SCFA Quantification in Fecal Samples

SCFA concentration in the feces was determined using gas chromatography (22 (link)). Stool (30 mg) was homogenized in 900 µL of 0.1 N hydrochloric acid. Phosphoric acid (200 µL at 25%) was then added, and the sample centrifuged at 14000 × g for ten min. The supernatant was added to an internal standard solution (500 μM of 4‐methyl‐valeric acid, Sigma‐Aldrich) and 5% phosphoric acid in a glass chromatography tube, well mixed, and kept at room temperature for 30 min. The supernatant was analyzed for SCFA using agas chromatograph (Agilent 7890A/5975C, USA) and Agilent DB‐WAX column (30m × 0.25mm × 0.25um). A flame‐ionization detector with an injector temperature of 150℃ and a detector temperature of 250℃ was employed. The concentration of acetic, propionic, butyric, iso‐butyric, valeric, isovaleric, and hexanoic acids was determined using standard curves.