## 4 Discussion
This could be useful for the management of pathogenic bacterial overgrowth during dysbiosis, which may occur under different disease conditions. Several studies using cellular or animal models have described the modulatory effects of CVL and CVL-rich essential oils (EOs) on gut microbiota [11]. EOs rich in CVL increased the relative abundance of some beneficial species such as _Bacilli_, _Lactobacillus_, and _Veillonelaceae_ in weaned piglets [39], and improved microbiota composition, as well as piglet health and performance [40]. Additionally, CVL increased the relative abundance of_Lactobacillus_ spp. in the chicken gut [41]. CVL supplementation also improved gut dysbiosis by enhancing the abundance of beneficial bacteria such as _Frimicutes_, and reducing the abundance of harmful bacteria such as _Proteobacteria_[42]. The treatment of germ-free zebrafish colonized by microbiota with CVL-rich EOs exerted an anti-inflammatory effect by reducing serum amyloid and interleukin 18, 8, and upregulated the expression of tight junction proteins such as claudin-1 and occludin-2 [43]. In intestinal cells, i.e., Caco-2 [44], Vero cells [45], and IPEC-J2 [46], CVL treatment resulted in a significant reduction of bacterial pathogenicity and toxicity in vitro. One additional aspect to consider is the possibility that the changes to the gut microbiota induced by _T. spicata_ can have beneficial effects on the gut bile acid pool and their overall effects as anti-inflammatory agents, and can act agonists of ileal membrane-associated and nuclear receptors. The effects of _T. spicata_ on microbiota are supported by metabolomic analyses of VOCs. This analysis has allowed us to highlight the differences in microbiota metabolism, and facilitated the detection and quantification of metabolites upon _T. spicata_ fermentation. The results indicate that _T. spicata_ stimulated specific microbial pathways, resulting in the increased production of hepatonic acid, medium-chain fatty acids (MCFAs), and SCFAs (mainly propanoic acid). Beneficial effects of SCFAs are expected on the gut barrier, as well as at a systemic (metabolic) level. The increase in MCFAs and SCFAs can be beneficial in patients with irritable bowel syndrome, where MCFAs and SCFAs are reduced [47]. Besides this, the partial increase in SCFAs could be attributed to the high content of fiber in _T. spicata_ leaves. The increase in other metabolites such as ethanone, 1-(3-hydroxy-4-methoxyphenyl)-, phenol, 2-methyl-5-(1-methylethyl)-, benzenemethanol and alpha,alpha,4-trimethyl- point to the modulation of microbial activities associated with phenolic metabolism and biotransformation. Notably, a decrease in trimethylamine is observed in the _T. spicata_ metabolome, which could be of great relevance to cardiovascular health, as trimethylamine is a precursor of trimethylamine N-oxide (TMAO), which has been linked to cardiovascular risk [48]. Regarding the effects of _T. spicata_ on the intestinal barrier function, ethanolic extract (TE) and CVL, as well as digested _T. spicata_ with or without fecal fermentation samples, were tested. We found that the apical addition of the TE (100 and 200 \(\mathrm{\SIUnitSymbolMicro mL}\)) and CVL (50 \(\mathrm{\SIUnitSymbolMicro mL}\)) transiently increased the ionic permeability without affecting the integrity of Caco-2 monolayers, as suggested by the biphasic increase in short circuit current (Isc) and absence of changes in transepithelial resistance (Rt). Interestingly, both TE and CVL exhibited a biphasic response with a significant increase in intestinal transepithelial resistance observed in the second phase. The biphasic response observed in Caco-2 monolayers exposed to 25 \(\mathrm{\SIUnitSymbolMicro mL}\) of CVL might be attributed to various molecular and cellular processes. The initial increase in short-circuit current could be linked to the activation of ion channels or transporters on the apical membrane of Caco-2 cells.