*5.3. Collagen–TiO<sup>2</sup> Hybrid Composite*

Collagen is a large, coherent, covalently crosslinked fibrillar network protein. Its main sources are porcine, bovine, and ovine with many applications in the food, cosmetics, pharmaceutical, and biomedical industries [34]. The disadvantages of collagen are poor thermal instability, poor mechanical properties, and the possible contamination by pathogenic bacteria and chemical substances [93]. Particularly, collagen has been combined with TiO<sup>2</sup> to improve its physicochemical properties [5]. Preparation of collagen–TiO<sup>2</sup> hybrid composites is usually by dip-coating, followed by freeze-drying for aerogel development. Furthermore, the nanoparticles used are commercially available or synthesized by the Sol–gel method with sizes ranging from 10 to 30 nm in its anatase phase, and in some cases in its rutile phase (Table 6).

> **Table 6.** Effect of TiO<sup>2</sup> incorporation on collagen-based materials.



**Table 6.** *Cont.*

\* Material composition was based on the best-reported results. NI: No information; MWCNTs: multiwalled carbon nanotubes; g-PMMA: poly(methylmethacrylate); GPTMS-TIP: (3-glycidoxypropyl)trimethoxysilane; PVP: poly(vinyl pyrrolidone); SM: synthesis method; (TiO2): concentration of titanium dioxide; CP: crystallite phase.

## 5.3.1. Biomedical Applications of Collagen–TiO<sup>2</sup> Hybrid Composite

Table 6 lists, works on collagen-based materials functionalized with TiO<sup>2</sup> for biomedical applications. Park et al. [5] evaluated the effect of collagen-multi-walled carbon nanotubes (MWCNTs) composite coating deposited on titanium, using a dip-coating method on osteoblast growth. Cell proliferation studies confirmed a strong dependence of the extent of cell proliferation on the amount of MWCNTs incorporated in the composite in a dose-dependent response. Collagen–MWCNT–Ti showed higher cell proliferation than the collagen–MWCNT composite, where TiO<sup>2</sup> was responsible for cell proliferation. Truc et al. [94] studied the interaction between fibroblast and collagen modified on titanium (Ti) surface by electrochemical deposition (ECD), to reduce dental implant failure. They found that the Ti/Collagen hybrid composite showed rapid cell adhesion and proliferation.

Nojiri et al. [95] evaluated the establishment of perpendicularly oriented collagen attachments on TiO<sup>2</sup> nanotubes (TNT), which exhibited significant binding resistance, and the chemically linked collagen–TiO<sup>2</sup> facilitated epithelial cell stretching and sheet formation. Similarly, Bishal et al. [96] informed that collagen–TiO<sup>2</sup> promotes human osteoblast growth and proliferation in a dose-dependent manner with no inflammatory response detected, which was associated with the ability of TiO<sup>2</sup> to interact with calcium and phosphate elements, suggesting that this material could be used for applications in bone tissue engineering. On the other hand, Vedhanayagam et al. [97] informed that the poly(methyl methacrylate)–collagen–PdO–TiO<sup>2</sup> hybrid scaffolds did not show toxic effects on MG 63 cells (human osteosarcoma), and enhanced the alkaline phosphatase activity during in vitro osteogenic differentiation by the secretion of the osteogenic protein, leading to bone formation. Moreover, the hybrid scaffold exhibited higher thermal stability (83.45 ◦C), and mechanical strength (Young's modulus 105.57 MPa) than the pure collagen scaffold (71.64 ◦C, 11.67 MPa, respectively), due to the chemical and physical interaction between collagen and Palladium oxide (PdO)–TiO2.

Additionally, collagen–silane–TiO<sup>2</sup> has also been used as a functional agent of Mg alloys. The hybrid composite promotes the formation of a stable Mg(OH)2/MgCO3/CaCO<sup>3</sup> structure that effectively protects its corrosion. Moreover, the collagen–silane–TiO<sup>2</sup> improved osteoblasts and fibroblasts proliferation compared to bare and silane–TiO2-coated alloys. In the long term, collagen–silane–TiO<sup>2</sup> is a viable strategy to prevent Mg alloy degradation due to the formation of a complex structure [93]. On the other hand, Li et al. [34] made 3D nanocomposite scaffolds composed of collagen, polyvinyl pyrrolidone (PVP), and TiO<sup>2</sup> nanoparticles, with good degradation resistance in PVP dose-dependent response for potential tissue engineering applications. Likewise, collagen–chitosan–TiO<sup>2</sup> scaffolds exhibited antimicrobial activity against *S. aureus* and improved permeability, stability to degradation, and cell aggregation to stop bleeding, which are suitable for the development of wound-healing materials [27].

Significant evidence shows that collagen functionalization with TiO<sup>2</sup> nanoparticles improved its biological properties for dental implants and bone and dermal regeneration.

## 5.3.2. Other Applications of Collagen–TiO<sup>2</sup> Hybrid Composite

Other researched applications of the collagen–TiO<sup>2</sup> hybrid composite include the development of packaging materials, catalysts, and electronics (Table 6). Erciyes et al. [98] proposed the use of leather solid wastes as a source of collagen hydrolyzed to make composites functionalized with TiO2. The hybrid film exhibited improved water vapor permeability, water-solubility, elongation at break, and tensile strength. The authors highlighted the potential reuse of collagen-waste to develop packaging materials.

Additionally, Luo et al. [99] informed that collagen–TiO2: Tb3+-doped hybrid material exhibited excellent photocatalytic performance against methyl orange (93.87%) dye after 6 h of exposure in UV-light irradiation (150 W).

Furthermore, Cheng et al. [100] proposed a facile synthetic strategy to engineer a one-dimensional (1D) hierarchically ordered mesoporous TiO<sup>2</sup> nanofiber bundles (TBs) by using low-cost natural collagen fibers as a bio-template. In general, the hybrid structure can offer shortened ion diffusion paths, ensuring an efficient electrolyte penetration for ion access without affecting its structural integrity. They conclude that the hybrid materials had excellent electrochemical lithium and sodium storage properties.

In general, the collagen–TiO<sup>2</sup> hybrid material exhibited potential applications such as food and non-food packaging, environmental remediation, and electrochemical studies.

#### *5.4. Soy Protein–TiO<sup>2</sup> Hybrid Composite*

Soy protein isolate (SPI) is a by-product attained from the manufacture of soybean oil with a complex mixture of proteins (β-conglycinin and glycinin) with a minimum protein content of 90% on a moisture-free basis [101,102]. It is readily available, biodegradable, and biocompatible for edible coatings [19] with potential usage on food packaging [72,103]. However, the main disadvantages of SPI-based films include weak mechanical properties and high sensitivity to humidity [102,104]. In that sense, SPI films have been functionalized with TiO<sup>2</sup> to enhance their physical properties, where the most common method for its preparation is evaporative casting. Furthermore, the nanoparticles used are commercially available in its anatase phase, with concentrations ranging from 0.5% to 2% in weight of total solid content (Table 7).



\* Material composition was based on the best-reported results. NI: No information; SM: synthesis method; (TiO2): concentration of titanium dioxide; CP: crystallite phase.

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

Table 7 lists the work on soy protein isolate–TiO<sup>2</sup> hybrid material for food and non-food packaging development with enhanced properties. Malathi et al. [102] informed that TiO<sup>2</sup> incorporation into an SPI film promotes an increase in thickness, opacity, tensile strength, and elongation at break of the cast film, which was associated with the hydrogen bonding or O–Ti–O bonding. Moreover, a strong charge and polar interaction between side chains of soy protein molecules restrict segment rotation and molecular mobility, leading to an increase in the elongation of the hybrid film. Furthermore, Lu et al. [101] reported that the functionalization of an SPI film with TiO<sup>2</sup> promoted a decrease in water vapor (from 5.43 to 4.62 g·mm·m−<sup>2</sup> ·day−<sup>1</sup> ·kPa−<sup>1</sup> ) and oxygen (from 0.470 to 0.110 g·cm−<sup>2</sup> ·day−<sup>1</sup> ) permeability, as well as an increase in tensile strength (from 6.6683 to 14.5642 MPa) in a TiO<sup>2</sup> concentration-dependent response. They argue that the presence of TiO<sup>2</sup> in protein structure significantly changes the hydrophilic nature of the film, due to the stable covalent (Si–O–C, Ti–O–C, and Si–O–Ti) and non-covalent (hydrogen bonds and Van der Waals forces) interactions between TiO<sup>2</sup> and SPI. Moreover, the hybrid film exhibited antimicrobial effects against *E. coli* (inhibition zone by agar test diffusion assay of 27.34 mm). Wang et al. [23] demonstrated the bactericidal efficiency of an SPI–TiO<sup>2</sup> hybrid film under UV-light (at 365 nm during two hours) against *E. coli* (reduction of 71.01% of viable cells) and *S. aureus* (reduction of 88.94% of viable cells), which was associated with the synergistic antimicrobial effect between TiO<sup>2</sup> and β-conglycinin and glycinin peptides present in the SPI [107].

Additionally, Wang et al. [19] informed that TiO<sup>2</sup> incorporation in an SPI film positively influences its tensile strength (90.79% higher than control). On the other hand, the addition of nano-TiO<sup>2</sup> reduced the flexibility (70.21% less than control), and water vapor (65.67% less than control), and oxygen (46.50% less than control) permeability in comparison with control groups. This was due to the strong hydrogen bonds formed between the two main components, which could prevent water and oxygen from diffusing through the films. The reduction in flexibility values could be associated with a collapse of the crystalline structure of the hybrid material by the formation of aggregates by an excess of TiO2.

The reported application of SPI–TiO<sup>2</sup> hybrid film includes fruit preservation and water-dye degradation. Zhang et al. [105] reported that SPI–TiO<sup>2</sup> hybrid film was effective to extend the shelf life of strawberries stored at 4 ◦C up to 8 days without significant weight losses (<17.3%) and color changes with stable microbial quality in comparison with the uncoated fruits. Similar trends were reported in grapes coated with an SPI–TiO<sup>2</sup> hybrid film by Hoseiniyan et al. [95], who reported that coated grapes exhibited good performance during cold storage (31 days at 4 ◦C) without significant effects in the total soluble solids, titratable acidity, and weight losses. The hybrid film prevents the fungal infection of the fruits, and the coated fruits also had a good appearance and marketability compared with the uncoated fruits.

In summary, the incorporation of TiO<sup>2</sup> into SPI significantly improved its physicochemical properties and exhibited good fruit preservation performance.

## 5.4.2. Other Applications of Soy Protein Isolate–TiO<sup>2</sup> Hybrid Composite

Calza et al. [108] fabricated a system composed of soybean peroxidase and TiO<sup>2</sup> nanoparticles for environmental remediation purposes (Table 7). They informed that the hybrid material effectively remove orange II dye (100%) and carbamazepine (100%) drug from aqueous solutions after 60 min of exposure compared with the soybean peroxidase structure (<80% and <10%, respectively, after 120 min of exposure), which was associated with the synergistic properties of peroxidase and TiO2. Further studies are needed to understand the removal and degradation mechanism of soybean peroxidase–TiO2, which could be used as an alternative for wastewater treatment.

## *5.5. Other Proteins Functionalized with TiO<sup>2</sup>*

Table 8 lists various non-conventional proteins functionalized with TiO2, such as zein, keratin, sodium caseinate, lactoferrin, and sesame, to enhance their physicochemical properties, where the most common method for their preparation is evaporative casting for films and freeze-drying for hydrogels and scaffolds. Furthermore, the nanoparticles used are commercially available with sizes ranging from 10 to 200 nm in its anatase phase, and in some cases in its rutile phase, using concentrations ranging from 0.5% to 10% in weight of total solid content.




#### **Table 8.** *Cont.*

\* Material composition was based on the best-reported results. NI: No information; CEO: cumin essential oil; PVA: polyvinyl alcohol; PEG: polyethylene glycol; BTCA: 1,2,3,4-butane tetracarboxylic acid; PLA: poly(Lactic acid); SM: synthesis method; (TiO2): concentration of titanium dioxide; CP: crystallite phase.

#### 5.5.1. Packaging Applications of Non-Conventional Proteins Functionalized with TiO<sup>2</sup>

Table 8 lists reports on the use of non-conventional protein materials functionalized with TiO<sup>2</sup> for food and non-food packaging development. Kadam et al. [24] evaluated the effect of TiO2:SiO<sup>2</sup> nanoparticles incorporation on the thermal and mechanical properties of a cast zein film. They reported that mechanical properties (tensile strength) of the hybrid film were enhanced; however, its flexibility was reduced two-fold compared with zein film, possibly associated with the formation of TiO<sup>2</sup> aggregates. Furthermore, the water contact angle, water vapor permeability, and thermal properties of the hybrid film were improved by the addition of inorganic nanoparticles, associated with the interaction between zein and TiO2:SiO2, which promotes a stable and strong hydrogen bonds formation. Similarly, Amjadi et al. [109] made zein–sodium alginate (90:10) film functionalized with TiO2–betanin (0.5%:1%) nanoparticles and informed that the hybrid film exhibited antioxidant properties (by the presence of bioactive compounds in betanin) and high antimicrobial effects (by agar test diffusion assay) against *E. coli* (15.4 mm of inhibition zone) and *S. aureus* (16.9 mm of inhibition zone), which was attributed to the antimicrobial properties of TiO2. Moreover, Böhmer-Maas et al. [33] developed a

zein–TiO<sup>2</sup> nanofiber as an ethylene absorber for cherry tomatoes preservation (25 ◦C). They reported that coated fruits with the hybrid film exhibited less ethylene concentration (9.38 µg·L −1 ·g −1 ·h −1 ) than those coated with a zein film (10.27 µg·L −1 ·g −1 ·h −1 ), which permits extended the shelf life of cherry tomatoes up to 22 days. According to the authors, the ethylene degradation occurs by the oxidation of ethylene into CO<sup>2</sup> and water by the OH radicals and reactive oxygen species generated by the photocatalytic ability of TiO2.

Montes-de-Oca-Ávalos et al. [30] investigated the effect of TiO<sup>2</sup> incorporation on the physicochemical properties of a sodium caseinate film. They informed that mechanical, thermal, water vapor permeability characteristics of the caseinate film were improved in a TiO<sup>2</sup> concentration-dependent way, associated with good dispersion of TiO<sup>2</sup> through the film polymeric matrix. According to the authors, the presence of TiO<sup>2</sup> avoids protein agglomeration due to the stable hydrogen bond formation. Additionally, Alizadeh-Sani et al. [11] informed that a sodium caseinate–guar gum film functionalized with TiO<sup>2</sup> (1% *w*/*w*) and cumin essential oil (2% *w*/*w*) showed remarkable antimicrobial activity against *L. monocytogenes* (16 mm of inhibition zone), *S. aureus* (15 mm of inhibition zone), *E. coli* O157:H7 (14 mm of inhibition zone), *S. enteritidis* (12 mm of inhibition zone) in a strain-dependent manner. These results were associated with the cell wall differences between bacteria (outer membrane) and the synergistic antimicrobial effect among TiO<sup>2</sup> and cumin essential oil. Moreover, the water vapor permeability, tensile strength, and flexibility of the combined film were improved by a synergistic effect of TiO<sup>2</sup> and cumin essential oil.

Additionally, Montazer et al. [22] informed that the incorporation of TiO<sup>2</sup> in a wool keratin film stabilized by butane tetracarboxylic acid (BTCA) exhibited excellent UV-barrier properties related to the C–N and N–H bonds promoted for TiO<sup>2</sup> and BTCA interactions, with an optimum concentration of 0.6 g·L <sup>−</sup><sup>1</sup> and 12.94% *w*/*v*. Similarly, Wu et al. [110], who informed that thermal stability, mechanical resistance, and water vapor permeability of the keratin–tris film were improved by its functionalization with TiO<sup>2</sup> that may act as a physical cross-linker agent.

According to evidence, functionalization of non-conventional proteins like zein, keratin, and sodium caseinate with TiO<sup>2</sup> nanoparticles exhibited interesting properties for food and non-food packaging development.

#### 5.5.2. Environmental Applications of Non-Conventional Proteins Functionalized with TiO<sup>2</sup>

Usage of zein, keratin, and sesame proteins as a supporting material of TiO<sup>2</sup> for the removal and degradation of water pollutants have been explored (Table 8). Babitha and Korrapati [113] made mesoporous microspheres formed by zein and TiO<sup>2</sup> as an alternative for acid yellow (AY110) and acid blue (AB113) dyes decolorization under UV-light irradiation. They reported that the hybrid microspheres (1 mg·mL−<sup>1</sup> ) showed a dye removal efficiency of 96% and 89% in AY110 and AB113, respectively, at lower dye concentration (10 mg·L −1 ) but decreased at higher concentrations (100 mg·L −1 ), which was associated with the saturation of active sites into the hybrid matrix.

Additionally, Villanueva et al. [111] fabricated a hydrogel combining keratin (from cow's horn) and TiO<sup>2</sup> to remove trimethoprim from wastewater. They reported that the hybrid material exhibited good degradation efficiency (>95%) against antibiotic removal from aqueous solution in a TiO<sup>2</sup> dose-dependent response, with an optimum TiO<sup>2</sup> concentration of 10% *w*/*w* with performance up to four consecutive cycles (90%). It was associated with the swelling and adsorptive abilities of the hybrid film and to the presence of active sites on the catalyst surface due to the strong attachment between keratin and TiO<sup>2</sup> through covalent and non-covalent interactions. Moreover, Siriorn and Jatuphorn [112] reported that a chicken feather keratin–poly(lactic acid)–TiO<sup>2</sup> nanofibers (0.05 g) <sup>e</sup>ffectively remove methylene blue (90%) dye from aqueous solution (5 <sup>×</sup> <sup>10</sup>−<sup>6</sup> M) under visible light due to the improved adsorptive properties of the hybrid nanofibers.

Fathi et al. [16] made a sesame protein isolate film functionalized with TiO<sup>2</sup> for water-dye removal purposes. They reported that the hybrid film (64 cm<sup>2</sup> ) effectively degraded 76% of methylene blue dye (10 mg·mL−<sup>1</sup> ) under UV-light irradiation after 120 min of exposure. Moreover, the hybrid material exhibited enhanced water vapor permeability, water resistance, water contact angle, and mechanical strength in a TiO<sup>2</sup> dose-dependent response with an optimum TiO<sup>2</sup> concentration of 3% *w*/*w* associated with the interaction chemical and physical interactions between sesame protein and TiO2. On the other hand, the morphological studies through scanning electron microscopy revealed that a high concentration of TiO<sup>2</sup> exhibited an inhomogeneous dispersion, causing aggregations in the protein matrix that negatively affects its functionality.

To summarize, non-conventional proteins like zein, keratin, and sesame functionalized with TiO<sup>2</sup> nanoparticles could be a viable, low-cost, and efficient alternative for environmental applications as photocatalysts for wastewater treatment.

#### 5.5.3. Other Applications of Non-Conventional Proteins Functionalized with TiO<sup>2</sup>

Other potential uses of non-conventional proteins functionalized with TiO<sup>2</sup> include bone regeneration, antimicrobial activity, and textiles (Table 8). Johari et al. [114] made a fluorated silk fibroin–TiO<sup>2</sup> hybrid scaffold for bone tissue engineering with non-toxic effects in human osteoblast cells (SaOS-2) and suitable cell attachment and spreading on the hybrid material, which was associated with the fluoridation of TiO<sup>2</sup> nanoparticles (TiO2–F). Moreover, the hybrid scaffold exhibited good porosity (200 to 500 µm), mechanical resistance (tensile strength of 1.7 MPa), and adequate biodegradation rate (from 1% to 5% of weight loss in 30 days) in a TiO<sup>2</sup> dose-dependent response due to the formation of Ti–O–C bonds and the partial substitution of OH groups present in the TiO<sup>2</sup> surface by fluorine anions, that significantly increase the functional properties of TiO2. On the other hand, with high amounts of TiO<sup>2</sup> (>15%), some agglomerates could appear that negatively affect the technological properties of the hybrid scaffold.

Mehrabani et al. [115] informed that a chitin–fibroin–TiO<sup>2</sup> hybrid composite did not show cytotoxic effects on a human Caucasian fetal foreskin fibroblast cell line at low TiO<sup>2</sup> concentrations (<1.5% *w*/*w*). Nonetheless, the hybrid material exhibited a porosity of 94%, a density of 3118 mg·mL−<sup>1</sup> , and water resistance with a swelling degree of 93% after 24 h. In addition, it showed antimicrobial properties against *E. coli*, *S. aureus*, and *C. albicans*, which are suitable for the development of wound-healing materials. According to Feng et al. [117], incorporation of TiO<sup>2</sup> into fibroin (mostly α-helix) matrix promotes structural changes that permit a strong interaction with the β-sheets changing from typical silk I to Silk II structure in a TiO2-dependent manner, attributed to the presence of hydroxyl groups on the TiO2. The enhanced properties of fibroin could be related to the conformational structure. On the other hand, the authors reported that a high concentration of TiO<sup>2</sup> might negatively affect the mechanical properties of the hybrid material associated with the damage of its microscopic structure mainly by the formation of TiO<sup>2</sup> agglomerates, and possibly to the extra water used for the preparation of the hybrid material.

Kazek-Kesik et al. [116] coated a lactoferrin–collagen composite on titanium alloys for bone replacement. It was found that the presence of lactoferrin and TiO<sup>2</sup> enhanced osteoblast-like effect on MG-63 cells after seven days of evaluation in comparison with collagen-treated cells, mainly by the ability of both components to promote cell adhesion.

According to the evidence, the functionalization of non-conventional proteins with TiO<sup>2</sup> nanoparticles exhibited interesting properties and applications. However, further studies are needed to validate their potential uses.

#### **6. Disadvantages of Protein–TiO<sup>2</sup> Hybrid Composites and Perspectives**

Despite the observation that protein–TiO<sup>2</sup> hybrid composites exhibited excellent technological and functional properties with great potential to be used in several applications, it is necessary to evaluate the safe use and implementation of this kind of hybrid composites, mainly due to the presence of TiO<sup>2</sup> in their composition.

In this context, it has been reported that pure TiO<sup>2</sup> exhibited toxicological and adverse effects in cell lines (HeLa and HaCaT), proteins (microtubule and bovine serum albumin), and animal models (Sprague–Dawley rats, Wistar rats, and mussel *Mytilus coruscus*) in a concentration-dependent response, typically at doses ranging from 0.4 to 100 mg·mL−<sup>1</sup> with direct application [118–123]. Nonetheless, the tested concentrations of TiO<sup>2</sup> in these works were higher than the recommended safe usage (<1% by weight) by international regulations in the use of TiO<sup>2</sup> as a food additive [124].

However, the amount of TiO<sup>2</sup> used as a functional agent to develop protein–TiO<sup>2</sup> hybrid composites ranges from 0.003 to 1 mg·mL−<sup>1</sup> , depending on its application. For example: in food packaging materials manufacturing, the amount average of TiO<sup>2</sup> employed is 0.28 mg·mL−<sup>1</sup> , while for packaging materials with non-food purposes it is 0.85 mg·mL−<sup>1</sup> . Moreover, for the development of scaffolds, dental implants, and wound-healing materials, the average amount of TiO<sup>2</sup> is 0.23 and 0.9 mg·mL−<sup>1</sup> for making hybrid materials for environmental remediation.

According to Xu et al. (2017) [123], the interaction between protein structure and TiO<sup>2</sup> plays a critical role in the safe use of these materials, which usually depends on the new properties of each hybrid composite and the used concentration of TiO<sup>2</sup> [125]. In this sense, there are a few reports on the toxicity status of protein–TiO<sup>2</sup> hybrid composites, which reported no toxicological or adverse effects on their use, associated with the low concentration of TiO<sup>2</sup> used for the functionalization of protein-based materials. However, most of the published reports cited in this document focused on in vitro evaluations. Therefore, further studies are needed to evaluate the possible human health and environmental risks on the usage of these hybrid composites.

#### **7. Concluding Remarks**

Significant evidence indicates that functionalization of protein-based materials by adding TiO<sup>2</sup> nanoparticles is a feasible approach to improve their thermal, mechanical, optical, water-resistance, gas-barrier, and adsorptive properties. The evaporative casting method is one of the most common procedures for the preparation of protein–TiO<sup>2</sup> hybrid films and coating and freeze-drying for hydrogels and scaffolds, using commercial TiO<sup>2</sup> with a particle size ranging from 10 to 200 nm (the most frequently used is 10–25 nm in size) in its anatase phase with a crystalline structure.

Protein–TiO<sup>2</sup> hybrid composites are an active research area for developing eco-friendly and active food and non-food packaging materials with antimicrobial and UV-protective effects. Furthermore, they are attractive and biocompatible materials to fabricate wound-healing patches, tissue engineering scaffolds, or biosensors for biomedical applications.

On the other hand, although the functionalization of protein-based materials with TiO<sup>2</sup> offers significant advantages, some limitations have been reported, especially those associated with the concentration of TiO2. Higher concentrations of TiO<sup>2</sup> could promote an inhomogeneous dispersion through the polymeric matrix, forming agglomerates that negatively affected the technological and functional properties of the hybrid material, particularly in flexibility and transparency. Likewise, the preparation method could negatively influence the properties of the hybrid material, associated with the physical and chemical interactions between components. For example, if there was no proper mixing ratio between protein and TiO2, a saturation of the available functional groups in the polymeric matrix can affect the physicochemical properties of the film. Additionally, other possible limitations of the protein–TiO<sup>2</sup> hybrid composites could be related to the type and source of protein and its possible structural changes by the presence of TiO<sup>2</sup> and its stability for diverse applications.

There are some challenges to be achieved for industrial applications; one of the most important is to obtain the correct amounts of protein and TiO<sup>2</sup> nanoparticles because different uses require different formulations with desirable properties. For example, the shelf life of climacteric fruits depends on the correct exchange of oxygen, carbon dioxide, and water vapor permeability. Meanwhile, products with high amounts of lipids require UV-protective effects to prevent their oxidation. On the other hand, wound-healing materials should exhibit high water and mechanical resistance but correct gas exchange, high adherence, and antimicrobial properties. Moreover, standardized protocols for their preparation are needed for industrial-scale implementation. It is also necessary to carry out in vivo tests to evaluate the possible human health and environmental risks on the usage and safe implementation of these

hybrid composites in diverse applications. Therefore, further research efforts should be dedicated to solving these challenges.

**Author Contributions:** Conceptualization, L.M.A.-E., Z.V.-d.l.M., D.A.L.-d.l.M., A.P.-L., and E.M.G.; methodology, L.M.A.-E., Z.V.-d.l.M., N.R.-B., T.S.-C., K.N., D.A.L.-d.l.M, A.P.-L., and E.M.-G.; investigation, L.M.A.-E., Z.V.-d.l.M., N.R.-B., T.S.-C., K.N., D.A.L.-d.l.M., A.P.-L., and E.M.-G.; writing–original draft preparation, L.M.A.-E., Z.V.-d.l.M., N.R.-B., T.S.-C., K.N., D.A.L.-d.l.M., A.P.-L., and E.M.-G.; writing–review and editing, L.M.A.-E., Z.V.-d.l.M., D.A.L.-d.l.M., A.P.-L., and E.M.-G.; supervision, L.M.A.-E., Z.V.-d.l.M., D.A.L.-d.l.M., A.P.-L., and E.M.-G. All authors have read and agreed to the published version of the manuscript.

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

**Acknowledgments:** The authors gratefully acknowledge the financial support from a scholarship (702634) from CONACYT-Mexico, as well as Acoyani Garrido-Sandoval for proofreading the English language of this research.

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

#### **References**


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