Multifuge x3

The films were produced by a casting method. Suspensions of the amaranth flour (4, 5 and 6%, m/V) in water were homogenized for 25 min, and the pH was adjusted to 10 using 2 M NaOH. The filmogenic solutions were then heated at 80 °C for 15 min and different mass fractions of glycerol (25, 30 and 35%, dry mass basis) were added. The amaranth flour and glycerol solutions were then centrifuged (Multifuge™ X3; Thermo Scientific™) at 698.75×g for 10 min. The chitosan nanoparticles loaded with the chia extract (0, 0.375 and 0.75%, based on the mass of amaranth flour) were dispersed in 500 µL of water and vortexed for 30 min. Samples were then added to the filmogenic solution. The dispersions were stirred for 10 min at room temperature. The filmogenic solutions were placed in Petri dishes with silicone molds (12.57 cm²) and dried at 40 °C for 12 h. The nanocomposite films were conditioned at 58% relative humidity (RH) using saturated NaBr solutions for 48 h before characterization.

Chia seed extract was obtained as reported by Morales-Olán et al. (23 (link)). Briefly, chia flour (0.5 g) was added to 80% aqueous ethanol solution (3 mL). The solution was stirred for 24 h at 25 °C and centrifuged (Multifuge™ X3; Thermo Scientific™, Madrid, Spain) at 1006.2×g for 10 min. The alcohol was then removed with a rotavapor (model R-114; Büchi Labortechnik AG, Flawil, Switzerland), and the water was removed by lyophilization (FreeZone 2.5-liter benchtop freeze dryer; Labconco, Kansas City, MO, USA).

After grinding with liquid nitrogen, each 0.3 g sample was added to 1 ml protein extraction pyrolysis liquid (Sangon) and placed in an ice box (PCR Cooler; Eppendorf, Hamburg, Germany) for ultrasonic crushing. Then, the centrifuged supernatant fluid (Multifuge X3; Thermo Fisher Scientific, Waltham, MA, USA) was determined quantitatively using the bicinchoninic acid assay. After the concentration was adjusted, 20 μl protein sample buffer was added to 20 μl sample and boiled for 10 minutes. The pretreated protein samples were separated by SDS-PAGE and transferred to PVDF membranes (Sangon). Then, the PVDF membranes were moved to TBST solution (Sangon) for blocking. The membrane was incubated with the primary antibody solution for 1 to 2 hours at 20°C on a shaking table. After being washed with TBST solution repeatedly, the membrane was incubated for 1 to 2 hours at 20°C with the conjugated antibody (Abcam, Cambridge, MA, USA). The bands were visualized using ECL (Sangon) and the pictures were conserved by a chemiluminescence imaging apparatus (SmartChemi 610; Sage, Beijing, China).