2.6. Fecal Short Chain Fatty Acids (SCFAs) Measurement

The quantification of fecal SCFAs was performed using gas chromatography as previously described [33]. Briefly, 40 mg of fecal samples were homogenized in 300 μL 0.1 N hydrochloric acid followed by the addition of phosphoric acid (50 μL, 0.25%). Samples were centrifuged at 3000× g for 10 min. Internal standard solution (150 mg of 4-methyl-valeric acid, S381810, Sigma-Aldrich, Saint Louis, MO, USA) was added to the supernatant and SCFAs were analyzed via Varian model 3400 Gas Chromatograph (Varian, Walnut Creek, CA, USA) with a Stabilwax-DA column (30-m × 0.25-mm i.d.; Restek, Bellefonte, PA, USA).