Cecal Metabolite Extraction and NMR Analysis

Cecal digesta was weighted to ≈150 mg. Metabolomics buffer (0.125 M KH₂PO₄, 0.00375 M NaN₃, and 0.375 M KF; pH 7.4) was added to the cecal digesta samples at a ratio of 2:1 (v/w) and homogenized using a Bullet Blender tissue homogenizer (Next Advance; Troy, NY, USA) with 150 mg of 2-mm-diameter zirconium oxide beads (Next Advance) at setting eight for 5 min. Samples were centrifuged for 5 min at 14,000× g, and supernatants were filtered through a 3000 MWCO Amicon Ultra-0.5 filter (Millipore Sigma, Oakville, ON, Canada) by centrifugation at 14,000× g for 30 min at 4 °C. An aliquot of 360 µL was mixed with 200 µL of metabolomics buffer and 140 µL of deuterium oxide containing 0.05% (v/v) trimethylsilylpropanoic acid (TSP) for a final volume of 700 µL; TSP was used as a chemical shift reference for ¹H-NMR spectroscopy. The solutions were vortexed and centrifuged at 12,000× g for 5 min at 4 °C, and an aliquot of 550 µL of the supernatant of each sample was loaded in a 5 mm NMR tube. Spectra were obtained as previously described [26 (link)].