Study On The Inactivation Effect And Mechanism Of Low-temperature Plasma Treatment On Aspergillus Flavus

Aspergillus flavus is widely distributed in nature and is a common saprophytic fungus.Aspergillus flavus is prone to contaminating food crops and damaging grain quality.At present,the main prevention and control methods for Aspergillus flavus include physical methods such as light,microwave,and radiation,chemical methods such as ammonia fumigation,ozone,natural compounds,and biological methods such as screening for antagonistic bacteria and their metabolic enzymes.Atmospheric low-temperature plasma is a new type of non hot food processing method that has broad application prospects in ensuring food safety and improving shelf life.Due to the complex composition and biological response of plasma,its inhibitory mechanism on microorganisms has not yet been fully elucidated,so further research is needed.This paper conducted experiments on the inhibitory effect of Aspergillus flavus after plasma treatment and preliminarily explored its antibacterial mechanism.(1)The inhibitory effect of low-temperature plasma on the growth of Aspergillus flavus and the inactivation effect of spores:Fresh Aspergillus flavus spores were used as materials and treated with low-temperature plasma for 0,15,30,60,and 90 seconds.Under in vitro conditions,treatment for 30 seconds can completely inhibit the germination of Aspergillus flavus spores and the growth of Aspergillus flavus mycelium.The scanning electron microscopy results showed that after lowtemperature plasma treatment,the spores of Aspergillus flavus underwent deformation and shrinkage,and the degree of deformation deepened with increasing treatment time.Under living conditions,60 s low-temperature plasma treatment can effectively inhibit the growth of Aspergillus flavus in peanut and corn kernels.(2)The effect of low-temperature plasma on the membrane structure of Aspergillus flavus spores:After low-temperature plasma treatment for 0,30,60,and 90 seconds,the p H of the spore suspension significantly decreased,and the active substances such as H2O2 and NO2-significantly increased.The solution showed strong oxidation and acidity.The leakage of cell contents and the change of cell microenvironment indicate that plasma destroys the cell membrane and changes the permeability of the cell membrane.The spores treated with plasma showed strong green fluorescence,with fluorescence intensities 2.01 ± 0.08 times,2.19 ± 0.24 times,and 3.89 ± 0.09 times higher than those of the control group,respectively.This confirms that low-temperature plasma induced oxidative damage to Aspergillus flavus spores,resulting in the accumulation of reactive oxygen species(ROS)in Aspergillus flavus.Under lowtemperature plasma stimulation,the activity of NADPH oxidase is significantly enhanced,rapidly catalyzing the generation of higher concentrations of ROS.In this process,the activities of superoxide dismutase(SOD),catalase(CAT)and peroxidase(POD)were inhibited,and the rate of ROS accumulation exceeded the rate of ROS clearance by the cell’s own antioxidant system,thus causing more ROS accumulation.A large number of free radicals act on lipids and proteins,and oxidative end products such as malondialdehyde(MDA)and protein carbonyl groups accumulate in large quantities,further leading to damage to cell membrane structure.(3)Gene expression profile analysis of Aspergillus flavus spores treated with lowtemperature plasma:A total of 3682 significantly differentially expressed genes(DEGs)were obtained through RNA sequencing,with up and down regulated genes accounting for 2001 and 1681 respectively.According to GO classification and enrichment results of differential genes,lowtemperature plasma mainly affects the synthesis and metabolism of Aspergillus flavus and the cell components such as organelle,cytoplasm and mitochondria.According to the classification and enrichment results of KEGG Pathway,low-temperature plasma mainly affects the ribosome,TCA cycle,glyoxylic acid and dicarboxylic acid metabolism,oxidative phosphorylation,protein processing in endoplasmic reticulum and other pathways of Aspergillus flavus.(4)The effect of low-temperature plasma on energy metabolism of Aspergillus flavus:Using the soluble protein,pyruvate,acetyl coenzyme A,and ATP content of Aspergillus flavus spores as indicators,the effect of low-temperature plasma on its metabolites under different treatment times was explored.The study found that the soluble protein content of Aspergillus flavus spores decreased by 27.3%,35.5%,and 40.9% compared to the control group after being treated with low-temperature plasma for 30 seconds,60 seconds,and 90 seconds,respectively,indicating a decrease in the catalytic activity of related enzymes in the cell body.The content of pyruvate increased by 60.8%,70.9%,and 103.3% compared to the control group,indicating that the downstream pathway of pyruvate may be inhibited.In addition,the content of acetyl Co A decreased by 56.5%,65.6%,and 79.4% compared to the control group,and the ATP content decreased by 43.0%,49.3%,and 54.9%,respectively,confirming that the downstream pathway of pyruvate was inhibited and the energy metabolism of Aspergillus flavus was abnormal.The total mitochondrial dehydrogenase activity decreased by 10.8%,26.9%,and 47.7%,respectively.The results further indicate that low-temperature plasma interferes with the energy metabolism pathway of Aspergillus flavus,thereby affecting ATP synthesis and achieving the effect of inhibiting Aspergillus flavus.