2.2. Fecal Metabolite Extraction

This protocol describes the simultaneous extraction of polar and non-polar metabolites from fecal samples. Respecting a decreasing gradient of solvent polarity, we performed a successive five-solvent-based (Ethanol-Ethyl, Acetate-Diethyl, Ether-Chloroform-Hexane) metabolite extraction protocol. All fecal samples were aseptically taken. Before analysis, fecal water was extracted via mixing 3 g of fresh stool with Ethanol (stored at −20 °C) in the ratio 3:20 (g:mL; feces:Ethanol). The mixtures were homogenized for 3 min and centrifuged at 4000 rpm for 20 min at 4 °C. The supernatants were transferred and filtered through a 0.45 µm Millex-GV Syringe Filter. An amount of 20 mL of Ethyl Acetate was added to the pellets. The mixtures were well shaken, vortexed for 3 min and centrifuged at 4000 rpm for 20 min at 4 °C. The same operation was repeated successively when adding Diethyl ether, Chloroform, and Hexane. A triplicate extraction was performed for every sample. All filtered fecal waters were dried to complete dryness of solvents, under reduced pressure in a speed vacuum at 10 °C, to get a pellet of concentrated metabolites. All the extracted metabolites were stored at −20 °C until analysis.