Screening And Characteristics Of Aflatoxin B1-degrading Microorganisms

Aflatoxin(Aflatoxin,AFT)have very strong carcinogenic and mutagenic and teratogenic nature which is secondary metabolites of aspergillus flavus and parasitic aspergillus.It widely exist in food,feed and agricultural products.It can not only make great harm to the health of human beings and animals,but also will bring huge economic losses at the same time and has been widely concerned in food safety.Till now several methods have been developed to remove and detoxicate AFB1.Although physical and chemical methods could reduce AFB1 to some extent,but most of these methods usually lead to adverse effects such as loss of product nutrition,or ganoleptic qualities,easy to produce by-products and not completely remove toxin as well as requiring expensive equipments,which limited their practical applications.Therefore,it is an urgent need to solve the problem to find a safe and effective detoxification methods.Much attention has been paid to the biodegradation of mycotoxins in recent years,especially using microbes.It was reported that the microorganisms could bioconvert aflatoxin into non-toxicity or low toxicity products under mild conditions and without using chemicals and losing nutritive value.In this thesis,an aflatoxin-degrading strain was screened and identified,using coumarin as sole carbon source and energy sources to screen microorganisms which can high efficient degradation AFB1.(1)In this study,coumarin as sole carbon source isolated 86 AFB1 degradation microorganisms,12 strains have been found the degradation of AFB 1 capacity from 25%to 95%by HPLC.(2)Through analyze the ITS DNA gene sequencing,these 12 isolates belong to Fusarium sp.,Penicillium sp.,Acremonium sp.,Band acremonium sp.,Altemaria sp.,Emericellopsis pallid sp.,respectively.(3)The cell free supernatantof Fusarium sp.WCQ3361 degradation effect stronger than living cells and intracellular extract.The degradation of AFB 1 was very quickly by cell free supernatant of Fusarium sp.WCQ3361.The degradation rate was 70.20%after been treatmented 1 min,the degradation rate was78.81%after been treatment 20 min.The optimum pH was 7.0.The best carbon source for corn flour,the best nitrogen source for the beef extract.The cell free supernatantof WCQ3361 very resistant to heat treatment,the degradation ability does not decrease and it was 80.57%after boiled 10 min.The degradation ability of fermented supernatant was reduced by 50%after treatmented by proteinase K..MTT experiments further confirmed the fermented supernatant can degrade the AFB1,it in conformity with the experimental results before.The fermented of WCQ3361was used in the feeds which polluted by AFB 1 was showed very good degradation effect,the degradation efficiency were above 60%.Through ammonium sulfate precipitation and anion exchange column separated the active substances with degradation AFBlability.The saturation of 60%ammonium sulfate can settle protein with highest degradation activity of 86.62%.Through anion exchange column separated crude protein with higher degradation activity of 57.47%.(4)The cell free supernatant of Acremonium sp.WCQ6A degradation effect stronger than living cells and intracellular extract.The degradation ability of fermented supernatant was reduced after treatmented by proteinase,but the degradation ability does not change after thermal treatment.The degradation rate was 74.43%after been treatmented 5 min,the degradation rate was79.36%after been treatment 48 h.The degradation rate was keep above 71.60%.The optimum pH was 8.0.The influence of degradation rate more noticeable by metal ion.Fe3+and Zn2+can promote the degradation of AFB1,Ni2+could significantly inhibit the degradation of AFB1.The best carbon source for lactose,the best nitrogen source for the ammonium nitrate.(5)The degradation effect of the cell free supernatant of Penicillium sp.5952 was strongest.The degradation ability of he cell free supernatant was reduced to 6.21%after treatmented Pancreatic enzyme,the degradation ability was reduced to 12.99%after after thermal treatment.The degradation ability of the cell free supernatant was relatively slow,the degradation rate was 95.92%after been treatment 48 h.The degradation rate was keep above 92.81%from 30? to 50?.The optimum pH was 7.0 and the degradation rate was 94.99%.Cu2+and Zn2+could significantly inhibit the degradation of AFB1.Strain 5952 shown the degradation ability after fermentation for 18 h,degradation rate was 94.91%after fermentation for 48 h.Use the glucose,maltose,lactose sugar,sorbitol,maize flour and sugar as carbon source the degradation rate above 89.19%.In addition to using beef extract as nitrogen source degradation rate is relatively lower,the rest of the peptone,tryptone,soybean peptone,yeast extract and ammonium nitrate as nitrogen source degradation efficiency were above 91.25%.