Db fatwax

The short chain fatty acid (SCFA) profiles were measured on fecal supernatant by GC- MS according to Eberhart et al.^{45 (link)} by use of a gas chromatography system Agilent GC 7890B − 5977B (Agilent Technologies, Palo Alto, CA, USA) fitted with a column DB- FATWAX, (30 m, 0 × 0.25 mm x 0.25 μm operated in split mode (20:1). The conditions were as follows: Oven temperature program: 100°C for 3 min, ramped to 100°C at a rate of 5°C min-1, to 150°C for 1 min, to 200°C at a rate of 20°C min-1, and finally held at 200 for 5 min. Helium was used as a carrier gas at a flow rate of 1 mL min-1; inlet temperature of 250°C.
Differences in the concentration of each SCFA according to the time period were determined by Kruskal–Wallis test. A CCA was also assessed to study the associations between the production of each individual SCFA with the general pattern of the specific decade. Specific Spearman’s rank correlations between microbial genera and SCFA were obtained and built by use of gplots package⁴⁶ after normalization of the microbiota data as relative abundance using the microbiome package.⁴⁷

SCFA analysis was performed using gas chromatography–mass spectrometry (GC-MS), following the method described by Eberhart et al. [59 (link)]. For each fecal sample, an aliquot of 100 mg was placed in a 15 mL vial. Then, 1000 μL of the internal standard solution (3-Methylvaleric acid) was added, followed by 2 mL of diethyl ether. The vial was capped, shaken vigorously by hand and vortexed for 10 s. Subsequently, approximately 5–10 g of sodium sulfate was added to the vial. The vial was tightly recapped and shaken vigorously by hand and then vortexed for another 10 s. The samples were then centrifuged at 4000 rpm for 2 min at 4 °C. Finally, the supernatant was injected. The analysis was performed using an Agilent GC 7890B–5977B GC-MS with a multipurpose sampler (Gerstel MPS, Mülheim, Germany). The GC column used was Agilent DB-FATWAX, 30 m × 0.25 mm × 0.25 μm, operated in split mode (20:1). The oven temperature program was set as follows: 100 °C for 3 min, ramped to 100 °C at a rate of 5 °C min⁻¹, then to 150 °C for 1 min, further ramped to 200 °C at a rate of 20 °C min⁻¹, and finally held at 200 °C for 5 min. Helium was used as the carrier gas at a flow rate of 1 mL min⁻¹, with an inlet temperature of 250 °C. The injection volume was two μL. Standards curves for acetate, butyrate, and propionate were used for quantifying the SCFAs in fecal samples.