Screening Of Aflatoxin B₁ Degrading Microorganisms And Study On The Degradation Mechanism

Mycotoxin contamination is a serious problem that has long plagued food safety and poses a great threat to the health of the people’s consumption.The contamination of food with mycotoxins has become increasingly serious in recent years due to the frequent occurrence of extreme weather.Therefore,blocking and controlling mycotoxin contamination and ensuring the quality and safety of food and oil is a major and urgent need of China.Among the mycotoxins that contaminate food,aflatoxins are hazards that cannot be ignored which are produced by Aspergillus strains and contain high toxicity and carcinogenicity.Furthermore,their chemical properties are stable.Therefore,how to reduce their harm is the current research hot spots and difficulties.At present,physical and chemical reduction technology research is extensive,and has achieved certain results.Biological reduction technology has become a current research hot spot because it is green,efficient and specific with no secondary pollution and other advantages.A strain of Meyerozyma guilliermondii（AF01）was selected from mycotoxin-contaminated cereals with both efficient adsorption and rapid degradation on aflatoxin B₁（AFB₁）.The degradation product was identified and its structure was characterized using a combination method of isotope tracing and nuclear magnetic resonance（NMR）identification.Further,the decoupling of the adsorption and degradation of AFB₁ by AF01 was achieved by the product localization method.The degradation enzyme gene was then mined and validated by a combination of bioinformatics and chemical analysis.The resolution of the degradation mechanism of AF01 was completed.Based on the above studies,the potential of AF01 to remove aflatoxin contamination in complex matrices was tested by fermentation experiments with highly toxic peanut meal.The main findings of the study are as follows:（1）Using enrichment culture and single bacterium screening,a new M.guilliermondii strain with stable detoxification effect on AFB₁ was isolated for the first time.Using 648 samples of mycotoxin-contaminated maize and rice collected from Funan County in Anhui Province and Weifang City in Shandong Province as materials,three strains that can stably remove AFB₁ were screened by enrichment culture using AFB₁ as the sole carbon source.After molecular identification and morphological analysis,the three bacteria were identified as M.guilliermondii,Rhodococcus erythropolis and Enterobacter hormaechei,which were numbered AF01,AF02 and AF02,respectively.Preliminary analysis of the AFB₁removal characteristics of the three bacteria showed that 85.90%,97.60%and 71.00%of AFB₁could be removed by the three strains inoculated into the toxin tube as a single bacterium,and the AF01 strain had both adsorption and degradation effects on AFB₁.This is the first report of the adsorption and degradation of AFB₁ by M.guilliermondii.（2）By rapidly targeting and determining the structure of AFB₁ degradation products by combining isotope tracing and NMR,the coupling problem of AFB₁ adsorption and degradation by AF01 was resolved,and a new degradation pathway of AFB₁ by AF01 was revealed.In order to decouple the adsorption and degradation of AFB₁ by the screened yeast（AF01）and to reveal the degradation pathway of AFB₁ by AF01,a combination of isotope tracing and multiple wave spectroscopy techniques including NMR was used.Firstly,based on a time-gradient sampling method combined with isotope tracer mass spectrometry analysis developed in this paper,the AFB₁ degradation products of the screened M.guilliermondii were rapidly and accurately targeted.Then,in order to accurately determine the structure of degradation products in this new degradation pathway of AFB₁ degradation by AF01,the degradation products were prepared and purified in large quantities with purity over 90%.Finally,the planar structure,spatial configuration and the agreement with the standards of the degradation products were determined by NMR,HPLC for the separation of chiral compounds and ultraviolet absorption spectroscopy scanning technique,respectively.The results showed that the composition of degradation products was AFL and epi-AFL,with AFL accounting for the majority.Based on the above findings,the product localization method was used to successfully resolve the type of action of each component of AF01 strain on AFB₁.The results showed that the function of cell-free fermentation supernatant of AF01 was adsorption;the function of intracellular lysate on AFB₁ was degradation;the function of living cells was mainly degradation,and the inactivated cells could only adsorb AFB₁.The degradation pathway of AFB₁ by M.guilliermondii revealed in this paper was firstly reported.（3）A novel AFB₁ degrading enzyme and its key encoding gene was discovered using bioinformatics analysis combined with inverse inference of degradation product structure.In order to reveal the mechanism of AFB₁ degradation by a new degradation pathway for AFB₁ by the screened yeast,M.guilliermondii（AF01）,the degradation enzyme and its encoding gene were screened by a combination of bioinformatics analysis and back-propagation of the chemical structure of the degradation product.The whole genome of AF01was sequenced,and then the key gene MG2-4 of the degradation was discovered and validated based on database comparison,protein function annotation and in vitro experiments.The results showed that the protein encoded by this gene was composed of 313 amino acids and belonged to the aldo-keto reductase（AKR）family.This AKR family degradation enzyme could significantly improve the AFB₁ degradation rate with the addition of coenzyme NADPH,and the reaction conditions were mild and could degrade more than 90%of AFB₁ at37°C.As the first reported microbial AKR family protein that degraded AFB₁,it provided a new enzyme and genetic resource for the biodegradation of AFB₁.（4）The potential for detoxification applications of the screened M.guilliermondii was verified by detoxification experiments using aqueous solution and solid-state fermentation of peanut meal.In order to test the application potential of M.guilliermondii（AF01）in practical production,detoxification experiments were carried out in aqueous solution and in a complex material（peanut meal）.In the detoxification experiments of aqueous solutions,the whole fermentation broth of strain AF01 and its different fractions were used to react with AFB₁.The experimental results showed that the whole fermentation broth of AF01 could remove AFB₁up to 95.55%in 72 h and this included degradation and adsorption,where adsorption was a strong adsorption that cannot be easily desorbed;the fermentation supernatant could adsorb AFB₁ up to 71.73%;the intracellular lysate could degrade AFB₁ up to 25.07%;the cell suspension could degrade AFB₁ up to 71.04%after the optimization of reaction conditions.In the experiments of peanut meal detoxification,the detoxification effect of AF01 strain in complex materials was tested step by step using three forms,namely shake flask,shallow tray and fermentation bags.The optimal conditions for AFB₁ removal from peanut meal by AF01were determined by single-factor and response surface experiments at natural p H with an inoculum of 5.5%for 75 h at 29°C.Shallow trays and fermentation bags were then used to simulate solid state fermentation in production.The results showed that AF01 could obtain68.85%and 70.31%of AFB₁ removal in the scaled-up experiment,respectively.And there was no significant change in the appearance and quality of peanut meal after fermentation.Combining the two,it can be concluded that the AF01 strain with a new degradation pathway for AFB₁ explored in this paper had significant potential for application and development.The new way of AFB₁ degradation by AF01 can be developed and deepened through the refinement of the degradation strain and degradation enzymes and the combination with other technologies,which can provide new materials and new ideas for the future joint removal of AFB₁ pollution by various biological methods.This study can provide technical support for the research related to aflatoxin removal by biotechnology,and provide microbial and genetic resources for the development and application of aflatoxin-degrading microorganism agents,which is of great significance to ensure the quality and safety of agricultural products in China.