Version 8

Sample preparation for pH measurement and NMR spectroscopy was performed according to previously published methods.^[49] The detailed methods for sample preparation (intestinal contents, feces, and serum) were provided in supplementary material (Method S3, Supporting Information). ¹H NMR spectra were acquired using a Bruker Avance IVDr NMR spectrometer equipped with a 5 mm ¹H‐optimized double resonance broad‐band probe operating at a frequency of 600.13 MHz (Bruker BioSpin, Rheinstetten, Germany). A one‐dimensional NOESY pulse sequence was adopted to acquire the ¹H NMR spectra with specific parameters including measurement temperature: 300 K, relaxation delay: 4 s, spectral width: 7212 Hz, data points: 32k, and scans: 32. A line‐broadening factor of 0.3 Hz was employed to process free induction decays before Fourier transformation. All spectra were baseline and phase corrected before further analysis. Identification and quantification of metabolites from the obtained NMR spectra were implemented in Chenomx (Version 8.6, Chenomx Inc., Alberta, Canada).

Baseline and phase corrections were conducted for raw spectra of intestinal contents and feces in Topspin 3.6.2 before processing in Matlab 2018b. Spectral processing followed our previous method.^[22] Multivariate data analysis (MVDA) was performed on the processed NMR spectra using SIMCA 16 (Sartorius, Umeå, Sweden). MVDA included unsupervised principal component analysis (PCA) and supervised orthogonal projections to latent structures discriminant analysis (OPLS‐DA). For OPLS‐DA models, a cross‐validation procedure using venetian blinds with seven segments was conducted. The S‐line plot of the OPLS‐DA models was used to visualize the differences in spectral signal intensity between two groups. The S‐line plot also reveals the correlations between absolute values of variables and predictive scores (p(corr)) by the color. p(corr)>0.6 indicates that a variable is important to the group discrimination.^[43] For the quantification of metabolites, Chenomx (Version 8.6, Chenomx Inc., Edmonton, Alberta, Canada) was used.