2.5.10. Antimicrobial Activity

The antibacterial activity of the films was assessed using a modified Japanese Industrial Standard (JIS) Z2801 [32]. The bacterial suspension was spread between the film samples (dimensions of 2 cm × 2 cm). The inoculated film samples were incubated at 95% RH and 25 ◦C for 24 h. Thereafter, the bacteria were recovered with PBS and plated onto MHA. The number of viable cells was determined after incubation at 37 ◦C for 24 h. The antibacterial activity of the films was defined by surface reduction of log10 CFU of the test culture during incubation. Surface reduction (R) was calculated according to the Equation (3):

$$R = \left[ \log\left(\frac{B}{A}\right) - \log\left(\frac{C}{A}\right) \right] = \log\left(\frac{B}{C}\right) \tag{3}$$

where A is the average of the viable bacterial counts on the control sample immediately after inoculation, B is the average of the viable bacterial counts on the control sample after 24 h and C is the average of the viable bacterial counts on the test sample after 24 h. According to the value of the surface reduction, the antibacterial activity of the films was evaluated with the following assessment: nonsignificant (R < 0.5), slight (0.5 ≤ R < 1), significant (1 ≤ R < 3), and strong (R ≥ 3).
