*3.5. Antimicrobial activity*

*S. aureus* and *E. coli* are common microorganisms associated with food-related diseases. Therefore, the incorporation of active substances in the design of packaging materials can be an important technology not only to avoid food waste but also to enhance food safety [80]. For the pure eugenol in its original liquid form, the MIC and BIC values for *S. aureus* were 1.25 μL/mL and 2.5 μL/mL, respectively, and for *E. coli* these values were 2.5 μL/mL and 5 μL/mL, respectively. The MCM-41 particles with eugenol presented a MIC value against *S. aureus* and *E. coli* of 10 μg/mL and 20 μg/mL, respectively, while the BIC values were 40 μg/mL for both bacteria. The higher value observed for *E. coli* can be ascribed to the greater bacterial resistance of G- bacteria than G+ ones [81], thus a higher dose of the antimicrobial was needed to obtain the same efficacy.

The antimicrobial activity of the film samples was evaluated using the JIS Z2801. The reduction values in the open system against *S. aureus* and *E. coli* are gathered in Tables 6 and 7, respectively. Tables 8 and 9 includes the values against *S. aureus* and *E. coli*, respectively, in the closed system. As expected, it can be observed that both the unfilled PHBV film and the different PHBV films containing MCM-41 without eugenol showed no inhibition effect on the bacterial growth (R ≤ 1). In contrast, the incorporation of MCM-41 with eugenol into the PHVB film exhibited significant antibacterial activity against both bacteria. In the open system, at the initial day, that is, for the tests carried out the same day of the film production, the bacterial reduction on the film surface gradually increased with the content of MCM-41 with eugenol. As it can be seen in Table 6 for *S. aureus*, at the lowest contents, that is, 2.5 and 5 wt.-% MCM-41 with eugenol, the films presented a slight antibacterial activity (R ≥ 1 and < 2). For the highest tested contents, that is, 7.5 and 10 wt.-% MCM-41 with eugenol, the films generated a significant surface reduction (R ≥ 1 and < 3). Although none of the films produced a strong reduction (R ≥ 3), materials with values of surface reduction in the 1–2 range are usually considered as bacteriostatic [82]. Therefore, electrospun PHBV films with 10 wt.-% MCM-41 with eugenol were able to provide a bacteriostatic effect against *S. aureus*. As also shown in the table, after 15 days, the films still kept a significant antibacterial activity. In particular, the films with 7.5 and 10 wt.-% MCM-41 with eugenol still presented significant values of reduction (R ≥ 1 and < 3) while these presented slight values (R ≥ 0.5 and < 1) for loadings of 2.5 and 5 wt.-%. This suggests that, although part of eugenol was released from the films, MCM-41 was still able to retain over time a significant amount of EO. Regarding *E. coli*, shown in Table 7, the required concentration of MCM-41 with eugenol to generate an antimicrobial effect in the open system was 15 wt.-%. At this content, the films presented a significant value of reduction, that is, R values of 1.30 and 1.40 at days 0 and 15, respectively. This supports the above-described higher antimicrobial resistance of *E. coli*, as a G- bacterium, which would need more exposure time to the active oil to render a similar antimicrobial activity.

The tested closed system was aimed to better represent the real conditions in a packaging material. In the case of *S. aureus*, which is shown in Table 8, the film with 10 wt.-% MCM-41 with eugenol was selected since this sample showed a high R value at a relatively low content of filler. One can observe that the antimicrobial activity was higher than that observed in the open system, showing R values of 1.35 and 1.64 for day 0 and 15, respectively. This confirms the high volatility of eugenol, which remained enclosed and still active in the system in comparison to the open one. In Table 9, for *E. coli*, the R values were 1.34 and 1.58 for day 0 and 15, respectively, in the closed system. Therefore, the here-achieved antimicrobial effect was somehow higher in the closed system than in the open one. This result has been recently ascribed to the volatile portion of active components accumulated in the system's headspace, which successfully contributed to decrease bacterial growth [83]. In any case, the differences in bacterial reduction in both tested packaging conditions, that is, the open and closed systems, for each type of bacteria was relatively low. This observation can be related to the use of MCM-41 that successfully performed as vehicles to control the release of eugenol and to render high antimicrobial activity.

Similar to this study, other authors have previously reported the antibacterial activity of eugenol in different biopolymer articles. For instance, PCL/gelatin electrospun membranes loaded with active peptide containing 30 wt.-% of eugenol successfully inhibited the growth of *E. coli* and *S. aureus* with inhibition rates of 71.6% and 78.6%, respectively [84]. In another study, compression-molded PHBV bilayer films were sprayed with four active components, among them eugenol, resulting in antimicrobial systems against G- and G+ bacteria such as *E. coli* and *Listeria innocua* (*L. innocua*) [65]. In this previous research, the added active agents were more effective against G- than G+, which in agreemen<sup>t</sup> with the present results. The benefit of loading antimicrobial agents in MCM-41 has been also studied elsewhere, both against bacteria and fungi. For instance, Park et al. [85] loaded allyl isothiocyanate, a natural antimicrobial, in MCM-41 as a novel controlled release vector against selected foodborne pathogenic microorganisms. In other studies, other volatile EOs were immobilized on the surface of mesoporous silica materials acting as antifungal agents and showing improved antimicrobial activity than the free compounds [39,40].


**Table 6.** Antibacterial activity against *Staphylococcus aureus* (*S. aureus*) in the open system for the electrospun films of poly(3-hydroxybutyrate-*co*-3-hydroxyvalerate) (PHBV) and PHBV/Mobil Composition of Matter (MCM)-41 with eugenol.


**Table 7.** Antibacterial activity against *Escherichia coli* (*E. coli*) in the open system for the electrospun films of poly(3-hydroxybutyrate-*co*-3-hydroxyvalerate) (PHBV) and PHBV/Mobil Composition of Matter (MCM)-41 with eugenol.

**Table 8.** Antibacterial activity against *Staphylococcus aureus* (*S. aureus*) in the closed system for the electrospun films of poly(3-hydroxybutyrate-*co*-3-hydroxyvalerate) (PHBV) and PHBV/Mobil Composition of Matter (MCM)-41 with eugenol.


**Table 9.** Antibacterial activity against *Escherichia coli* (*E. coli*) in the closed system for the electrospun films of poly(3-hydroxybutyrate-*co*-3-hydroxyvalerate) (PHBV) and PHBV/Mobil Composition of Matter (MCM)-41 with eugenol.

