Separation And Structure Identification Of Lipid Species In Chinese Mitten Crab (Eriocheir Sinensis)

Aldehydes from lipid degradation is the key odorants in aquaculture of Chinese mitten crab(Eriocheir sinensis),which is mostly come from phospholipids with rich unsaturated fatty acids as the lipidomics concerned.Phospholipid is a complex system in which the composition and distribution of fatty acids give the unique aroma to E.sinensis.Based on the analysis of lipid metabolomics during the crab steaming,the relationship among lipid fraction contents,non-volatile metabolic products of lipids and key odorant contents will be further studied and to deduce the possible pathways of key odorant formation by the lipids degradation and oxidation.Firstly,in order to figure out the composition of the fatty acid,lipase was used to enzymolyze the lipid and compared the fatty acids before and after enzymolysis.Then the lipid was purified by prep-HPLC and there formed single molecular species.Combine these steps to detect the composition of fatty acids in lipids.Using the gonad of female E.sinensis as the material due to its high content of unsaturated fatty acids,but considering the costing,the lecithin was used for the method establishment and finally verifying by PC in E.sinensis.The main results were listed as follows:(1)Compare the HPLC-UV detector with potentiometric titrator to analyze the processing of enzymolysis and eventually use the lipase A2 to design the experiment.The optimal condition were as follows: the concentration is 0.008g/mL,temperature 40°C,pH 6.5,enzyme concentration 0.1IU/mL,time 7h.(2)Use SHIMADZU LC-15 HPLC with 3 types of chromatographic column to separate PC under two mobile phase system: methanol/hexane/ammonium acetate/glycerol and methanol/acetonitrile/water/choline chloride.The result showed that the method with methanol/acetonitrile/water/choline chloride,ELET C18 chromatographic column,flow rate 2mL/min,UV detector 206 nm proved to be a reliable way to make the separation.(3)The PC in female E.sinensis was obtained by SPE method,which was used by the method established before to purify the single molecular species.On this basis,lipase A2 was used to enzymolyze the specific molecular species and finally figured out the structure,which has a C16:0 on Sn-1 position and a C20:4n6 on Sn-2 position.Besides,qualification and quantitation method was build,the data of recovery rate and relative standard deviation showed the method was useful.This study combined biology and chemical methods to identify the lipid molecular species structure of E.sinensis,which lay the foundation for detecting the oxidation degradation characteristics of artificially synthesized lipid and fatty acids and determination for the parts where the aroma comes from.