Screening,Identification And Evaluation Of Zearalenone And Aflatoxin B1 Degrading Strains

Zearalenone(ZEN)and aflatoxin B1(AFB1)are secondary metabolites with strong toxicity produced by several fungal strains.Mycotoxins contamination in feed and food cause a serious threat to human and animal health.Therefore,the search of a safe and efficient methods which can remove mycotoxins without side effects is a research hotspot.The purpose of this study is to screen those microorganisms that can efficiently degrade ZEN and AFB1 from moldy material and herbivorous animal manure,and to explore the mechanism and characteristics of degraded products.Experiment 1: Screening,identification and evaluation of zearalenone degrading microorganismsTotal 40 survival strains were isolated in the first screening using ZEN as the sole carbon and energy source,and the strain with the highest ZEN degradation rate at 85.37% was selected from rabbit manure in the secondary screening.Combining with morphological,physiological and biochemical characteristics and 16 S r RNA gene sequencing analysis,the strain was identified as the Bacillus amyloliquefaciens named H6.After fermentation culture of the strain H6 with 6% inoculation at 37 ℃,180 r/min and 72 h,the fermentation liquid degradation rates of 1 μg/m L,5 μg/m L,10 μg/m L,15 μg/m L and 20 μg/m L ZEN were 95.60%,91.38%,93.64%,88.60% and 85.80%,respectively;and at 12 h and 24 h,the degradation rates of 1 μg/m L ZEN were 86.69% and 89.34%,respectively,and the degradation rates of 20 μg/m L ZEN were 76.67% and 84.14%,respectively.At 37 ℃,180 r/min and 72 h,the supernatant and bacterial suspension of strain H6 could remove 1 μg/m L ZEN by 68.93% and 28.70%,respectively;however,there were almost no degradation activity after the supernatant treated with protease K,protease K+SDS and heating;the degradation rates of extracellular protein precipitated with 80% ammonium sulfate and supernatant concentrated 8 times by polyethylene glycol 6 000 were 59.68% and 93.80%,respectively.Compared with the ZEN group,the proliferation rate of MCF-7 cell was significantly reduced when treated with ZEN degradation product.The molecular weight of ZEN degradation product was detected as 398 by LC-MS analysis.The results showed that Bacillus amyloliquefaciens H6 screened from rabbits feces had high degradation activity for 1~20 μg/m L ZEN,and the extracellular enzyme played an important role in degradation activity.The results also indicated that the concentrated supernatant had a significant degradation effect,and ZEN was degraded into non-estrogen metabolite with molecular weight of 398.Experiment 2: Screening,identification and evaluation of aflatoxin B1 degrading strainTotal 42 survival strains were isolated in the first screening using coumarin as the sole carbon and energy source.and strain S1 with the highest AFB1 degradation rate at 52.64% was selected from sheep manure in the secondary screening.Combining with morphological,physiological and biochemical characteristics and 16 S r RNA gene sequencing analysis,the strain S1 was identified as the Bacillus subtilis.Fermentation culture of the strain S1 with 10% inoculation,the fermentation liquid degradation rates of 1 μg/m L AFB1 were 46.21% and 80.26% at 37 ℃,180 r/min,48 h and 72 h,respectively.At 37 ℃,180 r/min and 72 h,the supernatant of strain S1 could degrade 1 μg/m L AFB1 by 55.86%;however,the degradation activity of the supernatant after treated with protease K,protease K+SDS and heating be almost disappeared.The 1 μg/m L AFB1 degradation rates of extracellular protein precipitated with ammonium sulfate were 30.21%~33.13%,and the degradation rate of supernatant concentrated 8 times by polyethylene glycol 6 000 was 80.64%.The 200 μg/m L AFB1 degradation rate was 80.64% after AFB1 and fermentation liquid of S1 co-incubation at 37 ℃ and 180 r/min for 72 h.The expression levels of IL-6,TNF-α and Bax in emboryonic primary hepatocytes in AFB1 degradation group were significantly lower than AFB1 group(p<0.05).The results showed that Bacillus subtilis S1 screened from sheep feces had high degradation efficiency,and the extracellular enzyme secreted by the strain S1 played a key role in degradation process.The results also indicated that the concentrated supernatant had a good degradation effect,and the damage for emboryonic primary hepatocytes were weakened after AFB1 degrading.