The Preparation, Detection And Genetic Toxicity Evaluation Of Versicolorin A And A Feasibility Study Of Using Versicolorin A As A Biomarker To Predict Of Aflatoxin B₁ Contamination

Purpose:As it is well known that Aflatoxin(AFT) and Sterigmatocystin(ST) are the most prominent mycotoxins,which are serious toxicity.In respect of food safety,it is the imperative checking item for import and export trade,it also has been established the international maximum residue limit(MRL).If we can establish a prediction detective proposal,the risk report will be obtained before the toxins generate,and we have opportunity to adopt measure to prevent further economic losses.The objective of this research is to choose a biomarker molecule which is closely related with AFT during it's biosynthesis in order to predict the toxic contamination.Methods:(1) Establish a reliable method to detect the Versicolorrin A(VA) by HPLC,include extraction,purification,preparation of the standard VA,as well as the identification.(2) Evaluate the toxicity of the prepared pure VA.Ames test and human peripheral lymphocytes test have been carried out.(3) Detect VA,ST and AFTB₁ of the pure cultivation products of Aspergillus flavus and Aspergillus parasiticus.Detect the time course of VA and AFB₁ of the rice samples with the infection of Aspergillus parasiticus.The key issue and hard work of this project is purified and prepared the standard VA,and we need to obtain sufficient pure VA to set up the HPLC analysis method and to use as the standards for the sample analysis,as well as the enough pure VA for the toxicology test.Plan to use the mutagenic strain of Aspergillus parasiticus,which can enrich the VA to solve this problem.Result:(1) This program is successful to purify the 20mg VA standard substance with the purity of more than 98%.For the analysis,three modern detection technology,including HPLC,Pre-HPLC and MS have been used.The method is convenient,fast,and automatic with good reproducible.(2) The mutagenetic toxicity of VA is showed positive at the dose of 2.5μg/plate S₉⁺ and 5μtg/plate S₉^- with Salmonella typhimurium TA98 at Ames test.While in human peripheral lymphocytes test,VA presents a hereditary toxicity at the dose of 4800nmol/L.(3) At the 7^th and 10^th day,we can stably detect the VA from each culture medium of the two toxic molds.AFTB₁ only can be detected from PG medium at 10^th day of cultivation.In the rice samples,VA can be detected at the 5^th,7^th and 10^th day but AFTB₁ at 14^th day after the infection under 27℃(TLC methods). Under 28℃,VA can be detected at the 3^th,5^th,7^th and 10^th day but AFTB₁ at 7^th and 10^th day after the infection (HPLC methods).Conclusion:Has established a measurement method use HPLC to detect the VA in rice sample.VA potentially is a biomarker for the prediction of Aflatoxin contamination.Ames test showed VA is mutagenetic positive with and without rat liver extracts(S₉⁺ 2.5μg/plate;S₉^- 5μg/plate).Human peripheral lymphocytes test showed positive at(1.6μg/ml) so,the detection of VA in a foods sample may imply the sample is in a high-risk pollution with AFTB₁.Further more,the sample is contaminated with toxic compound in spite of non AFTB₁.