## 3 Discussion
However, very little information is available in the literature on the enantiomeric distribution of chiral compounds in oregano EOs, and the only paper reporting experimental data showed a quite different picture as compared to our results, both in terms of chemical profiles and enantiomeric distributions [25]. In that study, EOs from native populations of _Origanum vulgare_ collected in two different geographical locations in northwestern Himalaya and belonging to the linalool and cymyl chemotypes were investigated [25]. In that study, pairs of enantiomers were determined for four of the seven chiral compounds investigated in the present study, i.e., \(\alpha\)-pinene, linalool, terpinene-4-ol and \(\alpha\)-terpineol. In all cases, the enantiomeric distributions observed were markedly different between the two populations from Himalaya. Thus, the determination of enantiomeric distribution was confirmed to represent a valuable analytical tool for a detailed chemical characterization of oregano EOs, which may be useful for the recognition of geographical origin. Moreover, in that study, a marked effect of the distillation process on the observed enantiomeric distribution in the obtained EOs was postulated to explain some inversions of the ratios for some compounds between the two EOs investigated [25]. Data from the present study seemed to rule out the possibility of a marked effect of the distillation step on the enantiomeric distributions, while the minor differences observed between the EOs and dried aerial parts could be plausibly attributed mainly to differences in the analytical process, and, in particular, to the different concentration levels of individual VOCs in the isolates analyzed in the two cases. Moreover, an expected effect of the distillation process could be a higher extent of racemization, whereas on the contrary in this study, a slightly lower extent of racemization was observed in the EOs instead of the dried plant material. On the other hand, the analysis performed on several biological activities of the EOs from the two genotypes provided a characterization useful for highlighting their possible distinguishing properties among different oregano genetic resources and for evaluating their potential for food and pharmaceutical applications. All the EOs showed a marked dose-dependent antimicrobial activity against some pathogenic and spoilage bacteria, belonging to both Gram-negative and Gram-positive groups. In the direct contact in vitro test performed with CAR1 and THY5, a greater effect was observed against \(L\). _monocytogenes_ OH, a Gram-positive species, as compared to the Gram-negative \(S\). Typhimurium LT2. These data are coherent with the antibacterial mechanism elucidated for thymol and carvacrol: these oxygenated monoterpenes, bearing a phenolic functional group, are able to interfere with the phospholipid bilayers, damaging the bacterial cell membrane and causing a decrease in ATP levels until the cell dies [3; 31; 32], while the hydrophilic nature of the outer membrane of Gram-negative bacteria exerts a shielding action towards EOs [33; 34]. The antimicrobial activity of CAR1 and THY5 was also observed during a challenge test, in which a food matrix of minced beef was used to study the behaviour of a pathogenic or spoilage microorganism under specific storage conditions [35]. Although both CAR1 and THY5 showed antimicrobial activity, THY5's effect appeared to be slower and more persistent than CAR1's in the food contaminated by \(L\). _monocytogenes_ OH. The different dynamics of the two pathogens observed in this assay in the presence and absence of oil treatment could be partly ascribable to an inherent variability and complexity in the food matrix. The increase in microbial load observed after seven days of storage both in the presence and absence of the EOs could be due to multiple concomitant causes: e.g., recontamination of the ground meat or a decrease in the EO activity due to too long of a storage time.