## Results
### Effects of T. spicata on Fecal Volatile Organic Compounds (VOCs)  
With the aim of ascertaining the impact of _T. spicata_ on the metabolic activities of the main cultivable bacterial group from the gut microbiota, we performed HS-SPME GC-MS analyses to evaluate the VOCs. For this purpose, the volatile profiles were evaluated in the fecal medium and _T. spicata_ digest (FMTD), fecal slurry (FS) and fecal slurry and _T. spicata_ digest (FSTD) groups (Supplementary Table S1). In total, 94 VOCs were identified and divided into the following chemical classes: alcohols (n = 8), carboxylic acids (n = 9), hydrocarbons (n = 23), indoles (n = 3), phenols (n = 5) and terpen (n = 18), plus another 28 compounds not belonging to any of the classes previously mentioned. In addition, the results obtained were analyzed by multivariate analysis of the principal component (PCA). The unsupervised PCA, which explained 68.3% of the variance, showed that there was a difference distribution among samples containing fecal slurry and samples with only fecal medium (Figure 3).  
Figure 2: Log (Copy Number) based on DNA concentration and amplicon length after a qPCR of extracted DNA derived from the batch fermentation of FS and FSTD. Tested bacteria were: all bacteria, _Bifidobacterium genus_, _Escherichia coli_ subgroups, _Clostidium coccodies_, and _Lactobacillus genus_. _p_-value obtained by Student’s _t_-test.  
Figure 3: Principal component analysis (PCA) of volatile organic compounds (VOCs) in fecal medium and _T. spicata_ digest (FMTD), fecal slurry (FS) and fecal slurry and _T. spicata_ digest (FSTD) samples. The distributions of sample replicates (a,b,c) were plotted according to the first two components Dim 1 (50.2%) and Dim 2 (18.1%) (**A**), and the contributions of each variable are shown in the loading plot (**B**).  
The analysis has shown that the FMTD samples were placed in the first and fourth quadrants, while samples of fecal slurry with and without the addition of _T. spicata_ digest were placed in the second and third quadrants. To evaluate the statistical differences of the VOCs between those of FS and FSTD, a paired comparison was undertaken through the Wilcoxon rank-sum test and a fold change analysis (log2 FC > 2) (Figure 4).  
As shown in the Volcano plot, the addition of _T. spicata_ led to increases in tetradecanal; dodecanal; heptanoic acid; ethanone, 1-(3-hydroxy-4-methoxyphenyl)-; phenol, 2-methyl-5-(1-methylethyl)- and benzenemethanol, alpha,alpha,4-trimethyl-, and to decreases in phenol; nonanal; decane,2,4-dimethyl-; benzene, propyl-; butanal, 3-methyl-; trimethylamine and benzaldehyde.  
The concentrations of SCFAs and CVL (the main volatile phenolic compound in _T. spicata_) were also evaluated. As depicted in Table 4, higher concentrations of propanoic acid, butanoic acid, and isovaleric acid were observed in FSTD compared to FS, while in the FMTD, as expected, SCFAs were not detected.  
Figure 4: Volcano plot of statistically significant VOCs in fecal slurry. Increases (red) and decreases (blue) in VOC concentration in FSTD compared to FS. Non-statistically significant differences of VOCs are reported as grey dots. Wilcoxon rank-sum tests (\(p<0.05\)) combined with a fold change analysis (log2 FC > 2) were used.