Molecular Characteristics Study Of PA & DHA Structural Lipids In Tuna Viscera

Tuna viscera has plenty type of lipids,and rich in eicosapentaenoic acid(EPA)and docosahexaenoic acid(DHA),which is comprehensively utilized of marine bioprocessing waste in recent years.In this project,tuna viscera was used as raw material to prepare EPA and DHA structural lipids by transesterification.The lipidomics technology based on mass spectrometry can realize the comprehensive analysis of lipids.Therefore,this topic adopts the technology of liquid chromatography tandem mass spectrometry to characterize the structural lipids,the process of transesterification was dynamic analyzed from the molecular level and with combination of multivariate statistical analysis looks for differential metabolites.The main research results of this subject are as follows:1.Ultrasonic-assisted organic solvent extraction was used to extract fish oil.Comparing the extraction rate,PL yield and physicochemical indexes of fish oil with chloroform-methanol(2:1,V/V),95% ethanol,absolute ethanol,isopropanol,n-hexane,cyclohexane,ethyl acetate,and absolute ethanol was finally used as the extraction solvent in this experiment.Phospholipids and glycerides were separated by acetone sedimentation and used as raw materials for subsequent transesterification.2.The ethyl ester-type EPA and DHA(EPA & DHA-EE)were enriched by the urea adduction,and the optimal adduction conditions were obtained by single factor experiment optimization: the ratio of ethanol to fatty acid ethyl ester was 3:1,the ratio of urine to ester was 1.5:1,and the crystallization was in-5 ℃ lasting for 10 h.Under the optimal conditions,the yield of EPA & DHA-EE was 89.47%,and the recovery rate was 76.33%.Subsequently,the enzyme-catalyzed transesterification reaction system of TG and EE was studied in a solvent-free system.Under the conditions of a determined temperature of 60 °C and a catalytic time of 20 h,the lipase Novozyme 435(N435),Lipozyme RM IM(LRI),and Lipozyme TL IM(LTI)were optimized from three factors: water content,substrate ratio,and enzyme loading.The optimal catalytic conditions of N435 were: water content of 0.5%,substrate mass ratio of 1:2,and lipase loading of 20%.Under this conditions,the relative content of EPA and DHA enriched TAG(EPA & DHA-TAG)in the catalytic product reached68.74%.The optimal catalytic conditions of LRI were: water content of 1.5%,substrate mass ratio of 1:4,and 20% lipase loading.As a result,the relative content of EPA & DHA-TAG reached 73.26%.The optimal catalytic conditions for LTI were: water content of 2%,substrate mass ratio of 1:4,and enzyme loading of 25%.At this conditions,the relative content of EPA & DHA-TAG reached 56.13%.HPLC-Orbitrap-MS/MS used for qualitative and quantitative TAGs.Multivariate statistical analysis models were built by combination of the TAG molecule samples and their content,the potential biomarkers are TAG(16:0/22:6/22:6),TAG(20:5/22:5/22:6),TAG(16:1/20:5/22:5)and so on.3.EPA & DHA-EE was acidified to prepare free EPA and DHA(EPA & DHA-F),and used as acyl donors to undergo acid hydrolysis with tuna by-product phospholipids under the catalysis of lipase to enrich phospholipid-type EPA and DHA(EPA & DHA-PL).The addition amount of n-hexane was immobilized at 3 m L,the catalytic conducted at 60 °C for 24 h.The lipases of Novozyme 435(N435),Lipozyme RM IM(LRI)and Phospholipase A1(PLA1)were compared at different water content,substrate mass ratio and enzyme loading.The optimal catalytic conditions of Phospholipase A1 were as follows: water content of 0.5%,substrate mass ratio of 1:2,and enzyme loading of 20%.And the total content of EPA &DHA-PL in the catalytic product reached 186.39 mg/g.The optimal catalytic conditions of Lipozyme RM IM were as follows: water content 1%,substrate mass ratio 1:4,enzyme addition 20%,and the total content of EPA & DHA-PL in the product reached 168.10 mg/g.The optimal catalytic conditions of Novozyme 435 were as follows: water content 0.25%,substrate mass ratio 1:5,enzyme loading 20%,and the total content of EPA & DHA-PL in the product reached 114.13 mg/g.Using HILIC-Pre IS-MS in the precursor ion scanning mode that locked the m/z 301.6 and m/z 327.6 to qualitative and quantitative EPA & DHA-PL.And the catalytic products under the optimal conditions of the three enzymes were analyzed by multivariate statistical analysis models,and obtains the potential markers: PC(p-16:0/22:6),PC(18:0/20:5),PC(16:0/20:5),etc.