## 4 Discussion
### Chemical Metabolites Profiling the Microbial Fermentation
Polyamines (i.e., putrescine, cadaverine, spermidine, spermine) are ubiquitous and essential for growth and differentiation in the large intestine, and they participate in the immunomodulation of the human gut microbiota [113]. Their upregulation in the colon has been studied in animal models to suppress inflammation and improve longevity [114]. These small polycationic molecules are emerging players in bacteria-host interactions due to their core physiological functions, as well as their role in bacterial pathogenesis [115]. In the present work, putrescine was mainly detected during the first 24 h of colonic fermentation. The concentration values (>200 uM) were consistent with previous fecal culture fermentations [116]. The lack of significant quantities of spermidine and spermine suggested that putrescine was not used to produce these metabolites. Meanwhile, the biogenic amine cadaverine was largely produced during the 3-day fermentation (>300 \(\upmu\)M) (Figure 4a). Some authors observed that the cytotoxicity threshold of cadaverine in intestinal cells is twice that of putrescine [117]. However, there is scarce information reporting the expected values of these amines in the human intestinal context. Methylamines are directly related to gut microbiota metabolism [118]. The high levels of methylamine detected here at the end of the batch experiment (Table 1) could be a consequence of the microbial transformation of essential methyl donor nutrients. A low abundance of methyl-donor nutrients such as choline could also explain the high levels of _Gammaproteobacteria_ detected in the system [61]. The quantified productions of the aromatic 2-phenylethylamine and tyramine are probably associated with the abundant presence of _Enterococcus_ in the fecal fermented systems [119]. These biogenic amines are both considered ligands of the so-called trace amine-associated receptors (TAARs), whose outcome effects are deeply implicated with neurologic, cognitive, and psychiatric manifestations, including sensory perception and taste [120]. The TAARs are considered an emerging pharmacological target for the treatment of human disorders [121]. Tyramine and 2-phenylethylamine are presumed to exhibit direct effects on human gut epithelial cells through TAARs modulation [122]. Meanwhile, bacteria-derived production of the heteroaromatic neurotransmitter histamine was possibly below the quantification limit (<8.4 mM) of the analytical method used. Tryptamine (Figure 4b), derived from tryptophan metabolism, was detected throughout the whole fermentation process. Tryptamine, as well as its downstream metabolite indole acetic acid, are considered among the main tryptophan-derived ligands of AhR [123]. Interestingly, tryptamine is a CYP1A1 substrate, in line with the suggestion that AhR endogenous ligands are also regulated by CYP enzymes [124]. In general, the metabolism profiled through the production of distinct molecules in the static in vitro model allowed us to extract some relevant insights into the fermentative process in this class of experiment. First, the SCFA and BCFA, the aromatic amines tyramine, 2-phenylamine, and tryptamine, as well as the aliphatic cadaverine, are stably produced and detectable during the 72 h of the experiment. Therefore, this is probably a convenient and adequate setup to study these metabolites. Conversely, the production of some other aliphatic amines such as methylamine, spermine, spermidine, and putrescine might require no static but long-term fermentation models.