2.7. Analysis of Short-Chain Fatty Acids by Gas Chromatography

The analysis of SCFAs was performed by Gas Chromatography (GC) in the fecal culture supernatants to quantify acetic, propionic, butyric acid (major SCFAs), as well as isobutyric and isovaleric acid (branched chain fatty acids: BCFAs). 250 µL of culture supernatants collected at time 0 and 24 h of incubation were mixed with 0.3 mL methanol, 0.05 mL of the internal standard solution (2-ethylbutyric acid 1.05 mg/mL), and 0.05 mL of 20% (v/v) formic acid. The mixture was centrifuged, and the supernatant was collected for SCFA quantification in a system composed of a 6890N GC injection module (Agilent Technologies Inc., Palo Alto, Ca, USA) connected to a flame injection detector (FID) and a mass spectrometry (MS) 5973N detector (Agilent), as described previously [24,25]. Samples were analyzed in triplicate and results were expressed in µg/mL. Increments (∆) in the levels of these compounds at 24 h of incubation with respect to the basal conditions (time 0) were calculated for each fermentation batch with the different 2′FL preparations tested.