Extracted fibers from L. cyanescens, Coll fibers were prepared from the trimmings of cowhides collected from tannery at Central Leather Research Institute, Chennai [34], sulfuric acid (H₂SO₄,), n-octanal (n-C₈H₁₆), anhydrous sodium sulfite (Na₂SO₃), sodium chlorite (NaClO₂), sodium monochloroacetic acid (MCAA) (C₂H₃ClO₂Na), acetone (C₃H₆O), polyethylene glycol (PEG)(C₂H₆O₂), nitric acid (HNO₃), titanium(IV) isopropylate (TTIP) (Ti[OCH(CH₃)₂]₄), tannic acid (C₇₆H₅₂O₄₆), cetyltrimethylammonium bromide (CTAB) (C₁₉H₄₂BrN), ethanol (C₂H₆O), silver nitrate (AgNO₃), tatanic acid (C₇₆H₅₂O₄₆), acetic acid (C₂H₄O₂), methanol (CH₄O) and isopropyl alcohol (C₃H₈O). All chemicals were used as received.

20 g of dried extracted fiber [30] were boiled with 300 mL of 6% (w/v) hydroxide solution (NaOH) solution for 1 h for the pre-treatment process for the solubilization of surface impurity, hemicellulose, and partially lignin. The pre-treated fiber were washed and ground in a laboratory blender for 5 min to reduce the fiber size (similar to paper pulp). Then, the delignification process was carried out by bleaching with 300 mL of 0.7% (w/v) sodium chlorite under acidic conditions at pH = 4 adjusted with 5% acetic acid solution, and boiled for 3 h. The partially lignin-free fiber residue was washed with distilled water until neutral pH. The neutralized fiber residue was boiled with 300 mL of 5% (w/v) sodium sulfite solution for 5 h to completely remove lignin as well as partially remove of hemicellulose. The obtained holocellulose was boiled with 300 mL of 17.5% (w/v) NaOH solution for 5 h to remove hemicelluloses. The isolated cellulose was washed with distilled water until neutral pH and air-dried.

In a 500 mL round flask, 170 mL of distilled water and 10 mL of 0.1 M CTAB solution were stirred for 10 min at 30°C. To this solution, 10 mL of 0.1 M silver nitrate solution was added dropwise with continuous stirring for 10 min. 20 mL solution of water containing 1.4 g of previously dissolved tannic acid was added to initiate the reduction reaction. The color of the solution changed from colorless to yellowish brown. After adding a few drops of NaOH solution (0.1 M), the color of the solution changed to dark brown and the reaction was stirred at room temperature for 2 h. The mixture was centrifuged at 15.000 rpm for 15 min at 4°C. The residue was washed three times with distilled water and once with ethanol. The Ag NPs were dried in an air oven at 80°C for 4 h.

For the synthesis of TiO₂ nanoparticles, 5 mL TTIP was introduced into 20 mL of ethanol, and then the mixture was stirred at 500 rpm at a temperature of 60°C for 30 min. Next, a mixture composed of 20 mL of ethanol and distilled water (1:1) (v/v) was prepared, and then 2 mL of concentrated nitric acid (72%) was added dropwise under constant stirring. This mixture was gradually incorporated into the first solution, and the whole was stirred at 1000 rpm for 3 h at 100°C, which led to the formation of a gel. The obtained product was centrifuged at 10,000 rpm for 20 min. The precipitate was collected, washed successively with distilled water and then with ethanol to remove residual impurities. After drying at 100°C for 8 h in a hot air oven, the dried powder was calcined at 500°C for 2 h under nitrogen atmosphere to obtain TiO₂ NPs.

The preparation of the heterostructure Ag@TiO₂ nanocomposite was carried out according to the following protocol: a reaction mixture with a ratio of 1:0.2 of TTIP and Ag NPs was used. Briefly, 0.24 g of Ag NPs was dissolved in 10 mL of ethanol and stirred at 500 rpm for 10 min. Then, 1.2 g of TTIP was added dropwise to the above mixture and the reaction continued for 1 h 30 min at 60°C. The resulting colloidal solution was transferred to a crucible and dried at 55°C for 24 h, then calcined at 450°C for 2 h under nitrogen purge.

CMC was synthesized from cellulose extracted from L. cyanescens according to the method described by [9]. The synthesis reaction of CMC proceeds in two steps: alkalization, followed by etherification of cellulose under heterogeneous conditions. First step: 1 g of isolated cellulose powder was introduced into 100 mL Erlenmeyer flask, and 60 mL of water-isopropanol alcohol solvent mixture of ratio (1:4) was added. Alkalization was carried out by dropwise addition of 8 mL of NaOH solution at different concentrations (10%, 20%, 30% and 40%) (w/v) under magnetic stirring for 2 h at room temperature (~30°C). Afterwards in the second step, the carboxymethylation reaction was initiated by adding a variable amount of MCAA (0.4; 0.8; 1.2; 1.6 g) to the reaction mixture. The mixture was heated at 60°C under reflux and continuous magnetic stirring for 3 h. Then the precipitate was separated by filtration and placed in 100 mL of methanol for 40 min. Finally, a quantity of glacial acetic acid was added to neutralize the mixture. Then filtered and washed four times with 50 mL of 80% ethanol. To remove unwanted salts, the mixture was washed again with 50 mL of absolute methanol. The final product was dried in an air dryer at 50°C for 12 h. The recipe for the experiments (CMC1, CMC2, CMC3 and CMC4) of the synthesis of CMCs is summarized in Table 2.

The yield (%) of CMC is calculated according to the following relationship: (1)CMC(% )=Weight of CMC (g)Weight of cellulose (g)×100

In 100 mL beaker, 0.7 g of CMC based on L. cyanescens fiber was dissolved in 30 mL of distilled water under magnetic stirring of 1000 rpm at 80°C for 1 h. Meanwhile, in another 100 mL beaker, 0.3 g of Coll was also dissolved in 20 mL of 0.5 M acetic acid solution with an Ika homogenizer for 10 min. The formulation of the bioplastic was carried out by adding the Coll solution dispersion to the CMC solution at room temperature with 10% of PEG as a plasticizer and emulsifier to reduce crystallinity and improve the mechanical properties and thermal stability [35]. The mixture was stirred at 1200 rpm for 2 h. For the preparation of hybrid composite films, 5% of nanoparticles was added to the organic mixture. The homogeneous plastic product was cast into a plastic Petri dish and dried at room temperature for 24 h, and then at 40°C in an air oven for 30 min to obtain an organic-inorganic hybrid film. Table 3 summarizes the recipe for the experiments (Formulation 1, Formulation 2, Formulation 3 and Formulation 4).

The pathogenic bacterial strains (Staphylococcus aureus, Bacillus cereus and Salmonella typhi) previously stored at −80°C were recovered and cultured on a nutrient agar for bacteria. The agar was then incubated in an incubator at 37°C for 24 h in order to obtain young and viable cells. Then, the revived strains were cultured in a nutrient broth. The culture broth was subsequently incubated at 37°C for 24 h. The load of the obtained cultures was adjusted to 107 cell/mL using a spectrophotometer according to the method [36]. The antibacterial tests were conducted under direct solar light condition.

The Dynamic Light Scattering (DLS) was used to determine the average hydrodynamic diameter of the synthesized nanoparticles. Measurements were performed using a Litesizer 500 particle analyzer.

The UV-visible spectra of the nanoparticles were obtained using a UV-visible spectrophotometer (Shimadzu UV-1800). The spectrum was taken from 0 to 800 nm with a scanning interval of 0.5 nm.

The surfaces of the samples were analyzed using a scanning electron microscope field emission spectroscopy (FESEM, TESCAN, model-CLARA) operating at an accelerating voltage of 15.0 kV. Prior to SEM observation, the samples were sputtered with a thin layer of gold to avoid the charging effects of the electron beam during examination.

The degree of substitution (DS) of CMC from L. cyanescens fibers represents the average number of hydroxyl groups replaced by carboxymethyl and sodium carboxymethyl groups at C2, C3, and C6 in the cellulose structure. The DS was determined by titration according to ASTM-D1439-03 standard.

The mechanical properties of the composite films were determined using a Universal Testing Machine (UTM) (Zwick/Z010) in accordance with ASTM D638. All experiments were carried out at a cross-sectional speed of 5 mm/min with an initial length L0 of 140 mm. The tensile strength and elongation at break measurements were carried out with three (03) repetitions for each sample and the average value was recorded.

The contact angle of the synthesized films was determined using the sessile drop method using a Drop Shape Analyzer—DSA25 equipped with a monochrome camera B-CAM-21-BW (CCCIR) and an LED R60 lamp (Conrad). 4 μL of distilled water was deposited on the surface of each film and the contact angle was recorded after 30 s. One Touch Grabber and Image J software were used to calculate the contact angles. An average value was obtained for triplicate measurements.

The water absorption behavior of the synthesized film samples was examined by gravimetric method as reported by [37], then adapted to our study. Indeed, samples of film were cut into squares measuring 1 cm × 1 cm and mass m₀ previously known were immersed in 50 mL of distilled water, at room temperature for 48 h. Every 4 h the samples were removed from the water, cleaned using paper to remove excess residual water on the surface and weighed (m₁). Finally, the water absorption rate of samples was determined as follows: (2)water absorption rate  (% )=100×m1−m0m0

The absorption rate retained is the average of three values measured on 3 samples, for each formulation.

The demonstration of antibacterial activity was carried out according to the method recommended by Barefoot and Klaenhammer [38]. The Mueller-Hinton agar was prepared according to the manufacturer’s instructions and sterilized at 121°C for 15 min. The prepared media were poured into Petri dishes. After solidification, a volume of 100 µL of the microbial suspension (107 spores/mL) prepared previously was deposited on the surface of the agar and then spread. Film discs of 1 cm in diameter were placed on the agar which had been previously inoculated with the pathogenic strain. The Petri dishes were placed at 4°C for 1 h to allow good diffusion of the antimicrobial substance. The dishes were then incubated at 37°C. The presence of inhibition zones formed around the discs was examined after 24 h of exposure in solar light [39].

The average particle size and distribution size of TiO₂ NPs, Ag NPs, and heterostructure Ag@TiO₂ nanocomposite are shown in Fig. 1. The measured sizes are 46, 140, and 403 nm for TiO₂ NPs, Ag NPs, and Ag@TiO₂ nanocomposite, respectively. These results show that TiO₂ NPs are the smallest and most uniform. The Ag NPs exhibit a slightly broader distribution with relatively large size. In contrast, the particle size of Ag@TiO₂ nanocomposite is significantly larger, with a broader particle size distribution, indicating that the particles are probably agglomerated. It has been reported the size-dependent antibacterial efficacy of nanoparticles [40–44]. The antibacterial efficacy increases with the decreasing particles size. Beyond size, shape-dependent also plays an important role in the antibacterial performance of Ag NPs [40]. Ali et al. [45] compared antibacterial efficacy of Ag NPs, TiO₂ NPs and Ag@TiO₂ nanocomposite against Escherichia Coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and Enterobacter Cloacae. The authors concluded that, the heterostructure Ag@TiO₂ nanocomposite show enhanced antibacterial activity against bacterial strains because of its high surface area and best against bacterial adhesion lead to higher reactivity and releases high amount of Ag⁺ than that from Ag NPs and TiO₂ NPs [41,44,45].

The UV-visible absorption spectra of the synthesized nanoparticles (TiO₂, Ag and Ag@TiO₂) are plotted in Fig. 2. The spectrum of TiO₂ NPs shows an absorption band in the ultraviolet, with a maximum around 340 nm, characteristic of the electronic transitions of the anatase TiO₂ semiconductor [46]. This property is important for photocatalytic application, as it allows TiO₂ NPs to act as a self-cleaning or decontaminating agent under UV radiation [46]. Ag NPs, for their part, present a marked absorption peak around 420 nm, attributed to surface plasmon resonance (SPR) [47]. This optical phenomenon results from the collective vibration of electrons on the surface of the nanoparticles when exposed to light. The presence of this characteristic peak indicates that the Ag NPs are well formed. In addition, the position and intensity of this SPR peak provide important information on the relatively large size and non-agglomeration of the prepared Ag NPs, two essential elements to evaluate their antibacterial efficacy [47,48]. The spectrum of the heterostructure Ag@TiO₂ nanocomposite shows both signatures, an absorption in the UV (inherited from TiO₂ NPs), and a broadening of the absorption in the visible around 420 nm (related to Ag NPs). This superposition confirms that the Ag NPs have been well grafted onto the surface of the TiO₂ NPs, forming a heterostructure nanocomposite. The introduction of Ag NPs causes a red shift of the UV-visible spectra edges to longer wavelength compared to pristine TiO₂. The shift in the absorption of TiO₂ is favourable for the improvement of photocatalytic and photo-oxidized activities of TiO₂ under both UV and visible regions and expands the antimicrobial activity of Ag@TiO₂ nanocomposite response range under direct solar condition. The band gap of TiO₂ (~3.2 eV) is higher than that of Ag (~2.25 eV) leading to reducing the band gap of the heterostructure Ag@TiO₂ nanocomposite (~2.2–2.9 eV) [43,49] and promoting the transfer of electrons from valence band (VB) to the conduction band (CB). Because, the Ag CB is below the TiO₂ CB, while the Ag VB band is above the TiO₂ VB band. Consequently, when the heterostructure Ag@TiO₂ nanocomposite is exposed to solar light, electrons (e-) migrate from the CB of TiO₂ to the CB of Ag, while holes (h+) are transfer from the VB of TiO₂ to the VB of Ag preventing the reduction of electrons recombination as illustrated by the scheme in Fig. 3. The electron–hole pairs generated take part in a series of oxidation/reduction reactions with species adsorbed on the TiO₂ surface, to generate highly reactive oxygen species (ROS). Ag@TiO₂ nanocomposite exhibits enhanced ROS formation activity under visible light compared to pristine TiO₂, due to the SPR effect of Ag NPs [50]. ROS are natural by-products of cellular oxidative metabolism and play important roles in the modulation of cell survival, cell death, differentiation, cell signaling [50,51]. Thus, the photocatalytic activity of Ag@TiO₂ nanocomposite increases the level of ROS in the bacteria cells and disrupts the cellular oxidative metabolism, consequently, the cells endure very high oxidative stress that leads to the death of the bacteria. This type of assembly is known to be more stable and more effective against microbes, thanks to the combined action of TiO₂ NPs, which destroys germs with light, and Ag NPs, which kills microorganisms [48]. Thus, the UV-Visible results confirm the quality of the synthesized nanoparticles and validate their suitability for use in biodegradable functional films for food applications as antimicrobial.

The percentage yield of the synthesized CMC is presented in Fig. 4a and shows a characteristic evolution as a function of NaOH concentration. Indeed, the yield of CMC increases significantly when the NaOH concentration increases from 10% to 30% reaching a maximum of 91% for CMC3, before decreasing to 77% at the NaOH concentration of 40%. This phenomenon resulting from the decrease in CMC yield could be due to a limitation of MCAA as an etherifying agent to replace the OH groups of cellulose. The DS exhibits the same trends with the NaOH concentration (Fig. 4b) with the maximum of 0.95 at 30% of NaOH concentration. Thus, the 30% NaOH concentration seems to be an optimal balance point to obtain a high yield while maintaining the structural integrity of the modified cellulose. This behavior is explained by two reactions that occur simultaneously during the carboxymethylation process. The first (Eq. (2)) involves the reaction of a hydroxyl group of cellulose with MCAA in the presence of NaOH, which leads to the formation of CMC. The second, concurrent and unwanted, consists of the reaction between NaOH and MCAA, forming a by-product called sodium glycolate according to the equation: (3)NaOH +Cl-CH2 COONa →HO-CH2 COONa + NaCl

When the sodium hydroxide concentration is too high, this secondary reaction becomes dominant and consumes MCAA, thus reducing the amount available for the main reaction and resulting in the decreasing of the DS. This phenomenon was confirmed by the results of Joshi et al. [52].

The photographs and SEM morphological observation images of the films in Fig. 5 show a clear influence of the nature of the nanoparticles on the appearance and the surface morphology of films. The control film (Fig. 5a) without nanoparticles appears white and presents a smooth and homogeneous surface. The introduction of TiO₂ NPs (Fig. 5b) generates a yellowish film with a slight roughness and fine aggregates well integrated into the matrix, while the dark glossy black film with Ag NPs in Fig. 5c exhibits strong roughness with random dispersion and distribution of Ag NPs particles sizes on the surface. In contrast, the film containing the heterostructure Ag@TiO₂ nanocomposite (Fig. 5d) reveals a heterogeneous black with a white tone surface appearance marked by massive agglomerates, a sign of less efficient dispersion. These morphological differences suggest that the incorporation and type of nanoparticles strongly influence the microscopic structure of the films, which could affect their functional properties such as mechanical strength, permeability and antimicrobial activity.

The results of the water contact angles of the films are illustrated by the images presented in Fig. 6. The measured angles are 53°, 62°, 71° and 87° respectively for formulation 1, formulation 2, formulation 3 and formulation 4. A progressive increase in the contact angle from 53° to 87° is observed, reflecting a decrease in wettability and an increasing trend towards hydrophobicity. The film (Fig. 6a) without nanoparticles exhibits the lowest contact angle (53°), indicating strong hydrophilicity attributed to CMC, a naturally hydrophilic polymer. The addition of TiO₂ NPs and Ag NPs in formulation 2 and formulation 3 (Fig. 6b,c, respectively) significantly the contact angle, suggesting a reduction in wettability due to a change in surface roughness. Formulation 4, containing heterostructure Ag@TiO₂ nanocomposite reaches a maximum angle of 87° as shown in the Fig. 6d and close to hydrophobic behavior. It has been reported that the synergetic effect of bimetallic in collagen-cellulose sponge increases the water angle up to 123° [53]. Upon immobilizing collagen within the bacterial cellulose matrix via tannin, the water contact angle increased further due to the formation of hydrogen bonds between cellulose fibers, collagen, and tannin, reducing then the numbers of free hydroxyl groups with increase in the hydrophobicity of the composite film [19]. These results show a conformity with those obtained by the DLS test where the particle size increases with the surface roughness which is important for food packaging applications where a certain resistance to humidity is sought.

Fig. 7 illustrates the stress-strain curves obtained from the tensile tests on the biocomposite films (formulation 1 to formulation 4). In general, all the curves show a progressive increase in stress with deformation, until reaching a maximum value followed by a sudden rupture. This behavior indicates a certain capacity of the films to deform before rupture [54]. The control film (Control), free of nanoparticles, shows the lowest performance with a maximum stress of 9.08 MPa and a strain of 1.77%, reflecting low mechanical strength and limited elasticity. The addition of TiO₂ NPs leads to a significant improvement in mechanical properties, with the stress of 30.78 MPa and strain of 7.17%, which can be attributed to a good reinforcing effect of inorganic fillers in the polymer matrix [55]. The formulation 3 film containing only Ag NPs, shows an even higher strength (31 MPa) but a reduced strain (2.03%), suggesting brittleness of the material. This low strain could be explained by the agglomeration of Ag NPs in the matrix, leading to poor load distribution and the formation of stress concentration zones [56]. On the other hand, the formulation 4 film containing the Ag@TiO₂ nanocomposite, shows the best mechanical performance with a maximum stress of 38.77 MPa and a strain of 9% characteristic of a ductile material. This notable improvement can be interpreted as the result of a synergistic effect between the Ag NPs and TiO₂ NPs, allowing both a good load transfer, a reinforced interaction with the matrix and a better homogeneity in the load distribution [48]. These results confirm the interest of this hybrid biocomposite for packaging applications, where the combination of strength and flexibility is essential.

The kinetic study of water absorption of the different formulated films is illustrated in Fig. 8 and shows a characteristic two-phase evolution: an initial phase of rapid absorption followed by a plateau, reflecting the progressive saturation of the hydrophilic sites of the polymer matrix. The control film, formulation 1 composed of the organic matrix Coll-CMC, displays the highest water absorption capacity, almost 100% at 48 h. This behavior is attributable to the intrinsic hydrophilic nature of CMC, rich in hydroxyl groups, promoting water retention by hydrogen interactions and swelling of the macromolecular network [57–60]. The distribution pattern of the interconnected Coll and CMC formed three-dimensional structures and Coll fibrils tend to penetrate the CMC network to form a supramolecular double network structure composite material [60]. The high water absorption could be explain by the loose network structure of Coll fibrils within the composite. The introduction of inorganic nanoparticles into the organic matrix significantly affects the absorption kinetics and capacity. The formulation 2 film containing TiO₂ NPs, shows a notable reduction in the absorption rate to 80% at 48 h. This decrease is explained by the barrier effect created by the nanoparticles, which partially fill the pores and limit the diffusion of water into the matrix. In addition, the interactions between the hydroxyl groups of the CMC and the polar surfaces of TiO₂ contribute to a densification of the network, as highlighted by [29] in chitosan-based films reinforced with TiO₂. The formulation 3 film enriched with Ag NPs, reaches a maximum absorption of 65%. This intermediate value suggests that Ag NPs, although less effective than TiO₂ NPs as a physical barrier, nevertheless interact with the Coll-CMC matrix. These interactions (coordinated bonds or hydrogen bonding with the carboxylate groups) slightly limit the chain mobility and the availability of active sites for water adsorption. These effects have been reported by [47] who studied the modification of Ag NPs-based films in active packaging applications. Finally, the formulation 4 film containing the heterostructure Ag@TiO₂ nanocomposite shows the lowest water absorption, reaching 50% at 48 h. This result is consistent with a dual action: on the one hand, the dense and hydrophobic structure induced by the presence of TiO₂ NPs; on the other hand, the synergistic effects of Ag NPs that reinforce the cohesion of the matrix by the release of the metal ion Ag⁺ to formed intramolecular metal-ligand bonds stable to water in wet heterostructure Ag@TiO₂, nanocomposite [60]. Such synergy has been documented by Akhavan [48] in Ag/TiO₂ hybrid systems embedded in polysaccharide matrices, showing a significant reduction in water permeability and an improvement in dimensional stability. Kinetically, it is observed that the nanoparticle-loaded films reach their absorption plateau more slowly than the Coll-CMC film alone, indicating a slower diffusion of water within the reinforced organic matrices. This dynamics can be modeled by pseudo-first-order kinetics, suggesting that the imbibition rate is controlled both by water diffusion in the pores and by the availability of active sites on the surface of the polymer chains. In conclusion, the results obtained clearly show that the incorporation of nanoparticles, particularly the heterostructure Ag@TiO₂ nanocomposite can effectively reduce the hydrophilicity of Coll-CMC-based films. This strategy is promising for the formulation of barrier films for food, biomedical or smart packaging applications, where water resistance is crucial.

The standard disk diffusion method was used to evaluate the antibacterial activity of TiO₂ NPs, Ag NPs, and heterostructure Ag@TiO₂ nanocomposite against three bacterial strains. Fig. 9 displays the results of the test. Among the tested samples, heterostructure Ag@TiO₂ nanocomposite showed the most pronounced antibacterial effect on all bacteria. In contrast, TiO₂ NPs alone did not show a significant inhibition zone. This low efficacy could be attributed to their relatively smaller size, as reported by [61]. The significant improvement in antibacterial activity in the case of the Ag@TiO₂ nanocomposite could result from a synergistic effect between the Ag NPs and the TiO₂ NPs. Indeed, the presence of Ag NPs would amplify the antibacterial effect, in particular thanks to the increase in the size of the particles in the composite, promoting better interaction and adhesion with bacterial cells [61]. This enhanced activity can be explained by several mechanisms. On the one hand, the release of metal ions (especially Ag⁺) from the surface of nanoparticles would contribute to toxicity towards microorganisms. On the other hand, the generation of reactive oxygen species (ROS) would play a key role, particularly in the case of TiO₂ NPs, which triggers a photocatalysis process under natural sunlight irradiation and releases hydroxyl radicals (OH) and superoxides (O₂-) in an aqueous medium. These ROS attack cell membranes by degrading phospholipids and can also induce damage to bacterial DNA, leading to a pronounced antibacterial effect as mentioned earlier.