The milk oligosaccharide was extracted using the traditional Folch extraction with slight modification . Briefly, the milk was mixed with chloroform and methanol mixture (3:1, v/v) in a 1:4 (milk v/solvent v) ratio in the 50mL Nalgene™ Oak Ridge high-speed PTFE FEP centrifuge tubes. The mixture was shaken vigorously for 5 minutes until homogeneous, followed by 40 minutes of centrifuge at 4000 RPM. The upper layer of the solution was extracted and concentrated in the rotary evaporator under 55 ℃ until all solvents were evaporated. Total carbohydrate concentration was determined using the phenol-sulfuric acid assay . The absorbance of the carbohydrate assay was detected by microplate reader (BMG Labtech FLUOstar Omega). After that, the oligosaccharide extracts were stored at -20 ℃ until usage. Three extractions were performed on each milk type on different days.