| Aflatoxin is a kind of mycotoxin produced mainly by Aspergillus flavus and Aspergillus aspergillus.It is highly toxic and widely distributed.It can contaminate large quantities of food and feed,which poses a serious threat to human and animal safety.Traditional physical and chemical methods for the removal of aflatoxin have the disadvantages of secondary pollution and high cost,while biological methods for the removal of aflatoxin in grain have remarkable effect,low cost,no pollution and low impact on grain quality,which has gradually attracted wide attention.For the purpose of efficient degradation of aflatoxin B1(AFB1),strains that can effectively reduce AFB1 were isolated and screened from a variety of biomass contaminated with aflatoxin.Mixed bacteria were constructed through combination design,and conditions of efficient bacteria were optimized to study their degradation characteristics and degradation products.Finally,the optimal ratio and optimal conditions of AFB1 degradation compound bactericide were obtained,and the following results were obtained:1.Using coumarin as the only carbon source,three strains were isolated from fermented bamboo powder and sauerkraut in water,which had a degradation rate of AFB1 above 40%.which were named Z1,Z2 and K1.After isolation,purification and molecular biological identification,Z1 and K1 were identified as Bacillus subtilis,and Z2 were identified as Bacillus cereus.The conditions of these three bacteria were optimized and a complex bacterial agent was constructed.2.By optimizing the single factor,the fermentation conditions of the three strains were explored.After the environmental factors such as temperature,p H,fermentation time and toxin concentration were tested,it was found that the degradation rate of AFB1 of strain Z1 reached 58.59% when the fermentation time was 72 h and p H 6.0 at 31℃.The degradation rate of AFB1 of strain Z2 reached 54.32% at 31℃,p H7.0,fermentation time 72 h and initial toxin concentration of 20ng/m L.The degradation rate of AFB1 increased to 60.19% when K1 was treated at 31℃,p H 7.0,fermentation time 72 h and toxin concentration 20 ng/m L.3.In order to further enhance the effect of AFB1 degradation,the mixed bacterial agent was constructed by orthogonal design,and the proportion of fermentation broth of three strains was designed to explore the degradation ability of AFB1 under optimal conditions after mixed culture.It was concluded that when the ratio of the compound bacterial agent was Z1:Z2:K1: = 3:1:3,At 34℃,p H value 7,inoculation dosage 5% and glucose concentration 5g/L,the degradation rate of AFB1 reached 66.37%,which was significantly improved compared with the degradation effect of single strain.4.The content of AFB1 in the precipitation of three kinds of thalli was very low,indicating that the removal of AFB1 by thalli was degradation.The extracellular supernatant,bacterial suspension and intracellular fluid of the three kinds of bacteria were determined respectively,and the extracellular supernatant had the strongest degradation ability.Scanning electron microscopy(SEM)showed that the bacterial morphology changed after the degradation of the toxin,the surface crumpled,and the attached toxin sites could be seen.After the absorption and degradation of AFB1,the bacteria died and cracked.The analysis of five degradation products after degradation of AFB1 showed that the degradation effect of the compound bacterioides was achieved by destroying the dihydrogen double bond structure of AFB1 furan ring and vanillone ring structure,so as to achieve the detoxification effect.In this study,three kinds of bacteria were selected,which had a high effect on the degradation of AFB1,the preliminary study on its degradation characteristics and mechanism can provide theoretical basis and technical support for the degradation of AFB1 in food and feed,which is of great significance for the prevention and control of aflatoxin in agriculture. |
