Fecal SCFA Analysis Protocol

To analyze fecal short-chain fatty acid (SCFA) levels, 400–500 mg feces were first diluted 1:4 in water. Then, 0.1 ml of 50% ortho-phosphoric acid (AppliChem GmbH, Darmstadt, Germany) was added before the samples were homogenized and filtered using polyester syringe filters (REF: 729033; Macherey–Nagel GmbH & Co. KG, Düren, Germany). Afterwards, 1 µl filtrate was analyzed using a capillary gas chromatograph (HP6890 Series; Hewlett Packard Corp., Paolo Alto, California, USA) with a flame ionization detector using the column OPTIMA-FFAP (REF: 726344.10; Macherey–Nagel GmbH & Co. KG, Düren, Germany) with standards for all SCFAs (Merck Schuchardt OHG, Hohenbrunn, Germany). Fecal dry mass was assessed by drying 300–500 mg feces overnight at 103 °C [35 (link)]. The SCFA data are expressed in relation to dry mass to overcome bias due to differing fecal water contents.