Study On The Inhibitory Of Volatile Compounds From Zanthoxylum Schinifolium Pericarp Against Aspergillus Flavus Growth

Aspergillus flavus is a common spoilage fungus in post-harvest grains.Contamination by A.flavus can seriously affect the quantity and quality of stored grains such as peanuts,corn,rice,and wheat.It can even produce secondary metabolites aflatoxins,which poses a potential threat to the health of humans and animals.Therefore,preventing the growth of A.flavus in post-harvest grains is critical to ensuring grain quality and food safety.Plant volatile organic compounds（PVOCs）hold remarkable potential for controlling fungal decay in agro-and food products.The pericarp of Zanthoxylum schinifolium,a traditional Chinese spice,is conventionally buried in grain piles to prevent fungal spoilage of stored grains in China.However,the antifungal mechanism of VOCs from Z.schinifolium pericarp by which they inhibit fungal spoilage of grains remains unclear.In this study,the VOCs from Z.schinifolium pericarp were analyzed and the volatile components that played the main antifungal effect on A.flavus were clarified.And the inhibition mechanism of the major antifungal component against A.flavus growth was clarified through metabolomics,transcriptomics,and physiobiochemical analysis.The main conclusions are as follows:（1）Simulation storage experiments of wheat grains showed that the VOCs from Z.schinifolium pericarp could effectively inhibit the growth of A.flavus in 20%moisture wheat grains,and the GC-MS results showed that the two components with the highest content VOCs from Z.schinifolium pericarp were linalool（50.31%）and D-limonene（20.92%）.Vapor fumigation and liquid contact experiments showed that linalool was the main inhibitory component in the VOCs of Z.schinifolium,and the concentration of linalool that inhibited colony diameter and mycelial growth of A.flavus was 0.571 and 1.2μL/m L,respectively.（2）It was discovered that 1.2μL/m L linalool could induce damage to the A.flavus cell membrane and result in the leakage of cell contents by observing the changes in conductivity,A_260nm,and PI fluorescence staining of A.flavus mycelia.After 6 h of exposure to 1.2μL/m L linalool,metabolomic analysis revealed 90 differentially expressed metabolites in A.flavus mycelia,including 69 upregulated and 21 downregulated metabolites.These differentially expressed metabolites were mainly involved in biological pathways such as ABC transport,TCA cycle,linoleic acid metabolism,biosynthesis of amino acid and c AMP signaling pathway.Furthermore,linalool inhibited the mitochondrial ATPase and mitochondrial dehydrogenase activities of A.flavus,leading to a decrease in superoxide anion content and superoxide dismutase activity,and an increase in hydrogen peroxide content and catalase activity.It was speculated that linalool inhibited A.flavus growth by disrupting the permeability and integrity of cell membranes,tricarboxylic acid cycle,and ATP binding cassette transport and by inducing mitochondrial dysfunction and oxidative stress.（3）Linalool vapor at 800μL/L can effectively prevent A.flavus growth in 22%moisture wheat grains.The germination of A.flavus spores can be completely inhibited at a linalool concentration of 2μL/m L,and a linalool concentration of 10μL/m L can result in spore death.Scanning electron microscopy revealed that linalool treatment caused wrinkling and spore breakage.The transcriptomics results showed that 3806 genes were significantly differentially expressed in A.flavus spores exposed to 2μL/m L linalool,predominantly showing enrichment regarding the ribosome,DNA replication,glutathione metabolism,peroxisome,and MAPK signaling pathways.Flow cytometry showed that linalool treatment caused hyperpolarization of mitochondrial membrane potential.4’,6-Diamidino-2-phenylindole staining indicated that linalool caused DNA fragmentation in A.flavus spores,and monodansylcadaverine staining confirmed that linalool induced autophagy in A.flavus spores.Thus,we propose that linalool can inhibit A.flavus spore germination by damaging the plasma membrane,causing mitochondrial dysfunction and DNA damage,and inducing autophagy in A.flavus spores.In conclusion,this research found that linalool is the main volatile compound of the Z.schinifolium pericarp,which can effectively inhibit the growth of A.flavus.The target and molecular mechanism of linalool inhibiting the growth of A.flavus were investigated,which provides a theoretical basis for the development of a new natural antifungal agent for postharvest grain management.