Inhibition And Mechanism Of Tea Polyphenol Monomers On AFB₁ Production By Aspergillus Flavus

Aflatoxin B₁（AFB₁）is a strongly carcinogenic mycotoxin produced by fungi such as Aspergillus flavus（A.flavus）.Oxidative stress induced by ROS is the prerequisite for AFB₁ biosynthesis.Antioxidants can effectively inhibit the biosynthesis of AFB₁.Previous studies indicated that tea polyphenols can significantly reduce intracellular oxidative stress and inhibit AFB₁ synthesis of A.flavus,but the mechanism is unclear.Therefor,the main monomer that inhibited the synthesis of AFB₁ were identified by comparing inhibition effect of five tea polyphenol monomers（EGCG,EC,ECG,EGC,C）.And the relationship between monomers structure and inhibition was depicted after that.Then,the effect of the main monomer on the transcriptome of A.flavus was studied by RNA-Seq,to obtain genes associated with aflatoxin pathway and oxidative stress signaling pathway.Finally,the molecular mechanism of inhibition was elucidated by analyzing the inhibition effect from the aspects of gene transcription,protein expression and AFB₁ metabolic pathways.The main results in this study are as follows:1.Five tea polyphenol monomers had no obvious inhibition on the growth of A.flavus and didn’t damage the mycelia structure.However,they could effectively inhibit the biosynthesis of AFB₁ and the avtivity decreased along series:EGCG>EC>ECG>EGC>C.The results showed that EGCG exhibited the highest inhibitory activity at 2 mg/mL（92.00%）.Five tea polyphenol monomers were calculated using the DFT B3LYP method with 6-31G（d,p）basis set to explore the relationship between the structure and the inhibition of AFB₁biosynthesis.The results showed that ester type monomers had better inhibition activity than non-ester type.Compounds with the more hydroxyl groups and the larger positive charges number of hydroxyl H atom,had the higher antioxidant activity and inhibition effect,indicating that inhibition of AFB₁ synthesis related to the ability of scavenging radicals.In addition,the smaller LUMO energy of the molecules,the higher ability to inhibit the synthesis of AFB₁,showing that tea polyphenol monomers would clear electronics when they inhibited AFB₁synthesis.2.Analysis of the transcriptome data of A.flavus showed that there were927 differentially espressed genes（p<0.05）caused by EGCG,with 496down-regulated and 431 up-regulated.Up-regulated genes were mainly involved in the synthesis of antioxidant enzymes and lipids,and down-regulated genes were related to signaling pathways activated by stimulation of oxidative stress.Some of aflatoxin biosynthetic pathway genes were down-regulated by EGCG,including initial genes aflA,aflC,aflE,mid-term gene aflJ and transcription factor gene aflR.In addition,genes（mkk2,sakA,atf A）in oxidative stress-mediated MAPK signaling pathway were also down-regulated.It was speculated that EGCG might inhibit the transcription of aflatoxin biosynthetic pathway genes（afl A,afl C,aflE,aflJ,aflR）through the MAPK pathway and result in inhibition of AFB₁ synthesis.3.Based on the data analysis of the A.flavus transcriptome,the effects of EGCG on the following aspects were investigated:intracellular ROS levels in A.flavus,expression and phosphorylation of SakA in the cytoplasm and nucleus,transcription of aflatoxin biosynthetic pathway genes（aflA,aflC,afl E,aflJ,aflR）and transcription factor gene atfA,NOR and AFB₁ synthesis.The results showed that EGCG could significantly reduce the intracellular ROS level of A.flavus,and relieve the intracellular oxidative stress.EGCG also inhibited the activation of SakA by ROS,which decreased the phosphorylation level of the kinase in cytoplasm and nucleus by 38.92%and 78.64%,respectively.EGCG down-regulated the transcription of atf A,afl A,afl C,aflE,aflJ,and aflR,and reduced the synthesis of NOR by 70.91%,which in turn blocked the synthesis of AFB₁ with the inhibition of 87.50%.The above results indicated that EGCG inhibited the transcriptional activation of aflatoxin biosynthetic pathway genes（aflA,aflC,aflE,afl J,aflR）by inhibiting ROS-mediated SakA/AtfA signaling pathway and blocked the synthesis of AFB₁ by limiting the supply of synthesis precursors.The research contributed to deeply understanding the complex regulatory network of AFB₁ synthesis,which provided the theoretical foundation for finding and developing natural inhibitors to control AFB₁ contamination in food industry.