2.6. Analysis of pH and SCFAs

The liquid was collected after 0, 12 and 24 h of fermentation for pH detection. The fermentation product was centrifuged at 10,000× g for 10 min, and then the supernatant was transferred to a 10 mL screw-cap tube, and the pH value was measured with a pH S-3B instrument (Lai Chi Instrument Co., Ltd., Shanghai, China).

After fermentation, the sample was mixed thoroughly with 800 μL of deionized H₂O and 200 μL of 50% H₂SO₄. The mixture was added to 1 mL of diethyl ether and shaken by vortex for 10 s. The supernatant was collected after centrifugation (12,000× g, 10 min) and dehydrated by anhydrous CaCl₂. The resulting supernatant was analyzed on a gas chromatograph using a hydrogen flame detector (flame ionization detector, FID) equipped with an SH-Stabil wax-DA (30 m × 0.32 mm × 0.50 μm). A flame ionization detector was used with an injector temperature of 260 °C, followed by temperature programming by holding the temperature at 80 °C for 1.5 min, increasing it to 240 °C at a rate of 10 °C/min and holding at 260 °C for 20 min. Nitrogen was used throughout the measurement process as a carrier gas. The concentrations of acetate, propionate, butyrate, isobutyrate, valerate and isovalerate were calculated based on the peak area of standard samples (Sigma Aldrich, St. Louis, MO, USA). The injection volume was 1 μL, and the injection was repeated 3 times.