Study On Lipid Composition Of Plant And Aspergillus Flavus Based On Ultra-high Performance Liquid Chromatography With High-resolution Mass Spectrometry

In this paper,glycerolipids,phospholipids,and sphingolipids were used as materials,and based on the ultra-high performance liquid chromatography and high-resolution mass spectrometry technology,the cleavage laws of glycerolipids,phospholipids and sphingolipids in mass spectrometry were systematically characterized,which laid a foundation for subsequent qualitative and quantitative analysis of lipids.Using rapeseed oil,soybean oil,peanut oil and their corresponding oil crops as raw materials,a method for analyzing sphingolipids in vegetable oils and oil crops has been established,which provided a reference for exploring the composition characteristics of sphingolipids in vegetable oils and oil crops.In this paper,using Aspergillus flavus,which is widely present in vegetable oils and oil crops,as raw materials,according to the characteristics of the secretion of its secondary metabolites that are affected by the lipid composition of biofilms,a research method for the lipidomics of Aspergillus flavus was established,and three different types of lipid extraction method were compared.This paper explored the difference in lipid composition of Aspergillus flavus spores with different toxin-producing ability and the lipid composition characteristics of Aspergillus flavus mycelium.The main findings were as follows:（1）The cleavage law of glycerolipids,phospholipids and sphingolipids in mass spectrometry was systematically characterized.Among them,monoglycerides,diglycerides,and triglycerides will form[M+NH₄]⁺parent ions in the positive ion mode.Its characteristics Ionic fragments are mainly related to the loss of fatty acids attached to the glycerol backbone,and they do not respond in negative ion mode.Phospholipids will form the parent ion of[M+H]⁺in the positive ion mode,and its characteristic ion fragments are related to the loss of the head group part and the attached fatty acid.In the negative ion mode,the precursor ions formed by the three types of phospholipids PC,LPC and SM are[M+HCOO]^-,and the parent ions formed by other phospholipids are[M-H]^-.Other characteristic ion fragments are also the same as the head group part that it contains,and the loss of connected fatty acids is related.Sphingolipids are mainly a class of lipids with a long-chain sphingosine skeleton as the core structure.The difference in the cleavage law in mass spectrometry mainly exists in the different polar parts connected to the oxygen at the C1 position of the sphingosine skeleton.（2）The sphingolipid profiling method was established based on UPLC-Orbitrap MS.The types and contents of sphingolipid molecules of three main oil crops and their vegetable oils were comprehensively studied.24,22 and 24 sphingolipids were identified in the three oil crops,among which So（d16:0）and So（d18:0）,So（d20:0）and So P（d22:1）four sphingolipids were detected in the three oils,and the content reached more than 10%of their total sphingolipid content.11,14,and 18 sphingolipids were identified in rapeseed oil,soybean oil,and peanut oil,respectively.Among them,So P（d22:1）is the sphingolipid with the highest content of the three vegetable oils,reaching 83.37%,71.34%,and sphingolipids,respectively.84.00%.The sphingolipid analysis method established in this study will provide supporting technology for the evaluation of the nutritional function of vegetable oil and oilseeds.（3）The differences of the three lipid extraction methods are compared.In the positive ion mode,there are 871,592 and 854 kinds of lipids extracted by the MTBE method,Folch method and Bligh-Dyer method.From the relative abundance of the extracted substances（peak area）,the peak area lipid（>6×10⁷）extracted by the MTBE method and Bligh-Dyer method is the largest（MTBE:440;Folch:349;BD:478）.In the negative ion mode,the number of lipids extracted by the MTBE method,the Folch method and the Bligh-Dyer method were 469,507 and 285,respectively.The peak area（>6×10⁷）lipids extracted by the three lipid extraction methods of MTBE method,Folch method and Bligh-Dyer method were82,387 and 95 respectively.From the perspective of extraction repeatability,in the positive ion mode,654,485,and 488 lipids with peak area relative standard deviations less than 20%extracted by MTBE,Folch and Bligh-Dyer methods are 654,485 and 488,respectively,accounting for 75%,82%and 57%of the total amount.In the negative ion mode,the MTBE,Folch and Bligh-Dyer methods extract the number of lipids with a relative standard deviation of less than 20%of the peak area are 432,456 and 208,which account for 92%,90%and72%of the total lipids respectively.（4）Explore the difference in lipid composition of Aspergillus flavus spores with different toxin-producing ability.The study used the high resolution of the Thermo Orbitrap Fusion mass spectrometer to implement a high-coverage targeted mass spectrometry strategy for Aspergillus flavus spores.The results showed that in the positive ion mode,844 lipid molecules were detected,and in the negative ion mode,252 lipid molecules were detected.We used the PCA analysis method to clarify the differences between the different experimental groups,and used the OPLS-DA model to analyze the differential lipids of Aspergillus flavus spores with different toxin-producing abilities.Among the three kinds of Aspergillus flavus spores with different toxin-producing ability,68 different lipids were found in the positive ion mode,and 100 different lipids were found in the negative ion mode.Using ultra performance liquid chromatography tandem mass spectrometry can analyze the lipid composition characteristics of Aspergillus flavus mycelium with high throughput,high sensitivity and efficiency.A total of 385 lipid compositions were detected in the positive ion mode.A total of 187 lipid components were detected in the negative ion mode.In the positive ion mode,135 types of phospholipids,31 types of sphingolipids and 219 types of glycerolipids were mainly detected in Aspergillus flavus mycelium,which accounted for35.06%,8.05%and 56.88%respectively.In the negative ion mode,127 types of phospholipids,5 types of sphingolipids,and 18 types of other lipids were mainly detected,which accounted for 84.76%,3.33%,and 12%,respectively.