Preliminary Study On Screening, Identification Of Aflatoxin B1 Degrading Strain And The Degradation Mechanism

As one of the most toxic in aflatoxin found currently,aflatoxin B₁?AFB₁?has strong hepatotoxicity,high mutagenicity and high teratogenicity and is widely used in agricultural products and feeds.It has serious threats to human health and also causes serious economic loss on grains and animal husbandry.AFB₁ has very stable physicochemical properties,and it is difficult to degrade during food processing.Compared with the physical and chemical methods,the reaction conditions of biodegradation AFB₁ are mild,not easy to cause nutrient loss and no newly produced toxins.Bacterial degradation AFB₁ is one efficient,safe and environmental detoxification method.At present,there are few applications of biodegradation of AFB₁ and no related applications of bioenzyme degradation of AFB₁ have been found.Therefore,the separation of highly efficient degradation of AFB₁ bacteria has important practical value.Aiming at separating and identifying strain for AFB₁ efficient degradation,through screening bacteria efficiently degrading AFB₁,condition optimizing degradation and action mechanism equivalence aspect for strain degradation AFB₁,following results can be obtained:?1?Adopt indirect competition ELISA method to determine content AFB₁ in culture medium,relative coefficient for obtain standard curve is more than 0.9990,recovery rate is more than 98%,precision is less than 5%.Linearity is better within scope of 2.5?g/kg to 75?g/kg.This shows that methods established by the test are simple,convenient,accurate and reliable.It is applicable to detection of AFB₁ in culture medium.?2?Coumarin is used for replacing AFB₁ as only carbon source,for preliminary test for AFB₁ degrading strain;add AFB₁ standard solution,carry out secondary screening test.Through screening test,nice strains which can remove AFB₁ are obtained,wherein,the highest removal rate is YC2 strain separated from fish intestine.Through elution and extraction for YC2 strain cell,determine remaining amount of AFB₁ in cell,determine the process of the strain removing AFB₁ is degradation reaction.?3?Through combining with morphological observation,physiological and biochemical identification and 16S rRNA gene sequence determination analysis,authenticate strain YC2 with highest activity for degrading AFB₁.Bacterial colony is milk-white round one,edge is regular and thin,central part of thallus is slightly ridge,moundy;it is rod-shaped when observed under microscope,gram stain is positive.After 16S rRNA gene sequence and phylogenetic analysis,through preliminary determination,the strain is bacillus subtilis.?4?Study on YC2 strain degradation aflatoxin B₁ condition.Give comparison on pH,temperature,incubation time and influence of metal ion on YC2 strain degradation activity,results show that,the best condition for degradation reaction of YC2 strain separated from fish intestines for aflatoxin B₁ is as below:pH 7,temperature is about 35?,incubation time is 72h,without needing to add metal ion.?5?From measuring the degradation ability of AFB₁ in fermentation supernatant liquid and intracellular liquid,it was found that the ability to degrade AFB₁ in supernatant liquid was highest?82.6%?.We can judge that the strain secretes one extracellular substance for degrading aflatoxin B₁?The degradation rate of AFB₁decreased significantly after heat treatment and protease K treatment.We can infer that an extracellular protease secreted by YC2 has degradation function of AFB₁.