Enzyme-Catalyzed Synthesis Of Docosahexaenoic Acid Phosphatidylcholine(DHA-PC) And Phosphatidylglycerol(DHA-PG)

Docosahexaenoic acid-phospholipids(DHA-PLs)are phospholipids with the special structure containing DHA side chains.A large number of studies have shown that DHA-PLs have the common nutritional functions of DHA and PLs,and have obvious advantages in oxidation stability and bioavailability.DHA-Phosphatidylcholine(DHA-PC)has been used as a food nutrient fortifier,which has the effects of promoting infant retinal development,reducing the risk of neurological diseases,and anti-tumor.DHA-Phosphatidylglycerol(DHA-PG)can be used to regulate the growth of keratinocytes,relieve skin inflammation and reduce serum and liver lipids.However,natural DHA-PLs are mainly derived from marine animals such as antarctic krill and sea fish eggs,which have some problems such as raw material supply,allergens and heavy metal pollution.Therefore,the use of fermented microalgae oil as the DHA donor to synthesize DHA-PC has the value of research and development.In this paper,lipase was used to catalyze the reaction between DHA-rich triglycerides and soy phosphatidylcholine(SPC)to try to synthesize DHA-PC.A high performance liquid chromatography(HPLC)method for the determination of DHA-PC was established and it was compared with the traditional gas chromatography(GC)method for the determination of DHA-PC.The process conditions of enzyme-mediated synthesis of DHA-PC were studied.Further,the reaction process of phospholipase D(PLD)-mediated synthesis of DHA-PG was studied.The main findings were as follows:(1)An efficient HPLC was firstly proposed and successfully applied to the determination of DHA-PC.DHA-PC was pre-treated by alkaline hydrolysis,acidification and then the obtained components underwent derivatization usingα-bromoacetophenone as derivatization reagent,triethylamine as catalyst,to make them have ultraviolet(UV)absorption.In this paper,a gradient elution method was used to separate DHA-PC in phospholipid products.The effective separation of the target compound at low temperature was achieved by optimizing the gradient range and steepness.Compared HPLC with the traditional GC method for detecting DHA-PC in phospholipid products,it was found that there was almost no significant difference between them in determining the main fatty acids in DHA-PC.In addition,GC method had lower detection limits(LOD)and quantification limits(LOQ),and the raw materials used in the reaction were more expensive,so GC method was more conducive to the determination of DHA-PC in micro-produced phospholipid products.Therefore,this article chose the GC method to determine DHA-PC in phospholipids.(2)The main factors influencing the synthesis of DHA-PC by transesterification between DHA algae oil(Algal oil containing triglyceride of DHA,DHA-TAG)and PC mediated by CALB in the n-hexane-water system were investigated.Enzyme amount,temperature,the volume ratio of two-phase,the mass ratio of substrates,pH,and reaction time significantly affect the reaction yield.The orthogonal experiment was designed on the basis of single factor experiments.Then the optimal experimental combination conditions were obtained.The optimal conditions for the synthesis of DHA-PC were as follows:enzyme amount was 300 LU,temperature was 40 ℃,the volume ratio of two-phase(n-hexane:water)was 5:1,the mass ratio of substrates(PC:DHA-TAG)was 1:3,pH was 7.5,and reaction time was 24 h.Under optimal conditions,the highest yield of DHA-PC reached 21%,which could meet the requirements of most commercially available DHA-PLs supplements(approximately 12%).(3)The main factors influencing the synthesis of DHA-PG by transphosphatidylation between DHA-PC and glycerol mediated by PLD in the diethyl ether-water system were investigated.The volume ratio of two-phase,pH,reaction time,the mass ratio of substrates,and temperature significantly affect the reaction yield.The orthogonal experiment was designed on the basis of single factor experiments.The optimal conditions for the synthesis of DHA-PG were as follows:the volume ratio of two-phase(ether:water)was 1:1,pH was 5.5,reaction time was 4 h,the mass ratio of substrates(DHA-PC:glycerol)was 1:10,temperature was 33 ℃.Under optimal conditions,the highest yield of DHA-PG reached 77%.