2.3.4. Short-Chain Fatty Acid Measurement
Based on primary aims in the previous study which focused on children’s growth compared between baseline, 14 weeks, and 35 weeks of the intervention [Each stool sample, weighing one gram, was thoroughly mixed with 3 mL of internal standard solution (0.85 μM heptanoic acid) after thorough vortexing and subsequent centrifugation at 2000× The reaction mixture was analyzed using a GC- Flame Ionization Detector (GC-FID) on a model 7890A instrument (Agilent Technologies in Santa Clara, CA, USA) equipped with a capillary column (DB-FFAP, 30 m × 0.53 mm × 0.5 um). The initial oven temperature was set to 90 °C and held for 1 min, followed by an increase at a rate of 20 °C per minute until reaching 190 °C, where it was held for 25 min. The injector and detector temperatures were 210 °C, and the split vent purge was maintained at 20 mL per minute after 0.5 min. The gas flow and septum purge rates were 7.7 and 3.0 mL per minute, respectively. The H2 flow was 40 mL per minute, air flow was 400 mL per minute, and makeup flow was 45 mL per minute. The peak areas of the respective analyte production ions were measured against the peak areas of corresponding internal standard production ions for quantification. A standard SCFAs-phenol-p-cresol mixture containing acetic acid (acetate), propionic acid (propionate), butyric acid (butyrate), isobutyric acid (isobutyrate), valeric acid (valerate), isovaleric acid (isovalerate), caproic acid, hexanoic acids, phenol, and p-cresol (Sigma-Aldrich, Darmstadt, Germany) was utilized to determine the fatty acid concentration in µmol/mL by comparing their peak areas with the standard.