Investigation Of The Inhibitory Effect And Mechanism Of 2-ethylhexanol On Aspergillus Flavus

Aspergillus flavus(A.flavus)can contaminate a variety of food and oil crops,including maize,wheat,and peanut,causing them to have reduced food and economic value.In addition,aflatoxin B1(AFB1)produced by A.flavus is classified as a class I carcinogen by the International Agency for Research on Cancer,posing a serious threat to the lives of humans and animals.In the previous research results of the group,it was found that 2-ethylhexanol,a volatile organic substance produced by a strain of Bacillus subtilis,has an anti-mold effect on unhulled rice,but its anti-mold effect and mechanism are still unclear.In this thesis,we firstly revealed the preliminary effect of 2-ethylhexanol on A.flavus,followed by a preliminary investigation on the inhibition mechanism of its growth and toxicity production,and finally conducted a study on the application of 2-ethylhexanol in the storage and preservation of maize,wheat,and peanut with high moisture content.The main results are as follows:1.The effects of 2-ethylhexanol on the minimum inhibitory concentration(MIC)and minimum bactericidal concentration(MBC),spore germination,mycelium,and colony diameter of A.flavus were investigated by two different modes of action,the double petri dish method,and the 96-well plate gradient dilution method.The MIC values of 2-ethylhexanol against A.flavus were 0.169 and 6.25μL/mL measured by the double petri dish method and gradient dilution method,respectively.2-ethylhexanol at both concentrations significantly reduced the germination rate of spores,decreased the dry weight of mycelium,and inhibited the re-expansion of colony diameter.2.The effects of 2-ethylhexanol on A.flavus gene transcription were analyzed by combining the effects of intracellular material leakage and conductivity,ergosterol content,cell membrane integrity,reactive oxygen species(ROS)content,DNA damage level,AFB1 content,with SEM and TEM observations of cell morphology and microstructure,the transcript levels of related gene expression were analyzed using RTqPCR technique.The results showed that the ergosterol content of A.flavus decreased after 2-ethylhexanol treatment,the material leakage was severe,the extracellular conductivity increased,the cell membrane permeability increased,the level of ROS increased,the damage to DNA was intensified,the accumulation of AFB1 content was reduced,most of the genes related to the ergosterol synthesis pathway,the AFB1 synthesis pathway pre-regulatory gene aflD and the global regulator LeaA,VeA,and VelB genes were down-regulated,which furthermore prevented the growth and toxicity production of A.flavus.3.Maize,wheat,and peanut with high moisture content were used as the growth substrates of A.flavus,and the effect of 2-ethylhexanol fumigation was evaluated by measuring the total number of molds,fatty acid values and AFB1 content of maize,wheat,and peanut after inoculation with A.flavus under storage conditions at a temperature of 28±2℃,with a humidity of 75%.The results showed that 2ethylhexanol at a concentration of 0.169 μL/mL could provide good mildew protection for maize and wheat,while 2-ethylhexanol fumigation could significantly inhibit the increase of fatty acid values of maize,wheat,and peanut and effectively reduce the accumulation of AFB1 content,thus delaying the deterioration of quality.