| Aflatoxins are a class of secondary metabolites produced by fungi, which have very strong toxicity and carcinogenicity. The physical and chemical properties of aflatoxins are very stable, and thus it is difficult to degrade during food processing. In contrast to physical and chemical treatment methods, biological degradation of aflatoxins have many advantages, such as mild reaction conditions, no loss of nutrients and no newly produced toxins. Therefore, more and more attention has been paid on biological degradation method. To date, a variety of microbial species including bacteria and fungi have been reported as capable of degrading aflatoxins.In this study, bacterial strains that can degrade aflatoxin B1(AFB1) were screened and identified from the environment which is easily contaminated by aflatoxins. Using coumarin as the sole carbon and energy source, we identified 12 bacterial strains that can effectively degrade AFB1 from the animal manure, fermented foods, peanut soil and microbial strains stored in our laboratory. Sequencing of 16 S r DNA shows that these 12 strains belong to the genus of Bacillus. We determined the rate of AFB1 degradation using HPLC to examine AFB1 content of fermented supernatant liquid. The rates of AFB1 degradation of 9 strains were over 80%, and that of 7 strains were no less than 60%. The rate of AFB1 degradation of one strain isolated from natto food could reach 85.73%, and the degradation rate for the corresponding concentrated crude enzyme liquid is 91.4%. This strain was identified as Bacillus subtilis based on morphological, physiological, biochemical and phylogenetic analyses, and it was named as Bacillus subtilis Natto3.We further investigated the effect of fermentation time, reaction temperature, p H value and metal ions on the rate of AFB1 degradation using the concentrated fermentation liquid of Bacillus subtilis Natto3 as the enzyme source. We found that the optimal fermentation time, reaction temperature, and p H value for the degradation of AFB1 were 72 h, 37 oC and 8.5, respectively; and that the degradation activity could be inhibited to different extents by five metal ions including Zn2+, Mn2+, Mg2+, Cu2+ and Li+.Under the reaction condition of 37℃ and culture 72 h, the rates of AFB1 degradation for the culture, cell extract and fermented supernatant liquid of Bacillus subtilis Natto3 were 6.5%, 1.4% and 87%, respectively. We also determined if the rate of AFB1 degradation for the fermented supernatant liquid of Bacillus subtilis Natto3 was influenced by three different treatment methods including digesting with 1 mg m L-1 proteinase K, boiling for 10 min, and filtering with 0.22 um filteration membrane. We found that the former two methods could significantly lower the rate of AFB1 degration(to 8.6% and 15%, respectively), whereas filtering had no great impact on AFB1 degradation(83%). It was possible that AFB1 degradation was realized for Bacillus subtilis Natto3 to produce AFB1 degradation enzyme complex by secretion into medium. Using the contaminated peanut sample as the source of aflatoxins rather than AFB1 standard, we further investigated the effect of the amount of crude enzyme liquid, p H value and reaction temperature on the rate of AFB1 degradation. Under the reaction condition of 35℃,p H 8 and 3m L crude enzyme liquid, after incubation of crude enzyme liquid from Bacillus subtilis Natto3 for 10 days, AFB1 content in highly contaminated peanut sample was reduced from 344μg kg-1 to 25μg kg-1, and AFB1 content in moderately contaminated sample was reduced from 192μg kg-1 to 11.4μg kg-1, whereas it was greatly decreased in slightly contaminated sample from 51.3μg kg-1 to an undetectable level. These results demonstrate the ability of Bacillus subtilis Natto3 to degrade both the AFB1 standard and the virtually aflatoxin-contaminated peanut sample. We provide a preliminary study for the future application of food grade microorganisms in the biological degradation of aflatoxins. |
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