubbles in the bioplastic membranes as well as their clear transparency suggested the compatibility of the well-blended components [76] and consequently enhanced their mechanical properties. Simplifying illustration of the Figure 11, it presents the stress in mega Pascal units that affects the rheological endurance of each of the six bioplastic membranes expressed by the strain properties of the six blends. It is worth of mentioning that the mechanical relationship between stress and strain was determined for each of the ADB and NDB membranes.

**Figure 11.** Stress–strain graphs of the six bioplastic membranes fabricated from gum Arabic (GA) and polyvinyl alcohol (PVA) with different ratios for each of the air-dried (ADB) and nanodehydrated (NDB) membranes showing proportionality limit (PL) and ultimate stres (US).


**Table 5.** Proportionality limit (PL) and ultimate stress (US) of the six blends for each of the air-dried (ADB) and nanodehydrated (NDB) membranes.

As clear from Table 5 and Figure 11, the plotted stress-strain curves for the six blended membranes were differed concerning to their proportionality limit (PL) and ultimate strength (US). Regarding to sub-graphs of the bioplastic membranes in Figure 11a–f, both ADB (the red curve) and NDB (the blue curve) are similar in their ascending trend starting from the PL level up to the US. This behavior means that each membrane, else ADB or NDB was stressed through two stages: (1) in the 1st one, the stress was increased from zero up to the PL level, and (2) through the 2nd stage, each membrane transitioned from elastic to plastic nature as the load was increased from the PL up to the maximum load resulting the US stage.

Regarding to sub-graphs of the bioplastic membranes in Figure 11a–f, both ADB (the red curve) and NDB (the blue curve) are similar in their ascending trend starting from the PL level up to the US. This behavior means that each membrane, else ADB or NDB was stressed through two stages: (1) in the 1st one, the stress was increased from zero up to the PL level, (2) through the 2nd stage, each membrane transitioned from elastic to plastic nature as the load was increased from the PL up to the maximum load resulting the US stage.

Regarding to proportionality limit (PL) of the bioplastic membranes, it is higher for the NDB than that for the ADB for all the six blend ratios. This indicates that the NDB membranes has higher elasticity endurance compared to their analogous membranes.

#### 3.3.1. Ultimate Tensile Strength (UTS)

The observed curves of the UTS for the six polymeric blend membranes are presented in Figure 12.

The bioplastic membrane with the blend ratio of GA/PVA = 1:0.25 was found to have the highest UTS values (14.05 MPa and 15.44 MPa for ADB and NDB, respectively). It can also be seen from Figure 12 that the GA had a higher UTS' mean value than that for PVA (8.62 MPa and 8.74 MPa for ADB and NDB, respectively).

For more illustration, tensile stress increased as the GA content decreased from GA=100 % which has no PVA content (10.17 MPa, 10.75 MPa for ADB and NDB, respectively) up to the membrane with GA/PVA = 1:0.25 (14.05 MPa, 15.44 MPa for ADB and NDB, respectively). After that, the UTS decreased gradually with the consequent decrease in GA, thus increasing the PVA allocations in the bioplastic blend. Moreover, no significant difference was detected between the bioplastic membranes dehydrated by ordinary and nano-techniques (ADB and NDB) for all six bioplastic blends (Figure 12).

**Figure 12.** Ultimate tensile strength (UTS) of the six bioplastic membranes fabricated from gum Arabic (GA) and polyvinyl alcohol (PVA) with different ratios for each of the air-dried and nanodehydrated membranes.

#### 3.3.2. Modulus of Elasticity (MoE)

Regarding the MoE graph presented in Figure 13 for the six bioplastic blends, the blend ratio of GA/PVA = 1:0.25 had the highest MoE values (14.05 MPa and 15.44 MPa for ADB and NDB, respectively). Moreover, GA was slightly higher in its MoE mean value than that for PVA (19.14 MPa and 17.79 MPa for ADB and NDB, respectively).

Also from Figure 13, it is worth mentioning that both graphs of ADB and NDB are similar in their trend concerning to the MoE curve, in which they increase with decreasing GA's and increasing PVA's allocation in the blend up to the blend ratio of 1:0.25. For more illustration, the mean value of the MoE was increased from the GA, 100%, and zeroallocation of PVA (20.31 MPa and 20.77 MPa for ADB and NDB, respectively) up to the membrane with GA/PVA = 1:0.25. After that, the UTS was decreased by decreasing the GA and increasing the PVA allocations in the bioplastic blend. Moreover, MoEs' mean values were found to be statistically similar concerning the bioplastic membranes dehydrated by ordinary and nano-techniques (ADB and NDB) for all six bioplastic blends (Figure 13).

**Figure 13.** Modulus of elasticity (MoE) of the six bioplastic membranes fabricated from gum Arabic (GA) and polyvinyl alcohol (PVA) with different ratios for air-dried (ADB) and nanodehydrated membranes (NDB).

#### 3.3.3. Elongation at Failure (EaF)

The EaF data presented in Figure 14 indicates that adding PVA amounts to the bioplastic blends shows a significant increase in the EaF of the produced membranes for the GA/PVA's blinding ratios of 1:0.25, 1:0.5, 1:0.75, and 1:1. In addition, it can also be seen from Figure 14 that the PVA had higher EaF's mean values (227.09% and 237.91%, for ADB and NDB, respectively) than those for GA (144.04% and 145.25%, for ADB and NDB, respectively). Moreover, there are no significant differences in the UTS belonging to the bioplastic membranes dried by means of ordinary and nano-dehydration methods (ADB and NDB) for all six bioplastic blends (Figure 14).

**Figure 14.** The percentage of elongation at failure (EaF) of the six bioplastic membranes fabricated from gum Arabic (GA) and polyvinyl alcohol (PVA) with different ratios for each of the air-dried (ADB) and nanodehydrated (NDB) membranes.

#### *3.4. Bacterial and Fungal Biodegradation*

The microbial communities for the initial soil samples as well as the buried bioplastic sheets were found to be different in number and species (Table 6). Depending on the type of buried membrane, different types of bacteria and fungi were found. *Pseudomonas* spp. [112,113], *Bacillus* spp. [58,112,113], *Aspergillus* spp. [114], and *Penicillium* spp. [112,115] were the predominant species for the buried PVA. *Bacillus* spp. [59], *Pseudomonas* spp., *Aspergillus* spp., *Rhizorpous* spp., *Fusarium* spp., *Penicillium* spp., and yeast *Saccharomyces* [59] were additional important species for the buried GA.

Moreover, the (GA/PVA = 1:1) bioplastic blend's microbial populations included *Bacillus* spp. [58,116], *Pseudomonas* spp., *Aspergillus* spp., *Rhizorpous* spp., *Fusarium* spp., and *Penicillium* spp. In addition, more fungal species than bacteria were found, which is consistent with the findings of Hindi et al. [98], who discovered that fungal isolates had a higher ability to use the sheets as growth substrates than bacteria.

Table 6 contains information about the colony-forming units (CFUs) of different microbial species. The total counts of bacteria, fungus, and yeast were determined to be 2.28 × <sup>10</sup><sup>5</sup> and 1.88 × <sup>10</sup><sup>3</sup> CFU/mL, respectively, in the first soil sample, and they were higher than those for GA and PVA (Table 5). After 30 and 60 days, pure GA (100%) had a higher CFU than pure PVA (100%). The CFU values measured after 30 and 60 days for each of the six bioplastic sheets showed no discernible differences.

In addition, increasing colony-forming units (CFUs) over a defined period measured in hours was termed as hourly duplication (HD) of the CFU and was presented in Figures 15 and 16 for bacteria and fungi, respectively. For the prediction of the HD's values within the determined incubation periods, functional formulas were mathematically derived to reach this target, which is presented in Table 7.


**Table 6.** Colony-forming units (CFUs) of microbial populations for bacterial and fungal species in the buried NDBs blended from gum Arabic (GA) and polyvinyl alcohol (PVA) in different ratios for soil burying.

<sup>1</sup> Values within parentheses are standard deviations.

**Figure 15.** Hourly duplication (HD) in colony-forming units (CFUs) of the bacterial population in the buried NDBs blended from gum Arabic (GA) and polyvinyl alcohol (PVA) in different ratios.

**Figure 16.** Hourly duplication (HD) in colony-forming units (CFUs) of fungi populations in the buried NDBs blended from gum Arabic (GA) and polyvinyl alcohol (PVA) in different ratios.


**Table 7.** Functional relationships between the incubation period (IP) as an independent variable (x) and hourly duplication (HD) as a dependent variable (y) of bacteria and fungi populations in the buried NDBs blended from gum Arabic (GA) and polyvinyl alcohol (PVA) in different ratios.

<sup>1</sup> Incline angle in degrees.

Belonging to both bacterial (Figure 15) and fungi (Figure 16) communities, the HD values determined during and just after 800 h and up to 1400 h for each of the six bioplastic sheets showed the same ascending trend. For both trends, the HD's mean values for all the blends' ratios through the 1st duration (0–800 h) showed a slower duplication rate than those within the 2nd region (800–1400 h). These duplication rates can be noticed from the slop angle of the HD curves, as shown in Figures 15 and 16 [117,118].

In addition, it can also be seen when comparing the HDs of bacteria (Figure 15) with those for fungi (Figure 16) that the HDs' rate for fungi communities grown on the six bioplastic membranes was higher than those recorded for their analogous curves belonging to the bacteria. Moreover, the higher level of HD for the fungi community was more obvious at the 2nd stage of the incubation period. This finding can be observed by speculating the curves' slopes (tan θn, where n = 1–24).

#### **4. Discussion**

#### *4.1. Scientific Illustration of the Ease of Peeling the Bioplastic Membranes Away from the Acrylic Platform*

In addition to the issue of drying the bioplastic sheets facing all hydrophilic natural polymer-based membranes, peeling these sheets to be rolled up is a major problem in the hydrophilic bioplastic blend field. Studying the ease of peeling the bioplastic membrane away from the casting panel template was achieved by investigating the chemical and physical properties of each of three parameters, namely, the bioplastic blend (fluid phase), the PMMA's platform (solid phase), and the liquid/solid interface, as shown in Figure S4 [57,98,119–125].

Regarding the triboelectric series that classifies materials based on their propensity to take electrons (tribo-positive) or not (tribo-negative), it is important to note that PMMA is a biocompatible polymer, which due to its propensity for either donating or absorbing electrons, occupies the middle position on the triboelectric series [119].

The following examples show how simple it is to separate the bioplastic membrane from the acrylic platform:

a. Materials with relatively low surface energies are regarded as non-stick surfaces [125] and vice versa. As shown in Table S1, the acrylic platform exhibits modest surface energy (41 dynes/cm) and contact angle (82◦), both of which are indicative of a non-stick surface [126].

b. Acrylic is a powerful static generator in terms of electrostatic charge. When its surface is wiped back and forth, positive and negative surficial charges arise that draw and hold microscopic particles. Surficial charge variations have the potential to cause agglomerated particles to discharge in an unanticipated manner, endangering contamination-sensitive materials [127]. PMMA is positioned close to the middle of this empirical series for the surface potential and is regarded as a tribo-positive electron-donating material [119,127].

GA is composed of three distinct fractions, as shown in Figure S3, including the arabinogalactan–protein complex (MW 1500 kDa; approximately 10% of the total gum solids), arabinogalactan (MW 280 kDa; approximately 88% of the total gum solids), and glycoprotein (MW 250 kDa; approximately 2% of the total gum solids [128–130]. Due to their low molecular weight and branching pattern, arabinogalactan films are challenging to produce [131,132].

According to Winiewska et al. [133], PVA chains have a particular percentage of acetate groups (14%), which are the source of the polymer molecules negative charges. The structure of the PVA adsorption layer is impacted by even the comparatively modest portion of these groups. The presence of more acetate groups in the polymeric chains resulted in increased PVA adsorption levels, indicating that these groups are crucial to PVA adsorption [134]. As the pH of the solution rises, so does the contribution of charged acetate groups.

Due to the electrostatic attraction of negative charges present along the polymeric chains, the polymer chains extend further. The amount of PVA is directly influenced by the degree of development in the polymer macromolecules.

In general, plastics are categorized into four categories by Nuraje et al. [135]: super hydrophilic, hydrophilic, hydrophobic, and super hydrophobic, with contact angles (θ◦) of below 5◦, below 90◦, 90◦–150◦, and 150◦–180◦, respectively.

The liquid–fluid–solid system exhibits three different interfaces in its configuration when a liquid drop is placed on a solid surface (Figure S4), namely, liquid–fluid, solid–fluid, and liquid–solid. It is noticed that adhesive and cohesive forces are present at each interface as a result of the intermolecular forces at work there. Cohesive forces cause the drop to return to its spherical shape, whereas adhesion forces encourage it to spread out. The conflict between these two forces determines the contact angle [57,73,133–137]. It is feasible to establish a connection between the static contact angle and the interfacial stresses under equilibrium conditions. The Young–Dupre equation is the name of this relationship. By applying the Hild [126] formula, it was discovered that the spreading of a droplet of a bioplastic blend is equal to A − (B + C), where A is the surface tension of the bioplastic blend, B is the surface tension of the acrylic panel, and C is the surface energy of the interface between the bioplastic blend and the acrylic panel. While liquid will spread when the spreading is zero to positive, it will not if the spreading is negative.

#### *4.2. Scientific Illustration of the Nanodehydration of the Bioplastic Membranes*

It is well known that drying bioplastic membranes is a critical issue when manufacturing these products. This crucial problem arises from the highly hydrophilic nature of their natural-based precursors, such as the hydrophilic GA and PVA used in this study. Accordingly, a novel technique and device were invented to accelerate the dehydration process of such products [57]. The invention, termed the stratified nano-dehydrator (SND), is constructed from accurately selected materials, including perforated acrylic panels (poly- (methyl methacrylate)), polypropylene's non-woven textile, cellulosic cotton floss, and an effective dehydrant agent such as P2O5, which is the most effective dehydrant reagent, rather than calcium chloride, magnesium sulfate, aluminum oxide, lithium aluminum hydride, metallic sodium, or silica gel.

The reasons for choosing PMMA material as barrier panels within the dehydrator apparatus were due to its self-electrostatic charging property as well as its ability to force the evaporated water molecules to have higher surface tension, which facilitates their

escape outside the SND atmosphere and, subsequently, accelerates drying the bioplastic membranes. Furthermore, the cellulosic material (loosened Egyptian cotton floss) was selected for this task due to its high content of alpha cellulose, well-known for its high hydrophilicity, which is essential to attaining good affinity to both moisture molecules as well as dehydrant crystals.

The scientific concepts of ion dehydration reported by Pavluchkov et al. [138] can be used to explain the water molecules' diffusion, especially in the transition state, by converting the water liquid into gaseous or steam matter.

Polar covalent bonds between the O- and H-atoms were extensively reported to generate an asymmetric distribution of electrons in a water molecule, with two excess electrons on the O-atom's side of the molecule and two deficient electrons on the H-atom's side. This asymmetry accepts water molecules with both molecular cohesion that attracts water molecules to each other and/or adhesion that attracts water molecules to their neighboring asymmetrically charged materials like ions, polarized molecules, and charged surfaces, including but not limited to glass [139] and the PMMA's casting platform used in the present investigation [57].

Since water molecules have high polarity, their transportation (evaporation) from the bioplastic membranes upon the drying process can be viewed like the ion dehydration phenomenon that regulates ionic transport through sub-nanopores, which can permit selectivity between similar sized and charged ions, as referred to by Pavluchkov et al. [138]. Transition-state theory gives an idea about molecular activation parameters (enthalpy and entropy) that determine the interaction level between the transported species and the wet bioplastic blend, as well as the freedom of molecular motion within it. Since hydration and dehydration effects are characterized by substantial enthalpic (due to changes in the chemical bonds between the ion and its surroundings) and entropic (due to changes in the spatial structure of the ion) changes at the molecular level, this theory has been successfully suggested to explore dehydration-related transport phenomena in membranes [138].

The enclosed system within the SND was partially vacuumed to give mildly driven forces that accelerate the water vapor molecules' escape outside the dehydrator [57] and subsequently enhance the drying process itself.

#### *4.3. Chemical and Physical Properties of the Bioplastic Membranes* 4.3.1. FTIR

Different organic functional groups found in naturally occurring substances can be recognized using Fourier transform infrared (FTIR) spectroscopy. The complicated vibrational modes were seen in the FTIR spectra for the various bioplastic samples over a wide range of wavenumbers (Figure 4).

Figure 4 shows the strong and broad O-H stretching vibrations at 3416 cm−<sup>1</sup> dominating the primary FTIR spectra of the six bioplastic sheets. At 2939 cm<sup>−</sup>1, the C-H stretching modes are riding above the board peak. Along with the bulk ring mode at 1426 cm<sup>−</sup>1, the carbonyl stretching modes are seen at 1641 cm−1. At 1047 cm−1, the typical C–O–C antisymmetric stretching mode was found. These findings are modified from those attained by other researchers [108–111] for the study of biopolymeric materials.

For an additional illustration, the overall banding of the FTIR analysis showed a carbohydrate fingerprint at 900–1250 cm−<sup>1</sup> [138]; C–O–C anti-symmetric stretching at 1426 and 1047 cm−<sup>1</sup> [57,98,99]; COO<sup>−</sup> asymmetric stretching at 1402 cm−<sup>1</sup> [139]; an O–H in-plane bending band in carboxylic acids at 1625.4, 1627.4, 1430, 1436.91, and 1437 cm−<sup>1</sup> [132,133]; COO– symmetric stretching and carbonyl stretching modes at 1641 cm−<sup>1</sup> [57,98,99,101]; C–H stretching at 2800–3000, 2885, and 2939 cm−<sup>1</sup> [57,98,99,101,137,140–142]; vibrational modes of the C–H group at 2910.87 cm−<sup>1</sup> [141,142]; O–H stretching vibrations at 3261, 3416, and 3000–3600 cm−<sup>1</sup> [57,98,137,141,143], and the unique presence of O–H groups at 3526.35 cm−<sup>1</sup> [142,143].

The FTIR for the NDB used in the current work and the ADB created by Hindi et al. [98] have main functional groups that share chemical characteristics, according to the comparison. As a result, the chemical components of the bioplastic products have been preserved by the use of innovative casting blends, nano-dehydration, and membrane peeling.

#### 4.3.2. XRD

The GA-broad diffractogram's greatest intensity was recorded at 2 θ◦ = 20◦ (Figure 5), which supports the amorphous nature of gum Arabic [13]. Moreover, a typical peak for pure PVA, a semi-crystalline polymer detected at 2 θ◦ = 19.9◦ (Figure 4f), confirmed its semi-crystallinity feature [57,92,111].

With the increase in PVA allocation in the blend, the crystallinity index values increased. The growing CI of the bioplastic blends can be correlated to the increasing PVA allocation in the blend because the CI value of PVA (54.81%) was found to be greater than that of GA (19.4%).

#### 4.3.3. TGA

TGA analyzes the mass change behavior in bioplastic membranes that occurs as a function of temperature and time in a controlled environment. The best uses for it are to evaluate reaction kinetics, volatile contents, thermal stability, degradation traits, aging/lifetime breakdown, and degradation features.

The thermal deterioration of the samples (Figure 6) increased at higher temperatures (up to 500 ◦C) than at lower temperatures, according to a comparison of the mass losses between the temperature zones. Furthermore, a comparison of the mass losses across the temperature ranges revealed that PVA shed more weight in the higher temperature zones than GA. A mass loss of up to 100 ◦C can be attributed to the water molecule's large solvation capacity, which results in the evaporation of loosely bound moisture on the surface, or "free water". Furthermore, mass loss at temperatures up to 150 ◦C can be due to hygroscopic water evaporation [57,92].

#### 4.3.4. DTA

Similar information is provided by DTA, which measures the temperature difference between a sample and a reference due to thermal treatments in a material. The DTA typically provides phase transition information in addition to the TGA.

It is commonly known that two types of thermograms can be distinguished for a given material during thermal reactions: endothermic, which uses energy, and exothermic, which excludes energy. The depolymerization of the bioplastic materials themselves as a result of heat treatment causes exograms to occur (Figure 7). Moreover, the endotherm can be attributed to the fusing or melting of crystallites as well as the evaporation of free moisture (up to 100 ◦C) and hygroscopic moisture (up to 120 ◦C) [57].

As shown in Figure 7 and Table 3, GA had the lowest value of heat change (−1017.3 Vs/mg), but the endotherm of pure PVA absorbed the maximum amount of energy (2119.7 Vs/mg) among the other bioplastic blends. As a result, PVA is more thermally stable than GA because it absorbs heat more effectively, shielding the bioplastic sample from potential thermal degradation brought on by rising temperatures. Moreover, the enhanced PVA allocation in the blends boosted the thermal stability of the bioplastic sheets.

GA exhibits greater thermal stability than PVA at higher temperatures (about 350 ◦C). As a result, altering a bioplastic blend to increase PVA or decrease GA enhances the thermal stability of the resulting bioplastic membrane [57,92].

#### *4.4. Anatomical Ultrastructure of the Bioplastic Membranes*

#### 4.4.1. Surface Roughness and Nanometric Particle Size

While the pure PVA sheets (0/1 blend ratio) had the greatest PS values for both the ADB and NDM, the pure GA membrane (1/0) had the lowest PS values. Gradually raising the PVA concentration in the bioplastic blend increased the PS. The surface roughness (SR) features examined via atomic force microscopy (AFM), as shown in Table 4 and Figure 8, provide confirmation of this. The results obtained for PVA-based membranes and the median value for those cast from GA/PVA (1:1) are compared.

As a result, the presence of PVA causes the SR of the water-based polymeric blends to increase, generating a surface that is rougher. This is supported by the surface roughness features examined using atomic force microscopy (AFM), as shown in Table 4 and Figure 8.

Increases in PVA allocation make the blend's texture coarser since PVA membranes have a rougher structure than GA membranes. According to the comparison of the membranes, there is no statistically significant difference between the ADB and NDB in their PS, along with the six bioplastic blends. Additionally, analyses of the effects of different bioplastic blend ratios on a membrane showed that, in both cases, blends with higher PVA concentrations produced membranes with higher porosities (PD and VV).

It is important to note that smoother sheets are preferred for packaging over coarser ones because the latter tend to gather more dust on their surfaces. Although PVA is a crucial part of the bioplastic mixture that improves the quality of the final membrane and makes it easier for it to peel off the casting surface after drying, a careful balance must be taken into account to have the best quality and smoothest surfaces.

Moreover, there is no statistical distinction in the PS between the ADB and the NDB. Because of this, the unique techniques created to make it easier to cast their mixes, dry them faster, and peel membranes off easily using a self-electrostatic template did not alter the parent roughness properties. Because of this, the unique approaches employed in the current study did not alter the permeability of the membranes (PD and VV).

#### 4.4.2. Membrane Permeability

For the membrane ultrastructure presented in Figure 9, the GA membranes had the lowest PD and VV compared to those for PVA, which had the highest ones for both air-dried bioplastic (ADB) membrane and nanodehydrated transparent bioplastic (NDB) membrane. Accordingly, increasing the PVA allocation increased the membrane permeability, which facilitated the water evaporation from the blend during the nano-dehydration procedures.

When compared to PVA membranes, which had the highest PD and VV for both the air-dried bioplastic (ADB) and nanodehydrated (NDB) membranes, the GA membranes had the lowest ultrastructures. As a result, increasing the PVA allocation also increased the membrane permeability, which made it easier for the blend's water to evaporate throughout the nano-dehydration processes.

Moreover, there is no statistical difference between the ADB and NDB for each case of the PS, PD, or VV. Because of this, the unique approaches employed in the current study did not alter the permeability of the membranes (PD and VV), as shown in Figure 9 and Table 4.

#### *4.5. Mechanical Properties of the Bioplastic Membranes*

The evaluation of a film's capability and mechanical integrity heavily relies on its mechanical properties. The interactions between the blend's components had a significant impact on the matrices of blended films. The mechanical properties were reported to be solely dependent on the chemical structure, which could be best described by using UTS, MoE, and EaF [74]. In addition, it was reported by Gomaa et al. [77] that the internal molecular force, the crystallinity shape, and the content of the polymer all have a significant impact on the mechanical characteristics [77].

The findings understood from Table 5 and Figure 11 revealed that both ADB (the red curve) and NDB (the blue curve) are similar in their ascending trend starting from the PL level up to the US. This behavior means that each membrane, else ADB or NDB was stressed through two stages: (1) in the 1st one, the stress was increased from zero up to the PL level, (2) through the 2nd stage, each membrane transitioned from elastic to plastic nature as the load was increased from the PL up to the maximum load resulting the US stage.

In addition, regarding to proportionality limit (PL) of the bioplastic membranes, it is higher for the NDB than that for the ADB for all the six blend ratios. This indicates that the NDB membranes has higher elasticity endurance compared to their analogous membranes.

As clear from Table 5 and Figure 11, the plotted stress-strain curves for the six blended membranes were differed concerning to their proportionality limit (PL) and ultimate strength (US).

Regarding to sub-graphs of the bioplastic membranes in Figure 11a–f, the similarity between the ADB and NDB in their ascending trend starting from the PL level up to the US can be explained that these membranes was stressed through two stages: (1) in the 1st one, the stress was increased from zero up to the PL level, and (2) through the 2nd stage, each membrane transitioned from elastic to plastic nature as the load was increased from the PL up to the maximum load resulting the US stage.

Since the proportionality limit (PL) of the bioplastic membranes was found to be higher for the NDB than that for the ADB for all the six blend ratios. This indicates that the NDB membranes has higher elasticity endurance compared to their analogous membranes.

The highest values of the UTS (Figure 12) and the MoE (Figure 13) for the membranous sample at the blend ratio of 1:0.25 can be attributed to the strong interaction between the GA and PVA at this optimum blend ratio, which permitted complete miscible blending [76].

The EaF of the bioplastic film samples is explained by the maximum change in its length before failure or breaking as clear from Figure 14 [74].

As shown in Figure 14, adding the GA to the blends enhanced the EaF's membranes up to the blend ratio of GA/PVA of 0.5/0.5. This could be attributed to the good interfacial adhesion among the polymer components (GA and PVA). These findings of the mechanical study confirm the addition of gum acacia can improve mechanical properties, which decrease with an increase in the allocation of gum Arabic [74].

Adding polyvinyl alcohol to the gum Arabic for preparing the bioplastic blend films improved the mechanical properties of these membranes, especially in the blend ratio of GA/PVA of 1:0.25, which enhanced both UTS and MoE, while EaF was enhanced for the blend ratio's membrane GA/PVA of 1:1. Therefore, the results of this work may show that the functional properties of GA/PVA blend films are adequate for food packaging applications and in the pharmaceutical industry for controlled release of drugs [74].

#### *4.6. Microbial Biodegradation*

Biodegradation of the NDB material was confirmed significantly by its reduction in weight for all six NDB samples, and it was found that degradation commenced within 30 and 60 days [144–147].

The microbial communities in all the buried bioplastic sheets, including the control one, were different in number and species. The species of bacteria and fungi differed according to the type of buried sheet.

The microbiological study revealed that all six bioplastic sheets are able to be degraded, contrary to petroleum-based sheets.

Biodegradation is the process by which microorganisms can degrade bioplastic membrane materials, leading to a loss of weight after a period of time. Our results show that all blended bioplastic membranes have reduced weight, especially GA. Our results agree with those of Sasaki et al. [92], who prepared films of phenolic extracts incorporated into GA and found that the highest weight loss of films was 45.81%, compared with GA (26.87%) after 30 days.

Microorganisms can degrade bioplastic membranes through a process called biodegradation, which eventually causes the membranes to lose their weight. Our findings indicate that the weights of all blended bioplastic membranes, particularly GA, decreased. Our findings are consistent with those of Sasaki et al. [92], who created films using phenolic extracts mixed with GA and discovered that after 30 days, the weight loss of the films was higher than that of the GA (45.81%).

These findings were contrary to Ibrahim et al. [78], who discovered that for nanofiber membranes based on homogenous polymeric blends of gum Arabic, polyvinyl alcohol, and silver nanoparticles, the biodegradation tests of the generated nanofibers revealed that 99.09% of the material was broken down after 28 days (Table 7). These variations in the results can be explained by the fact that a variety of factors, including microbes, humidity, sunshine, and oxygen, can affect the bioplastic's capacity to degrade [80].

In addition, because it affects the microbial population and shapes it, the depth of the soil that bioplastic membranes are buried in is a crucial component for biodegradation [78]. In addition, as a result of using gum as a source of nutrients, the number of bacteria increased over time [82,98].

Our results proved that *Pseudomonas* spp., *Bacillus* spp., and *Micrococcus* spp. were the most commonly isolated bacterial strains appearing in different samples, while *Rhizobus* spp., *Penicillium* spp., and *Fusarium* spp. were the most commonly isolated fungus strains that appeared in our different samples. These findings agree with those found by Santos–Beneit et al. [93] and Sasaki et al. [92], which were isolates of *Bacillus cereus*, *Bacillus polymyxa*, *Bacillus licheniformis*, *Corynebacterium xerosis*, *Staphylococcus epidermis*, *Streptococcus bovis*, and the fungi *Penicillium notatum*, *Rhizopus nigricans*, *Aspergillus niger*, and *Fusarium moniliforme* from gum Arabic [68,87,89,90,92,144,145].

Belonging to comparisons within communities, it was found that the HD values determined during and just after 800 h and 1400 h for each of the six bioplastic sheets buried in the soil were similar in their trend concerning each of the bacteria (Figure 15) and fungi (Figure 16) as well as Table 7. This similarity in trends can be attributed to the constancy of the burying depth of the bioplastic membranes [78] and/or various factors, including microbes, humidity, sunshine, and oxygen, which can affect the bioplastic's capacity to degrade [80].

Moreover, a common trend was registered between the NDB products fabricated in the current investigation and the ADB synthesized by Hindi et al. [98] and Hindi and Albureikan [57]. Accordingly, the nano-dehydration invention did not affect the parent's ability to biodegrade the bioplastic membranous product.

#### **5. Conclusions and Future Perspectives**

Great success was achieved for the fabrication of bioplastic membranes from gum Arabic mixed with polyvinyl alcohol by applying a novel casting method, termed static vibrated-free horizontal flow, which produces free air bubble sheets. The novel nanodehydration technique gave the best solution for drying the bioplastic sheets and can be used for any water-based biopolymeric-based product. It is the first time that an acrylic (poly-(methyl methacrylate)) panel used as an ideal template surface features an electrostatically charged hydrophobic surface. As a result, peeling off its template surface is made simpler.

The most important properties of the nanodehydrated bioplastic membranes were studied using Fourier transform infrared spectroscopy, X-ray powder diffraction, thermogravimetric analysis, differential thermal analysis, and atomic force microscopy to ensure that the novel techniques did not distort the product quality. The nanodehydrated bioplastic membranes retained their parent properties, including chemical functional groups, crystallinity index, mass loss, thermal stability, ultrastructure features (surface roughness and permeability), and their ability for microbial biodegradation. PVA had a higher crystallinity index (CI), a greater mass loss at higher temperatures, higher thermal stability due to its higher heat content, and greater clearance of surface roughness due to its high particle size (PS), as well as higher permeability parameters, namely, pore diameter (PD) and void volume (VV), than those for GA. Accordingly, increasing the PVA allocation in the bioplastic blends could enhance their properties except for mass loss, whereas increasing the GA allocation in the NDB blend reduced its mass loss at elevated temperatures.

There is no statistical difference between the bioplastic membranes synthesized elsewhere with ordinary air drying or nano-dehydration in terms of their particle size and

permeability, indicating that the novel procedures used did not distort the parent properties examined as well as their ability for biodegradation. Adding polyvinyl alcohol to the gum Arabic for preparing the bioplastic blend films improved the mechanical properties of these membranes, especially in the blend ratio of GA/PVA of 1:0.25, which enhanced both UTS and MoE, while EaF was enhanced for the blend ratio's membrane GA/PVA of 1:1. Therefore, the results of this work may show that the functional properties of GA/PVA blend films are adequate for food packaging applications and in the pharmaceutical industry for controlled release of drugs [74]. The biodegradation of the nanodehydrated bioplastic membranes was confirmed significantly by the reduction in weight for all six blended samples, and degradation was found to start within 30 and 60 days. Pure GA was the most commonly biodegraded sample among the other bioplastic samples. The microbial communities in all of the buried bioplastic sheets, including the control sample, were different in number, species, and duplication rates. The microbiological survey revealed that all six bioplastic sheets are able to be degraded, contrary to petroleum-based sheets.

#### **6. Patent**

System, apparatus, and methods for manufacturing biodegradable biopolymeric materials (US Patent No. 11548192).

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/polym15153303/s1, Figure S1. The practical procedure used for the novel casting of the (NDB) membranes; Figure S2. The vibrational casting process of the polymeric blends into sheets; Figure S3. Chemical constituents of the polymers used to synthesize the nanodehydrated-bioplastic (NDB) membranes: (a) gum Arabic (GA) precursor, (b) polyvinyl alcohol (PVA) precursor, (c) (poly-(methyl methacrylate), PMMA); Figure S4. Visualization analysis of void volumes (VV, nm3) of the six nanodehydrated-bioplastic membranes (NBMs): (a) GA (100%); (b) GA/PVA = 1:0.25; (c) GA/PVA = 1:0.5; (d), GA/PVA = 1:0.75; and (e) GA/PVA = 1:1, and (f) PVA = 100% based on AFM-image analysis; Table S1. Surface energy and contact angle of the most important industrial polymers; Table S2. Calculating means of mass loss (ML) of the NDB membranes blended from gum Arabic (GA) and polyvinyl alcohol (PVA) in the six ratios and different temperature zones (T-zones).

**Author Contributions:** Conceptualization, S.S.H. and M.O.I.A.; methodology, S.S.H.; validation, S.S.H. and M.O.I.A.; formal analysis, S.S.H.; investigation, S.S.H. and M.O.I.A.; writing—original draft preparation, S.S.H. and M.O.I.A.; writing—review and editing, S.S.H. and M.O.I.A. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was funded by the Deanship of Scientific Research (DSR), KAU, Jeddah, under grant No. G: 85/155/1434.

**Institutional Review Board Statement:** Not applicable.

**Data Availability Statement:** The supporting data for the reported results, including a link to the publicly archived datasets analyzed or generated during this study, can be found under the following patent: US Patent for System, apparatus, and methods for manufacturing biodegradable biopolymeric materials (Patent #11548192, issued 10 January 2023)—Justia Patents Search, https: //patents.justia.com/patent/11060208 (accessed on 17 November 2022).

**Acknowledgments:** The P.I. author is deeply thankful to DSR, KAU, Jeddah for funding this research work. The project that revealed this invention was funded by the Deanship of Scientific Research (DSR), King Abdulaziz University, Jeddah, under grants no. G: 85/155/1434 and, respectively. The P.I. author therefore acknowledges with thanks the DSR for technical and financial support. Appreciation is given to the Center of Nanotechnology (CN) for its technical assistance. Deep thanks to Rakan A. Alanazi for his scientific assistance throughout this investigation.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **Nomenclature**


#### **References**


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### *Article* **Improving the Recyclability of an Epoxy Resin through the Addition of New Biobased Vitrimer**

**Antonio Veloso-Fernández 1,\*, Leire Ruiz-Rubio 1,2, Imanol Yugueros 1, M. Isabel Moreno-Benítez 3, José Manuel Laza <sup>1</sup> and José Luis Vilas-Vilela 1,2**

> <sup>1</sup> Grupo de Química Macromolecular (LABQUIMAC), Departamento de Química Física, Facultad de Ciencia y Tecnología, Universidad del País Vasco UPV/EHU, 48940 Leioa, Spain; leire.ruiz@ehu.eus (L.R.-R.); iyugueros004@ikasle.ehu.eus (I.Y.); and josemanuel.laza@ehu.eus (J.M.L.); joseluis.vilas@ehu.eus (J.L.V.-V.)

<sup>2</sup> BCMaterials, Basque Center for Materials, Applications and Nanostructures, UPV/EHU Science Park, 48940 Leioa, Spain


**Abstract:** In recent decades, the use of thermoset epoxy resins (ER) has spread to countless applications due to their mechanical properties, heat resistance and stability. However, these ERs are neither biodegradable nor recyclable due to their permanent crosslinked networks and usually, they are synthesized from fossil and toxic precursors. Therefore, reducing its consumption is of vital importance to the environment. On the one hand, the solution to the recyclability problems of epoxy resins can be achieved through the use of vitrimers, which have thermoset properties and can be recycled as thermoplastic materials. On the other hand, vitrimers can be made from natural sources, reducing their toxicity. In this work, a sustainable epoxy vitrimer has been efficiently synthesized, VESOV, by curing epoxidized soybean oil (ESO) with a new vanillin-derived Schiff base (VSB) dynamic hardener, aliphatic diamine (1,4-butanediamine, BDA) and using 1,2-dimethylimidazole (DMI) as an accelerator. Likewise, using the same synthesized VSB agent, a commercial epoxy resin has also been cured and characterized as ESO. Finally, different percentages (30, 50 and 70 wt%) of the same ER have been included in the formulation of VESOV, demonstrating that only including 30 wt% of ER in the formulation is able to improve the thermo-mechanical properties, maintaining the VESOV's inherent reprocessability or recyclability. In short, this is the first approach to achieve a new material that can be postulated in the future as a replacement for current commercial epoxy resins, although it still requires a minimum percentage of RE in the formulation, it makes it possible to recycle the material while maintaining good mechanical properties.

**Keywords:** sustainable materials; epoxy resin; Schiff base; epoxidized soybean oil; epoxy vitrimer; reprocessability; recyclability

#### **1. Introduction**

In recent years, special attention has been paid to the use of epoxy resins (ER). This type of thermoset polymer has distinguished properties, such as thermal stability, mechanical strength, creep resistance, electrical insulation, and chemical resistance [1–7]. These polymers are industrially synthesized to use as coatings, adhesives, electronic packaging materials or composites for automobile, aerospace or transportation industries [1,2,8,9]. In fact, the global annual production in 2020 reached almost 10 million tons [10].

Nevertheless, most of the current epoxy thermosets (~90%) are prepared from nonrenewable diglycidyl ether of bisphenol A (DGEBA) and cannot be reprocessed or recycled due to their permanent crosslinking, which causes significant waste and environmental problems after their service lifetime [11–13]. In addition to the non-renewability, bisphenol

**Citation:** Veloso-Fernández, A.; Ruiz-Rubio, L.; Yugueros, I.; Moreno-Benítez, M.I.; Laza, J.M.; Vilas-Vilela, J.L. Improving the Recyclability of an Epoxy Resin through the Addition of New Biobased Vitrimer. *Polymers* **2023**, *15*, 3737. https://doi.org/10.3390/ polym15183737

Academic Editor: Raffaella Striani

Received: 26 July 2023 Revised: 4 September 2023 Accepted: 5 September 2023 Published: 12 September 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

A and epichlorohydrin, which are the raw materials of DGEBA, are toxic, fossil derivatives and both are categorized as hazardous to living organisms [14].

Therefore, recently, more attention has been paid to designing sustainable epoxy thermoset resins from diverse renewable resources, such as epoxidized vegetable oils [15], cardanol [16], isosorbide [17], vanillin [18,19], etc. Specifically, vegetable oils are prime candidates to replace fossil-based derivatives in polymer materials due to: (i) their universal availability, (ii) low toxicity and (iii) low price [20]. Moreover, the presence of carbon-carbon double bonds enables them to be easily transformed into epoxidized vegetable oils (EVOs) through a curing process with hardening agents. Nevertheless, their highly crosslinked structure combined with slightly flexible backbones provides EVOs with poor mechanical strength fusing with poor ductility and low glass transition temperature (Tg) [21,22].

To address these issues, new studies to develop new epoxy resins that contain the remarkable properties of thermosets, as well as the intrinsic capacity of thermoplastics to be recycled after their useful life, are needed. One of the possible solutions is the development of covalent adaptable networks (CANs).

The so-called CANs are polymeric materials with permanent crosslinks, which can reversibly transform into dynamic crosslinks, allowing their chains to flow (analogous to thermoplastics) when they are induced by an external stimulus, such as temperature [23,24], exposure to ultraviolet light [25] or pH [26,27]. In the absence of this stimulus, their reticulated structure offers them stiffness and durability as thermosetting materials. Thus, these polymeric materials, which have thermosetting polymer properties due to their crosslinking networks, are recyclable and reusable due to the dynamic nature of these crosslinks.

In 2011, Leibler et al. reported [28] a new class of CANs called vitrimers, which resemble vitreous silica due to their change in viscosity and the fact that they also follow an Arrhenius relationship that increases with temperature. Vitrimers belong to a sub-class of CANs in which the crosslinking bonds have an associative nature, resulting in the ability of the material to change its topology via exchange reactions [29–33]. Certainly, vitrimer crosslink density can be recognized as almost constant regardless of external stimuli, resulting in two principal effects [34–38]. First, unlike dissociative CANs, which transform from a solid state more suddenly [39], an extended gummy/rubbery phase can be observed in vitrimers when heated. Second, some researchers have also remarked a greater creep/solvent resistance for vitrimers [37,40,41]. Moreover, vitrimers are distinguished according to their temperature-dependent viscoelastic behavior, as the covalent exchange rate is related to the transition temperature. At high temperatures, when the exchange reactions become fast enough, the viscosity of vitrimers is basically controlled by the exchange reactions, leading to a decrease in viscosity with the temperature that follows the Arrhenius law.

The viscoelastic behavior of vitrimers changes with the topology freezing temperature (Tv) [28]. The Tv is chosen by agreement as the temperature at which the viscosity reaches <sup>10</sup><sup>12</sup> Pa·s [42]. Below Tv, vitrimers behave as conventional thermosets, and above Tv, they can undergo creeping and relaxing stresses. The control of this temperature is essential since the exchange of covalent bonds and permanent crosslinking allows or does not allow thermal recycling [43–45]. Thus, epoxy vitrimers are a substantial advance for the replacement of current thermosets.

The developed vitrimers so far have been overviewed according to the nature of the dynamic exchange reaction. The most common dynamic interactions used in the design of vitrimers have been carboxylate transesterification [28], transamination of vinylogous urethanes [46], transalkylation of triazolium salts [47], disulfide exchange [48] or Schiff base (imine) exchange [49]. Among them, Schiff bases show great potential in the fabrication of epoxy vitrimers due to the presence of a reversible covalent bond since it can be hydrolyzed to aldehyde or ketone and to amine under acid conditions [50]. Furthermore, compounds obtained from natural resources can be used as reagents, which are more attractive and interesting compared with the existing materials. Among them, recent studies show the design of sustainable epoxy vitrimers with natural phenolic compounds such as vanillin (VAN) as the starting material due to the stiffness structure provided by the benzene ring leading to high-Tg epoxy vitrimers combined with superior mechanical strength and modulus [51–53]. It should be noted that VAN is one of the few compounds with a phenolic group manufactured on an industrial scale from biomass, especially from tannin and lignin [54]. Therefore, this reagent has the potential to become a key precursor for the synthesis of bio-based polymers as it presents an aromatic structure that can achieve good thermo-mechanical properties.

Taking into account all the premises described, in this work, a biovitrimer is developed from components obtained from natural resources, such as epoxidized soybean oil (ESO) and VAN. ESO is used as a bio-based monomer and a vanillin derivative as a new Schiff base to act as a biobased hardener. First, the Schiff base is prepared using VAN and aliphatic diamine (1,4-butandiamine, BDA). Second, ESO and the new Schiff base are combined to form the vitrimer. However, numerous studies have shown the susceptibility of vitrimers to creep substantially under use conditions [55–57]. For that reason, in order to improve the properties of this new material, a critical fraction of permanent crosslinks (30, 50 and 70 wt%) is added to the new biovitrimer, which has little or no detrimental effect on reprocessability [48,52,58,59]. During this work, the poly(bisphenol A-co-epichlorohydrin) glycidyl end-capped is used as a commercial epoxy resin (ER). Finally, in all the samples, thermal and mechanical properties, as well as their reprocessability or recyclability, have been investigated.

#### **2. Experimental Section**

#### *2.1. Reagents*

Vanillin (VAN, 99%), 1,4-butanediamine (BDA, 99%), 1,2-dimethylimidazole (DMI, 97%) and commercial epoxy resin (ER) poly(bisphenol A-co-epichlorohydrin) glycidyl endcapped (Mn~355 g mol−1) were purchased from Sigma Aldrich (Saint Louis, MO, USA). Methanol (MeOH, ≥99.5%) was obtained from PANREAC (Barcelona, Spain). Epoxidized soybean oil (ESO) EPOVINSTAB H-800-D was kindly supplied by Hebron S.A. (Barcelona, Spain). All the chemicals were used as received.

#### *2.2. Synthesis of Vanillin-Derived Schiff Base Curing Agent*

The vanillin-derived Schiff base (VSB) curing agent was synthesized by dissolving 10.0 g (66 mmol) of vanillin in 100 mL of methanol and mixed in a 500 mL single-necked round-bottomed flask with 3.3 mL (33 mmol) of 1,4-butandiamine. A yellow powdered product (VSB) was obtained (Scheme 1a) after solvent evaporation, which was washed with methanol and vacuum dried at 50 ◦C for 24 h.

#### *2.3. Synthesis of Vitrimers*

The VSB-cured ESO biovitrimer (VESOV) was synthesized by a two-stage procedure pre- and post-curing. To achieve the pre-curing stage, predetermined amounts of VSB and ESO were added to the reaction using an equivalent phenolic/epoxy hydroxyl ratio (simplified as X). Previous works performed by Zhao et al. [60] and Zeng et al. [61] demonstrated that the optimal ratio to obtain a vitrimer with a highly crosslinked network is X = 0.7. First, the VSB was heated until reaching its melting point in a 100 mL single-necked round-bottomed flask placed into an oil bath and stirred with a magnetic stirring bar under a nitrogen atmosphere. When the VSB was completely melted, the corresponding amount of ESO was added. After ESO and VSB were fully mixed, the catalyst 1,2-dimethylimidazole (DMI) (0.5 wt%) was added, allowing the resulting mixture to react until the magnetic stirrer could not turn due to the increase in the viscosity of the medium (Scheme 1b). Then, the post-curing stage started with the transfer of the resultant mixture to a 10 cm × 10 cm × 1.0 mm stainless steel mold, which was placed in a compression molding machine at 150 ◦C under 10 bar for 2 h. After allowing the sample to reach room temperature, a film was obtained. Scheme 1c shows in purple and red how the network

structure is reorganized by the thermal-induced exchange reaction of Schiff base in the crosslinking structure of VESOV, leading to the stress relaxation behavior.

**Scheme 1.** (**a**) Preparation of the dynamic curing agent (VSB), (**b**) curing reaction of ESO with VSB to form VESOV, and (**c**) exchange reaction of the Schiff base in the vitrimer, indicating in color the outcome exchange.

For the synthesis of VESOV+ER, the same procedure was used, adding the epoxy resin at the same time as the ESO. Thus, it was decided to add progressive amounts by weight of epoxy resin (30, 50 and 70 wt%) to determine the influence of the addition of epoxy resin to the VESOV. Additionally, to investigate the reaction between VSB+ER, the same procedure as ESO was followed, adding ER to the VSB. Therefore, the mechanical and thermal properties of five samples were studied: VESOV, VESOV+ER (30 wt% of ER), VESOV+ER (50 wt%), VESOV+ER (70 wt%) and VSB+ER.

#### **3. Characterization**

Fourier transform infrared (FTIR) spectra with wavelengths from 4000 to 400 cm−<sup>1</sup> were recorded by a Nicolet Nexus spectrophotometer (Thermo Fisher Scientific Inc., Madison, WI, USA), where the samples were measured within KBr pellets. The resolution and scanning number were 4 cm−<sup>1</sup> and 32 times, respectively. The data were analyzed using OMNIC 8.2 software.

Proton nuclear magnetic resonance (1H-NMR) spectra were recorded on a Bruker AV-600 (600 MHz) spectrometer (Billerica, MA, USA) using deuterated chloroform (CDCl3) as the solvent.

The melting temperature of VSB and the glass transition temperature of the vitrimers were measured by differential scanning calorimeter (DSC) with a DSC METTLER TOLEDO 822e instrument (Greifensee, Switzerland) equipped with STAR© v14.0 software. The samples (~10 mg) were placed in 100 μL aluminum crucibles. Samples were heated from −10 ◦C to 250 ◦C, in the case of the VSB, and from −10 ◦C to 150 ◦C for the vitrimers. A scanning rate of 10 ◦C·min−<sup>1</sup> under a nitrogen atmosphere with a flow rate of 20 mL·min<sup>−</sup>1.

The thermal stability of the samples (~10 mg) was measured by thermal gravimetric analysis (TGA) under a nitrogen atmosphere (20 mL·min−1) with a temperature range of 25–800 ◦C and a heating rate of 10 ◦C·min−<sup>1</sup> by a SHIMADZU DTG-60 thermal gravimetric analyzer (Kyoto, Japan). The statistic heat-resistant index temperature (Ts) is a characteristic of the thermal stability of the cured resin [62] and was calculated according to Equation (1):

$$\mathbf{T\_s} = 0.49 \times \left[ \mathbf{T\_{5\%}} + 0.6 \times \left( \mathbf{T\_{30\%}} - \mathbf{T\_{5\%}} \right) \right] \tag{1}$$

where T5% and T30% are the temperatures at, respectively, 5% and 30% weight loss. T5% was considered the onset decomposition temperature (To) of the sample.

Dynamic mechanical analysis (DMA) was carried out in the tensile mode by a DMA1- METTLER TOLEDO instrument (Greifensee, Switzerland) equipped with STAR© v14.0 software for curve analysis. Storage modulus (E ), loss modulus (E) and loss factors (tan <sup>δ</sup>) values were collected at 3 ◦C·min−<sup>1</sup> heating rate from −10 to 150 ◦C, displacement of 20 μm, and 1, 3 and 10 Hz frequencies. Rectangular-shaped testing bars with a width of 5.0 mm, length of 10.0 mm and thickness of 0.5 mm were prepared. The glass transition was assigned at the maximum of the loss factor (tan δ = E/E ).

Reprocessing tests were performed on the compression molding machine (20 TM Hot Plates Press, Hidrotecno S.L., Oiartzun, Spain). The films were cut into small pieces with scissors, placed into the 10 cm × 10 cm × 1.0 mm stainless steel mold, and reprocessed at 150 ◦C for 60 min at 10 bar. After cooling to room temperature, the reprocessed films were obtained, and their thermo-mechanical properties were measured.

#### **4. Results and Discussion**

Epoxy thermosets exhibit tremendous thermo-mechanical properties; however, they lack the capacity to be reprocessed after their use. The main objective of this work is to obtain a substitute for epoxy resins that can be considered biobased. In this regard, it must be taken into account that to consider a material as biobased, a minimum of 50% of the compounds incorporated into its formulation must be obtained from natural resources. For this, first, the Schiff base is synthesized using VAN and aliphatic diamine (1,4-butandiamine, BDA). Then, a sustainable epoxy vitrimer derived from vegetable oil ESO is prepared, and finally, to improve the thermo-mechanical properties of the reprocessable vitrimer, a small amount of commercial epoxy resin (ER) is added.

#### *4.1. Synthesis and Characterization of Vanillin-Derived Schiff Base (VSB) Hardener*

VSB hardener was synthesized by refluxing VAN and BDA in methanol for 24 h. The chemical structure of VSB was confirmed by FTIR and 1H-NMR. Figure 1a shows how the characteristic stretching peak of C=O on the aldehyde group (1670 cm−1) of the VAN and the broad stretching of N-H (between 3400 and 3250 cm<sup>−</sup>1) of BDA is not observed in the VSB FTIR spectrum. Instead, a new characteristic peak appears at 1655 cm−<sup>1</sup> for VSB, attributing to the formed Schiff base unit (the most significant FTIR signals were compiled in the Supporting Information). Moreover, 1H-NMR easily (Figures S1 and S2) allows the Schiff base identification via the disappearance of the signal from the aldehyde group of the VAN and the presence of a new signal in the VSB typical of an imine (Figure 1b). Precisely, the signals for the H proton of alcohol, imine, benzene ring, methoxy and butane were observed at shifts of 9.8, 8.2, 7.4–6.9, 3.9 and 1.8 ppm, respectively. These facts indicate

that the Schiff base bond has been successfully formed by the reaction between the amino and aldehyde groups. In addition, the DSC curve of VSB shows only one sharp melting peak at 153 ◦C (Figure 1c), observing that the VSB was successfully synthesized, as the correspondent melting peak of VAN (~86 ◦C) was not observed in the DSC curve of VSB. In short, it is corroborated that the vanillin-derived Schiff base was prepared successfully.

**Figure 1.** (**a**) FTIR spectra of VAN, BDA and VSB, (**b**) 1H-NMR spectrum of VSB and (**c**) DSC heating curve for VAN and VSB.

#### *4.2. Synthesis and Characterization of VESOV, VESOV+ER (Different Percentages) and VSB+ER*

Once the VSB was formed (Scheme 1a), using DMI as a catalyst, five different formulations of ESO and/or ER vitrimers were developed: VESOV, VESOV+ER (30 wt%), VESOV+ER (50 wt%), VESOV+ER (70 wt%) and VSB+ER. In all cases, the synthetic procedure was the same as described above; after a pre-curing stage where the VSB is reacted with the ESO, the ER or a mixture of both, the post-curing is accomplished (Scheme 1b). Subsequently, the thermo-mechanical properties of the samples were studied.

First, the thermal stability of the samples was studied to confirm that the material remains stable and does not suffer any degradation during the reprocessing procedure. The thermal stability profiles of the obtained vitrimers are displayed in Figure 2 and Tables 1 and S1. Clearly, all samples are thermally stable up to a temperature of at least 272–297 ◦C (onset decomposition temperature, To, in Table 1), demonstrating that they possess acceptable thermal stability under all conventional modeling approaches and that they are also thermally resistant during reprocessing, which is important for the recycling of the vitrimer through thermal processing [63].

**Figure 2.** Thermogravimetric curves for VESOV, VESOV+ER (with different weight ratio) and VSB+ER.

**Table 1.** To and Ts values determined by TGA Analysis.


From Table 1, it can be observed that the statistic heat-resistant index temperature values (Ts) show the same trend as To (or T5%) values. The results demonstrate that samples with low epoxy resin (ER) content exhibit higher thermal stability (VESOV has the highest), whereas increasing the ER content (VESOV+ER) decreases the thermal stability. The reason for this behavior could be associated with the higher amount of VSB in the network, involving a higher content of imine bonds. Moreover, the higher the oxirane ring content, the more ester and hydroxyl groups are created through the curing. These functions can also promote the thermal scissions of the networks [64].

The glass transition temperature (Tg), which usually acts as the upper limit use temperature for thermosetting materials, is a major parameter [65]. Figure 3a,b shows the DSC curves of epoxy vitrimers to make a comparison of the Tg-onset (simplified as Tg) between VESOV, VESOV+ER (different percentages) and VSB+ER.

In Figure 3a, it was observed that in the VESOV+ER samples (30 and 50 wt%), two glass transition temperatures coexist. The first one appears at the same range as the Tg peak of VESOV, whilst the other Tg appears a little more displaced at higher temperatures. This may be due to the formation of two polymeric networks that do not mix with each other; that is, a heterogeneous mixture was obtained. However, as noted below, it is most likely that the post-curing process (2 h at 150 ◦C) was not enough to obtain a fully cured vitrimer. In Table 2, the Tg of the different samples is summarized (Tg-onset).

As can be noticed in Table 2, when the fraction of epoxy resin increases, the Tg values rise significantly; that is, higher stiffness is achieved by making the fluidity of the chains more difficult. Therefore, the addition of a small amount of epoxy resin helps VESOV to improve its thermal properties. Indeed, VESOV has a Tg equal to 28.7 ◦C, while the addition of only 30 wt% of ER increases the Tg to 48.2 ◦C. However, by continuing to add more percentage in weight in ER (50 and 70 wt%), the increase that occurs in Tg is not as significant (Tg = 48.7 and 50.3 ◦C for 50 and 70 wt% in ER, respectively). That is, a larger increase in the added proportion of ER did not provide significantly higher Tg.

**Figure 3.** (**a**) Calorimetric curves of the original and (**b**) reprocessed vitrimers: VESOV, VESOV+ER (with different proportion by weight) and VSB+ER.

**Table 2.** Tg values obtained in the calorimetric curves (DSC) and the maximum peak of the tan δ (DMA) in the original and reprocessed samples.


Further, the Tg of the samples was determined via DMA. Figure 4 shows both the storage modulus (E´) and the loss factor (tan δ) (its maximum peak is the Tg of each sample) versus temperature. In addition, Table 2 shows the glass transition temperatures of all the samples obtained by DMA. All the curves with their corresponding numerical data are presented at a frequency of 3 Hz.

**Figure 4.** DMA curves obtained for (**a**) original and (**b**) reprocessed vitrimers at 3 Hz frequency: VESOV, VESOV+ER (with different weight proportion) and VSB+ER.

As in the DSC, it is observed that while increasing the amount of epoxy resin, the glass transition temperature of the sample increases progressively. On the other hand, in Figure 4a, the tan δ curve of VSB+ER exhibits two peaks, implying it is not completely cured, as not all the chains of the system have reacted effectively. That is, as said before, the post-curing process is not enough to reach a total curing, and samples need a greater postcuring. Furthermore, the difference in the storage modulus is notable amongst samples. Thus, while VESOV has a very low storage modulus, it is remarkably increased by adding a small amount of epoxy resin.

In conclusion, analyzing the results obtained so far, it can be said that adding a small amount of epoxy resin (30 wt%) considerably improves the thermo-mechanical properties of the VESOV vitrimer. Finally, the reprocessability of these vitrimers was studied.

#### *4.3. Reprocessability of VESOV, VESOV+ER (Different Percentages) and VSB+ER*

The dynamic character of the Schiff base exchanges improves the ability of the samples to be reprocessed. The reprocessing is performed as explained before: The samples were cut into small pieces with scissors, placed into a steel mold and reprocessed at 150 ◦C for 60 min at 10 bar. These reprocessed samples are shown in Figure 5, observing that VESOV has a considerable ability to be reprocessed (Figure 5a) since it contains dynamic covalent bonds. On the contrary, after carrying out this test with the VSB+ER (Figure 5e), incomplete recovery of the original shape is achieved, as it includes permanent crosslinks due to epoxy resin's nature as a thermoset. Figure 5b–d also shows the reprocessability of those vitrimers synthesized using different proportions of ESO and epoxy resin (VESOV+ER). In Figure 5b, it can be seen that only with the inclusion of 30 wt% of epoxy resin is it observed that the reprocessability of the sample substantially improves. In order to compare and verify if any degradation occurs after reprocessability, FTIR analysis was performed. In the supporting information, FTIR spectra of all the samples are included. No differences were observed between pre- and post-reprocessed spectra (Figure S3).

**Figure 5.** Digital photos demonstrating the reprocessability of (**a**) VESOV, (**b**) VESOV+ER (30 wt%), (**c**) VESOV+ER (50 wt%), (**d**) VESOV+ER (70 wt%) and (**e**) VSB+ER.

Yet, it is necessary to verify that this reprocessing does not diminish the good thermomechanical properties obtained for the original samples. For this, the characterizations via DSC and DMA of the different reprocessed samples were accomplished. Moreover, a comparison between the original and remolded samples is performed to evaluate how many times samples can be reprocessed without losing their thermo-mechanical properties.

In DSC, the reprocessed samples only present a Tg, showing that they are totally cured (Figure 3b). In addition, as seen in Table 2, all the glass transition temperatures obtained for the reprocessed samples are similar or even higher than those corresponding to the samples without reprocessing. The only exception is precisely the vitrimer synthesized only with ESO (VESOV), which showed a clear decrease in its Tg.

However, DMA measurements (Figure 4b) demonstrate that all the reprocessed samples slightly increase their glass transition temperatures (Table 2) because they undergo a post-curing process in which the crosslinking of their bonds increases. This fact is confirmed by the width of the tan δ peaks, as in the original samples, the width of the peak is greater, denoting that the samples are more heterogeneous. In the reprocessed samples, peak width decreases considerably, denoting that the crosslinking of the bonds has been superior and obtained a larger homogeneity of the system.

#### **5. Conclusions**

In conclusion, it has been possible to verify that 1,4-butandiamine is a suitable reagent to synthesize a vanillin-derived Schiff base (VSB) that can be used as a curing agent in the development of a new vitrimer from epoxidized soybean oil (VESOV). Furthermore, the inclusion of a commercial epoxy resin to the VESOV helped to improve its mechanical and thermal properties, observing by DSC and DMA techniques that when the percentage of epoxy resin increases, the Tg increases remarkably. However, these new epoxy materials must have a balance between their thermo-mechanical properties and their reprocessability to be considered vitrimers. In this way, a circular economy can be established, such as thermoplastics. Standing on this, it can be concluded that the VESOV+ER 30 wt% sample seems to be the most suitable to achieve this objective since it has the qualities of a thermoset (high storage modulus and Tg) and contains elements of a thermoplastic (good reprocessability), with this last property not attributable to conventional epoxy resins.

In short, this new dynamic hardener based on vanillin and 1,4-butandiamine obtained good properties as expected; a sustainable vitrimer was synthesize based only on epoxidized vegetable oil. The first approach to this objective has been made by developing a new material with remarkable features by adding a small amount of commercial epoxy resin (30 wt%), so the material can be considered biobased with good mechanical properties and the possibility of recycling. Therefore, this material can be postulated in the future, with slight improvements, to be a replacement for current commercial epoxy resins.

**Supplementary Materials:** The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/polym15183737/s1, Figure S1: 1H-NMR spectrum of 1,4-butandiamine. Figure S2: 1H-NMR spectrum of vanillin; Figure S3: FTIR spectra of pre- (black) and post-reprocessed (red) sample; Table S1: TGA results obtained for samples: T30% and final residual mass %.

**Author Contributions:** Conceptualization, A.V.-F. and J.M.L.; methodology, J.M.L.; validation, L.R.-R., M.I.M.-B. and J.L.V.-V.; formal analysis, A.V.-F.; investigation, I.Y.; resources, L.R.-R.; data curation, I.Y.; writing—original draft preparation, A.V.-F.; writing—review and editing, J.M.L.; visualization, M.I.M.-B.; supervision, J.L.V.-V.; project administration, J.L.V.-V.; funding acquisition, J.L.V.-V. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Basque Government ELKARTEK (KK-2023/00056; KK-2023/00016; KK-2022/0082; KK-2022/0040) and Grupos Consolidados (IT1756-22).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The authors are thankful for the funding from the Government of the Basque Country under the Grupos de Investigación del Sistema Universitario Vasco, (IT1756-22) program and the ELKARTEK program. The authors also thank the technical support of SGIker (UPV/EHU).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **Abbreviations**


#### **References**


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## *Article* **Investigating the Mechanical, Thermal, and Crystalline Properties of Raw and Potassium Hydroxide Treated Butea Parviflora Fibers for Green Polymer Composites**

**Abisha Mohan 1, Retnam Krishna Priya 1,\*, Krishna Prakash Arunachalam 2,\*, Siva Avudaiappan 3,4,5, Nelson Maureira-Carsalade <sup>6</sup> and Angel Roco-Videla 7,\***


**Abstract:** The only biotic factor that can satisfy the needs of human species are plants. In order to minimize plastic usage and spread an immediate require of environmental awareness, the globe urges for the development of green composite materials. Natural fibers show good renewability and sustainability and are hence utilized as reinforcements in polymer matrix composites. The present work concerns on the usage of Butea parviflora fiber (BP), a green material, for high end applications. The study throws light upon the characterization of raw and potassium hydroxide (KOH)–treated Butea Parviflora plant, where its physical, structural, morphological, mechanical, and thermal properties are analyzed using the powder XRD, FTIR spectroscopy, FESEM micrographs, tensile testing, Tg-DTA, Thermal conductivity, Chemical composition, and CHNS analysis. The density values of untreated and KOH-treated fibers are 1.238 g/cc and 1.340 g/cc, respectively. The crystallinity index of the treated fiber has significantly increased from 83.63% to 86.03%. The cellulose content of the treated fiber also experienced a substantial increase from 58.50% to 60.72%. Treated fibers exhibited a reduction in both hemicelluloses and wax content. Spectroscopic studies registered varying vibrations of functional groups residing on the fibers. SEM images distinguished specific changes on the raw and treated fiber surfaces. The Availability of elements Carbon, Nitrogen, and Hydrogen were analyzed using the CHNS studies. The tensile strength and modulus of treated fibers has risen to 192.97 MPa and 3.46 Gpa, respectively. Thermal conductivity (K) using Lee's disc showed a decrement in the K values of alkalized BP. The activation energy Ea lies between 55.95 and 73.15 kJ/mol. The fibers can withstand a good temperature of up to 240 ◦C, presenting that it can be tuned in for making sustainable composites.

**Keywords:** green composites; stem fiber; crystallinity; thermal behavior; reinforcement material

#### **1. Introduction**

For centuries, the distinctive characteristics of natural fibers have made them valuable for diverse purposes. The properties of natural fibers, including their mechanical, physical, and chemical attributes, are contingent on factors such as the specific fiber type, the plant

**Citation:** Mohan, A.; Priya, R.K.; Arunachalam, K.P.; Avudaiappan, S.; Maureira-Carsalade, N.; Roco-Videla, A. Investigating the Mechanical, Thermal, and Crystalline Properties of Raw and Potassium Hydroxide Treated Butea Parviflora Fibers for Green Polymer Composites. *Polymers* **2023**, *15*, 3522. https://doi.org/ 10.3390/polym15173522

Academic Editor: Raffaella Striani

Received: 24 June 2023 Revised: 13 August 2023 Accepted: 17 August 2023 Published: 24 August 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

species from which they are derived, and the environmental conditions in which they are cultivated. Natural fibers are categorized based on their chemical composition, which can be either cellulose-based or lignin-based. Cellulose-based fibers such as cotton, jute, flax, hemp, and sisal have high tensile strength, suitable flexibility, and low density, making them suitable for applications such as textiles, paper, and composites. Lignin-based fibers, such as wood fibers, have high stiffness and strength, making them suitable for applications such as building materials and composites. Due to their environmentally friendly and sustainable behavior, natural fibers are progressively being utilized as substitutes for synthetic fibers in a wide range of applications. The lowered density of natural fiber composites (NFCs), along with their advantageous tribological and insulating qualities, could increase the cargo capacity of aircraft. Boeing and Airbus, two aviation industry titans, applied considerable effort to learn more about the usage of natural fibers in airplane interiors [1].

Natural fiber composites (NFCs) are composite materials that are made from a combination of natural fibers and a matrix material. NFCs are becoming increasingly popular as a sustainable and environmentally friendly substitute for conventional composite materials, which predominantly rely on synthetic fibers. The natural fibers used in NFCs can come from plant, animal, or mineral sources. The matrix material can be made from a variety of materials such as bio-based polymers, thermosetting resins, or thermoplastics. The characterization of natural fibers are instrumental in developing and optimizing new applications. NFCs have numerous advantages over traditional composite materials. The efficient properties possessed by natural fibers are light weight, high aspect ratio, low density, soundproof, thermal, mechanical properties, and biodegradability [2–4]. The combined effect of cellulose, hemicellulose, lignin, and wax dictates the overall properties of fibers. However, the hydrophilicity of the fibers turn in as a threat while intriguing fibers in making composites [5,6]. Microwave drying systems using halogen lamps were employed to bring down the moisture absorption in bast fibers [7]. The inadequate interfacial bonding contributes to diminished mechanical properties, which can be influenced by factors such as contact angle, orientation of microfibrils relative to the cell axis, and the Young's modulus of the fiber [8]. By subjecting fibers to different treatments, it is possible to transit their hydrophilic nature to hydrophobic, resulting in improved performance and easier disposal [9–11]. Studies in the literature demonstrate that alkali treatment has caused notable changes in the mechanical properties of reinforcements [12–15]. Specifically, alkali–treated Borassus fruit fibers exhibited significant increase of 41% in tensile strength, 69% in modulus and 40% in elongation [16]. 5% NaOH action on Acacia Caesia bark fibers had removed amorphous constituents and improved its tensile nature [17]. KOH– treated Ijuk fibers displayed enhanced tensile and stiffness in the fabricated composites [18]. Furthermore, natural fibers typically require lower processing temperatures compared to synthetic alternatives, which could be overthrown by employing flame retardants like phosphates, phosphoric acids, N-methynol functional phosphorus esters, antimony-halogen combinations, boron and nitrogen compounds [1]. Generally practiced chemical treatment process are bleaching, benzoylation, acetylation, silane, permanganate, etc.

The present work focuses on the Butea Parviflora (BP) plant, which is native to most South East Asian countries, including India. It is one among the many plants of the Fabaceae family with the genus name 'Butea'. It has a trifoliate alternate spiral leaf arrangement and bears flowers and seeds. Seeds are imbibed with many pharmaceutical benefits and are crushed for oil [19]. Being a deciduous climbing shrub, it could extend up to 20 m in height. Long fiber strands are torned out for domestic utilities by localities.

The Butea parviflora (BP) fiber is believed to possess most characters, as found in other stem fibers discovered to date, and there has been limited research conducted on it. The BP plant has climbing branches twined strongly around each other. The roots are strongly fixed to the ground, thus rendering a mechanical support from retrieving its path of growth. They are scattered all over India mostly in the Western and Eastern ghats and are widely flourished from moist to arid region. Fibers for the present study are collected from the village of Thirunandikarai, Kanniyakumari District, Tamil Nadu. Characterization of BP fibers is necessary to understand their properties and potential applications. Raw and 0.1 M of KOH–treated Butea fibers are prepared for the characterization procedures including physical, mechanical, spectroscopic, thermal, crystalline, morphological, and chemical testing methods. Physical testing methods involve measurements of fiber diameter, length, density and aspect ratios. Mechanical testing techniques are employed to assess the strength and rigidity of fibers, while the spectroscopic, thermal, crystalline, and morphologies are studied using the FTIR, Thermogravimetric analysis (TG-DTA), X-ray diffraction (XRD), and Scanning Electron Microscopic studies (SEM) [20]. Chemical testing methods are used to identify the chemical composition of fibers, including the detection of impurities and extractives. The experimental data indicate that BP has the potential to serve as a superior reinforcing material in the formulation of sustainable composites.

#### **2. Materials and Methods**

#### *2.1. Material Extraction*

The collected BP fibers are mechanically removed from the branchy stems using a metal teeth. The peeled fibers are then dried in the absence of sunlight for about 7 days in a clean environment. Fibers are drenched in water and surface modification is attained by soaking it in 0.1 M of KOH environment for 30 min. Alkali pre-treatment is performed to eliminate impurities such as wax, oil, etc., from the fibers, while also inducing modifications to enhance their properties [21,22]. Fibers are kept at room temperature for over 10–15 days. Potassium hydroxide was chosen over NaOH in the current study, because it is less alkaline. In Ijuk fibers, KOH–treated fibers generated the highest tensile and stiffness than NaOH [18]. Given that KOH treatment was not performed prior on Butea fibers, KOH with 0.1 molarity was carried on Butea fibers. Fibers mercerized with 0.1 M KOH solution on other fibers demonstrated an enhancement in the mechanical properties [23]. Sisal fibers from the literature showed an improved hydrophobic behavior while treated with same molarity of alkali solution [24]. Alkali-treated fibers are proceeded with vacuum desiccating for 2 days [25]. Figure 1 shows the fibers extracted from Butea parviflora (BP).

**Figure 1.** Collected fibers from Butea parviflora plant.

*2.2. Physical Properties of Butea Parviflora (BP) Fiber*

The physical factors of unprocessed and alkali–treated Butea parviflora (BP) fibers are crucial in making composites. Randomly selected BP fibers 30 in number are taken to establish the physical aspects.

Diameter of BP is calculated using an optical microscope. KOH–treated fibers show a decrease in the diameter of the raw fibers. It can be believed that the interfacial strength decreases with an increase in diameter regardless of surface modifications [26].

Aspect ratios of natural fibers are found by calculating the ratio between the length and diameter. The aspect ratios of alkali–treated BP (203.91) are greater than raw BP (174.59). Higher the aspect ratios, more will be the compressive strength of composites. The addition of coconut and oil palm fibers to soil building blocks resulted in an augmentation of both compressive and tensile strength, which is correlated with higher aspect ratios of the fibers [27].

The linear density (LD) is a measure to determine the fineness of a fiber, and excellency in tensile strength is observed with higher LD values. An average length of 10 cm was chosen to calculate the fiber's LD using the equation [28].

$$\text{Linear density (LD)} = \frac{\text{massoffibers (grams)}}{\text{lengthffibers (meter)}} \tag{1}$$

Density plays a crucial role in determining the suitability of natural fiber composites for various applications. It is a prime factor that distinguishes and discriminates natural fiber composites from their synthetic counterparts. Density is analyzed using the liquid pycnometer method, with the immersion liquid toluene, using the equation [9,29],

$$\rho = \frac{(\text{mb} - \text{ma})}{[(\text{mc} - \text{ma}) - (\text{md} - \text{mb})]} \text{pt} \tag{2}$$

In the given context, ma represents the mass of the empty pycnometer (in kilograms), mb denotes the mass of the pycnometer filled with fibers (in kilograms), mc represents the mass of the pycnometer filled with toluene (in kilograms), and md indicates the mass of the pycnometer filled with both fibers and toluene (in kilograms).

The density of KOH–treated BP (1.340 g/cc) is higher than raw BP (1.238 g/cc). Less-dense extractives of fibers like lignin and hemicellulose, along with airspaces, might get removed by the alkalization. Hence, the density of treated BP has been incremented [28]. Density values of BP are comparable with other fibers like Thespesia populnea (1.412 g/cc) [30], carbon (1.40 g/cc), and aramid fibers (1.40 g/cc) and are much smaller than E-glass fibers (2050 g/cc) [31]. Physical aspects of Butea fibers are displayed in Table 1.

**Table 1.** Comparison made between the physical and chemical attributes of untreated and alkalized BP fibers, alongside other types of fibers.


#### **3. Characterization Studies**

*3.1. X-ray Diffraction (XRD) Analysis*

The crystalline nature of Butea fibers was measured using powder X-ray diffraction. The analysis was conducted using a D8 Advance Model diffractometer from the manufacturer, Bruker AXS, Karlsruhe, Germany. Recording the spectrum for 2θ values was taken between 3◦ and 80◦ under 40 kV and a current supply of 35 mA. The Segal empirical formula was utilized to calculate the crystallinity index of BP fibers [34,35].

$$\text{CI} = \frac{\text{I}\_{200-\text{I}\_{\text{am}}} \ast 100\text{\textdegree}}{\text{I}\_{200}} \ast 100\text{\textdegree} \tag{3}$$

where I200—maximum intensity of the crystalline diffraction peak at 2θ angle range of 22◦ to 23◦, and Iam—minimum intensity of an amorphous peak at 2θ angle of 18◦. Additionally, the crystallite size was calculated utilizing Scherrer's equation [36].

$$\text{CS} = \frac{\text{K}\lambda}{\beta\_{200}\cos\Theta} \tag{4}$$

where K—Scherrer's constant, λ—wavelength of X-rays (0.154 nm), β200—the peak's full width at half maximum, and θ—Bragg angle.

#### *3.2. Scanning Electron Microscopy (SEM)*

Scanning electron microscopy gives outstanding results in identifying the morphological features; thereby, the fundamental characters of the fibers are lit up with detailed clarity. The surface images of fibers were scanned with the working voltage from 0.5 to 30 kV, using an instrument, Jeol 6390LA/OXFORD XMXN, from JEOL India PVT LTD; South Delhi, India, a subsidiary company of JEOL Limited, Tokyo, Japan.

#### *3.3. Thermogravimetric Analysis*

Heat resistance is very much needed for making composites [37]. By indulging fibers in thermal analysis, the nature of samples under various environments of heating and cooling, along with inert oxidation-reduction atmospheres, can be cited. The change in mass is adjoined with a variety of reactions such as decomposition, degradation, adsorption, vaporization, oxidation, reduction, etc. Tg-dta and DSC analyses were carried out using the Perkin Elmer STA 6000 Model, from the manufacturer Perkin Elmer Inc., Mumbai, India. The heating process was monitored at a rate of 20 ◦C per minute under a dynamic nitrogen atmosphere within the temperature range of 40–800 ◦C.

#### *3.4. Thermal Conductivity Using Lee's Disc Method*

Thermal conductivity was assessed using Lee's disc method, wherein the mass, diameter, and thickness of Lee's disc were measured using a digital weighing machine, Vernier caliper, and screw gauge. At the onset of steady temperature, the disc is let to cool down, and dropping temperatures are noted. The thermal conductivity was determined by employing a specific equation for the calculation process [38].

$$\mathbf{k} = \frac{\text{mxd}(\mathbf{r} + 2\mathbf{h})}{\pi \mathbf{r}^2 (\mathbf{T} \mathbf{l} - \mathbf{T} \mathbf{2}) (2\mathbf{r} + 2\mathbf{h})} dT / dt \,\mathcal{W} / m / \mathbf{K} \tag{5}$$

The various parameters involved are: m represents the mass of the Lee's disc, d refers to the sample thickness, x denotes the specific heat, r represents the radius of the Lee's disc, h signifies the thickness of the Lee's disc, and *dT/dt* represents the tangential slope. Additionally, T1 represents the steady temperature of the vapor chamber, and T2 represents the steady temperature of the Lee's disc.

#### *3.5. CHNS Analyzer*

CHNS elemental analysis offers a quick method to determine the levels of carbon, hydrogen, nitrogen, and sulfur in organic samples and various other materials, including volatile or viscous samples. The analysis was performed using the model Elementar Vario EL III, Micro Cube manufactured by Elementar, Langenselbold, Germany with a precision > 0.1% absorbance.

#### *3.6. Single Fiber Tensile Testing*

The tensile strength of BP fibers were measured using single fiber strength and elongation (Zwick/Roell) from the Physical Testing Laboratory, SITRA, Coimbatore. All analyses were conducted at a controlled temperature of approximately 21 ◦C with a tolerance of ±1 ◦C, along with a relative humidity of 65%. The gauge length was set at 50 mm, and the transverse rate was maintained at 30 mm/min. The tensile strength of BP fibers was determined using [39]

$$\text{Tensile strength}(\sigma) = \frac{\text{Tensile force (F)}}{\text{cross sectional area of fibers (A)}} \tag{6}$$

The microfibril angles of BP fibers are calculated using the global deformation equation [40].

$$
\varepsilon = \ln\left(1 + \frac{\Delta \mathcal{L}}{\mathcal{L}}\right) = -\ln\left(\cos\alpha\right) \tag{7}
$$

where ε—strain developed, α—microfibril angle (MFA), L—fiber length, and ΔL—elongation at the time of breaking.

#### *3.7. FTIR Analysis*

The FTIR spectrometer (Model FTIR-8400S spectrum, SHIMADZU, Kyoto, Japan) was employed to identify the functional groups present in both untreated and alkali–treated fibers. The analysis was conducted using a KBr matrix with a scan rate of 45 scans per minute and a resolution of 4 cm<sup>−</sup>1, within a wavenumber range of 400 cm−<sup>1</sup> to 4000 cm<sup>−</sup>1.

#### **4. Results and Discussion**

#### *4.1. Determination of Chemical Composition*

The presence of cellulose, lignin, hemicellulose, and wax content in the fiber sample was determined through chemical analysis. Extraction methods, maturity of plant parts, and the habitat of plants would have a direct outcome on the cellular compositions [41]. Standardized methods were followed to find the cellular composition. Percentage of cellulose and hemicellulose was found from the acid and neutral detergent method. Lignin content was found using the Klason method, and moisture quantity was measured by drying the sample. The wax percentage was determined using the Soxhlet extraction method, where the chosen solvent's vapor dissolves wax from the fiber samples. The variance between the extracted mass and the dried mass calculates the wax% present in the samples.

The cellulose content of 0.1 M KOH–treated BP was 60.72%, which is higher than the raw fiber (58.5%) and is thought to withstand hydrostatic pressure gradients of the fibers. After alkali treatment, fibers showed a visible improvement to serve as reinforcement material [42]. The cellulose values are in agreement with Kenaf (53.14%) [43] and Okra fibers (60–70%) [44]. Hemicellulose in alkalized BP deeply declined to 19.2% from 40.13%. There are almost no comprehensive treatment methods to extract hemicellulose completely without dissolving the cell components [45]. Complete removal of hemicelluloses could potentially lead to a reduction in composite strength while enhancing its stiffness [46]. Molecular weights of hemicellulose are lower than cellulose, and also, the alkali treatment on BP has eliminated a high degree of hemicelluloses, and because of that, physical properties such as density, aspect ratio, and linear density show an increase [47].

Furthermore, the complete removal of hemicellulose or lignin through alkalization may not be foolproof due to the presence of hydrogen bonding between residual hemicellulose and cellulose fibrils [48,49]. Lignin contributes to the structural integrity of fibers. A higher lignin percentage (18.09%) of BP can possibly favor excellent rigidity compared to other fibers. The physical properties of BP fibers were not negatively influenced by lignin. However, the presence of lignin impacted the thermal stability of BP fibers by stretching its degradation temperature [50]. The cellulose/lignin ratio in BP fibers was almost around 3:1. It is necessary to obtain a high cellulose/lignin ratio in samples to receive better crystalline, structural, and physical properties while introducing these fibers for composite making [51]. Modifying the cellulose/lignin ratio through diverse oxidative treatments is essential for these fibers, as this approach could yield improved fiber properties, namely (higher thermal stability, high mechanical strength), beyond those observed in the current study.

The amount of wax housed in the BP fibers (0.31%) was minimized to 0.25% using KOH action, and hence, initial flushing of samples prior to alkali treatment was considered optional. Dewaxing occurred during the alkali action had introduced a rough surface, which is shown in the SEM images. Moreover, the elimination of wax and other contaminants contributed to the enhancement of the tensile properties of the BP fibers [52]. A comparison of the chemical composition between BP fibers and other natural fibers is presented in Table 2 [40].


**Table 2.** Comparison of chemical characteristics of raw and alkalized BP with other fibers.

#### *4.2. X-ray Diffraction (XRD) Analysis*

The XRD analysis revealed the crystalline nature of the BP fibers in Figure 2. The lattice planes at (110) and (200) belong to the crystallographic plane group of celluloses [44]. It turns out that the crystallinity index (CI) of 0.1 M KOH–treated BP (86%) was more than the untreated BP (83%). SEM images also display an ordered arrangement of cellular components in the alkalized fiber. High CI indicates a better orientation of cellulose around the fiber axis, which attributes to the higher strength of fibers [53]. Additionally, the thermal degradation of fibers is also toggled to higher temperatures with the rise in CI. The CI for BP fiber is greater than other fibers and is tabulated in Table 3. Under certain conditions, it is possible for the crystalline regions to undergo rearrangement, leading to an increased level of crystallinity in the fiber [54]. Meanwhile, the crystallite size of the alkalized BP has risen from 7.5 nm to 8.04 nm. The CS of Butea fibers is smaller than the Sida cordifolia stem (18 nm). The increment of CS in the treated BP is suspected owing to the varying strain caused by the intrusion of K+ ions on the cellular arrangement during treatment [39].

**Figure 2.** X-ray diffractogram of raw and 0.1 M KOH–treated Butea parviflora (BP).

**Table 3.** Comparison of crystallinity index of raw and alkalized BP with other fibers.


#### *4.3. CHNS Analysis*

The presence of elements like carbon, nitrogen, hydrogen, and sulfur in Butea fibers can be detected using the CHNS analyzer. The analysis employs finely chopped raw and alkali-treated fibers. Samples with a high carbon content are regarded advantageous when used as fillers in strengthening composites [12]. The low heat conductivity values obtained from Lee's disc setup of alkalized BP can be accredited due to its high carbon content. Table 4 shows the weight percent of carbon, hydrogen, and nitrogen.

**Table 4.** CHNS analysis of BP fiber.


ND—not detected.

#### *4.4. FESEM Analysis*

The surface characteristics of both untreated and 0.1 M alkali-treated BP fibers are depicted in Figure 3a–f. SEM analysis is highly used to question the failure approach at the micro level [57]. The presence of small peaks against the long stripes is seen in the raw fiber. Epidermal projections appear on the longitudinal surface. The clouded irregularities in Figure 3a could be part of non-cellulosic debris [58]. This imperfection is removed in the alkalized fibers. It is assumed that the KOH treatment has washed away most of the oil and waxy impurities tied up with the microfibrils, generating a rough interface on the top of the fibers. The elimination of non-cellulosic structures, mainly wax and hemicellulose, could have created fine grooves along the axis. This might greatly improve the expansive adhesion with the matrix interface [41]. The axial arrangement of treated fibrils is more coordinated than the raw fiber.

**Figure 3.** (**a**–**c**) SEM photographs of raw BP, (**d**–**f**) SEM photographs of 0.1 M KOH treated BP.

#### *4.5. Thermogravimetric Analysis*

The thermal nature of BP was monitored between 40 and 800 ◦C at a heating rate of 20 ◦C/min. The Tg-dta and DSC curves are provided in Figure 4a,b. Three-step thermal degradation was observed in both fibers. The initial stage of mass loss is anticipated due to the evaporation of moisture present in the fiber [59,60]. The degradation pattern observed in the dtg graph of both fibers between 200 and 260 ◦C is because of the elimination of hemicellulose. The quick dismissal of cellulose occurs around 240–350 ◦C leaving anhydro cellulose and levoglucosan [61]. A mass loss of 50 and 45.06% was registered for the raw and treated BP in the second stage, which concerns the exclusion of hemicellulose, lignin, and a tiny fraction of celluloses. The swift reaction is cascaded to the next step with the huge dismissal of hemicellulose. Lignin degradation is registered between the range 280 and 500 ◦C [62]. Patterns of mass loss noted around specific temperatures are shown in Table 5.

**Figure 4.** (**a**). Thermogravimetry plot of untreated and alkalized Butea fiber; (**b**). differential thermogravimetry plot of untreated and alkalized BP; (**c**). differential scanning calorimetry curve of raw and alkalized BP.


**Table 5.** Mass loss with temperature from TG.

DTG shows that the maximum degradation peak for the alkali-treated fibers has been backtracked to 324 ◦C compared to that of the raw fiber, which was marked at 365 ◦C. Alkali action might have dismissed lignin, and hence, the treated fibers have noticed an early decomposition. Minor peaks were noted for the raw and alkalized BP between 400 and 500 ◦C. Removal of lignin could have occurred within this limit. Weight loss of fibers was stabilized around 500 ◦C leaving the residues [63]. Other cellulosic fibers like Eucalyptus grandis and Pinus taeda spotted their maximum decomposition temperatures at 353 ◦C and 360 ◦C [61].

#### *4.6. Differential Scanning Calorimetry*

The DSC curves are plotted in Figure 4c. As the temperature increases, notable peaks appear, signaling various thermal events or transitions taking place within the fiber. A prominent endothermic peak was obtained for the KOH-treated fibers at 486 ◦C. It indicates the pyrolysis and exclusion of lignified compounds, leaving behind char. For the untreated profile, a peak was spotted at 503 ◦C, owing to the loss of diversified functional groups of lignin. This peak value clearly correlates with the elevated decomposition temperature indicated in the DTG curve. A minor peak was spotted at 360 and 330 ◦C in the raw and treated BP, marking the removal of cellulose and hemicelluloses. A small hump seen initially around 100 ◦C in both fibers is because of moisture removal [64]. All the outcomes show that BP fiber can be signed in for making fiber reinforcement composites as long as its thermal stand-by temperature does not exceed 240 ◦C.

#### *4.7. Activation Energy of Fibers*

The kinetic activation energy (Ea) of BP was determined using the Coast–Redfern method [65].

$$\log\left[\frac{-\log(1-\alpha)}{\text{T}^2}\right] = \log\frac{\text{AR}}{\text{\(\beta\)}\text{Ea}}\left[1-\frac{2\text{RT}}{\text{Ea}}\right] = \frac{\text{Ea}}{\text{2.303RT}}\tag{8}$$

Ea was estimated through linear interpolation of data points between log[−log(1 − <sup>α</sup>)/T2] and 1000/T. The plot is shown in Figure 5. It speaks more about the aptness of the fibers to be used in composite making. Ea of cellulose fibers show different patterns due to variations in the fiber contents and structure [66].

The activation energy calculated for the raw BP (Ea = 73.15 kJ/mol) was higher than for alkalized fiber (Ea = 55.95 kJ/mol). The activation energy has its impact more on the untreated fiber rather than the alkalized BP. The thermal stability of green fibers is primarily determined by their decomposition temperature. The kinetic activation energy (Ea) values of other fibers are: Prosopis juliflora (76.72 kJ/mol), C. quadrangularis (74.18 kJ/mol), and Coccinia grandis (82.3 kJ/mol) [33,67]. The thermal outcomes of Butea fibers are shown in Table 6.

**Table 6.** Thermal outcomes of Butea parviflora fibers.


#### *4.8. Thermal Conductivity*

Natural fiber-based materials are highly influential because of their potential insulation behavior. The thermal conductivity (K) of untreated and alkalized BP fiber, found using Lee's disc method, was K = 0.029 Wm<sup>−</sup>1k−<sup>1</sup> and K = 0.020 Wm<sup>−</sup>1k−1. Thermal conductivity plots of BP fibers are shown in Figure 6. K values of BP fibers are much lower than woodbased thermal insulation foam (k = 0.038 Wm−1k−1) [68]. The activity was performed at two Lee's disc setups at room temperature, with the fibers woven tightly without void spaces. The steady temperature of the untreated and 0.1 M KOH-treated fibers are at 73.5 ◦C and 67.4 ◦C. Based on the observations, it can be deduced that as the material

thickness decreases, its conductivity also reduces, resulting in improved thermal insulation properties [69].

**Figure 6.** A linear plot of heat transport of raw and KOH-treated BP fibers.

The K value of the treated BP fibers is comparatively lower than other plant fibers, like corn stalks (K = 0.121 Wm<sup>−</sup>1k−1) and Areca husk fiber (K = 0.021 Wm−1k−1) [70]. The reduced K value of the alkalized BP accounts for the amorphous content dwelling in the fiber. Lowered heat conducting behavior of BP fibers may lay a path to act as a better thermal insulator, or it can appease the synthetic thermal insulators the least.

#### *4.9. Single Fiber Tensile Test*

Tensile properties of fiber predominantly gear on a number of things, like the maturity of plant parts, habitat, fibers chosen for testing, and so on. The presence of cellulose is a crucial factor influencing the mechanical behavior of fiber composites, as it exhibits a diverse range of polymeric actions [71,72]. The tensile strength of the alkalized fiber increased by 192.97 MPa compared to the raw fiber's value of 92.64 MPa. Additionally, the treated fiber exhibits a high tensile modulus of 3.462 GPa, whereas the raw fiber had 2.164 GPa. The removal of amorphous components resulted in a more organized alignment of microfibrils along the fiber axis, thereby significantly enhancing the strength of the fibers.

Higher MFA (α) might result in poor fiber orientation. The tensile values are on the rise when the MFA is low and vice versa [40]. The elongation at break and strain experienced by the fibers play a crucial role in enhancing the MFA (microfibril angle). Higher MFA introduces higher ductility of fibers, which is also dependent on the orientation of microfibrils. Meanwhile, the MFA (α) of treated fiber (19.67 ± 10.49◦) is lower than the raw fiber (21.11 ± 14.08◦). The range of MFA values of BP appease with the other fibers and can be introduced for composite reinforcements. A semiempirical relation shown in Equation 9 was formulated by Satyanarayanan et al. It relates to MFA and fiber elongation, and the relation agrees with the BP fibers as well [73].

$$
\varepsilon = 2.78 + 7.28 \times 10^{-2} \theta + 7.7 \times 10^{-3} \theta^2 \tag{9}
$$

where ε is the % elongation, and *θ* is the MFA with the cellulose content. These tensile values of Butea fibers were compared with various fibers in Table 7.


**Table 7.** Tensile properties of BP and other natural fibers.

#### *4.10. FTIR Analysis*

Spectroscopic investigation on fibers gives a detailed account of the structure and presence of constituents binding with the fiber arrangements like cellulose, hemicellulose, pectin, lignin, and others [78]. The FTIR absorption peak of the raw and treated fibers are provided in Figure 7, and the spectroscopic assignments are listed in Table 8. The presence of a prominent band in the range of 3600–3000 cm−<sup>1</sup> can be attributed to the stretching of hydrogen-bonded O–H groups in cellulose and/or hemicellulose [79]. A strong peak at 2922 and 2916 cm−<sup>1</sup> of fibers is the outcome of the C–H stretching vibration of cellulose [80,81]. Due to the free vibration of the carboxyl group, a peak is visible in both fibers at 1640 cm−<sup>1</sup> [44].

**Figure 7.** FTIR image of raw and 0.1 M KOH-treated BP.

**Table 8.** Spectroscopic vibrations in BP fibers.


Asymmetric stretching of C–O–C in lignin caused a vibration in the raw fiber at 1383 cm−1, whereas the vibration was removed in the treated fibers. Alkaline reagents facilitate the breakdown of lignin into smaller, low-molecular-weight compounds [61,82]. An observable peak split was noticed around 1064 cm−<sup>1</sup> in the raw BP due to O–H vibrations [83,84]. A glitch noted at 847 cm−<sup>1</sup> in the raw BP has been unseen in the treated BP. Slight differences in the vibrations of functional groups were observed between the raw and alkalized BP. These variances can be attributed to the removal of specific chemical groups during the alkalization process.

#### **5. Conclusions**

The aptness of raw and 0.1 M KOH-treated Butea parviflora (BP) fiber to be consumed for green composites was examined, and the following observations were drawn. The density and fineness of the alkalized fiber have risen to (1.34 g/cc) and 346 tex, then the raw fiber, which is (1.23 g/cc) and 312 tex. The chemical composition of fibers clearly witnessed the changes in the levels of cellulose and hemicellulose between the raw and alkalized fibers cellulose hiked to 60.72% while hemicellulose dropped from 40 to 19% in the alkalized BP. The elimination of wax and pectin has a significant impact on the semicrystalline fiber, resulting in enhanced crystallinity. XRD analysis revealed a substantial increase in cellulose content (up to 86.03%) and an enlargement in crystallite size (8.04 nm) after the treatment. FTIR assignments marked slender vibrational changes in the raw and KOH-treated fiber.

The SEM images neatly distinguish the presence and absence of components on the fiber surface, aiding in the analysis of their effective bonding with the matrix phase. Due to their low thermal conductivity (K = 0.020 W/mK), BP fibers are suitable to act as thermal insulators in structural applications. Choosing natural fibers for thermal insulation would significantly reduce carbon footprints compared to synthetic insulators.

Complete analysis of the Tg-dta and DSC studies provided insights into the mass loss of cellulosic and amorphous components at specific temperatures. From the DTG curves, degradation peaks for Butea fibers were observed. The maximum temperature up to which the fibers can stay active was noted to be around 240 ◦C. The activation energy of the raw fiber (Ea = 73.15 kJ/mol) was higher than that of the treated fiber, indicating that the thermal potentials of the raw fibers are better than the treated BP fibers.

Increments in the crystallinity values and cellulose content directly influence the tensile behavior, showing an abrupt rise in the tensile values of raw BP from 92.64 to 192.97 MPa for the alkalized BP. Only the thermal behavior of raw fibers showed a trifling swiftness than the KOH-treated BP. However, all the properties of treated fibers, except the thermal outcome, surpass those of the untreated fiber.

Summing up the text, the present work highlights the enormity of Butea parviflora fiber through various studies and analyses. The low density, high crystallinity, and thermal stability of BP fibers differentiate its novelty from other green fibers available in the market. It can stand as a suitable contender in the global market of composites in minimizing carbon emissions and safeguarding green territory. The findings provide a positive way to introduce fiber as a reinforcement material in composite making. The impeccable assets of the plant fiber can be further harvested by subjecting them to various treatments along with assessing their properties.

**Author Contributions:** A.M.—Conceptualization, Data curation, Formal analysis, Methodology, Validation, Visualization, Writing—original draft, Writing—review and editing. R.K.P.—Conceptualization, Data curation, Formal analysis, Methodology, Supervision, Validation, Visualization, Writing—original draft, Writing—review and editing. K.P.A.—Conceptualization, Data curation, Formal analysis, Methodology, Supervision, Validation, Visualization, Writing—review and editing. S.A.—Data curation, Formal analysis, Methodology, Supervision, Validation, Visualization, Writing—review and editing. N.M.-C.—Data curation, Funding acquisition, Project administration, Validation, Writing—review and editing. A.R.-V.—Data curation, Project administration, Validation, Project administration, Visualization. All authors have read and agreed to the published version of the manuscript.

**Funding:** The author thanks Vicerrectoria de Investigacion y Desarrollo (VRID) y Direccion de Investigacion y Creacion Artistica DICA, Proyecto presentado al Concurso VRID-Iniciación 2022, VRID N◦2022000449-INI, Universidad de Concepción, Concepción, Chile. Centro Nacional de Excelencia para la Industria de la Madera (ANID BASAL FB210015 CENAMAD), Pontificia Universidad Católica de Chile, Vicuña Mackenna 7860, Santiago, Chile, and Dirección de Investigación de la Universidad Católica de la Santísima Concepción, Concepción, Chile.

**Institutional Review Board Statement:** Not Applicable.

**Data Availability Statement:** Will be provided on request.

**Acknowledgments:** The authors gratefully appreciate the support provided by the Research scholar, M. Abisha (Reg. No. 20213042132004) PG & Research Department of Physics, Holy Cross College (Autonomous) Nagercoil, Affiliated to Manonmaniam Sundaranar University, Tirunelveli, 627012, Tamil Nadu, India.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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## *Article* **Marginal Micro-Seal and Tensile Bond Strength of a Biopolymer Hybrid Layer Coupled with Dental Prosthesis Using a Primerless-Wet System**

**Morakot Piemjai 1,\*, Onusa Waleepitackdej <sup>1</sup> and Franklin Garcia-Godoy 2,3**


**Abstract:** The aim of this study is to compare the marginal seal and tensile bond strength (TBS) of prostheses fixed to enamel-dentin using different adhesive systems. Resin-composite inlays directly fabricated from Class V cavities of extracted human molars/premolars and mini-dumbbellshaped specimens of bonded enamel-dentin were prepared for microleakage and tensile tests, respectively. Four adhesive systems were used: primerless-wet (1-1 etching for 10-, 30-, or 60-s, and 4-META/MMA-TBB), primer-moist (All-Bond2 + Duolink or Single-Bond2 + RelyX ARC), self-etch (AQ-Bond + Metafil FLO), and dry (Super-Bond C&B) bonding. Dye penetration distance and TBS data were recorded. Failure modes and characteristics of the tooth-resin interface were examined on the fractured specimens. All specimens in 10-, 30-, and 60-s etching primerless-wet, Super-Bond, and AQ-Bond had a microleakage-free tooth-resin interface. Primer-moist groups showed microleakage at the cementum/dentin-resin margin/interface. Significantly higher TBSs (*p* < 0.05) were recorded in primer-less-wet and Super-Bond groups with the consistent hybridized biopolymer layer after the chemical challenge and mixed failure in tooth structure, luting-resin, and at the PMMA-rod interface. There was no correlation between microleakage and TBS data (*p* = −0.148). A 1–3 μm hybrid layer created in the 10–60 s primerless-wet technique, producing complete micro-seal and higher tensile strength than enamel and cured 4-META/MMA-TBB, may enhance clinical performances like Super-Bond C&B, the sustainable luting resin.

**Keywords:** primerless-wet bonding; resin adhesive system; hybrid layer; tensile bond strength; micro-seal; luting resin; dental prosthesis; fixed prosthodontics

#### **1. Introduction**

Dental enamel naturally protects the dentin and pulp from invasion by external stimuli. Therefore, non- or minimally invasive restorations or prostheses that protect the enamel from tooth reduction, recurrent caries, or tooth fracture are crucial in maintaining healthy dentin and pulp. High tensile bond strength adhesives are required when restorations or prostheses are not sufficiently resistant to displacement under functional loading [1]. Severe tooth reduction to gain more retention, resistance form, or strength for restorations/prostheses removes dentin, especially when restoring with non-hybrid layer formation materials, such as amalgam restorations and dental prostheses fixed with acid-base cement.

The total-etch concept was developed to simplify bonding to both enamel and dentin by etching the entire cavity with 40% phosphoric acid gel [2]. Strong phosphoric acid demineralizes enamel deeper than mild acid [3]. Thus, demineralized enamel might remain after resin polymerization allowing oral acid penetration. However, monomer diffusion

**Citation:** Piemjai, M.; Waleepitackdej, O.; Garcia-Godoy, F. Marginal Micro-Seal and Tensile Bond Strength of a Biopolymer Hybrid Layer Coupled with Dental Prosthesis Using a Primerless-Wet System. *Polymers* **2023**, *15*, 283. https:// doi.org/10.3390/polym15020283

#### Academic Editor: Raffaella Striani

Received: 18 November 2022 Revised: 23 December 2022 Accepted: 25 December 2022 Published: 5 January 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

into etched enamel is more accessible than demineralized dentin, as phosphoric acidconditioned dentin collapses when air-dried [4]. Therefore, phosphoric acid demineralized dentin cannot provide adequate permeability for complete monomer impregnation in either dry or moist systems [4,5]. In addition, it leads to a leakage pathway [6,7], post-operative hypersensitivity, and secondary caries [8,9].

Ferric ions in an acid conditioner can aggregate glycosaminoglycan (GAG) in demineralized dentin and provide permeability for potential monomers to diffuse through completely in dry or wet conditions [6–11]. Therefore, a hybridized dentin with a leakagefree interface was formed [6–9]. Self-etch bonding systems and self-adhesive cement were introduced to simplify the bonding steps and minimize aggressive phosphoric acid etching on dentin. However, bonded restorations using these self-etching or self-adhesive systems could not reliably provide a leakage-free dentin-resin interface [12–15] because of the limitation of monomer diffusion through any smear layer into the intact dentin [14,16]. Ferric chloride (1%) in 1% citric acid aqueous conditioner (1-1), a mild acid for smear layer removal, and 4-methacryloyloxyethyl trimellitate anhydride in methyl methacrylate initiated by tri-n-butyl borane resin (4-META/MMA-TBB) can provide reliable hybridized dentin when wet bonding with primer in the long-range periods (10–60 s) of conditioning [10]. A 1–3 μm hybridized dentin layer suggested that 1-1 conditioned dentin was sufficiently permeable for water to evaporate and for monomers to impregnate. Thus, only blot-drying with or without primer (primerless-wet bonding) can produce a complete hybrid layer that reinforces the dentin [10,17] and prevents dye penetration of direct restorations [7,17].

Dental clinical failures are often found in direct or indirect restorations and fixed partial prostheses due to secondary caries, especially at the cementum/dentin margin [18–21]. Detachment of restorations or prostheses is a minor complication that leads to failure [21–24]. The demineralized dentin, the defect, remains in restored-dentin, which may lead to a leakage pathway and recurrent caries [6,8,9], strongly influencing the failure of restorations or prostheses [18–21]. The hybrid layer formed by dry bonding using 10% citric acid and 3% ferric chloride aqueous solution (10-3) conditioned for 10 s and 4-META/MMA-TBB resin (Super-Bond C&B, Sun Medical, Shiga, Japan; a sustainable luting resin since 1983), provides a significantly higher 15-year survival with less secondary caries and prosthesis detachment complication rates of full coverage retainers than those of acid-base cement [21]. However more extended 10-3 etching period of 30–60 s creates demineralized dentin too deep (>4 μm) to be fully impregnated by the monomers before starting polymerization. Thus, exposed demineralized dentin remains to allow leakage, caries, or pulp infection [8,21]. A tensile test using a mini-dumbbell-shaped bonded specimen [2,25] and a microleakage test [6,8] can detect this remaining demineralized dentin, the weakening part of the restored dentin.

We hypothesized that primerless-wet bonding could create a reliable hybrid layer on enamel-dentin and provide a complete micro-seal and tensile bond strength comparable with Super-Bond C&B. Moreover, a complete seal might not relate to the tensile bond strength of tooth-resin interface luting with various resin adhesives.

The objective of this study was to compare the dye penetration distance and the tensile bond strength at the tooth-resin interface of a prosthesis fixed to enamel-dentin using different adhesive systems: dry (Super-Bond C&B), moist with primer (All-Bond2 + Duoink or Single-Bond2 + RelyX ARC), self-etch (AQ-Bond + Metafil FLO), and primerless-wet (1-1 conditioner and 4-META/MMA-TBB resin) bonding.

#### **2. Materials and Methods**

Previously frozen extracted human molars and premolars without caries, restorations, or cracked lines were collected and stored in water at −20 ◦C for 2–3 months. Then, all teeth were randomly divided into two experimental groups of 7 premolars and 14 molars for micro-seal evaluation and 42 molars to prepare the mini-dumbbell-shaped specimens for tensile testing. The primary experimental steps are illustrated in Figure 1.

**Figure 1.** An illustrated diagram for the steps carried out in this experiment.

#### *2.1. Micro-Seal Evaluation Using Dye Penetration*

Class V cavities at the cementoenamel junction (CEJ) on the buccal and lingual surfaces of seven premolars and all axial surfaces for fourteen molars were outlined. A box cavity of 2 × 3 mm and 1.5 mm depth with approximately 5◦ divergent axial walls was prepared with occlusal and gingival margins on enamel and cementum, respectively, using a diamond bur with an air-water sprayed high-speed handpiece. Resin composite inlays of 2 × 3 × 1.5 mm were directly fabricated from the cavities with light-cured resin composite (Metafil CX, Sun Medical, Shiga, Japan). Each inlay was light-cured for 60 s on both outer and inner surfaces. All cavities were randomly divided into 7 groups of 10 specimens (1 premolar and 2 molars) for different tooth conditionings and/or resin cement. Primerless-wet bonding using 1-1 conditioning for 10 s, 30 s, 60 s (Groups 1-1-10s, 1-1-30s, 1-1-60s respectively) and 4-META/MMA-TBB resin; and commercially available adhesive resin cement: Super-Bond C&B (Sun Medical, Shiga, Japan), All-Bond2 + Duolink (Bisco, Schaumburg, IL, USA), Single-Bond2 + RelyX ARC (3M ESPE, Saint Paul, MN, USA), or AQ-Bond Plus + Metafil FLO (Sun Medical, Shiga, Japan) was used to fix an inlay prosthesis into the cavity. The manipulation of commercial systems followed manufacturers' recommendations, as shown in Table 1, and the main chemical composition of luting adhesives and resin composite inlay, as shown in Table 2. Fine diamond burs in a high-speed handpiece were used to finish the restored margins after the polymerization of adhesives. After storing in water at 37 ◦C for 24 h, all tooth surfaces except an area of the inlay and 1 mm away from the occlusal (enamel) and gingival (cementum) margins were coated with two layers of nail varnish (Pias, Bangkok, Thailand). Specimens were then immersed in 0.5% basic fuchsin dye for 24 h. After soaking, all specimens were cleaned with tap water before being vertically sectioned at the center of each restoration using a diamond disc with a slowspeed handpiece. The distance of dye penetration was measured under a stereomicroscope (ECLIPSE E400 POL, Nikon, Japan) at ×50–×200 magnifications.


#### **Table 1.** Manipulation of tooth-conditioning, luting adhesive, and prosthesis cementation.

**Table 2.** The main chemical composition of luting adhesives and resin composite inlay.


#### *2.2. Tensile Bond Strength Test*

Forty-two extracted sound human molars without cracks were root-embedded in acrylic blocks (Formatray, Kerr, Orange, CA, USA). A 2 mm occlusal portion was horizontally removed using a sectioning machine (Isomet 1000 series 15, Buehler, Lake Bluff, IL, USA) to expose a surface which was then ground with a wheel diamond bur (111 Intensiv, Grancia, Switzerland). A prepared surface of 2 mm in width (0.5 mm of enamel/DEJ and 1.5 mm of dentin) and 4 mm in length was outlined with double-sided tape. One of the tooth conditionings and adhesive systems, as previously mentioned in the microleakage test (Table 1), was randomly selected to bond that area with a square PMMA rod (7 × 7 × 4 mm) to form a handle for tensile testing. A 2.0-mm thick vertical section

was prepared using the sectioning machine. A mini-dumbbell bonded specimen with a cross-section of 3.0 × 2.0 mm was shaped using a diamond fissure bur (B11, GC Dental Industrial Co., Tokyo, Japan) operated in a high-speed handpiece under the air-water spray. All specimens were stored in 37 ◦C water for 24 h prior to tensile testing (n = 6) [2,10,16]. Each mini-dumbbell specimen was securely bonded to disposable PMMA jigs using 1-1-10s bonding on the tooth surface and self-cured acrylic (Unifast, Trad, GC Int. Co., Tokyo, Japan) on the PMMA surface to facilitate tensile testing [16]. An assembled specimen was aligned in a universal testing machine (Instron 8872, Norwood, MA, USA) and vertically loaded in tension at a crosshead speed of 1.0 mm/min. The force at failure was recorded in Newtons. The mode of failure, the cross-sectional area of the fractured surface, and the enamel and dentin area were examined under a stereomicroscope and SEM. Tensile bond strengths were calculated in MPa.

#### *2.3. Characteristics Evaluation of Tooth-Resin Interfacial Biopolymer Layer*

Fracture specimens from each bonding system were randomly selected and vertically sectioned (without epoxy embedding) into 1 mm thick pieces. The tooth-resin interface surface to be examined was finished with #600 and #1200 grit abrasive papers and finally polished with 0.05 μm alumina paste and then ultrasonically cleaned for 15 min. The chemical challenge, either soaking in 6 mol/L HCl for 30 s or soaking in 6 mol/L HCl for 30 s followed by 1% NaOCl for 60 min, was carried out to test the resistance of acidic and proteolytic degradation, akin to caries formation. For SEM examination, all polished and chemically soaked specimens were desiccated and gold-sputtered. The characteristics of the newly formed interfacial biopolymer layer between the tooth and cured resin were examined at ×35 to ×5000 magnifications.

#### *2.4. Statistical Analysis*

Normal distribution and homoscedasticity of dye penetration distance and tensile bond strength data were analyzed using one-sample Kolmogorov-Smirnov and Levene tests, respectively. In addition, Pearson correlation between leakage distance and tensile bond strength data was performed using SPSS for Windows version 22 (IBM Corporation, Somers, NY, USA). The significant difference was set at α = 0.05.

#### **3. Results**

Means and standard deviations (SD) of dye penetration distance, tensile bond strength, and mode of failure for all groups are summarized in Table 3. No dye penetration at the cementum/dentin-resin interface was found in the primerless-wet groups (1-1-10s, 1-1-30s, 1-1-60 s) (Figure 2), Super-Bond C&B (Figure 3a), and AQ-Bond (Figure 3b) specimens and at the enamel-resin interface in all groups. No statistically significantly different dye penetration distance at the dentin-resin interfaces was found between All-Bond2 and Single-Bond2 when analyzed using a t-test. All specimens in these moist bonding with primer groups leaked at the dentin-resin interface (Figure 4).


**Table 3.** Mean ± SD of dye penetration distance at tooth-resin interface (n = 10), tensile bond strength, and failure modes of enamel/DEJ/dentin-resin dumbbell-shaped specimens (n = 6) for all groups.

0 = No dye penetration. E/DEJ/D = cohesive failure in enamel, DEJ or dentin, R = cohesive failure in luting resin, R/PMMA = failure at the resin-PMMA-rod interface, DD = failure at demineralized dentin-resin interface, HsE = failure in hybridized suspended enamel smears. There was no significant difference between groups connected with a vertical line (*p* > 0.05).

(**a**) (**b**) (**c**)

**Figure 2.** No dye penetration at the cementum/dentin-resin interface (arrowed) of primerless-wet bonding groups: (**a**) 1-1-10s, (**b**) 1-1-30s, (**c**) 1-1-60s (original ×200, D = dentin, R = resin-composite in-lay).

**Figure 3.** No dye penetration at the cementum/dentin-resin interface (arrowed) of Super-Bond (**a**) and AQ-Bond (**b**) specimens (original ×200, D = dentin,R=resin-composite inlay).

**Figure 4.** Dye penetration at the cementum/dentin-resin interface (arrowed) of moist bonding with primer groups: (**a**) All-Bond2, (**b**) Single-Bond2 (original ×200, D = dentin, R = resin-composite inlay).

As a significant difference was found in the test of homogeneity of variances, Brown-Forsythe and Tamhane multiple comparisons were used to reveal a significant difference in tensile bond strength between groups. No significant difference in tensile bond strength was found among 1-1-10s, 1-1-30s, 1-1-60s, Super-Bond, and Single-Bond2; Single-Bond2, All-Bond2, and AQ-Bond groups. Cohesive failure originated in enamel followed by either dentino-enamel junction (DEJ), dentin, cured luting resin or adhesive failure at resin-PMMA rod interfaces mainly occurred in fractured specimens of primerless-wet and Super-Bond groups (Figure 5). In contrast, failure occurring in demineralized dentin or at the resin-demineralized dentin interface was found in Single-Bond2 (Figure 6a), and All-Bond2 fractured specimens (Figure 6b). The lowest tensile bond strength was measured in AQ-Bond specimens, where the original failure was found at the suspended resin-smear layer of the enamel-resin interface (Figure 6c).

**Figure 5.** Stereo and SEM micrograph of the fractured surface showing cohesive failure originating in enamel followed by either DEJ, dentin, cured resin, or adhesive failure at resin-PMMA rod interfaces (R/PMMA) primarily found in primerless-wet and Super-Bond groups: sagittal view at ×50 magnification (**a**) and cross-sectional view of 1-1-60s (**b**) and Super-Bond (**c**) specimens (D = dentin, E = enamel, R = luting resin).

A consistent thickness of hybridized enamel or hybridized dentin after loading and the chemical challenge was found in primerless-wet (Figure 7) and dry bonding (Super-Bond C&B) (Figure 8) systems. A detached or degraded enamel- or dentin-resin interfacial layer was found in moist with primer (All-Bond2 and Single-Bond2) (Figure 9) and self-etch (AQ-Bond) (Figure 10) systems. The correlation between the dye penetration distance and the tensile bond strength data for the enamel and dentin-bonded interface was very weak (Pearson correlation = −0.148)

(**a**) (**b**) (**c**)

**Figure 6.** SEM micrograph of the fractured surface showing failure: in the remaining demineralized dentin of Single-Bond2 (**a**) and at the demineralized dentin-resin interface of All-Bond2 (**b**) moist bonding with primer specimens, and in the hybridized suspended smears at the enamel-resin interface of AQ-Bond (**c**) specimen (DD = demineralized dentin, HsE = hybridized suspended enamel smears, R = luting resin).

**Figure 7.** SEM micrographs of fractured specimens after chemical challenge demonstrating: the stable hybridized enamel (**a**) and hybridized dentin (**b**) of 1-1-30s primerless-wet specimens (H = hybrid layer,R=resin, ME = modified enamel, MD = modified dentin).

(**a**) (**b**)

**Figure 8.** SEM micrographs of fractured specimens after chemical challenge demonstrating: the stable hybridized enamel (**a**) and hybridized dentin (**b**) of Super-Bond C&B specimens (H = hybrid layer,R=resin, ME = modified enamel, MD = modified dentin).

**Figure 9.** SEM micrographs of fractured specimens after chemical challenge demonstrating: the degraded hybridized enamel (**a**) and the detached and degraded dentin-resin interface (black arrow) of All-Bond2 specimens (**b**) (H = hybrid layer, R = resin, ME = modified enamel, MD = modified dentin).

**Figure 10.** SEM micrograph of fractured specimens after chemical challenge demonstrating: the degraded hybridized enamel (white arrow) (**a**) and the hybridized dentin (**b**) of AQ- Bond specimens (H = hybrid layer,R=resin, ME = modified enamel, MD = modified dentin).

(**a**) (**b**)

#### **4. Discussion**

The complete marginal seal, no significant differences in TBSs, and the same failure mode among primerless-wet and Super-Bond groups suggest that the milder acid of 1-1 conditioner using primerless-wet bonding could adequately prepare the etched enamel-dentin for 4-META/MMA-TBB resin to entirely impregnate as well as that of the 10-3 conditioner in dry bonding (Figures 2, 3a and 5, Table 3). Furthermore, long etching periods of 10–60 s of 1-1 dissolved less content of calcium ions, therefore even blot-drying without primer could provide the permeability of acid-etched enamel-DEJ-dentin for 4-META/MMA-TBB to penetrate completely before being polymerized to form a 1–3 μm hybrid layer. Therefore, no adhesive failure at the tooth-resin interface was noticed with the average strength like dry bonding using 10-3 solution for 10 s etching of Super-Bond C&B.

The mode of failure originating on the enamel surface suggests that resin infiltration into acid-etched enamel-DEJ-dentin using primerless-wet bonding and dry bonding using Super-Bond C&B could provide a tensile bond strength higher than that of the tensile strength of enamel itself (Figure 5). The complete hybridization of resin into the total etched enamel-DEJ-dentin depends on the demineralized tooth substrate's permeability and the monomers' diffusion potential. Non-detachment with consistent thickness hybridized layers against loading force for failure and chemical challenge found in the primeless-wet and Super-Bond groups (Figures 7 and 8) suggest the high resin content encapsulates the tooth component in the hybrid layer. Therefore, the enamel- and dentin-resin hybrid layer, created using a primerless-wet bonding with 10 s to 60 s 1-1 conditioning, 4-META/MMA-TBB, and PMMA powder could be a sustainable biopolymer to provide a complete microseal and high tensile bond strength comparable with that of Super-Bond C&B. The longrange conditioning period of 1-1 for 10 s to 60 s ensures more safety manipulation in the clinical situation.

The adhesive failure at the demineralized dentin-resin interface or cohesive failure in the remaining demineralized dentin found in Single-Bond2 and All-Bond2 fractured specimens minimized the tensile bond strength and was probably the cause of the leakage (Figures 4 and 6a,b). This demineralized dentin is the leakage pathway for dye or lactic acid to penetrate [6,8,9]. After tensile loading and chemical challenge, the in-consistent enamel-resin interface and the detached and degraded dentin-resin interface confirmed monomers' incomplete impregnation into the demineralized tooth substrate (Figure 8). These results suggest that moist bonding using 32% or 35% phosphoric acid for a 15 s etching period, kept moist and either primed and bonded using one or separate steps cannot provide a complete marginal seal of cementum/dentin and a stable hybrid layer.

Although achieving a complete seal for the enamel and cementum/dentin margin/interface (Figure 3b), AQ-Bond specimens had a significantly lower tensile strength than the primerless-wet and Super-Bond groups. The fracture mode originated in the hybridized suspended smear layer of the enamel-resin interface (Figure 6c); the thin hybridized enamel with degradation and the detached hybridized dentin after chemical immersion (Figure 10) suggest the remaining smear and the low resin content of the hybrid layer. These results imply that scrubbing this self-etch monomer for 20 s could not sufficiently remove all the smear layer to provide high adhesion to enamel and dentin. Therefore, careful removal of more smear layers by aggressively air-blowing off or an additional scrubbing application [9] is recommended for cavities with no retentive form and require higher retention, such as a large wedge shape abrasion lesion.

As the primer and bonding agents of all groups contain the methacrylate monomers with hydrophobic and hydrophilic groups, the significantly different factor is the conditioned tooth surface of each system. This study's results suggest that the permeability of conditioned tissue of the adhesive system that provides the durable biopolymer hybridized dentin influences the complete micro-seal and higher tensile bond strength. Moreover, the complete micro-seal or dye penetration distance was unrelated to the TBS data. Therefore, luting resin or resin adhesives that provide a complete marginal seal should be primarily considered to protect dentin and pulp for long-term function. In other words, a complete seal margin with an impermeable hybrid layer is more reliable than a high tensile bond strength adhesive with the leaked margin in preventing recurrent or secondary caries [8,9,21], the most common dental restoration failure, ensuring the lifelong survival of restored vital teeth. In clinical cases where high retention and completely sealed dentin is required, i.e., a short clinical crown or severe tooth wear and partial coverage retainers, a complete hybrid layer with high tensile strength and micro-seal margins can extend the long-term survival of vital teeth with less invasive treatment or without intentional pulp removal [21,26,27]. The results of this study support the hypothesis.

The novelty of this study is that a primerless-wet system using mild acid (1-1) conditioning for 10–60 s and blot-drying to remove all smears and water is less aggressive and safer than a dry bonding system using a 10-3 conditioner. Furthermore, its total etching creates durable hybridized enamel and dentin, providing the micro-seal and tensile bond strength (TBS) better than a primer-moist system. In addition, its TBS is higher than the self-etch system. However, an in-vivo study should be carried out to evaluate the effect of dentinal fluid in a vital tooth before introducing this system into the market. In the future, dentists can use this adhesive system as long-term dentin protection to treat patients at home.

#### **5. Conclusions**

Primerless-wet bonding using 1-1 conditioning for 10 s to 60 s and 4-META/MMA-TBB luting resin provided a reliable hybrid layer, a biopolymer, with a marginal micro-seal and tensile strength of the bonded enamel/DEJ/dentin similar to that of a dry system using Super-Bond C&B and higher than that of enamel itself. It can be a sustainable luting resin or adhesive agent with a sustainable hybrid layer. A basic fuchsin dye penetration was found when demineralized cementum/dentin was left underneath to provide a leakage pathway. To successfully prevent biological failure, a luting resin providing a complete marginal seal is preferable to the one with a leaked margin, even with high bond strength, as there is no correlation between complete marginal micro-seal and TBS data.

**Author Contributions:** Conceptualization, M.P.; data curation, M.P. and O.W.; formal analysis, M.P. and O.W.; investigation, M.P. and O.W.; methodology, M.P. and O.W.; project administration, M.P.; resources, M.P. and O.W.; supervision, M.P.; validation, M.P. and O.W.; visualization, M.P. and O.W.; writing—original draft, M.P.; writing—review and editing, M.P. and F.G.-G. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no funding.

**Institutional Review Board Statement:** The study was conducted in accordance with the Declaration of Helsinki, and approved by the Institutional Review Board of Faculty of Dentistry, Chulalongkorn University. The study did not require ethical approval.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The authors would like to express their appreciation to Nobuo Nakabayashi for his support and comments; John Harcourt, The University of Melbourne, for assistance with English clarity.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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## *Review* **Modification of Starches and Flours by Acetylation and Its Dual Modifications: A Review of Impact on Physicochemical Properties and Their Applications**

**Edy Subroto \*, Yana Cahyana , Rossi Indiarto and Tiara Aray Rahmah**

Department of Food Industrial Technology, Faculty of Agro-Industrial Technology, Universitas Padjadjaran, Bandung 45363, Indonesia; y.cahyana@unpad.ac.id (Y.C.); rossi.indiarto@unpad.ac.id (R.I.); tiara16005@mail.unpad.ac.id (T.A.R.)

**\*** Correspondence: edy.subroto@unpad.ac.id

**Abstract:** Various modification treatments have been carried out to improve the physicochemical and functional properties of various types of starch and flour. Modification by acetylation has been widely used to improve the quality and stability of starch. This review describes the effects of acetylation modification and its dual modifications on the physicochemical properties of starch/flour and their applications. Acetylation can increase swelling power, swelling volume, water/oil absorption capacity, and retrogradation stability. The dual modification of acetylation with cross-linking or hydrothermal treatment can improve the thermal stability of starch/flour. However, the results of the modifications may vary depending on the type of starch, reagents, and processing methods. Acetylated starch can be used as an encapsulant for nanoparticles, biofilms, adhesives, fat replacers, and other products with better paste stability and clarity. A comparison of various characteristics of acetylated starches and their dual modifications is expected to be a reference for developing and applying acetylated starches/flours in various fields and products.

**Keywords:** acetylation; starch; flour; physicochemical properties; modification

#### **1. Introduction**

Native starches/flours generally have several drawbacks related to functional, pasting, and physicochemical properties, which can limit their use in various applications. These limitations include low swelling ability, absorption capacity, solubility, starch clarity, and freeze stability [1–4]. Most of the starches tend to retrograde easily when the starch paste is stored at low temperatures. This is caused by the amylose chains that had previously come out of the granules binding to each other again to form a crystalline structure [5,6]. Retrogradation causes an increase in starch viscosity, crystallinity, gel structure, and gel texture [7,8]. Various treatments and modifications have been used to improve these properties, one of which is by modifying acetylation. Acetylation is a modification involving the substitution of hydroxyl groups with acetyl groups; the number of substituted acetyl groups affects the characteristics of starch/flour [9–11].

The modification of acetylation in starch/flour has been reported to increase swelling ability, clarity of starch paste, and stability of starch against retrogradation [12–14]. Acetylated starch is often applied to improve the texture and appearance of products whose quality may decrease due to damage during processing or retrogradation. Acetylated starch can also provide a good thickening effect in various foods. However, acetylated starch is unstable to thermal processes characterized by increased breakdown viscosity [15–17]. This can be overcome by combining acetylation with other modifications that can improve thermal stability, such as cross-linking modifications and hydrothermal treatments, such as heat moisture treatment (HMT) and annealing (ANN). Hydrothermal modification can

**Citation:** Subroto, E.; Cahyana, Y.; Indiarto, R.; Rahmah, T.A. Modification of Starches and Flours by Acetylation and Its Dual Modifications: A Review of Impact on Physicochemical Properties and Their Applications. *Polymers* **2023**, *15*, 2990. https://doi.org/10.3390/ polym15142990

Academic Editor: Raffaella Striani

Received: 13 June 2023 Revised: 6 July 2023 Accepted: 7 July 2023 Published: 9 July 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

improve the formation of amylose-lipid complexes and the regularity of the crystalline matrix to control the swelling capacity and increase stability to heating and friction [18–20].

Modifying acetylation combined with cross-linking or hydrothermal treatment can improve the clarity of the paste, texture, and thermal stability [21,22]. Several studies have stated that hydrothermal treatment is able to increase the effectiveness of acetylation reactions with starch molecules so that more acetyl groups are substituted and can minimize the use of chemicals for the acetylation process [10,23]. However, several dual-modified treatments have contradictory effects, so the resulting characteristics depend on the dominant treatment [10,22].

Changes in the characteristics of starch/flour due to the modification of acetylation are highly dependent on the degree of substitution (DS) and treatment conditions such as the source of starch, type of reagent, pH, temperature, and time [12,15,24]. The starch source determines the amylose-amylopectin content, which determines the amorphous and crystalline structures of the starch granules. This type of acetylation reagent generally uses acetic acid, vinyl acetate, or acetic anhydride, which can be catalyzed using bases such as NaOH and KOH. At the same time, temperature and time reaction determine the level of DS obtained, which greatly affects the characteristics of the starch/flour produced. This review describes studies on modifications of various types of starch and flour by acetylation or dual modification of their physicochemical characteristics, as well as their applications in various fields/products, so that they can become a reference for the development of starches/flours.

#### **2. Applications of Acetylated Modified Starch/Flour**

Modifying a starch affects its characteristics, and the changes that occur depend on the type of modification applied. Chemical modifications such as acetylation weaken the starch's structure, thereby increasing its hydration capacity and reducing its tendency to retrograde [25,26]. Meanwhile, the combination of acetylation with cross-linking and hydrothermal modification (HMT and ANN) can increase the orderliness of the crystalline matrix so that the gelatinization process becomes slower and granule swelling is limited [1,27,28]. Therefore, acetylated modified starch or its dual modification is often used to improve the hydration quality and thermal stability of the product [24,29]. However, the application of acetylated starch and its dual modifications has been developed in various fields and various products. Some applications of acetylated modified starches/flours for various products and fields can be seen in Table 1.


**Table 1.** Some applications of acetylated modified starches/flours for various products and fields.


#### **Table 1.** *Cont.*


**Table 1.** *Cont.*

Acetylation modification can increase the functionality of starch and its applications, especially in foods. The high hydration ability of acetylated starch has the potential to be used as a thickening agent. Several studies also reported that acetylation modification has good stability and resistance to retrogradation and syneresis, so it has the potential to be applied as a stabilizer in products that require low-temperature storage [13,45]. In addition, acetylation modification can increase OAC so that it can be applied as a filming agent. Acetylated starch has many applications in the food industry, some of which are in products such as retorted soups, sauces, canned pie fillings, frozen food, baby food, and salad dressings [15,24,46].

Acetylated modified starch, especially in nanocrystal form, has been effectively applied for encapsulation and as a delivery system for various drugs and other active compounds [47]. de Oliveira et al. [30] applied acetylated cassava starch as starch-based nanoparticles for the encapsulation of antioxidants; it was reported that acetylated cassava starch interacted well with antioxidant compounds, especially BHT and protected antioxidants from the degradation process, and increased the thermal stability of nanoparticles. Liu et al. [32] applied acetylated debranched waxy corn starch as a nanocarrier for curcumin, and it was reported that curcumin micelles of acetylated starch had a spherical shape with a particle size of about 50–100 nm and could accommodate curcumin until the concentration of 0.45 mg/mL. Gangopadhyay et al. [33] applied retrograded acetylated corn starch to drug (budesonide) delivery, and it was reported that tablets from retrograded acetylated corn starch were able to release the drug in ileocolonic by 81.38% and were potentially suitable for the treatment of ileocolonic diseases. Meanwhile, Xiao et al. [31] reported that acetylated rice starch nanocrystals could be used for protein (BSA) delivery by significantly slowing BSA protein release.

Acetylated modified starch, especially in nanocrystal form, has been applied to improve the quality of bioactive films. Promhuad et al. [34] applied acetylated cassava starch and Maltol Incorporated in active biodegradable film/packaging fabrication; it was reported that film based on acetylated cassava starch, which was incorporated with 10% maltol, reduced molecular mobility, hydrophilicity, elongation, and tensile strength. The active film based on acetylated cassava starch inhibited the fungal growth by up to six times longer and maintained the flavor of bakery products. Fitch-Vargas et al. [35] applied acetylated corn starch in the manufacture of starch-based bioplastics; it was reported that acetylated corn starch improved homogeneity and mechanical properties, while the solubility of starch-based bioplastics decreased to 24.9–28.2%. Meanwhile, Nasseri et al. [36]

reported that acetylated corn starch could be applied to biodegradable polymers poly(lactic acid) for packaging materials, where acetylated corn starch can increase the thermal stability of biodegradable polymers and improve mechanical properties such as toughness and tensile strength.

Acetylated modified starch and its dual modification can also be used as a stabilizer in various emulsion products and can act as a fat replacer. Yao et al. [37] applied acetylated cassava starch nanoparticles as an emulsion stabilizer; it was reported that acetylated starch nanoparticles improved hydrophobicity and improved emulsion capacity by improving the droplet size and homogeneity so that the storage stability increased up to 35 days. Cui et al. [38] applied cross-linked acetylated cassava starch to the manufacture of set yogurt, and it was reported that cross-linked acetylated cassava starch improved the stability, viscous modulus, and elastic modulus of the set yogurt. Meanwhile, Osman et al. [16] utilized acetylated corn starch as a fat replacer in beef patties, and it was found that it was suitable as a fat replacer for meat products. At the same time, the use of 15% acetylated corn starch improved the acceptance of organoleptic, microstructural, and physicochemical properties in beef patties.

Acetylated starch can also be applied directly in the manufacture of various food products such as bread and noodles. For some food products, such as bread, retrogradation causes bread stalling, where the quality of the bread decreases in the form of a harder texture [48]. Acetylated starch could slow down and improve retrogradation, thereby improving the texture and quality of bread. Rahim et al. [39] applied acetylated arenga starches to bread making; it was reported that adding acetylated starch up to 50% was able to improve the quality of the bread produced, which included sensorial properties, oven spring, oil absorption, and oil holding capacity. Meanwhile, Lin et al. [40] applied acetylated corn starch to the manufacture of noodles, and it was reported that acetylated corn starch increased the brightness and reduced the tensile properties, chewiness, adhesion, and hardness of noodles. Acetylated starch was also reported to increase resistant starch and slowly digestible starch in noodles. Wang et al. [41] also reported that acetylated rice starch and potato starch improved the gut microbiota fermentation by producing more SCFA and were easier to use and more quickly fermentable by the gut microbiota.

Acetylated modified starch and its dual modification can also be used in the chemical field, namely as a waste treatment coagulant and as an adhesive, especially to increase its water resistance and shear strength [49]. Gu et al. [42] applied acetylated-crosslinked corn starch as a wood-based panel adhesive, where the adhesive had better water resistance up to 1 MPa, and the adhesive was also heat-resistant so that it could be used in hightemperature pressing. Wang et al. [43] applied acetylated waxy corn starch as a wood adhesive, and it was found that the adhesive's resistance to water increased up to 61.1%, and the shear strength increased up to 321% in the wet state and 59.4% in the dry state. Meanwhile, Posada-Velez et al. [44] applied acetylated potato starch and corn starch as coagulants for wastewater treatment, and it was found that acetylated starch from both corn and potato starch had good effectiveness as a coagulant for wastewater treatment by significantly reducing pH, color, turbidity, and electrical conductivity.

#### **3. Acetylation Modification Process in Starch/Flour**

#### *3.1. Mechanism of Acetylation*

The characteristics of starches and flours could be improved by chemical, physical, and combination or dual modifications. These modifications aim to change some of the properties of starch by altering its original structure through physical treatment or changing the hydroxyl groups in starch through chemical reactions such as oxidation and esterification [50–53]. The modification of starch includes the use of heat, oxidizing agents, alkalis, acids, and other chemicals that will generate new chemical groups, resulting in changes in size, morphology, molecular structure, and other physicochemical characteristics [54,55].

Chemical modification can change the significant characteristics of starch and flour. Chemical modifications can be carried out through acid hydrolysis, oxidation, cross-linking, and the addition of functional groups such as acetylation. In general, chemical modification adds new functional groups to the starch, which then affect the physicochemical properties of the modified starch [52,56–58]. The chemical modification of acetylation has been widely applied to various food industries. Several studies have been developed by combining acetylation modification with physical or other chemical modifications, such as acetylation + hydrothermal and acetylation + crosslinking, in order to increase starch's functional value and expand its application [26,59,60].

Acetylation is a chemical modification technique conducted through the esterification of starch using acetic anhydride, acetic acid, and vinyl acetate reagents and alkali (NaOH, KOH, Ca(OH)2, and Na2CO3 as catalysts [61,62]. The basic principle of the acetylation reaction is the substitution of starch-free hydroxyl groups with acetyl groups (Figure 1) by weakening the bonds between starch molecules to produce starch that is amphiphilic (hydrophilic and hydrophobic) [63,64]. Acetylation is an indirect esterification process, so it is necessary to add a catalyst so that the reaction can take place. Before the reaction, the starch is first conditioned in an alkaline state to form the starch base. An acetate reagent is then added to form starch acetate [30,65–67]. The basic principle of the acetylation reaction by the substitution of starch-free hydroxyl groups with acetyl groups can be seen in Figure 1.

**Figure 1.** The basic principle of the acetylation reaction by the substitution of starch-free hydroxyl groups with acetyl groups [67], with permission from Elsevier, 2015.

Figure 1 shows that the acetylation reaction occurs via the substitution of the acyl group with the free hydroxyl group portion of the glucose monomers as a constituent of starch molecules. There are many hydroxyl groups, so more free hydroxyl groups being substituted with acetyl groups will result in a greater degree of substitution. Acetylation can reach equilibrium, and if the reverse reaction occurs, it indicates the hydrolysis reaction of the ester bond. The rate of the acetylation reaction is affected by several factors as well as other esterification reactions, especially the structures of the acids and alcohols that react and the catalyst used [68]. The catalyst usually uses strong bases such as KOH and NaOH using anhydride reagents such as acetic anhydride. However, using organic acids or carboxylic acids as organocatalytics is also competitive to produce safer and more environmentally friendly processes. Organocatalytics that can be used include Laspartic, citric, L-tartaric, L-malic, L-lactic, glycolic, fumaric acids, and L-proline. These

organic catalysts can also produce high esterification reaction rates and high degrees of substitution [69,70].

The degrees of substitution (DS) affect the physicochemical and functional characteristics of acetylated modified starch/flour [71,72]. The DS values ranged from 0.01 to 3, describing the number of substituted acetyl groups in one glucose unit. Starch acetate with DS 0.01 indicates that there is one substituted acetyl group in 100 units of glucose. In contrast, starch acetate with DS 3 indicates that there are 300 substituted acetyl groups in 100 units of glucose. This is based on the theory that acetylation reactions can substitute three free hydroxyl groups of glucose units on C2, C3, and C6 atoms with acetyl groups [16,73]. Acetic starch with low DS has been widely applied in the food industry for many years. The Food and Drug Administration (FDA) stipulates that the maximum permissible limit for the percentage of acetyl groups in foodstuffs is 2.5% or the equivalent of a DS value of 0.1, but generally, for commercial food products, starch acetate is used with a low DS (<0.1) or medium DS (0.1–1.0) [15,74].

#### *3.2. Effect of Acetylation Methods on Properties of Starch/Flour*

The acetylation method involving reactant types and concentrations can affect the acetylation reaction's efficiency. The DS of starch acetate is greater when the reactant concentration is high [75]. Generally, the ability and efficiency of acetic anhydride in substituting acetyl groups were greater than that of vinyl acetate at the same conditions and concentrations. In addition, the type of catalyst and reaction medium (solvent) also affect the efficiency of the reaction. Solvents that can be used in acetylation reactions include water, pyridine, and DMSO. Pyridine and DMSO have greater efficiency than water but can have adverse environmental and health impacts [24]. Thus, the number of substituted acetyl groups is affected by several factors, including the source or type of starch, the concentration of the reactants, pH, reaction time, and the catalyst used [65,76]. The starch type with many amorphous parts, such as starch from tubers, is more easily penetrated or substituted by acetyl groups. An example is the acetylation of potato starch, which produced a higher DS (DS: 0.180–0.238) than corn starch (DS: 0.133–0.184) [77]. The use of reactants with higher concentrations can produce higher DS; for example, the use of an acetylation of sweet potato starch using acetic anhydride at concentrations of 2, 4, 6, and 8% produced DS of 0.032, 0.059, 0.091, and 0.123 respectively [78]. pH can also affect DS, where the use of a higher pH could increase DS; for example, the acetylation of yellow pea starch at pH 9–10 produced a higher DS (DS: 0.071) than pH 7.5–9.0 (DS: 0.066) [62]. Longer reaction times can increase DS; for example, a chestnut starch acetylation at 30, 60, and 90 min reaction times resulted in the DS being 0.010, 0.020, and 0.024, respectively [14]. The type of catalyst used can also affect the DS; for example, the use of catalysts of Ca(OH)2, NaOH, and KOH 1 N in the acetylation of waxy cornstarch produced DS of 0.077, 0.081, and 0.085, respectively [61]. The DS value can be determined by several techniques, including headspace gas chromatography (HS-GC), infrared spectroscopy or FT-IR, nuclear magnetic resonance (NMR), and titration [53]. Several methods of modifying acetylation on various types of starch/flour and their effect on DS values can be seen in Table 2.

**Table 2.** Several methods of modifying acetylation on various types of starch/flour and their effect on DS values.



#### **Table 2.** *Cont.*


**Table 2.** *Cont.*

Infrared spectroscopy or FT-IR is also frequently used to determine the DS value of acetylated modified starch/flour. An example of the effect of acetylation modification on the FTIR spectra of starch can be seen in Figure 2. The FTIR spectrum of acetylated starch is shown by the appearance of several peaks or new absorption bands at wave numbers 1240, 1375, 1435, and 1754 or 1742 cm−1, which interprets C–O carbonyl stretching vibrations, CH3 symmetry deformation vibrations, CH3 antisymmetric deformation vibrations, and carbonyl C=O, respectively [98–100]. The appearance of these new absorption peaks indicates the occurrence of an esterification reaction, namely the substitution of hydroxyl groups by acetyl groups. The greater intensity of the absorption peaks indicates that the DS value is also greater and is followed by a weakening of the peaks at wave numbers 3421, 1082, and 1014 cm−1, which interprets the reduction of hydroxyl groups. Additionally, the spectrum of acetylated starch also shows that the anhydroglucose units tend to shift towards high wave numbers [100].

**Figure 2.** FTIR spectra of native corn starch and acetylated corn starches at different DS (a) 0.85, (b) 1.78, (c) 2.89 [100], with permission from Elsevier, 2008.

The DS value of acetylated starch can also be determined based on nuclear magnetic resonance (NMR) spectra, which generally use 1H NMR, 13C NMR, or 13C–1H COSY. NMR spectra can show changes in anhydroglucose units due to the substitution of hydroxyl groups by acetyl groups. An example of the effect of acetylation modification on the NMR spectra of starch can be seen in Figure 3.

**Figure 3.** Single Pulse-13C NMR spectra of native pea starch (NPS) and acetylated pea starch (APS) by organocatalytic acetylation at different reaction times. Signal 1 corresponds to the carbon of the alkyl group, and signal 6 corresponds to the carbon of the ester groups of starch [101].

The hydroxyl (-OH) groups of C2, C3, and C6 show a proton signal from the anhydroglucose unit between 4.4 and 5.6 ppm; the signal at 5.10 ppm corresponds to the anomeric proton of the link (α-1, 4), whereas the anomeric proton of the junction (α-1,6) exhibits a small signal at 4.86 ppm [101,102]. Figure 3 shows that in acetylated starch, an addition signal between 1.8 and 2.2 ppm indicates a methyl proton from the acyl group, thus indicating that the acetylation process was successful. The level of acetylation increases with the length of the reaction time. The spectrum of native starch shows four main signals (Signals 2, 3, 4, and 5). In comparison, in acetylated pea starch, there are two additional signals, namely signal 1 at 16.4 ppm and signal 6 at 166.6 ppm, which interpret the carbon of the methyl proton from the acyl group (-CH3) and the ester group (C=O). The longer the reaction time, the greater the area of the two signals, which means that the DS value of the acetylation increases [101].

Some of the advantages of acetylation modification include increasing starch clarity, lowering the gelatinization temperature, increasing the stability of frozen storage, and being more resistant to retrogradation [24,103]. Therefore, acetylated starch is commonly used in the food industry for the production of salad dressings, retorted soups, frozen foods, baby food, and snacks [86,104]. Chang and Lv [105] reported that acetylation reactions could also produce resistant starch in the form of RS type 4, which has a low glycemic index. In addition to its application in food ingredients, several studies have stated that acetylated starch with high DS is commonly used as a packaging material or in cigarette filters and has several applications in the pharmaceutical field [96,106]. Although it can improve the characteristics of starch, this modification tends to produce starch that is less resistant to high-temperature heating. Therefore, several researchers carried out a combination of physical modification + acetylation to improve the thermal stability of starch. Several modification combinations have been carried out, including annealing-acetylation [10,97] and sonication + acetylation [63].

#### **4. Characteristics of Acetylated Modified Starch/Flour**

#### *4.1. Functional Properties of Acetylated Modified Starch/Flour*

Functional characteristics are the properties of starch/flour that affect the usability of starch when applied. The functional characteristics of starch/flour can be viewed through several parameters, including amylose leaching/solubility, oil absorption capacity (OAC), water absorption capacity (WAC), freeze-thaw stability (FTS), and swelling power (SP). Swelling power is related to the amount of water absorbed, where the greater the swelling power value, the more water is absorbed. Starch granules will swell when heated in water, which is caused by the breaking of hydrogen bonds between starch molecules so that starch molecules will bind with water [54,107].

The swelling of granules is closely related to the release of starch molecules from granules or what is known as amylose leaching. As gelatinization proceeds, the water present in the starch suspension enters the outer amorphous and crystalline regions (located near the amorphous lamellae) [108,109]. The process of entry of water into the amorphous region causes granule swelling and weakens the hydrogen bonds between amylose and amylopectin chains. The continuous heating process takes place, causing starch molecules dissolved in water to easily move in and out of the solution system. Starch molecules that dissolve in hot water (amylose) will come out with the water, causing amylose leaching [110,111]. Solubility and swelling power are affected by several factors, including the amylose-lipid complex, molecular weight, granule size, amylose/amylopectin ratio, distribution of amylopectin chain lengths, amylose-amylopectin chain interactions, the molecular structure of starch granules, and crystal arrangement [28,112–115]. The solubility is more affected by the amylose content, while the swelling power is affected by the amylopectin component [116].

Water absorption capacity (WAC) is defined as the ability of granules to absorb water. WAC determines the amount of water available for starch gelatinization during cooking [117]. The gel formation cannot reach optimum conditions if the amount of water is lower. The greater the WAC, the more starch constituent material is lost, while the lower the WAC, the more compact the structure. Solubility, swelling power, and WAC are related to other parameters, namely viscosity, and crystallinity. Increases in solubility, SP, and WAC are associated with decreased crystallinity of starch and cause an increase in viscosity [118,119].

Starch paste stored at freezing temperatures will cause the water contained in the paste to form ice crystals, and these ice crystals will melt during thawing and release of water from the granules, which is called syneresis [120]. Freeze-thaw stability (FTS) describes the ability of pasta to withstand repeated freezing and thawing cycles without any physical changes. FTS is correlated with retrograde tendencies. The increased aggregation of starch molecules causes an increase in the release of water molecules from starch granules, so the syneresis increases, and FTS decreases [6,89]. Modification treatments, both acetylation and hydrothermal, can affect the interactions between starch molecules, which can affect the stability of the paste in frozen storage [74,121].

Retrogradation occurs when starch components that have been gelatinized re-associate. This association causes the starch structure to become more compact, making the paste more turbid. The modification of acetylation in corn starch was reported to increase starch's stability against retrogradation and increase the clarity of starch paste [15,87]. Based on the level of clarity, the paste is classified into three types, namely transparent, moderately transparent, and cloudy. The clarity of the starch paste can be observed through a spectrophotometer by measuring the % transmittance, where the higher the %transmittance, the more transparent the paste. The level of clarity of the paste varies depending on the type of paste, solubility, swelling power (SP), and the aggregation of starch molecules [122,123]. Starch with high clarity and viscosity is suitable for application as a thickening agent, while some food products, such as salad dressings, require opaque starch [9,15,124].

Starch with high solubility and hydration ability is needed in several processed food industries, such as in the manufacture of noodles, bakery products, jelly, and many more [56,125]. Acetylation modification can increase SP and WAC, while hydrothermal modification generally causes a decrease in SP and WAC. Information regarding the effect of acetylation modification and its dual modification on the functional properties of starch/flour is available in Table 3.

**Table 3.** The effect of acetylation and its dual modification on the functional properties of starch/flour.



**Table 3.** *Cont.*

Modification by acetylation could increase the swelling power, solubility, and WAC as the DS increases. The modification of acetylation causes an increase in SP in sweet potato starch [128], potato starch [10,77,83], purple yam, white yam, and cocoyam starches [84,85], sword bean starch [90], chickpea and yellow pea starches [62], corn and waxy corn starches [10,77,87], rice and waxy rice starches [10,88], sago starch [93], wheat starch [92], and waxy barley starch [10]. This increase is due to the substitution of the hydrophilic (acetyl) group, which facilitates water penetration into the starch granules [87,89].

Improvements in starch hydration and water absorption capacity after modification by acetylation were found in sweet potato starch [128], potato starch [83], purple yam, white yam, and cocoyam starches [84,85], sword bean starches [90], oat starch [89], rice starch [88], and sago starch [93]. The increased hydration power of the granules due to the substitution of acetyl groups causes increased flexibility of the structure, making it easier to bind water [46]. Increased starch solubility due to modification of acetylation was found in sweet potato starch [128], potato starch [77,83], sword bean starch [90], corn and waxy corn starch [77,87], rice starch [88], and sago starch [93].

The modification of acetylation can also affect the solubility of starch and flour. Acetylation will generally increase solubility, mainly due to the addition of acetyl groups that disrupt the interactions between starch molecules, which in turn increases the affinity of starch molecules to dissolve in water [88]. The increase in starch solubility due to the substitution of acetyl groups will weaken the hydrogen bonds that connect starch on an intermolecular level as well as inhibit intermolecular associations so that the starch molecules will bind with water and dissolve along with the water coming out of the starch granules [84]. Nevertheless, several studies have found a decrease in WAC in acetylated potato and corn starch [77], a decrease in solubility in acetylated cocoyam starch [85], and a decrease in SP in acetylated japonica and indica rice starch [91]. The effect of acetylation modification on the hydration and solubility of granules is influenced by the number of substituted acetyl groups (DS); the higher the DS, the greater the changes that occur [88,93].

Several studies reported that the combination of modification by esterification (acetylation) + hydrothermal treatment was able to increase SP, WAC, and starch solubility [10,23,97]. This is because the esterification modification is related to the substitution of ester groups (acetyl/hydroxypropyl), which weakens the granule structure, thereby facilitating the penetration of water into the granules and increasing the interaction of starch molecules with water [10,23].

The acetylation modification can also reduce retrogradation tendencies and increase the clarity level of starch paste [46,59,77,85,128,129,131]. Increasing the clarity of the paste is related to the hydration ability of the granules. The greater the hydration ability of the granules, the higher the level of clarity. The substitution of acetyl groups will limit the intermolecular interactions of starch, thereby reducing the tendency for starch syneresis [87,89,129]. The decrease in syneresis tendencies in acetylated starch is related to the substitution of acetyl groups, which limits intermolecular interactions when the paste is stored at low temperatures [46]. In addition, the modification of acetylation and its combination can also affect the whiteness of starch, where most of the modification treatments cause a decrease in whiteness [10,26].

Modification by acetylation can lead to an increase in the hydration ability of starch granules. This is due to the disorganization of the intragranular structure followed by the following events: (1) disruption of the intermolecular hydrogen bonds of the starch and increased penetration of water into the amorphous regions [75,88], (2) the presence of repulsive forces rejecting intermolecular starch due to the substitution of acetyl groups, (3) the partial depolymerization of the amylopectin structure, which causes a decrease in molecular weight (MW) [88,132], (4) a decrease in starch crystallinity [92,132,133] and the limitation of starch intermolecular interactions [84].

The acetyl group can have two properties, namely hydrophilic and hydrophobic. Acetylation at low DS makes starch tend to be hydrophilic, while acetylation at high DS makes starch tend to be hydrophobic [93,134]. Therefore, several studies reported that acetylation causes an increase in OAC [85,89,90,93]. The high OAC value has the potential to improve the flavor and mouthfeel of food products, such as whipped cream, sausages, chiffon cakes, and various processed desserts [135].

Several studies have reported that acetylation can reduce the tendency of starch to experience retrogradation and syneresis and shows a better level of clarity and transparency of starch than natural starch [77,85,89]. This is due to the substitution of acetyl groups which will hinder the association between starch molecules [88]. The clarity of starch is related to the ability of starch hydration (WAC and SP). The increased hydration ability of the granules causes more water molecules to be trapped in the granules so that when observed using a spectrophotometer, more light can be transmitted by the paste [136].

Acetylation can reduce the occurrence of syneresis in starch. A decrease in the percentage of syneresis indicates an increase in the stability of the paste against frozen storage [13,15]. Syneresis is related to retrogradation events in which reassociation occurs between starch molecules when stored at low temperatures. Syneresis causes a decrease in the water content of amylose/amylopectin, which can affect product characteristics. The substitution of acetyl groups can prevent the reassociation of the starch molecules so that the retrogradation tendency decreases. In addition, the substitution of acetyl groups also increases the water storage capacity so that the retention power of granules to water is also greater [10,46,83,88,130]. Mendoza et al. [84] reported that limiting the intermolecular associations of starch due to acetylation was necessary to produce starch with high hydration, good storage stability, and high clarity.

Acetylation modifications can also be combined with other modifications. The combination of acetylation with hydrothermal treatments (ANN and HMT) can increase the hydration and solubility of starch [26,97]. A single hydrothermal modification generally reduces the hydration power and solubility of starch. The addition of esterification modifications (acetylation or hydroxypropylation) can increase the hydration ability of granules and starch solubility. This statement is supported by several studies, which reported that the combination of acetylation + ANN modification in waxy cereal and potato starches had a higher swelling power (SP) than native starch and ANN starch, while the combination with HMT could increase SP, but this combination decreased the solubility of starch [10].

The substitution of ester groups (acetyl/hydroxypropyl) can weaken the crystalline structure of granules so that water penetration into the granules becomes easier and SP increases [10,23]. Nonetheless, Sitanggang et al. [97] reported that the combination of acetylation + ANN modification in black bean and pinto bean starches reduced SP and starch solubility. This decrease was due to an increase in starch crystallinity due to ANN modification, so the hydration power of the granules decreased [28]. In addition, the substitution of the acetyl group in this dual-modified starch causes the starch to become more hydrophobic [86,93].

Egodage [10] reported that the combination of acetylation + ANN modification in waxy rice and waxy potato starches increased the percentage of transmittance, which indicated an increase in the clarity of the paste. Low-temperature storage conditions caused a decrease in the clarity of the paste caused by the retrogradation process. The decrease in clarity of acetylated and acetylated + ANN-modified starch paste was not as big as that of natural starch and ANN-modified starch. The substitution of the acetyl group prevented amylopectin molecules from aggregating in the paste so that the retrogradation tendency decreased [10,77]. Reducing the retrogradation tendency could reduce the level of paste syneresis. Abedi et al. [130] reported that the combination of acetylation + sonication modifications reduced the percentage of syneresis due to the substitution of acetyl groups, which prevented the retrogradation process. Thus, the combination of acetylation + hydrothermal modification can improve the functional characteristics of starch. Changes in the functional properties of starch depend on the dominant modification treatment. Acetylation modification tends to weaken the crystalline structure of starch, so if this modification is combined with hydrothermal-modified starch, it can weaken the perfect structure. The substitution of ester groups with low DS tends to cause hydrophilic starch, thereby increasing the hydration ability of the modified starch, but if the DS is too low, the esterification reaction does not change the characteristics of the starch [93,137].

#### *4.2. Pasting Properties of Acetylated Modified Starch/Flour*

Pasting properties are indicators that determine starch properties during processing, which affect the cooking quality and functionality. Pasting properties described as an amylographic curve can be used to determine the application of starch in food

ingredients [138,139]. Starch with low viscosity is suitable for liquid-based foodstuffs, while starch with high viscosity is suitable for use as a thickening agent [140–142].

Based on the amylographic curve, starch is classified into four types, namely types A, B, C, and V [143,144]. Starch type A shows high swelling of starch granules followed by a rapid decrease in viscosity during cooking, commonly found in potato starch, cassava starch, and several types of cereals. Type B starch, which has almost the same curved shape as type A but has a lower viscosity, is usually found in cereals. Type C starch, which exhibits limited swelling of the granules, has no peak viscosity, and tends to be heat-stable, is commonly found in modified starches and legumes. Type V starch, showing limited swelling of the granules, is usually found in starch that interacts with alcohol or fatty acid [144,145]. The effect of acetylation modification and its dual modification on the pasting properties of starch/flour is presented in Table 4.

**Table 4.** The effect of acetylation and its dual modification on the pasting properties of starch/flour.



**Table 4.** *Cont.*

In general, acetylation modification causes a decrease in starch gelatinization temperature so that it facilitates product application in terms of energy saving and its utilization in products that are susceptible to heat [128]. Several studies reported that the modification of acetylation causes a decrease in gelatinization temperature in sweet potato starch [128]; potato starch [147]; yam starches [84,85]; cowpea, yellow pea, and chickpea starches [62]; pinto bean and black bean starches [129]; rice starch [88,91]; waxy cornstarch [61]; and wheat starch [92], as well as decreased setback viscosity in sweet potato starch [104,128]; potato starch [127]; sword bean starch [90]; yellow pea, and chickpea starches [62]; black bean and pinto bean starches [129]; and rice starch [88]. A decrease in SB indicates a decreased retrogradation tendency, making the starch more stable at low-temperature storage. Meanwhile, most studies report that acetylation causes an increase in viscosity followed by an increase in starch hydration ability [59,62,88,90,92].

Table 4 also shows that acetylation modification combined with hydrothermal treatment can improve the pasting properties of starch to cover the weaknesses in hydrothermal modification. Sitanggang et al. [97] reported that the combination of acetylation + ANN modification could increase PT and SB and reduce PV and BD. The addition of the esterification treatment (acetylation/hydroxypropylation) was able to weaken the crystalline matrix so that it had a higher viscosity and a lower gelatinization temperature when compared to the single hydrothermal treatment [23,148,149].

The substitution of acetyl groups causes weak intermolecular forces, which decreases the gelatinization temperature of starch acetate. This statement is supported by several researchers who reported a decrease in gelatinization temperature after the modification of acetylation, including sweet potato starch [128], potato starch [147], yam starches [84,85], cowpea, yellow pea, and chickpea starches [62], pinto bean and black bean starches [129], rice starch [88,91], waxy maize starches [61], and wheat starch [92]. A decrease in the pasting temperature of the acetylated starch indicates that a large amount of energy is not required for the starch to gelatinize. This can be used to save energy in cooking or processing using acetylated starch [128].

The substitution of the acetyl group weakens the intermolecular bonds between amylose and amylopectin, thereby facilitating the penetration of water into the amorphous region, which is followed by an increase in the ability to absorb water during gelatinization [59,85,88,90]. Increasing the hydration ability of starch granules during gelatinization can increase the viscosity of the paste. Several studies reported an increase in paste viscosity after the modification of acetylation in sweet potato starch [127], purple yam starch [84], sword bean starch [90], yellow pea cowpea, and chickpea starches [62], buckwheat starch [59], waxy maize starch [61], rice starch [88], and wheat starch [92]. However, the effect of acetylation modification on starch viscosity is not always the same. Several studies reported that acetylation can have a different effect on each type of starch. The difference in results obtained is influenced by several factors, including the distribution of amylopectin chains, amylose content, molecular arrangement of granules, and the degree of substitution of acetyl groups [62,84].

Acetylation modification can reduce the setback viscosity (SB), which indicates an increase in starch storage stability, especially at cold temperatures. This statement is reinforced by several studies which reported a decrease in SB after modification of acetylation [62,88,90,104,127,129]. Acetyl group substitution inhibits intermolecular interactions of starch, thereby minimizing the tendency of starch retrogradation [88,94,104].

The modification of acetylation with different materials and types of starch can produce different pasting properties as well. Acetylation with vinyl acetate and acetic anhydride in starch can cause a decrease in the gelatinization temperature due to the weakening of the intermolecular interactions of starch [25,150]. However, acetylation with vinyl acetate in yellow pea and chickpea starches changed the type of starch gelatinization from type C to type B. In contrast, acetylation with acetic anhydride did not change the gelatinization pattern. The peak viscosity of all types of starch increased after acetylation modification with vinyl acetate. In contrast, acetylation with acetic anhydride caused a decrease in the peak viscosity of cowpea and chickpea starches. Both types of reagents caused a decrease in the SB of cowpea starch but an increase in SB in yellow pea starch. Meanwhile, acetylation with either acetate anhydride or vinyl acetate did not change the SB chickpea starch. Thus, the modification of acetylation with different reagents and starch sources has a different effect on its pasting properties [62,84,127].

Hydrothermal modification can increase the starch gelatinization temperature followed by a decrease in peak, BD, and SB viscosity, while acetylation causes a decrease in PT and SB followed by an increase in PV and BD [59,151,152]. Changes in the pasting properties of hydrothermally modified starch are contradictory to acetylation modifications. Sitanggang et al. [97] reported that the combination of acetylation + ANN modification led to an increase in PT and SB, followed by a decrease in peak viscosity and BD. This indicates that ANN modification is dominant because the resulting characteristics resemble ANN starch. In other studies, the dual modification of retrogradation + acetylation [94] and sonication + acetylation [130] produced starch with characteristics like acetylated starch. Yu et al. [94] reported that acetylation of retrograded starch caused a greater decrease in viscosity. This may be due to the substitution of acetyl groups causing starch to be hydrophobic. Referring to Rahim et al. [93], the greater the substituted acetyl group, the more hydrophobic the starch. In addition, acetylation treatment can increase the PV and BD and decrease the pasting temperature (PT). This is due to the substitution of acetyl

and acetylated distarch adipate (acetic anhydride and adipic acid)

Acetylated (pH 8.0–8.4, NaOH 3%, 10 and 240 min)

Acetylated (pH 8, NaOH 1 M,

60 min) Corn starch

groups weakening the crystalline structure so that water penetration into the granules is easier [11,41]. Thus, the pasting properties of the dual-modified starch will lead to the dominant treatment.

#### *4.3. Starch Granule Morphology of Acetylated Modified Starch/Flour*

The shape and size of starch granules vary depending on the starch source. Most of the tuber starches have oval-shaped granules, but some have round, polygonal, and spherical ones, and some are irregular; the size varies from 1 to 110 μm depending on the type of starch [153]. For example, sweet potato starch has polygonal granules [154,155], round, hexagonal, and spherical with a size of 4–26 μm and a smooth surface without cracks [104].

Changes in the morphology of starch granules are commonly found in modified starches. Several studies have stated that acetylation modification causes granule damage (deformation, fusion, cracking, resulting in holes) [84,88,94]. However, some modifications, such as annealing, generally do not cause significant changes in granule morphology. The morphological characteristics of modified starch may vary in each sample and are influenced by several factors, including the source of starch, the type of modification, and the modification conditions (time, temperature, and reagents used). The morphological characteristics of starch granules can be analyzed in several ways, including light microscopy, scanning electron microscope (SEM), transmission electron microscope (TEM), atomic force microscope (AFM), and confocal laser scanning microscope (CLSM) [27,156,157]. Information on the morphological characteristics of acetylated and its dual modification of starch/flour can be seen in Table 5.


**Table 5.** The effect of acetylation and its dual modification on the morphological characteristics of starch/flour.

> starch. There was no significant change, but the modified acetylated distarch adipate starch had a more compact surface.

Holes appeared, but starch granules tended to

Granule aggregation occurred; the aggregation between granules was getting bigger along with the greater concentration of acetic anhydride.

retain their shape [84]

[158]

[87]

Potato starch

Purple yam (PY) starch White yam (WY) starch **Table 5.** *Cont.*


In general, acetylation can cause the fusion and aggregation of starch granules [73,77, 88,147]. This is related to the substitution of acetyl groups, which causes the disintegration of the structure and the more porous nature of the granules [104,159]. Xu et al. [73] reported

422

that the substitution of acetyl groups in high numbers caused granule fragmentation, where the starch granules melted to form a fiber-like structure.

In addition, several studies have stated that acetylation reactions can also cause the formation of holes or pores so that the surface of the granules becomes rough [89,90,158]. These changes may occur due to partial hydrolysis by acids and reactions with alkalis. Fornal et al. [158] reported that the formation of holes or pores could be associated with the gelatinization of the granule surface due to the neutralization reaction with alkali (NaOH) in the acetylation modification process. However, several studies stated that acetylation modification at low DS did not cause changes in granule morphology [10,59,83,84]. Examples of morphological changes in acetylated starch can be seen in Figure 4.

**Figure 4.** Morphological changes in acetylated modified chestnut starch [14] and barley starch [160], with permission from Elsevier, 2015.

Figure 4 shows that the modification of acetylation can cause starch granules to deform to form small fragments. The substitution of acetyl groups into starch molecules weakens the inter-/intramolecular bonds of starch and causes starch granules to lose their integrity [88,159]. The integrity of the starch is weakened, and its structure becomes increasingly porous during the acetylation reaction; then, the starch granules experience fragmentation and aggregate with each other [15,159].

Changes in the morphology of starch granules due to acetylation modifications can have different results. Several factors affect the morphological diversity of acetylated starch, namely internal factors such as the content of amylose-amylopectin and external factors such as type of reactant, concentration of reactant, reaction time, temperature, type of alkali, and concentration of alkali. Morphological changes will increase with the greater substitution of acetyl groups [126,160]. This may be due to the low DS acetylation reaction only taking place in the amorphous area of the granule surface so that it does not cause changes in the granule structure, whereas, at high DS, acetylation reactions can take place in the internal structure of the granules which causes greater damage [84,159].

The combination of acetylation with other modifications (dual modifications), such as hydrothermal treatment, also produces a variety of granule morphology. Acetylation modifications generally cause granule aggregation and fusion, whereas hydrothermal modifications (HMT and ANN) are more likely to maintain their integrity [10,161,162]. Therefore, in the dual treatment, the resulting modification of the morphological characteristics depends on the dominant modified treatment. However, there are dual-modified treatments that give a synergistic effect, while others depend on the most dominant treatment. The dual modification treatment that is synergistic includes sonication-acetylation modification. The sonication modification facilitates the acetylation reaction so that the effectiveness of the acetylation reaction in starch granules increases [130].

The dual modification of ANN-acetylation can produce starch with morphological characteristics resembling acetylated starch. This indicates that the modification of esterification/acetylation has a dominant effect [97]. Egodage [10] reported that the use of 5% ANN-Acetylation treatment did not cause granule changes, but 10% ANN-Acetylation treatment caused morphological changes resembling acetylated modified starch. This was due to the 5% acetylation of substituted acetyl groups that are too low to change the morphology of the granules. Although there was a slight change in the acetylated and dualmodified starch granules, the sizes and shapes of the granules did not change significantly; this indicates that the integrity of the granules was maintained during modification [10]. Different results were reported by Yu et al. [94], who stated that the dual modification of acetylation-retrogradation in starch and sweet potato flour caused granule deformation where the granules undergo fusion and aggregation. This could lead to granule damage when the ANN modification is smaller than during the retrogradation modification, so the ANN-acetylated starch is more stable in maintaining its structure than the retrogradationacetylated starch. The differences in the characteristics produced are influenced by several factors, including the type of modification, the type of starch and its structure, as well as the conditions and treatment of the modification [56,163].

#### *4.4. Starch Crystallinity of Acetylated Modified Starch/Flour*

The crystallinity of starch can be determined by observing the X-ray diffraction pattern. The X-ray diffraction pattern is related to the formation of semicrystalline regions during modification so that the amorphous and crystalline areas can be identified [164,165]. The basic principle of this test is the exposure of X-rays to the sample by scanning the diffraction area at an angle of 2θ from 4◦. The diffractogram pattern will produce a series of diffraction peaks with varying relative intensities along a certain value (2θ). Amorphous and crystalline regions can be distinguished by making curves and linear lines. The curve is made by connecting each point of minimum intensity; the area above the curve is known as the crystalline region (αc). The linear line is made by connecting two intensity points at 4◦ and 37◦ (2θ); the area that lies between the curve and the linear line is known as the amorphous region (αa). The ratio of the upper area (αc) to the total diffracted area (α<sup>c</sup> + αa) is known as the degree of crystallinity or relative crystallinity [165–168].

Based on the intensity peaks formed, the starch crystallinity is divided into three types, namely types A, B, and C. The type A diffraction pattern has a distinctive pattern with peaks of 23◦, 18◦, 17◦, and 15◦ (2θ), commonly found in cereal starch (rice) [169,170], and sweet potatoes [153–155,171]. The type B starch diffraction pattern is characterized by a small peak at 5.6◦ (2θ) and double peaks at 24◦ and 22◦ (2θ), commonly found in fruits, tubers, and high amylose starch [172,173]. Type C starch is a combination of different type A and type B crystal structures and is further classified into type CA (close to type A) and CB (close to type B). Type C starch showed strong diffraction peaks at 17◦ and 23◦ (2θ) and some small peaks around 5.6◦ and 15◦ (2θ). CA-type starch showed a shoulder peak at 18◦ (2θ), while CB type showed two shoulder peaks at 22◦ and 24◦ (2θ) [167]. Type C starch is commonly found in beans and sweet potatoes. Besides types A, B, and C, there is type V, which is formed due to the presence of amylose-fat complexes. Lopez-Rubio et al. [174] V-type crystals show diffraction peaks at points 7◦, 13◦, and 20◦ (2θ). Guo et al. [141], in their research, stated that sweet potato has a type C diffraction pattern. The acetylation modification process and its combination can affect starch crystallinity. Information regarding the effect of acetylation modification and its combination on various types of starch on crystallinity can be seen in Table 6.


**Table 6.** The effect of acetylation and its dual modification on the crystallinity of starch/flour.


**Table 6.** *Cont.*

The type of starch crystallinity due to modification treatment can change, as indicated by the change in diffraction intensity. The acetylation treatment conditions and the dual modifications applied can change the polymorphic properties of starch. In general, acetylation at low DS can weaken the diffraction intensity, but no change in the crystalline diffraction pattern was found. This is supported by several studies, which stated that starch acetate weakened diffraction intensity but still retained its type of crystallinity [10,61,62,73,77,83–86,175]. However, the results obtained in each study were not always the same. Shah et al. [89] reported that acetylation did not change the type of starch crystallinity but caused a decrease in the diffraction intensity peak and found an increase in the peak 2θ of 20◦. This peak may reflect the formation of amylose complexes with other compounds, such as amylose-lipids. In comparison, Adebowale et al. [90] reported that acetylation modification changed the crystallinity type of sword bean starch from type B to type C, indicating that polymorph A began to form during the modification.

The modification treatment of both acetylation and dual modification can increase, decrease, or not change the relative crystallinity (RC) of starch. The rearrangement of the double helix structure in starch granules can increase starch crystallinity, while the decrease occurs due to the partial gelatinization of starch granules [28,176]. The modification of starch caused an increase in RC, as seen in studies of acetylation modification of potato and cassava starch [83,147] and on the dual ANN-acetylation modification in mung bean starch [97]. Meanwhile, a decrease in RC occurred in the modified acetylation of white and purple yam starch [84], oat starch [89], high amylose maize starch [73], Banggai yam starch [175], waxy maize starch [86], waxy barley, waxy corn, waxy potato, waxy rice starch [10], and corn starch [79]. RC reduction also occurred in dual modifications acetylation-ANN of waxy potato starch, waxy barley starch, waxy rice starch, and waxy corn starch [10]. However, no RC changes were found in the modified acetylation of peas starch [62], cocoyam starch [85], and waxy maize starch [61].

The effect of acetylation on crystallinity depends on the type of starch and the treatment conditions. Differences in starch crystallinity are affected by several internal components of starch, including the interaction of double helices in crystals, the arrangement of double helices in the crystalline area, and the number of crystalline areas, which is affected by amylopectin content and chain length, and crystal size [164,165]. The polymorphic type and crystallinity of starch are also strongly affected by the internal components of starch and external factors such as environmental conditions and the presence of fat, which can form amylo-lipid complexes [28,177,178].

The substitution of hydroxyl groups with acetyl groups can weaken the hydrogen bonds that connect between starch molecules, which then causes a decrease in starch crys-

tallinity [159]. This statement is supported by several studies, which stated that acetylation modification caused a decrease in RC [10,73,79,84,89,175]. Nonetheless, the crystallinity index of cassava and potato starch increased after acetylation modification [83,147]. This was due to the weakening of the amorphous area of the granules followed by an increase in the crystalline area. Meanwhile, in several studies, acetylation with low DS only took place in the amorphous area of the granules, so it did not cause any changes in the crystalline area [61,62,85]. The effect of acetylation modification on the X-ray diffractogram profile can be seen in Figure 5.

**Figure 5.** XRD profiles of acetylated modified chestnut starch at different reaction times (NS = native starch, ACS-1 = acetylated starch 30 min, ACS-2 = acetylated starch 60 min, ACS-3 = acetylated starch 90 min) [14].

Figure 5 shows that the acetylation process does not cause changes in the diffraction peaks. Acetylation reactions with a low degree of substitution tend to attack amorphous areas so that no significant changes are found in the crystalline structure and do not change the diffraction pattern [14,85,175]. In Figure 5, two types of peaks are observed, namely the B-type peaks at 5.6◦ (2θ) and A-type peaks at 17.0◦ and 23.0◦ (2θ). The acetylation modification did not significantly change the A-type polymorphs at 17.0◦ and 23.0◦ (2θ) because these A-type polymorphs have a strong crystalline structure that is difficult to penetrate by acetylating reagents. However, the longer acetylation was able to reduce the number of B-type polymorphs at 5.6◦ (2θ), which had a weak crystalline structure. This could be due to the longer acetylation increasing the substitution of acetyl groups and reducing the hydroxyl groups in the amylose and amylopectin molecular chains, thereby damaging the long-range order of double helices so that the intra- and intermolecular bonds of starch weakened and caused a decrease in crystallinity [14]. However, an increase in crystallinity was found after 50 min of acetylation. This may have been due to the acid residue left after modification. This residue can cause the degradation of starch amorphous areas [175]. Wang et al. [179] reported an increase in crystallinity after the modification of acid hydrolysis because acid tends to attack amorphous areas. Acetylation with high DS can leave more acid residues and cause damage to amorphous areas. Thus, DS on acetylation modification in starch and flour greatly affects the crystallinity of the resulting starch.

Acetylation modification can be combined with several other modifications, especially hydrothermal modification, to obtain the desired crystallinity characteristics. In general, the combination of acetylation and hydrothermal modification (HMT, ANN) did not cause

a change in the crystalline diffraction pattern, but a change in the RC did occur. The combination of ANN + acetylation modification caused a decrease in crystallinity in waxy (rice, barley, corn, and potato) starches [10]. Nonetheless, the dual ANN + AS modification of mung bean starch caused an increase in crystallinity, but the crystallinity was lower when compared to the single ANN treatment. This indicated that acetylation modification could disrupt the crystal structure of ANN starch, which was already perfect. The dual modification treatment could also weaken the effect of changing one of the treatments, such as the ANN + acetylation modification in mung bean starch [97].

The modification of acetylation and the combination of ANN + acetylation did not cause any changes in the starch crystalline diffraction pattern. However, the modification of acetylation could cause a decrease in RC and increases in DS. This was due to the substitution of acetyl groups weakening the formation of intermolecular hydrogen bonds, causing a weakening of the crystalline structure [86]. The dual combination of ANN + acetylation modification could cause a greater decrease in RC than ANN modification. This decrease indicated that the effects of these two modifications are opposite to each other. Structural changes that occurred during ANN led to an increase in the mobility of the amylopectin chains in the amorphous lamellae and the movement of molecules in the crystalline region, facilitating the entry of acetyl groups into the granules. This increase in acetyl group substitution then caused greater damage [10,15].

#### *4.5. Comparison of Acetylated Modified Starch/Flour with Other Modifications*

The modification of acetylation in starch/flour has several advantages, including a relatively easy modification process that can produce starch/flour, which has a high swelling ability, clear starch paste, and good stability against retrogradation [14,98,100,102]. However, acetylation modification has several drawbacks because it requires a process to clean up chemical residues, which is quite expensive; the potential for waste is not environmentally friendly; and acetylated starch is unstable to thermal processes [15–17]. In addition, different characteristics may occur depending on the type of starch/flour and the processing conditions. The general comparison of acetylated modified starch/flour with other modifications can be seen in Table 7.


**Table 7.** The general comparison of acetylated modified starch/flour with other modifications.

#### **5. Conclusions and Future Research**

The modification of acetylation in starch/flour generally causes the fusion of starch granules, increasing the granule hydration ability, solubility, paste viscosity, storage stability and decreasing the gelatinization temperature and retrogradation stability. Acetylation generally does not change the crystalline structure of starch because it takes place in the amorphous areas of the granules, and some of them reduce the degree of crystallinity. Changes in starch/flour characteristics due to acetylation are strongly affected by the degree of acetyl group substitution. Dual acetylation modification with hydrothermal treatments such as HMT/ANN or cross-linking can produce starch/flour with better cooking and storage stability than native starch or single-modified starch and broaden its application to various products.

The modification of acetylation in starch/flour is continuing to develop, especially to increase the efficiency of the modification process and the application of acetylated starch in various fields and products. The efficiency of the modification process is being improved, including via pre-treatment through the formation of porous starch, such as by ultrasonication, partial hydrolysis, or oxidation. Acetylated starch/flour was also developed through dual modifications, including cross-linking and hydrothermal treatments, to increase thermal stability. The application of acetylated starch has also been developed more broadly to produce starch nanoparticles, which can then be used for encapsulation, starch-based composite, biofilms, drug delivery systems, and other applications.

**Author Contributions:** Conceptualization, E.S. and Y.C.; methodology, E.S., R.I. and T.A.R.; software, R.I.; validation, Y.C., R.I. and T.A.R.; formal analysis, E.S. and T.A.R.; investigation, E.S. and T.A.R.; resources, Y.C. and R.I.; data curation, R.I. and T.A.R.; writing—original draft preparation, E.S. and T.A.R.; writing—review and editing, Y.C. and R.I.; visualization, E.S. and R.I.; supervision, Y.C.; project administration, E.S. and R.I.; funding acquisition, E.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This article was supported by Universitas Padjadjaran, Indonesia.

**Institutional Review Board Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The authors would like to thank the Rector of Universitas Padjadjaran and The Ministry of Education, Culture, Research, and Technology of the Republic of Indonesia.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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## *Article* **Optimization of Cellulose Nanofiber Loading and Processing Conditions during Melt Extrusion of Poly(3-hydroxybutyrate-***co***-3-hydroxyhexanoate) Bionanocomposites**

**Siti Shazra Shazleen 1, Fatimah Athiyah Sabaruddin 1, Yoshito Ando <sup>2</sup> and Hidayah Ariffin 1,3,\***


**Abstract:** This present study optimized the cellulose nanofiber (CNF) loading and melt processing conditions of poly(3-hydroxybutyrate-*co*-3-hydroxyhexanoate) P(HB-*co*-11% HHx) bionanocomposite fabrication in twin screw extruder by using the response surface methodology (RSM). A face-centered central composite design (CCD) was applied to statistically specify the important parameters, namely CNF loading (1–9 wt.%), rotational speed (20–60 rpm), and temperature (135–175 ◦C), on the mechanical properties of the P(HB-*co*-11% HHx) bionanocomposites. The developed model reveals that CNF loading and temperature were the dominating parameters that enhanced the mechanical properties of the P(HB-*co*-11% HHx)/CNF bionanocomposites. The optimal CNF loading, rotational speed, and temperature for P(HB-*co*-11% HHx) bionanocomposite fabrication were 1.5 wt.%, 20 rpm, and 160 ◦C, respectively. The predicted tensile strength, flexural strength, and flexural modulus for these optimum conditions were 22.96 MPa, 33.91 MPa, and 1.02 GPa, respectively, with maximum desirability of 0.929. P(HB-*co*-11% HHx)/CNF bionanocomposites exhibited improved tensile strength, flexural strength, and modulus by 17, 6, and 20%, respectively, as compared to the neat P(HB-*co*-11% HHx). While the crystallinity of P(HB-*co*-11% HHx)/CNF bionanocomposites increased by 17% under the optimal fabrication conditions, the thermal stability of the P(HB-*co*-11% HHx)/CNF bionanocomposites was not significantly different from neat P(HB-*co*-11% HHx).

**Keywords:** poly(3-hydroxybutyrate-*co*-3-hydroxyhexanoate); cellulose nanofiber; bionanocomposite; melt-extrusion processing; optimization; response surface methodology

### **1. Introduction**

The growing use of plastics around the world has led to an increase in plastic waste. In Malaysia, plastic waste constituted 19% of the total waste generated where most of the commodity plastics are derived from petroleum, and they are single use, i.e., they are be discarded after being used only once [1]. This leads to the accumulation of disposal plastic waste that mostly ends up in landfills or dumps in the open environment. According to Jambeck et al. [2], Malaysia was placed eighth among the world's top 10 countries for having poorly managed plastic waste. In light of the environmental damage caused by plastic waste pollution, and also the difficulties of managing that waste on land and in water, there is indeed an urgent need to establish sustainable and cost-effective solutions. Therefore, recent advancements in biodegradable and recyclable polymers are essential, considering the uncertainty of petroleum usage worldwide. Manufacturing industries are transitioning to more eco-friendly, sustainable economic production as a consequence of the

**Citation:** Shazleen, S.S.; Sabaruddin, F.A.; Ando, Y.; Ariffin, H. Optimization of Cellulose Nanofiber Loading and Processing Conditions during Melt Extrusion of Poly(3-hydroxybutyrate-*co*-3 hydroxyhexanoate) Bionanocomposites. *Polymers* **2023**, *15*, 671. https://doi.org/10.3390/ polym15030671

Academic Editor: Raffaella Striani

Received: 10 November 2022 Revised: 2 December 2022 Accepted: 30 December 2022 Published: 28 January 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

intense pace of scientific and technological advancement. Today, the most well-known and widely used polymers in a multitude of areas are polylactic acid (PLA), polycaprolactone (PCL), polyglycolide (PGA), and polyhydroxyalkanoates (PHAs). Among all, PHA has drawn significant attention as one of the most viable substitutes for synthetic polymers. This is because PHAs are more compostable and biodegradable in marine conditions than PLA. Although PLA is compostable, it may remain in the ocean for up to 1000 years before it can be composted [3]. PCL and PGA are considered to be non-toxic, yet because of their higher crystallinity they degrade more slowly than PLA [4]. Moreover, PHA properties are comparable to most non-degradable materials [5], making PHA suitable for industrial uses, particularly in packaging.

PHA is recognized as a sustainable alternative among the most prominent synthesized and commercialized biodegradable polymers as it can be converted into water and carbon dioxide in the presence of oxygen, or into methane under anaerobic conditions without forming toxic products, by microorganisms found in water and soil [6]. PHAs are a type of linear biopolyester made up of hydroxyalkanoate (HA) units organized in a basic structure produced via bacterial fermentation and are currently being marketed as a means of creating a more sustainable future [7]. Their properties differ significantly depending on the structure and composition of their monomers [8]. PHAs offer several benefits over petroleum-based polymers, including the ability to be synthesized from renewable carbon sources, processability, and biodegradability. The most widespread and extensively studied member of this family is the homopolymer poly(3-hydroxybutyrate) (PHB) and the copolymers poly(3-hydroxybutyrate-*co*-3-hydroxyvalerate) (PHBV) and poly(3-hydroxybutyrate-*co*-3-hydroxyhexanoate) (PHBHHx).

PHBHHx is one of the most promising biodegradable aliphatic polyesters of the PHA family due to the fact that it has unique combination properties including full anaerobic degradability, moisture resistance, and good barrier properties [9]. PHBHHx has higher elastic characteristics and a wider processing window than PHB and PHBV due to the relatively long alkyl side chain, making it a suitable biopolymeric source for developing biocomposites with increased flexibility [10,11]. However, despite their potential, the effective utilization of PHBHHx-based materials is exacerbated by their low mechanical properties and difficulties in processing as compared to synthetic polymers [12]. In addition, PHBHHx-based materials are still hindered by their high production costs and are dependent on the performance of bacterial fermentation [13]. Their high manufacturing cost has surpassed the cost of manufacturing conventional plastics. These limitations have restrained the applicability of these materials in a wide range of applications. The incorporation of nanofillers, particularly bio-based nanofillers in PHBHHx, is seen as an ideal strategy for developing bio-based nanocomposites with superior mechanical properties.

Nanofillers have a higher aspect ratio than micro-sized fillers, giving them better reinforcement effects. Recent studies have focused on the use of nanocellulose, particularly cellulose nanofiber (CNF) as reinforcing bio-based nanomaterials. CNF has been known for its outstanding properties such as high mechanical properties and thermal stability, large specific surface area, renewability, biodegradability, and biocompatibility properties that can be produced by mechanical or chemical treatments which are advantageous for reinforced polymers [14]. CNF has a low coefficient of thermal expansion of 0.1 ppm/K; an estimated strength of 2–3 GPa, which is five times stronger than mild steel; and a high Young's modulus of 130–150 GPa [15]. Recently, the effect of CNF as a reinforcement material for PHBHHx has been widely reported [14–17]. Most studies agreed that the addition of CNF can enhance the mechanical properties of PHBHHx bionanocomposites significantly.

Nonetheless, CNF loading beyond a certain percentage can be detrimental as it may lead to significant nanofiller agglomerations. Several studies have documented that the improvement of polymer nanocomposite may endure immense difficulty attributable to the dispersion of nanofibers [18–22]. The hypothesis is that if the nanofibers are evenly distributed throughout the polymer matrix, the optimal nanocomposite properties can be attained. It should be highlighted at this stage that proper alignment and control of nanofiber dispersion have remained a key problem for many years [23]. The effectiveness of load transfer between the nanofibers and the polymer matrix and subsequently the mechanical properties of nanocomposite are both governed by the strength of adhesion in the interface region [23]. Consequently, the characteristics of the nanocomposite deteriorate if there is inadequate adhesion at the interphase.

In complement to the CNF reinforcing effect, in practice, the applied melt-processing process and conditions can have a profound effect on the mechanical properties [12]. Nonetheless, studies on the mechanical properties of PHBHHx/CNF bionanocomposites under the influence of processing temperature and shear stress have been scarcely reported. To our knowledge, there are no studies that explicitly correlate variations in mechanical properties to the practical processing parameters and CNF loading used in melt extrusion of PHBHHx. Identifying the optimal values for these parameters to enhance the mechanical properties of the bionanocomposite is a challenging and complicated task as there are so many design parameters to consider. In light of this, the prediction and assessment of design parameters is vital for the optimum design of bionanocomposites for a particular application. However, few studies to date have quantitatively optimized the mechanical characteristics of PHBHHx/CNF bionanocomposites depending on the design parameters. Hence, the present in-depth research was performed with the purpose of improving the mechanical characteristics of P(HB-*co*-11% HHx) through the optimization of CNF loading and processing conditions. Through the use of Design-Expert software, mathematical models were generated between the aforementioned parameters and responses. The validation experiment was conducted to verify whether the obtained optimal conditions result in the intended mechanical properties for P(HB-*co*-11% HHx)/CNF bionanocomposites.

#### **2. Materials and Methods**

#### *2.1. Materials*

Poly(3-hydroxybutyrate-*co*-3-hydroxyhexanoate) with 11 mol% of HHx unit, P(HB-*co*-11% HHx) as determined by 1H-NMR was supplied by ©KANEKA Biodegradable Polymer TM (Kaneka Co., Osaka, Japan). Spray-dried CNF was purchased from ZoepNano Sdn. Bhd., (Serdang, Malaysia) and used in this experiment as nanofiller. The CNF powder had an average particle size of less than 100 nm.

#### *2.2. P(HB-co-11% HHx) Bionanocomposite Fabrication and Molding*

P(HB-*co*-11% HHx)/CNF bionanocomposite was fabricated by using twin-screw extruder (Labtech Engineering Co., Ltd., Bangkok, Thailand) with the screw diameter of 16 mm. Prior to mixing, P(HB-*co*-11% HHx) powder was dried in an oven at 60 ◦C for 24 h to remove moisture because it is very essential to minimize the hydrolytic degradation during the processing at high temperature [24]. The P(HB-*co*-11% HHx) and CNF powder were mechanically mixed before being fed into the extruder. After the extrusion, the obtained filament was granulated by a pelletizer (SHEER SGS 25–E4, MAAG Group manufactures, Zurich, Switzerland) and then molded into 11 × 11 cm film with thickness of 1 mm by direct compression molding using a hydraulic hot press at temperature of 160 ◦C and 110 kg.cm−<sup>2</sup> pressure for 10 min. Cooling was then performed for 30 min under the same pressure.

#### *2.3. Characterization of P(HB-co-11% HHx) Bionanocomposite* 2.3.1. Mechanical Analysis

Mechanical properties of bionanocomposites were analyzed using an Instron Universal Testing Machine–Instron 5566 (Instron, Norwood, MA, USA) with load cell of 10 kN at room temperature. Five dog-bone-shaped specimens for tensile strength were tested according to the standard method of ASTM D 638-05 with crosshead speed of 1 mm/min. Meanwhile, flexural strength and modulus tests were performed at 1.21 mm/min speed according to the standard method of ASTM D790. One-way ANOVA and Duncan's multiple range test were

used to statistically evaluate the mechanical properties of the fabricated bionanocomposites following the validation experiment.

#### 2.3.2. Experimental Design and Optimization

A face-centered central composite design (CCD), comprising three different factors, was used to run the experiment: CNF content (X1) (1 to 9 wt.%), rotational speed (X2) (20 to 60 rpm), and temperature (X3) (135 to 175 ◦C). In this study, CCD was used to determine the optimum melt-extrusion conditions in fabrication of P(HB-*co*-11% HHx)/CNF bionanocomposites with maximum mechanical properties. The temperature was set between 135 and 175 ◦C in consideration of the onset melting point of P(HB-*co*-11% HHx) from differential scanning calorimetry (DSC), which is approximately 155 ◦C, and onset degradation of CNF and P(HB-*co*-11% HHx) from thermogravimetric (TG) analysis around 280–290 ◦C. These variables were studied at five different levels coded as –α, −1, 0, +1, and +α, where α = 2. Actual and coded values of the variables are summarized in Table 1. The CCD consists of 19 runs including five replications of center points to determine pure error and reduce the variability in the data collection. The mechanical properties of the tensile strength (Y1), flexural strength (Y2), and flexural modulus (Y3) were recorded as responses. The experimental design arrangement was randomized to prevent systematic error and minimize the effects of uncontrolled factors.


**Table 1.** Central composite design matrix of coded and actual factor level.

The experimental data were analyzed, and response surface plots were generated using Design-Expert statistical software (Version 7.0, Stat-Ease Inc., Minneapolis, MN, USA). Analysis of variance (ANOVA) was used to determine the significance of each factor and the regression coefficient of the linear, quadratic, and interaction terms with a confidence level over 95% or a *p*-value lower than 0.05. The influence of the factors on the responses was illustrated using a contour plot, and the optimal levels were identified. Actual experimentation was performed to verify and validate the predicted optimal conditions obtained from software for CNF content, rotational speed, and temperature. Data were fitted to a second-order polynomial equation as shown in Equation (1), where Y1, Y2, and Y3 are the responses; X1, X2, and X3 are the varied factors ranging from –2 to 2, which influence the response Y; β<sup>0</sup> is the constant coefficient; β1, β2, and β<sup>3</sup> are linear coefficients; β11, β22, and β<sup>33</sup> are quadratic coefficients; and β12, β13, and β<sup>23</sup> are interaction coefficients. The validity

and adequacy of the regression models were verified by comparing the experimentally obtained data with the fitted value predicted by the models.

$$\mathbf{Y} = \boldsymbol{\beta}\_0 + \boldsymbol{\beta}\_1 \mathbf{X}\_1 + \boldsymbol{\beta}\_2 \mathbf{X}\_2 + \boldsymbol{\beta}\_3 \mathbf{X}\_3 + \boldsymbol{\beta}\_{11} \mathbf{X}\_1^2 + \boldsymbol{\beta}\_{22} \mathbf{X}\_2^2 + \boldsymbol{\beta}\_{33} \mathbf{X}\_3^2 + \boldsymbol{\beta}\_{12} \mathbf{X}\_1 \mathbf{X}\_2 + \boldsymbol{\beta}\_{13} \mathbf{X}\_1 \mathbf{X}\_3 + \boldsymbol{\beta}\_{23} \mathbf{X}\_2 \mathbf{X}\_3 \tag{1}$$

#### 2.3.3. Thermal Stability Analysis

Thermal stability of neat P(HB-*co*-11% HHx) and P(HB-*co*-11% HHx)/1.5% CNF bionanocomposites were analyzed using a thermogravimetric analyzer (TGA 4000, Perkin Elmer, Waltham, MA, USA). The samples weighing around 8–12 mg were placed on a ceramic pan and heated from 50 to 500 ◦C at a heating rate of 10 ◦C/min under nitrogen flow of 100 mL/min.

#### 2.3.4. X-ray Diffraction Analysis

X-ray diffraction (XRD) spectroscopy was used to quantify the crystallinity of bionanocomposites. An automated Shimadzu 6000 X-ray diffractometer (Tokyo, Japan) operating at 40 kV with a current of 20 mA and Cu radiation of =1.5406 between 2T = 10 and 50◦ was used for the experiment.

#### **3. Results and Discussion**

#### *3.1. Response Surface Model Analysis*

The design matrix generated by Design-Expert software included data on tensile strength, flexural strength, and flexural modulus allowed regression analysis to be performed to identify the best-fit model for the experimental data, with the derived regression equation being used to predict a particular response at points that are not included in regression [25]. The tensile strength, flexural strength, and flexural modulus were indicated to be correlated with CNF loading, rotating speed, and temperature by regression analysis of the experimental data. Different parameters including the model F value, the lack of fit F value, the coefficient of determination R2, adjusted R2, press value, and coefficient of variation (CV) were used to assess the model's adequacy. The experimental and predicted values of responses are summarized in Table 2.

**Table 2.** The experimental and predicted values of responses.


\* Exp.: experimental; \*\* Pred.: predicted.

Full quadratic models were adopted as the best-fitting model for all responses as detailed in Table 3. The models were chosen using an ANOVA with a sufficient coefficient of determination (R2) (above 80%), insignificant lack-of-fit probability (*p* > 0.005), and significant model probability (*p* < 0.05). A significant *p*-value and an insignificant lack of fit, respectively, indicate a good model and a good fit of the model to the data [2,3]. The linear (X1, X2, X3), interactive (X1X2, X1X3, X2X3), and quadratic (X1 2, X2 2, X3 2) *p*-values are presented in the same table. A lower *p*-value (*p* < 0.05) suggests that the corresponding coefficient is more significant.


**Table 3.** Analysis of variance (ANOVA) for response surface quadratic model.

\* Statistically significant at *p* < 0.05 for model; \*\* statistically insignificant at *p* > 0.05 for the lack of fit.

The *p*-values for the lack-of-fit of tensile strength, flexural strength, and modulus were 0.1462, 0.6977, and 0.8991, respectively, which were higher than 0.05, signifying that the model had insignificant lack-of-fit. This is a good indicator that the proposed model fits the experimental data, and the factors have a significant effect on the responses. If the model exhibits significant lack-of-fit, it should not be applied to predict a particular response as it fails to represent data at points that were not included in the regression [25,26]. Ghelich et al. [27] mentioned that the significant lack-of-fit may be related to (i) replicate measurements with repetitive center point data that are consistent with each other, (ii) missing significant higher order non-standard terms or engagement in the model, (iii) larger residual errors compared to the pure error, or (iv) inadequate equal error at any points, i.e., heteroscedasticity (significant disparity between sizes of the observations), implying a more appropriate model fitting.

The coefficient of determination R<sup>2</sup> measures the quality of experimental data fitting to the model where the value was approximately near 1, highlighting that the dependent variable was predicted with less error than the independent variables of CNF loading, rotational speed, and temperature. The R2 values for tensile strength, flexural strength, and modulus were 0.9092, 0.9883, and 0.9619, respectively, signaling a high proportion of variability predicted by the models of 91%, 99%, and 96%, respectively, from CNF loading, rotational speed, and temperature of P(HB-*co*-11% HHx)/CNF bionanocomposite fabrication, as seen in Table 3. Moreover, a high R2 value close to 1 displays good agreement between predicted and reported results within the experimental range [28]. Figure 1 shows the plot of experimental by predicted values, where the proximity of the points scattered along the fitted line demonstrates agreement between experimental and predicted values, evidencing the adequacy of models to estimate the mechanical properties of P(HB-*co*-11%

HHx)/CNF bionanocomposites prepared at varying CNF loadings, rotational speeds, and temperatures. Hence, these findings affirm that all the responses are affected by experimental factors.

The mathematical relationship between the response and variable process parameters can be established using response surface modeling. The final regression equations (in terms of coded factors) to predict the effect of factors on the responses are shown in Equations (2)–(4), where Y1, Y2, and Y3 represent tensile strength, flexural strength, and flexural modulus, respectively; X1, X2, and X3 are CNF loading, rotational speed, and temperature, respectively.

$$\begin{array}{c} \text{Y}\_1 = 21.35 - 0.61 \,\text{X}\_1 - 0.17 \,\text{X}\_2 - 0.44 \,\text{X}\_3 - 0.020 \,\text{X}\_1^2 - 0.11 \,\text{X}\_2^2 - 1.15 \,\text{X}\_3^2 + 0.12 \,\text{X}\_1 \text{X}\_2 - 0.02 \,\text{X}\_3^2 - 0.08 \,\text{X}\_1 \text{X}\_3\\ \text{ 0.22 } \text{X}\_1 \text{X}\_3 - 1.15 \,\text{X}\_2 \text{X}\_3 \end{array} \tag{2}$$

$$\begin{array}{c} \text{Y}\_2 = 31.49 - 0.62 \,\text{X}\_1 - 0.23 \,\text{X}\_2 - 0.18 \,\text{X}\_3 + 0.29 \,\text{X}\_1^2 + 0.077 \,\text{X}\_2^2 - 1.54 \,\text{X}\_3^2 + 0.045 \,\text{X}\_1 \text{X}\_2 \\ \text{ + } 0.43 \,\text{X}\_1 \text{X}\_3 - 0.33 \,\text{X}\_2 \text{X}\_3 \end{array} \tag{3}$$

$$\begin{aligned} \mathbf{Y}\_3 &= 1.06 + 0.014 \,\mathbf{X}\_1 + 0.003375 \,\mathbf{X}\_2 + 0.038 \,\mathbf{X}\_3 - 0.007963 \,\mathbf{X}\_1^2 - 0.004713 \,\mathbf{X}\_2^2 - 0.037\\ &\quad \mathbf{X}\_3^2 + 0.0085 \,\mathbf{X}\_1 \mathbf{X}\_2 + 0.012 \,\mathbf{X}\_1 \mathbf{X}\_3 - 0.007963 \,\mathbf{X}\_2 \mathbf{X}\_3 \end{aligned} \tag{4}$$

**Figure 1.** *Cont.*

The polynomial equation in terms of uncoded variables of factors was obtained by exchange of coded variables with actual values as shown in Equations (5)–(7).

Y1 = −292.88125 + 1.20304 (CNF content) + 0.95185 (Rotational speed) + 3.82790 (Temperature) <sup>−</sup> 0.00499155 (CNF content)2 <sup>−</sup> 0.00106216 (Rotational speed)2 <sup>−</sup> 0.011550 (Temperature)2 + 0.006125 (CNF content)(Rotational speed) − 0.011 (CNF content)(Temperature) − 0.0059 (Rotational speed)(Temperature) (5) Y2 = − 333.88437 − 4.40841 (CNF content) + 0.41136 (Rotational speed) + 4.78479 (Temperature) + 0.071529 (CNF content)2 + 0.000773649 (Rotational speed)2 <sup>−</sup>0.015414 (Temperature)2 + 0.00225 (CNF content)(Rotational speed) + 0.021250 (CNF content)(Temperature) − 0.00327 (Rotational speed)(Temperature) (6) Y3 = − 8.61259 − 0.079280 (CNF content) + 0.013608 (Rotational speed) + 0.11970 (Temperature) <sup>−</sup> 0.0019907 (CNF content)2 <sup>−</sup> 0.0000471284 (Rotational speed)2 <sup>−</sup> 0.000373378 (Temperature)2 + 0.000425 (CNF content)(Rotational speed) + 0.000575 (CNF content)(Temperature) − 0.000075 (Rotational speed)(Temperature) (7)

#### *3.2. Effect of Melt-Extrusion Processing Conditions on Mechanical Properties of P(Hb-Co-11% HHx)/CNF Bionanocomposites*

The effect of each processing factor, namely CNF loading, rotating speed, and temperature, on the mechanical characteristics of P(HB-*co*-11% HHx)/CNF bionanocomposite was assessed using a quadratic regression model. The contour and response surface plots generated from the empirical predicted model in Equations (2)–(4) can be used to better assess the whole relationship between the independent variable X and the response variable Y, as depicted in Figures 2–4. The response surface plots, which are shown from the pairwise combination of targeted variables by keeping other variables at their center point level, demonstrate the mutual interaction of the independent factors. The response surface plots were converted into a three-dimensional (3D) diagram to determine the optimal conditions for each variable towards maximum mechanical properties. Thus, in all response surface and 3D contour plots, the fixed variable is held at a rotational speed of 30 rpm.

**Figure 2.** The 3D and response surface contour plots for the dependence of P(HB-*co*-11% HHx)/CNF bionanocomposite's tensile strength on CNF loading and temperature as significant factors.

**Figure 3.** The 3D and response surface contour plots for the dependence of P(HB-*co*-11% HHx)/CNF bionanocomposite's flexural strength on CNF loading and rotational speed as significant factors.

**Figure 4.** The 3D and response surface contour plots for the dependence of P(HB-*co*-11% HHx)/CNF bionanocomposite's flexural modulus on CNF loading and temperature as significant factors.

Figure 2 depicts the 3D and response surface contour plots for the effects of CNF loading and temperature on tensile strength based on Equation (1). Results indicate that these variables are the most important factors influencing tensile strength (Table 3). It was

observed that further increment in CNF loading of more than 3 wt.% reduced the tensile strength of the bionanocomposites. This might be due to the agglomeration of the CNF within P(HB-*co*-11% HHx) matrix which disrupted the compactness and the spherulite structure of the bionanocomposites [29,30]. Meanwhile, the tensile strength of P(HB-*co*-11% HHx)/CNF bionanocomposites increased as temperature rose from 135 to 155 ◦C before declining from 155 to 175 ◦C. This result may be explained by the fact that P(HB-*co*-11% HHx) twin-screw extrusion at high temperature can result in a reduction in molecular weight owing to thermal degradation [10,24]. It has been discovered that random chain scission is the process of degradation causing PHAs to rapidly lose molecular weight when exposed to heat [31].

Figure 3 the depicts 3D and response surface contour plots for the effects of CNF loading and rotational speed on flexural strength based on Equation (3). CNF loading (linear and quadratic) had a significant effect (*p* < 0.05) on flexural strength, as well the linear effect of rotational speed, interaction effect of CNF loading-rotational speed, and CNF loading-temperature and also the quadratic effect of temperature (Table 3). In comparison to the quadratic effect of temperature, the quadratic effect of CNF loading was less pronounced, with the coefficient of each factor of 1.54 and 0.29, respectively (Equation (3)). It was discovered that CNF dispersion in P(HB-*co*-11% HHx) was unaffected by rotating speed, as demonstrated by insignificant changes in tensile strength and flexural modulus. Conversely, a significant linear effect was noticed for flexural strength (Table 3), where a minor improvement could be noticed while processing P(HB-*co*-11% HHx) bionanocomposite at a slower rotational speed (Figure 3). Flexural strength decreases with the increase in CNF loading and rotational speed from 1 to 9 wt.% and 20 to 60 rpm, respectively.

Similar to flexural strength, significant quadratic effects of both CNF loading and temperature were observed against flexural modulus with no significant interactions between all factors (Table 3). Flexural modulus decreased with the increase in processing temperature from 155 to 175 ◦C, which is similar to the results of tensile and flexural strength. As aforementioned, fabricating bionanocomposites at high processing temperature leads to polymer degradation due to random chain scission, resulting in brittleness of the bionanocomposites [32,33]. Since the polymer molecular weight substantially decreases at temperatures above 155 ◦C, processing at those temperatures seems unsuitable. However, different from tensile and flexural strength, the addition of CNF up to 9 wt.% did not negatively affect the flexural modulus (Figure 4). In this study, CNF distribution in P(HB-*co*-11% HHx) was found to be unaffected by rotational speed, as proved by in Table 3, where no significant interaction can be seen between CNF loading and screw speed.

#### *3.3. Response Surface Optimization of P(HB-co-11% HHx)/CNF Bionanocomposites*

Numerical optimization was performed in accordance with the design and analysis, taking each criterion into consideration (Table 4). Due the severity of the effect on tensile and flexural strength as well as flexural modulus, these responses were set at a maximum value. As shown in Table 4, the optimal CNF loading, rotational speed, and temperature for P(HB-*co*-11% HHx)/CNF bionanocomposite fabrication were 1.5 wt.% CNF, 20 rpm, and 160 ◦C, respectively. For these ideal conditions, it was predicted that the tensile strength, flexural strength, and modulus would be 22.96 MPa, 33.91 MPa, and 1.022 GPa, respectively, with a maximum desirability of 0.929.

#### *3.4. Validation Experiment*

The mechanical properties of the P(HB-*co*-11% HHx)/CNF bionanocomposite fabricated at the proposed parameter were consistent with the predicted value throughout the validation experiment as tabulated in Table 5, with the exception of the tensile strength, which was 9% higher. This finding was very good and favorable in light of the objective of this study, which was to attain high mechanical properties.


**Table 4.** The settings and solutions of the numerical optimization criterion.

**Table 5.** Comparison between predicted and experimental values of P(HB-*co*-11% HHx)/CNF bionanocomposites fabricated at optimal conditions.


The mechanical properties of the neat P(HB-*co*-11% HHx) and P(HB-*co*-11% HHx)/CNF bionanocomposites prepared under these ideal conditions are shown in Table 6. This result demonstrated the ability of CNF to increase tensile strength, flexural strength, and flexural modulus by 17, 6, and 20%, respectively.

**Table 6.** Mechanical properties of neat P(HB-*co*-11% HHx) and P(HB-co-11% HHx)/CNF1.5 bionanocomposites.


All data are means of five replicates ± S.D. The superscript letters indicate significant difference (*p* < 0.05) according to Duncan's multiple range test.

#### *3.5. Effect of CNF on Thermal Stability and Crystallinity Properties of P(HB-co-11% HHx)/CNF Bionanocomposites*

The thermal stability of spray dried-CNF, neat P(HB-*co*-11% HHx) and P(HB-*co*-11% HHx)/CNF1.5 bionanocomposites was investigated by thermogravimetric analysis. The TG and DTG curves are presented in Figure 5, and the thermal degradation data are summarized in Table 7. T10 represents the temperature at which 10% of mass reduction was recorded, while Tmax represents the temperature at maximum mass loss, which was taken from the DTG thermogram.

**Figure 5.** TG and DTG curves for neat P(HB-*co*-11% HHx) and P(HB-*co*-11% HHx)/CNF1.5 bionanocomposites (Red arrow shows the zoom in peak for SD-CNF for DTG).

**Table 7.** Thermal degradation temperatures of neat P(HB-*co*-11% HHx) and P(HB-*co*-11% HHx)/CNF at 10 wt.% of weight loss (T10) and maximum degradation temperature (Tmax).


P(HB-*co*-11% HHx) thermal decomposition commenced at 280 ◦C and was completed at 320◦C with single step degradation profile. As seen from Table 7, the T10 and Tmax of neat P(HB-*co*-11% HHx) were 294 and 307 ◦C, respectively. Spray dried-CNF was less thermally stable at the beginning, where some weight loss started to occur at temperature around 100 ◦C, indicating the removal of moisture. Nevertheless, the Tmax value was higher compared to P(HB-*co*-11% HHx). The addition of 1.5 wt.% CNF did not change the thermal stability of neat P(HB-*co*-11% HHx).

X-ray diffraction (XRD) analysis was performed to determine the crystallinity properties of neat P(HB-*co*-11% HHx) and optimized P(HB-*co*-11% HHx)/CNF1.5 bionanocomposites. Figure 6 displays the XRD patterns for both bionanocomposite samples, and the crystallinity index calculated is tabulated in Table 8.

**Table 8.** Crystallinity index (CI) of neat P(HB-*co*-11% HHx) and P(HB-*co*-11% HHx)/CNF bionanocomposites.


**Figure 6.** XRD patterns of neat P(HB-*co*-11% HHx) and optimized P(HB-*co*-11% HHx)/CNF1.5 bionanocomposites.

Sharp crystal peaks are visible in this sample, and their intensity was noticeably higher than that seen in the neat P(HB-*co*-11% HHx) sample, in accordance with the pattern of a bionanocomposite film containing 1.5 wt.% of CNF. This can be linked to the effective CNF distribution in the film matrix. This finding demonstrated that the addition of CNF promotes the growth of crystalline areas in the P(HB-*co*-11% HHx) matrix as evidenced by the increase in crystallinity index from 25.1 to 30.1%. As proved in Table 6, it has been suggested that an increase in crystallinity leads to an increase in the strength and modulus of bionanocomposites. Additionally, there is no peak shift in the XRD pattern of P(HB-*co*-11% HHx)/CNF bionanocomposites, indicating that the melt compounding by the twin screw extrusion process did not alter their crystal structures [24].

#### **4. Conclusions**

P(HB-*co*-11% HHx)/CNF bionanocomposites fabricated using twin screw extruders were evaluated and predicted, and their melt-extrusion processing parameters were optimized using response surface modeling analysis based on the CCD method. The individual and interaction effects of three important melt-extrusion processing conditions, namely CNF loading, rotation speed, and temperature, on mechanical properties of P(HB-*co*-11% HHx)/CNF bionanocomposites (tensile strength, flexural strength, and modulus) were adequately modeled and optimized. It was discovered that CNF loading and temperature have a substantial effect on the mechanical properties of the P(HB-*co*-11% HHx)/CNF bionanocomposites; however, rotational speed has a less influential effect on mechanical properties except flexural modulus. At optimum CNF loading, rotational speed, and temperature of 1.5 wt.%, 20 rpm, and 160 ◦C, tensile strength, flexural strength, and flexural modulus are reported to be 22.96 MPa, 33.91 MPa, and 1.022 GPa, respectively. The response values obtained from the validation experiment were in good agreement with the predicted values. Validation tests proved that the response surface equations were adequate for predicting responses. The results of the TG and DTG study indicated that the thermal stability of the P(HB-*co*-11% HHx) matrix did not differ significantly from neat P(HB-*co*-11% HHx) when the optimum amount of CNF was introduced. The XRD analysis revealed that the addition of CNF increased crystallinity, which promotes the formation of crystalline regions in the P(HB-*co*-11% HHx) matrix. This research highlights the importance of optimal melt-extrusion processing conditions and their influence on the mechanical properties of P(HB-*co*-11% HHx)/CNF bionanocomposites. The findings of this research are anticipated to aid in the invention of novel P(HB-*co*-11% HHx) materials for packaging and other applications, taking into consideration the relevance of the correlation between melt-extrusion process parameters and the potential for process optimization to

enhance the mechanical properties of P(HB-*co*-11% HHx). This process optimization can be further used as one of the technical developments for bio-based nanocomposites derived from green materials with excellent prospects in novel high performance food packaging materials. With the optimum process conditions, the properties such as high mechanical performance with additional desired function such good thermal and barrier properties can be achieved. In addition, the application of bio-based materials to replace the synthetic fossil-based materials provides a range of benefits for various economic entities. Positive feedback has also been reported concerning ecological, economic, and social aspects associated with the transition from traditional plastic to bio-based plastics that can be widely used as packaging materials and act as a significant driver of the industry's growth.

**Author Contributions:** The manuscript was completed through the contributions of all authors. Conceptualization, H.A. and S.S.S.; methodology, H.A. and S.S.S.; software, H.A.; formal analysis, S.S.S. and F.A.S.; investigation, S.S.S. and F.A.S.; data curation, H.A.; writing—original draft, S.S.S.; writing—review and editing, H.A. and F.A.S.; visualization: S.S.S. and F.A.S.; supervision, H.A. and Y.A.; project administration, H.A.; funding acquisition, H.A. and Y.A. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the UPM-Kyutech Matching Grant (UPM-Kyutech/2020/9300471).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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