Preparation Of Immobilized Lipase PCL And Its Application In The Synthesis Of ?-linolenic Acid-rich Diacylglycerol

Studies have shown that?-linolenic acid-rich diacylglycerol has a good therapeutic effect on reducing visceral fat area,body mass index and serum triglyceride concentration in individuals with overweight.At present,studies on the?-linolenic acid diacylglycerol are mainly focused on their physiological activities,while reports on their biosynthesis are few.A mono-and diacylglycerol lipase PCL from Penicillium carmenii only decompose or synthesize mono-and diacylglycerol,but is inactive to triacylglycerol.This unique substrates specificity makes it great potential for the synthesis of high purity diacylglycerol.In this study,PCL obtained by fermentation was immobilized and then applied to the esterification to prepare?-linolenic acid-rich diacylglycerol.The product was separated and purified by molecular distillation,and high-purity of?-linolenic acid-rich diacylglycerol was obtained.Then basic physicochemical properties of?-linolenic acid-rich diacylglycerol were studied.The main research results obtained are as follows:1.Fermentation and immobilization of lipase PCL:Lipase PCL obtained by fermentation was immobilized on six kinds of resins separately.The results showed that the resin ECR8806 was the best support material,and the esterification activity of immobilized PCL was 598.15 U/g under the optimal immobilization conditions.Fourier transform infrared spectroscopy?FT-IR?and confocal laser scanning microscope?CLSM?further confirmed the successful adsorption of lipase PCL onto resin ECR8806.Studied on the enzymatic properties of immobilized PCL,and it was found that the optimum temperature of the immobilized PCL remained unchanged compared with the free PCL,but its temperature tolerance was significantly improved.2.Production of?-linolenic acid-rich diacylglycerol by immobilized PCL-catalyzed esterification:Comparison of the catalytic ability of immobilized PCL during esterification of?-linolenic acid or ethyl linolenate with glycerol,and it was found that?-linolenic acid has a higher esterification degree.The reaction conditions of?-linolenic acid and glycerol in a solvent-free system have been further studied,and the results showed that when the molar ratio of?-linolenic acid to glycerol was 1:4,the enzyme loading was 5%?w/w,with resepect to total reactants?,and the reaction temperature of 40°C,the esterification degree of?-linolenic acid reached 89.24%,and the content of diacylglycerol in the products was54.49%.Moreover,the immobilized PCL retained 91.60%of its initial activity after the tenth cycle,which indicates that immobilized PCL has a good operational stability.3.Separation and purification of?-linolenic acid-rich diacylglycerol and the studis on their basic properties:Under the above optimized reaction conditions,?-linolenic acid-rich diacylglycerol was prepared in a scale-up reaction.After 9 hours,the content of diacylglycerol was 52.98%.Then the reaction mixture was further purified using molecular distillation,and the purity of diacylglycerol was up to 99.28%?1,3-diacylglycerol,67.76%;1,2-diacylglycerol,31.52%?at a distillation temperature of 170°C.The content of?-linolenic acid in diacylglycerol was 78%.The acid value,peroxide value,iodine value and saponification value of the high purity of?-linolenic acid-rich diacylglycerol were 0.21 mg KOH/g,2.83 mmol/kg,201.13 g/100 g?I₂?and 193.34 mg KOH/g,respectively.In addition,studies have found that adding the antioxidant TBHQ to high purity of?-linolenic acid-rich diacylglycerol,the oxidation of?-linolenic acid-rich diacylglycerol can be effectively inhibited.