Development and Characterization of Sustainable Antimicrobial Films Incorporated with Natamycin and Cellulose Nanocrystals for Cheese Preservation

: Environmental pollution and food safety are both issues of global concern. In this sense, sustainable and antimicrobial nanocomposites based on cellulose/poly (vinyl alcohol) blend incorporated with natamycin and cellulose nanocrystals (CNC) were manufactured and characterized. The developed ﬁlms were evaluated according to their mechanical and optical properties, and their barrier to oxygen and water vapor permeation. The antimycotic activity was evaluated in vitro against fungi and yeasts. The ﬁlm’s potential to act as an active packaging for Minas cheese preservation was also assessed. The incorporation of CNC increased the ﬁlms’ tensile strength; however, it did not inﬂuence the barrier properties to water vapor (4.12 × 10 − 7 g · cm.m − 1 · h − 1 · Pa − 1 ) and oxygen (3.64 × 10 − 13 g · cm · m − 1 · h − 1 · Pa − 1 ). The incorporation of natamycin, on the other hand, resulted in ﬁlms that were more opaque (around 24%) and of a yellowish color. The active nanocomposites developed showed antimicrobial effects against all analyzed fungi and yeasts (approximately 35 mm of inhibition zone) and were able to control the growth of S. cerevisiae in cheese, reducing a log cycle until the 12th day of storage. Since they performed well in vitro and on food, it was concluded that the ﬁlms showed potential to be applied in Minas cheese preservation.


Introduction
Cheese production is one of the most important activities of the dairy industry in Brazil [1]. In order to guarantee a final product with microbiological quality, improve the product shelf life, minimize risks to consumers' health, as well as to reduce economic losses due to food waste, certain precautions are required throughout the production chain, such as the implementation of good hygiene practices and food safety management systems [2,3]. Besides that, several innovative technologies have been developed and used to preserve perishable goods and to ensure food safety.
Among these new technologies, it is worth noting the active packaging with the incorporation of antimicrobial substances [4,5]. An important advantage of this kind of technology is that the addition of the preservative agents into the polymer matrix of the packaging enables reducing the addition of preservatives directly into the food formulation, which is in accordance with emerging consumer trends such as lower additive content in foods [6,7]. Moreover, since most microbial growth takes place on the food surface, the presence of antimicrobial agents in the packaging may improve their preservative action [8].
obtained from the culture collection of the Food Packaging Laboratory, Federal University of Viçosa.

Experimental Design
The experimental design was completely randomized, and a two-factor five-level rotatable central composite design (RCCD) with five repetitions at the central point, totaling thirteen trials, was implemented to study the effect of the incorporation of CNC and natamycin in the optical, mechanical, barrier, and antimycotic properties of the MC/PVOH blend. The coded levels and their respective real values of concentrations of natamycin and CNC are displayed in Table 1. The % was based on the final polymers' mass.

Elaboration of the Polymeric Blend Incorporated with CNC and Natamycin
Different concentrations of CNCs (Table 1, Section 2.2.1) were dispersed in 200 mL of distilled water, homogenized in ultraturrax (5 min, 20,000 rpm, model T25, IKA), and ultrasonicated (450 W, Unique Group, Rio de Janeiro, RJ, Brazil) for 5 min. After, 2.60 g of PVOH and 6.25 g of MC were added to the CNC dispersion and heated at 70 • C for 4 h under continuous stirring until solubilization, followed by gelification. Different concentrations of natamycin were added subsequently (Table 1, Section 2.2.1), followed by the incorporation of the plasticizer glycerol (30% wt./wt.). All concentrations were calculated based on the total polymer mass. The obtained dispersions were continuously stirred for 20 min and poured on glass plates (18 cm × 34 cm) until solvent evaporation.

Thickness and Mechanical Properties
Film thickness was measured, in µm, with a digital micrometer (model 547-401, Mitutoyo, Kawasaki, Japan). Ten specimens of each treatment were analyzed, and the measurement was realized in ten random points of each sample [9]. The films were also evaluated according to their mechanical properties of ultimate tensile strength (UTS, in MPa), elongation at break (EB, in %), and modulus of elasticity (Young's modulus, YM, in MPa) using a Universal Testing Machine (model 3367, Instron Corporation, Norwood, MA, USA) equipped with a 1 kN load cell. Five specimens (25 mm × 150 mm) of each treatment were evaluated. The initial distance of grids separation was 100 mm, and the standardized rate of separation was 50 mm·min −1 [23].

Color and Opacity Measurement
The color of the films was assessed by a Color Quest XE spectrophotometer (Hunter Lab, Reston, VA, USA), working with D65, 10 • angle, and using the CIELAB scale for the L*, a* and b* coordinates. Opacity (OP), yellowness index (YI), and b* (tendency to yellow) were the parameters investigated. Two samples were analyzed for each treatment, and five measurements were performed at random points of each sample [24].

Water Vapor Permeability (WVP)
WVP was investigated by the gravimetric method according to ASTM E96/E96M-10 [25] with modifications [9]. The obtained films were cut into circles (Ø = 83 mm) and sealed with paraffin in poly(methyl methacrylate) cups containing a saturated solution of lithium chloride (RH = 12% ± 5% at 25 • C ± 2 • C). After, the cups were placed in desiccators containing a saturated solution of sodium chloride (RH = 75% ± 5% at 25 • C ± 2 • C). The cups were weighted periodically, enough to provide ten data points after the steady state was reached. The results were expressed as g·cm·m −1 ·h −1 ·Pa −1 .

Oxygen Permeability (O 2 P)
The permeability to oxygen was verified in a VAC-V1 gas permeability tester (Labthink Instruments, Jinan, China). The experiment was carried out at 23 • C ± 2 • C and 50% RH. The exposed area of the films was 38.48 mm 2 , and the analysis was conducted for 8 h. The transmission rate of oxygen through film was expressed as g·cm·m −1 ·h −1 ·Pa −1 .

In Vitro Assessment of the Antimycotic Activity of the Films
The yeasts S. cerevisiae and K. lactis were grown on Sabouraud broth, incubated for 24 h at 32 • C, plated on acidified PDA, and incubated once more for 7 days at 25 • C ± 2 • C. After, isolated colonies were selected, suspended in a 5 mL 0.85% (wt./wt.) saline solution, and adjusted to 0.5 McFarland standard (around 1.0 × 10 6 cells·mL −1 ) [26]. Regarding the molds, the conidia were grown on Sabouroud broth for 7 days at 25 • C ± 2 • C, plated on acidified PDA, and incubated again at the same conditions. Subsequently, the conidia were collected and transferred to 5 mL 0.85% (wt./wt.) saline solution and adjusted to optical density (OD 530 ) of 0.10 (GBC UV/Vis 918), which represented around 5 × 10 4 conidia·mL −1 [26].
After, aliquots of 100 µL of the suspensions prepared were spread on acidified PDA, and samples of the films (Ø = 10 mm) were placed at the center of each plate. The plates inoculated with molds or yeasts were incubated for 7 days at 25 • C ± 2 • C, and the inhibition zone verified was measured in mm.

Assessment of the Active Films on Minas Cheese Preservation
The surface of the food sample (25 g) was decontaminated with alcohol 70% (v/v). Subsequently, aliquots of 100 µL of S. cerevisiae (10 4 CFU·mL −1 ) (Section 2.2.7) were spread on cheese surface, and, right after, the samples were left to dry for 15 min [27]. The inoculated samples were packaged with the following elaborated films: film A (5% of CNC and 2% natamycin) and film B (5% of CNC and 0% of natamycin, taken as the control). The samples were stored at 4 • C for 15 days. Every three days, for a 15-day period, the samples (25 g) were homogenized with 225 mL of peptone water (0.1% v/v), diluted accordingly, and spread plated on PDA agar. The plates were incubated for 7 days at 25 • C ± 2 • C, and the results were expressed as log of colony forming unit of S. cerevisiae per gram of cheese (Log CFU·g −1 ) [9].

Statistical Analysis
The models representing the parameter's behavior as a function of CNC and/or natamycin concentrations were obtained using analysis of variance (ANOVA) with a 5% level of significance followed by regression analysis. The software Minitab 17 was used.

Films' Appearance
The MC and PVOH blends incorporated with CNC and natamycin were successfully achieved, as can be seen in Figure 1. Macroscopically, they presented a smooth and homogeneous aspect. Natamycin incorporation, especially, resulted in films with a yellowish color and less transparent than films incorporated with CNC only. Complementarily, Table 2 shows the results obtained for opacity, yellow index, and tendency to yellow.  (5), 5% of CNC and 2.5% of natamycin (6), 2.5% of CNC and 0% of natamycin (7), 2.5% of CNC and 5% of natamycin (8), and 2.5% of CNC and 2.5% of natamycin (9). From the results for optical properties, it was possible to adjust models (non-significant lack-of-fit) that explained the behavior of the three parameters analyzed as a function of the concentration of natamycin only, as displayed in Figure 2. CNC concentration, in turn, did not impact these parameters, suggesting a good dispersion of the nanomaterial in the polymer matrix. The yellowish color of films containing natamycin is possibly due to the slightly yellow color of the powder. In addition, the increased opacity verified when higher amounts of natamycin were added into films was probably the result of the hydrophobic portion of the peptide and its poor solubility in water, which affected the antimicrobial dispersion in the hydrophilic matrix and resulted in opaquer films [14,28].  (5), 5% of CNC and 2.5% of natamycin (6), 2.5% of CNC and 0% of natamycin (7), 2.5% of CNC and 5% of natamycin (8), and 2.5% of CNC and 2.5% of natamycin (9). Table 2. Results obtained for optical parameters (tendency to yellow, b*, opacity, OP, and yellow index, YI), thickness, mechanical properties (ultimate tensile strength, UTS, elongation at break, EB, Young's modulus, YM), water vapor permeability (WVP), and oxygen permeability (O 2 P) of films elaborated with methyl cellulose, poly(vinyl alcohol), cellulose nanocrystals, and natamycin.  From the results for optical properties, it was possible to adjust models (non-significant lack-of-fit) that explained the behavior of the three parameters analyzed as a function of the concentration of natamycin only, as displayed in Figure 2. CNC concentration, in turn, did not impact these parameters, suggesting a good dispersion of the nanomaterial in the polymer matrix. The yellowish color of films containing natamycin is possibly due to the slightly yellow color of the powder. In addition, the increased opacity verified when higher amounts of natamycin were added into films was probably the result of the hydrophobic portion of the peptide and its poor solubility in water, which affected the antimicrobial dispersion in the hydrophilic matrix and resulted in opaquer films [14,28].

Mechanical Properties
When developing new materials for food packaging, it is crucial to evaluate their mechanical properties since these features will allow us to infer about the packaging behavior when under certain conditions, such as mechanical stress. The results for thickness, UTS, EB, and YM are also displayed in Table 2 Complementarily to the adjusted model, Figure 3 shows the surface response obtained for UTS.

Mechanical Properties
When developing new materials for food packaging, it is crucial to evaluate their mechanical properties since these features will allow us to infer about the packaging behavior when under certain conditions, such as mechanical stress. The results for thickness, UTS, EB, and YM are also displayed in Table 2. It was possible to adjust a regression model for only UTS, and the behavior was a function of both CNC (X1) and natamycin (X2) concentrations, as demonstrated in Equation (1). The other parameters (thickness, EB, and YM) were not explained by any models, therefore, we considered only the global mean value: thickness of 172 µm; 68.74% of EB; YM of 7.89 MPa.
Complementarily to the adjusted model, Figure 3 shows the surface response obtained for UTS. The findings indicated that both CNC and natamycin impacted the films' UTS, which assumed a parabolic shape. This suggested that, beyond certain concentrations, the additive's incorporation had a negative effect on the UTS parameter. Furthermore, the term X1X2 in Equation (1) suggested that, for this parameter, there was an interaction between CNC and natamycin: the UTS was likely to increase when higher concentrations of CNC and lower concentrations of natamycin were used. Other research also verified an increase in the tensile strength of sustainable films of PVOH incorporated with CNC [29,30]. This behavior may be explained by the high amount of -OH groups on the CNC structure, which are able to strongly interact with the hydrophilic polymeric matrices (MC and PVOH), increasing the compaction of the chains. This more homogenous structure may play an important role in stress redistribution all over the chains. Besides that, the high specific surface area and aspect ratio are CNCs' features that allow their application as potential reinforcing materials [31]. Natamycin, on the other hand, contributed less to the mechanical parameters, which may be due to its hydrophobic region and lower compatibility with both polymers.

Water Vapor and Oxygen Permeability
The results obtained for WVP and O2P are presented in Table 2. In the food packaging field, it is of utmost importance to ensure that gas exchanges between the food and the external environment are minimal. While water vapor permeation can adversely affect both the product's shelf life and its sensorial quality, oxygen permeation triggers oxidation, which in turn leads to undesirable changes in flavor, texture, and appearance.
It was not possible to adjust a regression model for either WVP or O2P as a function of CNC and natamycin concentrations (significant lack of fit, indicating not suitable models). Due to this, we considered only the global mean values: 4.12 × 10 −7 g·cm·m −1 ·h −1 ·Pa −1 for WPV and 3.64 × 10 −13 g·cm·m −1 ·h −1 ·Pa −1 for O2P. Although it is said in the literature that CNC is able to improve the barrier properties of biodegradable packaging, hindering the flow of molecules through tortuous paths, contrary to our expectations, neither CNC nor natamycin influenced the gas permeability of the elaborated films [32,33].

In Vitro Antimycotic Activity
At first, the films' antimycotic activity was assessed in vitro against four molds of importance in food preservation, as well as two yeasts. Photographs of the inoculated plates after the incubation period of seven days are displayed in Figure 4. The findings indicated that both CNC and natamycin impacted the films' UTS, which assumed a parabolic shape. This suggested that, beyond certain concentrations, the additive's incorporation had a negative effect on the UTS parameter. Furthermore, the term X 1 X 2 in Equation (1) suggested that, for this parameter, there was an interaction between CNC and natamycin: the UTS was likely to increase when higher concentrations of CNC and lower concentrations of natamycin were used. Other research also verified an increase in the tensile strength of sustainable films of PVOH incorporated with CNC [29,30]. This behavior may be explained by the high amount of -OH groups on the CNC structure, which are able to strongly interact with the hydrophilic polymeric matrices (MC and PVOH), increasing the compaction of the chains. This more homogenous structure may play an important role in stress redistribution all over the chains. Besides that, the high specific surface area and aspect ratio are CNCs' features that allow their application as potential reinforcing materials [31]. Natamycin, on the other hand, contributed less to the mechanical parameters, which may be due to its hydrophobic region and lower compatibility with both polymers.

Water Vapor and Oxygen Permeability
The results obtained for WVP and O 2 P are presented in Table 2. In the food packaging field, it is of utmost importance to ensure that gas exchanges between the food and the external environment are minimal. While water vapor permeation can adversely affect both the product's shelf life and its sensorial quality, oxygen permeation triggers oxidation, which in turn leads to undesirable changes in flavor, texture, and appearance.
It was not possible to adjust a regression model for either WVP or O 2 P as a function of CNC and natamycin concentrations (significant lack of fit, indicating not suitable models). Due to this, we considered only the global mean values: 4.12 × 10 −7 g·cm·m −1 ·h −1 ·Pa −1 for WPV and 3.64 × 10 −13 g·cm·m −1 ·h −1 ·Pa −1 for O 2 P. Although it is said in the literature that CNC is able to improve the barrier properties of biodegradable packaging, hindering the flow of molecules through tortuous paths, contrary to our expectations, neither CNC nor natamycin influenced the gas permeability of the elaborated films [32,33].

In Vitro Antimycotic Activity
At first, the films' antimycotic activity was assessed in vitro against four molds of importance in food preservation, as well as two yeasts. Photographs of the inoculated plates after the incubation period of seven days are displayed in Figure 4. Films elaborated without natamycin (T7) did not show antimicrobial activity, as can be seen in Figure 4. When incorporated with natamycin, all the films displayed antimicrobial properties, suggesting that they could be used as active films for food preservation against these microorganisms. Regardless of the natamycin concentration, the inhibition zones verified for each mold or yeast investigated were similar, and the mean values obtained are presented in Table 3. The only exception was when the films were tested against A. alternata, in which it was possible to adjust a polynomial model of the inhibition zone as a function of natamycin concentration (Table 3). Table 3. Inhibition zones obtained when the elaborated films with different concentrations of natamycin (0.7%, 2.5%, 4.3%, and 5.0%) were assessed against molds and yeasts of importance in food preservation.

Microorganism
Inhibition Natamycin is a preservative allowed for dairy products, and it has been the object of study of several pieces of research regarding active food packaging. Similar to the present study, Chakravartula et al. (2020) [34] verified that blend films elaborated with cassava Films elaborated without natamycin (T7) did not show antimicrobial activity, as can be seen in Figure 4. When incorporated with natamycin, all the films displayed antimicrobial properties, suggesting that they could be used as active films for food preservation against these microorganisms. Regardless of the natamycin concentration, the inhibition zones verified for each mold or yeast investigated were similar, and the mean values obtained are presented in Table 3. The only exception was when the films were tested against A. alternata, in which it was possible to adjust a polynomial model of the inhibition zone as a function of natamycin concentration (Table 3). Table 3. Inhibition zones obtained when the elaborated films with different concentrations of natamycin (0.7%, 2.5%, 4.3%, and 5.0%) were assessed against molds and yeasts of importance in food preservation.

Microorganism
Inhibition Natamycin is a preservative allowed for dairy products, and it has been the object of study of several pieces of research regarding active food packaging. Similar to the present study, Chakravartula et al. (2020) [34] verified that blend films elaborated with cassava starch, chitosan, and natamycin presented potential to be used as active packaging due to the antifungal activity displayed by the films. Films incorporated with natamycin were also manufactured by Grafia et al. (2018) [16]. The authors tested the active films in vitro against A. niger previously isolated from onions and achieved interesting outcomes.
Literature shows that the mechanism of action of natamycin against fungi involves interference in the cells' vacuole and a preferred binding to ergosterol, a sterol molecule found in fungi cells membrane responsible for cell integrity [35][36][37]. Besides that, natamycin is also referred to as an efficient inhibitor of the transport of nutrients, compromising cell function [37].

Application of the Active Packaging in Minas Cheese
The central composite design allowed us to optimize the packaging system and define the film formulation that should be tested on food based on the outcomes found for the investigated properties. Minas cheese, a traditional-Brazilian product, was chosen as the food sample in this step since preliminary tests indicated that the film remained well preserved after contact with the food. The active films tested on the Minas cheese samples were elaborated with 5% of CNC and 2% of natamycin (film A), and 5% of CNC (film B, control). In this sense, cheese samples previously inoculated with S. cerevisiae were packaged with the films and stored for up to 15 days at low temperatures (4 • C). Regression models were adjusted for each case, and they are presented below (Equations (2) and (3)): in which Y A is the yeast growth, in log CFU·g −1 , when the film A was used, Y B is the yeast growth, also in CFU·g −1 , when the film B was used. For both equations, T represents the time, in days. In Figure 5, it is possible to observe the yeast counts in Minas cheese during the storage time.
Polysaccharides 2023, 4, FOR PEER REVIEW 10 starch, chitosan, and natamycin presented potential to be used as active packaging due to the antifungal activity displayed by the films. Films incorporated with natamycin were also manufactured by Grafia et al. (2018) [16]. The authors tested the active films in vitro against A. niger previously isolated from onions and achieved interesting outcomes. Literature shows that the mechanism of action of natamycin against fungi involves interference in the cells' vacuole and a preferred binding to ergosterol, a sterol molecule found in fungi cells membrane responsible for cell integrity [35][36][37]. Besides that, natamycin is also referred to as an efficient inhibitor of the transport of nutrients, compromising cell function [37].

Application of the Active Packaging in Minas Cheese
The central composite design allowed us to optimize the packaging system and define the film formulation that should be tested on food based on the outcomes found for the investigated properties. Minas cheese, a traditional-Brazilian product, was chosen as the food sample in this step since preliminary tests indicated that the film remained well preserved after contact with the food. The active films tested on the Minas cheese samples were elaborated with 5% of CNC and 2% of natamycin (film A), and 5% of CNC (film B, control). In this sense, cheese samples previously inoculated with S. cerevisiae were packaged with the films and stored for up to 15 days at low temperatures (4 °C). Regression models were adjusted for each case, and they are presented below (Equations (2) and (3) in which YA is the yeast growth, in log CFU·g −1 , when the film A was used, YB is the yeast growth, also in CFU·g −1 , when the film B was used. For both equations, T represents the time, in days. In Figure 5, it is possible to observe the yeast counts in Minas cheese during the storage time. It can be observed that the film incorporated with natamycin impacted the growth of S. cerevisiae in cheese in approximately one log cycle until the 12th day of refrigerated It can be observed that the film incorporated with natamycin impacted the growth of S. cerevisiae in cheese in approximately one log cycle until the 12th day of refrigerated storage when compared to the film incorporated with only CNC. This means that a reduction of approximately 90% of the yeast population was achieved. Although S. cerevisiae is an important fermenting agent for the food industry, mostly involved in the manufacturing of bakery products and alcoholic beverages, it is considered a contaminant microorganism in cheese, usually related to product spoilage [38,39] To date, fungal spoilage in cheese is one of the major challenges faced by the dairy industry [40]. In a study with 61 cheesemakers from the United States, 71% reported that the growth of undesirable mold on cheese surfaces was one of the most common issues in the area [41]. The occurrence of strange color and pigment development, probably caused by microbial growth, was also mentioned by around 54% of the respondents [39]. In addition, 28% of the interviewed cheesemakers mentioned that around 5 to 10% of their production was lost due to quality issues, indicating an economic loss that can be significant.
In order to minimize contaminants in dairy, it is essential to ensure the quality of the milk, as well as invest in and enforce good hygiene practices throughout the entire production chain. The adoption of novel technologies, such as active packaging, could be a complementary strategy to enhance quality and contribute to prolonging the product's shelf life. In this sense, these findings suggest that the application of a sustainable packaging MC/PVOH-based incorporated with CNC and natamycin in Minas cheese could play an important role in product preservation. Although CNC does not present antimicrobial activity, the material can be added as a nanofiller to enhance the mechanical properties of the packaging, while natamycin functions as the active component effective against molds and yeasts.

Conclusions
In the present study, an MC/PVOH/CNC/natamycin blend film was successfully manufactured and showed potential to be used as an antimicrobial film in Minas cheese preservation. Initially, it was hypothesized that the presence of CNC would improve the films' mechanical and barrier properties; however, only the UTS parameter was influenced by the CNC concentration. Nevertheless, this was an important finding since CNC did act as a mechanical reinforcing material. Future studies should be more focused on enhancing the barrier properties of the film since it is a significant parameter of food preservation. Furthermore, the presence of CNC did not impact the optical properties of the elaborated packaging, which was also a positive result. Natamycin, on the other hand, had a negative effect on the optical parameters and on the mechanical properties of the films, probably due to lower compatibility among the antimicrobial and the polymers. Although it enabled the film to act as an antimicrobial packaging against yeasts and molds, as well as displayed a good performance when tested on Minas cheese, its compatibility and interaction with the polymers should be studied in greater depth to allow the obtainment of active films with not only antimicrobial properties, but also better mechanical, optical, and barrier properties.