Metabolomics Method Of Aspergillus Flavus Research Based On LC-MS/MS

The problem of aflatoxin contamination is a worldwide problem.Researchers from various countries have used various advanced techniques to study the aflatoxin biosynthesis and its regulation.That research area has made great progress.This achievements contribute to people's understanding of aflatoxin biosynthesis,gene regulation and other aspects.The issue of aflatoxin contamination has become a major risk for the quality and safety supervision of agricultural products,for instance,peanuts.Currently in China,it is proposed to carry out the large-scale warning model of aflatoxin based on meteorological factors and the molecular warning research based on biomarkers.Warning research is an important link in the control of aflatoxin.This study aim to achieve the discovery of Aspergillus flavus biomarkers and study on accumulation of aflatoxin,in this paper,we carried out the following researches:1.The analytical methods of 12 compounds,such as norsolorinic acid,were established by using LC-HRMS analysis platform and D-optimal mixing design.The detection limit of the analytical method was 0.002?g/kg-0.1?g/kg,the limit of quantification?LOQ?is in the range of 0.008?g/kg-0.35?g/kg,and the intra-day precision is less than 5%,which indicates that the method has high sensitivity and stability.We used this methods to investigate the temporal metabolic changes of compounds on the passway of aflatoxin biosynthesis.The results showed that the total amount of AFB₁ was increased in 1to 5 days,reached the highest level on the fifth day,however,AFB₂ reached the peak on the sixth day,and then the peak of AFB₁ was increased.Aflatoxin content began to decline,and maintain a stable trend.The trend of aflatoxin precursor compounds was also synchronized with AFB₁ and AFB₂,but the peak appeared on the fourth day and then began to decrease.2.The MIL-101 was used to synthesize the macromolecular nano-organometallic material MIL-101 for the enrichment and reduction of matrix effect of aflatoxin B₁,B₂,G₁,G₂,OMST,ST.The preparation process and instrumental analysis conditions were optimized.The LC-HRMS method for the determination of toxic compounds on the biosynthetic pathway of Aspergillus flavus was established.The detection limit was 0.002-0.016?g/kg,the limit of quantification was 0.008-0.05?g/kg,and the linear range was 0.01-500?g/kg,the recovery was 71.2-91.9%,the correlation coefficient was0.9990-0.9998,the established method was highly sensitive,the matrix effect was not obvious after purification,and The intraday and inter-day precision was below 5%,and the reproducibility was good.The results show that the method is simple,accurate and reliable,and it is suitable for the simultaneous analysis of the accumulation of aflatoxin.3.The LC-HRMS mass spectrometry platform was used to optimize the pretreatment method of non-targeted metabolomics of Aspergillus flavus,including the key steps of sample quenching and metabolite extraction.The quenching solvents of cold glycerol buffer solution?-30??was confirmed.The extraction solvents of was optimized by the D-optimal Mixture Design.The optimum conditions were as follows:MeOH:ACN:EA:HCOOH?0.245:0.241:0.257:0.01 v/v/v/v/v?.This optimum solution also compared with other common solution.We find this mix solution is a optimum solution.The instrument conditions and data processing parameters was optimized.This method of non-targeted metabolomics of Aspergillus flavus was established,which provided a way to drive the discovery of potential biomarkers of Aspergillus flavus.4.The non-target metabolic analysis of Aspergillus flavus was performed under the conditions of low temperature 18?,optimum temperature of 28?and high temperature of 38?.The samples of Aspergillus flavus were prepared,data aquisition,raw data pretreatment and multivariate statistical analysis,potential biomarker screening,differential metabolite identification,Aspergillus flavus metabolic pathway analysis,etc.we identified a total of 20 substantially potential biomarkers under the temperature disturbance.we found that the temperature mainly affects the tricarboxylic acid cycle,amino acid metabolism,fatty acid biosynthesis,glucose metabolism and other energy metabolic pathways.Additional,It also regulated the biosythesis of aflatoxin,cyclopiazonic acid,kojic acid,ditryptophenaline and other secondary metabolic pathways.The results showed that kojic acid could be used as a candidate biomarker to verify.This work provided a candidate reference for the study of aflatoxin risk assessment and provided new insight into aflatoxin molecular warning.