## 2 Methods
### Fecal Microbiota Characterization
#### 2.4.4 Fecal Batch Volatile Organic Compound (VOC) Analysis  
For the analysis of volatile organic compounds (VOCs), 2 mL each of the fecal medium and _T. spicata_ digest, fecal slurry and fecal slurry, and _T. spicata_ digest samples was taken and placed inside 20 mL vials that were closed with a magnetic cap with polytetrafluoroethylene septa (PTFE)/silicone. As internal standard, 10 \(\upmu\)L of 4-methyl-2-pentanol was added. A Clarus 680 gas chromatograph (Perkin Elmer, Beaconsfield, UK) was used for GC-MS analysis. For the extraction of volatile compounds, a CombiPAL injector sample system (CTC Analytucs, Zwingen, Switzerland) and a 50/30 \(\upmu\)m DVB/CAR/PDMS (Supelco, Bellefonte, PA, USA) were used. Helium was used as a carrier gas with a flow rate of 1 mL/min. Firstly, the samples were equilibrated for 15 min at 45 \({}^{\circ}\)C. To extract the volatile compounds, the fiber was exposed to 45 \({}^{\circ}\)C for 45 min in the headspace of the vials. The injection took place by heating the fiber inside the heated injection port to 220 \({}^{\circ}\)C in splitless mode. An Rtx-WAX column 30 m \(\times\) 0.25 mm \(\times\) 0.25 \(\upmu\)m (Restek, Bellafonte, PA, USA) and coupled Clarus SQ8 MS (Perkin Elmer) were used for VOCs separation with the source set at a temperature of 250 \({}^{\circ}\)C and the mass transfer line MS at a temperature of 230 \({}^{\circ}\)C. The oven temperature was initially 35 \({}^{\circ}\)C for 8 min and then increased to 60 \({}^{\circ}\)C with an increase of 4 \({}^{\circ}\)C/min, 160 \({}^{\circ}\)C with an increase of 6 \({}^{\circ}\)C/min and finally a temperature of 200 \({}^{\circ}\)C with an increase of 20 \({}^{\circ}\)C/min, at which it was maintained for 15 min. Electron ionization masses were recorded at 70 eV in the mass-to-charge ratio range from m/Z 34 to 350. For peak identification, chromatograms were analyzed using the NIST/EPA/NIH Mass Spectral Library with Search Program--Data ver. 2.3. Gaithersburg, MD, USA: National Institute of Standards and Technology (NIST); 2017) library. A peak area threshold of >1,000,000 and a probability of match of 85% or greater was used to identify the compounds. The relative concentration (expressed in \(\upmu\)g/g of 4-methyl-2-pentanol) was estimated by the interpolation of the relative area with the air of the internal standard. The absolute  
\begin{table}
\begin{tabular}{c c c c c c c} \hline \hline
**Target** & **Primers** & **Primer Sequence (5\({}^{\circ}\)–3)** & **Gene** & **Product** & **T Annealing** & **Reference** \\ \hline All Bacteria & Uni331F & TCCTACCGGAGCAGCAT & & & & \\ \cline{2-6}  & Uni797R & GGACTACCCGATTCATCTG & & & & \\ \hline \multirow{2}{*}{_Lactobacillus_ genus} & Lp-F & AAAATCATGCGTGGCGTAC & & & & \\ \cline{2-6}  & Lp-R & ATGTTGCGTGCTTCGCT & & & & \\ \cline{2-6}  & Bifid-F & CTCCTGAACGGGGTG & & & & \\ \cline{2-6}  & Bifid-R & GGTGTCTTCCCGATATCTA & & & & \\ \hline _Escherichia coli_ subgroup (_E. coli_, _Haria alvi,_ _Shigella_ spp.) & Eco-F & GTTAAATACTTGCATTG & & & & \\ \cline{2-6}  & Eco-R & ACCAGGGTATCAATCCTGT & & & & \\ \hline \multirow{2}{*}{_Clostidium coccoides_ group} & Eco-F & AAAATGACGGTACCTGACTA & & & & \\ \cline{2-6}  & CoCo-R & CCTTTGAGTTTCATTTCTGGGA & & & & \\ \hline \hline \end{tabular}
\end{table}
Table 1: List of primers and related features.  
concentrations of short chain fatty acids (SCFAs) and CVL (main phenolic and volatile compounds in _T. spicata_) in the samples were detected using standard curves.