GC-MS analysis of bacterial SCFAs

After measuring the optical density (OD_600nm) of the overnight bacterial culture, supernatants were collected after centrifugation (15 min at 6000 rpm) and kept at −20 °C. SCFA were measured as previously described [35 (link),38 (link)] with slight modifications. Briefly, after the addition of the SCFA standards, each sample was acidified and then extracted using diethyl ether (Biosolve, Dieuze, France), with gentle stirring for 1 h and centrifugation for 2 min at 5000 rpm at 4 °C. The organic layers were derivatized using tert-butyldimethylsilyl imidazole (Sigma-Aldrich), and samples were incubated for 30 min at 60 °C before analysis by gas chromatography-mass spectrometry (GC-MS 7890A-5975C; Agilent Technologies, Montpellier, France) using a 30 m × 0.25 mm × 0.25 µm capillary column (HP1-MS; Agilent Technologies), as previously described [35 (link)]. The SCFA concentrations were reported at the mean concentrations divided by the optical density of the culture and expressed as mean ± SEM.