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)].
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