Intestinal Short-Chain Fatty Acid Analysis

SCFAs including acetate, propionate, butyrate, isobutyrate, valerate, and isovalerate were analysed as described previously67 (link). To ensure the homogenicity of the intestine content sample, the freeze-dried samples were prepared using a Vacuum freeze-dryer (Hrist ALPHA 2-4/LSC, Germany) at −80 °C. Briefly, freeze-dried samples (0.5–0.6 g) were weighed into 10 ml centrifuge tubes and mixed with 8 ml ddH₂O, homogenised, and centrifuged in sealed tube at 7,000 g and 4 °C for 10 min. A mixture of the supernatant fluid and 25% metaphosphoric acid solution (0.9 and 0.1 ml, respectively) was centrifuged at 20,000 g and 4 °C for 10 min after standing in a 2 ml sealed tube at 4 °C for over 2 h. The supernatant portion was then filtered through a 0.45-μm polysulfone filter and analysed using Agilent 6890 gas chromatography (Agilent Technologies, Inc, Palo Alto, CA, USA) with a flame ionisation detector and a 1.82 m × 0.2 mm I.D. glass column that was packed with 10% SP-1200/1% H₃PO₄ on the 80/100 Chromosorb W AW (HP, Inc., Boise, ID, USA). The concentration of NH₃-N in the supernatant fluid was measured at 550 nm using a UV-2450 spectrophotometer (Shimadzu, Kyoto, Japan)68 . The bioamines including 1,7-heptyl diamine, cadaverine, phenylethylamine, putrescine, trytamine, tyramine, spermidine, and spermine, as well as the indoles and skatoles, were analysed as described previously69 .