Quantification of Fecal Short-Chain Fatty Acids

The fecal SCFAs were determined using gas chromatography according to our previous method [12 (link)]. In brief, 100 mg of fecal samples were dissolved in water at a ratio of 1:9 (w/v). After centrifugation (12,000× g, 4 °C, 10 min), the supernatants were filtered using 0.45 μm membranes and mixed with 0.03 mL of 2-ethlbutyric acid (0.04 M) and 0.02 mL of HCL (6 M). Then, the supernatant of the mixtures was analyzed for the levels of SCFAs through gas chromatography (GC-2010 Plus system, Shimadzu, Japan) that had a DB-WAXetr column (30 cm × 0.25 mm × 0.25 μm, Agilent Technologies, Stockport, UK) and a flame ionization detector. Standard calibrations were used to estimate the contents of SCFAs by determining the peak areas.