G2010plus

Fecal SCFAs were analyzed by using gas chromatography (Shimadzu G2010Plus, Kyoto, Japan) equipped with a DB-FFAP chromatographic capillary column (30 m × 0.530 mm × 1.00 μm; Agilent, Santa Clara, CA, USA). The pre-treatment of samples was based on a method described before with some modifications [64 (link)]. In brief, fresh feces (100–150 mg) were dissolved in 0.8 mL 0.2 M HCl (0.02 mg/mL 2-ethylbutyric acid as internal standard) and 0.2 mL 0.15 M oxalate. The mixture was vortexed for 1 min and centrifuged at 12,000× g and 4 °C for 15 min. The supernatant was filtrated using a 0.22 μm filter for further analysis. N2 was supplied as carrier gas at a flow rate of 10 mL/min. The flow rates of air, H2, and N2 as make up gas were 260, 30, and 30 mL/min, respectively. The oven temperature was increased from 100 °C to 160 °C at 5 °C/min and then held at this temperature for 4 min. The SCFAs contents, including acetate, propionate, isobutyrate, butyrate, valerate, and caproate were quantified using standard curves.

SCFA contents, including acetic, propionic, butyric, valeric, isobutyric, and isovaleric acids in the fermentation fluids, were analyzed by using gas chromatography (GC; Shimadzu G2010Plus, Kyoto, Japan) on a DB-FFAP chromatographic capillary column (30 m × 0.530 mm × 1.00 μm; Agilent, Santa Clara, CA, USA), with calculated standard curves based on the method described in Tian et al. [26 (link)], with minor modifications. A total of 200 μL of the fermentation samples was mixed with 200 μL of 0.3 mg/mL 2-ethylbutyric acid and 50 μL of 0.15 M oxalic acid. The mixture was filtered at 0.22 μm into a vial for analysis.