The Inhibitory Effect Of Plant-based Active Ingredients On Aspergillus Flavus And Its Application In Peanut Butter

As a harmful fungus,Aspergillus flavus（A.flavus）can easily infect food crops such as corn,peanuts and wheat,which not only damages grain quality,but also affects economic benefits.It is easy to infect humans and animals,leading to aspergillosis in humans and animals;more importantly,A.flavus can produce a large number of harmful metabolites,among which aflatoxin B₁（AFB₁）is the most common one and has been listed as a class I carcinogen by the International Agency for Research on Cancer.For the prevention and control of A.flavus,for the prevention and control of A.flavus,the current common measures are:a.physical methods such as microwave,radiation and light regulation;b.chemical methods such as potassium sorbate,sodium benzoate and ozone;c.Antagonistic bacteria and its enzyme production,Bacillus and its enzyme production and other biological methods.However,the above methods are limited in the use process because of their high operation difficulty,easy to produce drug resistance and low application.Plant-derived natural compounds have attracted more and more researchers’attention due to their advantages of green,high efficiency and safety.This paper mainly studies the inhibitory effect of plant derived active ingredients from different sources,such as thymol,carvacrol,o-vanillin,citral,eugenol,pomegranate peel and ginger essential oil（GEO）,on Aspergillus flavus and the application of ginger essential oil chitosan film（GEO-C）in food.The main research results are as follows:（1）Five plant derived active ingredients were selected and compounded in pairs.When the concentration ratio of citral and eugenol was 1/4MIC+1/4MIC,the fractional inhibitory concentration index（FIC）was 0.5,which had synergistic antifungal effect on A.flavus.Propidium iodide（PI）staining experiments showed that the combination of citral and eugenol destroyed the cell membrane integrity of A.flavus.The results of cell content release experiment,relative conductivity and extracellular pH value showed that the leakage of nucleic acids,ions and other substances in cells and the content of extracellular acidic substances were high.Fluorescence white staining experiments showed that the internal diaphragm of A.flavus mycelium was destroyed.Treatment of peanuts with 1/4MIC+1/4MIC citral and eugenol at 72 h can completely inhibit the growth of A.flavus on peanuts,providing a scientific basis for reducing the use of antifungal agents and reducing costs.（2）Pomegranate peel ethanol extract has a strong antifungal effect on A.flavus.The minimal inhibitory concentration（MIC）of pomegranate peel extract against A.flavus was 10mg/mL.PI staining experiments showed that pomegranate peel extract destroyed the cell membrane integrity of A.flavus and the degree of damage was positively correlated with the concentration of pomegranate peel extract.The determination of cell content release,relative conductivity and extracellular pH value showed that with the increase of pomegranate peel extract concentration,OD₂₆₀ and relative conductivity increased significantly,and pH value decreased,indicating that pomegranate peel extract increased the permeability of A.flavus cell membrane,resulting in the leakage of nucleic acids,ions and other substances in the cell.The total lipid content in the cell membrane decreased,indicating that the pomegranate peel extract destroyed the cell membrane components of A.flavus.At the same time,in vitro antifungal experiments showed that pomegranate peel extract had the potential to be applied to grain preservation and could achieve the effect of reuse of peel waste.（3）GEO was extracted from ginger by steam distillation.GEO was analyzed by gas chromatography-mass spectrometry（GC-MS）.A total of 104 compounds were detected,of which the highest content wasα-zingiberene 25.3%.The MIC of GEO against A.flavus was 8μl/mL.PI staining experiment,cell content release experiment,relative conductivity and extracellular Ph value determination experiments showed that GEO destroyed the integrity of the cell membrane of A.flavus,resulting in the leakage of intracellular nucleic acids and acidic substances.The determination of total lipid and ergosterol content showed that GEO destroyed the cell membrane components of A.flavus mycelium.In vitro antifungal experiments,MIC group could completely inhibit the growth of A.flavus on peanut and corn.（4）Dissolved 3 g of chitosan in 150 mL 1.5%glacial acetic acid,stirred at 40°C until complete dissolution,and add 0.45 g of Tween and different concentrations of GEO and Tween80 to prepare GEO-C.GEO-C was prepared and the thickness,swelling,water solubility,volatile component content,and transmittance of the film were measured.The results showed that the film thickness increased significantly with the increase of GEO concentration,while the swelling,water solubility,volatile component content,and transmittance decreased significantly with the increase of GEO concentration;DPPH and ABST free radical scavenging experiments showed that with the increase of GEO concentration,the free radical scavenging rate of GEO-C significantly increased,indicating that GEO-C has antioxidant activity.Applying GEO-C to peanut butter and studying the effects of this film on the antifungal effect,pH value,texture,acid value,peroxide value,thiobarbituric acid,and carbonyl content of peanut butter during storage,as well as comparing it with peanut butter without any treatment,the results showed that GEO-C can inhibit the growth of A.flavus in peanut butter without significant changes in pH value,reducing the viscosity of peanut butter,and reducing the rate of lipid oxidation and protein oxidation,Thereby improving the quality of peanut butter.