4 methylvaleric acid

Aliquots (0.4 mL) from tubes were collected for SCFA analysis. Immediately after sampling, aliquots were mixed with 100 μL of the internal standard mixture (157.5 μL of 4-methylvaleric acid (nr 277827–5G, Sigma-Aldrich Inc., St. Louis, MO, USA), 1.47 mL of 85% phosphoric acid, 39 mg of copper sulfate pentahydrate) and the resulting blend was brought to 25 mL of final volume with purified water and 400 μL of copper sulphate solution (2.75 mg/mL) to halt fermentation. Samples were stored at −80 °C until analysis.
Defrosted samples were centrifuged at 3000× g for 10 min (Microfuge^® 20R, Beckman Coulter, Brea, CA) and 4 μL was prepared for injection into a gas chromatograph (model 5890 Series II, Hewlett Packard, Palo Alto, CA, USA) equipped with a fused silica capillary column (NukolTM, Supelco nr 40369-03A, Bellefonte, PA, USA) and a flame ionization detector (GC-FID 7890A, Agilent Technologies, Inc., Santa Clara, CA, USA). SCFA was assayed and identified as previously described [36 (link)] using acetate, propionate and butyrate relative to 4-methyl valeric acid as standards (Supelco, Bellefonte, PA, USA).

The quantification of fecal SCFAs was performed using gas chromatography as previously described [33 (link)]. Briefly, 40 mg of fecal samples were homogenized in 300 μL 0.1 N hydrochloric acid followed by the addition of phosphoric acid (50 μL, 0.25%). Samples were centrifuged at 3000× g for 10 min. Internal standard solution (150 mg of 4-methyl-valeric acid, S381810, Sigma-Aldrich, Saint Louis, MO, USA) was added to the supernatant and SCFAs were analyzed via Varian model 3400 Gas Chromatograph (Varian, Walnut Creek, CA, USA) with a Stabilwax-DA column (30-m × 0.25-mm i.d.; Restek, Bellefonte, PA, USA).

Samples collected from the bottles at the end of fermentation were centrifuged, and supernatant from each bottle was analyzed for SCFA. Concentrations of SCFA in post-fermentation solution were determined using a GC system. Briefly, 0.8 mL of the test sample (supernatant from centrifuged at 2500×g for 10 min at 4 °C) were added in a tube with 0.2 mL of 25% phosphoric acid and 0.2 mL of internal standard solution (150 mg of 4-methyl-valeric acid, S381810, Sigma-Aldrich) and vortexed thoroughly. Samples were analyzed for SCFA (i.e., acetate, propionate, butyrate, isobutyrate, valerate, isovalerate, and caproate) using a GC system (TRACE™ 1300 gas chromatograph; Thermo Scientific, Waltham, MA, USA) with a Stabilwax-DA column (30-m × 0.25-mm internal diameter; Restek, Bellefonte, PA, USA). A flame-ionization detector was used with an injector temperature of 170 °C and a detector temperature of 190 °C. Branched-chain fatty acids (BCFA) content was calculated as the sum of iso-butyrate and iso-valerate.