Preparation Of N-3 Polyunsaturated Fatty Acid Glycerides By Lipase-catalyzed Selective Methanolysis

n-3 Polyunsaturated fatty acids（n-3 PUFA）have many physiological functions.Compared with n-3 PUFA in the form of ethyl esters（FAEE）and free fatty acids（FFA）,n-3 PUFA in the form of glycerides has better bioavailability and stability,but its contents in natural oils are generally low,leading to its limited applicability in high value-added products.Although the enzymatic enrichment of n-3 PUFA has the advantages of good reaction selectivity and mild conditions,it still has the problem of low enrichment efficiency.Therefore,this study investigated the concentration of n-3 PUFA-enriched glycerides by lipase-catalyzed selective methanolysis of natural oils.The main contents are as follows.First,six kinds of oil containing n-3 PUFA（including five fish oils and one alga oil）were analyzed.Afterwards,lipase-catalyzed selective methanolysis of 1812 fish oil was performed,saturated fatty acids（SFA）and monounsaturated fatty acids（MUFA）released from the fish oil were converted to FFA or their methyl esters,leading to the production of glycerides rich in n-3 PUFA.The reaction conditions of selective methanolysis were optimized.The results showed that the lipase A from Candida antarctica（CAL-A）showed the strongest discriminatory properties against n-3 PUFA in fish oil;the reaction system was methanol（Me）aqueous solution,the water content was 47%（w/v）,the molar ratio of methanol to oil was 46:1,the lipase dosage was 480 U/g oil（w/w,0.08%）,the temperature was 57°C,and the content of n-3PUFA in the glycerides increased from the initial 39.88%to 71.41%after 11 h,the total content of DHA and EPA increased to 1.81 times of the original,and the yield of n-3 PUFA was 73.67%.These results indicated that selective methanolysis catalyzed by CAL-A had a high enrichment efficiency.Thirdly,the substrate adaptability of the CAL-A-catalyzed selective methanolysis was verified.Under the optimal reaction conditions,the selective methanolysis of five kinds of oil（including 2000 fish oil,salmon oil,anchovy oil,tuna oil and DHA algal oil）was performed,and then the purified products were analyzed.The results showed that after enzymatic methanolysis,the n-3 PUFA contents in the glycerides of the five oils increased to 1.5 times and above of the original one,where the n-3 PUFA contents in the glycerides were above 60%when tuna oil and DHA algae oil were used as raw materials.The triglyceride（TAG）contents（50.04%～68.61%）,acid values（0.04～0.26 mg KOH/g）and anisidine values（6.99～19.37）of the final six products met the requirements of European pharmacopoeia standards,and the peroxide values ranged from 5.12 mmol/kg to 7.34 mmol/kg.The results indicated that the selective methanolysis has good substrate adaptability and the product quality basically met the requirements.Finally,using 1812 fish oil as the substrate,the effects of the fish oil and methanol concentration on the initial rate of methanolysis reaction（v）were investigated under the optimal reaction conditions,and the maximum release rates（V_m）of SFA,MUFA and n-3 PUFA and the inhibition constant of methanol(K_{i M})on SFA,MUFA and n-3 PUFA were calculated separately to investigate the mechanism of selective enrichment of n-3 PUFA by CAL-A-catalyzed selective methanolysis.Our findings revealed that the CAL-A-catalyzed methanolysis of 1812fish oil followed a Ping-Pong Bi Bi mechanism with inhibition by methanol.Therefore,rate equation for the methanolysis under the optimal reaction conditions could be stated as:The V_m of SFA was 40.84 mmol/（L·min）,the V_m of MUFA was 6.18 mmol/（L·min）,the V_m of n-3 PUFA was 0.26 mmol/（L·min）,the K_{i M} of the n-3 PUFA（0.30 mmol/L）was lower than that of the SFA and MUFA（219.64 mmol/L and 79.71 mmol/L）.These results indicated that the CAL-A had higher transesterification activity with SFA than that with MUFA and n-3 PUFA.The methanol reaction system promoted the release of SFA and MUFA from TAG,and inhibited the release of n-3 PUFA from TAG.In summary,n-3 PUFA-enriched glycerides were prepared by enzymatic methanolysis reaction using natural oils as raw materials.The kinetic study of methanolysis showed that CAL-A had a strong selectivity for SFA and MUFATAG in methanolysis reaction system,and the methanol reaction system inhibited the release of n-3 PUFA from TAG,resulting in the enrichment of n-3 PUFA in glycerides.The enzymatic methanolysis has high fatty acid selectivity,good substrate adaptability,high enrichment efficiency,and the final product conformed to the pharmacopoeia standard,which has strong application prospects.