2.5.7. Water Solubility

The films solubility in water was defined by the content of dry matter solubilized after 24 h of immersion in water. The initial dry matter content of each film was determined by drying to constant weight in an oven at 105 ◦C. Two disks of films (2 cm of diameter) were cut, weighed and immersed in 50 mL of water. After 24 h of immersion at 20 ◦C with occasional magnetic stirring, the pieces of films were taken out (by filtration) and dried to constant weight in an oven at 105 ◦C, in order to determine the weight of dry matter which was not solubilized in water [24]. The measurement of solubility of the films was determined as follows:

S(%) = [(mi − mf)/(mi)] × 100%, where S is the solubility, mi is the initial mass and mf the final mass.

#### 2.5.8. Antioxidant Activity

The antioxidant activity of the films was evaluated by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay and the β-carotene bleaching test.

For the DPPH free radical scavenging assay, 3 disks of the film (6 mm of diameter) were added to 2.9 mL of a DPPH methanolic solution (0.1 mM). Then, the absorbances were measured at 517 nm every 30 min, for 5 h, against a blank of methanol [13].

For the β-carotene bleaching test, 500 μL of a β-carotene solution (20 mg/mL in chloroform) were added to 40 μL of linoleic acid, 400 μL of Tween 40 and 1 mL of chloroform. The chloroform was then evaporated under vacuum, and 100 mL of oxygenated distilled water were added to the mixture to form an emulsion. Then, 5 mL of this emulsion were pipetted into test tubes containing 3 disks of the film (6 mm of diameter). Finally, the tubes were shaken and placed at 50 ◦C in a water bath for 1 h. The absorbances of the samples were measured at 470 nm [13].

#### 2.5.9. Antibacterial Properties

The antibacterial properties of the films against two foodborne pathogens (*Enterococcus faecalis* ATCC 29212 and *Listeria monocytogenes* LMG 16779) were evaluated by solid diffusion assay. For this test, inoculums were prepared by suspending bacteria in a sterile saline solution (NaCl; 0.85%; w/v) to a cell suspension of 0.5 McFarland (1–2 <sup>×</sup> 108 colony-forming units/mL (CFU/mL)). Disks of the films (6 mm of diameter) were prepared under aseptic conditions. Then, the Müeller-Hinton agar (MHA) plates were inoculated and allowed to dry, being the disks of films placed over the inoculated culture medium. Finally, the plates were incubated at 37 ◦C for 24 h. After the incubation, all the plates were visually checked for inhibition zones, being their diameters measured using a pachymeter. This assay was performed three independent times [13,21].
