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.