| Aspergillus flavus pollution and aflatoxins(AFs)pollution in pepper powder is a serious problem,which not only causes great economic losses,but also poses a serious threat to the health of consumers.For the prevention and control of A.flavus pollution in pepper powder,physical sterilization methods,such as ultraviolet disinfection and pasteurization,are low in efficiency.In addition,chemical synthetic fungicides may cause drug-resistant strains and environmental pollution.Therefore,it always is a focus of researchers to find appropriate measures to reduce the risk of A.flavus pollution in pepper powder.At present,a large number of studies have shown that many microorganisms can produce volatile organic compounds(VOCs)with antibacterial activity.VOCs have wide coverage,high efficiency and no direct contact with food,which have broad application prospects in the field of fungal pollution control of agricultural products.Lactic acid bacteria are recognized as safe food-grade probiotics.Its metabolites such as organic acids,antimicrobial peptides and enzymes with antibacterial activity have been reported to effectively inhibit the growth of harmful microorganisms in food.In recent years,a large number of studies have shown that VOCs produced by lactic acid bacteria also have excellent antibacterial effects,and have broad application prospects in the control of spoilage microorganisms and toxigenic fungi in food.This paper focuses on the inhibitory effect of VOCs produced by six lactic acid bacteria strains on the growth of A.flavus,studies the inhibitory effect of the main antifungal active compounds in VOCs on A.flavus,and analyzes the antifungal mechanism of the antifungal active compounds in combination with transcriptomics,and provides theoretical support for the development of new antifungal drugs suitable for the prevention and control of A.flavus pollution in pepper powder.Our study revealed the following:1、A mouth-to-mouth sealed plate assay method was used to investigate the inhibitory effect of VOCs released by six lactic acid bacteria strains on the growth of A.flavus,and gas chromatography-mass spectrometry(GC-MS)was used to analyze the composition and relative content of VOCs.Partial least squares analysis(PLS)was used to analyze the correlation between the antifungal effect and the relative content of VOCs,which showed that among the VOCs released by six lactic acid bacteria strains,1-pentanal,1-pentanol,glycidol,1-hexanol and 1-heptanol were the five compounds most related to the antifungal effect.The pure substances test in vitro showed that the five compounds can effectively inhibit the mycelium growth of A.flavus.In addition,the inhibition rate of AFs is positively related to the concentration of 1-pentanal and 1-pentanol,have a positive correlation with the concentration of,while glycidol,1-hexanol and 1-heptanol have the effect of low promotion and high inhibition on the production of AFs.The inhibitory effect of the binary combination of five antifungal compounds on A.flavus was determined by checkerboard method.The result that the minimum inhibitory concentration(MIC)of the combination of 1-pentanal and 1-heptanol was only half of which used alone showed that they had additive effect against A.flavus,while other combinations were irrelevant or antagonistic.2、A mouth-to-mouth sealed plate assay method was also used to investigate the inhibition mechanism of 1-pentanal,1-heptanol and their combination(C-VOCs)on A.flavus.The results showed that the morphology and structure of A.flavus cells changed significantly after treatment with VOCs,the mycelia twisted,deformed,and the spores sunken and cracked;The permeability of cell membrane of A.flavus increased,leading to the leakage of intracellular substances such as nucleic acid,protein and sugar,and the integrity of cell membrane of A.flavus was destroyed,the content of main components of cell membrane such as lipid and ergosterol decreased,while the content of malondialdehyde(MDA)increased,leading to membrane lipid peroxidation;The respiration and energy metabolism of A.flavus were inhibited,and the intracellular ATP content,ATPase activity,succinate dehydrogenase(SDH)activity and malate dehydrogenase(MDH)activity decreased.3、Samples of A.flavus fumigated with 1-pentanal,1-heptanol and C-VOCs were analyzed with transcriptomic techniques,and antifungal mechanism of VOCs against A.flavus was further revealed from a molecular biology perspective.Our results showed that A.flavus treated with 1-pentanal,1-heptanol and C-VOCs generated a significant number of differentially expressed genes(DEGs).Gene Ontology(GO)enrichment analysis showed that the attribute classification of DEGs in the cell component was closely related to the membrane function,which further confirmed that the cell membrane was main target of 1-pentanal,1-heptanol and CVOCs against A.flavus.Kyoto encyclopedia of genes and genomes(KEGG)enrichment analysis showed that multiple metabolic pathways of A.flavus treated with 1-pentanal,1-heptanol and C-VOCs were affected,and the significantly different metabolic pathways were mostly related to sugar metabolism and amino acid metabolism.VOCs affected the expression of key enzymes in these metabolic pathways,which maked the whole metabolic pathway unable to operate normally.According to the above experimental results,the VOCs produced by the tested lactic acid bacteria can effectively inhibit the growth of A.flavus.1-pentanal,1-heptanol,glycidol,1-hexanol and 1-pentanol are the key antifungal factors.Meanwhile,1-pentanal and 1-heptanol has additive effect against A.flavus;The combination of 1-pentanal and 1-heptanol can destroy A.flavus cellular morphology and structure,can damage the function of cell membrane,disrupt energy metabolism and block the normal expression of related genes and key enzymes in metabolic pathway,thus achieving the inhibition effect against A.flavus.Therefore,the combined use of 1-pentanal and 1-heptanol is expected to become a new control method to effectively control A.flavus pollution in agricultural products. |
PDF Full Text Request