| Aspergillus flavus is a common aerobic fungus,not only easy to contaminate food crops(peanuts,corn),but also infest humans and animals caused by Aspergillosis,its production of aflatoxin(Afaltoxin)is a strong toxicity and strong carcinogenicity.Staphylococcus aureus is an important foodborne pathogen,and its secretion of enterotoxins and enterotoxin-like can cause serious food safety problems,and has a very high mortality and morbidity rate.In addition,S.aureus is highly resistant to drugs and is prone to develop resistance to antibiotic drugs.Currently,efficient control of pathogenic bacteria is mainly achieved by three methods: physical(adsorption,radiation,etc.),chemical(ammonia fumigation,alkali treatment,etc.),and biological(competitive inhibition and antagonism of microorganisms).Among them,natural products of plant origin have attracted much attention by virtue of green,safe and efficient.In this paper,we focus on the inhibitory effect and inhibition mechanism of hinokitiol against Aspergillus flavus and Staphylococcus aureus,and the main findings are as follows:(1)To investigate the inhibitory effect of 46 natural compounds against Aspergillus flavus and to screen them.Sorbic acid,as a commonly used chemical fungicide,has broad-spectrum bacterial inhibitory properties and good inhibition effect.Using sorbic acid as a positive control,the best inhibitory effect of hinokitiol on Aspergillus flavus was screened by the inhibition circle method and the medium with drug method,followed by 4-propylphenol,both of which were significantly better than sorbic acid.On this basis,the MICs of 4-propylphenol on the growth of Aspergillus flavus under contact and fumigation conditions were determined to be 0.35 and 0.04mg/m L,respectively.in the in vitro inhibition experiments,fumigation concentration of 1.1 mg/m L of 4-propylphenol could completely inhibit the growth of Aspergillus flavus on peanut,and fumigation at 1.7 mg/m L for 24 h could completely kill the spores of Aspergillus flavus on peanut.The inhibition effect of hinokitiol was better than that of 4-propylphenol,and the inhibition rate of hinokitiol and 4-propylphenol on at 0.2mg/m L concentration was 100% and 54.10%,respectively.(2)Inhibition of Aspergillus flavus and Staphylococcus aureus by hinokitiol.The inhibitory effect of hinokitiol on the two test bacteria was studied.The MIC and MFC of hinokitiol were 0.08 and 0.64 mg/m L for Aspergillus flavus and 0.04 and 0.08mg/m L for Staphylococcus aureus under the test conditions with drug medium;the MIC and MFC of positive control sorbic acid were 0.45 and 0.8 mg/m L for Aspergillus flavus and 0.8 and 1.4 mg/m L for Staphylococcus aureus,respectively.Under the closed fumigation condition,the MIC and MFC of hinokitiol for A.flavus were 0.016 and 0.032 mg/m L,and the MIC and MBC of S.aureus were 0.012 and 0.016 mg/m L,respectively,and sorbic acid had no fumigation inhibition effect on A.flavus and S.aureus.The above data showed that the bacterial inhibition effect of hinokitiol was significantly better than that of sorbic acid,and showed better fumigation inhibition.Further tests showed that hinokitiol can significantly inhibit Aspergillus flavus contamination of stored peanuts,fumigation concentration of 0.256 mg/m L,the growth of Aspergillus flavus on peanuts was completely inhibited,0.512 mg/m L fumigation for 24 h can be completely killed Aspergillus flavus spores on peanuts.(3)Mechanism of the inhibitory effect of hinokitiol on Aspergillus flavus and Staphylococcus aureus.The effects of hinokitiol on cell membrane integrity and permeability were determined by scanning electron microscopy,Annexin V-FITC/PI double staining and extracellular conductivity,and the effects of hinokitiol treatment on the inhibition of three oxidoreductase activities,ATP content,total dehydrogenase activity,reactive oxygen species(ROS)content and the addition of reactive oxygen species scavenger L-cysteine(Cys)in the cells of the test bacteria were also determined.The effect of the addition of reactive oxygen scavenger L-cysteine(Cys)on the inhibition effect.The results showed that the scanning electron microscopy results showed that the cell morphology of Aspergillus flavus and Staphylococcus aureus was changed,the cells were folded and depressed,and even ruptured after the treatment with hinokitiol;the PI staining and extracellular conductivity results showed that hinokitiol disrupted the integrity and permeability of the cell membrane of Aspergillus flavus and Staphylococcus aureus,and the intracellular material was leaked.The intracellular ROS content of both bacteria increased after hinokitiol treatment,and the inhibitory effect of hinokitiol on both bacteria was weakened by the addition of Cys(ROS scavenger),and the intracellular ROS accumulation was significantly inhibited,indicating that ROS was significantly correlated with the inhibitory effect of hinokitiol;meanwhile,the superoxide dismutase,catalase and peroxidase activities in the cells of the test bacteria were significantly reduced,and the ATP content decreased.At the same time,the superoxide dismutase,catalase and peroxidase activities,ATP content decreased,and total dehydrogenase activity decreased.It was shown that hinokitiol interfered with the redox system of the test cells,resulting in a surge of ROS,which caused oxidative damage and the accumulation of ROS that could not be cleared in time,further interfering with the energy metabolic pathway and affecting ATP synthesis,thus inhibiting the growth of A.flavus and S.aureus.(4)Transcriptomic analysis of the inhibitory effects of hinokitiol on A.flavus and S.aureus.The effect of hinokitiol on A.flavus resulted in 3407 differentially expressed genes,1639 up-regulated genes and 1768 down-regulated genes;the effect of hinokitiol on S.aureus resulted in 1316 differentially expressed genes,1255 up-regulated genes and 61 down-regulated genes.Jungianol mainly affected genes related to ergosterol synthesis and antioxidant system of A.flavus.As shown by GO enrichment analysis,hinokitiol mainly affected components such as cellular processes,metabolic processes and organelles in A.flavus,and cellular components such as synthetic processes,metabolic processes and cellular membranes in S.aureus.The results of KEGG Pathway showed that hinokitiol mainly affected the cellular metabolism,membrane transport and glycolysis,TCA cycle,steroid biosynthesis pathway of Aspergillus oryzae,and amino acid and energy metabolism pathway and membrane transport pathway of Staphylococcus aureus. |
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