Abstract: 【Objective】 This study was aimed to screen out strains that could efficiently degrade alflatoxin, and to detect the degrading activity and residual toxicity, in order to provide solutions for the prevention and control of alflatoxin B₁(AFB₁) pollution.【Method】 Samples were collected from feces of herbivorous animal such as yak and sheep, as well as moldy feed from livestock farms.The strain with the highest efficiency in degrading AFB₁ was screened using coumarin, a structural analog of AFB₁, as the sole carbon source, and the strains were identified by morphology, physiology and biochemistry and 16S rDNA sequencing.AFB₁ was mixed with an appropriate amount of fermentation broth, the final concentration of AFB₁ was 1 μg/mL, and AFB₁ was cultured at 37 ℃ at 150 r/min, and the degradation rate of AFB₁ at different time was measured.The supernatant, bacterial suspension and bacterial cells were prepared by differential centrifugation, and the bacterial cells were ultrasonically lysed to obtain an intracellular solution, and the degradation rates of different components to AFB₁ were determined.The supernatant was subjected to proteinase K, proteinase K+SDS and heat treatment to detect the degradation rate of AFB₁.Precipitation of the supernatant with different concentrations of saturated ammonium sulfate, and the supernatant concentrate solutinon was prepared using 7 ku dialysis bags, that were for the measurement of degradation efficiency of AFB₁.So as to determine the nature and distribution of the active ingredients of the strain.Chicken hepatocytes were divided into AFB₁, AFB₁-strain, strain and blank control groups.The relative expression of the inflammatory factor interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α) and Bcl-2-associated X protein (Bax) genes were evaluated.【Result】 After initial screening and rescreening, one strain was isolated from the sheep feces sample numbered S₁, which had the highest activity of degrading AFB₁ with a degradation rate of 60.36%.The strain S₁ was cultured on the LB solid medium for 12 h, and pale yellow opaque colonies could be observed, which had smooth surface and bumps, the microscopic examination was Gram-positive and rod-shaped.The 16S rDNA gene was amplified by PCR and further sequenced, and the sequence identity with LC55966.1 was 100%.Based on nucleotide sequence analysis and physiological and biochemical results, the strain was comprehensively identified as Bacillus subtilis.The bacterial fermentation broth was reacted with AFB₁ (1 μg/mL) for 4, 12, 24, 48 and 72 h, and the degradation rates reached 10.98%, 25.36%, 46.24%, 52.65% and 80.84%, respectively.The degradation rate of AFB₁ in the supernatant was significantly higher than that in the bacterial suspension and intracellular solution (P<0.05).The degradation activity of the supernatant was decreased after heat treatment, while the degradation activity was basically lost after treatment with proteinase K and SDS.There was no significant difference in the degradation rate of supernatants treated with different concentrations of saturated ammonium sulfate (P>0.05).However, the degradation rate of AFB₁ in the supernatant after dialysis concentration was significantly higher than that in the supernatant precipitated by ammonium sulfate (P<0.05).In chicken embryonic liver primary cells, the expressions of IL-6, TNF-α and Bax induced by degradation product of AFB₁ with Bacillus subtilis S₁ were significantly lower than that in AFB₁ group (P<0.05).【Conclusion】 The strain of Bacillus subtilis S₁ was screened out, which could degrade AFB₁ efficiently.Its degradation activity was from a protein mainly distributed in the culture medium supernatant, and its residual toxicity of AFB₁ was significantly reduced after degradation.

Key words: aflatoxin B₁; Bacillus subtilis; detoxification