Shotgun Lipidomics And Hydrolysis Mechanism Of Phospholipids From Aquatic Product During Storage

Phospholipids are present in all organisms. Aquatic products are good sources of phospholipid rich in n-3 polyunsaturated fatty acids(n-3 PUFA-PL). Compared to glyceride rich in n-3 polyunsaturated fatty acids, PUFA-PL has better oxidative stability and faster absorption. In this paper we established the method to analyze phospholipids (PLs) by direct injection electrospray ionization tandem mass spectrometry (ESI-MS/MS). Using the method, the profile of PL from fifteen aquatic products were analyzed. A deep insight of the research was tracking PL profiling and the activity phospholipases from Gadus macrocephalus, Litopenaeus vannamei and Crassostrea plicatula during storage at 4℃ and-20℃. The third part of the paper was extracting the hydrolytic enzymes from aquatic product and investigating the hydrolysis mechanism of PLs from aquatic product during storage by shotgun lipidomics analysis method. This paper provide a theoretical foundation and scientific basis for the utilization and thefollow-up study of aquatic product.Firstly, the methods for analyzing PL by ESI-MS/MS was established. Lipids were extracted with a modified Folch method, and then the analysis was taken directly into ESI source by injection pump. The MS/MS experiments combining precursor ion scan and neutral loss scan were performed to monitor different classes of PL (phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol). The research strategy of shotgun lipidomics was simple, fast and accurate for qualitative and quantitative, with excellent stability and reproducibility, thus making it would be suitable to analyze PL from various biological samples for lipidomics research. The profiling of 15 auqatic product were analyzed. The result showed that the molecular species and the content of PL from different samples are different. The most abundant content of PLs are from marine fish, which could account for 33.91-42.12% of the total lipids. The content of PLs from freshwater fish are the least abundant, which could only account for 12.85-18.33% of the total lipids. Shrimps have the most PLs species, and molluscs are rich in EPA, DHA and plasmalogens. The content of plasmalogens from oysters could account for 11.23% of the PLs. The lysophospholipids (LPL) from fresh aquatic products are in low content.Secondly, we investigated the PL profiling of muscle from Gadus macrocephalus, Litopenaeus vannamei and Crassostrea plicatula during storage at 4 ℃ and-20 ℃ for different time. The result showed that hydrolysis is the main causes for the deterioration of PLs storage at 4℃ and-20℃. The most content of PL molecular species decreased gradually during the whole storage in all 3 kinds of samples. The content of LPLs increased slightly first and then decreased, which showed relevance to the changes of PLs. The deterioration of PL storage at 4℃ is much faster than that at-20℃. And content of PL and LPL from samples storage at-20℃ for 12 weeks did not have significantly change. It indicated that the PL could stay stability during low temperature storage for a long time. Throughout storage, the correlation analysis suggested that the degradation of PLs were correlated to various phospholipases (including lipase, phospholipase A1, phospholipase A2, phospholipase C, phospholipase D), especially the changes of the activity of PLA2(r>0.9). The the hydrolytic enzymes are more active at 4℃ than at-20℃, which means low temperature may inhibit the activities of the hydrolytic enzymes.Finally, we purified the phospholipase from Litopenaeus vannamei and established the model system of hydrolysis was preliminary. Through analyzing changes of intramuscular phospholipids and free fatty acids (FFA)before and after reaction, model system of phospholipases hydrolysis and control experiment, hydrolysis characteristic was researched.The PC from Litopenaeus vannamei and egg yolk were used as the substrate. The content of PC from Litopenaeus vannamei decreased from 516.45mg/g to 146.14mg/g, while the content of PC from egg yolk decreased from 517.22mg/g to 408.20mg/g. The content of PUFA increased significantly, with an increasing of 280.40mg/g(p<0.001) and 79.89mg/g(p<0.001) from Litopenaeus vannamei and egg yolk respectively. The results indicated the hydrolysis mechanism of PLs from aquatic products during storage was correlated to the lipid hydrolytic enzyme activities, which were mentioned for the first time.