# _Bacillus subtilis_ Simultaneously Detoxified Aflatoxin B1 and Zeralenone
## 4 Discussion  
Both AFB1 and ZEN are the two most prevalent mycotoxins that contaminate food and animal feed, posing a serious threat to human and animal health. There are various biodegradation methods for mycotoxins. Microbial degradation is considered an attractive method for removing mycotoxins [42]. [PERSON] [43] isolated the _P. aeruginosa_ M-4 strain, which achieved a degradation rate of 90.57% for AFB1 in maize. Active substances from _Pseudomonas_ strains found in seawater were capable of degrading AFB1 [44]. Both _P. episeptica_ and _P. fluorescens_ demonstrated the ability to degrade AFB1 [45]. Furthermore, a combination of _Lactobacillus plantarum_, _L. bulgarius_, and _L. acidophilus_ effectively inhibited the growth of _A. flavus_ in silge while also degrading its toxic metabolites. Moreover, _Rhodococcus_ species exhibited effective degradation of ZEN [46; 47; 48; 49], with intracellular enzymes being the main active substances involved in degradation, resulting in a degradation rate of 90%. _Acinetobacter calcinetoacetate_ isolated from the moldy-stained root mass of radix Daustellariae has also exhibited efficient ZEN degradation [50]. Currently, probiotic _Bacillus_ species are extensively utilized as dietary supplements in food and animal feed to modulate the gut microbiota and improve health and immunity [51]. Furthermore, various _Bacillus_ species, including _B. subtilis_, _B. velez_, _B. schafferii_, and _B. amylolytica_ have demonstrated the ability to degrade AFB1 and ZEN [52; 53; 54]. The primary bioactive substances are commonly found in bacterial fermentation supernatants. ZEN-phosphotransferase of _B. subtilis_ Y816 [55] and CotA laccase of _B. subtilis_[56] exhibit potential in reducing ZEN toxicity. Additionally, BsDyP peroxidase from _B. subtilis_ SCK6 expressed in _Escherichia coli_ BL21/pG-Tf2 cells efficiently degrades ZEN and other mycotoxins [57]. Active substances of _B. amylolytic_ FS-3 and _B. amylolytic_ H6 were found to degrade ZEN, while the thioesterase from the H6 strain was able to detoxify ZEN and reduce its estrogen toxicity [58; 59]. Additionally, extracellular enzymes produced by  
Figure 7: Degradation of AFB1 (0.8 mg/kg) and ZEN (3 mg/kg) in corn gluten meal by _B. subtilis_ ZJ-2019-1 and a mold remover (the antifungal agent). **(A)** Degradation of AFB1 in corn gluten meal. **(B)** Degradation of ZEN in corn gluten meal. Different letters indicate significant differences among the means according to [PERSON]’s test (_p_ < 0.05). _B. amylolyticus_ 7D3-2 effectively degraded ZEN [60], whereas _Bacillus_ T-246 and _B. pumilus_ T-420 removed ZEN through a combination of adsorption and degradation by extracellular enzymes [61]. In our study, _B. subtilis_ ZJ-2019-1 demonstrated the ability to simultaneously degrade AFB1 and ZEN while exhibiting greater thermal and alkaline stability (Figures 1 and 2). The supernatants of _B. subtilis_ ZJ-2019-1 achieved a residual rate of 40.55% for 10 mg/L AFB1 and 48.75% for ZEN, which was lower than that observed in cells (Figure 1). The cell-free supernatants of _B. subtilis_ ZJ-2019-1 showed superior efficacy in detoxifying both AFB1 and ZEN compared to the bacterial cells, suggesting that the fractions of _B. subtilis_ ZJ-2019-1 responsible for degrading these mycotoxins may be secreted into the culture media. _B. subtilis_ ZJ-2019-1 appears to eliminate AFB1 by degradation and detoxify ZEN by degradation and adsorption. Similar findings were observed for AFB1 degradation by the culture supernatants (over 90%) of _B. licheniformis_ CFR1, which exhibited higher efficiency than the cell lysates (12.26%) [29]. Notably, following thermal treatment in an autoclave at 121 \({}^{\circ}\)C for 15 min or boiling water at 100 \({}^{\circ}\)C for 3 h, the supernatants of _B. subtilis_ ZJ-2019-1 promoted the degradation of AFB1 and ZEN (Figures 1 and 4). This indicates that the fractions responsible for degrading AFB1 and ZEN from _B.