2.2. Carotenoid Extraction and Saponification

The extraction and purification were conducted in the shortest possible time, to avoid degradation and isomerization [21]. In the first step, 1 kg of fruit pulp was separated and homogenized with 1 L hexane using a mechanical homogenizer (Daihan Scientific, Wonju-si, Korea). Homogenized samples were transferred to 200 mL centrifuge bottles, and centrifuged at 7000× g for 10 min at 4 °C, and the supernatant containing carotenoids was then recovered. The pelleted sample was repetitively (2–3 times) extracted using hexane, until they were colorless. The collected supernatants were pooled, partitioned, and the upper hexane phase was collected. The partitioning between upper hexane and the lower water phase was improved by adding ~10% (v/v) of 1 M sodium chloride. The hexane fraction was evaporated in a vacuum-rotary evaporator (Büchi, Switzerland) at 35 °C. The obtained extract was dissolved in 20 mL hexane, mixed with equal volume of 10% methanolic-potassium hydroxide (KOH; w/v), flushed with nitrogen (N₂) gas (to minimize the oxidation), and incubated at 55 °C for 45 min for saponification. The mixture was transferred into a separating funnel, extracted thrice with hexane containing 10% diethyl ether, and the upper lipophilic hexane solutions were pooled, and then washed three times with water to remove traces of KOH. Diethyl ether was added to improve the polarity of hexane solution and enhance the solubility of non-esterified β-cryptoxanthin. The lipophilic hexane solution was dried under vacuum (<35 °C) using a rotary evaporator, and the residue was re-dissolved in 10 mL acetone.