Ethyl butyric acid

SCFA analysis was performed as described by Bakker-Zierikzee et al. (30 (link)). Briefly, SCFAs were extracted from fecal PBS homogenate. The homogenates were mixed in formic acid, ethyl butyric acid (1.25 g/L; Sigma USA), and MilliQ water. Some fecal SCFAs such as acetic, propionic, n-butyric, iso-butyric, n-valeric, and iso-valeric acids were detected through gas chromatography with capillary column (Varian 3800, Varian Inc., Walnut Creek, CA, USA).

Using the previously described methods, the content of SCFA was calculated from the cecal content [44 (link)]. Briefly, a mixture of 400 µL of deionized water and 100 mg of cecal content was homogenized. Samples were homogenized using phosphoric acid, left for 30 min and then centrifuged (12,000× g for 15 min). The internal standard used to precisely determine the experimental concentrations was ethyl butyric acid (Sigma, Sigma Aldrich, St. Louis, MO, USA). Capric acid (Himedia, Mumbai, India) was added to each sample to a final concentration of 0.0375 mM. Samples were analyzed using BIOvision chemical kits.

Sheep milk and cow milk used in this work were provided by commercial dairy farms and were frozen before use. Baby feces were donated and stored at −80 °C shortly after collection.
Chemicals used were as follows: KCl (99.5%), KH₂PO₄ (99.5%), MgCl₂(H₂O)₆, orthophosphoric acid (85%), and diethyl ether (98%) were sourced from BDH Laboratory Supplies, Dorset, England. NaCl (99.6%) and CaCl₂ (99%) were sourced from ThermoFisher Scientific, (Waltham, MA, USA). NaHCO₃ (99%) was sourced from JT Baker, (Phillipsburg, NJ, USA). Pancreatin (37452 FIP-U/mg) was sourced from AppliChem GmbH, (Darmstadt, Germany). D₂O (99.8%) was sourced from Cambridge Isotope Labs Inc, (Tewksbury, MA, USA). (NH₄)₂CO₃ (99%), pepsin, gastric lipase (20,000 U/mg), cysteine (97%), bile salts, ethylbutyric acid (98%), N-methyl-N-t-butyldimethylsilyltrifluoroacetamide (95%), and imidazole (99%) were sourced from Sigma-Aldrich, (St. Louis, MO, USA).