## 3 Results
### Colon Microbiota-Derived Metabolic Production
#### 3.3.2 Biogenic Amines Profile  
The colon microbiota-derived production of nine biogenic amines was analyzed. The measured concentrations of the two polyamines, putrescine and cadaverine, and the tryptophan metabolite tryptamine, are presented in Figure 4a,b, respectively.  
Figure 3: Production of short-chain fatty acids (SCFA) and branched-chain fatty acids (BCFA) and acidification during the in vitro fermentation. Concentrations are expressed in mM for: (**a**) C2: acetate, (**b**) C3: propionate, (**c**) C4: butyrate, (**d**) C5: valerate, (**e**) iC4: isobutyrate, (**f**) iC5: isovalerate, (**g**) total quantification of SCFA, and (**h**) BCFA, all quantified using SPME-GC-MS. (**i**) Recorded consumptions (mL/day) of base (NaOH) and acid (HCl). Data are presented as mean \(\pm\) SD, and statistical significances (one-way ANOVA comparisons, followed by [PERSON]’s post-test) are shown as \(p<0.05\) (*), \(p<0.001\) (***), and \(p>0.05\) (ns).  
As shown in Figure 4a, cadaverine was abundantly produced (\(\sim\)400-600 uM) by the intestinal microbiota. The structural polyamine analogous putrescine was detected above 200 uM, but only in samples from the first day of fermentation. In addition, between 10-30 uM of tryptamine were produced by the microbiota after 24 h and 72 h (Figure 4b), which was significantly higher than the concentration observed at the time of the inoculation.  
The results of the UPLC-FLD quantification of the remaining six biogenic amines (i.e., methylamine, spermidine, spermine, 2-phenylethylamine, tyramine, histamine) are shown in Table 1.  
After 48 h of fermentation (see Table 1), a concentration of 35.75 uM of methylamine was detected in one of the replicates. By the end of the colonic simulation, greater levels of methylamine were identified in all samples, yet with a very high standard deviation between replicates. The polyamine spermidine was identified at the time of the inoculation  
\begin{table}
\begin{tabular}{c c c c c c c} \hline \hline
**Class** & **Amines** & **Structure** & **0 h** & **24 h** & **48 h** & **72 h** \\ \hline \multirow{4}{*}{Aliphatic amines} & Methylamine & **NH2** & - & - & 35.75 uM \({}^{\dagger}\) & 283.86 uM \(\pm\) 128.76 \\ \cline{2-7}  & Spermidine & **NH2** & 0.75 uM \({}^{\ddagger}\) & - & 0.12 uM \({}^{\dagger}\) & - \\ \cline{2-7}  & Spermine & **NH2** & **NH2** & - & - & - \\ \hline \multirow{4}{*}{Aromatic amines} & 2- & & 5.20 uM \(\pm\) 0.96 & 3.13 uM \(\pm\) 4.38 & 4.58 uM \(\pm\) 1.62 \\ \cline{2-7}  & & **HO** & - & 27.42 uM \(\pm\) 19.87 & 25.42 uM \(\pm\) 20.79 & 28.85 uM \(\pm\) 7.93 \\ \cline{1-1} \cline{2-7}  & Histamine & & 7.51 uM \({}^{\ddagger}\) & - & - & - \\ \hline \hline \end{tabular} \({}^{\dagger}\) Only detected in one replicate, (-) below the limit of quantification (LOQ).  
\end{table}
Table 1: Biogenic amines produced during the in vitro colonic simulation.  
Figure 4: Microbiota-derived production of (**a**) putrescine and cadaverine and (**b**) tryptamine. Data are expressed as mean \(\pm\) SD. Statistically significant differences evaluated using one-way ANOVA comparisons followed by [PERSON]’s post-test are indicated as \(p\) < 0.05 (\({}^{\star}\)), \(p\) < 0.01 (\({}^{\star\star}\)), \(p\) < 0.001 (\({}^{\star\star}\)), and \(p\) > 0.05 (ns).  
(0 h), and after 48 h of the experiment, at concentrations lower than 1 \(\upmu\)M, but in one of the technical replicates only. Its derivative spermine was not detected in the fermentation samples. The monoamine neurotransmitter histamine was exclusively detected in one of the replicates at 0 h.  
After 24 h, and until the end of the colonic fermentation, productions of the aromatic amines 2-phenylethylamine and tyramine were identified. Though concentrations of 2-phenylethylamine ranged between 3-5 \(\upmu\)M, tyramine was produced in greater concentrations. However, important variations in tyramine production were identified between replicates.