Study On Inhibitory Effect Of O-vanillin Against Aspergillus Flavus

Aspergillus flavus(A.flavus)is one of the important causes of food pollution.It not only easily pollutes peanut,corn and other food crops,but also produces carcinogenic aflatoxin.Therefore,it is necessary to study the effective control methods of A.flavus pollution.At present,the control of A.flavus has carried out a lot of research,such as physical,chemical,biological methods.Physical method is not suitable for routine use because of its complicated operation and high cost.In chemical methods,synthetic preservatives(such as benzoic acid and its sodium salt,p-hydroxybenzoate,etc.)are not conducive to the environment,but also can not guarantee human health.Compared with synthetic chemicals,natural products from plants and microorganisms have attracted much attention due to their high yield,low residue,high efficiency,environmental and human health and safety.o-Vanillin is a natural plant product.In this paper,o-vanillin was taken as the research object to study its inhibitory effect on A.flavus and explore its antifungal mechanism.The main research results are as follows:(1)The minimum inhibitory concentration(MIC)of o-vanillin on spore germination and mycelial growth of A.flavus were 100?g/m L.By scanning electron microscopy(SEM)and transmission electron microscopy(TEM),it was observed that 100?g/m L of o-vanillin could damage the surface and internal microstructure of A.flavus mycelia,resulting in the folding and deformation of mycelia surface,serious or even fracture,and the reduction of fiber modifications on the outer wall of cells.The internal structure was disorganized,the plasma membrane was separated,the cytoplasm was lost and the cell wall was thinned.The results showed that o-vanillin could completely inhibit the growth of A.flavus on peanuts and corn at 100?g/m L within 72 h.(2)To study the effect of o-Vanillin on the cell wall of A.flavus.By Fourier transform infrared(FTIR)and X-ray photoelectron spectroscopy(XPS),it was found that o-Vanillin changed the OH group and the protein on the cell wall.In addition,o-vanillin significantly reduced the content of?-1,3-glucan in cell wall,but had no significant effect on the content of chitin.(3)To study the effect of o-Vanillin on cell membrane of A.flavus.The effect of pyridine iodide(PI)staining on cell membrane integrity was determined.The influence of content,relative conductivity and p H,Na~+,K~+on membrane permeability was determined.The contents of total lipid,trehalose,ergosterol,glycerol,reactive oxygen species(ROS),catalase(CAT),peroxidase(POD),superoxide dismutase(SOD),malondialdehyde(MDA)and glutathione(GSH)were determined in cell membrane.The results were as follows:After high concentration o-vanillin treatment,PI fluorescence was strong,which destroyed the integrity of cell membrane.o-Vanillin resulted in the increase of the content and relative conductivity The content of Na~+remained unchanged,but K~+increased,which indicated that o-vanillin increased the permeability of cell membrane,caused ion leakage and affected the potassium channel.The p H value decreased with the increase of o-vanillin concentration,indicating that o-vanillin induced A.flavus mycelium to release acid ions.The content of total lipid,trehalose and ergosterol decreased in cell membrane,while the content of glycerol increased,which indicated that o-vanillin could destroy the structure of cell membrane.ROS,MDA and GSH increased,while the activities of CAT,POD and(SOD decreased,which indicated that o-vanillin caused ROS accumulation and weakened the ability of cell to scavenge free radicals.(4)To study the effect of o-vanillin on A.flavus mitochondria.By measuring acetone acid and citric acid synthase(CS),isocitrate dehydrogenase(ICDH)and?-ketone glutarate dehydrogenase(?-KGDH),succinate dehydrogenase(SDH)and malate dehydrogenase(MDH)activity,the results showed that the pyruvate decreased,the activities of CS,ICDH,?-KGDH and SDH decreased,and the activity of MDH increased after the treatment of o-vanillin,which indicated that o-vanillin could destroy the tricarboxylic acid cycle and cause mitochondrial dysfunction.(5)The results of transcriptome sequencing showed that there were 5600 differential genes induced by o-vanillin in A.flavus,among which 3822 genes were up-regulated and1778 genes were down-regulated.It mainly affects genes related to the synthesis of?-1,3-glucan in cell wall and trehalose and ergosterol in cell membrane.The GO function enrichment results of different genes showed that o-vanillin mainly affected the metabolism,transmembrane transport and cell membrane of A.flavus.At the same time,the results of KEGG pathway enrichment showed that the down-regulated gene enrichment pathways were mainly the tricarboxylic acid cycle,ergosterol synthesis and lipid metabolism pathways.We believe that o-vanillin can affect the cell wall,cell membrane and mitochondria of A.flavus.