Protein–TiO2: A Functional Hybrid Composite with Diversified Applications

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 TiO2 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–TiO2 hybrid materials are biodegradable and exhibit improved tensile strength, elasticity, thermal stability, oxygen and water resistance in a TiO2 concentration-dependent response. Nonetheless, they showed enhanced antimicrobial and UV-protective effects with good biocompatibility on different cell lines. The main applications of protein–TiO2 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–TiO2 hybrid composites exhibited potential applications; however, standardized protocols for their preparation are needed for industrial-scale implementation.


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., TiO 2 -Ag), organic-organic (e.g., wheat gluten-cellulose), and organic-inorganic (e.g., collagen-TiO 2 ) [3][4][5], and they can be synthesized by spin and dip coating, slot-casting, electrochemical self-assembly, and chemical vapor, atomic or molecular layer Table 1. Potential applications of some protein-based materials.

Protein Source Application
Ref.
Yellow pea protein isolate Food and non-food packaging [36] Whey protein Food and non-food packaging [37] Corn zein Food and non-food packaging [38] Soy protein isolate Food and non-food packaging [39] Rice bran Food and non-food packaging [40] Wheat gluten Food and non-food packaging [41] Gelatin Food and non-food packaging [42] Gelatin Biomedical [43] Keratin Biomedical [44] 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 × 10 −10 g·m −1 ·s −1 ·Pa −1 ) 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. 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 (-NH 2 , -OH, -COOH, and -SH) of the protein structure [6,29].
Additionally, usage of TiO 2 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 TiO 2 , which could be developed using diverse methodologies.

Possible Structural Interaction between R-Groups Amino Acid with TiO 2 Nanoparticles
A major understanding of the interactions between proteins and TiO 2 surfaces will be a potential core for many applications in bio-nanotechnology [58]. Ranjan et al. [59] in silico observed that the TiO 2 (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 2 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 2 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 2 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.

Preparation of Functionalized Protein-TiO 2 Materials
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].

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].

Preparation of Functionalized Protein-TiO2 Materials
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 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].

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].

Dip Coating 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 thin coatings with high purity, good adhesion, high surface, and uniformity. However, this methodology requires high sintering temperatures and thermal expansion mismatch [61][62][63].

Dip Coating 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 thin coatings with high purity, good adhesion, high surface, and uniformity. However, this methodology requires high sintering temperatures and thermal expansion mismatch [61][62][63].

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].

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].

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].

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].

Applications of Protein-TiO 2 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 2 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.

Gelatin-TiO2 Hybrid Composite
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 shown in Table 4.

Gelatin-TiO 2 Hybrid Composite
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 2 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 2 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.

Food and Non-Food Packaging Applications of Gelatin-TiO 2 Hybrid Composite
The potential use of gelatin-based materials functionalized with TiO 2 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 2 nanoparticles with antimicrobial properties against S. aureus and E. coli, associated with the physical and chemical interactions of TiO 2 with the bacteria cell membrane. Incorporation of TiO 2 at low concentrations (5% w/w) decreases the water vapor (from 8.90 to 1.61 × 10 11 g·m −1 ·s −1 ·Pa −1 ), and oxygen permeability (from 214 to 95 cm 3 ·µm/m 2 ·day) of protein-based film. Similarly, Qingyan et al. [26] informed that gelatin-TiO 2 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 2 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 2 (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 2 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 2 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-TiO 2 -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 2 concentration of 0.5% w/w, which was attributed to the chemical interaction of TiO 2 and C=O groups in the protein structure. On the other hand, the hybrid film exhibited antimicrobial activity in strain-and TiO 2 dose-dependence, where the Gram-negative bacteria were less susceptible than Gram-positive. At low concentrations of TiO 2 (<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 TiO 2 :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 TiO 2 :Ag concentration-dependent response. Furthermore, the carboxymethyl cellulose (CMC)-gelatin-TiO 2 : Ag exhibited good photocatalytic degradation of ethanol, benzene, and ammonia [18]. Furthermore, the incorporation of TiO 2 : 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 TiO 2 , 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 TiO 2 , it could cause agglomerations interfering with the organization and interaction of protein and TiO 2 [16].
Similar results were reported in a fish gelatin-chitosan film functionalized with TiO 2 :Ag nanoparticles, where the improved antibacterial activity (E. coli, S. aureus, and Botrytis cinerea), optical, water-related, and mechanical properties were in a TiO 2 :Ag dose-dependent response [3]. The addition of TiO 2 :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 2 film could be negatively affected by a high concentration of TiO 2 (>0.5 g 100 mL −1 ), 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 2 nanoparticles effectively delays fish oil photo-and auto-oxidation up to 18 days. They reported that hybrid film containing 2% of TiO 2 could control fish oil oxidation due to the enhanced UV-protective and oxygen-barrier properties associated with the physicochemical characteristics of TiO 2 .
According to the results, incorporation of TiO 2 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.

Biomedical Applications of Gelatin-TiO 2 Hybrid Composite
Gelatin-TiO 2 hybrid composites have been used for biomedical purposes. Lai et al. [80] immobilized gelatin onto TiO 2 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 2 hybrid material. They argued that extracellular matrix protein-based plays an important role in bone mineralization, while TiO 2 present in the hybrid matrix facilitates osteoblast differentiation. Ferreira et al. [71] fabricated a macroporous TiO 2 -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 2 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 2 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 2 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 2 hybrid hydrogel in an open and infected with S. aureus methicillin-resistant at 5 × 10 7 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-TiO 2 -superoxide dismutase biosensor supported in Pt surface for O 2 •− detection. They reported that the biopolymer blend (CMC and gelatin), provided a biocompatible environment for super oxide dismutase-TiO 2 , 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 2 •− detection in healthy and cancerous brain tissue (coefficient of determination or R 2 of 0.991). In this context, functionalization of gelatin-based materials with TiO 2 exhibited potential biomedical applications, associated with its enhanced biological properties.

Other Applications of Gelatin-TiO 2 Hybrid Composite
Other investigated applications of the gelatin-TiO 2 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 TiO 2 , 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 2 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 × 10 −3 mpy) in comparison with gelatin-coated (corrosion rate 10.10 × 10 −3 mpy) and uncoated (corrosion rate 9.94 × 10 −3 mpy) steel. The results were associated with the formation of a dense and stable network structure formed by the gelatin and TiO 2 nanoparticles. Furthermore, Bin Liu et al. [84] used gelatin as a template to fabricate TiO 2 mesoporous microspheres for hydrogen production. They reported that the assistance of gelatin positively influenced the morphology and physicochemical characteristics of TiO 2 nanoparticles (surface area of 98.3 m 2 ·g −1 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 2 hybrid material exhibited pharmaceutical, anti-corrosive, and hydrogen production applications.

Whey Protein-TiO 2 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 2 to improve its technological and functional properties [29]. The most common method for preparing whey protein-TiO 2 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.

Food and Non-Food Packaging Applications of Whey Protein-TiO 2 Hybrid Composite
The potential use of whey protein-TiO 2 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 TiO 2 . It was found that technological properties such as UV-protective, mechanical, and water-resistance properties were improved in a TiO 2 dose-dependent response, associated with the intramolecular connections of protein and TiO 2 through covalent and non-covalent interactions. Moreover, the authors argued that at low concentrations of TiO 2 , a reinforcement of whey protein-TiO 2 structure occurs. Meanwhile, self-assembly of TiO 2 -TiO 2 interactions are detected at high TiO 2 concentrations, influencing its technological and functional properties, mainly associated with a reduction in the crystalline structure of TiO 2 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 TiO 2 , where an excessive amount of TiO 2 in the polymeric matrix affected its functionality because TiO 2 may act as an anti-plasticizer agent [31,90]. Moreover, in a combined chitosan-whey protein film reinforced with sodium laurate-TiO 2 nanoparticles. Zhang et al. [89] reported that sodium laurate-modified TiO 2 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 2 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 2 nanoparticles. Moreover, the TiO 2 -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 TiO 2 .
Alizadeh-Sani et al. [28] informed that a whey protein isolate-cellulose nanofiber-TiO 2 -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 −1 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 2 (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 TiO 2 . 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 2 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 2 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 2 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 TiO 2 .
According to the evidence, the incorporation of TiO 2 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 2 exhibited antimicrobial properties for potential food and non-food packaging.

Other Applications of Whey Protein-TiO 2 Hybrid Composite
Ortelli et al. [92] fabricated a hybrid cotton fabric with anti-fire properties incorporating a whey protein-TiO 2 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 2 acts as a physical reinforcement agent to fix whey protein to cotton fabrics in a stable way with the hydroxyl groups.

Collagen-TiO 2 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 2 to improve its physicochemical properties [5]. Preparation of collagen-TiO 2 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 lists, works on collagen-based materials functionalized with TiO 2 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 2 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 2 nanotubes (TNT), which exhibited significant binding resistance, and the chemically linked collagen-TiO 2 facilitated epithelial cell stretching and sheet formation. Similarly, Bishal et al. [96] informed that collagen-TiO 2 promotes human osteoblast growth and proliferation in a dose-dependent manner with no inflammatory response detected, which was associated with the ability of TiO 2 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 2 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)-TiO 2 .
Additionally, collagen-silane-TiO 2 has also been used as a functional agent of Mg alloys. The hybrid composite promotes the formation of a stable Mg(OH) 2 /MgCO 3 /CaCO 3 structure that effectively protects its corrosion. Moreover, the collagen-silane-TiO 2 improved osteoblasts and fibroblasts proliferation compared to bare and silane-TiO 2 -coated alloys. In the long term, collagen-silane-TiO 2 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 2 nanoparticles, with good degradation resistance in PVP dose-dependent response for potential tissue engineering applications. Likewise, collagen-chitosan-TiO 2 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 2 nanoparticles improved its biological properties for dental implants and bone and dermal regeneration.

Other Applications of Collagen-TiO 2 Hybrid Composite
Other researched applications of the collagen-TiO 2 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 TiO 2 . 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.
Furthermore, Cheng et al. [100] proposed a facile synthetic strategy to engineer a one-dimensional (1D) hierarchically ordered mesoporous TiO 2 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 2 hybrid material exhibited potential applications such as food and non-food packaging, environmental remediation, and electrochemical studies.

Soy Protein-TiO 2 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 2 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). The hybrid composite showed antimicrobial activity. [23] * Material composition was based on the best-reported results. NI: No information; SM: synthesis method; (TiO 2 ): concentration of titanium dioxide; CP: crystallite phase. Table 7 lists the work on soy protein isolate-TiO 2 hybrid material for food and non-food packaging development with enhanced properties. Malathi et al. [102] informed that TiO 2 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 2 promoted a decrease in water vapor (from 5.43 to 4.62 g·mm·m −2 ·day −1 ·kPa −1 ) and oxygen (from 0.470 to 0.110 g·cm −2 ·day −1 ) permeability, as well as an increase in tensile strength (from 6.6683 to 14.5642 MPa) in a TiO 2 concentration-dependent response. They argue that the presence of TiO 2 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 2 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 2 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 2 and β-conglycinin and glycinin peptides present in the SPI [107].

Food and Non-Food Packaging Applications of Collagen-TiO 2 Hybrid Composite
Additionally, Wang et al. [19] informed that TiO 2 incorporation in an SPI film positively influences its tensile strength (90.79% higher than control). On the other hand, the addition of nano-TiO 2 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 TiO 2 .
The reported application of SPI-TiO 2 hybrid film includes fruit preservation and water-dye degradation. Zhang et al. [105] reported that SPI-TiO 2 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 2 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 2 into SPI significantly improved its physicochemical properties and exhibited good fruit preservation performance.

Other Applications of Soy Protein Isolate-TiO 2 Hybrid Composite
Calza et al. [108] fabricated a system composed of soybean peroxidase and TiO 2 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 TiO 2 . Further studies are needed to understand the removal and degradation mechanism of soybean peroxidase-TiO 2 , which could be used as an alternative for wastewater treatment. Table 8 lists various non-conventional proteins functionalized with TiO 2 , 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 lists reports on the use of non-conventional protein materials functionalized with TiO 2 for food and non-food packaging development. Kadam et al. [24] evaluated the effect of TiO 2 :SiO 2 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 2 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 TiO 2 :SiO 2 , which promotes a stable and strong hydrogen bonds formation. Similarly, Amjadi et al. [109] made zein-sodium alginate (90:10) film functionalized with TiO 2 -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 TiO 2 . Moreover, Böhmer-Maas et al. [33] developed a zein-TiO 2 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 2 and water by the OH radicals and reactive oxygen species generated by the photocatalytic ability of TiO 2 .

Other Proteins Functionalized with TiO 2
Montes-de-Oca-Ávalos et al. [30] investigated the effect of TiO 2 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 2 concentration-dependent way, associated with good dispersion of TiO 2 through the film polymeric matrix. According to the authors, the presence of TiO 2 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 2 (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 2 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 2 and cumin essential oil.
Additionally, Montazer et al. [22] informed that the incorporation of TiO 2 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 2 and BTCA interactions, with an optimum concentration of 0.6 g·L −1 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 2 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 2 nanoparticles exhibited interesting properties for food and non-food packaging development.

Environmental Applications of Non-Conventional Proteins Functionalized with TiO 2
Usage of zein, keratin, and sesame proteins as a supporting material of TiO 2 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 2 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 −1 ) 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 2 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 2 dose-dependent response, with an optimum TiO 2 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 2 through covalent and non-covalent interactions. Moreover, Siriorn and Jatuphorn [112] reported that a chicken feather keratin-poly(lactic acid)-TiO 2 nanofibers (0.05 g) effectively remove methylene blue (90%) dye from aqueous solution (5 × 10 −6 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 2 for water-dye removal purposes. They reported that the hybrid film (64 cm 2 ) effectively degraded 76% of methylene blue dye (10 mg·mL −1 ) 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 2 dose-dependent response with an optimum TiO 2 concentration of 3% w/w associated with the interaction chemical and physical interactions between sesame protein and TiO 2 . On the other hand, the morphological studies through scanning electron microscopy revealed that a high concentration of TiO 2 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 2 nanoparticles could be a viable, low-cost, and efficient alternative for environmental applications as photocatalysts for wastewater treatment.

Other Applications of Non-Conventional Proteins Functionalized with TiO 2
Other potential uses of non-conventional proteins functionalized with TiO 2 include bone regeneration, antimicrobial activity, and textiles (Table 8). Johari et al. [114] made a fluorated silk fibroin-TiO 2 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 2 nanoparticles (TiO 2 -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 2 dose-dependent response due to the formation of Ti-O-C bonds and the partial substitution of OH groups present in the TiO 2 surface by fluorine anions, that significantly increase the functional properties of TiO 2 . On the other hand, with high amounts of TiO 2 (>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 2 hybrid composite did not show cytotoxic effects on a human Caucasian fetal foreskin fibroblast cell line at low TiO 2 concentrations (<1.5% w/w). Nonetheless, the hybrid material exhibited a porosity of 94%, a density of 3118 mg·mL −1 , 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 2 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 TiO 2 -dependent manner, attributed to the presence of hydroxyl groups on the TiO 2 . 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 2 might negatively affect the mechanical properties of the hybrid material associated with the damage of its microscopic structure mainly by the formation of TiO 2 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 2 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 2 nanoparticles exhibited interesting properties and applications. However, further studies are needed to validate their potential uses.

Disadvantages of Protein-TiO 2 Hybrid Composites and Perspectives
Despite the observation that protein-TiO 2 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 2 in their composition.
In this context, it has been reported that pure TiO 2 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 −1 with direct application [118][119][120][121][122][123]. Nonetheless, the tested concentrations of TiO 2 in these works were higher than the recommended safe usage (<1% by weight) by international regulations in the use of TiO 2 as a food additive [124].
However, the amount of TiO 2 used as a functional agent to develop protein-TiO 2 hybrid composites ranges from 0.003 to 1 mg·mL −1 , depending on its application. For example: in food packaging materials manufacturing, the amount average of TiO 2 employed is 0.28 mg·mL −1 , while for packaging materials with non-food purposes it is 0.85 mg·mL −1 . Moreover, for the development of scaffolds, dental implants, and wound-healing materials, the average amount of TiO 2 is 0.23 and 0.9 mg·mL −1 for making hybrid materials for environmental remediation.
According to Xu et al. (2017) [123], the interaction between protein structure and TiO 2 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 2 [125]. In this sense, there are a few reports on the toxicity status of protein-TiO 2 hybrid composites, which reported no toxicological or adverse effects on their use, associated with the low concentration of TiO 2 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.

Concluding Remarks
Significant evidence indicates that functionalization of protein-based materials by adding TiO 2 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 2 hybrid films and coating and freeze-drying for hydrogels and scaffolds, using commercial TiO 2 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 2 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 2 offers significant advantages, some limitations have been reported, especially those associated with the concentration of TiO 2 . Higher concentrations of TiO 2 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 TiO 2 , 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 2 hybrid composites could be related to the type and source of protein and its possible structural changes by the presence of TiO 2 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 2 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.