| Mycotoxins are toxic secondary metabolites produced by fungal species,about 25%of food crops are contaminated by mycotoxins worldwide each year.Aflatoxin B1(AFB1),classified as a Group 1 human carcinogen by the International Agency for Research on Cancer(IARC).Sterigmatocystin(STC),as the precursor of AFB1,is usually regarded by IARC as a potential carcinogen in Group 2.Up to now,regulation levels for STC in food have not yet been defined,which could be caused by limited occurrence data.To command differences in the distribution of two mycotoxin in variety of foods,this research analyses distribution characteristics of AFB1 and STC in different foods collected from China's mainland,by establishing a high-efficient,accuracy,sensitive and fast method for the simultaneous detection of AFB1 and STC.Meanwhile,due to share the same biosynthtic pathway,the effect of STC on A.flavus growth and aflatoxin B1 production is studied and transcriptome and proteomic are used to explore possible mechanism.The main content and results of this research were as follows:1.The quantitative analysis method of AFB1 and STC in foods was established by HPLC-MS/MS.The extraction parameters of the QuEChERS(Quick,Easy,Cheap,Effective,Rugged and Safe)method have been optimized including ration and volume of extraction solvent,ultrasound time and clean-up adsorbents.This novel method for the detection of AFB1 and STC in food has been validated according to SANCO/12571/2013 and(EC)No 401/2006 regulation.Results showed good linear relationships and selectivity.Mean recoveries for two tested analytes were in the range of 77.7-119.7%,the range of RSD was 1.3-15.4%.In addition,the average recoveries and RSD in beer were 92.7-103.6%and 4.0-18.0%,respectively.The limit of detection(LOD)was 0.02-0.18 ?g/kg and limit of quantity(LOQ)was 0.5?g/kg for AFB1 and STC in different matrix.The proposed method can meet the trace analysis requirements of AFB1 and STC in foods.2.A total of 1153 food samples collected from 18 provinces in China's mainland and 315 stale rice samples stored in the laboratory for one-two years were detected for AFB1 and STC.The detectable rate for AFB1 in samples were in range of 0-40%,and 0.2%for grains,7.8%for grains-based products,8.7%for nuts,respectively.Two nuts samples exceed the limits and none of beer samples was contaminated by AFB1,The detectable rate of Jiangxi province was the highest with 40%,which samples were peanut.Sichuan and Guangxi was following with 18.75%and 14.29%,respectively.and none of AFB1 was detected in Heilongjiang province.STC was detected in 12.9%of all samples with average range 0.39?g/kg.amongst 2.6%for grains,28.3%for grains-based products,4.3%for nuts and 0%for beer was contaminated by STC.The positive rate of Sichuan,Shandong and Zhejiang were 47.92%,23.81%and 23.68%,respectively.Walnuts from Yunnan and Shanxi as well as peanuts from Hunan province were not contaminated with STC.Compared with samples from supermarkets and farmer-market,the stored rice for one-two years was higher for positive rate with 4.4%and 84.8%,the mean levels was 0.84?g/kg and 2.38 ?g/kg.3.The aim of this chapter was to investigate the effect of STC on A.flavus growth and AFB1 production in PDB(potato dextrose agar)medium at different concentration.Dry weight of mycelium in different days was measured and AFB1 extracted from cultures was quantified by HPLC-MS/MS.The result indicated that A.flavus was not affected by the presence of STC.Moreover,the content of AFB1 in two medium was gradually increasing with the extension of the cultural time,but the levels in cultures with STC were reduced compared to control cultures and a significant difference between concentrations was performed.This means when STC is present the production of AFB1 by A.flavus in PDB medium is inhibited.4.High-throughput sequencing was applied to analyze the transcriptome of A.flavus cultured in PDB with and without STC.Result demonstrated that 3377 differentially expressed gene were identified between treatment and control,including 1182 up-regulated and 2195 down-regulated.GO enrichment analysis showed that these genes mainly took part in cellular component organization or biogenesis,intracellular organelle,organelle,macromolecular complex.KEGG enrichment shown that the pathway was mainly valine,leucine and isoleucine biosynthesis and aflatoxin biosynthesis.Furthermore,there is lesser impact on secondary metabolic gene clusters in response to STC.However,30 genes in aflatoxin biosynthesis gene cluster were in different degree down-expressed and norB was inhibited totally.Enhancing oxidoreductase activity,promoting fungal growth and improving branched chain amino acid biosynthesis is the posiible mechanism to inhibit AFs biosynthesis.5.The isobaric tagging for relative and absolute quantitation(iTRAQ)technique was used to investigate the effect of STC on the proteomic profile of A.flavus.A total of 2733 proteins were identified,and 331 proteins were differentially expressed in response to STC.Among these 331 proteins,48 were up-regulated and 283 were down-regulated.According to GO and KEGG analysis for all differentially expressed proteins,the cell part,protein-containing complex,catalytic activity,antioxidant activity and organelle part were enriched.And the differentially expressed proteins were mainly participated in the metabolic pathways about aflatoxin biosynthesis,glycolysis/gluconeogenesis,glutathione metabolism and carbon metabolism.Above all,the up-regulated expression of enzyme activity in carbohydrate metabolism and down-regulated expression of enzyme in glutathione metabolism may be the potential mechanism to inhibit AFs biosynthesis.Moreover,12 proteins were identified in aflatoxins biosynthesis,7 of which were down-expressed.AflG,the fold change of was the largest in aflatoxin biosynthesis,may be the key enzyme that STC inhibits the synthesis of aflatoxin. |
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