Preparation Of High-purity1,3-dioleoylglycerol

Diacylglycerol (DAG) is a kind of structured lipid that one acyl in the sn-1,2,3positionof triacylglycerol (TAG) is replaced by hydroxyl, which is the esterification product byglycerol and two fatty acids, a natural minor component of various edible oils and theintermediate metabolite of lipid. It is recognized as security (GRAS) food component, as wellas a multifunctional additive, which has broad application prospects in the food,pharmaceutical, chemical and other industries.Diacylglycerol has two isomers1,3-diacylglycerol and1,2(2,3)-diacylglycerol.1,3-diacylglycerol has a different metabolic pathways from ordinary oil in human body.Dietary1,3-diacylglycerol does not accumulate in the body can reduce visceral fats and bloodlipid, alleviate diabetes, suppress the increase of body weight. Due to the limited amount ofdiacylglycerol existing in natural products, the synthesizing technique of high-purity1,3-diacylglycerol has been attracting increasing attention from home and aboard. In order toobtain high purity1,3-diacylglycerol, the esterification of oleic acid and glycerol catalyzed byLipozyme RM IM in solvent-free system and the separation and purification of esterificationproduct by molecular distillation associated with silica gel column chromatographytechnology were studied in the present study.Firstly, the production of1,3-dioleoylglycerol through esterification of glycerol witholeic acid using immobilized lipase as a catalyst in solvent-free system was analyzed.Lipozyme RM IM was selected as the optimum catalyzer from immobilized lipases includingLipozyme RM IM, Lipozyme TL IM and Novozymes435. Then comparerd methods ofvacuum dehydration and molecular sieve dehydration, the vacuum dehydration was chosen asthe proper method for this study. We also investigated the influences of substrate molar ratio,enzyme load and reaction temperature on the conversion rate of oleic acid and the content of1,3-dioleoylglycerol. According to response surface optimization, the optimum technicalconditions were established as follows:(oleic acid:glycerol)2.1:1for substrate molar ratio,6.9%for enzyme load and63oC at1.9h for reaction temperature and time, under theseconditions the content of1,3-dioleoylglycerol were61.20%.Secondly, esterified compounds were distilled by two-stage molecular distillation whilethe preheating temperature, feed rate, scraper speed and distillation temperature were undercontrol: preheating temperature40oC, feed rate2mL/min, scraper speed100r/min, first-stagedistillation temperature180oC, second-stage distillation temperature200oC and vacuumpressure1.0×10-2mbar. After two-stage molecular distillation, the purity of1,3-dioleoylglycerol was increased from61.20%to76.41%and the recovery of1,3-dioleoylglycerol was69.02%.Lastly, for further purification silica gel column chromatography was used. The resultsshowed that D was selected as the best eluant system from four different eluant systerms A、B(different ratios of n-hexane:ethyl ether:acetic acid) and C、D (different ratios of n-hexane:ethyl acetate: acetic ether:acetic acid) thought thin layer chromatography silica gel plate.Subsequently, As the recovery ratio and purity of1,3-dioleoylglycerol were established asindicators, the optimum conditions of silica gel column chromatographic separation were determined. mobile phase flow rate1.5mL/min, sample concentration75mg/mL and silicagel load30g. Under the above optimal conditions, the purity and recovery of1,3-dioleoylglycerol were98.03%and69.90%respectively.