Screening,Identification And Degradation Mechanism Of Aflatoxin B₁ Degrading Bacteria

Aflatoxin B₁（AFB₁）is a class of secondary metabolites produced by Aspergillus flavus,Aspergillus terreus and Aspergillus parasiticus containing a bifuran ring and oxanonaphthalenone structure,is a potent fungal toxin with mutagenic,hepatotoxic and immunosuppressive properties as well as carcinogenicity and teratogenicity AFB₁is often detected in products such as soil,grains,nuts,dairy products and edible oils.Feed and food products are highly susceptible to contamination when exposed to high temperatures and high humidity.Animals consuming AFB₁-contaminated toxins harm other animals by contaminating feed,animal by-products such as milk and dairy products,and thus entering the food chain.This leads to wider spread and serious consequences Therefore,the search for safe and effective methods for AFB₁degradation already cutting-edge research AFB₁can be degraded by physical,chemical and biological methods,and biological degradation has a high specificity,is less harmful to the quality of food,and can even completely degrade the toxin where appropriate Therefore,biologic degradationis slowly becoming the most appropriate detoxification method.The study was carried out in terms of screening and identification of efficient AFB₁degrading strains,optimization of fermentation industry,localization analysis of degrading active substances,analysis of degradation products and heterologous expression of degrading enzyme genes,and the main results are as follows:1.Screening and identification of AFB₁-degrading bacteria:In this study,303 morphologically different strains were initially isolated from different samples,and the AFB1 degradation rate was used as an index for re-screening.4 strains with good AFB1 degradation ability were obtained through re-screening,among which strain A6isolated from corn field soil had the strongest AFB1 degradation ability,and the AFB1degradation rate could reach 90.10%after co-incubation for 72 h.It was named as HNGD-A6 for subsequent experimental study.The strain HNGD-A6 was identified as Bacillus megaterium.2.Optimization of fermentation conditions for strain HNGD-A6:Optimization of medium types:The optimal medium for strain HNGD-A6 was LB medium（tryptone 1%,yeast extract 0.5%,sodium chloride 1%）through the study of the effect of different mediums on the degradation rate of AFB₁.Single-factor optimization of fermentation conditions:The optimal fermentation conditions for strain HNGD-A6 were0.01%inoculum,pH 8.0,temperature 37℃,loading volume 50 mL,and fermentation time48 h.Response surface test study:A response surface test design was conducted with fermentation temperature,pH,and loading volume as factors,and the optimal fermentation conditions for strain HNGD-A6 were predicted to be 0.01%inoculum,pH 7.9,temperature37.4℃,and loading volume.The optimal fermentation conditions were predicted to be0.01%inoculum,pH 7.9,37.4℃,52.2 mL loading volume,48 h fermentation time,and94.66%AFB₁degradation rate at 72 h.The degradation rate of AFB₁was 91.72%after PK and PK+SDS treatment,and the degradation effect was almost unchanged after heat treatment.The degradation activity was judged to be extracellular enzyme and heat resistant.3.Structural analysis of degradation products and toxicological evaluation:The degradation solution of AFB₁degraded by supernatant of strain HNGD-A6 was examined by UPLC-HRMS and two major degradation products were found,P1-AFD₁(C₁₆H₁₄O₅),and P2(C₁₄H₁₆N₂O₂).Among them,P1 was obtained from the opening of the lactone ring of the main virulence site of AFB₁;based on the structure of P2,it is known that all three virulence sites of AFB₁were destroyed.Salmonella typhimurium TA98 and TA100were used as the test strains,and the Ames test was performed on the degradation products of AFB₁by strain HNGD-A6.After 72 h of AFB₁degradation,the number of mutagenic colonies of the test strains was significantly lower compared with the number of colonies under the action of AFB₁,indicating that AFB₁had been mostly degraded after 72 h of degradation,and its mutagenicity was greatly reduced,and the test results were negative for mutagenicity.Zebrafish larvae were tested for the teratogenicity and hepatotoxicity of AFB₁degradation products by strain HNGD-A6.The survival rate of zebrafish larvae was determined by the zebrafish larvae survival rate test,and the induction concentration of AFB₁was 80ng/mL,and the survival rate of zebrafish larvae in both degradation product and reagent groups was 100%.After the induction of AFB₁,the liver of zebrafish larvae was obviously shrunken,the yolk sac was underdeveloped and showed obvious signs of blackening,and the morphology showed obvious teratogenic phenomena such as curved spine and curved tail,while there was no teratogenic phenomenon in the degradation product group.There was no significant difference between the degradation product group and the control group in the determination of ALT,AST and LDH,indicating that the degradation products were not toxic to the livers of zebrafish larvae.4.Genome-wide analysis and mining of degradase genes:After sample quality testing,library construction,library quality testing,library sequencing and information analysis,strain HNGD-A6 was analyzed by whole genome sequencing,and the genome sequence length was 5321281 bp obtained by sequencing and assembling,among which the ratio of guanine and cytosine（GC）was The total number of encoded proteins was 5366,115 tRNA genes and 39 rRNA genes,and the integrity of the genome assembly was good.The strain HNGD-A6 was sequenced and then subjected to functional annotation of universal databases,including Nr,GO,KEGG and other databases.The amino acid sequences of strain HNGD-A6 were library-built and then compared with known AFB1 degrading enzyme genes using Blast P function to obtain five AFB1 degrading enzyme candidate sequences.A heterologous recombination method was used to construct a protein expression vector using pET-28a（+）as the vector.The successfully constructed recombinant vector plasmid was introduced into E.coli expression strain BL21,and two candidate genes were successfully expressed by low temperature induction using IPTG at16℃.The purified AttM and CueO were incubated with AFB₁at different pH and temperature conditions for 24 h,and then tested for degradation effect.The degradation of AFB₁was best by AttM at pH 9 with a degradation rate of 57.71%and at 80℃with a degradation rate of 80.77%;the degradation of AFB₁was best by CueO at pH 9 with a degradation rate of 45.53%and at 80℃with a degradation rate of 65.62%.Both recombinant proteins degraded best at 80℃,which indicated that both of them have certain high temperature resistance properties.