Screening Of AFB₁-degrading Microbial Consortium And Study On Its Degradation Mechanism

Aflatoxin B₁（AFB₁）is the most toxic mycotoxin found to be extremely carcinogenic,teratogenic,nephrotoxic,and hepatotoxic,which is widely found in mold contaminated peanut,corn,cereals and other agricultural products and food.AFB₁ is an important factor causing food safety accidents.Therefore,the problem of AFB₁ pollution has attracted widely attention,and it is becoming more and more urgent to find ways to remove aflatoxin B₁ effectively and safely.Using microorganism to degrade aflatoxin efficiently and then to reduce and control the harm of aflatoxin,which has the advantages of environmental protection,high efficiency and no toxicity,is an effective way to solve the pollution of aflatoxin and become a research hot spot.At present,there are many researches studing on the effect of microbial degradation of AFB₁ all over the world,although some research progresses have been obtained,the detailed mechanism of microbial degradation of AFB₁and the research of specific degradation enzyme are still not deep enough.In addition,the research on microbial aggregates with special advantages in the degradation of AFB₁ is equally lacking,so it is necessary to continue further research to promote its wide application.In this study,the microorganisms were selected from the soil at the compost site of Ya’an campus of Sichuan Agricultural University as the material through the“external amoy method”to construct the microbial consortium"MBCA（Microbial Consortium A）"for the efficient degradation of AFB₁.Furthermore,the degradation effect of MBCA on AFB₁was investigated,and the microbial structure composition of MBCA was analyzed.The main findings are as follows:1.MBCA showed good degradation effcet of AFB₁,the optimum culture conditions were 55℃,pH 7.0,and the degradation rate of AFB₁ for 500μg/L for 120 h under this condition was up to 92%.The degradation ability of MBCA to AFB₁ is stable and has good environmental adaptability.The results of amplification and sequencing of the MBCA V3-V4 regions by Ion S5^TMXL high-throughput platform sequencing showed that the three microorganisms,Bacillus thermoamylovorans,Tepidimicrobiumsp GRM1 and Aeribacillus pallidus,might be key microorganisms for the degradation of AFB₁ by MBCA.2.MBCA degraded AFB₁ by secreting extracellular enzymes.The optimum degradation conditions of its extracellular enzyme crude extract were pH 8.0,55℃.The degradation rate of AFB₁ for 500μg/L was up to 91.2%for 72 h under this condition.Cu²⁺can promote the degradation ability of the crude enzyme extract.3.The AFB₁ degrading enzyme of MBCA was separated and purified by ammonium sulfate grading precipitation and ion exchange chromatography.The purified enzyme molecular weight was 66.2 kDa,the specific enzyme activity was 16.73×10³ U/mg,and the purification multiple was 15.64.The results of SDS-PAGE showed that the purification effect was good,and the apparent Km value of the enzyme was 6.91×10^-5 mol/L by double reciprocal mapping.The optimum reaction conditions of the enzyme were pH 6.0 and 60℃,and Fe²⁺promoted the enzyme activity to some extent.