*2.6. Antimicrobial Assays*

The antibacterial activity of the neat eugenol, the eugenol-containing MCM-41 particles, and the electrospun films with MCM-41 with eugenol was evaluated against *S. aureus* CECT240 (ATCC 6538P) and *E. coli* CECT434 (ATCC 25922). These strains were obtained from the Spanish Type Culture Collection (CECT, Valencia, Spain) and stored in phosphate buffered saline (PBS) with 10 wt.-% tryptic soy broth (TSB, Conda Laboratories, Madrid, Spain) and 10 wt.-% glycerol at −80 ◦C. Previous to each study, a loopful of each bacteria was transferred to 10 mL of TSB and incubated at 37 ◦C for 24 h. A 100-μL aliquot from the culture was again transferred to TSB and grown at 37 ◦C to the mid-exponential phase of growth. An approximate count of 5 × 10<sup>5</sup> colony-forming units (CFU)/mL of a culture resulted in an absorbance value of 0.20, as determined by optical density at 600 nm (UV 4000 spectrophotometer, Dinko Instruments, Barcelona, Spain).

The minimum inhibitory concentration (MIC) and minimum bactericide concentration (MBC) of eugenol against the selected foodborne bacteria was tested following the plate micro-dilution protocol, as described in the Methods for Dilution Antimicrobial. Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard Tenth. Edition (M07-A10) by the Clinical and Laboratory Standards Institute (CLSI). For this, a 96-well plate with an alpha numeric coordination system (columns 12 and rows A-H) were used, where 10 μL of the tested samples were introduced in the wells with 90 μL of the bacteria medium. In the wells corresponding to A, B, C, E, F, and G columns different concentrations of eugenol, that is, 0.312, 0.625, 1.25, 2.5, 5, 10, 20, 40, 80, 160 μL/mL, were tested, in triplicate, from rows 1 to 10. Columns D and H were used as control of eugenol in TSB without bacteria. Row 11 was taken as positive control, that is, only TSB, and row 12 was used as negative control, that is, *S. aureus* and *E. coli* in TSB. The plates were incubated at 37 ◦C for 24 h. Thereafter, 10 μL of resazurin, a metabolic indicator, was added to each well and incubated again at 37 ◦C for 2 h. Upon obtaining the resazurin change, the wells were read through color difference. The MIC value was determined as the lowest concentration of eugenol presenting growth inhibition.

The antimicrobial performance of the films was evaluated by using a modification of the Japanese Industrial Standard JIS Z2801 (ISO 22196:2007). A microorganism suspension of *S. aureus* and *E. coli* was applied onto the test films of PHBV/MCM-41 with eugenol and also PHBV/MCM-41, as negative control without eugenol, both sizing 2 × 2 cm2. After incubation for 24 h at 24 ◦C and at a RH of at least 95%, bacteria were recovered with PBS, 10-fold serially diluted and incubated at 37 ◦C for 24 h in order to quantify the number of viable bacteria by conventional plate count. The antimicrobial activity was evaluated from 1 (initial day), 8, and 15 days. The antibacterial activity was taken as the test surface reduction (R) using the equation 1:

R = [log(B/A) − log(C/A )] = log(B/C), (1)

where A is the mean of bacterial counts of the control sample immediately after inoculation, B is the mean of bacterial counts of the control sample after 24 h, and C is the mean of bacterial counts of the test sample after 24 h. Antimicrobial activity was evaluated with the following assessment: Nonsignificant (R < 0.5), slight (R ≥ 0.5 and <1), significant (R ≥ 1 and <3), and strong (R ≥ 3) [46].
