Gelatin-Based Biofilms with FexOy-NPs Incorporated for Antioxidant and Antimicrobial Applications

Currently, gelatin-based films are regarded as promising alternatives to non-environmentally friendly plastic films for food packaging. Nevertheless, although they have great biodegradability, their weak mechanical properties and high solubility limit their applications. In this way, the use of nanoparticles, such as FexOy-NPs, could improve the properties of gelatin-based biofilms. Thus, the main objective of this work was to include different concentrations of FexOy-NPs (0.25 and 1.0%) manufactured by green synthesis (GS) and chemical synthesis (CS) into gelatin-based biofilms in order to improve their properties. The results show that FexOy-NPs can be distributed throughout the biofilm, although with a greater concentration on the upper surface. In addition, the incorporation of FexOy-NPs into the biofilms improves their physicochemical, mechanical, morphological, and biological properties. Thus, it is possible to achieve suitable gelatin-based biofilms, which can be used in several applications, such as functional packaging in the food industry, antioxidant and antimicrobial additives in biomedical and pharmaceutical biomaterials, and in agricultural pesticides.


Introduction
A film is a very thin (thickness < 1 mm), transparent, and, in many cases, stretchable plastic with different uses [1]. It is normally made of polyethylene (PE) or polypropylene (PP), which offers great flexibility, making it perfect for wrapping products of different shapes and sizes [2]. The most interesting properties of these films are their transparency (which allows one to see inside the package), their flexibility and adaptation to all kinds of shapes and sizes, and their impermeability, which prevents the passage of air and moisture, acting as a barrier and protecting the interior [3]. These properties are very important in the food industry, as they help to extend the shelf life of food by preventing oxidation-reduction reactions and interaction with microbes [4].
In recent years, the film sector has been affected by an increase in demand due to two fundamental factors: the need for safety in the transport of products, thereby increasing the need for wrapping the merchandise, and the current trend towards better presented products, which has considerably increased the use of films as an element with hygienic and aesthetic characteristics [5]. Nevertheless, the low biodegradability of these packaging materials is generating a great pollution problem, being unsuitable for the food industry. In this way, natural biopolymer-based films are currently being investigated, such as those made of gelatin, chitosan, cellulose, or cellulose derivatives, which confer them good biodegradability without releasing toxic substances [6,7]. Among the raw materials, gelatin has great potential to be used in diverse industrial applications: (1) in the food industry, to protect food from certain factors such as drying, light, and oxidation, since it may be used in biofilms to incorporate a wide range of additives, including antioxidants,

Materials
The gelatin protein used in this study was food gelatin type B 200/220 g blooms supplied by Manuel Riesgo, S.A. (Madrid, Spain), being a food gelatin that contains sulfur dioxide (<10 ppm). Gallic acid (C 7 H 6 O 5 ) and DPPH (2,2-diphenyl-1-picrylhydrazyl) were purchased from Sigma Aldrich (Darmstadt, Germany). All the reagents were of analytical grade. Fe x O y -NPs were synthesized according to a previous work with slight modifications [37]. Briefly, it consists of colloidal precipitation in which 20 mL of Phoenix dactylifera L. extract, which is rich in polyphenols (green) or NaOH (chemical) (used as reductors) were mixed with 20 mL of FeCl 3 ·6H 2 O (used as a precursor). The resulting 40 mL of mixture was heated under continuous stirring for 2 h at 50 • C. Then, the obtained precipitate was filtered, washed, and dried in an oven for 8 h at 100 • C. Finally, they were calcinated in a muffle for 5 h at 500 • C. CS

Biofilm Processing Method
Biofilms were fabricated by the casting procedure [3]. To this end, gelatin was firstly dissolved in distilled water (2% w/v), subjecting it to magnetic stirring for 2 h at 60 • C and 600 rpm. Subsequently, different concentrations of Fe x O y -NPs (0.25 and 1.0% w/w) with respect to the initial gelatin were dispersed in the solutions by ultrasound for 0.25 h. Finally, a constant volume (42.7 mL) of the solution was cast into Teflon plates (7.6 cm of diameter) and dried at room conditions (22 ± 1 • C and 35 ± 1% RH) for 3 days. The biofilms were peeled off and kept in a desiccator for further characterization. A reference biofilm was manufactured without the dispersion of NPs. Figure 1 shows a scheme of the different steps of this process.
FexOy-NPs were synthesized according to a previous work with slight modifica [37]. Briefly, it consists of colloidal precipitation in which 20 mL of Phoenix dactylif extract, which is rich in polyphenols (green) or NaOH (chemical) (used as reductors) mixed with 20 mL of FeCl3·6H2O (used as a precursor). The resulting 40 mL of mi was heated under continuous stirring for 2 h at 50 °C. Then, the obtained precipitate filtered, washed, and dried in an oven for 8 h at 100 °C. Finally, they were calcinate muffle for 5 h at 500 °C. CS FexOy-NPs had a mean size of 49 ± 2 nm, a 2.20 Fe2O3:Fe3O4 ratio, and 47% cr linity. GS FexOy-NPs had a mean size of 32 ± 1 nm, a 0.84 Fe2O3:Fe3O4 ratio, and 69% tallinity.

Biofilm Processing Method
Biofilms were fabricated by the casting procedure [3]. To this end, gelatin was f dissolved in distilled water (2% w/v), subjecting it to magnetic stirring for 2 h at 60 °C 600 rpm. Subsequently, different concentrations of FexOy-NPs (0.25 and 1.0% w/w) respect to the initial gelatin were dispersed in the solutions by ultrasound for 0.25 nally, a constant volume (42.7 mL) of the solution was cast into Teflon plates (7.6 c diameter) and dried at room conditions (22 ± 1 °C and 35 ± 1% RH) for 3 days. The bio were peeled off and kept in a desiccator for further characterization. A reference bi was manufactured without the dispersion of NPs. Figure 1 shows a scheme of the diff steps of this process.

Water Solubility
For the determination of this parameter, the samples (2 × 2 cm 2 ) were firstly weig (Wi) and then placed in an oven at 105 °C for 24 h. Later, the samples were immers 50 mL of distilled water for 24 h. Finally, the films were taken out and redried at 1 for 24 h to obtain the final dry weight (Wf). The weight loss or water solubility perce (WS%) was calculated with Equation (1) [26,29]:

Water Solubility
For the determination of this parameter, the samples (2 × 2 cm 2 ) were firstly weighted (W i ) and then placed in an oven at 105 • C for 24 h. Later, the samples were immersed in 50 mL of distilled water for 24 h. Finally, the films were taken out and redried at 105 • C for 24 h to obtain the final dry weight (W f ). The weight loss or water solubility percentage (WS%) was calculated with Equation (1) [26,29]:

Optical Properties
Another essential property of biofilms is their transparency, which restricts light transmission while allowing visibility through the packaging material. To determine Materials 2022, 15, 1966 4 of 13 this property, UV-vis spectroscopy was used. Thus, 1 × 2 cm 2 samples were measured in a UV-vis spectrophotometer (Model 8451A, Hewlett Packard Co., Santa Clara, CA, USA) at 600 nm. A blank was carried out with air [24,38]. The results were indicated as transmittance (amount of light that can pass through the system).

Mechanical Properties
Static tensile tests were performed in order to evaluate the mechanical properties of the biofilms. For this, a modification of the ISO 527-3:2019 standard [39] was used. The samples were subjected to an increasing axial force at a speed of 10 mm/min until break in an MTS Insight 10 Universal Testing Machine (Berlin, Germany). During these tests, temperature and relative humidity were constant at 22 ± 1 • C and 35 ± 1%, respectively. The maximum stress (σ max ), strain at break (ε max ), and Young's modulus of each biofilm were analyzed. Scanning electron microscopy (SEM) was used to determine the microstructure of the samples. The biofilms were analyzed on both sides (bottom and upper surfaces). In addition, their thickness was measured using ImageJ software. The samples were firstly covered with a thin gold layer to improve their conductivity and, thus, the quality of the micrographs. Later, they were observed in a Zeiss EVO microscope (Pleasanton, CA, USA) with an acceleration voltage of 10 kV and magnification of 3 KX [40].

Energy Dispersive X-ray Spectroscopy (EDX)
The elemental composition distribution of the biofilms was also evaluated. For this, a EDX detector was attached to an SEM microscope [41]. In this way, the Fe distribution was analyzed in both surfaces of each biofilm through mapping (by converting to 8-bit grayscale and then Auto Threshold, where the obtained percentage was representative of the color area of Fe).
2.6. Functional Properties 2.6.1. Antioxidant Activity Antioxidant activity of the biofilms was determined using the protocol described by Mehmood et al. (2020) with slight modifications [29]. Thus, 1 mL of film solution was mixed with 1 mL of DPPH solution dissolved in methanol (40 ppm). This mixture was kept in the dark for 30 min at 25 • C. Finally, the absorbance of each solution was read at 517 nm in a spectrophotometer. Gallic acid was used as the positive control. DPPH inhibition (IP) was calculated using Equation (2).
where A and B are the DPPH absorbance without and with antioxidant agent, respectively.

Antimicrobial Activity
The antimicrobial activity of the different biofilms was evaluated using an agar diffusion experiment [29]. In this way, cylindrical biofilms (9 mm of diameter) were firstly sterilized by immersion in 96% (v/v) ethanol for 2 min, after which they were rinsed thrice with sterile phosphate buffered saline (PBS). Then, they were placed in agar gels inoculated with Staphylococcus aureus (S. au) and Escherichia coli (E. col). The antibacterial activity was determined as the inhibition zone (diameters) surrounding the biofilm after 24, 48, and 72 h of incubation at 37 • C using ImageJ software.

Statistical Analysis
At least 3 replicates of each sample were performed for each measurement. The results were presented as mean values with standard deviations, which were calculated using IBM SPSS statistic software. In addition, the significant differences were evaluated using a one-way ANOVA with 95% confidence level (p < 0.05).

Water Solubility
Water solubility (WS) is a critical parameter for food packaging applications. In this sense, biofilms must be insoluble in water to improve water resistance and product safety [26,29]. Table 1 shows the water solubility (WS) values of the different biofilms. As can be seen, the reference biofilm (neat gelatin) reached the highest WS value (80.9%). Higher WS of neat gelatin biofilms was due to the hydrophilic nature of gelatin [42]. Thus, the incorporation of Fe x O y -NPs into the system improved its water resistance. Nevertheless, the increment in the Fe x O y -NP concentration did not significantly improve their water resistance. Therefore, it could be concluded that the incorporation of NPs could decrease the solubility in water, regardless of the incorporated concentration. These results could be due to the formation of strong hydrogen bonds between the biopolymer chains and NPs, as has already been reported in previous works [42]. Likewise, Wongphan et al. (2022) reported that the incorporation of enzymes into polymers could cause an interaction via hydrogen bonding that enhances the hydrophobic groups by reducing the contrast, which results in a decrease in the solubility [43]. These results are similar for GS and CS NPs. The transmittance of each biofilm is also presented in Table 1. The reference system (neat gelatin) had 60% transmittance, which showed the great transparency of these biofilms. The incorporation of Fe x O y -NPs into gelatin-based biofilms reduced the transmittance values of the systems. Thus, this decrease was noted linearly with the increase of Fe x O y -NP concentration. Moreover, GS NPs led to a lower reduction than CS NPs, although this difference vanished at the highest NP content. The decrease in transparency brought about by NPs could be associated with the increase in solid material, which hindered the mobility of the biopolymer chains. Thus, the dispersion of fillers filled up free space preventing the passage of the light through it. Similar results were obtained in other works with gelatin-based biofilms containing silver or magnetic iron oxide NPs [24,29,44,45]. Figure 2 shows the tensile profile of the different biofilms. As can be observed, the neat gelatin system presented a short elastic zone followed by a plastic one. This plastic zone decreased as the amount of Fe x O y -NPs in the biofilm increased. The comparison of the different systems is better defined through the mechanical parameters shown in Table 1. In this way, the addition of the Fe x O y -NPs generated an increase in Young's modulus and maximum stress (σ max ), being more pronounced in GS Fe x O y -NPs than in CS Fe x O y -NPs. This result is due to the fact that the presence of immiscible particles caused non-homogeneous networks and reduced extensibility of the films [45]. In contrast, no significant differences were found between the incorporated GS and CS-NPs at the same concentration for the strain at break (ε max ) of the different films studied. Thus, the incorporation of Fe x O y -NPs stiffened the biofilms, probably due to the increasing presence of solid material in the biofilms and the increase of their thickness [29]. The incorporation of GS and CS-NPs may reduce the cohesion between the polymer chains and result in the reduction of strain at break [46]. On the other hand, the GS Fe x O y -NPs achieved greater maximum stress and Young's modulus than the CS Fe x O y -NPs. This result could be due to the size of the NPs. In this way, smaller NPs generated a better interconnection in the structure, which highlighted this effect [47]. Thus, the incorporation of NPs always improved the mechanical resistance of the biofilms due to the strong network between NPs and biopolymer chains. Figure 2 shows the tensile profile of the different biofilms. As can be observed, the neat gelatin system presented a short elastic zone followed by a plastic one. This plastic zone decreased as the amount of FexOy-NPs in the biofilm increased. The comparison of the different systems is better defined through the mechanical parameters shown in Table  1. In this way, the addition of the FexOy-NPs generated an increase in Young's modulus and maximum stress (Ϭmax), being more pronounced in GS FexOy-NPs than in CS FexOy-NPs. This result is due to the fact that the presence of immiscible particles caused nonhomogeneous networks and reduced extensibility of the films [45]. In contrast, no significant differences were found between the incorporated GS and CS-NPs at the same concentration for the strain at break (εmax) of the different films studied. Thus, the incorporation of FexOy-NPs stiffened the biofilms, probably due to the increasing presence of solid material in the biofilms and the increase of their thickness [29]. The incorporation of GS and CS-NPs may reduce the cohesion between the polymer chains and result in the reduction of strain at break [46]. On the other hand, the GS FexOy-NPs achieved greater maximum stress and Young's modulus than the CS FexOy-NPs. This result could be due to the size of the NPs. In this way, smaller NPs generated a better interconnection in the structure, which highlighted this effect [47]. Thus, the incorporation of NPs always improved the mechanical resistance of the biofilms due to the strong network between NPs and biopolymer chains. Nevertheless, the increase in NP concentration showed much lower effects as compared to their mere incorporation. Thus, no significant increase in Young's modulus could be observed for either of the two types of NPs, while the effect on depended on the NPs used. The maximum stress was reduced when increasing GS NP content or increased when CS was used. These contradictory results could be attributed to the occurrence of two opposite effects. On one hand, an increase in NPs may induce an enhancement of the mechanical properties of the biofilms by promoting NP-protein hydrogen bonding. On the other hand, it may also favor NP agglomeration, which may interfere with the Nevertheless, the increase in NP concentration showed much lower effects as compared to their mere incorporation. Thus, no significant increase in Young's modulus could be observed for either of the two types of NPs, while the effect on σ max depended on the NPs used. The maximum stress was reduced when increasing GS NP content or increased when CS was used. These contradictory results could be attributed to the occurrence of two opposite effects. On one hand, an increase in NPs may induce an enhancement of the mechanical properties of the biofilms by promoting NP-protein hydrogen bonding. On the other hand, it may also favor NP agglomeration, which may interfere with the formation of these NP-protein domain arrangements [29]. Likewise, aggregation of nanoparticles in biodegradable polymers was possible with increasing nanofiller concentrations, which may lead to changes in the mechanical and barrier properties of the films [48]. Figure 3 shows the bottom and upper surface of the different biofilms. As can be seen, the biofilm without NP incorporation (neat gelatin) presented a smooth and homogeneous structure on both surfaces. On the other hand, the incorporation of NPs generated rough surfaces with fissures and holes. Thus, the incorporation of NPs seemed to alter the nanostructure of biofilms, being more evident in the case of green nanoparticles (GS Fe x O y -NPs). This effect could be due to the functional groups present in GS Fe x O y -NPs (as they come from the polyphenols used in their synthesis), which could alter the structure of the biopolymer chains in the biofilms by charge interaction [49]. Furthermore, Fe x O y -NPs seemed to form more aggregates, probably due to the increase in concentration [48]. This effect was more evident in CS Fe x O y -NPs among films, which may be due to the non-availability of stabilizing agents [32]. Figure 3 shows the bottom and upper surface of the different biofilms. As can be seen, the biofilm without NP incorporation (neat gelatin) presented a smooth and homogeneous structure on both surfaces. On the other hand, the incorporation of NPs generated rough surfaces with fissures and holes. Thus, the incorporation of NPs seemed to alter the nanostructure of biofilms, being more evident in the case of green nanoparticles (GS FexOy-NPs). This effect could be due to the functional groups present in GS FexOy-NPs (as they come from the polyphenols used in their synthesis), which could alter the structure of the biopolymer chains in the biofilms by charge interaction [49]. Furthermore, FexOy-NPs seemed to form more aggregates, probably due to the increase in concentration [48]. This effect was more evident in CS FexOy-NPs among films, which may be due to the nonavailability of stabilizing agents [32].   Figure 4 shows this distribution in both surfaces (bottom and upper). Nevertheless, the colored area in each image was calculated to improve the comparison between the systems ( Table 2). As can be seen, there was a homogeneous distribution of NPs in both surfaces of the biofilms, since Fe could be observed in the whole images. Nevertheless, there was a higher NP concentration on the upper surface than on the bottom surface, being more evident in the higher NP percentage (1%). This behavior can be explained by the hydrophobicity of the NPs, which made them migrate to the surface where they presented less steric repulsion, as well as by their density, which made them float instead of sinking into the film solution [50]. Additionally, this was explained by the strengthened Van Der Waals forces as particle size and concentration increased or that larger particles would exhibit higher surface tension, causing them to settle on the surface [50]. On the other hand, a higher predisposition to the agglomerations or aggregations of NPs could be observed at higher NP concentrations, causing them to fall by gravity. Among the different NPs, CS NPs presented greater precipitate than GS NPs, possibly due to their larger size. In addition, the highest concentration of CS Fe x O y -NPs showed agglomeration of NPs in certain areas of the biofilm. This behavior was already reported by Rufus et al. (2017) and Hosseini et al. (2015), who attributed it to an entropic process [26,36].

Scanning Electron Microscopy (SEM)
to improve the comparison between the systems ( Table 2). As can be seen, there was a homogeneous distribution of NPs in both surfaces of the biofilms, since Fe could be observed in the whole images. Nevertheless, there was a higher NP concentration on the upper surface than on the bottom surface, being more evident in the higher NP percentage (1%). This behavior can be explained by the hydrophobicity of the NPs, which made them migrate to the surface where they presented less steric repulsion, as well as by their density, which made them float instead of sinking into the film solution [50]. Additionally, this was explained by the strengthened Van Der Waals forces as particle size and concentration increased or that larger particles would exhibit higher surface tension, causing them to settle on the surface [50]. On the other hand, a higher predisposition to the agglomerations or aggregations of NPs could be observed at higher NP concentrations, causing them to fall by gravity. Among the different NPs, CS NPs presented greater precipitate than GS NPs, possibly due to their larger size. In addition, the highest concentration of CS FexOy-NPs showed agglomeration of NPs in certain areas of the biofilm. This behavior was already reported by Rufus et al. (2017) and Hosseini et al. (2015), who attributed it to an entropic process [26,36].   As can be seen in Table 1, the incorporation of Fe x O y -NPs produced an increase in antioxidant activity. In addition, the higher the Fe x O y -NP concentration, the higher the antioxidant activity. Thus, these nanoparticles act as antioxidants, as was reported in similar articles [29,51]. Regarding the different nanoparticles, the GS Fe x O y -NPs presented higher DPPH inhibition than the CS Fe x O y -NPs. This behavior could be due to the greater amount of antioxidant groups in them, conferred during their synthesis by polyphenols. In this way, green synthesis did not only display advantages in the synthesis process by reducing toxic waste and lowering costs, but it also allowed the generation of nanoparticles that provided greater functionality in future applications. These results showed that the gelatin-based Fe x O y -NPs displayed higher antioxidant activity than others reported in the literature, such as those obtained by Zafar et al. (2020) using iron oxide nanoparticles (IONPs) [29], and Shiv et al. (2019) using melanin nanoparticles [7]. It is worth being mentioned that the IP% of the positive control (gallic acid) was from 94.9 to 98.4%.

Antibacterial Activity
The inhibition area produced by the different biofilms is collected in Table 3 through its significant measure (diameter). An image of these inhibition areas can be observed in the supplementary materials. As can be seen, the neat gelatin biofilms showed antibacterial activity against Staphylococcus aureus, since a small zone of inhibition could be observed for the first 24 h. This could be due to the presence of sulfur dioxide (<10 ppm), as it is food gelatin. Nevertheless, this effect was lost in time when the bacteria grew. In this way, neat gelatin only had the ability to act on the peptidoglycan layer of bacteria to generate their lysis. However, it did not show the ability to act on the lipid layer, thus it only affected gram-positive bacteria and not gram-negative bacteria [52]. The inclusion of Fe x O y -NPs improved the antibacterial activity of biofilms. In this way, higher inhibition areas could be observed, as well as an effect on both types of bacteria. Nevertheless, although the difference in nanoparticle concentration on each of the surfaces of the biofilms was different, the antibacterial activity did not seem to have significant differences on each of the surfaces, making it a fully functional material. Regarding the synthesis of NPs, the GS Fe x O y -NPs presented higher antibacterial activity than the CS Fe x O y -NPs. This could be due to the smaller size of the green NPs, which conferred the biofilms a better capacity to inhibit the replication of bacterial DNA [53,54], as well as the higher crystallinity shown by GS Fe x O y -NPs [30]. Higher crystallinity allowed the released of Fe +2 /Fe +3 to collide with the negatively charged membranes of bacteria, destroying their protein structure and causing them to die [55]. The incorporated antimicrobial function into biopolymer films could be an efficient method to enhance the shelf-life extension capacity for biodegradable films [56]. On the other hand, all the samples showed a decrease in their inhibitory capacity over time, being more notable in CS Fe x O y -NPs.

Conclusions
The incorporation of Fe x O y nanoparticles into gelatin-based biofilms improved their properties even at a low concentration. Thus, the 0.25% (w/w) led to a reduction in the water solubility and to an improvement in the antioxidant activity and mechanical properties. An increase in NPs concentration led to opaquer or less transparent biofilms with greater thickness, but not to a significant improvement in the other properties evaluated, which may be due to the tendency of the nanoparticles to agglomerate at higher concentrations caused by their hydrophobic nature. Therefore, their inclusion reduced the water solubility of the biofilms and improved their mechanical resistance and functional properties. Furthermore, the dispersion of the nanoparticles incorporated by the casting method led to their heterogeneous distribution with differences in the distribution in both upper and bottom surfaces of the biofilms. However, these differences did not alter the antibacterial properties.
Finally, the green nanoparticles (GS Fe x O y -NPs) showed better antioxidant and antibacterial activities than the CS Fe x O y -NPs, which must improve the added value of these biofilms. This antioxidant activity of (GS Fe x O y -NPs) highlighted high antioxidant activity (PI% = 84.7% compared to gallic acid as a standard with PI% = 96.65). A control on the distribution of the nanoparticles in the biofilms and the use of the green nanoparticles led to sustainable biofilms with a significant improvement of the functional properties. All this makes these biofilms highly applicable for use as functional packaging to preserve food, antioxidant and antimicrobial additives in multiple applications such as biomedical and pharmaceutical biomaterials, as well as in agricultural pesticides. Nevertheless, further characterization of these biopolymer films, including a biodegradability study, will be required in future works as a previous stage to their use in packaging applications.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/ma15051966/s1. Figure S1: Image of inhibition area over time of neat gelatin-based biofilm without Fe x O y -NPs incorporated. Figure S2: Image of inhibition area over time of gelatin-based biofilm with 1.0% GS Fe x O y -NPs incorporated. Figure S3

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