# _Bacillus subtilis_ Simultaneously Detoxified Aflatoxin B1 and Zeralenone
## 3 Results
### Identification of Active Components of B. subtilis ZJ-2019-1  
To determine the fraction responsible for degrading mycotoxins, we collected the supernatants and cellular contents of _B. subtilis_ ZJ-2019-1, which were then subjected to treatments involving heating, SDS or protease K. Subsequently, these treated samples were incubated with 1 mg/L AFB1 or ZEN. As shown in Figure 4A, the degradation rate of AFB1 by the autoclaved and boiled supernatants of _B. subtilis_ ZJ-2019-1 reached 79.85% and 79.34%, respectively, higher than that of the untreated supernatants (58.03%). However, no degradation of AFB1 was observed with the cellular contents of _B. subtilis_. Figure 3: Degradation of ZEN by _B. subtilis_ ZJ-2019-1 under different conditions. (**A**) Effect of temperature on ZEN degradation. (**B**) Effect of pH on ZEN degradation. (**C**) Effect of toxin concentration on ZEN degradation. (**D**) Effect of incubation time on ZEN degradation. Different letters indicate significant differences among the means according to [PERSON]’s test (\(p<0.05\)). Figure 2: Degradation of AFB1 by _B. subtilis_ ZJ-2019-1 under different conditions. (**A**) Effect of temperature on AFB1 degradation. (**B**) Effect of pH on AFB1 degradation. (**C**) Effect of toxin concentration on AFB1 degradation. (**D**) Effect of incubation time on AFB1 degradation. Different letters indicate significant differences among the means according to [PERSON]’s test (\(p<0.05\)). Similarly, the degradation rate of ZEN by the autoclaved supernatants of _B. subtilis_ ZJ-2019-1 reached 100%, which was superior to that of the boiled supernatants (68.53%), untreated supernatants (59.77%), and cellular contents (33.66%) (Figure 4B). After treatment with protease K, the cell-free supernatants of _B. subtilis_ ZJ-2019-1 exhibited enhanced degradation efficiency towards AFB1 (74.02%) and ZEN (61.64%) (Figure 4). However, after treatment with 1% SDS, the degradation capacity of the supernatants of _B. subtilis_ ZJ-2019-1 decreased to 29.65% for AFB1 and 9.36% for ZEN. Similarly, co-treatment with proteinase K and 1% SDS resulted in a further reduction in degradation activity to 26.00% for AFB1 and 4.23% for ZEN. Effects of Different Factors on AFB1 and ZEN Degradation by the Crude Enzymes of B. subtilis ZJ-2019-1  
As shown in Figures 5A and 6A, the degradation rate of mycotoxins by the crude enzymes of _B. subtilis_ ZJ-2019-1 increased with temperature rising from 30 \({}^{\circ}\)C to 70 \({}^{\circ}\)C. The maximum degradation rate of AFB1 and ZEN was 100% and 94.29%, respectively, at a temperature of 70 \({}^{\circ}\)C. The degradation rate of AFB1 and ZEN by the crude enzymes increased as the pH values ranged from 4.0 to 9.0, with the highest degradation rates of 73.91% and 46.11%, respectively. Some proteases may require EDTA as a co-factor for the degradation of mycotoxins [41]. The degradation rate of AFB1 by the crude enzymes of _B. subtilis_ ZJ-2019-1 markedly decreased from 54.94% to 6.35% with an increase in EDTA concentration from 0 to 50 mM (Figure 5C). Similarly, the degradation rate of ZEN by the crude enzymes decreased from 56.12% to 0.00% as the EDTA concentration increased from 0 to 50 mM (Figure 6C). To evaluate the impact of mycotoxin concentrations on degradation, different concentrations of AFB1 and ZEN (1, 5, 10, and 20 \(\mu\)g/mL) were added to the crude enzymes from _B. subtilis_ ZJ-2019-1 and incubated at 37 \({}^{\circ}\)C for 72 h. The results showed that different mycotoxin concentrations did not exert significant effects on the degradation of AFB1 and ZEN by the crude enzymes of _B. subtilis_ ZJ-2019-1; the maximum degradation rates of AFB1 or ZEN were 76.46% and 46.23% at a concentration of 1 \(\mu\)g/mL (Figures 5D and 6D). The impact of different incubation time on mycotoxin degradation by crude enzymes from _B. subtilis_ ZJ-2019-1 was also investigated. It was found that degradation rates of AFB1 and  
Figure 4: Effects of different treatments of _B. subtilis_ ZJ-2019-1 fractions on mycotoxin (1 mg/L) degradation.