Synthesis Of Structured Phosphatidylcholine Enriched With DHA/EPA By Immobilized Phospholipase A₁-Catalyzed Transesterification

Phospholipids are a class of naturally presented functional lipids that have good physicaland chemical properties and physiological functions. They are widely applied in food,pharmaceutical, and cosmetics industries. Phospholipids with different structures havedifferent physiological functions, thus the study of modification to obtain various functionalphospholipids is important. The modificaton of phospholipids can be realized by many ofmethods. However, enzymatic of phospholipids that could maintain their naturalconfigurations is more interesting in research. In the other hand, eicosapentaenoic acid(EPA)and docosahexaenoic acid(DHA) are functional fatty acids, which have a beneficialbiochemical and pharmacological effects on human health and treatment/prevention of somediseases. So far, the DHA/EPA can be ingested as in the form of various free fatty acids(FFA),ethyl esters, triglycerides and phospholipids(PL). It has been demonstrated that the easiestabsorption of EPA and DHA for man is in the form of free fatty acids. However, the free fattyacids EPA and DHA can also be easily oxidized to hydro-peroxides and normally have soursmells that inhibit their utilization. In recent years, it has been found that DHA/EPA could beeasily absorbed and have higher bioavailability in the body as PL than as the correspondingtriglycerides or ethyl esters. This research presents the synthesis of structuredphosphatidylcholine(PC) enriched with DHA/EPA by transesterification of DHA/EPA-richethyl esters with PC using immobilized phospholipsase A1(PLA1) in solvent-free medium.The results are as follows:1. Immobilization of phospholipsase A1. It was found that D380 was the best carrier andthe best conditions for the adsorption were: carrier/PLA1 ratio(w/v) of 1:3, p H of 5,adsorption temperature of 30℃ and adsorption time of 6h. At the moment, the protein loadand phospholipase activity of immobilized PLA1 were 36.96 mg/g and 720.95U/g,respectively.2. Immobilized PLA1 was employed to catalyze the transesterification of PC and ethylesters to produce DHA/EPA-rich PC and LPC. The maximal incorporation was 30.7% underthe optimized conditions: enzyme loading at a concentration of 15%(w/w, with respect tototal reaction mixture), PC/ethyl esters mass ratio of 1:6, temperature of 55 °C, and waterdosage of 1.25%(w/w, with respect to total reaction mixture), and it was found that theproduct were a mixture of PC(16.5%), 1-LPC(26.3%), 2-LPC(31.4%) and GPC(25.8%).Additionally, the reusability of the immobilized enzyme was evaluated under the optimalreaction conditions. The activity of immobilized PLA1 decreased to 48.9% after the fifth cycle(after it had been used to be carried out a total of six reactions trials). It indicated thatimmobilized PLA1 had a good operational stability in the transesterification of PC and ethylester-DHA/EPA.3. Response surface methods(RSM) was used to estimate the transesterification reactionof PC and DHA/EPA-rich ethyl esters. The results show that when only the incorporation ofDHA/EPA into PC and LPC were considered, the optimal levels of the three factors were:reaction temperature 57.5℃, water addition 1.28% and substrate mass ratio(PC/ethyl esters)1:6.8, the maximal incorporation was 31.0%, the corresponding PL composition of reactionproduct included 15.2% PC, 58.1% LPC, and 26.7% GPC. When both the incorporation andthe total yield of PC are considered, the optimum reaction temperature, water addition andsubstrate mass ratio(PC/ethyl esters) were 56.9℃, 1.10% and 1:7, respectively. The maximalincorporation of DHA/EPA and PL composition were 29.6%, 21.7% PC、54.6% LPC、23.7%GPC, respectively. When both the incorporation of DHA/EPA and the total yield of LPC areconsidered, the optimum reaction temperature, water addition and substrate mass ratio(PC/ethyl esters) were 57.4℃, 1.36% and 1:6.4, respectively, the corresponding incorporationof DHA/EPA and PL composition were 30.7%, 12.8% PC 、 59.0% LPC 、 28.2% GPC,respectively. Analysis of the data presented in Figs.1, 2, 3 and scheme 4-1 indicates thatDHA/EPA-enriched PC and 1-LPC dominate in the reaction mixture during the early stages ofthe reaction(before 12h), whereas at later stages highly DHA/EPA-enriched 2-LPC and GPCbecome dominant.