Fecal samples (n = 5 per group) were stored frozen at the end of the experiment. A fixed amount was weighed (0.5 g) and suspended in ultra-pure water (2.5 mL) in a 15 mL tube, vortexed briefly, and then a total of 7.5 mL 1.33% HCl/ethanol solution was added. The samples were homogenized for 2 min and centrifuged at 17,968 g for 10 min at room temperature. Supernatant was promptly transferred to a 2 mL sample vial. This step was repeated three times. A quantity of the pooled extract containing acetate, propionate and butyrate were transferred into a 2 mL glass vial and loaded onto an Agilent 7890 gas chromatograph (GC) system with automatic loader/injector (Agilent Technologies, Santa Clara, CA, United States). Pure fatty acid standards (cod. 71251 Acetic Acid, cod. 94425 Propionic Acid, cod. 19215 Butyric Acid Merck Life Science, Milano, Italy) were also prepared to prepare a calibration curve and each sample was analyzed three times on the same day (De Caro et al., 2020 (link)).
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