| Summary: | <i>Bacillus</i> sp. H16v8 and <i>Bacillus</i> sp. HGD9229 were identified as Aflatoxin B<sub>1</sub> (AFB<sub>1</sub>) degrader in nutrient broth after a 12 h incubation at 37 °C. The degradation efficiency of the two-strain supernatant on 100 μg/L AFB<sub>1</sub> was higher than the bacterial cells and cell lysate. Moreover, degradations of AFB<sub>1</sub> were strongly affected by the metal ions in which Cu<sup>2+</sup> stimulated the degradation and Zn<sup>2+</sup> inhibited the degradation. The extracellular detoxifying enzymes produced by co-cultivation of two strains were isolated and purified by ultrafiltration. The molecular weight range of the detoxifying enzymes was 20–25 kDa by SDS-PAGE. The co-culture of two strains improved the total cell growth with the enhancement of the total protein content and detoxifying enzyme production. The degradation efficiency of the supernatant from mixed cultures increased by 87.7% and 55.3% compared to <i>Bacillus</i> sp. H16v8 and HGD9229, individually. Moreover, after the degradation of AFB<sub>1</sub>, the four products of the lower toxicity were identified by LC-Triple TOF-MS with the two proposed hypothetical degradation pathways.
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