2.9. Antimicrobial Properties

Staphylococcus aureus BCTC13962 and Escherichia coli BCRC10675 were obtained from the laboratory of the brewing (Wuxi, China). First, the test strain was aseptically inoculated into Tryptone Soy Broth (TSB; Solarbio Sci. & Tec. Co., Ltd., Beijing, China) and incubated overnight at 37 °C. The concentrations of the S. aureus and E. coli suspensions were ≈1 × 10⁹ CFU/mL and ≈5 × 10⁹ CFU/mL, respectively. To reach an initial concentration between 10⁷ and 10⁸ CFU/mL, 200 µL of bacterial culture was aseptically taken to 4.8 mL of TSB broth containing 2.5 mg of the film sample. The mixture was then incubated at 37 °C for 3 h with shaking at 100 rpm. To plate with Tryptone Soy Agar (TSA) for S. aureus and Luria–Bertani (LB; Qingdao Hope Bio-Technology Co., Ltd., Shandong, China) agar for E. coli, 0.1 mL of the mixture was sampled. Colonies were counted after the plates had been cultured for 1.5 days at 37 °C. As a control, bacterial suspension (0.5 mL) devoid of film sample was employed. The percentage of inhibition was determined by Equation (9):