*2.1. Materials*

Corn starch (Grain Processing Corporation, Muscatine, IA, USA) and chitosan (90% deacetylation degree, Alfadelta Materias Primas, Naucalpan, Mexico) were used without modification. Glycerol (J.T. Baker, Ciudad de Mexico, Mexico) as a plasticizing agent. Glacial acetic acid was acquired at Productos Quimicos Monterrey (Monterrey, Mexico). Cellulose nanocrystal (CNC) powder was provided by CelluForce Inc. (spray-dried powder, needleshaped, nano-sized 7.5 nm <sup>×</sup> 150 nm, named CelluForce NCC®. Montreal, QC, Canada). To simulate environments with different relative humidities (11, 22, 32, 43, 57, 75, 84, and 90%), supersaturated salt solutions of LiCl, CH3COOK, MgCl2, K2CO3, NaBr, NaCl, KCl, and BaCl<sup>2</sup> were used, which were purchased from Jalmek Cientifica (Monterrey, Mexico). Trypticase soy agar, casein peptone, and trypticase soy broth manufactured by MCD LAB S.A. of C.V. (Oaxaca, Mexico) were used for the cultivation of microorganisms in antimicrobial tests.

### *2.2. Film Forming Solutions Preparation*

For the preparation of the polymeric matrix, solutions of acetylated corn starch (5% *w*/*v* dispersed in water) and chitosan at 1% (*w*/*v*) dissolved in acetic acid (1% *v*/*v*) were used. A 50:50 ratio of polymeric solutions and 30% glycerol was used; these concentrations were determined according to the results obtained in a previous study [13,26]. First, the starch dispersion was prepared, and the plasticizer was added. Then, the temperature was increased. When the temperature reached 50 ◦C, the chitosan solution (prepared by dissolving the polymer at room temperature and stirring for 24 h) was added. Cellulose nanocrystals (CNC) were added at concentrations of 0, 0.5, 2.5, 5, 7.5, and 10% (*w*/*w*) with regard to the total biopolymer content. The composite film-forming solutions were constantly stirred and maintained at a temperature of 90 ◦C for 10 min to achieved adequate gelatinization of the corn starch. They were then left to stand for 30 min to eliminate air bubbles that had formed during the process. The polymer nanocomposite solutions (0.75 mL/cm<sup>2</sup> ) were poured into acrylic molds and dried in an oven (Thermolyne, Blue M, Blue Island, IL, USA) at 65 ◦C for 5 h. Composite films with 0% CNC were used as the control.

#### *2.3. Rheology*

The apparent viscosity of the filmogenic solutions was determined as a function of the nanocrystal content. A controlled stress rheometer (MCR 501 model, Anton Paar Physics, Graz, Austria) with a stainless steel cone-plate geometry of 50 mm diameter and angle of 2◦ was used, at a shear rate from 0.01 to 1000 s−<sup>1</sup> [27]. The analysis was performed at 25 ◦C.

#### *2.4. Antimicrobial Activity*

Pathogenic bacteria *Staphylococcus aureus* and *Listeria monocytogenes*, which are of significant concern in the food industry, were used to determine the antimicrobial capacity of the composite films with various contents of CNC. The bacterial strains were inoculated following the methodology proposed by Gómez-Aldapa, Díaz-Cruz [28]. For the evaluation of the films, the disc diffusion technique was used, using film samples of 5 mm in diameter placed directly on the inoculated trypticase soy agar plates. The plates were incubated at 35.5 ◦C for 24 h. The evaluations were carried out in triplicate, and the results are expressed as mean and standard deviation.
