Free fatty acid preparation by saponification of SPO
SPO (20 g) was combined with 2.2 g KOH and 40 mL 95% ethanol, and the
saponification mixture was stirred at 75 °C in a water bath for 2 h.
After the completion of the reaction, distilled water (100 mL) and
hexane (200 mL) were added, and the mixture was transferred to a
separating funnel to stand for separation. The unsaponifiable matters in
the hexane phase were removed, and the collected lower layer containing
soaps was acidified with 3 M hydrochloric acid to pH 2 to release free
fatty acids. Subsequently, the solution was extracted three times with
total 300 ml hexane. The upper layer containing free fatty acids was
collected, and trace water was removed using anhydrous sodium sulfate.
Then the extract was concentrated to dryness at 50 °C on a rotary
evaporator to obtain SPOMFs. The final product was stored in a sealed
aluminum
pot at -20 °C before use.