4.3. Short Chain Fatty Acids Analysis

An aliquot of 100 mg of frozen fecal sample was diluted in 1 mL of 5% phosphoric acid, followed by homogenization and freezing of the fecal homogenates for dry matter precipitation. Stool samples were then thawed and centrifuged for 5 min at 112× g (Jouan Centrifuge A14, Saint Herblain, France). The SCFA acetic, propionic, butyric, isobutyric, valeric, and isovaleric acids were quantified in the supernatants by gas chromatography and flame ionization detection (GC-FID, Agilent 6890A, Agilent Technologies, Waldbronn, Germany). The capillary chromatographic column used was a DB-WAXtr column (100% polyethylene glycol, 60 m, 0.325 × 0.25) and helium was used as the carrier gas at 1.5 mL/min. Injection was made in splitless mode, with an injection volume of 1 μL and a temperature of 260 °C. Methyl valeric was used as an internal standard, and the standard curve was prepared in a similar way to the samples. The detector temperature was 260 °C. The column was heated at 50 °C for 2 min, followed by an increase of 15 °C every min to 150 °C, 5 °C every min to 200 °C, and finally 15 °C every min to 240 °C. The different SCFAs were identified by the retention time of the standard compounds.