Extraction And Purification Of Euphausia Pacifica Phospholipids And Enzymatic Preparation Of Phospholipids Enriched In N-3Polyunsaturated Fatty Acids

The n-3polyunsaturated fatty acids, especially DHA and EPA have importantphysiological functions, such as enhancing the development of visual system andnervous system, prevention of cardiovascular diseases, anti-inflammatory functionand so on. Natural n-3polyunsaturated fatty acids have two forms, in the form oftriglycerides and in the form of phospholipids. The products of n-3PUFA in thetriglyceride form have always been drawn on great attention for the physiologicalfunctions. While latest researches show that products of n-3PUFA in thephospholipids form are superior to products of n-3PUFA in the triglyceride form indigestion, absorption, stability and bioavailability. However, the content of n-3PUFAis usually low in natural phospholipids, besides, the sources are limited. Therefore, itis necessary to focus on the research of increasing the content of n-3PUFA thatesterified into phospholipids. The phospholipids with high content n-3PUFA arepromising products. Currently, researches on enzymatic preparation of n-3PUFAphospholipids are mainly focus on soybean phospholipids and yolk phospholipids,researches on enzymatic enrichment of high content n-3PUFA phospholipids that arefrom marine creatures have not been reported so far.In this work, Euphausia pacifica was used as the raw material, the extraction andpurification process of Euphausia pacifica phospholipids was studied firstly. Then theprocess of enzymatic preparation of phospholipids enriched in n-3PUFA was studied.Finally, the oxidation stability of the prepared n-3PUFA phospholipids was evaluated.Specific research contents and results were as follows:Firstly, ethanol was used to extract phospholipids from Euphausia pacifica and afurther purification by acetone, then analysis the components in phospholipids byhigh-performance liquid chromatograph equipped with an evaporative light scatteringdetector. Based on the results of single factor experiment and orthogonal experiment，the optimal process conditions were as follows：95%ethanol，6to1ratio of solvent tokrill sample，a four-hour extraction time and an extraction temperature of40℃. Undersuch conditions，the reasonable phospholipids yield of17.58%and purity of46.09% were obtained. The purity reached78.85%with a further purification by acetone at4℃. HPLC-ELSD were used to analyze the components in phospholipids. Resultsshowed that the main components were phosphatidycholine(PC) and phosphatidy-lethanolamine(PE), PC accounted for75.92%, PE accounted for11.28%.Subsequently, the transesterification (acidolysis) reaction between Euphausiapacifica phospholipids and free n-3PUFA was studied. The results of pre-reaction andpost-reaction were compared. By comparing the results of different enzymes, differentreaction systems, and single factor experiment, orthogonal experiment were used tooptimize the major factors affecting the transesterification reaction. The optimalprocess conditions were as follows：in the solvent free reaction system, usingimmobilized phospholipase A1as the catalyst, adding30%(total weight ofphospholipid) enzyme, reaction time24h, reaction temperature53℃, with a substratemass (free fatty acids to krill phospholipids) ratio of7.6. Under such conditions, theobtained phospholipids contained31.05%DHA,13.88%EPA, the total content ofDHA and EPA was44.93%. DHA content increased20.52%, EPA content increased4.62%, the total content of DHA and EPA increased25.14%by comparing with thecontent of DHA and EPA in the raw material phospholipids, indicating that thismethod was effective in increasing the n-3PUFA content in Euphausia pacificaphospholipids and can be used for the preparation of high content n-3PUFAphospholipids.Finally, the main physical and chemical indexes for the raw materialphospholipids and product phospholipids were analyzed. The effects of temperature,air and the addition of antioxidants on the oxidation stability were also evaluated.Analysis results showed that the acid value, iodine value and peroxide value of theproduct phospholipids were slightly higher than that of raw material phospholipids.The research results showed that the lower temperature, the lower auto-oxidation rateof product phospholipids, product phospholipids were more stable under airtightcondition than exposure placed, the increase antioxidant capacity order of the additionof three kinds of antioxidants on the phospholipids was TBHQ> VE> VC. As aresult, the high content in n-3PUFA phospholipids should be stored at lowtemperature, sealing, add right amount of TBHQ.