2.5.7. Antimicrobial Activity

*Staphylococcus aureus* (*S. aureus*) CECT240 (ATCC 6538p) and *Escherichia coli* (*E. coli*) CECT434 (ATCC 25922) 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 (99.5% purity, Sigma Aldrich S.A. Madrid, Spain) at –80 ◦C. Prior to each study, a loopful of 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 the TSB and grown at 37 ◦C to the mid-exponential phase of growth. The approximate count of 5 × 10<sup>5</sup> CFU/mL of culture had an absorbance value of 0.20, as determined by the optical density at 600 nm (UV 4000 spectrophotometer, Dinko Instruments, Barcelona, Spain).

The minimum inhibitory concentration (MIC) and minimum bactericide concentration (MBC) of the OEO, RE, and GTE against the selected food-borne 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) was used, where 10 μL of the tested samples were introduced into 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 the natural products, 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 the natural extracts in the TSB without bacteria. Row 11 was taken as a positive control, that is, only the TSB, and row 12 was used as a negative control, that is, *S. aureus* and *E. coli* in the TSB. The plates were incubated at 37 ◦C for 24 h. Thereafter, 10 μL of resazurin sodium salt (MP biologicals, Illkirch, France), 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 the color difference. The MIC and MBC values were determined as the lowest concentration of the natural products presenting bacteriostatic and bactericide effects, respectively [41].

The antimicrobial performance of the electrospun PHBV films was evaluated using a modification of the Japanese Industrial Standard (JIS) Z2801 (ISO 22196:2007) [42]. To this end, a microorganism suspension of *S. aureus* and *E. coli* was applied onto the test films, that is, containing the natural extracts, and the negative control film, that is, without the natural extracts, all sizing 1.5 × 1.5 cm2. Tests were performed in either hermetically closed or open vials with a volume of 20 mL. After incubation at 24 ◦C and at a relative humidity (RH) of, at least, 95% for 24 h, the 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 a conventional plate count. The antimicrobial activity was evaluated after 1 (initial day), 8, and 15 days. The value of the antimicrobial reduction (R) was calculated following Equation (4):

$$R = \left[ \log\left(\frac{B}{A}\right) - \log\left(\frac{C}{A}\right) \right] = \log\left(\frac{B}{C}\right) \tag{4}$$

where *A* is the average of the number of viable bacteria on the control film sample immediately after inoculation, *B* is the average of the number of viable bacteria on the control film sample after 24 h, and *C* is the average of the number of viable bacteria on the test film sample after 24 h. Three replicate experiments were performed for each sample and the following assessment was conducted: Nonsignificant (R < 0.5), slight (R ≥ 0.5 and <1), significant (R ≥ 1 and <3), and strong (R ≥ 3) as in Reference [43].

## 2.5.8. Antioxidant Activity

The DPPH inhibition assay was used to evaluate the free radical scavenging activity of the neat OEO, RE, GTE in their oil forms, in the electrospun PHBV fibers (at day 1) and their corresponding films (at 1, 8, and 15 days). Samples were weighed in triplicate in cap vials and then an aliquot of the DPPH solution (0.05 g/L in methanol) was added to each one. Vials without samples were also prepared as controls. All the samples were prepared and immediately stored at room temperature for 2 h in darkness. After this, the absorbance of the solution was measured at 517 nm in the UV 4000 spectrophotometer from Dinko Instruments. Results were expressed as the percentage of inhibition to DPPH following Equation (5) [44] and μg equivalent of trolox per gram of sample, employing a previously prepared calibration curve of trolox.

$$\text{Inhibition DPPH (\%)} = \frac{A\_{\text{Control}} - \left(A\_{\text{sample}} - A\_{\text{blank}}\right)}{A\_{\text{control}}} \ast 100\tag{5}$$

where *Acontrol*, *Ablank*, and *Asample* are the absorbance values of the DPPH solution, methanol with the test sample, and the test sample, respectively.

## 2.5.9. Statistical Analysis

The solution properties, color, transparency, and opacity values, and contact angle values were evaluated through analysis of variance (ANOVA) using STATGRAPHICS Centurion XVI v 16.1.03 from StatPoint Technologies, Inc. (Warrenton, VA, USA). Fisher's least significant difference (LSD) was used at the 95% confidence level (*p* < 0.05). Mean values and standard deviations were also calculated.
