7890 gas chromatograph

Fecal samples were lyophilized, derivatized and analyzed by gas chromatography time-of-flight mass spectrometry (GC-TOF-MS) (Agilent 7890 gas chromatograph and LECO Pegasus III time-of-flight mass spectrometer) as previously described [23 (link),24 (link)]. MS-DIAL software [25 (link)] and FiehnBin base database were used for raw peaks exacting, the data baselines filtering, and calibration of the baseline [26 (link)]. Peaks detected in ≤50% of QC samples or <50% samples of every group were removed, except QC group or RSD>30% in QC samples [27 (link)]. SIMCA-P v13.0 (Umetrics, Umea, Sweden) was used for partial least squares-discriminant analysis (PLS-DA) and orthogonal projections to latent structures-discriminant analysis (OPLS-DA). The first principal component of variable in importance projection (VIP) was obtained to refine the analysis. VIP > 1.5 was first selected as “changed metabolites”. Obtained metabolites were validated by searching in the Kyoto Encyclopedia of Genes and Genomes (KEGG) [28 (link)].

To detect phenolics and vanillin production from enzymatic reactions, we used a quantitative GC–MS analysis. The resulting phenolics from enzymatic reactions were extracted by adjusting the pH of the samples to below 2 with 6 mol l⁻¹ HCl and addition of butyl acetate (1:1, v:v) and then derivatized [59 (link)]. The derivatized samples (1 µl) were analyzed on an Combi-PAL autosampler (Agilent Technologies GmbH, Waldbronn, Germany) coupled to an Agilent 7890 gas chromatograph in split less mode coupled to a Leco Pegasus two time-of-flight mass spectrometer (LECO, St. Joseph, MI, USA) as described by Weckwerth et al. [60 (link)]. The chromatograms were exported from the Leco ChromaTOF software (version 3.25) to the R software. Peak detection, retention time alignment, and library matching were performed using the Target Search R-package [61 ]. Metabolites were quantified by the peak intensity of a selective mass. The intensity normalization procedure was performed by dividing the fresh weight, followed by sum of the total ion count and global outlier replacement [62 (link), 63 (link)].