Methods Development Of The Extraction Of Phospholipids From Three Marine By-products And The Novel Lipidomics Analysis

With the continuous development of the marine fishery industry,the production of marine products in China has increased year by year,but the massive waste of by-products cannot be ignored.Phospholipids widely exist in various organisms.The sn-2 position of the phospholipids which come from marine organisms are often linked with the ?-3 polyunsaturated fatty acid chains.This kind of phospholipids not only has the physiological functions of phospholipids,but also has the functions of ?-3 polyunsaturated fatty acids.The utilization and analysis of marine organisms' phospholipids can reduce the waste of by-products and can also deepen the research and exploration of marine source foodsThis project focuses on the two parts as followingFirst,an improved Folch method that replaces chloroform with 1,2-dichloroethane was established to extract the phospholipids of shrimp heads,squid viscera and tuna viscera.The effect of the relative extraction volume and the composition of phospholipids were compared between the improved Folch method and the traditional methods like Bligh&Dyer method,Folch method,MTBE method,and ethanol extraction methodAmong the four traditional methods,the MTBE method had the highest relative extraction volume of phospholipids of shrimp head,squid viscera and tuna viscera.Among the 1,2-dichloroethane-methanol method with different solvent ratios,the 1,2-dichloroethane-methanol(1:2,v/v)method had the highest relative extraction volume of phospholipids of the samples.Compared with the MTBE method,the phospholipids extraction rate of the 1,2-dichloroethane-methanol(1:2,v/v)method was slightly higher than MTBE method,and the rest were lower than MTBE method.The 1,2-dichloroethane method and the ethanol extraction method both had poor phospholipids extraction effects on the above three samples.Apart from these two methods,the effects of other methods on the composition of phospholipids of three marine by-products were discussed by liquid chromatography-mass spectrometry.The types of phospholipids extracted from the same by-products by the six extraction methods were basically the same.There were 48 kinds of phospholipids detected from the shrimp head.There were 47 kinds of phospholipids detected from the squid viscera.And there were 53 kinds of phospholipids detected from the tuna viscera.In the phospholipid sub-classes,the types of phospholipids containing unsaturated fatty acid chains extracted from the same by-products by the six extraction methods were basically the same.This result also applied to phospholipids containing ?-3 polyunsaturated fatty acid chains.All the above methods had a high extraction rate for ?-3 polyunsaturated fatty acid chain phospholipids.This indicates that these methods could not only extract phospholipids from marine by-products,but also extract phospholipids containing ?-3 polyunsaturated fatty acid chains efficiently.In summary,the MTBE method and the 1,2-dichloroethane-methanol(1:2,v/v)method were both can be used to extract the phospholipid from marine by-products.The MTBE method can be used for more kinds of samples,and the 1,2-dichloroethane-methanol(1:2,v/v)method is safer and more suitable for the food processing industry.Subsequently,three lipidomics methods including liquid chromatography-mass spectrometry,multi-dimensional tandem mass spectrometry and iKnife intelligent monopolar electrode mass spectrometry are been established to detect and analyse the phospholipids of marine by-products.Meanwhile,this study compares the advantages and disadvantages of these lipidomics methods and explores their scope of application.This project established three lipidomics methods including liquid chromatography-mass spectrometry,multi-dimensional tandem mass spectrometry and iKnife intelligent monopolar electrode mass spectrometry.The squid viscera phospholipids were identified and quantified by liquid chromatography-mass spectrometry in the negative-ion full scan mode.A total of 17 kinds of PC,10 kinds of PE,9 kinds of PI,11 kinds of PS were detected.The phospholipids obtained by this extraction method contained many highly unsaturated fatty acyl chains.For the multi-dimensional tandem mass spectrometry,the obtained phospholipids from the fishery by-products were injected directly by a syringe pump and identified and quantified by tandem mass spectrometry in the precursor-ion scan and neutral-loss scan modes.The results showed that 69 kinds of phospholipid molecules were detected in the phospholipid extract.Phospholipids with at least one eicosapentaenoic acyl/docosahexaenoic acyl chain were frequently found.An iKnife intelligent monopolar electrode mass spectrometry method was established for real-time lipidomics profiling of the viscera of tuna.The results showed that a total of 41 molecular species of phospholipids was found and identified in the m/z range of 699.50 and 911.56.After methodological verification,the above three lipidomics methods were found to be good in accuracy,and the results can be repeated.By comparison,the three methods have their own characteristics.Liquid chromatography-mass spectrometry is suitable for qualitative and quantitative analysis when the amount of sample is large.This method has a low detection speed,but its results are highly accurate.Multi-dimensional tandem mass spectrometry is suitable for qualitative and quantitative analysis when the amount of sample is small,and this method can be used to establish a rapid sample detection method.iKnife intelligent monopolar electrode mass spectrometry is suitable for lipid profile analysis,and this method can identify unknown samples in real time.Through this project,we can further deepen the research on the extraction of phospholipids from the marine by-products,make full use of the physiological and biochemical characteristics of phospholipids,expand its economic value.This research is also helpful in reducing the waste of by-product resources and plays a significant role in maintaining the ecological environment.The development and establishment of lipidomics methods can make it more applicable in food science,provide new ideas for food science research,and make the discipline develop in a more diversified direction.