Study On The Inhibition Of Carvacrol On The Growth And Toxin Production Of Aspergillus Flavus And Its Application

Aspergillus flavus is a kind of saprophytic fungi,which is a common mold in the natural environment,and the growth rate is fast.It often exists in moldy peanut,nut,corn and other food products.A.flavus can produce harmful secondary metabolites,mainly aflatoxins?AFT?,which has strong carcinogenic,teratogenic,mutagenic and immunosuppressive effects on human and animal liver.At present,it is known that aflatoxin B₁?AFB₁?is the most destructive and polluting of aflatoxins.AFB₁ has extreme toxicity,with 10 times stronger than potassium cyanide and 68 times higher than arsenic,which can cause acute hepatitis,liver cell necrosis,liver cancer and other diseases.A great number of studies have proved that plant essential oil has a good growth inhibition and toxic effect on A.flavus,and most of the plant essential oil has a high degree of biosafety,which meets the requirements of people for the safety of food preservatives,non-toxic and no side effects.Carvacrol,the main component of essential oil from Origanum and thyme,has good antibacterial effect and can inhibit the growth of microorganisms and mites.In this study,we used carvacrol to treat A.flavus,and studied the effect of carvacrol on apoptosis and gene expression of A.flavus.The main results are as follows:1.Inhibitory effect of carvacrol on spore germination and toxin production of A.flavusWhen the concentration of carvacrol was 300?g/ml,the germination of A.flavus spores could be completely inhibited,and the minimum inhibitory concentration of carvacrol on the growth of A.flavus was 37.5?g/ml,and it could stably inhibit the growth of A.flavus in 96 hours.The morphology of the mycelium was abnormal and lost the ability of absorbing water again.The dry weight of mycelium was inversely proportional to the concentration of carvacrol.It was found by SEM that 300?g/ml of carvacrol could lead to abnormal mycelium morphology,deformation,rupture,folding and curling.2.Apoptosis of A.flavus induced by carvacrolROS and NO are the signal molecules related to apoptosis.The amount of ROS produced by A.flavus was detected by fluorescence probe method.The results showed that 37.5?g/ml carvacrol could significantly induce the amount of ROS,and the amount of ROS produced was positively correlated with carvacrol concentration.It was found that the activity of NR and NOS catalyzing the synthesis of NO was up-regulated in different degrees after the action of 300?g/ml carvacrol,and the up-regulated range of NR was significantly higher than that of NOS.It was concluded that carvacrol could activate these two catalyzing pathways rapidly to produce more NO.At the same time,annexin V-FITC/PI double staining method was used to determine the apoptosis of A.flavus mycelium.It was found that 300?g/ml carvacrol could cause obvious cells apoptosis.Then,the enzyme activity of caspase-9,a key enzyme of apoptosis inducing pathway,was determined.It was found that 300?g/ml carvacrol treatment at 4 hours could significantly increase the activity of caspase-9 in cells,thus inducing apoptosis.3.Effect of carvacrol on gene expression of A.flavusThe transcriptome of A.flavus showed that the clean data of all samples reached6.18 Gb,and the percentage of Q30?base recognition accuracy 99.9%?base was91.79%or more.Transcriptome analysis showed that AFLR,PKS/NRPS,VEA,aflg,Aflc,AFLD,aflf,aflk,AFLP,aflq,AFLS,aflu,aflw and other toxin synthesis related genes were down-regulated,Rho2.Atf21,alcohol dehydrogenase,sulfite oxidase,nitric acid reductase and other growth and development related genes were also down-regulated in varying degrees.It can be seen that the effect of carvacrol can change the expression level of toxin synthesis related genes and growth-related genes,thus affecting the normal physiological metabolism and the synthesis of secondary metabolites.4.The inhibitory effect of carvacrol on toxin synthesis in simulated granaryAccording to the specific application of carvol and the storage mode of simulated grain,the granary model was designed and established.The volatile of carvol was used to inhibit the toxin synthesis of A.flavus.The results showed that the concentration of 1.5 ml/250 ml,0.75 ml/250 ml for corn,9 ml/19008 cm³,4.5ml/19008 cm³ for peanut can effectively inhibit the synthesis of aflatoxin,and has the characteristics of long-term stability;The concentration of 0.375 ml/250 ml for corn,2.25 ml/19008 cm³ for peanut is good in a short time,but it is not suitable for long-term storage.In the later stage,the amount of toxin synthesis is high and the safety degree is low.At the same time,according to the data of the control group,humidity is the influencing factor of aflatoxin synthesis.In conclusion,the study of the antibacterial effect of carvacrol can provide a theoretical basis for further exploring the antibacterial mechanism of plant essential oil and the application of carvacrol.