Sample Preparation for Short-Chain Fatty Acid Analysis

Each collected sample was thawed and weighted (weight range 500–800 mg), then added sodium bicarbonate 10 mM (1:1 w/v) in a 1.5 mL centrifuge tube. The obtained suspension was then mixed with the aid of a sterile wooden stick and briefly shaken in a vortex apparatus, extracted in ultrasonic bath (15 min) and then centrifuged at 4 °C at 13.000 rpm for 90 min. The supernatant was collected, transferred in 1.5 mL centrifuge tube and stored at − 20 °C until use. For the analysis, these supernatant samples were thawed, briefly centrifuged at 5000 rpm and resuspended for 5 min in an ultrasonic bath. The SCFAs and MCFAs were then extracted as follows: an aliquot of 100 μL of sample solution (corresponding to 0.1 mg of stool sample) was added with 10 μL of internal standard (ISTD) mixture, 1 mL of tert-butyl-methyl ether and 50 μL of 1.0 M HCl solution in 1.5 mL centrifuge tube. Afterwards, each tube was shaken in a vortex apparatus for 2 min, centrifuged at 10,000 rpm for 5 min; the solvent layer was finally transferred in auto-sampler vial and analysed by Gas-Chromatography–Mass Spectrometry (GC–MS) method, using an Agilent GC–MS system composed with 5971 single quadrupole mass spectrometer, 5890 gas-chromatograph and 7673 autosampler^{72 (link)}.