2.8. Short-Chain Fatty Acids (SCFAs) Composition Analysis

The composition and concentration of SCFAs were quantified from frozen fecal samples using gas chromatography. A total of 50 mg of feces was weighed in a centrifuge tube to which 250 µL of ultrapure water was added (n = 8 per group; feces from mice were randomly selected). Then the suspension was vortexed for 5 min, and 10 µL of 5 mol L⁻¹ HCl was added into the centrifuge tube and vortexed again for 1 min. The suspension was then incubated for 10 min at room temperature, with intermittent shaking. Prior to chromatographic analysis, the fecal suspension was centrifuged at 12,000 rpm for 20 min. Then, 200 µL of supernatant was transferred to a new centrifuge tube and supplemented with 2-ethylbutyric acid to 1 mmol L⁻¹. Afterward, 1 µL of injection solution was analyzed using a capillary gas chromatograph (GC-2014, SHIMADZU, Kyoto, Japan) with the column DB-FFAP (J&W Scientific, Agilent Technologies Inc., Santa Clara, CA, USA). The specific operation parameters were as follows: flame ionization detector, 240 °C; injection port, 200 °C; temperature raising program: 100 °C for 30 s, 8 °C min⁻¹ until 180 °C (1 min), 20 °C min⁻¹ until 200 °C (15 min); nitrogen, hydrogen, and air flow rate: 20, 30 and 300 mL min⁻¹, respectively.