Quantification of Fecal Short-Chain Fatty Acids

The SCFAs acetate, propionate, isobutyrate, butyrate, isovalerate, and valerate were determined in faeces through gas chromatography (GC), using a system composed of a 6890NGC injection module (Agilent Technologies Inc., Palo Alto, CA, USA) connected to a flame injection detector (FID) and a mass spectrometry (MS) 5973N detector (Agilent), as previously described. Briefly, cell-free supernatants (100 μL) from faecal homogenates were mixed with 450 μL methanol, 50 μL internal standard solution (2-ethylbutyric 1.05 mg/mL), and 50 μL 20% v/v formic acid. Following centrifugation, the supernatants of this mixture were used for SCFA quantification by GC as previously described [18 (link)].