Gelatin/Chitosan Films Incorporated with Curcumin Based on Photodynamic Inactivation Technology for Antibacterial Food Packaging

Photodynamic inactivation (PDI) is a new type of non-thermal sterilization technology that combines visible light with photosensitizers to generate a bioactive effect against foodborne pathogenic bacteria. In the present investigation, gelatin (GEL)/chitosan (CS)-based functional films with PDI potency were prepared by incorporating curcumin (Cur) as a photosensitizer. The properties of GEL/CS/Cur (0.025, 0.05, 0.1, 0.2 mmol/L) films were investigated by evaluating the surface morphology, chemical structure, light transmittance, and mechanical properties, as well as the photochemical and thermal stability. The results showed a strong interaction and good compatibility between the molecules present in the GEL/CS/Cur films. The addition of Cur improved different film characteristics, including thickness, mechanical properties, and solubility. More importantly, when Cur was present at a concentration of 0.1 mM, the curcumin-mediated PDI inactivated >4.5 Log CFU/mL (>99.99%) of Listeria monocytogenes, Escherichia coli, and Shewanella putrefaciens after 70 min (15.96 J/cm2) of irradiation with blue LED (455 ± 5) nm. Moreover, Listeria monocytogenes and Shewanella putrefaciens were completely inactivated after 70 min of light exposure when the Cur concentration was 0.2 mM. In contrast, the highest inactivation effect was observed in Vibrio parahaemolyticus. This study showed that the inclusion of Cur in the biopolymer-based film transport system in combination with photodynamic activation represents a promising option for the preparation of food packaging films.


Introduction
In recent years, the increase in the level of consumption escalated the demand for goods and the development of the packaging industry. The world production of packaging materials has increased at an alarming rate of 8% per year [1,2]. However, more than 90% of these materials are discarded. Since most of them are petroleum-based plastic, they are difficult to degrade [3], resulting in serious environmental pollution and destruction of biodiversity. In addition, they may affect human health through water, soil, and air pollution. Therefore, in order to find the perfect substitute, natural polymers, such as proteins, carbohydrates, and lipids, as well as their derivatives, which are biodegradable and edible substances, have been used as packaging materials, attracting substantial interest from researchers in recent decades.

Films Preparation
In the present investigation, GEL/CS/Cur films were prepared according to the procedures published by Reference [33], but with slight modifications. For this purpose, GEL/CS/Cur film-forming solutions (FFSs) were obtained. In the first step, the GEL FFS (1.5%, m/v) was prepared by dissolving GEL in deionized water under continuous stirring for 1 h at 55 • C. The CS FFS (1%, m/v) was obtained by dissolving CS in an acetic acid solution (1% v/v), under continuous stirring at 55 • C for 2 h. Later, a 0.01 mmol/mL Cur solution was prepared by adding the proper amount of Cur in 95% ethanol. The mixture was stirred at 300 rpm until Cur was dissolved. Subsequently, the GEL FFS and the CS FFS were mixed according to Table 1, and glycerol (0.3%, m/v) was added. Magnetic stirring was applied for 3 h to achieve homogenization and remove bubbles. Different concentrations of FFSs are shown in Figure 1. Finally, all films were obtained by adding 30 mL of the FFSs to polystyrene Petri dishes (10 cm × 10 cm × 1 cm) and dried in an oven with the air-flow circulation at 40 • C for 24-36 h. Samples were stored at 25 • C and 55% RH in a brown desiccator before further analyses.

Films Preparation
In the present investigation, GEL/CS/Cur films were prepared according procedures published by Reference [33], but with slight modifications. For this p GEL/CS/Cur film-forming solutions (FFSs) were obtained. In the first step, the G (1.5%, m/v) was prepared by dissolving GEL in deionized water under continuous for 1 h at 55 °C. The CS FFS (1%, m/v) was obtained by dissolving CS in an ace solution (1% v/v), under continuous stirring at 55 °C for 2 h. Later, a 0.01 mmol/ solution was prepared by adding the proper amount of Cur in 95% ethanol. The was stirred at 300 rpm until Cur was dissolved. Subsequently, the GEL FFS and FFS were mixed according to Table 1, and glycerol (0.3%, m/v) was added. M stirring was applied for 3 h to achieve homogenization and remove bubbles. D concentrations of FFS solutions are shown in Figure 1. Finally, all films were obta adding 30 mL of the FFSs to polystyrene Petri dishes (10 cm × 10 cm × 1 cm) and an oven with the air-flow circulation at 40 °C for 24-36 h. Samples were stored and 55% RH in a brown desiccator before further analyses. The microstructure of the surface and cross-section of the films was acquired by using SEM (Quanta FEG 250, Hillsboro, OR, USA). The composite films were fixed on a stainlesssteel support with a double-sided adhesive, and the analysis was conducted in low vacuum (0.6 Torr), at an acceleration voltage of 20 and 10 kV respectively.
Four bacterial suspensions (1 mL) were centrifuged for 10 min at 4000× g. The supernatants were discarded, and the pellets were mixed with 500 µL of glutaraldehyde (2.5%) and formaldehyde (4%) in 0.1 M cacodylate buffer for 8 h at 4 • C. Subsequently the samples were dehydrated in serial dilutions of ethanol solutions (30%, 50%, 70%, 90%, and 100%) for 10 min. The samples were separately placed on the support, with a double-sided adhesive, and coated with gold. The microstructures of cells were observed by using SEM.

Color
Color parameters were measured by using a portable Minolta colorimeter (JZ-300, Osaka, Japan) with a standard white color plate (L 0 = 99.44, a 0 = −0.28, b 0 = 0.54) as the background reference. Results of L* (lightness), a* (red to green), and b* (yellow to blue) were directly read from the colorimeter. The total color difference (∆E) of the films was calculated according to Equation (1) [34]: where ∆L*, ∆a*, and ∆b* are the differences between each color value of the standard color plate and film specimen, respectively. Values were expressed as the means of ten measurements on different areas of each film.

UV-Visible Spectra
The UV-Vis transmission spectra of the composite films (1 cm × 4 cm) were obtained in order to evaluate the effect of the addition of Cur on the barrier properties of films to ultraviolet (UV) and visible (Vis) light. Spectra were recorded by using a UV spectrophotometer (UV-3600, Shimadzu, Tokyo, Japan). The film opacity was calculated by using Equation (2) [35]: where T 600 is the transmittance at 600 nm, and x is the film thickness (mm).

Thickness and Mechanical Properties
The thickness of film samples was measured by randomly taking the average of 10 points on the film, using a micrometer caliper (Mitutoyo, Japan) with a precision of 0.001 mm. The tensile strength (TS) and elongation at break (EB) of the films were obtained by using an Auto Tensile Tester (XLW-EC, PARAM, Jinan, China). Before testing, film samples were cut into strips (15 mm × 100 mm) and mounted in the tensile grip at an initial distance of 65 mm. Later, samples were stretched at a cross-head speed of 50 mm/min until breaking occurred. At least five replicates were tested for each film. The film pieces (2 cm × 2 cm, n = 3, M 1 ) were dried in an oven at 105 • C for 24 h to reach a constant weight (M 2 ). Samples were then completely immersed in centrifuge tube with 30 mL distilled water. Tubes were shaken at 180 r/min and 26 • C for 24 h. Later, samples were filtered to remove excess water and dried at 105 • C for 24 h until constant weight (M 3 ). The MC and WS (%) of film samples were calculated by using Equations (3) and (4) [36].
Polymers 2022, 14, 1600 The water vapor permeability (WVP) of the films was measured gravimetrically according to E96-05 (ASTM, 2005), but with some modifications. In this assay, the glass weighing bottle was filled with 20 mL of distilled water (100% RH). Later, the film sample was placed over the circular opening and sealed tightly with parafilm to prevent the leakage of water vapor. The glass weighing bottles were maintained at a constant temperature of 25 • C. Weight changes of the glass weighing bottles were monitored at intervals of 2 h for a total of 12 h. The slope of weight changes versus time plot was obtained by using linear regression (r 2 > 0.99). WVTR and WVP were calculated according to Equations (5) and (6) [37].
where ∆W/∆t indicated the weight change as a function of time (g/h), A is the area of the exposed film surface (m 2 ), L corresponds to the mean film thickness (m), ∆p is the water vapor pressure difference (kPa) between two sides of the film, and ∆RH is the relative humidity gradient across the film (%).

Fourier-Transform Infrared (FTIR) Spectroscopy
Fourier-transform infrared (FTIR) spectroscopy analysis was performed to obtain chemical information of the films' surface. The spectra of Cur and composite films were obtained by using a spectrophotometer (Thermo IS10, Thermo Fisher, MA, USA) system from 400 to 4000 cm −1 , with a resolution of 4 cm −1 and 32 scans.

X-Ray Diffraction (XRD)
In order to examine the crystalline structure of the films, X-ray diffraction patterns (XRD) were recorded by using a Rigaku Ultima IV X-ray diffractometer (RINT2000, Tokyo, Japan), equipped with a Cu-Kα radiation, at 40 kV voltage and 30 mA current. Samples were scanned over the 2θ range of 5-50 • , at a speed of 2 • /min (RINT2000, Tokyo, Japan).

Thermogravimetric Analysis (TGA)
The thermal properties of the composite films were determined by using a thermal analyzer (NETZSCH STA 449C, Selb, German). The measured samples were kept in the range of 30-800 • C, and the heating rate was 10 • C/min. Nitrogen was used as the protective gas [38].

Light-Emitting Diodes (LEDs) System
The blue LEDs (10 W, 450-460 nm, 30 cm) were used as the light source for the photodynamic treatment [39,40]. These LEDs were surrounded by deep photo accessories to prevent the interference of external light sources. The film samples were cut into squares (2 cm × 2 cm) and placed directly on 6-well plates. The distance was adjusted to 5 cm between the light source and film samples. The blue light intensity was 3.8 mW/cm 2 , which was determined by using a PM100D energy meter console (Newton, MA, USA). The obtained energy dosage of each composite film sample was calculated by using Equation (7) [41].
where E = dose (energy density) in J/cm 2 , P = irradiance (power density) in W/cm 2 , and t = time in s.

Antimicrobial Activity
The bacterial suspension (100 µL) was evenly distributed on the surface of the GEL/CS/Cur films containing different concentrations of Cur (0, 0.025, 0.05, 0.1, and 0.2 mM). Later, films were exposed to light for 70 min (15.96 J/cm 2 ) and then maintained in the dark for another 10 min to ensure that the bacteria were able to attach to the film before irradiation. In addition, GEL/CS/Cur films containing 0.1 mM Cur were irradiated for 30 min (6.84 J/cm 2 ), 50 min (11.4 J/cm 2 ), 70 min (15.96 J/cm 2 ), and 90 min (20.52 J/cm 2 ) in order to explore their potential use in PDI of food-borne pathogenic bacteria. After treatment, the films containing bacteria were homogenized with sodium chloride (0.85%, w/v) for 5 min. The antibacterial effect of the GEL/CS/Cur films was investigated by spreading 100 µL of the suspension onto agar plates and incubated for 12-48 h. Viable cells were quantified as Log CFU/mL. Herein, samples without light treatment and Cur were labeled as negative control (L−C−). In addition, samples with light treatment but without Cur were labeled as illumination control (L+C−). Moreover, samples with Cur but without light treatment were identified as Cur control (L−C+). All the experiments were performed in triplicate.

Statistical Analysis
The experimental data were analyzed by using SPSS (SPSS 17.0 Software, Inc., Chicago, IL, USA). One-way analysis of variance (ANOVA) was used to compare differences between pairs of means (p < 0.05).

Optical Properties of Films
Transparency and color of edible food packaging materials are critical properties that influence consumer acceptance, as they directly affect the appearance of the product. The color values (L*, a*, and b*), total color difference (∆E*), opacity, and picture of the GEL/CS film and GEL/CS /Cur films are shown in Table 2. The L* values of the composite film were in the range of 86.09-93.38, and the brightness of the composite films were not affected by the presence of Cur. However, the a* and b* values of the GEL/CS/Cur composite films were higher than those of the GEL/CS films, which indicated that the yellowness and redness of the composite films significantly improved with the addition of Cur. As a result, the total color difference (∆E*) of the curcumin-added composite films increased as compared to GEL/CS. Data in Table 2 indicated that the increase in Cur concentration resulted in an increase in film opacity, for which the values were lower than 5 at 600 nm in all cases. Thus, we can conclude that all the films prepared in the present study were transparent. The higher the opacity value, the lower the transparency of the film.  Figure 2 presented the UV-visible light transmission spectra (200-800 nm) of the composite films with and without Cur. The packaging material with good light-barrier properties effectively prevents light transmission and reduces light-induced oxidation of packaged foods, consequently inhibiting the lipid oxidation, nutrient loss, and degradation of active compounds [42]. All composite films exhibited high protection a,b,c,d,e Different superscript letters between columns indicate significant difference between the results (p < 0.05) (ANOVA). Figure 2 presented the UV-visible light transmission spectra (200-800 nm) of the composite films with and without Cur. The packaging material with good light-barrier properties effectively prevents light transmission and reduces light-induced oxidation of packaged foods, consequently inhibiting the lipid oxidation, nutrient loss, and degradation of active compounds [42]. All composite films exhibited high protection against UV light, which was probably the result of the excellent UV-absorbing properties of aromatic amino acids found in the GEL [43,44]. It was also observed that light absorption by the GEL/CS/Cur composite films decreased in the visible light wavelength region of 390-450 nm. This probably occurred because Cur absorbs light in the range of 400-500 nm, which is similar to the tested wavelength range. Since the phenolic compounds in Cur display excellent light absorption properties, the addition of Cur improved the lightbarrier characteristics of the films. With the increase of the Cur concentration, the light transmittance of the composite films decreased. However, all the analyzed films presented good transparency.

Microstructure of Films
The surface topography and cross-section are used to characterize the microstructure of packaging materials. This information is helpful in determining different properties, including sealing and flexibility. The surface and cross-section of the GEL/CS film and GEL/CS/Cur composite films were observed by using SEM (Figure 3). GEL/CS, GEL/CS/Cur 0.025 and GEL/CS/Cur 0.05 films presented homogeneous and smooth surfaces. However, with increasing Cur concentrations, slight protuberances appeared on the surface of GEL/CS/Cur films. In addition, when films contained Cur, the cross-section of the composite films appeared slightly rough in contrast to the control films. However, the films also displayed a uniform thickness and regular texture. Moreover, no macroscopic phase separation was observed, indicating that Cur was properly dispersed in GEL/CS FFSs. This mainly occurred because of the good materials compatibility. Roy et al. (2017) also found that Cur was well distributed in GEL/Cur composite films [30,45].

Microstructure of Films
The surface topography and cross-section are used to characterize the microstructure of packaging materials. This information is helpful in determining different properties, including sealing and flexibility. The surface and cross-section of the GEL/CS film and GEL/CS/Cur composite films were observed by using SEM (Figure 3). GEL/CS, GEL/CS/Cur 0.025 and GEL/CS/Cur 0.05 films presented homogeneous and smooth surfaces. However, with increasing Cur concentrations, slight protuberances appeared on the surface of GEL/CS/Cur films. In addition, when films contained Cur, the cross-section of the composite films appeared slightly rough in contrast to the control films. However, the films also displayed a uniform thickness and regular texture. Moreover, no macroscopic phase separation was observed, indicating that Cur was properly dispersed in GEL/CS FFSs. This mainly occurred because of the good materials compatibility. Roy et al. (2017) also found that Cur was well distributed in GEL/Cur composite films [30,45]. Polymers 2022, 14, x FOR PEER REVIEW 9 of 17

Mechanical Properties
Because of the low content of Cur, the thickness of the films varied in the range of 0.33-0.38, with little difference. Data in Table 3 indicate that the tensile strength (TS) and elongation at break (EB) of the composite films increased with respect to controls. The mechanical properties of the fabricated GEL/CS/Cur films were affected by the addition of Cur. It was believed that the hydrogen bond interaction between Cur and GEL/CS was responsible for the improved TS of the films [30]. These results agree with the XRD data. Other studies have reported that EB increases because Cur improves the adhesion between the filler and the polymer [46,47].

Mechanical Properties
Because of the low content of Cur, the thickness of the films varied in the range of 0.33-0.38, with little difference. Data in Table 3 indicate that the tensile strength (TS) and elongation at break (EB) of the composite films increased with respect to controls. The mechanical properties of the fabricated GEL/CS/Cur films were affected by the addition of Cur. It was believed that the hydrogen bond interaction between Cur and GEL/CS was responsible for the improved TS of the films [30]. These results agree with the XRD data. Other studies have reported that EB increases because Cur improves the adhesion between the filler and the polymer [46,47].

Moisture Content (MC), Water Solubility (WS), and Water Vapor Permeability (WVP)
Water sensitivity plays an important role in the wide applications of biodegradable films. The results of the moisture content (MC), water solubility (WS), and water vapor permeability (WVP) of GEL/CS and GEL/CS/Cur films were presented in Table 3.
According to our data, GEL/CS films displayed an MC of 21.96% higher as that quantified in GEL/CS/Cur films. Cur is a hydrophobic molecule with a very small capacity for water retention. This resulted in a continuous MC decrease from 21.29 to 18.54%. The UV-Vis spectra indicated that Cur-containing films absorbed UV-Vis irradiation between 390 and 450 nm, which is in agreement with the maximum absorption of Cur that occurs between 400 and 500 nm. As the Cur concentration gradually increased, the maximum absorption shifted to blue. This explains why the WS of GEL/CS/Cur films increased by increasing the concentration of Cur. Gómez-Estaca et al. [48] also observed a blue shift in the absorption spectrum of Cur, which occurred because of the formation of a Cur-GEL complex. This complex is responsible for the increase in Cur solubility in water. In addition, the increase in WS could be explained by the fact that the GEL swell in water and was partially soluble at 25 • C. As a result, with the increase in Cur content, the WS value of the GEL/CS/Cur films exhibited a continuous increase, reaching values between 20.46 and 24.75%.
The water barrier properties meet the requirements for food preservation. Therefore, a lower WVP should be considered in the application of biodegradable films. In the present study, by increasing the concentration of Cur, the overall value of WVP increased, with small differences. The complex composition of polymers results in weak cohesion between their components, leading to a less disordered crystal structure [49]. This phenomenon was the possible cause of the WVP increase.

Physicochemical and Structural Properties of Films
The chemical nature of the interactions in the GEL/CS/Cur films were investigated by using FTIR (Figure 4). The Cur spectra showed a peak at 3510 cm −1 , which was attributed to the phenol O-H stretching vibrations. Additional peaks at 1630 and 1505 cm −1 , corresponding to C=O and C=C stretching vibrations of the Cur structures, were also observed. Furthermore, peaks at 1275 cm −1 referred to ether C-O stretching vibration, and those at 810 and 964 cm −1 represented the C-H bending vibration, which was consistent with the alkene structure in Cur [30]. In the GEL/CS films, a broad band at 3287 cm −1 was attributed to the overlapped −OH stretching vibration of CS and GEL. The peak at 1536 cm −1 corresponded to the amide II C=O stretching vibration of CS, which overlapped with that of GEL [6]. Moreover, the spectra of GEL/CS showed bands corresponding to different amide types present in GEL (A, B, I, II, and III). These peaks were observed at 3285, 2936, 1637, 1541, and 1242 cm −1 , respectively, which indicated the existence of N-H, −CH 2 , and C=O stretching vibration, with N-H bending coupled to C-N stretching, and C-N stretching coupled to N-H bending [50]. Interestingly, after adding different Cur concentrations during the preparation of the GEL/CS/Cur composite films, the spectra of these materials displayed similar main peaks as those of the GEL/CS films. This indicated that the addition of Cur preserved the chemical structures originally present in GEL/CS films. Thus, the addition of Cur did not produce new chemical structures [51]. Moreover, changes in peak intensities were the result of physical interactions, non-covalent interactions, and hydrogen bonding [52,53].
Polymers 2022, 14, x FOR PEER REVIEW concentrations during the preparation of the GEL/CS/Cur composite films, the these materials displayed similar main peaks as those of the GEL/CS films. Thi that the addition of Cur preserved the chemical structures originally present films. Thus, the addition of Cur did not produce new chemical structures [51]. changes in peak intensities were the result of physical interactions, no interactions, and hydrogen bonding [52,53]. The crystalline structures of the composite films were discussed by XR ( Figure 5). All composite films presented a similar wide diffraction peak at 2θ = was considered to be the random coiled conformation of GEL, and this meant t with Cur added was amorphous [54]. The XRD data of the GEL/CS/Cur comp showed that the Cur addition resulted in an increased crystallinity. For this diffraction peak intensity increased [55]. However, according to the literatur Cur was a crystalline material that presented a series of diffraction peaks betw 30°. The disappearance of Cur peaks in the GEL/CS/Cur XRD pattern probab because Cur was present at low concentrations. Thus, the characteristic peaks overlapped with that of Cur [46]. The crystalline structures of the composite films were discussed by XRD analysis ( Figure 5). All composite films presented a similar wide diffraction peak at 2θ = 20 • , which was considered to be the random coiled conformation of GEL, and this meant that the film with Cur added was amorphous [54]. The XRD data of the GEL/CS/Cur composite films showed that the Cur addition resulted in an increased crystallinity. For this reason, the diffraction peak intensity increased [55]. However, according to the literature [56], pure Cur was a crystalline material that presented a series of diffraction peaks between 7 • and 30 • . The disappearance of Cur peaks in the GEL/CS/Cur XRD pattern probably occurred because Cur was present at low concentrations. Thus, the characteristic peaks of GEL/CS overlapped with that of Cur [46]. Polymers 2022, 14, x FOR PEER REVIEW 12 of 17

Thermal Properties
The thermal stability of the composite films was investigated by using thermogravimetric analysis (TGA) and derivative thermogravimetric analysis (DTG), and the results are shown in Figure 6. Cur is a hydrophobic molecule with a very low capacity for water retention and is stable until 200 °C. After this point, Cur is continuously degraded before 450 °C. However, it reaches the maximum degradation rate at 375 °C, and the residue rate was 35% at 800 °C. These results were consistent with the reported literature [48]. The weight loss rates of the GEL/CS/Cur films were faster than those of Cur and showed the similar weight-loss characteristics of the GEL/CS films. The weight-loss process was divided into two main stages: (1) water loss in the range between 50 and 150 °C, and (2) degradation of composites at 200-800 °C [57]. The maximum degradation rate occurred at about 300 °C, and the rapid degradation of Cur disappeared at 375 °C. In addition, the decomposition temperature for Cur was higher than that for composite films. This proved that Cur had good compatibility with GEL and CS, and that Cur was well embedded in GEL/CS films, forming a uniform system [58]. When the Cur was added at a 0.05 mM concentration, the residual rate of the film was the highest one (32%), which also indicated that the thermal stability of GEL/CS/Cur film was improved [57].

Thermal Properties
The thermal stability of the composite films was investigated by using thermogravimetric analysis (TGA) and derivative thermogravimetric analysis (DTG), and the results are shown in Figure 6. Cur is a hydrophobic molecule with a very low capacity for water retention and is stable until 200 • C. After this point, Cur is continuously degraded before 450 • C. However, it reaches the maximum degradation rate at 375 • C, and the residue rate was 35% at 800 • C. These results were consistent with the reported literature [48].

Thermal Properties
The thermal stability of the composite films was investigated by using thermogravimetric analysis (TGA) and derivative thermogravimetric analysis (DTG), and the results are shown in Figure 6. Cur is a hydrophobic molecule with a very low capacity for water retention and is stable until 200 °C. After this point, Cur is continuously degraded before 450 °C. However, it reaches the maximum degradation rate at 375 °C and the residue rate was 35% at 800 °C. These results were consistent with the reported literature [48]. The weight loss rates of the GEL/CS/Cur films were faster than those of Cur and showed the similar weight-loss characteristics of the GEL/CS films. The weight-loss process was divided into two main stages: (1) water loss in the range between 50 and 150 °C, and (2) degradation of composites at 200-800 °C [57]. The maximum degradation rate occurred at about 300 °C, and the rapid degradation of Cur disappeared at 375 °C. In addition, the decomposition temperature for Cur was higher than that for composite films. This proved that Cur had good compatibility with GEL and CS, and that Cur was well embedded in GEL/CS films, forming a uniform system [58]. When the Cur was added at a 0.05 mM concentration, the residual rate of the film was the highest one (32%), which also indicated that the thermal stability of GEL/CS/Cur film was improved [57]. The weight loss rates of the GEL/CS/Cur films were faster than those of Cur and showed the similar weight-loss characteristics of the GEL/CS films. The weight-loss process was divided into two main stages: (1) water loss in the range between 50 and 150 • C, and (2) degradation of composites at 200-800 • C [57]. The maximum degradation rate occurred at about 300 • C, and the rapid degradation of Cur disappeared at 375 • C. In addition, the decomposition temperature for Cur was higher than that for composite films. This proved that Cur had good compatibility with GEL and CS, and that Cur was well embedded in GEL/CS films, forming a uniform system [58]. When the Cur was added at a 0.05 mM concentration, the residual rate of the film was the highest one (32%), which also indicated that the thermal stability of GEL/CS/Cur film was improved [57].

In Vitro Antimicrobial Properties
In the present research, we evaluated the antibacterial potency of the composite films against E. coli, L. monocytogenes, V. parahaemolyticus, and S. putrefaciens (Figures 7 and 8). For this purpose, we selected blue LED illumination and Cur concentration as study variables. As it was shown in Figure 7, individual LED illumination (L+C−) and Cur treatment (L−C+) did not cause significant changes in the antimicrobial activity of the films as compared to the negative control (L−C−).

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In Vitro Antimicrobial Properties
In the present research, we evaluated the antibacterial potency of the composite films against E. coli, L. monocytogenes, V. parahaemolyticus, and S. putrefaciens (Figures 7 and 8). For this purpose, we selected blue LED illumination and Cur concentration as study variables. As it was shown in Figure 7, individual LED illumination (L+C−) and Cur treatment (L−C+) did not cause significant changes in the antimicrobial activity of the films as compared to the negative control (L−C−).  When the illumination time was increased, the irradiation dose increased and the antibacterial activity improved. These results were shown in Figure 7A. Obviously, the fabricated GEL/CS/Cur films exhibited good antibacterial activity when the Cur concentration was treated by 0.1 mM after 30 min of irradiation (6.84 J/cm 2 ). In the case of V. parahaemolyticus cells, a decrease from 8.55 to 4.76 Log CFU/mL was observed. Moreover, after the GEL/CS/Cur film with 0.1 mM Cur was irradiated for 70 min (15.96 J/cm 2 ), no bacterial cells were detected. Furthermore, L. monocytogenes and S. putrefaciens cells were killed and could not be detected after 90 min of irradiation (20.52 J/cm 2 ). It was also observed that, when the PDI illumination time increased from 30 min (6.84 J/cm 2 ) to 90 min (20.52 J/cm 2 ), E. coli cells presented a continuous decrease from 8.15 to 2.64 Log

In Vitro Antimicrobial Properties
In the present research, we evaluated the antibacterial potency of the composite films against E. coli, L. monocytogenes, V. parahaemolyticus, and S. putrefaciens (Figures 7 and 8). For this purpose, we selected blue LED illumination and Cur concentration as study variables. As it was shown in Figure 7, individual LED illumination (L+C−) and Cur treatment (L−C+) did not cause significant changes in the antimicrobial activity of the films as compared to the negative control (L−C−).  When the illumination time was increased, the irradiation dose increased and the antibacterial activity improved. These results were shown in Figure 7A. Obviously, the fabricated GEL/CS/Cur films exhibited good antibacterial activity when the Cur concentration was treated by 0.1 mM after 30 min of irradiation (6.84 J/cm 2 ). In the case of V. parahaemolyticus cells, a decrease from 8.55 to 4.76 Log CFU/mL was observed. Moreover, after the GEL/CS/Cur film with 0.1 mM Cur was irradiated for 70 min (15.96 J/cm 2 ), no bacterial cells were detected. Furthermore, L. monocytogenes and S. putrefaciens cells were killed and could not be detected after 90 min of irradiation (20.52 J/cm 2 ). It was also observed that, when the PDI illumination time increased from 30 min (6.84 J/cm 2 ) to 90 min (20.52 J/cm 2 ), E. coli cells presented a continuous decrease from 8.15 to 2.64 Log CFU/mL.
The effects of the Cur concentration on the inactivation of the four bacterial cells were evaluated in Figure 7B. In all cases, an increase in Cur concentration from 0.025 to 0.2 mM led to a significant decrease in the number of bacterial cells. Among the four species, V. parahaemolyticus was the most affected, as the number of cells decreased to 2.63 Log CFU/mL after 70 min of irradiation (15.96 J/cm 2 ). When Cur concentration in the When the illumination time was increased, the irradiation dose increased and the antibacterial activity improved. These results were shown in Figure 7A. Obviously, the fabricated GEL/CS/Cur films exhibited good antibacterial activity when the Cur concentration was treated by 0.1 mM after 30 min of irradiation (6.84 J/cm 2 ). In the case of V. parahaemolyticus cells, a decrease from 8.55 to 4.76 Log CFU/mL was observed. Moreover, after the GEL/CS/Cur film with 0.1 mM Cur was irradiated for 70 min (15.96 J/cm 2 ), no bacterial cells were detected. Furthermore, L. monocytogenes and S. putrefaciens cells were killed and could not be detected after 90 min of irradiation (20.52 J/cm 2 ). It was also observed that, when the PDI illumination time increased from 30 min (6.84 J/cm 2 ) to 90 min (20.52 J/cm 2 ), E. coli cells presented a continuous decrease from 8.15 to 2.64 Log CFU/mL.
The effects of the Cur concentration on the inactivation of the four bacterial cells were evaluated in Figure 7B. In all cases, an increase in Cur concentration from 0.025 to