Synthesis Of Palmitoleic Acid-enriched Monoglycides From Seabuckthorn Fruit Oil By Sn-1,3 Specific Lipase Enzymatic Reaction

Palmitoleic acid（POA）is a 16-carbon monounsaturated fatty acid with multiple physiological effects such as lowering blood sugar,lowering blood lipids,anti-inflammatory and promoting wound healing,which has a broad application prospect in food and medicine products.Some existing chemical methods to enrich palmitoleic acid were accompanied with certain negative impacts,such as high energy consumption,harmful by-products,which limited the application in medical product or high value-added food.Enzymatic preparation of functional esters has become a research hotspot in the field of oil modification due to its advantages of mild reaction conditions,environmental protection and low cost.Compared with free lipase,immobilized lipase can not only avoid the defect of free enzyme,but also maintain its high efficiency,specificity and mild enzyme catalyzed reaction,so it is widely used in various fields.In recent years,it has been found that seabuckthorn fruit oil is rich in palmitoleic acid,and the content at Sn-2 is high.However,the enrichment of Sn-2 palmitoleic acid in seabuckthorn fruit oil by lipase catalytic reaction has not been reported.In this study,seabuckthorn fruit oil was used as raw material,the feasibility of palmitoleic acid-enriched glycerides by selective hydrolysis and glycerolysis with Lipozyme RM IM（Sn-1,3 specific immobilized lipase）was investigated.The main research contents and results of this paper are as follows:1.Determination of fatty acid composition and physicochemical indexes of seabuckthorn fruit oil.The physicochemical indexes and fatty acid composition of the fruit oil of seabuckthorn with large fruit and small fruit were analyzed.The results showed that the seabuckthorn fruit oil used met the requirements of the industry standard T/ISA004-2021 seabuckthorn fruit oil.After saponification and methylation,the fatty acid composition of sea buckthorn fruit oil was analyzed by gas chromatography-mass spectrometry,which was found that there was little difference in fatty acid composition between small and large fruit seabuckthorn oil,and the unsaturated fatty acid content of small fruit oil was higher than that of large fruit oil.The content of palmitoleic acid in the two kinds of seabuckthorn fruit oil was 27.11%and 34.52%,respectively,and both of them could be used as ideal raw materials to enrich palmitoleic acid-enriched monoglycides and other unsaturated fatty acid functional esters.2.Synthesis of palmitoleic acid-enriched monoglycides from seabuckthorn fruit oil by Lipozyme RM IM hydrolysisLipozyme RM IM（immobilized product of free enzyme Rhizomucor miehei lipase）was used to catalyze the hydrolysis of small seabuckthorn fruit oil to synthesis monoglycerides rich in palmitoleic acid.The effects of water-oil mass ratio,p H value and temperature on the hydrolysis rate of seabuckthorn fruit oil were investigated by single factor experiment with enzyme dosage of 10%wt.%.On this basis,response surface method was used to optimize the process conditions of enzymatic hydrolysis of seabuckthorn fruit oil,and the relationship between the hydrolysis rate and the content of monoglycide was studied under the optimal hydrolysis conditions.The results showed that the optimal hydrolysis conditions were as follows:temperature is 55.10℃,p H is 7.03 and water-oil mass ratio is 1.03:1,when enzyme dosage was 10 wt.%（seabuckthorn fruit oil）.Under this condition,the hydrolysis rate reached79.59%after 12 h reaction.In the first 10 h of the optimal hydrolysis condition reaction,the hydrolysis rate was positively correlated with the content of monoglyceride,and the coefficient of correlation was 0.999.After that,the monoglyceride began to be hydrolyzed if the reaction continued.When the hydrolysis rate of enzyme reaction was 66.76%for 10 h,the maximum content of monoglyceride was 61.11%.The results of GC-MS showed that the content of palmitoleic acid increased from 27.11%to 40.84%,the content of linoleic acid increased from4.51%to 33.34%,and the total amount of two functional fatty acids reached 74.18%.3.Synthesis of palmitoleic acid-enriched monoglycerides from seabuckthorn fruit oil by Lipozyme RM IM glycerolysisLipozyme RM IM,a Sn-1,3 specific lipase,was used to catalyze glycerolysis of seabuckthorn fruit oil to synthesis palmitoleic acid-enriched monoglycerides.Taking seabuckthorn fruit oil as the research object,single factor experiment and response surface test were used to investigate the optimal conditions of glycerolysis under the enzyme dosage of 10%wt.%.The results showed that Lipozyme RM IM enzyme dosage of 10 wt.%（seabuckthorn fruit oil）catalyzed the glycerolysis of seabuckthorn fruit oil under the optimum conditions:reaction temperature is 51.94℃,time is 12 h,molar ratio of（TAG/Gly）substrate is 1:3.Under this condition,the yield of monoglyceride was 70.03%after 12 h reaction.¹³CNMR was used to detect the composition of glycerides in products by enzymatic glycerolysis,and it was found that the monoglycerides were mainly Sn-1 monoglycerides（1-MAG）,while the obtained diglycerides were mainly Sn-1 and 3 diglycerides（1,3-DAG）.A total of 6 fatty acids were detected in the purified MAG by GC-MS,and palmitoleic acid was the main fatty acid,accounting for 55.92%.A total of 5 fatty acids were detected in DAG,and oleic acid was the main fatty acid,accounting for 59.57%.Lipozyme RM IM catalyzed glycerolysis reaction to synthesis palmitoleic acid-enriched monoglycerides and oleic acid-enriched diglycerides.