*2.10. Water Vapor Permeability*

Water vapor permeability (WVP) was evaluated according to the methodology proposed by Jiménez-Regalado, Caicedo [13] and following the desiccant method [32]. A glass permeability test dish containing silica gel was used, on which the biodegradable films were mounted. The test dish was placed in a desiccator with NaBr, which generated a pressure gradient of 2854.23 Pa. The test dishes were weighed every 40 min for 8 h. WVP are reported as the mean and standard deviation of three replicates.

#### *2.11. Statistical Analysis*

Experimental data were analyzed for statistical significance by analysis of variance (ANOVA) and Tukey's test with a *p* < 0.05 significance level with the help of statistical software OriginPro 8.5.0 SR1 (OriginLab Corporation, Northampton, MA, USA).

#### **3. Results and Discussion**

#### *3.1. Rheology Behavior of Film Forming Solutions*

A rheological study of the biopolymeric film forming solutions was carried out to examine the behavior of the nanocrystals and their effect on viscosity. Figure 1 shows the apparent viscosity behavior as a function of the shear rate of the filmogenic solutions. The results showed that the apparent viscosity of the filmogenic solutions of starch–chitosan mixtures strongly depended on the shear rate they were subjected to. The decrease was due to the disentangling of polymeric chains. Similar behavior was observed in other formulations based on chitosan, both alone and in mix with other biopolymers [33]. The addition of the cellulose nanocrystals increased the apparent viscosity of the filmogenic solutions when these did not exceed a concentration of 5%. This was probably due to crosslinks generated by the CNC forming networks that caused resistance to flow. At higher percentages of nanomaterial, the viscosity no longer increased; it remained in a range similar to that of low concentrations of CNC, but always higher than that of the control film. *Polymers* **2022**, *14*, x FOR PEER REVIEW 6 of 17 percentages of nanomaterial, the viscosity no longer increased; it remained in a range similar to that of low concentrations of CNC, but always higher than that of the control film.

**Figure 1.** Log-log viscosity versus shear rate of film forming solutions of corn-starch and several ratios of CNC (*w*/*w*). **Figure 1.** Log-log viscosity versus shear rate of film forming solutions of corn-starch and several ratios of CNC (*w*/*w*).

#### *3.2. Antimicrobial Activity of the Nanocomposite Films*

*3.2. Antimicrobial Activity of the Nanocomposite Films*  A package or film that is going to be in contact with food, in addition to protecting it from mechanical damage, can also prevent or delay the growth of spoilage microorganisms and some pathogenic microorganisms. In this study, the antimicrobial capacity of CNCs was evaluated when they were incorporated into corn starch–chitosan composite films. Two pathogenic microorganisms, *Listeria monocytogenes* and *Staphylococcus aureus*, were used due to their relevance to the food industry. The size of the inhibition halos (clear areas around the disks) generated by the nanocomposite film samples are presented in Table 1. An evident inhibition of the growth of both microorganisms was observed when the percentage of nanocomposites was low (0 to 5% *w*/*w*). The inhibitory effect of chitosan in the corn starch–chitosan composite film was confirmed in both *L. monocytogenes* and *S. aureus*; this test was consistent with results presented in other reports [34,35]. A package or film that is going to be in contact with food, in addition to protecting it from mechanical damage, can also prevent or delay the growth of spoilage microorganisms and some pathogenic microorganisms. In this study, the antimicrobial capacity of CNCs was evaluated when they were incorporated into corn starch–chitosan composite films. Two pathogenic microorganisms, *Listeria monocytogenes* and *Staphylococcus aureus*, were used due to their relevance to the food industry. The size of the inhibition halos (clear areas around the disks) generated by the nanocomposite film samples are presented in Table 1. An evident inhibition of the growth of both microorganisms was observed when the percentage of nanocomposites was low (0 to 5% *w*/*w*). The inhibitory effect of chitosan in the corn starch–chitosan composite film was confirmed in both *L. monocytogenes* and *S. aureus*; this test was consistent with results presented in other reports [34,35]. Chitosan is a biopolymer that has been shown to have some antibacterial capacity against both gram-positive and

> Chitosan is a biopolymer that has been shown to have some antibacterial capacity against both gram-positive and gram-negative bacteria [36,37]. This good antimicrobial effect has improved biodegradable materials made from natural polymers such as starch from sev-

> most extensive diameter inhibition halo (12.52 ± 0.01 mm.) when a concentration of 0.5% CNC was used. In this case, the antimicrobial effect of the content of CNC was demonstrated in contrast to the control. In *S. aureus*, on the other hand, although the evaluations showed an inhibitory effect of the starch–chitosan films with CNC concentrations lower than 5.0%, the impact was not more significant than that shown in the control films, re-

vealing a difference in the sensitivity of both gram-positive microorganisms.

gram-negative bacteria [36,37]. This good antimicrobial effect has improved biodegradable materials made from natural polymers such as starch from several sources [12,38].

**Table 1.** Antimicrobial effect of cellulose nanocrystals (CNC) incorporated in corn starch–chitosan biodegradable films.


Mean <sup>±</sup> standard deviation of three replicas. Values with different letters (a, b, c) in the same column denote significant differences (Tukey test; *p* < 0.05). (--): No inhibition halo was observed.

The maximum inhibitory effect was observed on *L. monocytogenes*, which showed the most extensive diameter inhibition halo (12.52 ± 0.01 mm.) when a concentration of 0.5% CNC was used. In this case, the antimicrobial effect of the content of CNC was demonstrated in contrast to the control. In *S. aureus*, on the other hand, although the evaluations showed an inhibitory effect of the starch–chitosan films with CNC concentrations lower than 5.0%, the impact was not more significant than that shown in the control films, revealing a difference in the sensitivity of both gram-positive microorganisms.

The nanomaterials (NM) or nano reinforcement composition, shape, and size play an essential role in their antibacterial capacity. The smaller the NMs, the greater their specific surface area, with the latter increasing the probability of interacting with and crossing the bacterial cell membrane [23,26,39,40].

The mechanism by which nanoparticles generate toxicity is unknown; however, efforts are still being made to elucidate it. One possible mechanism is adhesion by the electrostatic interaction of nanoparticles to the bacterial cell membrane, affecting its structural integrity [25,38]. Some NMs can also cause oxidative stress through the formation of free radicals, which alters the permeability of the cell membrane, damaging it and causing its death [24,25]. The use of nanometric-sized compounds incorporated in packaging materials has been investigated in recent years due to their interesting functional properties.

The use of nanomaterials with antimicrobial effects offers several advantages and has broad applications. Benefits include prolonged antimicrobial activity, very low environmental or health toxicity and the absence of resistance of microbes to antibiotics and other drugs [38]. These antimicrobial biopolymeric formulations can be used to develop materials for the food packaging industry, as well as in the development of various medical devices.
