2.9. Antioxidant Activity

For this assay, 2.9 mL of a DPPH (2,2-diphenyl-1-picrylhydrazyl) (Sigma-Aldrich, St. Louis, MO, USA) solution (0.1 mM in methanol) were mixed with 3 disks of the biocomposite films (6 mm in diameter). Then, the absorbances of these mixtures were measured every 30 min for 5 h at 517 nm against methanol (Sigma-Aldrich, St. Louis, MO, USA) as a blank. The mixture of 2.9 mL of the DPPH solution with 100 µL of methanol was used as the control sample. The antioxidant activity of the biocomposite films was calculated using the following equation [13,19]:

where A_control is the absorbance of the control, and A_sample is the absorbance of the samples (films).

Firstly, 50 µL of a β-carotene solution (20 mg/mL in chloroform (Sigma-Aldrich, MO, USA)) were mixed with linoleic acid (40 µL), Tween 40 (400 µL), and chloroform (1 mL), the chloroform being evaporated under vacuum. Then, oxygenated distilled water (100 mL) was added to the residue, forming an emulsion. Subsequently, 5 mL of the emulsion were mixed with 3 disks of the biocomposite films (6 mm in diameter) in test tubes. Finally, the tubes were placed in a 50 °C environment for 1 h. The absorbances of these samples were measured at 470 nm against a blank containing an emulsion prepared without the β-carotene. The mixture of 5 mL of the emulsion with 300 µL of methanol was used as the control sample. The antioxidant activity of the biocomposite films was calculated as the percentage of inhibition of β-carotene oxidation using the following equation [13,19]:

where A^t=1h is the absorbance of the samples (films) or the control at the final time of reaction, and A^t=0h is the absorbance of the control at the initial time of reaction.