Screening,Identification And Degradation Mechanism Analysis Of T-2 Toxin-degrading Strain

T-2 toxin belongs to Class A trichothecene toxin,which is extremely toxic,widely pollutes grain,feed and food,and seriously endangers the life and health of human beings and animals.The purpose of this study is to screen T-2 toxin-degrading strains from wheat samples,explore their degradation characteristics and mechanisms,and optimize the enzyme-producing conditions of the degrading strains.The main contents and results are as follows:(1)From 50 wheat samples,T-2 toxin-free strains were screened and identified with T-2 toxin as substrate.Firstly,30 mixed bacteria samples with the removal rate of T-2 toxin above 90%were obtained,and 48 single bacteria were obtained after separation and purification.Ten single bacteria with removal rate above 90%were further screened,and finally two high-efficiency T-2 toxin-degrading strains AFJ-2 and AFJ-3 were obtained.Through morphological analysis and 16S rDNA molecular biological analysis,strain AFJ-2 was preliminarily identified as a Curtobacterium sp.and strain AFJ-3 was identified as Bacillus sp.(2)The detoxification characteristics of T-2 toxin-degrading strains AFJ-2 and AFJ-3 were explored,including the determination of detoxification curve,the study of adsorption,the localization of degrading active substances and the influence of enzyme inhibitors.The results showed that strains AFJ-2 and AFJ-3 had high degradation efficiency for T-2 toxin,and the concentration of T-2 toxin was 5 μg·mL-1 in 7 h and 12 h,respectively.Excluding adsorption,T-2 toxin-degrading by two strains belongs to biodegradation,and the degradable active substances are located in cells.Enzyme inhibitors SDS,PMSF and proteinase K can significantly inhibit the degradation of T-2 toxin by the two strains,indicating that the degrading active substance is intracellular degrading enzyme.(3)The metabolites of T-2 toxin degradation by strains AFJ-2 and AFJ-3 were investigated by UPLC-Q-TOF-MS/MS.Their degradation mechanism were studied through analyzing their primary and secondary mass spectra by software Mass Hunter,and comparing with the standard samples.The results showed that strain AFJ-2 transformed T-2 toxin into HT-2 and T-2 triol,and strain AFJ-3 transformed T-2 toxin into NEO.It was speculated that the interaction of AFJ-2 and AFJ-3 could transform T-2 toxin into 4-deacetylation-NEO.It is reported that the toxicity of HT-2,T-2 triol and NEO is lower than that of T-2 toxin,but the toxicity of 4-deacetyl-NEO has not been reported.(4)The fermentation process of T-2 toxin-degrading strain AFJ-3 was optimized by single factor experiment,climbing experiment and response surface experiment.When the fermentation medium is yeast extract 5 g·L-1,MgSO4·7H2O 16 g·L-1,soybean cake powder 5 g·L-1,the fermentation conditions are initial pH5.8,temperature 36.5℃,inoculum size 0.5%,and rotation speed 200 rpm,the maximum enzyme activity reaches 1674.68 U·mL-1,which is 47%higher than that before optimization.