# _Bacillus subtilis_ Simultaneously Detoxified Aflatoxin B1 and Zeralenone
## 2 Materials and Methods
### Localization of B. subtilis ZJ-2019-1 Active Substances for Degradation of AFB1 and ZEN  
To determine the active ingredients of _B. subtilis_ ZJ-2019-1 to degrade mycotoxins, the strain was cultured until reaching a stable stage, followed by centrifugation at 4 \({}^{\circ}\)C for 20 min (8000 rpm) to obtain the supernatants. These supernatants were then collected and filtered through 0.22 \(\upmu\)m filters to obtain cell-free supernatants. The bacterial precipitate was resuspended in sterile PBS and underwent three rounds of centrifugation and washing with sterile PBS. Subsequently, ultrasonication was performed for 5 min (600 W, working 5 s and intermittent 5 s). The resulting bacterial mixture was further centrifuged at 4 \({}^{\circ}\)C for 20 min (10,000 rpm, and the sediment obtained was resuspended in sterile PBS as cellular contents [36; 37; 38].  
The cell-free supernatants were autoclaved at 121 \({}^{\circ}\)C for 15 min and boiled in a water bath at 100 \({}^{\circ}\)C for 3 h following previous studies with slight modifications [35; 36; 37]. Specifically, the heated supernatants were supplemented with 1 mg/L of AFB1 or ZEN and incubated at 37 \({}^{\circ}\)C (200 rpm) for 48 h. To investigate whether the degradation of mycotoxins was enzymatic in nature, the cell-free supernatants were treated with protease K (2 mg/mL), SDS (1%), or both, at a temperature of 58 \({}^{\circ}\)C for 3 h [32]. All experiments were conducted in triplicate. The degradation of AFB1 and ZEN was determined using HPLC as described above.  
Effects of Different Factors on the Degradation of AFB1 and ZEN by the Crude Enzymes of B. subtilis ZJ-2019-1  
To investigate the impact of temperature on degradation rate, 10 mg/L AFB1 or ZEN was added into the crude enzymes of _B. subtilis_ ZJ-2019-1, followed by incubation at temperatures ranging from 30 to 70 \({}^{\circ}\)C for 72 h. Subsequently, samples were collected, and mycotoxin degradation rates were determined using HPLC analysis. For pH effect assessment, 10 mg/L AFB1 or ZEN was added to the crude enzymes of _B. subtilis_ ZJ-2019-1 and incubated for 72 h at 37 \({}^{\circ}\)C under varying pH conditions (4.0, 5.0, 6.0, 7.0, 8.0, or 9.0) [39]. Similarly, in the metal chelator treatment test, different concentrations of EDTA solutions (10, 20, 30, 40, or 50 mmol/L) were added into the crude enzymes of _B. subtilis_ ZJ-2019-1, followed by incubation at 37 \({}^{\circ}\)C for 72 h following previous studies with slight modifications [39].  
To evaluate the effect of mycotoxin concentration on degradation rate, different concentrations of AFB1 or ZEN (1, 5, 10, 15, and 20 mg/L) were added into the crude enzymes of _B. subtilis_ ZJ-2019-1. The resulting mixture was incubated at 37 \({}^{\circ}\)C for 72 h. To examine the impact of metal ions on degradation rate, diverse metal ion solutions (such as Co\({}^{2+}\), Cu\({}^{2+}\), Fe\({}^{2+}\), Fe\({}^{3+}\), Mg\({}^{2+}\), Mn\({}^{2+}\), Ni\({}^{2+}\), and Zn\({}^{2+}\)) were added to the crude enzymes of _B. subtilis_ ZJ-2019-1 and incubated at 37 \({}^{\circ}\)C for 72 h following previous studies with slight modifications [34]. Each metal ion had a final concentration of 5 mmol/L. Furthermore, regarding reaction time, the crude enzymes of _B. subtilis_ ZJ-2019-1 were subjected to incubation with either AFB1 or ZEN at a concentration of 10 mg/L at 37 \({}^{\circ}\)C for 24, 48, 72, 96, or 144 h. All experiments were conducted in triplicate. Following incubation, samples were collected and analyzed using HPLC to determine mycotoxin degradation rates.