**Protein–TiO2: A Functional Hybrid Composite with Diversified Applications**

**Luis Miguel Anaya-Esparza 1,2 , Zuamí Villagrán-de la Mora <sup>3</sup> , Noé Rodríguez-Barajas <sup>2</sup> , Teresa Sandoval-Contreras <sup>1</sup> , Karla Nuño <sup>4</sup> , David A. López-de la Mora 4,\* , Alejandro Pérez-Larios 2,\* and Efigenia Montalvo-González 1,\***


Received: 4 November 2020; Accepted: 2 December 2020; Published: 7 December 2020

**Abstract:** Functionalization of protein-based materials by incorporation of organic and inorganic compounds has emerged as an active research area due to their improved properties and diversified applications. The present review provides an overview of the functionalization of protein-based materials by incorporating TiO<sup>2</sup> nanoparticles. Their effects on technological (mechanical, thermal, adsorptive, gas-barrier, and water-related) and functional (antimicrobial, photodegradation, ultraviolet (UV)-protective, wound-healing, and biocompatibility) properties are also discussed. In general, protein–TiO<sup>2</sup> hybrid materials are biodegradable and exhibit improved tensile strength, elasticity, thermal stability, oxygen and water resistance in a TiO<sup>2</sup> concentration-dependent response. Nonetheless, they showed enhanced antimicrobial and UV-protective effects with good biocompatibility on different cell lines. The main applications of protein–TiO<sup>2</sup> are focused on the development of eco-friendly and active packaging materials, biomedical (tissue engineering, bone regeneration, biosensors, implantable human motion devices, and wound-healing membranes), food preservation (meat, fruits, and fish oil), pharmaceutical (empty capsule shell), environmental remediation (removal and degradation of diverse water pollutants), anti-corrosion, and textiles. According to the evidence, protein–TiO<sup>2</sup> hybrid composites exhibited potential applications; however, standardized protocols for their preparation are needed for industrial-scale implementation.

**Keywords:** proteins; titanium dioxide; functionalization; hybrid composites

#### **1. Introduction**

Nowadays, the development of eco-friendly materials with advanced characteristics and diverse applications is an active research area [1,2]. Hybrid compounds are composites that consist of combining inorganic–inorganic (e.g., TiO2–Ag), organic–organic (e.g., wheat gluten–cellulose), and organic–inorganic (e.g., collagen–TiO2) [3–5], and they can be synthesized by spin and dip coating, slot-casting, electrochemical self-assembly, and chemical vapor, atomic or molecular layer

deposition methods [6]. In general, hybridization or functionalization of organic compounds by incorporating inorganic compounds is a strategy that enables the attainment of hybrid materials with beneficial properties and new functionalities [6,7]. Recently, titanium dioxide (TiO2) has been used as a reinforcement agent to develop organic–inorganic hybrid materials with improved physicochemical, mechanical, UV- and gas-barrier, water resistance, and antimicrobial properties [1,3,8–11].

TiO<sup>2</sup> is an amphoteric, inert, non-toxic, biocompatible metal oxide that exhibited thermal and chemical stability for diverse applications with a relatively low cost of production [12]. The wide use of TiO<sup>2</sup> is to support its photocatalytic, adsorptive, UV-blocking, and antimicrobial properties [13–15]. It has been employed for environmental remediation in dye removal from aqueous media [16]. Moreover, TiO<sup>2</sup> is the main source of white pigments for food, pharmaceutical, and cosmetic applications in compliance with the recommended safe dosage [17–19]. Currently, there is a great interest in combining protein-based materials with inorganic compounds like titanium dioxide to fabricate protein–TiO<sup>2</sup> hybrid structures with improved physical and chemical properties, which open new opportunities and applications [1].

In the last decade, protein–TiO<sup>2</sup> hybrid composites and their potential applications have been explored [4,20–24]. Wang et al. [23] developed a soy protein isolate film combined with TiO<sup>2</sup> with antimicrobial properties against *Escherichia coli* and *Staphylococcus aureus*. Similar trends were reported when a marine algae protein-based film functionalized with TiO<sup>2</sup> was used [25]. Fathi et al. [16] informed that the sesame protein–TiO<sup>2</sup> hybrid film showed photocatalytic degradation of the methylene blue dye under UV-light radiation. Qingyan et al. [26] made gelatin film reinforced with TiO<sup>2</sup> with improved mechanical and UV-protective properties. Fan et al. [27] fabricated a collagen–chitosan–TiO<sup>2</sup> porous scaffold for wound-healing purposes. Meanwhile, He et al. [27] developed an active packaging with marine alga (*Gracilaria lemaneiformis*) protein isolate and TiO<sup>2</sup> for cherry tomatoes preservation, while a whey protein–TiO<sup>2</sup> hybrid film was used to prolong the shelf life of chilled and lamb meats [28,29]. Furthermore, the incorporation of TiO<sup>2</sup> in diverse protein-based materials (collagen, gelatin, soy, hey, marine alga, kefiran, zein, sesame, sodium caseinate, and wheat gluten) had a positive impact on the technological (mechanical, water resistance, and gas-barrier) and functional (antimicrobial and UV-protective) properties, which exhibited potential uses for various applications [16,17,20,24,25,30,31].

This review summarizes the advantages and limitations of protein-based material functionalization by adding TiO<sup>2</sup> nanoparticles, offers and provides an overview of their photocatalytic and antimicrobial properties, environmental remediation, potential food and non-food packaging, pharmaceutical, cosmetics, textile, and biomedical applications.

#### **2. Proteins: Applications and Limitations**

Proteins are biological molecules composed of α-amino acids connected by peptide bonds, which can be obtained from plant-derived or animal origins [32]. For example, zein and gluten are cereal proteins [4,33], meanwhile, other proteins can be obtained from legumes such as soy [19]. Collagen and gelatin are extracted from meat, fish, and poultry by-products [1,34], whereas whey protein is a by-product of dairy manufacturing [29]. They exhibited interesting technological and functional properties for diversified applications, associated with their composition and structure [2]. Moreover, they are non-toxic, abundant, readily available, biodegradable, low-cost, and biocompatible to combine with enzymes, microorganisms, and organic and inorganic compounds [6,32,35]. Most of the applications described in the literature for protein-based materials are focused on developing packaging materials for food and non-food purposes, or biomedical applications such as wound-healing materials, as shown in Table 1.


**Table 1.** Potential applications of some protein-based materials.

Acquah et al. [36] fabricated a yellow pea (*Pisum sativum*) protein-based film with potential food and non-food packaging applications. It exhibited moderate water solubility (36.5%), good mechanical properties (elongation of 65%, a tensile strength of 0.65 MPa, and elastic modulus of 6.65 MPa), as well as good thermal properties (glass transition of 95.5 ◦C), but high moisture uptake (82%) due to its hydrophilic nature (contact angle of 60◦ ), affecting its quality as a packaging material. Agudelo-Cuartas et al. [37] mentioned that whey protein-based films showed great potential for packaging purposes (good mechanical properties); however, their high-water solubility (59%) and water vapor permeability (1.4 <sup>×</sup> <sup>10</sup>−<sup>10</sup> <sup>g</sup>·m−<sup>1</sup> ·s −1 ·Pa−<sup>1</sup> ) limit their uses in foods with high water content (e.g., meat). According to Guo et al. [38], the protein-based film's mechanical properties are influenced by storage conditions (temperature and relative humidity). They found that tensile strength and elongation at break of a zein film were negatively affected when relative humidity and temperature increased from 34% to 80% and from 5 to 35 ◦C, respectively. They argue that the available –SH groups in the protein structure decreased gradually during storage by water absorption, implying new and weak interactions.

Su et al. [39] reported that soy protein isolate film exhibited good biodegradability and gas-barrier properties against oxygen and carbon dioxide when relative humidity was low, which are suitable featuring for the development of packaging materials. Wang et al. [40] suggested that modification of protein structure by alkaline conditions could be an alternative to improve the technological properties of protein-based films. They reported that the formation of protein aggregates in a rice bran film treated at pH 11 improved their physical, mechanical, and thermal properties, associated with an increase in the β-sheet content and non-covalent interactions, due to the modification of the protein structure.

Additionally, gelatin-based films exhibited great potential for fabricating food packaging or wound-healing materials; however, due to their hygroscopic nature, they needed to be combined with a crosslinking or plasticizer agent (organic or inorganic) to improve their water resistance and thermal stability [42,43]. It has been reported that keratin films are too rigid, and the addition of glycerol improved their flexibility and mechanical resistance, which are suitable for biomedical applications [44]. Similar trends were reported in a wheat gluten film by adding glycerol, but its thermal stability was improved and could be used for packaging purposes [41].

In general, protein-based films exhibited great potential applications; however, their functionality depends on their molecular characteristics, complexity, superficial charge, denaturation tendency, water resistance, and thermal stability [35]. Therefore, the incorporation of organic and inorganic materials in the protein matrix is a viable strategy to enhance their functional and technological properties [29,32,45]. Table 2 shows some protein-based materials functionalized with organic and inorganic compounds to form hybrid composites with potential applications.


NPs: nanoparticles; ZnO: Zinc oxide; REO: rosemary essential oil.

According to the evidence, the incorporation of organic and inorganic compounds improves the technological (water and thermal resistance, mechanical, and adsorptive) and functional (antimicrobial activity and biocompatibility) properties of protein-based materials, associated with their ability to form intramolecular bonds through covalent and non-covalent interactions with the functional groups (–NH2, –OH, –COOH, and –SH) of the protein structure [6,29].

Additionally, usage of TiO<sup>2</sup> as a functional agent to enhance the technological properties of diverse protein-based materials has been widely explored in the last years, mainly for the chemical and physical interactions between protein structure and TiO2, which could be developed using diverse methodologies.

## **3. Possible Structural Interaction between R-Groups Amino Acid with TiO<sup>2</sup> Nanoparticles**

A major understanding of the interactions between proteins and TiO<sup>2</sup> surfaces will be a potential core for many applications in bio-nanotechnology [58]. Ranjan et al. [59] in silico observed that the TiO<sup>2</sup> (1.09 nm) nanoparticles bind to 13 immunological proteins (Table 3), using a docking simulation program (AutoDock 4.0), a computed atlas of surface topography of proteins (CASTp) and PyMol software (version 1.5.0.4). They observed that nano-TiO<sup>2</sup> bound with a positively charged R-group (lysine, arginine, and histidine) and nonpolar aliphatic R-groups amino acid (proline, glycine, alanine, valine, leucine, methionine, and isoleucine) containing amino acids, most frequently with lysine and proline. On the other hand, TiO<sup>2</sup> had less affinity with the aromatic R-group (phenylalanine, tyrosine, and tryptophan), polar uncharged R-groups (serine, threonine, cysteine, asparagine, and glutamine), and negatively charged R-group (aspartate and glutamate)-containing amino acids. According to the authors, the affinity of TiO<sup>2</sup> with the amino acids depends on the ability to form stable hydrogen bonds, which depend on the binding and intermolecular energy of each amino acid. These interactions have been exploited to develop packaging, scaffolds, wound-healing, and dental implant materials with enhanced properties, and to remove and degrade water pollutants, among others.


**Table 3.** Some immunological proteins–TiO<sup>2</sup> interaction.


**Table 3.** *Cont.*

Adapted from Ranjan et al. [59]. NR: No reported. **4. Preparation of Functionalized Protein–TiO2 Materials** 

#### **4. Preparation of Functionalized Protein–TiO<sup>2</sup> Materials** The functionalization of protein-based materials through the introduction of organic (ascorbic

The functionalization of protein-based materials through the introduction of organic (ascorbic acid, cellulose, and starch) and inorganic (metallic or metal oxide) compounds is an attractive way to fabricate protein-based hybrid materials with enhanced properties, which has seen a significant increase in the last few years [6]. The most common methods for developing functionalized protein-based materials are evaporative casting, dip-coating, layer-by-layer assembly, freeze-drying, electrospinning, and electrochemical through protein denaturation by gelation-coagulation process [6,45]. acid, cellulose, and starch) and inorganic (metallic or metal oxide) compounds is an attractive way to fabricate protein-based hybrid materials with enhanced properties, which has seen a significant increase in the last few years [6]. The most common methods for developing functionalized proteinbased materials are evaporative casting, dip-coating, layer-by-layer assembly, freeze-drying, electrospinning, and electrochemical through protein denaturation by gelation-coagulation process [6,45].

#### *4.1. Evaporative Casting Method 4.1. Evaporative Casting Method*

The evaporative casting method is generally accepted and commercially used for its simplicity, flexibility, and applicability to large-scale production. It consists of preparing a viscous solution by mixing the components, casting them in a plate, and evaporating them under controlled temperature and vacuum conditions to remove the solvent solution and form film and coatings (Figure 1). In general, it is a relatively low-cost method (one-third to half of the other methods); however, its main limitations are the difficulty in achieving a uniform distribution of the reinforcement agent, the presence of air bubbles, and possible reactions between the polymeric matrix and functional agent [60]. The evaporative casting method is generally accepted and commercially used for its simplicity, flexibility, and applicability to large-scale production. It consists of preparing a viscous solution by mixing the components, casting them in a plate, and evaporating them under controlled temperature and vacuum conditions to remove the solvent solution and form film and coatings (Figure 1). In general, it is a relatively low-cost method (one-third to half of the other methods); however, its main limitations are the difficulty in achieving a uniform distribution of the reinforcement agent, the presence of air bubbles, and possible reactions between the polymeric matrix and functional agent [60].

**Figure 1.** Schematic representation to laboratory scale of an evaporative casting method to prepare protein-based hybrid materials (adapted from Fan et al. [27], Al-Zoubi et al. [6]) (figure created with BioRender.com). **Figure 1.** Schematic representation to laboratory scale of an evaporative casting method to prepare protein-based hybrid materials (adapted from Fan et al. [27], Al-Zoubi et al. [6]) (figure created with BioRender.com).

#### *4.2. Dip Coating Method 4.2. Dip Coating Method*

*4.3. Layer-by-Layer Deposition Method* 

Dip-coating is a technique widely used in many industrial fields to deposit onto any substrate. The process could be defined as depositing aqueous-based liquid phase coating solutions onto the surface of any substrate and is divided into five stages: immersion, start-up, deposition, drainage, and evaporation. It is achieved at low processing temperatures and is a low-cost method to develop Dip-coating is a technique widely used in many industrial fields to deposit onto any substrate. The process could be defined as depositing aqueous-based liquid phase coating solutions onto the surface of any substrate and is divided into five stages: immersion, start-up, deposition, drainage, and evaporation. It is achieved at low processing temperatures and is a low-cost method to develop thin

The layer-by-layer deposition is a common method for coating substrates to develop functional thin films. It is a cyclical process in which a charged material is adsorbed onto a substrate, and after washing, an oppositely charged material is adsorbed on the surface of the first layer. This constitutes a single bilayer film with a thickness generally on the order of nanometers, and the deposition process can be repeated until a multilayer film is obtained. This method offers advanced composites with coatings with high purity, good adhesion, high surface, and uniformity. However, this methodology requires high sintering temperatures and thermal expansion mismatch [61–63].

## *4.3. Layer-by-Layer Deposition Method*

The layer-by-layer deposition is a common method for coating substrates to develop functional thin films. It is a cyclical process in which a charged material is adsorbed onto a substrate, and after washing, an oppositely charged material is adsorbed on the surface of the first layer. This constitutes a single bilayer film with a thickness generally on the order of nanometers, and the deposition process can be repeated until a multilayer film is obtained. This method offers advanced composites with exceptional properties (mechanical, electrical, optical, and biological) unavailable by other means, but this deposition process is complex, and the need for multiple dipping cycles hampers its usage in microtechnologies and electronics [64,65].

#### *4.4. Freeze-Drying Method*

Freeze-drying is a process that consists of removing the solvent from a frozen suspension containing mixed components. First, the gels are frozen, transforming the gel to a solid; then, sublimation of the solvent (mainly water) is then achieved at low pressure, avoiding the formation of the vapor–liquid interface. This method is widely used for aerogel preparation with highly porous and large specific surface area structures that allow rapid disintegration. However, this procedure requires sophisticated equipment compared to the evaporative casting method [66,67].

#### *4.5. Electospinning Method*

Electrospinning is a simple method to produce ultra-thin fibers with high surface area, highly porous structure, and small pore size. In this method, the mixed solution is pumped through a capillary conductive needle to form a droplet; under suitable conditions, solvent evaporation occurs, and the compound contracts into solid polymeric materials instead of fibers. It has the advantages of mild experimental conditions, low cost, easy operation and function, and a wide range of raw materials. The spinning process is controllable, and the parameters can be adjusted according to the different requirements in various research fields. However, electrospinning with raw materials that have a low molecular weight is difficult [68].

#### *4.6. Electrochemical Method*

Electrochemical methods are widely used for the preparation of thin films and coatings through anodic or cathodic techniques. Both processes are commonly used to prepare coatings by electrodeposition which include: electrophoretic process (EPD) using deposition of charged particles in a stable colloidal suspension on a conductive substrate, acting as one of the two oppositely charged electrodes in the EPD cell, and the electrolytic process (ELD), which starts from solutions of metal salts. They exhibit some advantages like low-cost, ability to coat complex shapes, speed, uniform coating thickness, rapid deposition rates, and the ability to coat complex substrates; however, it is difficult to produce crack-free coatings, it requires high sintering temperatures, and the bonding strength between coating and substrate is not strong enough [61,69].

#### **5. Applications of Protein–TiO<sup>2</sup> Hybrid Composites**

Protein-based materials exhibited a wide range of applications. However, most of their potential uses are limited by their poor physicochemical properties [35]. Thus, their functionalization with TiO<sup>2</sup> is a viable alternative to improve the technological and functional properties of protein-based materials such as gelatin, wheat gluten, kefiran, zein, and soy and whey protein isolates for several applications [49] (Figure 2), as discussed below.

*Coatings* **2020**, 10, x FOR PEER REVIEW 7 of 30

**Figure 2.** Protein–TiO2 hybrid composites (**a**) with enhanced mechanical and reduced gas exchange (**b**) and their applications: as food and non-food packaging with UV-protective and antimicrobial properties (**c**), photocatalytic activity for dye removal and degradation (**d**), wound-healing material (**e**), tissue engineering scaffolds (**f**), and for the development of biosensors (**g**) (adapted from Lin et al. [3], Fan et al. [27], Alizadeh-Sani et al. [28], Emregul et al. [70], Ferreira et al. [71]) (figure created with BioRender.com). **Figure 2.** Protein–TiO<sup>2</sup> hybrid composites (**a**) with enhanced mechanical and reduced gas exchange (**b**) and their applications: as food and non-food packaging with UV-protective and antimicrobial properties (**c**), photocatalytic activity for dye removal and degradation (**d**), wound-healing material (**e**), tissue engineering scaffolds (**f**), and for the development of biosensors (**g**) (adapted from Lin et al. [3], Fan et al. [27], Alizadeh-Sani et al. [28], Emregul et al. [70], Ferreira et al. [71]) (figure created with BioRender.com).

#### *5.1. Gelatin–TiO2 Hybrid Composite 5.1. Gelatin–TiO<sup>2</sup> Hybrid Composite*

shown in Table 4.

Food and nonfood packaging

Evaporative casting/Film

In the last years, the number of applications of gelatin-based materials has considerably increased. Gelatin is a protein obtained from the hydrolysis of collagen from mammalian sources, mainly pork and cattle. It is non-toxic, biodegradable, and biocompatible [72]. However, its main disadvantage for industrial applications (e.g., food packaging) is its hydrophilicity [73]. Therefore, the incorporation of TiO2 into the gelatin matrix is a viable strategy to improve its technological and functional properties [74]. The most common method for the preparation of gelatin–TiO2 hybrid composites is evaporative casting for films and coatings and freeze-drying for aerogels. Furthermore, the nanoparticles used are commercially available with sizes ranging from 10 to 25 nm in its anatase phase, in some cases in its rutile phase, using concentrations ≤1% in weight of total solid content, as In the last years, the number of applications of gelatin-based materials has considerably increased. Gelatin is a protein obtained from the hydrolysis of collagen from mammalian sources, mainly pork and cattle. It is non-toxic, biodegradable, and biocompatible [72]. However, its main disadvantage for industrial applications (e.g., food packaging) is its hydrophilicity [73]. Therefore, the incorporation of TiO<sup>2</sup> into the gelatin matrix is a viable strategy to improve its technological and functional properties [74]. The most common method for the preparation of gelatin–TiO<sup>2</sup> hybrid composites is evaporative casting for films and coatings and freeze-drying for aerogels. Furthermore, the nanoparticles used are commercially available with sizes ranging from 10 to 25 nm in its anatase phase, in some cases in its rutile phase, using concentrations ≤1% in weight of total solid content, as shown in Table 4.



Commercial (TiO2:Ag): 0.4% *w*/*w* Size: 20 nm

TiO2 improved the technological and photocatalytic properties of gelatin film.

[18]

CMC (1 g 100 mL−1), gelatin (1 g 100 mL−1)


#### **Table 4.** *Cont.*


#### **Table 4.** *Cont.*

\* Material composition was based on the best-reported results. NI: No information; CMC: carboxymethyl cellulose; PVA: polyvinyl alcohol; GO: graphene oxide; β-tP: β-tricalcium phosphate; Hap: hydroxyapatite; SM: synthesis method; (TiO2): concentration of titanium dioxide; CP: crystallite phase.

#### 5.1.1. Food and Non-Food Packaging Applications of Gelatin–TiO<sup>2</sup> Hybrid Composite

The potential use of gelatin-based materials functionalized with TiO<sup>2</sup> nanoparticles as food and non-food packaging material has been extensively explored [26,76]. Nassiri and Nafchi [76] developed a bovine gelatin film reinforced with TiO<sup>2</sup> nanoparticles with antimicrobial properties against *S. aureus* and *E. coli*, associated with the physical and chemical interactions of TiO<sup>2</sup> with the bacteria cell membrane. Incorporation of TiO<sup>2</sup> at low concentrations (5% *w*/*w*) decreases the water vapor (from 8.90 to 1.61 <sup>×</sup> <sup>10</sup><sup>11</sup> <sup>g</sup>·m−<sup>1</sup> ·s −1 ·Pa−<sup>1</sup> ), and oxygen permeability (from 214 to 95 cm<sup>3</sup> ·µm/m<sup>2</sup> ·day) of protein-based film. Similarly, Qingyan et al. [26] informed that gelatin–TiO<sup>2</sup> film exhibited antimicrobial activity against *E. coli* (54% inhibition of viable cells) and *S. aureus* (44% inhibition of viable cells) under UV-light irradiation (365 nm) after 120 min of exposure. The above, associated with the photocatalytic properties of TiO<sup>2</sup> and its ability to generate reactive molecules (hydrogen peroxide, hydroxyl radical, and superoxide anions) with antimicrobial properties by affecting the cell viability. Moreover, the addition of TiO<sup>2</sup> (1% *w*/*w* of total solid content) in the gelatin film promoted an increase in its mechanical and thermal properties. It decreased water solubility, moisture uptake, water vapor permeability, and transparency due to the formation of hydrogen and Ti–O–C bonds and electrostatic interactions between protein and inorganic nanoparticles [12].

Azizi-Lalabadi et al. [74] made a hybrid film composed of gelatin and polyvinyl alcohol, reinforced with TiO<sup>2</sup> nanoparticles previously embedded in 4A-zeolite. The enhanced physicochemical (optical, gas-barrier, and water resistance) were attributed to the interaction of the N–H functional group present in the protein structure, with TiO<sup>2</sup> through hydrogen bonds. Moreover, the hybrid film exhibited antimicrobial properties especially against Gram-negative bacteria (*E. coli* and *P. fluorescens*). Moreover, the hybrid film effectively extended the shelf life of white shrimp (up to 12 days) compared to uncoated samples (6 days), without significant changes in sensory attributes [75]. Likewise, Riahi et al. [1] fabricated an active gelatin–TiO2–grape seed extract film for food packaging purposes and found that water contact angle, water vapor permeability, mechanical properties, and UV-protective effect

improved in a dose-dependent response with an optimum TiO<sup>2</sup> concentration of 0.5% *w*/*w*, which was attributed to the chemical interaction of TiO<sup>2</sup> and C=O groups in the protein structure. On the other hand, the hybrid film exhibited antimicrobial activity in strain- and TiO<sup>2</sup> dose-dependence, where the Gram-negative bacteria were less susceptible than Gram-positive. At low concentrations of TiO<sup>2</sup> (<3% *w*/*w*), the hybrid film showed a bacteriostatic effect against *E. coli* and *L. monocytogenes*, while at 5% *w*/*w* exhibited bactericidal action.

Pirsa et al. [77] evaluated the antioxidant and antimicrobial properties of a carboxymethyl cellulose–gelatin film reinforced with TiO2:Ag-doped nanoparticles. The hybrid film exhibited better mechanical properties (greater flexibility) in comparison with the control group. Moreover, it showed antioxidant activity and antibacterial effect against *E. coli* and *S. aureus* in a TiO2:Ag concentration-dependent response. Furthermore, the carboxymethyl cellulose (CMC)–gelatin–TiO2: Ag exhibited good photocatalytic degradation of ethanol, benzene, and ammonia [18]. Furthermore, the incorporation of TiO2: Ag-doped nanoparticles improved the antioxidant, mechanical, UV-barrier, water resistance, and mechanical properties of a *Rhinobatos cemiculus* gelatin film in a dose-dependent manner. At a low concentration of TiO2, it can disperse uniformly and insert in the amorphous region of soy protein isolate (SPI), leading to a major interaction between both components; however, at high concentrations of TiO2, it could cause agglomerations interfering with the organization and interaction of protein and TiO<sup>2</sup> [16].

Similar results were reported in a fish gelatin–chitosan film functionalized with TiO2:Ag nanoparticles, where the improved antibacterial activity (*E. coli*, *S. aureus*, and *Botrytis cinerea*), optical, water-related, and mechanical properties were in a TiO2:Ag dose-dependent response [3]. The addition of TiO2:Ag-doped nanoparticles did not alter the typical structure of biopolymers, but instead promoted stronger intramolecular hydrogen bonds formation [16,85]. On the other hand, it has been reported that the improved UV-protective effects, water-related, and mechanical properties of a fish gelatin–agar–TiO<sup>2</sup> film could be negatively affected by a high concentration of TiO<sup>2</sup> (>0.5 g 100 mL−<sup>1</sup> ), mainly by an inhomogeneous dispersion and saturation of nanoparticles in the protein structure [21].

Additionally, Vejdan et al. [78] informed that a gelatin–agar bilayer film functionalized with TiO<sup>2</sup> nanoparticles effectively delays fish oil photo- and auto-oxidation up to 18 days. They reported that hybrid film containing 2% of TiO<sup>2</sup> could control fish oil oxidation due to the enhanced UV-protective and oxygen-barrier properties associated with the physicochemical characteristics of TiO2.

According to the results, incorporation of TiO<sup>2</sup> into the gelatin-matrix improved its mechanical, thermal, UV-protective, gas-barrier, and water-related properties with antioxidant and antimicrobial performance, both desirable characteristics for the development of food and non-food packaging materials.

#### 5.1.2. Biomedical Applications of Gelatin–TiO<sup>2</sup> Hybrid Composite

Gelatin–TiO<sup>2</sup> hybrid composites have been used for biomedical purposes. Lai et al. [80] immobilized gelatin onto TiO<sup>2</sup> nanotubes to modulate osteoblast behavior for orthopedic and dental applications. The authors found that cell spreading, proliferation, and differentiation of osteoblasts were improved by gelatin–TiO<sup>2</sup> hybrid material. They argued that extracellular matrix protein-based plays an important role in bone mineralization, while TiO<sup>2</sup> present in the hybrid matrix facilitates osteoblast differentiation. Ferreira et al. [71] fabricated a macroporous TiO2–functional hydroxyapatite–gelatin scaffold loaded with multipotent adult progenitor cells for bone regeneration applications in calvaria defects. They informed that a hybrid scaffold promoted osteointegration and enhanced bone regeneration with complete closure defect. The result was associated with the ability of TiO<sup>2</sup> to form complexes with calcium ions, promoting the adsorption of calcium-binding extracellular matrix proteins and Argine-Glycine-Aspartate specific peptide sequences.

Additionally, hydroxyapatite–gelatin–graphene oxide composite deposited on TiO<sup>2</sup> nanotubes by electrochemical deposition exhibited excellent biocompatibility with MC3T3-E1 cells, promoting a better cellular integration [81]. Moreover, Urruela-Barrios et al. [79] mentioned that a sodium alginate–gelatin hydrogel 3D printing functionalized with nano-TiO<sup>2</sup> and β-tricalcium phosphate exhibited a potential use for tissue engineering application. The hybrid material fabricated with the micro-extrusion process, exhibited adequate porosity (pore size ranged from 150 to 240 µm), and mechanical resistance (13 MPa) to promote cell proliferation and cartilages.

Nikpasand and Reza-Parvizi [73] evaluated in vivo the wound dressing properties of a gelatin–TiO<sup>2</sup> hybrid hydrogel in an open and infected with *S. aureus* methicillin-resistant at 5 <sup>×</sup> <sup>10</sup><sup>7</sup> colony forming units (CFU) by excision-type wound-healing study in rats. They found that the hybrid composite exhibited a good wound-healing effect (wound area closure of 100% after 21 days), in comparison with gelatin-wound treatments (wound area closure of 71% after 21 days). Nonetheless, animals treated with the hybrid composite did not show wound infection by pathogenic bacteria after 14 days of evaluation and exhibited accelerated re-epithelization through fibroblast proliferation without inflammatory response after 21 days, which could be considered for wound therapies. On the other hand, Emregul et al. [70] developed a carboxymethyl cellulose–gelatin–TiO2–superoxide dismutase biosensor supported in Pt surface for O<sup>2</sup> •− detection. They reported that the biopolymer blend (CMC and gelatin), provided a biocompatible environment for super oxide dismutase–TiO2, which acts as a nanoscale electrode, enhancing the electron transfer rate through the Pt electrode. The hybrid sensor exhibited high analytical performance with a wide linear range of 1.5 nM to 2 mM, and high sensitivity and fast response time (1.8 s) for O<sup>2</sup> •− detection in healthy and cancerous brain tissue (coefficient of determination or *R* <sup>2</sup> of 0.991). In this context, functionalization of gelatin-based materials with TiO<sup>2</sup> exhibited potential biomedical applications, associated with its enhanced biological properties.

#### 5.1.3. Other Applications of Gelatin–TiO<sup>2</sup> Hybrid Composite

Other investigated applications of the gelatin–TiO<sup>2</sup> hybrid composite include pharmaceutical (development of empty capsule shells), anti-corrosive material, and hydrogen storage. Hosokawa et al. [82] evaluated the application of UV-laser irradiation (at 355 nm) to print hard gelatin capsule shells with TiO2, and it was found that hybrid capsules could be printed gray in a laser power-dependent response.

Additionally, Hayajneh et al. [83] studied the effect of gelatin–TiO<sup>2</sup> hybrid coating on the corrosion resistance of AISI 304 stainless steel, in a simulated marine environment (solution with NaCl at 3.5% *w*/*v*) through potentiodynamic polarization studies. The presence of hybrid coating improved the corrosion resistance of steel material (corrosion rate 2.63 <sup>×</sup> <sup>10</sup>−<sup>3</sup> mpy) in comparison with gelatin-coated (corrosion rate 10.10 <sup>×</sup> <sup>10</sup>−<sup>3</sup> mpy) and uncoated (corrosion rate 9.94 <sup>×</sup> <sup>10</sup>−<sup>3</sup> mpy) steel. The results were associated with the formation of a dense and stable network structure formed by the gelatin and TiO<sup>2</sup> nanoparticles.

Furthermore, Bin Liu et al. [84] used gelatin as a template to fabricate TiO<sup>2</sup> mesoporous microspheres for hydrogen production. They reported that the assistance of gelatin positively influenced the morphology and physicochemical characteristics of TiO<sup>2</sup> nanoparticles (surface area of 98.3 m<sup>2</sup> ·g <sup>−</sup><sup>1</sup> and pore size of 11.9 nm), enhancing the hydrogen adsorption capacity and hydrogen storage performance of hybrid microspheres. However, its hydrogen adsorption mechanism remains unclear. According to these data, the gelatin–TiO<sup>2</sup> hybrid material exhibited pharmaceutical, anti-corrosive, and hydrogen production applications.

## *5.2. Whey Protein–TiO<sup>2</sup> Hybrid Composite*

Whey protein is a by-product obtained from dairy processing during cheese production. It is used to develop edible films and coatings with good biodegradability and lower gas permeability for diverse applications [29]. However, the potential uses of whey protein-based materials are limited by their higher hydrophilicity due to polar residues outside the globular structure, which causes softening when they come in contact with high-moisture environments [86]. On the other hand, it exhibited good biocompatibility to interact with inorganic compounds like TiO<sup>2</sup> to improve its technological and functional properties [29]. The most common method for preparing whey protein–TiO<sup>2</sup> hybrid composites is evaporative casting. Furthermore, the nanoparticles used are commercially available

with sizes ranging from 10 to 25 nm in its anatase phase and using concentrations ≤1% in weight of total solid content, as listed in Table 5.


**Table 5.** Effect of TiO<sup>2</sup> incorporation on whey protein matrix properties.

\* Material composition was based on the best-reported results. NI: No information; WPI: whey protein isolate; REO: rosemary essential oil; MMT: montmorillonite; ZMEO: *Zataria multiflora* essential oil.; SM: synthesis method; (TiO2): concentration of titanium dioxide; CP: crystallite phase.

5.2.1. Food and Non-Food Packaging Applications of Whey Protein–TiO<sup>2</sup> Hybrid Composite

The potential use of whey protein–TiO<sup>2</sup> hybrid material for food packaging purposes has been investigated [28], as shown in Table 5. Zhou et al. [87] prepared a biodegradable whey protein film functionalized with TiO2. It was found that technological properties such as UV-protective, mechanical, and water-resistance properties were improved in a TiO<sup>2</sup> dose-dependent response, associated with the intramolecular connections of protein and TiO<sup>2</sup> through covalent and non-covalent interactions. Moreover, the authors argued that at low concentrations of TiO2, a reinforcement of whey protein–TiO<sup>2</sup> structure occurs. Meanwhile, self-assembly of TiO2–TiO<sup>2</sup> interactions are detected at high TiO<sup>2</sup> concentrations, influencing its technological and functional properties, mainly associated with a reduction in the crystalline structure of TiO<sup>2</sup> by its incorporation in a polymeric matrix and its tendency to form agglomerates at higher concentrations [17,31]. Similar trends were informed in a kefiran–whey protein film functionalized with TiO2, where an excessive amount of TiO<sup>2</sup> in the polymeric matrix affected its functionality because TiO<sup>2</sup> may act as an anti-plasticizer agent [31,90]. Moreover, in a combined chitosan–whey protein film reinforced with sodium laurate–TiO<sup>2</sup> nanoparticles. Zhang et al. [89] reported that sodium laurate-modified TiO<sup>2</sup> incorporation influenced the transparency, water vapor permeability, and mechanical and thermal properties of the hybrid film in a dose-dependent manner, and its intermolecular interaction with the available functional groups of the chitosan–whey protein matrix. Gohargani et al. [91] fabricated a chitosan–whey protein film, functionalized with TiO<sup>2</sup> and *Zataria multiflora* essential oil (ZMEO) nanoparticles with enhanced antimicrobial properties against foodborne pathogenic bacteria such as *L. monocytogenes*, *S. aureus*, and *E. coli.* Results were attributed to the synergistic effect of bioactive compounds present in the ZMEO and TiO<sup>2</sup> nanoparticles. Moreover, the TiO2–ZMEO incorporation into the hybrid film, improved water vapor permeability, and tensile strength with a significant decrease in the film's transparency and color, associated with the physicochemical properties of TiO2.

Alizadeh-Sani et al. [28] informed that a whey protein isolate–cellulose nanofiber-TiO2–rosemary essential oil (REO) effectively preserved quality (microbial deterioration and sensory attributes) of refrigerated meat during cold storage. They reported that lamb meat treated with the hybrid film showed microbial stability (4.1 log·CFU·g <sup>−</sup><sup>1</sup> of viable cells) for 6 days at 4 ◦C storage without changes in sensory attributes (color, odor, texture, and overall acceptability). Moreover, the treated meat exhibited reduced lipid oxidation during storage, ascribed to antioxidant properties of REO (80% of radical scavenging) [88]. Furthermore, the TiO<sup>2</sup> (1% *w*/*w*) and REO (2% *w*/*w*) addition in the whey protein isolate/cellulose nanofiber hybrid film, improved mechanical (tensile strength, elongation at break, and elastic modulus) and water-related properties (moisture uptake, water solubility, and water vapor permeability), with a decrease in its transparency in a dose-dependent response in comparison with whey protein-based film, associated with the UV-scattering ability of TiO2. Furthermore, the hybrid film showed an antimicrobial effect against foodborne bacteria (*E. coli* O157:H7, *L. monocytogenes*, *P. fluorescens*, and *S. enteritidis*) in a strain-dependent manner. It was associated with antimicrobial properties of TiO<sup>2</sup> and bioactive compounds (1,8-cineole, α-pinene, and camphor) in the REO; which can alter the cell membrane and finally cause cell death [86]. Nonetheless, they informed that a low content of TiO<sup>2</sup> migrated from the polymeric matrix to the meat product, under the Food and Drug Administration limit recommendations (<1% *w*/*w*) [88]. Similarly, Feng et al. [29] informed that a whey protein–TiO<sup>2</sup> hybrid film is effective in extending the shelf life of chilled meat (up to 15 days) without significant changes in its quality parameters (weight loss less than 7.87%, reduced lipid peroxidation, and microbial stability) during cold storage (4 ◦C). Moreover, the hybrid film exhibited enhanced mechanical, optical, and water-related properties associated with the physical and chemical interactions between carboxylic and sulfhydryl groups of some amino acids present in the protein matrix with TiO2.

According to the evidence, the incorporation of TiO<sup>2</sup> into whey protein-based materials can improve the thermal, UV-barrier, mechanical, and water-related properties through physical and chemical interactions. Furthermore, whey protein films functionalized with TiO<sup>2</sup> exhibited antimicrobial properties for potential food and non-food packaging.

## 5.2.2. Other Applications of Whey Protein–TiO<sup>2</sup> Hybrid Composite

Ortelli et al. [92] fabricated a hybrid cotton fabric with anti-fire properties incorporating a whey protein–TiO<sup>2</sup> coating by the dip-pad-dry-cure process (Table 5). In general, the hybrid cotton material showed major durability (resistance to washing) and flame-resistant compared with the control group because TiO<sup>2</sup> acts as a physical reinforcement agent to fix whey protein to cotton fabrics in a stable way with the hydroxyl groups.
