*3.5. X-ray Diffraction*

Figure 4 shows the X-ray diffraction patterns of biodegradable films based on starch– chitosan with several proportions of CNC. In the starch–chitosan biodegradable films, the presence of a peak at 22.42◦ was an indication of the crystalline nature of the CNC nanoparticle [6,20,42], being more visible when the concentration of nanocrystals was 2.5% (*w*/*w*) and higher. These nanocrystals correspond mainly to type I cellulose, which has a high crystallinity index, as observed through a diffraction peak at 20.5◦ [43], which was visible in the films developed in this study with CNC contents of 5, 7 and 10% (*w*/*w*). In the control films, a behavior corresponding to a more amorphous material was observed, which became more crystalline, that is, more ordered with the incorporation of the CNC. The crystalline nature of nanocellulose helps to generate packaging materials with a more ordered structure; this is reflected in the physicochemical characteristics of the material, i.e., mainly in the thermal, mechanical, and water absorption properties, which are described in Sections 3.4, 3.6 and 3.7.

#### *3.6. Water Vapor Adsorption Isotherms*

The adsorption isotherm graphically represents the relationship between water activity (or the equilibrium relative humidity of air in the environment) and material moisture content under conditions of equilibrium and constant temperature. It also allows us to know how water binds to the material [13,44]. Various mathematical models provide information on parameters that could effectively monitor a material or food during its storage [30]. The films composed of starch and chitosan presented a water absorption capacity that changed depending on the concentration of CNC.

**Figure 4.** X-ray diffractograms of starch–chitosan films reinforced with CNC (0 to 10% *w*/*w*). **Figure 4.** X-ray diffractograms of starch–chitosan films reinforced with CNC (0 to 10% *w*/*w*).

*3.6. Water Vapor Adsorption Isotherms*  The adsorption isotherm graphically represents the relationship between water activity (or the equilibrium relative humidity of air in the environment) and material moisture content under conditions of equilibrium and constant temperature. It also allows us to know how water binds to the material [13,44]. Various mathematical models provide information on parameters that could effectively monitor a material or food during its storage [30]. The films composed of starch and chitosan presented a water absorption capacity that changed depending on the concentration of CNC. By incorporating CNC into biobased starch–chitosan materials, their water absorption capacity decreased, except when 7.5% CNC was added (Figure 5), as illustrated in the relevant X-ray diffraction pattern. The higher the crystallinity of the material, the lower the water absorption capacity, because water molecules first interact with the amorphous part of the material. However, the shape or type of isotherm was not modified with an increase in the content of nanomaterials, with each film maintaining the same behavior or slope in the total range of relative humidity used in the analysis of the water adsorption isotherms. It has been reported that when the proportion of chitosan increases in starchbased formulations, the shape of the isotherm and the moisture content that these materials can absorb increase significantly when subjected to RH conditions higher than 60% or *aw* 0.6 [13]. According to Brunauer [45], the isotherms of the films evaluated in this study corresponded to those of type 2, where the adsorbate (water molecules) covers the film (adsorbent) until a monolayer is formed and the adsorption process continues in the form of a multilayer. This is a common profile in physical adsorption processes in which interactions are not very specific. Therefore, to produce this type of behavior, the affinity of the By incorporating CNC into biobased starch–chitosan materials, their water absorption capacity decreased, except when 7.5% CNC was added (Figure 5), as illustrated in the relevant X-ray diffraction pattern. The higher the crystallinity of the material, the lower the water absorption capacity, because water molecules first interact with the amorphous part of the material. However, the shape or type of isotherm was not modified with an increase in the content of nanomaterials, with each film maintaining the same behavior or slope in the total range of relative humidity used in the analysis of the water adsorption isotherms. It has been reported that when the proportion of chitosan increases in starch-based formulations, the shape of the isotherm and the moisture content that these materials can absorb increase significantly when subjected to RH conditions higher than 60% or *a<sup>w</sup>* 0.6 [13]. According to Brunauer [45], the isotherms of the films evaluated in this study corresponded to those of type 2, where the adsorbate (water molecules) covers the film (adsorbent) until a monolayer is formed and the adsorption process continues in the form of a multilayer. This is a common profile in physical adsorption processes in which interactions are not very specific. Therefore, to produce this type of behavior, the affinity of the adsorbate for the adsorbent must be higher than the affinity of the adsorbate for itself [45,46]. In this type of isotherm, two identifiable regions (i.e., two different slopes on the curve) can be observed: one around *a<sup>w</sup>* 0.1 to 0.4 and the other around *a<sup>w</sup>* 0.6 to 0.9. This represents typical behavior of a regular food product [46]. All materials showed an increase in the amount of water they could absorb when subjected to high relative humidity, i.e., mainly in regions with *a<sup>w</sup>* from 0.8 to 0.99. This range of *a<sup>w</sup>* is a critical point for the preservation of food products due to the presence of free water, which is no longer directly linked to the material and is more available so that pathogenic and deteriorating bacteria can perform their vital functions and reproduce [46,47]. Foods within this range of *aw*, such as eggs, meats, vegetables, and fresh fruits, are therefore potentially more susceptible to harboring pathogenic bacteria.

adsorbate for the adsorbent must be higher than the affinity of the adsorbate for itself [45,46]. In this type of isotherm, two identifiable regions (i.e., two different slopes on the curve) can be observed: one around *aw* 0.1 to 0.4 and the other around *aw* 0.6 to 0.9. This represents typical behavior of a regular food product [46]. All materials showed an increase in the amount of water they could absorb when subjected to high relative humidity, i.e., mainly in regions with *aw* from 0.8 to 0.99. This range of *aw* is a critical point for the In this study, a reduction in the content of water that the material could adsorb was observed with an increase of CNC content in the structural matrix, with the sample containing 7.5% CNC being able to absorb even more moisture than the control film. This indicated that in this formulation, the polymeric chains were free, interacting little with each other, making them more available to bond with water molecules. This was reflected in the parameters calculated by the GAB mathematical model, as shown in Table 3.

preservation of food products due to the presence of free water, which is no longer directly linked to the material and is more available so that pathogenic and deteriorating bacteria can perform their vital functions and reproduce [46,47]. Foods within this range of *aw*, such

harboring pathogenic bacteria.

**Figure 5.** Water adsorption isotherms of starch–chitosan films reinforced with CNC (0, 0.5, 2.5, 5, 7.5, and 10% *w*/*w*) at 25 °C. Experimental data (symbols); fitted to the GAB mathematical model (solid and dotted lines). **Figure 5.** Water adsorption isotherms of starch–chitosan films reinforced with CNC (0, 0.5, 2.5, 5, 7.5, and 10% *w*/*w*) at 25 ◦C. Experimental data (symbols); fitted to the GAB mathematical model (solid and dotted lines).

as eggs, meats, vegetables, and fresh fruits, are therefore potentially more susceptible to

In this study, a reduction in the content of water that the material could adsorb was observed with an increase of CNC content in the structural matrix, with the sample con-**Table 3.** GAB mathematical model parameters and regression coefficient, R<sup>2</sup> , calculated for composite starch–chitosan films with CNC.


had a bound water molecule [46]. This water content, present in the monolayer phase, was much higher than that of the control film, as well as that absorbed by the films containing lower percentages of CNC, although its binding energy was lower, as expressed by the *K* parameter. **Table 3.** GAB mathematical model parameters and regression coefficient, R2, calculated for composite starch–chitosan films with CNC. The value of *Xm* for the sample with 7.5% CNC presented the highest value, with a water content of 17% forming the monolayer of water that interacted directly with the material. In the monolayer, it was assumed that each hydrophilic group on the material had a bound water molecule [46]. This water content, present in the monolayer phase, was much higher than that of the control film, as well as that absorbed by the films containing lower percentages of CNC, although its binding energy was lower, as expressed by the *K* parameter.

#### **CNC Content (***w***/***w***) in Starch–Chitosan Films** *Xm C K* **R2** *3.7. Mechanical and Gas Barrier Properties*

*3.7. Mechanical and Gas Barrier Properties* 

0% 14.67 151.59 0.53 0.984 0.5% 13.46 6283.33 0.50 0.993 2.5% 11.57 106.25 0.58 0.997 5.0% 14.32 7692.23 0.53 0.987 In addition to their antimicrobial capacity, nanocomposites have been reported to improve the barrier properties against gases (water vapor and oxygen) and the mechanical properties of natural polymer-based packaging materials [16,48]. Table 4 shows the mechanical properties (tensile strength and elongation at break) and water vapor permeability of corn starch–chitosan films reinforced with CNC.

In addition to their antimicrobial capacity, nanocomposites have been reported to improve the barrier properties against gases (water vapor and oxygen) and the mechani-

7.5% 17.04 2863.44 0.43 0.991 10.0% 9.73 958.54 0.62 0.987


**Table 4.** Physicochemical properties of starch–chitosan biodegradable films reinforced with cellulose nanocrystals (CNC).

Values with different letters (a, b, c, d, e) in the same column denote significant differences (Tukey test; *p* < 0.05). Values are given as mean ± standard deviation (n = 8 for mechanical properties, and n = 4 for WVP).

A significant improvement in the mechanical properties of tension and elongation at fracture was observed with the addition of cellulose nanocrystals. Without presenting any trend or pattern of behavior as CNC concentration increased, in the present study, something similar to what was observed in other characterizations of these materials was observed. The addition of CNC at concentrations of 2.5% to 10% significantly increased the strength of the material, with the sample with 7.5% showing the highest value, i.e., 13.61 MPa, while the control (0% CNC) was 3.49 MPa. Regarding the elongation of the material, the film without CNC presented an elongation of 74.67%. When 0.5 and 5.0% of CNC were added to the formulation, the material increased its extension up to 140 and 145%, respectively. However, it was found that when nanocrystals were added in a proportion of 7.5%, the material was significantly affected, reducing its elongation to a value of 5.32%. In cassava starch films obtained by the casting method, it was found that incorporating only 1.5% of CNC (modified with stearic acid) improved the tensile strength of the materials by more than 300% [20]. This result was attributed to the fact that cellulose in micro- and nano-sizes has a high resistance to tension. When nanomaterials are compatible with the polymer matrix, a strong matrix-filler interaction is generated due to Van der Waals forces that can transfer stress, thus improving the resistance of the materials [49]. Nonetheless, the mechanical performance of the starch–chitosan-CNC films still cannot be compared to those of some films of synthetic origin, such as polyethylene [50].

As observed in the SEM microphotographs in Figure 2b–e, it is likely that the nanomaterials agglomerated when added in higher proportions [51]. These agglomerates may have influenced the mechanical properties of the materials. An excess of nanocomposites such as CNC negatively modified the mechanical properties of packaging materials [20], so it would be necessary to determine the optimal concentration according to the results or properties sought for a specific material.

According to the results obtained by Maradini, Oliveira [52], the tensile strength and Young's modulus of polyester can be improved with the addition of 1% (*w*/*w*) of CNC; however, when the concentration is increased to 2%, the mechanical performance is negatively affected. Although the addition of 4.5% CNC in PLA-gelatin multilayer films significantly reduced the tensile strength and elongation, this nanocomponent was not a viable alternative to reinforce this polymeric matrix [22]. When the nature of the reinforcement material and the polymeric matrix are not the same, this low compatibility can be seen as a weakness in terms of mechanical properties, i.e., the same effect as that generated when the nanomaterials have not been uniformly dispersed in the structural matrix material [53].

Water vapor permeability (WVP) is directly influenced by various material parameters, such as the hydrophilic or hydrophobic nature, the presence and type of additives, as well as the morphology of the resulting materials. Table 4 shows the WVP of the films made in this study based on their NCN content. Films containing 2.5% CNC (*w*/*w*) showed a lower WVP (2.46 <sup>×</sup> <sup>10</sup>−<sup>10</sup> <sup>g</sup>·m−<sup>1</sup> s <sup>−</sup>1Pa−<sup>1</sup> ) than the control film and those with other nanocrystal concentrations. This behavior can be attributed to the hydrophobic and highly crystalline

nature of the cellulose nanocrystals, in contrast to those of starch and chitosan. The addition of nanometric-sized additives can improve the structural matrix of biopolymers, generating a tortuous path and making it difficult for water molecules to pass. Other authors have reported similar results for starch-based biodegradable films reinforced with different nanometric particles [16,54]. However, a large amount of CNC could be considered excess, encouraging these nanomaterials to agglomerate and giving rise to a less homogeneous film, causing gases to flow more freely through the polymer chains and increasing their permeability [26,39]. Azeredo, Mattoso [55] reported that the presence and increase in the concentration of cellulose nanofibers in chitosan-glycerol films caused a significant reduction in WVP. A similar result was reported by Chou, Shi [56], who noted that the WVP of PVOH films decreased with the addition of CNC at 2.5% (*w*/*w*). Meanwhile, for biodegradable films made from a mixture of cassava starch, chitosan and gallic acid, the incorporation of 7.5% (*w*/*w*) cellulose nanofibers generated the material with the best water vapor barrier capacity [39].

#### **4. Conclusions**

This study made described a method for the production of biobased films from a mixture of corn starch and chitosan, which served as carriers of reinforcing nanocomposites, such as cellulose nanocrystals (CNC). It was observed that the cellulose nanomaterials were arranged differently with the polymeric chains of starch and chitosan, as reflected in their mechanical, rheological, thermal, and gas barrier performance. The small size of the CNCs helped them mix easily with the polymeric matrix; however, this interaction was limited to a certain extent. It was observed that when more nanomaterials were added, they could not disperse uniformly, which negatively affected mechanical performance, as manifest in a decrease in tensile stress. According to the results of the calorimetric analysis, a relationship was observed in the 0.5% and 5% CNC samples that presented greater elongation due to the plasticity effect and structural arrangements that allowed the structure recover under tension. The starch–chitosan films with CNC concentrations of between 0.5 and 5% presented the best functional properties, perhaps due to a more uniform distribution of the nanomaterials. The maximum inhibitory effect of the CNC nanocomposite films was observed with *Listeria monocytogenes* bacteria when 0.5% CNC was added. In comparison, the inhibitory impact on *Staphylococcus aureus* was very similar to that observed with the control films, which themselves showed a significant antimicrobial effect.

Among the critical factors in the selection of food packaging materials are the permeability and the speed of transmission of gases (water, light, oxygen, ethylene, aromas) and the mechanical performance that determines if the material will be able to maintain the integrity of the product. The films developed in this study were found to be permeable to water vapor to different degrees. The concentration of nanoparticles that can generate useful materials based on starch and chitosan can be determined according to the data presented herein. This study may be the starting point for the production of new nanocomposite materials; such antimicrobial films are currently being tested to preserve fresh fruits which are of economic importance but which are susceptible to the action of bacteria.

**Author Contributions:** Conceptualization, C.A.D.-C., E.J.J.-R. and R.Y.A.-L.; methodology, C.A.D.-C., E.J.J.-R. and R.Y.A.-L.; formal analysis, C.C., C.A.D.-C., E.J.J.-R. and R.Y.A.-L.; investigation, E.J.J.-R. and R.Y.A.-L.; writing—original draft preparation, C.C., C.A.D.-C., R.L.-G., R.D.d.L., E.J.J.-R. and R.Y.A.-L.; writing—review and editing, C.C., C.A.D.-C. and R.Y.A.-L.; visualization, E.J.J.-R. and R.Y.A.-L.; supervision, E.J.J.-R. and R.Y.A.-L.; project administration, R.Y.A.-L.; funding acquisition, C.C. and R.Y.A.-L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Centro de Investigación en Química Aplicada (CIQA) under internal project 6610 (call 2021) and by Dirección General de Investigaciones (DGI) of Universidad Santiago de Cali under call no. 01-2022.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

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

**Acknowledgments:** Thanks to the technicians M.C. Blanca Huerta Martinez, M.C. Maria Guadalupe Mendez-Padilla, M.C. Myrna Salinas Hernández and Jesus Francisco Lara Sánchez for DRX, thermal, and SEM characterizations, respectively. Rocio Yaneli Aguirre-Loredo (R.Y.A.-L.) give thanks to CONACYT for their nomination as Investigadora por Mexico assigned to CIQA. Carolina Caicedo (C.C.) acknowledges financial support from DGI of Universidad Santiago de Cali under project No. 939-621120-2148.

**Conflicts of Interest:** The authors declare no conflict of interest.
