2.6 Optimization of conditions for the synthesis of pine sterol oleate
Using the immobilized CRL as a catalyst, the process conditions such as the amount of catalyst, the molar ratio of the substrate between pine sterol and oleic acid, reaction time, and reaction temperature were optimized for the synthesis of pine sterol oleate by direct esterification of pine sterol with oleic acid. In a 15 mL reaction tube on an automatic temperature-controlled magnetic stirrer, 1.0 mmol of pine sterol, and 4.0-8.0 mmol of oleic acid were added as substrate, catalyzed by the immobilized enzyme of 5.0-9.0 U·g-1(relative to pine sterol mass), and the reaction temperature was 35-55°C for 12-60 h.
2.7 Gas Chromatography Analysis
The quantitative analysis of pine sterol and sterol ester was performed by gas chromatography (GC) with FID detector and dichloromethane as the solvent, using a DB-5HT (0.1 μm, 0.25×15 m) column with N2 as the carrier gas and a flow rate of 1.5 mL/min. The inlet temperature and detector temperature were 350°C, and the initial temperature of the column temperature chamber was 180°C, ramped up to 240°C at 10°C/min. The sample volume was 1 μL and the shunt ratio was 20:1.