3.6.1. Antimicrobial Activity

The EOs and NEs obtained from aromatic plants are constituted by a wide range of active compounds that are responsible for antimicrobial and antioxidant activity, which has promoted their application in active food packaging [1,41,42]. Table 4 shows the MIC and MBC values of the neat OEO, RE, and GTE against strains of *S. aureus* (Gram positive, G+) and *E. coli* (Gram negative, G-). The results

showed that the OEO presented the highest antibacterial effect against both bacterial strains, having achieved identical MIC and MBC values, that is, 0.625 μL/mL, against *E. coli*, and 0.312 μL/mL, against *S. aureus*. The fact that the MIC and MBC values were identical can be related to the high effectivity of the natural compounds that were achieved, at the same time, the inhibition of microbial growth and the elimination of 99.9% of the microorganisms [63]. The antimicrobial activity of the OEO has been mainly ascribed to its high content in carvacrol and thymol [64,65]. RE presented MIC values of 10 μL/mL and 5 μL/mL against *E. coli* and *S. aureus*, respectively, whereas its MBC values were 20 μL/mL, against *E. coli*, and 10 μL/mL, against *S. aureus*. The main compounds responsible for the antimicrobial activity of RE were ∝-pinene, myrcene, camphor, 1,8-cineole, and camphene [29,66,67]. Likewise, GTE showed the lowest antimicrobial performance. This NE presented MIC values of 160 μL/mL, against *E. coli*, and 80 μL/mL, against *S. aureus*. Its MBC values were 160 μL/mL, against *E. coli*, and 40 μL/mL, against *S. aureus.* Gallic acid (GA), theobromine, chlorogenic acid, and caffeic acid are known to be responsible for its antimicrobial activity [32,68]. Most research related to MIC and MBC determination has been conducted with these EOs and NEs, finding that these compounds have a broad inhibition spectrum against G+ bacteria, but they are not as efficient against some G- bacteria [69,70]. The values of the MBC and MIC for OEO were the same, while for RE and GTE, the MBC values were higher than the MIC values. This fact is related to the effectiveness of the active compounds, the susceptibility of the microorganisms, and the variation in the penetration rate of the extracts through the cell wall and the structures of the cell membrane [71,72].

**Table 4.** Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of oregano essential oil (OEO), rosemary extract (RE), and green tea tree extract (GTE) against *S. aureus* and *E. coli*.


The antimicrobial properties of the electrospun PHBV films containing OEO, RE, and GTE were also evaluated using the JIS Z2801 against *S. aureus* and *E. coli* bacteria, in both an open and closed system, for 1, 8, and 15 days. In relation to the open system, as shown in Table 5, one can observe that the films containing OEO showed the strongest inhibition. These films provided a strong reduction (*R* ≥ 3), that is, with a reduction of 3 log units, against *S. aureus,* and also a high antimicrobial effect, though slightly lower, presenting *R* values of 2.7–2.9 against *E. coli*. In the case of the films containing RE and GTE, the films yielded a bacteriostatic effect (1 ≤ R < 3) against both bacteria. The antimicrobial effect of RE was also approximately 1 log units higher than that observed for GTE. These results agreed with the MIC and MBC described above, where OEO inhibited the growth of *E. coli* and *S. aureus* at lower contents, whilst RE and GTE showed higher MIC and MBC values. It is also worthy to mention that in all cases, the bacterial reduction slightly increased over the days, which can be related to the slow release of the active compounds to the surface of the films. In comparison to the previous results of electrospun antimicrobial films reported by Jeong-Ann Parka and Song-Bae Kim [73] in open systems, it was observed that the inhibition of *S. aureus* increased from a 0.6 log of reduction, at the initial time, to a 1.2 log of reduction, after 120 min. In another study, Figueroa et al. [42] also reported an increase in the bacterial inhibition with the passage of storage days, showing a 3.9 log of reduction after 10 days against *S. aureus*.


**Table 5.** Antibacterial activity against *S. aureus* and *E. coli* of the electrospun poly(3-hydroxybutyrate*co*-3-hydroxyvalerate) (PHBV) films containing oregano essential oil (OEO), rosemary extract (RE), and green tea tree extract (GTE) in the open system for up to 15 days.

As shown in Table 6, for all the samples, the reduction was slightly higher in the closed system than in the open one. While the films containing OEO presented the strongest inhibition (*R* ≥ 3) after 15 days of storage in the closed system against the two bacterial strains, the films that contained RE and GTE showed a significant inhibition (1 ≤ R < 3). This result could be attributed to the accumulation of volatile active compounds in the headspace of the closed chamber. There are a limited number of studies reporting the antimicrobial performance of the active films in closed systems, which indeed are more practical from the point of view of packaging and the design of containers to avoid deterioration of food products during storage. For instance, Torres-Giner et al. [74] developed a multilayer system, based on an electrospun coating of zein composite nanofibers containing thymol on a polylactide (PLA) film, that was evaluated against *Listeria monocytogenes* in a closed atmosphere in desiccators. It was reported that a concentration as low as 1.6 ppm was able to produce a decrease in the CFU of about 3 log units, whereas above 6.1 ppm, no CFU were detected. The high antimicrobial performance achieved was ascribed to the capacity of the electrospun material to release the bioactive in a sustained manner. The results are of potential interest in packaging applications, since the antimicrobial effect was not only successfully achieved in open packaging systems, but it also prolonged and improved over time in closed packaging systems, thereby extending the shelf life of perishable foods [75,76].


**Table 6.** Antibacterial activity against *S. aureus* and *E. coli* of the electrospun poly(3-hydroxybutyrate*co*-3-hydroxyvalerate) (PHBV) films containing oregano essential oil (OEO), rosemary extract (RE), and green tea tree extract (GTE) in the closed system for up to 15 days.
