2.3. Lipid extraction from breast milk
Human milk fat was extracted using the classical Röse–Gottlieb method(Crocker et al., 1955). Briefly, 1 mL of ammonium hydroxide solution was added into 5 mL breast milk. The mixture was heated to 65 ± 5 ℃ using electric heating constant temperature oscillation water bath for 20 min. After cool to room temperature, 5 mL anhydrous ethanol, 10 mL anhydrous ether, and 10 mL petroleum ether were added into the mixture with full shaking and stand for 2 h. The supernatant was separated from water and then collected. The solvent of the supernatant was removed by nitrogen. The above operation was repeated for the second extraction. The resulting supernatant was combined with the first extract, and the solvent was blown dry to constant weight with nitrogen to obtain human milk fat. The human milk fat content was calculated with the results were expressed as g/100 mL.