Nukon

Fecal samples of ten mice per group were collected after ten weeks of treatment and homogenized in carbonate–phosphate buffer (one part feces with three parts buffer). Samples were centrifuged at 13,000 rpm for 10 min. The supernatant (400 µL) was combined with 100 µL of internal standard (5% phosphoric acid—containing 50 mM of 4-methylvaleric acid and 8% of copper sulfate) and SCFA analysis was performed by gas chromatography equipped with a fused silica capillary column (NukonTM, Supelco No: 40369-03A, Bellefonte, PA, USA) and a flame ionization detector (GC-FID 7890A, Agilent Technologies, Inc., Santa Clara, CA, USA) under the conditions described previously [44 (link)]. SCFA quantification was assessed through measurements of the peak areas for acetate, propionate, and butyrate relative to 4-methyl valeric acid.

At 6, 12, 24, 36, and 48 h time points after inoculation, we measured total gas production from fermentation as overpressure using a graduated syringe and passing a needle through the rubber stopper prior to unsealing the tubes. For pH measurements, the supernatant was transferred to a separate 15 ml Falcon tube and measured using a pH meter. We collected two aliquots from each tube, one for SCFA (0.4 ml) and other for DNA extraction (1 ml) and stored the aliquots at −80°C. An internal standard (157.5 μl of 4-methyl valeric acid, 1.47 ml of 85% phosphoric acid, 39 mg of copper sulfate pentahydrate in a total volume of 25 ml) was immediately added to the SCFA aliquots samples before vortexing.
We measured SCFAs as previously described (Tuncil et al., 2018b (link)). Briefly, 4 μl of the supernatants were analyzed on a fused-silica capillary column (Nukon^TM, SUPELCO No: 40369-03A, Bellefonte, PA, United States) using a gas chromatograph (GC-FID 7890A, Agilent Technologies, Inc.) (Tuncil et al., 2017 (link)). We used 4- methylvaleric acid (Fisher Scientific) as an internal standard and acetate, propionate, and butyrate (Fisher Scientific, Hampton, NH, United States) to generate standard curves.

The samples from the − 80 °C freezer were defrosted at room temperature and prepared for SCFA analysis as previously described²⁹ . The internal standard mixture for SCFA analysis was prepared using 4-methylvaleric acid, 85% phosphoric acid, and copper sulfate pentahydrate. Acetate, propionate, and butyrate were used as external standards. Samples were analyzed using a gas chromatograph (GC-FID 7890A, Agilent Technologies, Inc.) equipped with a fused silica capillary column (NukonTM, Supelco No: 40369-03A, Bellefonte, PA, USA) under the following conditions: injector temperature, 230 °C; detector temperature 230 °C; initial oven temperature, 100 °C; temperature program, 8 °C /min to 200 °C with a hold for 3 min at final temperature; carrier gas, helium at 0.75 ml/min. Quantification of short chain fatty acids was calculated from the peak areas of the acids relative to the internal standard. Total SCFA was measured as the sum of acetate, propionate, and butyrate, and relative proportions of each were determine.