Inhibitory Effect Of Metabolites Of Bacillus Subtilis J-15 On The Growth And Toxin Production Of Aspergillus Flavus

Aflatoxins,a highly toxic and carcinogenic metabolite secreted by Aspergillus flavus,can cause mildew in dairy products,corn,peanut and other food and feed,and endanger the safety and health of the body.The development of effective control against Aspergillus flavus and aflatoxin pollution has attracted more attention than before.Bacillus subtilis J-15 obtained from cotton field soil in Xinjiang in the early stage has broad-spectrum antifungal activity and can inhibit the growth of Aspergillus flavus.It is confirmed that its antagonistic active substance is mycosubtilin(hereinafter referred to as Myco).Based on clarifying the inhibitory effect of metabolites of Bacillus subtilis J-15 on Aspergillus flavus,this paper further studied the mechanism of antifungal subtilisin inhibiting Aspergillus flavus at the cellular and transcriptome levels,The inhibition of Aspergillus flavus polluted by peanut and corn was studied.The main results are as follows:1.The half lethal concentration of antifungal subtilisin on the fungal model fungus Saccharomyces cerevisiae(4 ?g / ml)is the dose treatment of Aspergillus flavus spores.The spore surface is concave and shrunk into a "bowl" shape.With the extension of treatment time,the depression and shrinkage are more significant,and the spores tend to apoptosis and death with treatment.2.Take 4 ?g / ml When mycosubtilin was used to treat Aspergillus flavus mycelium,the smoothness of mycelium decreased,shrunk and sporulation capacity decreased with the extension of time.3.Take 4 ?g/mL?20 ?g/m L mycosubtilin treated Aspergillus flavus spores respectively.Transcriptome analysis showed that mycosubtilin could induce significant changes in the expression of genes related to energy metabolism and material metabolism of Aspergillus flavus,in which the expression of autophagy related protein atg22 was significantly up-regulated,and the expression of genes related to cell wall synthesis(such as Chitinase)was significantly up-regulated,but the expression of genes related to material transport was down-regulated,so it was down-regulated as a whole,The results were positively correlated with the treatment time and the concentration of mycosubtilin.In addition,the expression of afl R?afl E ?afl H?afl J?afl M ? afl P,the key regulatory genes synthesized with aflatoxin family,was significantly down regulated.4.0-400?g/mL concentration(100 ?g/ml as gradient)J-15 antifungal extract containing antifungal subtilisin was used to treat corn and peanut inoculated with Aspergillus flavus,and the effects of antifungal subtilisin on grain polluted by Aspergillus flavus and aflatoxin production were discussed.The results showed that with the increase of the concentration of J-15 antifungal crude extract,the inhibitory effect on the expansion of Aspergillus flavus plaque inoculated with corn and peanut was more significant,and J-15 antifungal crude extract could significantly inhibit the greening of plaque.It was further analyzed by HPLC post column derivatization 0,100 and 200 ?g/ml(100?g/ml as gradient)J-15 antifungal crude extract treatment on the accumulation of aflatoxin in peanut samples inoculated with Aspergillus flavus for a week.The results showed that with the increase of treatment concentration,the content of aflatoxin B1 decreased significantly,and aflatoxin G2 did not produce at all.This is consistent with the results of transcriptome analysis after mycosubtilin treatment of Aspergillus flavus.It may be that mycosubtilin inhibits the expression of key enzyme genes of aflatoxin synthesis.In summary,the metabolite of Bacillus subtilis,mycosubtilin,can interfere with the transcription and translation and material synthesis of Aspergillus flavus,damage the cell wall of Aspergillus flavus,increase the permeability of cell membrane,inhibit the synthesis of Aspergillus flavus and reduce the spore production of Aspergillus flavus by inhibiting the gene expression of the key enzyme of aflatoxin synthesis.It is a good resource for preventing aflatoxin pollution and reducing the accumulation of aflatoxin toxin in grain storage,It has great development potential.