Biodetoxification of both AFB1 and ZEN by Bacillus subtilis ZJ-2019-1 in gastrointestinal environment and in mice

Biodetoxification of both AFB1 and ZEN by Bacillus subtilis ZJ-2019-1 in gastrointestinal environment and in mice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Biodetoxification of both AFB1 and ZEN by Bacillus subtilis ZJ-2019-1 in gastrointestinal environment and in mice Jianwen Wu, Wei An, Zhenlong Wang, Boquan Gao, Jiaxue Wang, Ya Zhao, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4590182/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Aflatoxin B1 (AFB1) and zearalenone (ZEN) are the most prevalent mycotoxins in production, posing a serious threat to the food and feed industry and resulting in a substantial economic burden. Therefore, finding a safe and efficient method for biodegradation of mycotoxins is of utmost important in addressing this issue. Bacillus subtilis ZJ-2019-1, capable of degrading AFB1 and ZEN, was isolated and cultured in our laboratory in vitro. In this study, we conducted detoxification tests on AFB1 and ZEN using B. subtilis ZJ-2019-1 in gastrointestinal environment and in mice, respectively. Our findings demonstrate that B. subtilis ZJ-2019-1 can effectively degrade both mycotoxins present in feed within gastrointestinal environment. Following degradation by B. subtilis ZJ-2019-1, the toxicity of the AFB1 and ZEN product decreased compared to their original levels. Furthermore, B. subtilis ZJ-2019-1 exhibited excellent detoxification effects on AFB1 and ZEN when tested in mice. These results indicate that B. subtilis ZJ-2019-1 possesses the ability to degrade both AFB1 and ZEN, making it suitable for application in the food and feed industry as a means to reduce economic losses caused by mycotoxins. biodegradation aflatoxin zearalenone Bacillus subtilis gastrointestinal environment mice Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Mycotoxins are toxic compounds produced by molds, which are widely distributed in nature, particularly in food and feed (Monika et al. 2019; Hamilton 2022). There exist various types of mycotoxins, with aflatoxin (AFB), zearalenone (ZEN), ochratoxin (OTA), and deoxynivalenol (DON) being the most well-known ones. These mycotoxins pose a serious threat to human and animal health (Assunção et al. 2020; Patrícia et al. 2021). Among them, both aflatoxin B1 (AFB1) and zearalenone (ZEN) are commonly co-occurring mycotoxins in cereals. AFB1, the most potent member of the aflatoxin family, is produced by Aspergillus flavus . AFB1 poses a significant global health threat, particularly in developing countries with favorable climatic conditions for A. flavus growth and AFB production (Awuchi et al. 2021; Frisvad et al. 2019). The toxicity of AFB1 mainly arises from its metabolites, notably AFB1-8, 9-epoxide, a highly reactive compound that binds to DNA and proteins, resulting in cellular genetic material and protein synthesis inhibition (Cheng et al. 2018; Guengerich et al. 1998). These effects ultimately lead to apoptosis, tissue damage, gene mutation, and tumor formation (Guindon-Kezis et al. 2014; Rotimi et al. 2021), notably influencing the liver due to its role as the primary site for AFB1 metabolism and detoxification; prolonged or high-dose exposure to AFB1 can cause hepatocyte injury, jaundice, hepatitis, liver cirrhosis, and even liver cancer (Guindon et al. 2007; Zhang et al. 2021; Jaskiewicz et al. 1988). Additionally, AFB1 also inhibit immune system function, compromising the body's resistance to pathogens. It diminishes immunoglobulin production by affecting various immune cells, thereby attenuating the immune response (Sun et al. 2016; Hao et al. 2015; Hussein and Brasel. 2001; Robens and Richard 1992). ZEN is a secondary metabolite produced by Fusarium spp., which are widely distributed in corn, wheat, barley, and other grains and their products. Due to its estrogenic activity and ability to mimic the role of estrogen in the body, ZEN is referred to as "environmental estrogens in feed". Given its widespread presence in food and feed, ZEN poses potential risks to human and animal health, particularly the reproductive system (Takemura et al. 2007; Wang et al. 2022; Vance et al. 2019). ZEN and its metabolites can imitate the biological effects of estrogen by binding to estrogen receptors and activating estrogen-dependent gene expression. In animals, ZEN can induce various physiological and pathological effects, including abnormal development of reproductive organs, reduced reproductive ability, and alterations in hormone levels (Malir et al. 2023; Deng et al. 2023; Yu et al. 2005; Ruzsás et al. 1979; Su et al. 2018). Furthermore, ZEN has the potential to impact the immune system (Yang et al. 2019; Vlata et al. 2006; Cai et al. 2017). It has been also demonstrated that ZEN and its metabolites possess cytotoxicity and genotoxicity properties capable of causing DNA damage, cell cycle arrest, as well as apoptosis (Liu et al. 2020; Tian and Li. 2017; Zhou et al. 2020), all potentially associated with the carcinogenic risk posed by ZEN. Notably, the simultaneous presence of AFB1 and ZEN in grain-based food and animal feed poses significant health hazards to both human and animal health due to their potent mutagenic, cytotoxic, and carcinogenic properties. The prevention and control of co-occurring mycotoxin pollution present a global challenge that requires comprehensive measures throughout all stages of crop growth, harvest, storage, and processing. This includes utilizing mildew-resistant varieties, improving storage conditions, implementing biological and chemical methods to mitigate mold growth and toxin production. Among the various strategies available for mycotoxin removal, the biodegradation approach stands out due to its some advantages, such as environmental friendliness, practical feasibility, and economic viability (Pang 2021). Eshelli et al. identified three types of AFB1-degrading actinomycetes, namely Rhodococcus ATCC4277, Streptomyces lividans TK 24, and Streptmyces aureus ATCC10762, with degradation rates of 95.95%, 87.95%, and 86.10%, respectively (Eshelli et al. 2015). S. cacaoi degraded 88.34% AFB1. Adebo et al. isolated three strains of Pseudomonas aeruginosa , P. aeruginosa , and staphylococci which could degrade AFB1 from gold aquifers, with degradation rates of 47.7%, 51.7%, and 56.8%, respectively (Harkai et al. 2016). Kumar et al., on the other hand, isolated a salt-tolerant Bacillus albus YUN5 capable of degrading AFB1 from traditional Korean food "doenjang", where its cell-free supernatant incubated with 2 mg/L AFB1 for 48 h achieved a degradation rate of 54.96% AFB1 (Kumar et al. 2023). Furthermore, A. niger RAF106 demonstrated the ability to degrade AFB1 at 30℃ and pH 4–8, achieving a maximum degradation rate of 54.96% (Fang et al. 2020). B. amylolyticus Fu2-3 derived from soil as well as B. amyloliquefaciens ZDS-1 were able to degrade more than 95% and 97.8% of ZEN, respectively (Zhang et al. 2016; Xu et al. 2016). Moreover, Rhodococcus SYA13 obtained from oil-contaminated soil efficiently degraded ZEN by 87.1% (He 2021). The Pseudomonas TH-N1 strain was found to degrade 79% ZEN at 37℃ and pH 4.5 (Tan et al. 2014). Wu et al. discovered that Saccharomyces CLY01 achieved a degradation rate of 96.79% for ZEN (Wu et al. 2009). R. mucilaginosa could degrade over 90% of ZEN (Guo et al. 2021). However, few bacteria have been reported to simultaneously degrade both AFB1 and ZEN. Our previous study showed that B. subtilis ZJ-2019-1 can simultaneously degrade AFB1 and ZEN in vitro (Wu et al. 2024). The present study aimed to investigate the detoxication of AFB1 and ZEN by B. subtilis ZJ-2019-1 gastrointestinal environment and in vivo, while also exploring the cytotoxicity of mycotoxin degradation products. Materials and methods Bacterial source, chemicals, and reagents The B. subtilis ZJ-2019-1 strain was isolated from pig feces collected from pig houses in Xingtai, Hebei Province (Vlata et al. 2006). The strain has been stored at the General Microbiology Center of the China Microbiological Culture Preservation and Management Committee (CGMCC No. 23636). The small intestinal and colonic fluid was purchased from Coolaber (Beijing). The mice were purchased from Vital River (Beijing). All chemical reagents were of analytical grade. Preparation of B. subtilis ZJ-2019-1 powder The monoclonal colonies of B. subtilis ZJ-2019-1 were isolated from LB solid medium, inoculated in LB liquid medium, and cultured in a constant temperature shaker. Once B. subtilis ZJ-2019-1 reached a stable growth phase, the bacterial solution was centrifuged and the supernatant was discarded. The cell pellet was then re-suspended with freeze-drying protectant (cell:protectant = 1:4) and frozen overnight at -80℃. Subsequently, the bacteria were subjected to freeze-drying for 48 h using a freeze-dryer and stored in a refrigerator at -80℃. Degradation of AFB1 and ZEN by B. subtilis ZJ-2019-1 in small intestinal fluid simulation Both corn gluten meal (0.999 g) and bacterial powder (0.01 g) were weighed in a 50 mL flask, followed by the addition of 16 mL gastric buffer. The mixture was then incubated in water bath shaker at 39℃ and 180 rpm for 4 h. Subsequently, 4 mL small intestinal buffer was added and incubated for 5 min; 2 mL small intestinal fluid was then added and incubated for 14 h. Finally, 2 mL colonic fluid was added and incubated for 24 h. The control samples were prepared accordingly, consisting of 0.999 g of corn gluten meal and 0.01 g of skim milk powder. Each treatment was repeated three times. Following the reaction, samples were collected and subjected to high performance liquid chromatography (HPLC) analysis to determine the degradation rate of mycotoxins by B. subtilis ZJ-2019-1. Detection of the degradation of AFB1 and ZEN by B. subtilis ZJ-2019-1 The degradation of AFB1 and ZEN by B. subtilis ZJ-2019-1 were detected using an Agilent SB-C18 (150 mm × 4.6 mm, 5 µm) column. The mobile phase consisted of a mixture of methanol, acetonitrile, and water (22:22:56 for AFB1 and 8:46:46 for ZEN). Prior to use, the mobile phase was filtered through a 0.22 µm membrane. The AFB1 or ZEN samples were added into methanol (1:3) and mixed on a rotator for 30 s. After centrifugation at room temperature for 1 min at 12,000 r/min, the supernatant was collected using a 2 mL syringe, filtered through a 0.22 µm aseptic filter membrane. For AFB1 analysis, the column temperature was maintained at 40℃, while for ZEN analysis it was kept at 30℃. The injection volume was set at 10 µL with a flow rate of 1.0 mL/min. Fluorescence detection utilized an excitation/emission wavelength of 365 nm/430 nm for AFB1 and 235 nm/460 nm for ZEN. Cytotoxicity of AFB1 and ZEN before and after degradation by B. subtilis ZJ-2019-1 The monoclonal colony of B. subtilis ZJ-2019-1 was inoculated in LB liquid medium and cultured in a constant temperature shaker. Once B. subtilis ZJ-2019-1 reached a stable growth phase, the culture was centrifuged at 4℃ for 15 min at 8,000 rpm. The resulting supernatant was then filtered through 0.22 µm membrane to obtain acellular supernatant. A volume of 5 mL of cell-free supernatant was separately mixed with AFB1 and ZEN at concentrations of 5, 10, 15, and 20 mg/L, respectively and incubated on a shaker at 37℃. The samples were collected at 0 h, 24 h, 48 h, and 72 h, respectively. Anhydrous ethanol was added to each sample followed by filtration using 0.22 µm membrane to remove miscellaneous bacteria. The RAW264.7 cells were cultured in DMEM medium (10% fetal bovine serum, 0.1% penicillin-streptomycin), and then seeded into a 96-well plate at a density of 5 × 10 4 cells per well (100 µL). Once the number of cells reached 70–80%, the cell-free supernatant was added for further culture, with each sample have three replicates. After incubation for 24 h, 10% of CCK-8 reagent was added to each well and incubated for 1 h. The absorbance at wavelength of 450 nm was measured. Experimental animals and management The 40 KM mice with, with an average body weight of (20 ± 2) g, were randomly divided into five groups with eight replicates in each group and one mouse per group. The five groups included the blank group, AFB1 group, ZEN group, AFB1 intervention group (AFB1 + B. subtilis ZJ-2019-1), and ZEN intervention group (ZEN + B. subtilis ZJ-2019-1). The adaptive culture period lasted for 7 d followed by a 21-d experimental period. Animals are raised in accordance with the animal welfare set forth by the Chinese Academy of Agricultural Sciences (CAAS). The mouse experiment was performed in accordance with the Animal Care and Use Committee of Institute of Feed Research (IFR) at the Chinese Academy of Agricultural Sciences (CAAS), and it received approval from the Laboratory Animal Ethical Committee and its Inspection of the Institute of Feed Research of CAAS (AEC-CAAS-20090609). Degradation of AFB1 and ZEN by B. subtilis ZJ-2019-1 in mice The mice were fed a normal diet. They received intragastric administration of 200 µL twice daily, with an interval of 1 h and at 18:00 every day. The blank group was administered aseptic phosphate buffer. The first sample of the AFB1 challenge group contained aseptic phosphate buffer with 0.5 mg/L AFB1, while the second sample consisted of sterilized phosphate buffer. The ZEN challenge group's sample contained aseptic phosphate buffer with 5 mg/L ZEN, and the second gavage used sterilized phosphate buffer. In the AFB1 intervention group, the first intragastric administration included aseptic phosphate buffer with 0.5 mg/L AFB1, the second involved B. subtilis ZJ-2019-1 (7.2×10 10 CFU/mL). Similarly, in ZEN intervention group, the first intragastric administration comprised aseptic phosphate buffer with 5 mg/L ZEN, followed by B. subtilis ZJ-2019-1 (7.2×10 10 CFU/mL). Sample collection of mice The mice were subjected to a 12-h fasting period prior to the execution of the experiment. The blood samples were collected before dissection, incubated at room temperature for 30 min and then centrifuged at 8,000 r/min for 10 min. The resulting serum was separated and transferred to 1.5 mL centrifuge tube, which was stored at -20℃ for subsequent biochemical analysis. A portion of the liver, uterus, and duodenum were immersed in a 4% tissue fixation solution for tissue sectioning. All animal handling procedures adhered to the protocols approved by the Animal Protection and Use Committee of the CAAS. Statistical analysis The statistical analysis was conducted using one-way analysis of variance (ANOVA) within a 95% confidence level, followed by either Student's test or Duncan's test, using the statistical software SPSS. Results Degradation of AFB1 and ZEN by B. subtilis ZJ-2019-1 in gastrointestinal simulation The ability of B. subtilis ZJ-2019-1 to degrade mycotoxins in animals was investigated by simulating the gastrointestinal environment of monogastric animals using naturally molded corn gluten meal. As shown in Fig. 1 , skim milk powder had no impact on AFB1 and ZEN degradation, whereas B. subtilis ZJ-2019-1 exhibited degradation capabilities for AFB1 and ZEN, with the degradation rate of 14.71% and 19.53%, respectively. These findings suggest that B. subtilis ZJ-2019-1 can effectively degrade AFB1 and ZEN within the gastrointestinal environment, highlighting its potential as a detoxifier of mycotoxins in animals. Cytotoxicity of AFB1 and ZEN degradation products by B. subtilis ZJ-2019-1 The B. subtilis ZJ-2019-1 strain exhibits in vitro degradation of mycotoxin and demonstrates potential for in vivo mycotoxin degradation; however, the toxicity of its degradation products requires further investigation. To assess the toxicity, we examined the effects of the products of AFB1 and ZEN degraded by B. subtilis ZJ-2019-1 on the proliferation of RAW264.7 cells. It was observed that there was a decrease in RAW264.7 cell proliferation rate with increasing concentrations of AFB1 (Fig. 2 A) and ZEN (Fig. 2 B). When the concentration of AFB1 and ZEN was 20 mg/L, the proliferation rate of RAW264.7 cells was 18.83% and 2.77%, respectively; however, after incubation with B. subtilis ZJ-2019-1 for 72 h, the degradation products of AFB1 and ZEN remarkably enhance the proliferation rate of RAW264.7 cells, with the proliferation rate of 65.24% and 62.03%, indicating their ability to promote cell growth. The results demonstrate that the degradation products of AFB1 and ZEN by B. subtilis ZJ-2019-1 exhibit much lower cytotoxicity towards RAW264.7 cells compared to AFB1 and ZEN. Effects of B. subtilis ZJ-2019-1 on serum biochemical indices of mice exposed to AFB1 and ZEN When exposed to AFB1, mice exhibited an elevation in the rate of aspartate aminotransferase/alanine transaminase (AST/ALT), alkaline phosphatase (ALP), total bile acids (TBA), triglyceride (TG), and total cholesterol (TC) levels in the serum, indicating potential liver inflammation. In contrast, oral administration of B. subtilis ZJ-2019-1 reduced these indices (Fig. 3 A, 3 C, 3 D, 3 F, 3 G). Upon exposure to AFB1, mice experienced a decrease in γ-glutamyltransferase (γ-TGase), amylase (AMY), and glucose (GLU) levels in the serum; however, oral administration of B. subtilis ZJ-2019-1 increased their levels (Fig. 3 B, 3 E, 3 H). When exposed to ZEN, mice exhibited an elevation in the rate of AST/ALT, γ-TGase, ALP, AMY, TG, TC, and GLU levels in the serum (Fig. 4 A- 4 C, 4 E- 4 H); oral administration of B. subtilis ZJ-2019-1 reduced their levels except γ-TGase. Upon exposure to ZEN, mice reduced TBA level; oral administration of B. subtilis ZJ-2019-1 enhanced the levels of its level (Fig. 4 D). These results suggest that B. subtilis ZJ-2019-1 may improve the inflammatory response. Effects of B. subtilis ZJ-2019-1 on organ tissues of mice exposed to AFB1 In the blank group, hepatocytes were observed centrally within irregular polygonal shapes and arranged in a cord-like structures. The hepatocyte cords exhibited a radial arrangement around the central vein, while the hepatic sinusoid space appeared uniformly without any dilatation (Fig. 5 A). No pathological changes such as hemorrhage, necrosis, and inflammation were detected in the liver tissue, indicating a normal tissue structure. Conversely, mice challenged with AFB1 displayed focal necrosis of hepatocytes accompanied by localized infiltration of inflammatory cells within the liver tissue (Fig. 5 B). Notably, administration of B. subtilis ZJ-2019-1 to the AFB1 intervention group resulted in liver tissue resembling that of the blank group (Fig. 5 C), suggesting its potential to prevent hepatocyte necrosis and inflammation induced by AFB1. The intestinal villous epithelial cells in the blank group exhibited intact and orderly arrangement. The distinct presence of the intestinal mucous layer, mucosal muscle layer, submucosa, and muscle layer indicated the absence of pathological changes such as necrosis, bleeding, and inflammation within this group (Fig. 5 D). Conversely, in the AFB1 challenge group, the intestinal villous epithelial cells displayed necrosis and exfoliation, while locally exposing the lamina propria of intestinal villi (Fig. 5 E). Notably, administration of B. subtilis ZJ-2019-1 to the AFB1 intervention group demonstrated normal intestinal tissue characteristics (Fig. 5 F), indicating that B. subtilis ZJ-2019-1 effectively prevent AFB1-induced necrosis and exfoliation of intestinal epithelial cells. Effects of B. subtilis ZJ-2019-1 on organ tissues of mice exposed to ZEN In the blank group, it exhibited round hepatocytes arranged in a cord shape around the center vein within an irregular polygonal structure. The hepatic sinusoid space was uniform and devoid of pathological changes such as hemorrhage, necrosis, and inflammation in the liver tissue (Fig. 5 G). Conversely, mice challenged with ZEN displayed focal necrosis of hepatocytes accompanied by inflammatory cell infiltration, edema, and swelling of some cells, light cytoplasmic staining and irregular vacuoles (Fig. 5 H). In comparison to the ZEN challenge group, liver tissue from mice treated with B. subtilis ZJ-2019-1 showed reduced hepatocyte necrosis and inflammation while exhibiting features, such as hepatocyte edema, cell swelling, cytoplasmic light staining, and irregular vacuoles without any pathological changes like hemorrhage or necrosis (Fig. 5 I). These findings suggest that B. subtilis ZJ-2019-1 can reduce the effects of ZEN-induced hepatocyte damage. The blank group exhibited diffuse infiltration of inflammatory cells in the interglandular space of the endometrium, along with a significant presence of fibroblasts in the interstitial space of the endometrial gland (Fig. 5 J). In the ZEN challenge group, there was slight edema and cell swelling observed in the endometrial glandular cells, accompanied by cytoplasmic light staining and irregular vacuoles within these cells. Additionally, a substantial proliferation of fibroblasts was noted in the endometrial glandular space, along with diffuse infiltration of inflammatory cells and necrotic/exfoliated cells masses within the uterine cavity (Fig. 5 K). Comparatively, mice in the B. subtilis ZJ-2019-1 intervention group displayed significantly improved uterine conditions compared to those in the ZEN challenge group. Specifically, mild edema and cellular swelling were observed in their endometrial glandular cells, along with lightly stained cytoplasm and irregular vacuoles within these cells (Fig. 5 L). Furthermore, diffuse infiltration of inflammatory cells could be seen within their endometrial glandular space. These findings suggest that B. subtilis ZJ-2019-1 can mitigate necrotic exfoliation caused by ZEN. Discussion The present study aimed to investigate the degradation effect of B. subtilis ZJ-2019-1 on mycotoxins-AFB1 and ZEN in naturally moldy corn gluten meal by simulating the gastrointestinal environment of monogastric animals. The findings revealed that B. subtilis ZJ-2019-1 exhibited remarkable potential for detoxification in feed in gastrointestinal environment and in vivo. AFB1 can induce autophagy by stimulating macrophages to generate reactive oxygen species (ROS), while ZEN can promote apoptosis by inducing mitochondrial dysfunction in macrophages (Wu et al. 2009; Guo et al. 2021). Other studies have indicated that exposure to AFB1 can lead to peritoneal macrophage damage and oxidative stress in mice, which is associated with iron homeostasis imbalance (Abid-Essefi et al. 2003). In our previous study, the ability of B. subtilis ZJ-2019-1 to degrade AFB1 and ZEN in vitro has been demonstrated, although the toxicity of its degradation products remains to be tested. In this study, the impact of the degradation products of AFB1 and ZEN by B. subtilis ZJ-2019-1 on the proliferation of RAW264.7 cells was assessed using the CCK8 method, along with effect of mycotoxins on RAW264.7 cell proliferation. The results revealed a decrease in RAW264.7 cell proliferation rate significantly with increasing concentrations of AFB1 and ZEN, and the degradation products of AFB1 and ZEN significantly increased the proliferation rate of RAW264.7 cells compared to the toxin group (Fig. 2 ). This suggests a significant reduction in toxicity when B. subtilis ZJ-2019-1 interacts with AFB1 and ZEN. Previous studies have demonstrated that AFB1 can induce hepatocyte disorder, cellar swelling, and increased apoptosis rate in mice (Wang et al. 2018). Similarly, ZEN has been shown to elicit various inflammatory reactions in liver cells and cause damage to uterine tissue structure accompanied by edema (Wang et al. 2018; Shi et al. 2023). Furthermore, the impact of AFB1 on porcine intestinal epithelial cells can trigger apoptosis, lead to intestinal injury, and reduce nutritional absorption rates (Ji 2017). In this study, administration of AFB1 to mice resulted in disrupted secretion of AST and ALT, as well as impaired metabolism and transport of TG in serum biochemical index analysis. Additionally, significant liver damage was observed (Fig. 3 , Fig. 5 A). Liver injury often leads to abnormal secretion of AST and ALT along with an increase in TG levels (An 2017). Moreover, it was found that AFB1 also induced intestinal damage in mice which caused a decrease in blood GLU content (Fig. 3 , Fig. 5 B). Administration of ZEN to mice similarly altered AST/ALT rate and elevated TG level (Fig. 4 ), indicating hepatic injury and its estrogenic properties by binding to the estrogen receptor (Ji 2020), causing insulin resistance and a significant increase in blood GLU content, which may contribute to the development of diabetes. The probiotic B. subtilis is commonly used as a feed additive (Yan et al. 2004). In our previous experimental study, the B. subtilis ZJ-2019-1 strain demonstrated in vitro capability to simultaneously degrade AFB1 and ZEN (Wu et al. 2024). In this work, oral administration of B. subtilis ZJ-2019-1 significantly alleviated liver injury of mice induced by AFB1 and ZEN exposure (Fig. 5 C, 5 I). Compared to the groups exposed solely to AFB1 or ZEN, treatment with B. subtilis ZJ-2019-1 notably reduced focal necrosis of hepatocytes and focal infiltration of inflammatory cells. Moreover, B. subtilis ZJ-2019-1 effectively mitigated intestinal injury induced by AFB1 through prevention of necrosis and exfoliation of intestinal villus epithelial cells (Fig. 5 F). Meanwhile, administration of B. subtilis ZJ-2019-1 could reduce mouse uterus damage induced by ZEN while preventing necrotic exfoliation of uterine cells caused by this toxin (Fig. 5 L). Therefore, oral administration of B. subtilis ZJ-2019-1 exhibits significant efficacy in preventing AFB1- and ZEN-induced liver, intestinal, and uterus damage in mice. Conclusions In this study, B. subtilis ZJ-2019-1 effectively degraded AFB1 and ZEN in corn gluten meal by simulating the gastrointestinal environment. The toxicity of the degradation products was significantly reduced to that of the original mycotoxin. Moreover, B. subtilis ZJ-2019-1 successfully restored the abnormal serum biochemical indexes caused by mycotoxins in mice, mitigated liver and intestine damage induced by AFB1, and alleviated liver and uterus injury caused by ZEN. The results demonstrate that the capability of B. subtilis ZJ-2019-1 to effectively degrade AFB1 and ZEN simultaneously, both in vitro and in vivo, thereby highlighting its potential as a promising detoxification agent for animals. Declarations Competing interests The authors declare no competing interests. Funding This work was funded by the Science and Technology Innovation Engineering Fund of Institute of Feed Research of Chinese Academy of Agricultural Sciences (CAAS-ASTIP-2023-IFR-14). Author Contribution Xiumin Wang and Jin Quanwang provided the design and concept of the experiment, and provided material support for the experiment. Jian Wenwu and Wei An jointly completed the experiment and wrote the main manuscript text. Zhenlong Wang helped to draw Figures 1-5, while Boquan Gao, Jiaxue Wang, Ya Zhao, and Yaping Guo assisted in the dissection and organ processing of experimental mice. Bing Han and Hui Tao made revisions to the article and finalized it. Acknowledgments We want to thank all the scientists, without their dedicated work, this review would not have been possible. We would also like to thank the editor and the reviewers for their critical reading, thoughtful comments, and constructive suggestions. We also thank the Science and Technology Innovation Engineering Fund of Institute of Feed Research of Chinese Academy of Agricultural Sciences for funding the research work. Data availability Data supporting the findings of this study are available within the paper. Additional data can be provided by the corresponding author upon reasonable request. References Abid-Essefi S, Baudrimont I, Hassen W, Ouanes Z, Mobio TA, Anane R, Creppy EE, Bacha H (2003) DNA fragmentation, apoptosis and cell cycle arrest induced by zearalenone in cultured DOK, Vero and Caco-2 cells: prevention by Vitamin E. Toxicology 192:237–248. An YN (2017) Mechanisms of aflatoxins b1-induced autophagy and extracellular traps in macrophages. Dissertation, Jilin University, Jilin. Assunção R, Viegas S (2020) Mycotoxin exposure and related diseases. Toxins 12(3):17. Awuchi CG, Ondari EN, Ogbonna CU, Upadhyay AK, Baran K, Okpala COR, Korzeniowska M, Guiné RPF (2021) Mycotoxins affecting animals, foods, humans, and plants: Types, occurrence, toxicities, action mechanisms, prevention, and detoxification strategies-a revisit. Foods 10(6):1279. Cai GD, Sun K, Xiang ZL, Wang L, Zhou H (2017) Effect of zearalenone on activation and proliferation of mouse T lymphocyte in vitro. Chin J Anim Vet Sci 48:1357–1364. Cheng CL, Zhao XH, Jiang HM, Fang J, Tian Y (2018) Research progress of aflatoxin and its control technology. Food Ind 39:296–300. Cheng XY (2022) Autophagy mediated by TRPML1 as a target for aflatoxins B1-induced injury and repair of porcine intestinal epithelial cells. Dissertation, Jiangxi Agricultural University, Jiangxi. Deng ZF, Wang LJ, Wu XQ, Yang HH, Li TY (2023) Research progress on metabolism and molecular toxicity of zearalenone in animals. China Anim Husb Vet Med 50:5194–5203. Eshelli M, Harvey L, Edrada-Ebel R, McNeil B (2015) Metabolomics of the bio-degradation process of aflatoxin B1 by actinomycetes at an initial pH of 6.0. Toxins 7(2):439–456. Fang QA, Du M, Chen J, Liu T, Zheng Y, Liao Z, Zhong Q, Wang L, Fang X, Wang J (2020) Degradation and detoxification of aflatoxin B1 by tea-derived Aspergillus niger RAF106. Toxins 12(12):777. Frisvad JC, Hubka V, Ezekiel CN, Hong SB, Nováková A, Chen AJ, Arzanlou M, LarsenTO, Sklenář F, Mahakarnchanakul W, Samson RA, Houbraken J (2019) Taxonomy of Aspergillus section Flavi and their production of aflatoxins, ochratoxins and other mycotoxins. Stud Mycol 93:1–63. Guengerich FP, Johnson WW, Shimada T, Ueng YF, Yamazaki H, Langouët S (1998) Activation and detoxication of aflatoxin B1. Mutat res 402:121–128. Guindon KA, Bedard LL, Massey TE (2007) Elevation of 8-hydroxydeoxyguanosine in DNA from isolated mouse lung cells following in vivo treatment with aflatoxin B(1). Toxicology 98(1):57–62. Guindon-Kezis KA, Mulder JE, Massey TE (2014). In vivo treatment with aflatoxin B1 increases DNA oxidation, base excision repair activity and 8-oxoguanine DNA glycosylase 1 levels in mouse lung. Toxicology 321:21–26. Guo Q, Zhou H, Hao J, Ye J, Cao Y, Liu X, Hu H (2021) Research on the removal effect and mechanism of tibet Rhodotorula mucilaginosa on zearalenone. Sci Technol Cereals Oils Foods 29:192–197. Hamilton D (2022) Understanding mycotoxin-induced illness: Part 1. Altern Ther Health Med 28(5):8–11. Hao S, Pan S, Hu J, Gan F, Huang K (2015) Aflatoxin B1 suppressed T-cell response to anti-pig-CD3 monoclonal antibody stimulation in primary porcine splenocytes: A role for the extracellular regulated protein kinase (ERK1/2) MAPK signaling pathway. J Agric Food Chem 63(26):6094–6101. Harkai P, Szabó I, Cserháti M, Csilla K, Risa A, Radó J, Balázs A, Berta K, Kriszt B (2016) Biodegradation of aflatoxin-B1 and zearalenone by Streptomyces sp. collection. Int Biodeterior Biodegrad 108:48–56. He R (2021) Screening, identification and degradation characteristics of zearalenone-degrading bacteria. Dissertation, Henan University of Technology, Henan. Hussein HS, Brasel JM (2001) Toxicity, metabolism, and impact of mycotoxins on humans and animals. Toxicology 167:101–134. Jaskiewicz K, Close PM, Thiel PG, Cole RJ (1988) Preliminary studies on toxic effects of cyclopiazonic acid alone and in combination with aflatoxin B1 in non-human primates. Toxicology 52:297–307. Ji J (2017) Combined toxicity of deoxynivalenol and zearalenone on ANA-1 metabolome of Kunming mice and macrophages. Dissertation, Jiangnan University, Jiangsu. Ji Q (2020) The toxic effect of AFB1 on primary mouse peritoneal macrophages and the mechanism of iron homeostasis. Dissertation, Northeast Agricultural University, Heilongjiang. Kang JG, Li Y, Jiang GJ, Wang YB, Ma ZF (2022) Lycopene Cuscuta chinensis and Taishan panshi powder can alleviate the reproductive toxicity of zearalenone in pregnant rats. Chin J Vet Sci 42:1895–1901, 1908. Kumar V, Bahuguna A, Lee JS, Sood A, Han SS, Chun HS, Kim M (2023) Degradation mechanism of aflatoxin B1 and aflatoxin G1 by salt tolerant Bacillus albus YUN5 isolated from 'doenjang', a traditional Korean food. Food Res 165:112479. Liu SS, Shi JB, Li Q, Cai GD, Zhou H, Gu JH, Yuan Y, Liu XZ, Liu PZ, Bian JC (2020) Effects of zearalenone on activation, proliferation and differentiation of mouse lymphocyte B in vitro. Chin J Vet Sci 50:1602–1608. Malir F, Pickova D, Toman J, Grosse Y, Ostry V (2023) Hazard characterisation for significant mycotoxins in food. Mycotoxin Res 39:81–93. Monika C, Philippe D (2019) Mycotoxin migration in moldy foods. Curr Opin Food Sci 29:88–93. Pang JC (2021) Study on the application of Bacillus subtilis in the elimination of feed toxin. Fuzhou University. Patrícia V, Guilherme LDS, Isadora ZE, Maria CD, Daiane H, Danielle MP, Lucélia H, Maria LS, Patricia VNF (2021) Effects of infestations of the storage mite Tyrophagus putrescentiae (Acaridae) on the presence of fungal species and mycotoxin production in stored products. J Stored Prod 94:101883. Robens JF, Richard JL (1992) Aflatoxins in animal and hman health. Rev Environ Contam. Toxicology 127:69–94. Rotimi OA, Onuzulu CD, Dewald AL, Ehlinger J, Adelani IB, Olasehinde OE, Rotimi SO, Goodrich JM (2021) Early life exposure to aflatoxin B1 in rats: Alterations in lipids, hormones, and DNA methylation among the offspring. IJERPH 18(2):589. Ruzsás C, Biró-Gosztonyi M, Wöller L, Mess B (1979) Effect of the fungal toxin (zearalenone) on the reproductive system and fertility of male and female rats. Acta Biol Acad Sci Hung 30(4):335–345. Shi AM, Wang Y, Ma WQ, Liu N, Zhou ZH (2023) Analysis of liver biochemical indexes and metabolism related factors in patients with non-homogeneous fatty liver. Chin J Hepatol 28:1346–1350. Song C, Fu CQ, Huangfu HP, Zhang AG, Wang YK, Shi DM, Wang JD, Peng W (2023) Effects of curcumin on zearalenone-induced oxidative stress and NLRP3 inflammasome activation in mice liver. Chin J Vet Sci 43:1898–1904. Su Y, Sun Y, Ju D, Chang S, Shi B, Shan A (2018) The detoxification effect of vitamin C on zearalenone toxicity in piglets. Ecotoxicol Environ Saf 158:284–292. Sun XQ, Cui XX, Li W, Han MY, Ishfaq M (2016) Establishment and evaluation of subchronic liver injury model induced by aflatoxins B in AA broilers. China Poul 38:28–32. Takemura H, Shim JY, Sayama K, Tsubura A, Zhu BT, Shimoi K (2007) Characterization of the estrogenic activities of zearalenone and zeranol in vivo and in vitro. J Steroid Biochem Mol Biol 103:170–177. Tan H, Hu Y, He J, Wu L, Liao F, Luo B, He Y, Zuo Z, Ren Z, Zhong Z, Peng G, Deng J (2014) Zearalenone degradation by two Pseudomonas strains from soil. Mycotoxin Res 30:191–196. Tian SL, Li W (2017) Research progress and control of zearalenone toxin. Cereal Feed Ind 12:36–40. Vance CK, King EH, Bowers SD, Ryan P, Walters K, Shappell NW (2019) Reproductive Performance of Mares Fed Dietary Zearalenone. Front Vet Sci 6:423. Vlata Z, Porichis F, Tzanakakis G, Tsatsakis A, Krambovitis E (2006) A study of zearalenone cytotoxicity on human peripheral blood mononuclear cells. Toxicol Lett 165:274–281. Wan BY (2022) Effect of zearalenone on ovarian development in weaned piglets via SIRT1/PGC − 1α pathway. Dissertation, Shandong Agricultural University, Shandong. Wang S, Fu W, Zhao X, Liu H, Zhou L, Li J, Cheng R, Wu X, Li X, Sun C (2022) Zearalenone disturbs the reproductive-immune axis in pigs: the role of gut microbial metabolites. Microbiome 10:234. Wang YR, Bai HY, Wang GY, Yang BH, Liao GZ (2018) Effect of feed supplemented with Bacillus coagulans and Bacillus subtilis on growth performance of growing pigs. Feed Res 9:1–5. Wu H, Xian J, Liu D (2009) Preliminary study on a zearalenone elimination yeast. Sci Technol Food Ind 30:215–216, 220. Wu JW, Wang ZL, An W, Gao BQ, Li CX, Han B, Tao H, Wang JQ, Wang XM, Li HR. (2024) Bacillus subtilis simultaneously detoxified aflatoxin B1 and zearalenone. Appl Sci 14:1589. Xu J, Wang H, Zhu Z, Ji F, Yin X, Hong Q, Shi J (2016) Isolation and characterization of Bacillus amyloliquefaciens ZDS-1: Exploring the degradation of Zearalenone by Bacillus spp. Food Control 68:244–250. Yan JC, Zheng YF, Zeng QL, Zhu HJ, Zhu XQ (2004) Effect of zearalenone on gap junctional communication. Toxicology 3:160–162. Yang ML, Wu FY, Liu J.H, Liu YJ, Chen BJ (2019) Research progress on toxicity of zearalenone. Feed Res 42:74–77. Yu ZL, Zhang LS, Wu DS (2005) Effects of zearalenone on proliferation and apoptosis of breast cancer cell line MCF-7. Chin J Prev Med 5:34–37. Zhang M, Zhao J, Xu JR, Cao L, Ma SB (2021) Toxic effect of AFB1 on canine hepatocytes and protective effect of NAC. J JNWAFU 49:1–10. Zhang Q, Xiong J, Zhao C, Wang Y, Zhang X (2016) Screening of zearalenone virus-free strain and preliminary study on virus-free mechanism. Sci Technol Cereals Oils Foods 24:76–81. Zhao CM, Xie WT, Lin HY, Hu YX, Ma WA, Li Y, Chen ZB (2024) Protective effect of Inonotus obliquus extract on liver injury induced by aflatoxins B1 in mice. China Anim Husb Vet Med 51:864–874. Zhou JC, Shi DH, Ji C (2020) Research progress on toxicity of zearalenone and deoxynivalenol to animals. Chin J Anim Nutr 32:2460–2466. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4590182","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":321679210,"identity":"494a59a3-94bd-4c78-a538-1aa115a15f79","order_by":0,"name":"Jianwen Wu","email":"","orcid":"","institution":"Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Jianwen","middleName":"","lastName":"Wu","suffix":""},{"id":321679211,"identity":"767c0080-2a0c-4620-ad4c-dc0f8eef1079","order_by":1,"name":"Wei An","email":"","orcid":"","institution":"Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"An","suffix":""},{"id":321679212,"identity":"4cbdb82e-f774-435d-99f0-1222d69af146","order_by":2,"name":"Zhenlong Wang","email":"","orcid":"","institution":"Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Zhenlong","middleName":"","lastName":"Wang","suffix":""},{"id":321679213,"identity":"95dcf4ef-143b-4208-9ee5-cffa9abb7cae","order_by":3,"name":"Boquan Gao","email":"","orcid":"","institution":"Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Boquan","middleName":"","lastName":"Gao","suffix":""},{"id":321679214,"identity":"b176721d-d5fb-481b-aa44-f8bf8da02873","order_by":4,"name":"Jiaxue Wang","email":"","orcid":"","institution":"Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Jiaxue","middleName":"","lastName":"Wang","suffix":""},{"id":321679215,"identity":"97fedbcd-9570-494e-a0ef-16d23bbb49a1","order_by":5,"name":"Ya Zhao","email":"","orcid":"","institution":"Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Ya","middleName":"","lastName":"Zhao","suffix":""},{"id":321679216,"identity":"861fca8e-fc45-481f-ab8e-ddfa549e6069","order_by":6,"name":"Bing Han","email":"","orcid":"","institution":"Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Bing","middleName":"","lastName":"Han","suffix":""},{"id":321679217,"identity":"73aa0931-c408-47a7-83fd-bd9baccff010","order_by":7,"name":"Hui Tao","email":"","orcid":"","institution":"Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Hui","middleName":"","lastName":"Tao","suffix":""},{"id":321679218,"identity":"979077d0-0120-41d0-9c0a-a89775c158dd","order_by":8,"name":"Yaping Guo","email":"","orcid":"","institution":"Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Yaping","middleName":"","lastName":"Guo","suffix":""},{"id":321679219,"identity":"5a351112-1d67-4e2b-a99d-7a94871d48ab","order_by":9,"name":"Jinquan Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA00lEQVRIiWNgGAWjYLACCQMwxfiAZC3MBiRbxiZBlDKD42cPv7AouCO74fjZYxU/auzsGdjPHsCv5UxemoWEwTPjDUDGzZ5jyYkNPHkJeLWYHcgxM5AwOJy4Aci4zcDGnMAgwYPfT2bn30C1ABnFDP/q7QlruZFj/ACs5UaOGTNj22HGBkJa7G+8MQMG8mHjmTfeGEv29h1PbOPJwa9Fsj/H+LPEn8OyfedzDD/8+FZtz89+hmD8sEkD44OxAc4lpB4ImD9+QNYyCkbBKBgFowAdAACnDUduTrDahQAAAABJRU5ErkJggg==","orcid":"","institution":"Chinese Academy of Agricultural Sciences","correspondingAuthor":true,"prefix":"","firstName":"Jinquan","middleName":"","lastName":"Wang","suffix":""},{"id":321679220,"identity":"e3959fac-0574-436e-8da3-8602243881a5","order_by":10,"name":"Xiumin Wang","email":"","orcid":"","institution":"Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Xiumin","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2024-06-16 14:57:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4590182/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4590182/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":59831840,"identity":"a4907aba-818c-41c9-ace9-86c130f80022","added_by":"auto","created_at":"2024-07-08 07:38:48","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":493443,"visible":true,"origin":"","legend":"\u003cp\u003eDegradation of AFB1 and ZEN in corn gluten meal by \u003cem\u003eB. subtilis \u003c/em\u003eZJ-2019-1 in gastrointestinal environment. A: Degradation of AFB1 in corn gluten meal by ZJ-2019-1 in gastrointestinal environment; B: Degradation of ZEN in corn gluten meal by ZJ-2019-1 in gastrointestinal environment.\u003c/p\u003e","description":"","filename":"Figure1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4590182/v1/bc8146c69f27af73c0647b81.jpeg"},{"id":59831332,"identity":"6e84397d-b8a5-4d71-8684-80d49d83e449","added_by":"auto","created_at":"2024-07-08 07:30:47","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":490433,"visible":true,"origin":"","legend":"\u003cp\u003eCytotoxicity of AFB1 and ZEN towards RAW264.7 cells before and after degradation by \u003cem\u003eB. subtilis \u003c/em\u003eZJ-2019-1. A: Cytotoxicity of AFB1 and its degradation products by ZJ-2019-1. B: Cytotoxicity of ZEN and its degradation products by ZJ-2019-1. Different letters indicate significant differences among the means according to Duncan’s test (p \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Figure2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4590182/v1/3e54973a6abd6f6d0cfbfa67.jpeg"},{"id":59831839,"identity":"3be48ed5-a5b7-4882-810d-5a4b3418eaac","added_by":"auto","created_at":"2024-07-08 07:38:47","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":67631,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of \u003cem\u003eB. subtilis \u003c/em\u003eZJ-2019-1 on serum biochemical indices of mice exposed to AFB1. Different letters indicate significant differences among the means according to Duncan’s test (p \u0026lt; 0.05). AST/ALT: aspartate aminotransferase/alanine transaminase; ALP: alkaline phosphatase; TBA: total bile acids; TG: triglyceride; TC: total cholesterol; γ-TGase: γ-glutamyltransferase; AMY: amylase; GLU: glucose.\u003c/p\u003e","description":"","filename":"Figure3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4590182/v1/67c0102f80d41399383be478.jpeg"},{"id":59831334,"identity":"7ae7be4a-780e-435a-93aa-899db08d0ed4","added_by":"auto","created_at":"2024-07-08 07:30:47","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":69224,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of \u003cem\u003eB. subtilis \u003c/em\u003eZJ-2019-1 on serum biochemical indices of mice exposed to ZEN. Different letters indicate significant differences among the means according to Duncan’s test (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Figure4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4590182/v1/b03a01eb13e24bc28d14037a.jpeg"},{"id":59831330,"identity":"e4e3d6d7-055f-4f3b-aaae-9447c64b0db7","added_by":"auto","created_at":"2024-07-08 07:30:47","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":316502,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of \u003cem\u003eB. subtilis \u003c/em\u003eZJ-2019-1 on organ tissues of mice exposed to AFB1 and ZEN. A: Liver of mice fed with a basic diet; B: Liver of mice fed with AFB1-contaminated diet; C: Liver of mice fed with AFB1-contaminated diet and \u003cem\u003eB. subtilis \u003c/em\u003eZJ-2019-1; D: Duodenumof mice fed with a basic diet; E: Duodenum of mice fed with AFB1-contaminated diet; F: Duodenum of mice fed with AFB1-contaminated diet and \u003cem\u003eB. subtilis \u003c/em\u003eZJ-2019-1; G: Liver of mice fed with a basic diet ; H: Liver of mice fed with ZEN-contaminated diet; I: Liver of mice fed with ZEN-contaminated diet and \u003cem\u003eB. subtilis \u003c/em\u003eZJ-2019-1; J: Uterus of mice fed with a basic diet; K: Uterus of mice fed with ZEN-contaminated diet; L: Uterus of mice fed with ZEN-contaminated diet and \u003cem\u003eB. subtilis \u003c/em\u003eZJ-2019-1.\u003c/p\u003e","description":"","filename":"Figure5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4590182/v1/144ef12b72552213b5f5cbe8.jpeg"},{"id":65357048,"identity":"1268a017-422e-42c6-8216-9891cbf50e6f","added_by":"auto","created_at":"2024-09-26 12:24:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2026646,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4590182/v1/7b05d1a1-43e9-4825-9938-6d226f078704.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Biodetoxification of both AFB1 and ZEN by Bacillus subtilis ZJ-2019-1 in gastrointestinal environment and in mice","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMycotoxins are toxic compounds produced by molds, which are widely distributed in nature, particularly in food and feed (Monika et al. 2019; Hamilton 2022). There exist various types of mycotoxins, with aflatoxin (AFB), zearalenone (ZEN), ochratoxin (OTA), and deoxynivalenol (DON) being the most well-known ones. These mycotoxins pose a serious threat to human and animal health (Assun\u0026ccedil;\u0026atilde;o et al. 2020; Patr\u0026iacute;cia et al. 2021).\u003c/p\u003e \u003cp\u003eAmong them, both aflatoxin B1 (AFB1) and zearalenone (ZEN) are commonly co-occurring mycotoxins in cereals. AFB1, the most potent member of the aflatoxin family, is produced by \u003cem\u003eAspergillus flavus\u003c/em\u003e. AFB1 poses a significant global health threat, particularly in developing countries with favorable climatic conditions for \u003cem\u003eA. flavus\u003c/em\u003e growth and AFB production (Awuchi et al. 2021; Frisvad et al. 2019). The toxicity of AFB1 mainly arises from its metabolites, notably AFB1-8, 9-epoxide, a highly reactive compound that binds to DNA and proteins, resulting in cellular genetic material and protein synthesis inhibition (Cheng et al. 2018; Guengerich et al. 1998). These effects ultimately lead to apoptosis, tissue damage, gene mutation, and tumor formation (Guindon-Kezis et al. 2014; Rotimi et al. 2021), notably influencing the liver due to its role as the primary site for AFB1 metabolism and detoxification; prolonged or high-dose exposure to AFB1 can cause hepatocyte injury, jaundice, hepatitis, liver cirrhosis, and even liver cancer (Guindon et al. 2007; Zhang et al. 2021; Jaskiewicz et al. 1988). Additionally, AFB1 also inhibit immune system function, compromising the body's resistance to pathogens. It diminishes immunoglobulin production by affecting various immune cells, thereby attenuating the immune response (Sun et al. 2016; Hao et al. 2015; Hussein and Brasel. 2001; Robens and Richard 1992). ZEN is a secondary metabolite produced by \u003cem\u003eFusarium\u003c/em\u003e spp., which are widely distributed in corn, wheat, barley, and other grains and their products. Due to its estrogenic activity and ability to mimic the role of estrogen in the body, ZEN is referred to as \"environmental estrogens in feed\". Given its widespread presence in food and feed, ZEN poses potential risks to human and animal health, particularly the reproductive system (Takemura et al. 2007; Wang et al. 2022; Vance et al. 2019). ZEN and its metabolites can imitate the biological effects of estrogen by binding to estrogen receptors and activating estrogen-dependent gene expression. In animals, ZEN can induce various physiological and pathological effects, including abnormal development of reproductive organs, reduced reproductive ability, and alterations in hormone levels (Malir et al. 2023; Deng et al. 2023; Yu et al. 2005; Ruzs\u0026aacute;s et al. 1979; Su et al. 2018). Furthermore, ZEN has the potential to impact the immune system (Yang et al. 2019; Vlata et al. 2006; Cai et al. 2017). It has been also demonstrated that ZEN and its metabolites possess cytotoxicity and genotoxicity properties capable of causing DNA damage, cell cycle arrest, as well as apoptosis (Liu et al. 2020; Tian and Li. 2017; Zhou et al. 2020), all potentially associated with the carcinogenic risk posed by ZEN.\u003c/p\u003e \u003cp\u003eNotably, the simultaneous presence of AFB1 and ZEN in grain-based food and animal feed poses significant health hazards to both human and animal health due to their potent mutagenic, cytotoxic, and carcinogenic properties. The prevention and control of co-occurring mycotoxin pollution present a global challenge that requires comprehensive measures throughout all stages of crop growth, harvest, storage, and processing. This includes utilizing mildew-resistant varieties, improving storage conditions, implementing biological and chemical methods to mitigate mold growth and toxin production. Among the various strategies available for mycotoxin removal, the biodegradation approach stands out due to its some advantages, such as environmental friendliness, practical feasibility, and economic viability (Pang 2021). Eshelli et al. identified three types of AFB1-degrading actinomycetes, namely \u003cem\u003eRhodococcus\u003c/em\u003e ATCC4277, \u003cem\u003eStreptomyces lividans\u003c/em\u003e TK 24, and \u003cem\u003eStreptmyces aureus\u003c/em\u003e ATCC10762, with degradation rates of 95.95%, 87.95%, and 86.10%, respectively (Eshelli et al. 2015). \u003cem\u003eS. cacaoi\u003c/em\u003e degraded 88.34% AFB1. Adebo et al. isolated three strains of \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e, \u003cem\u003eP. aeruginosa\u003c/em\u003e, and staphylococci which could degrade AFB1 from gold aquifers, with degradation rates of 47.7%, 51.7%, and 56.8%, respectively (Harkai et al. 2016). Kumar et al., on the other hand, isolated a salt-tolerant \u003cem\u003eBacillus albus\u003c/em\u003e YUN5 capable of degrading AFB1 from traditional Korean food \"doenjang\", where its cell-free supernatant incubated with 2 mg/L AFB1 for 48 h achieved a degradation rate of 54.96% AFB1 (Kumar et al. 2023). Furthermore, \u003cem\u003eA. niger\u003c/em\u003e RAF106 demonstrated the ability to degrade AFB1 at 30℃ and pH 4\u0026ndash;8, achieving a maximum degradation rate of 54.96% (Fang et al. 2020). \u003cem\u003eB. amylolyticus\u003c/em\u003e Fu2-3 derived from soil as well as \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e ZDS-1 were able to degrade more than 95% and 97.8% of ZEN, respectively (Zhang et al. 2016; Xu et al. 2016). Moreover, \u003cem\u003eRhodococcus\u003c/em\u003e SYA13 obtained from oil-contaminated soil efficiently degraded ZEN by 87.1% (He 2021). The \u003cem\u003ePseudomonas\u003c/em\u003e TH-N1 strain was found to degrade 79% ZEN at 37℃ and pH 4.5 (Tan et al. 2014). Wu et al. discovered that \u003cem\u003eSaccharomyces\u003c/em\u003e CLY01 achieved a degradation rate of 96.79% for ZEN (Wu et al. 2009). \u003cem\u003eR. mucilaginosa\u003c/em\u003e could degrade over 90% of ZEN (Guo et al. 2021). However, few bacteria have been reported to simultaneously degrade both AFB1 and ZEN.\u003c/p\u003e \u003cp\u003eOur previous study showed that \u003cem\u003eB. subtilis\u003c/em\u003e ZJ-2019-1 can simultaneously degrade AFB1 and ZEN in vitro (Wu et al. 2024). The present study aimed to investigate the detoxication of AFB1 and ZEN by \u003cem\u003eB. subtilis\u003c/em\u003e ZJ-2019-1 gastrointestinal environment and in vivo, while also exploring the cytotoxicity of mycotoxin degradation products.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eBacterial source, chemicals, and reagents\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eB. subtilis\u003c/em\u003e ZJ-2019-1 strain was isolated from pig feces collected from pig houses in Xingtai, Hebei Province (Vlata et al. 2006). The strain has been stored at the General Microbiology Center of the China Microbiological Culture Preservation and Management Committee (CGMCC No. 23636). The small intestinal and colonic fluid was purchased from Coolaber (Beijing). The mice were purchased from Vital River (Beijing). All chemical reagents were of analytical grade.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePreparation of\u003c/b\u003e \u003cb\u003eB. subtilis\u003c/b\u003e \u003cb\u003eZJ-2019-1 powder\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe monoclonal colonies of \u003cem\u003eB. subtilis\u003c/em\u003e ZJ-2019-1 were isolated from LB solid medium, inoculated in LB liquid medium, and cultured in a constant temperature shaker. Once \u003cem\u003eB. subtilis\u003c/em\u003e ZJ-2019-1 reached a stable growth phase, the bacterial solution was centrifuged and the supernatant was discarded. The cell pellet was then re-suspended with freeze-drying protectant (cell:protectant\u0026thinsp;=\u0026thinsp;1:4) and frozen overnight at -80℃. Subsequently, the bacteria were subjected to freeze-drying for 48 h using a freeze-dryer and stored in a refrigerator at -80℃.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDegradation of AFB1 and ZEN by\u003c/b\u003e \u003cb\u003eB. subtilis\u003c/b\u003e \u003cb\u003eZJ-2019-1 in small intestinal fluid simulation\u003c/b\u003e\u003c/p\u003e \u003cp\u003eBoth corn gluten meal (0.999 g) and bacterial powder (0.01 g) were weighed in a 50 mL flask, followed by the addition of 16 mL gastric buffer. The mixture was then incubated in water bath shaker at 39℃ and 180 rpm for 4 h. Subsequently, 4 mL small intestinal buffer was added and incubated for 5 min; 2 mL small intestinal fluid was then added and incubated for 14 h. Finally, 2 mL colonic fluid was added and incubated for 24 h. The control samples were prepared accordingly, consisting of 0.999 g of corn gluten meal and 0.01 g of skim milk powder. Each treatment was repeated three times. Following the reaction, samples were collected and subjected to high performance liquid chromatography (HPLC) analysis to determine the degradation rate of mycotoxins by \u003cem\u003eB. subtilis\u003c/em\u003e ZJ-2019-1.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDetection of the degradation of AFB1 and ZEN by\u003c/b\u003e \u003cb\u003eB. subtilis\u003c/b\u003e \u003cb\u003eZJ-2019-1\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe degradation of AFB1 and ZEN by \u003cem\u003eB. subtilis\u003c/em\u003e ZJ-2019-1 were detected using an Agilent SB-C18 (150 mm \u0026times; 4.6 mm, 5 \u0026micro;m) column. The mobile phase consisted of a mixture of methanol, acetonitrile, and water (22:22:56 for AFB1 and 8:46:46 for ZEN). Prior to use, the mobile phase was filtered through a 0.22 \u0026micro;m membrane. The AFB1 or ZEN samples were added into methanol (1:3) and mixed on a rotator for 30 s. After centrifugation at room temperature for 1 min at 12,000 r/min, the supernatant was collected using a 2 mL syringe, filtered through a 0.22 \u0026micro;m aseptic filter membrane. For AFB1 analysis, the column temperature was maintained at 40℃, while for ZEN analysis it was kept at 30℃. The injection volume was set at 10 \u0026micro;L with a flow rate of 1.0 mL/min. Fluorescence detection utilized an excitation/emission wavelength of 365 nm/430 nm for AFB1 and 235 nm/460 nm for ZEN.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCytotoxicity of AFB1 and ZEN before and after degradation by\u003c/b\u003e \u003cb\u003eB. subtilis\u003c/b\u003e \u003cb\u003eZJ-2019-1\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe monoclonal colony of \u003cem\u003eB. subtilis\u003c/em\u003e ZJ-2019-1 was inoculated in LB liquid medium and cultured in a constant temperature shaker. Once \u003cem\u003eB. subtilis\u003c/em\u003e ZJ-2019-1 reached a stable growth phase, the culture was centrifuged at 4℃ for 15 min at 8,000 rpm. The resulting supernatant was then filtered through 0.22 \u0026micro;m membrane to obtain acellular supernatant. A volume of 5 mL of cell-free supernatant was separately mixed with AFB1 and ZEN at concentrations of 5, 10, 15, and 20 mg/L, respectively and incubated on a shaker at 37℃. The samples were collected at 0 h, 24 h, 48 h, and 72 h, respectively. Anhydrous ethanol was added to each sample followed by filtration using 0.22 \u0026micro;m membrane to remove miscellaneous bacteria. The RAW264.7 cells were cultured in DMEM medium (10% fetal bovine serum, 0.1% penicillin-streptomycin), and then seeded into a 96-well plate at a density of 5 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells per well (100 \u0026micro;L). Once the number of cells reached 70\u0026ndash;80%, the cell-free supernatant was added for further culture, with each sample have three replicates. After incubation for 24 h, 10% of CCK-8 reagent was added to each well and incubated for 1 h. The absorbance at wavelength of 450 nm was measured.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eExperimental animals and management\u003c/h2\u003e \u003cp\u003eThe 40 KM mice with, with an average body weight of (20\u0026thinsp;\u0026plusmn;\u0026thinsp;2) g, were randomly divided into five groups with eight replicates in each group and one mouse per group. The five groups included the blank group, AFB1 group, ZEN group, AFB1 intervention group (AFB1\u0026thinsp;+\u0026thinsp;\u003cem\u003eB. subtilis\u003c/em\u003e ZJ-2019-1), and ZEN intervention group (ZEN\u0026thinsp;+\u0026thinsp;\u003cem\u003eB. subtilis\u003c/em\u003e ZJ-2019-1). The adaptive culture period lasted for 7 d followed by a 21-d experimental period. Animals are raised in accordance with the animal welfare set forth by the Chinese Academy of Agricultural Sciences (CAAS).\u003c/p\u003e \u003cp\u003e The mouse experiment was performed in accordance with the Animal Care and Use Committee of Institute of Feed Research (IFR) at the Chinese Academy of Agricultural Sciences (CAAS), and it received approval from the Laboratory Animal Ethical Committee and its Inspection of the Institute of Feed Research of CAAS (AEC-CAAS-20090609).\u003c/p\u003e \u003cp\u003e \u003cb\u003eDegradation of AFB1 and ZEN by\u003c/b\u003e \u003cb\u003eB. subtilis\u003c/b\u003e \u003cb\u003eZJ-2019-1 in mice\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe mice were fed a normal diet. They received intragastric administration of 200 \u0026micro;L twice daily, with an interval of 1 h and at 18:00 every day. The blank group was administered aseptic phosphate buffer. The first sample of the AFB1 challenge group contained aseptic phosphate buffer with 0.5 mg/L AFB1, while the second sample consisted of sterilized phosphate buffer. The ZEN challenge group's sample contained aseptic phosphate buffer with 5 mg/L ZEN, and the second gavage used sterilized phosphate buffer. In the AFB1 intervention group, the first intragastric administration included aseptic phosphate buffer with 0.5 mg/L AFB1, the second involved \u003cem\u003eB. subtilis\u003c/em\u003e ZJ-2019-1 (7.2\u0026times;10\u003csup\u003e10\u003c/sup\u003e CFU/mL). Similarly, in ZEN intervention group, the first intragastric administration comprised aseptic phosphate buffer with 5 mg/L ZEN, followed by \u003cem\u003eB. subtilis\u003c/em\u003e ZJ-2019-1 (7.2\u0026times;10\u003csup\u003e10\u003c/sup\u003e CFU/mL).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eSample collection of mice\u003c/h2\u003e \u003cp\u003eThe mice were subjected to a 12-h fasting period prior to the execution of the experiment. The blood samples were collected before dissection, incubated at room temperature for 30 min and then centrifuged at 8,000 r/min for 10 min. The resulting serum was separated and transferred to 1.5 mL centrifuge tube, which was stored at -20℃ for subsequent biochemical analysis. A portion of the liver, uterus, and duodenum were immersed in a 4% tissue fixation solution for tissue sectioning. All animal handling procedures adhered to the protocols approved by the Animal Protection and Use Committee of the CAAS.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe statistical analysis was conducted using one-way analysis of variance (ANOVA) within a 95% confidence level, followed by either Student's test or Duncan's test, using the statistical software SPSS.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eDegradation of AFB1 and ZEN by\u003c/b\u003e \u003cb\u003eB. subtilis\u003c/b\u003e \u003cb\u003eZJ-2019-1 in gastrointestinal simulation\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe ability of \u003cem\u003eB. subtilis\u003c/em\u003e ZJ-2019-1 to degrade mycotoxins in animals was investigated by simulating the gastrointestinal environment of monogastric animals using naturally molded corn gluten meal. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, skim milk powder had no impact on AFB1 and ZEN degradation, whereas \u003cem\u003eB. subtilis\u003c/em\u003e ZJ-2019-1 exhibited degradation capabilities for AFB1 and ZEN, with the degradation rate of 14.71% and 19.53%, respectively. These findings suggest that \u003cem\u003eB. subtilis\u003c/em\u003e ZJ-2019-1 can effectively degrade AFB1 and ZEN within the gastrointestinal environment, highlighting its potential as a detoxifier of mycotoxins in animals.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eCytotoxicity of AFB1 and ZEN degradation products by\u003c/b\u003e \u003cb\u003eB. subtilis\u003c/b\u003e \u003cb\u003eZJ-2019-1\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eB. subtilis\u003c/em\u003e ZJ-2019-1 strain exhibits in vitro degradation of mycotoxin and demonstrates potential for in vivo mycotoxin degradation; however, the toxicity of its degradation products requires further investigation. To assess the toxicity, we examined the effects of the products of AFB1 and ZEN degraded by \u003cem\u003eB. subtilis\u003c/em\u003e ZJ-2019-1 on the proliferation of RAW264.7 cells. It was observed that there was a decrease in RAW264.7 cell proliferation rate with increasing concentrations of AFB1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) and ZEN (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). When the concentration of AFB1 and ZEN was 20 mg/L, the proliferation rate of RAW264.7 cells was 18.83% and 2.77%, respectively; however, after incubation with \u003cem\u003eB. subtilis\u003c/em\u003e ZJ-2019-1 for 72 h, the degradation products of AFB1 and ZEN remarkably enhance the proliferation rate of RAW264.7 cells, with the proliferation rate of 65.24% and 62.03%, indicating their ability to promote cell growth. The results demonstrate that the degradation products of AFB1 and ZEN by \u003cem\u003eB. subtilis\u003c/em\u003e ZJ-2019-1 exhibit much lower cytotoxicity towards RAW264.7 cells compared to AFB1 and ZEN.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEffects of\u003c/b\u003e \u003cb\u003eB. subtilis\u003c/b\u003e \u003cb\u003eZJ-2019-1 on serum biochemical indices of mice exposed to AFB1 and ZEN\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWhen exposed to AFB1, mice exhibited an elevation in the rate of aspartate aminotransferase/alanine transaminase (AST/ALT), alkaline phosphatase (ALP), total bile acids (TBA), triglyceride (TG), and total cholesterol (TC) levels in the serum, indicating potential liver inflammation. In contrast, oral administration of \u003cem\u003eB. subtilis\u003c/em\u003e ZJ-2019-1 reduced these indices (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). Upon exposure to AFB1, mice experienced a decrease in γ-glutamyltransferase (γ-TGase), amylase (AMY), and glucose (GLU) levels in the serum; however, oral administration of \u003cem\u003eB. subtilis\u003c/em\u003e ZJ-2019-1 increased their levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWhen exposed to ZEN, mice exhibited an elevation in the rate of AST/ALT, γ-TGase, ALP, AMY, TG, TC, and GLU levels in the serum (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE-\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH); oral administration of \u003cem\u003eB. subtilis\u003c/em\u003e ZJ-2019-1 reduced their levels except γ-TGase. Upon exposure to ZEN, mice reduced TBA level; oral administration of \u003cem\u003eB. subtilis\u003c/em\u003e ZJ-2019-1 enhanced the levels of its level (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). These results suggest that \u003cem\u003eB. subtilis\u003c/em\u003e ZJ-2019-1 may improve the inflammatory response.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEffects of\u003c/b\u003e \u003cb\u003eB. subtilis\u003c/b\u003e \u003cb\u003eZJ-2019-1 on organ tissues of mice exposed to AFB1\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn the blank group, hepatocytes were observed centrally within irregular polygonal shapes and arranged in a cord-like structures. The hepatocyte cords exhibited a radial arrangement around the central vein, while the hepatic sinusoid space appeared uniformly without any dilatation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). No pathological changes such as hemorrhage, necrosis, and inflammation were detected in the liver tissue, indicating a normal tissue structure. Conversely, mice challenged with AFB1 displayed focal necrosis of hepatocytes accompanied by localized infiltration of inflammatory cells within the liver tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Notably, administration of \u003cem\u003eB. subtilis\u003c/em\u003e ZJ-2019-1 to the AFB1 intervention group resulted in liver tissue resembling that of the blank group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC), suggesting its potential to prevent hepatocyte necrosis and inflammation induced by AFB1.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe intestinal villous epithelial cells in the blank group exhibited intact and orderly arrangement. The distinct presence of the intestinal mucous layer, mucosal muscle layer, submucosa, and muscle layer indicated the absence of pathological changes such as necrosis, bleeding, and inflammation within this group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Conversely, in the AFB1 challenge group, the intestinal villous epithelial cells displayed necrosis and exfoliation, while locally exposing the lamina propria of intestinal villi (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Notably, administration of \u003cem\u003eB. subtilis\u003c/em\u003e ZJ-2019-1 to the AFB1 intervention group demonstrated normal intestinal tissue characteristics (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF), indicating that \u003cem\u003eB. subtilis\u003c/em\u003e ZJ-2019-1 effectively prevent AFB1-induced necrosis and exfoliation of intestinal epithelial cells.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffects of\u003c/b\u003e \u003cb\u003eB. subtilis\u003c/b\u003e \u003cb\u003eZJ-2019-1 on organ tissues of mice exposed to ZEN\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn the blank group, it exhibited round hepatocytes arranged in a cord shape around the center vein within an irregular polygonal structure. The hepatic sinusoid space was uniform and devoid of pathological changes such as hemorrhage, necrosis, and inflammation in the liver tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG). Conversely, mice challenged with ZEN displayed focal necrosis of hepatocytes accompanied by inflammatory cell infiltration, edema, and swelling of some cells, light cytoplasmic staining and irregular vacuoles (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH). In comparison to the ZEN challenge group, liver tissue from mice treated with \u003cem\u003eB. subtilis\u003c/em\u003e ZJ-2019-1 showed reduced hepatocyte necrosis and inflammation while exhibiting features, such as hepatocyte edema, cell swelling, cytoplasmic light staining, and irregular vacuoles without any pathological changes like hemorrhage or necrosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI). These findings suggest that \u003cem\u003eB. subtilis\u003c/em\u003e ZJ-2019-1 can reduce the effects of ZEN-induced hepatocyte damage.\u003c/p\u003e \u003cp\u003eThe blank group exhibited diffuse infiltration of inflammatory cells in the interglandular space of the endometrium, along with a significant presence of fibroblasts in the interstitial space of the endometrial gland (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eJ). In the ZEN challenge group, there was slight edema and cell swelling observed in the endometrial glandular cells, accompanied by cytoplasmic light staining and irregular vacuoles within these cells. Additionally, a substantial proliferation of fibroblasts was noted in the endometrial glandular space, along with diffuse infiltration of inflammatory cells and necrotic/exfoliated cells masses within the uterine cavity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eK). Comparatively, mice in the \u003cem\u003eB. subtilis\u003c/em\u003e ZJ-2019-1 intervention group displayed significantly improved uterine conditions compared to those in the ZEN challenge group. Specifically, mild edema and cellular swelling were observed in their endometrial glandular cells, along with lightly stained cytoplasm and irregular vacuoles within these cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eL). Furthermore, diffuse infiltration of inflammatory cells could be seen within their endometrial glandular space. These findings suggest that \u003cem\u003eB. subtilis\u003c/em\u003e ZJ-2019-1 can mitigate necrotic exfoliation caused by ZEN.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe present study aimed to investigate the degradation effect of \u003cem\u003eB. subtilis\u003c/em\u003e ZJ-2019-1 on mycotoxins-AFB1 and ZEN in naturally moldy corn gluten meal by simulating the gastrointestinal environment of monogastric animals. The findings revealed that \u003cem\u003eB. subtilis\u003c/em\u003e ZJ-2019-1 exhibited remarkable potential for detoxification in feed in gastrointestinal environment and in vivo.\u003c/p\u003e \u003cp\u003eAFB1 can induce autophagy by stimulating macrophages to generate reactive oxygen species (ROS), while ZEN can promote apoptosis by inducing mitochondrial dysfunction in macrophages (Wu et al. 2009; Guo et al. 2021). Other studies have indicated that exposure to AFB1 can lead to peritoneal macrophage damage and oxidative stress in mice, which is associated with iron homeostasis imbalance (Abid-Essefi et al. 2003). In our previous study, the ability of \u003cem\u003eB. subtilis\u003c/em\u003e ZJ-2019-1 to degrade AFB1 and ZEN in vitro has been demonstrated, although the toxicity of its degradation products remains to be tested. In this study, the impact of the degradation products of AFB1 and ZEN by \u003cem\u003eB. subtilis\u003c/em\u003e ZJ-2019-1 on the proliferation of RAW264.7 cells was assessed using the CCK8 method, along with effect of mycotoxins on RAW264.7 cell proliferation. The results revealed a decrease in RAW264.7 cell proliferation rate significantly with increasing concentrations of AFB1 and ZEN, and the degradation products of AFB1 and ZEN significantly increased the proliferation rate of RAW264.7 cells compared to the toxin group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). This suggests a significant reduction in toxicity when \u003cem\u003eB. subtilis\u003c/em\u003e ZJ-2019-1 interacts with AFB1 and ZEN.\u003c/p\u003e \u003cp\u003ePrevious studies have demonstrated that AFB1 can induce hepatocyte disorder, cellar swelling, and increased apoptosis rate in mice (Wang et al. 2018). Similarly, ZEN has been shown to elicit various inflammatory reactions in liver cells and cause damage to uterine tissue structure accompanied by edema (Wang et al. 2018; Shi et al. 2023). Furthermore, the impact of AFB1 on porcine intestinal epithelial cells can trigger apoptosis, lead to intestinal injury, and reduce nutritional absorption rates (Ji 2017). In this study, administration of AFB1 to mice resulted in disrupted secretion of AST and ALT, as well as impaired metabolism and transport of TG in serum biochemical index analysis. Additionally, significant liver damage was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Liver injury often leads to abnormal secretion of AST and ALT along with an increase in TG levels (An 2017). Moreover, it was found that AFB1 also induced intestinal damage in mice which caused a decrease in blood GLU content (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Administration of ZEN to mice similarly altered AST/ALT rate and elevated TG level (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), indicating hepatic injury and its estrogenic properties by binding to the estrogen receptor (Ji 2020), causing insulin resistance and a significant increase in blood GLU content, which may contribute to the development of diabetes.\u003c/p\u003e \u003cp\u003eThe probiotic \u003cem\u003eB. subtilis\u003c/em\u003e is commonly used as a feed additive (Yan et al. 2004). In our previous experimental study, the \u003cem\u003eB. subtilis\u003c/em\u003e ZJ-2019-1 strain demonstrated in vitro capability to simultaneously degrade AFB1 and ZEN (Wu et al. 2024). In this work, oral administration of \u003cem\u003eB. subtilis\u003c/em\u003e ZJ-2019-1 significantly alleviated liver injury of mice induced by AFB1 and ZEN exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI). Compared to the groups exposed solely to AFB1 or ZEN, treatment with \u003cem\u003eB. subtilis\u003c/em\u003e ZJ-2019-1 notably reduced focal necrosis of hepatocytes and focal infiltration of inflammatory cells. Moreover, \u003cem\u003eB. subtilis\u003c/em\u003e ZJ-2019-1 effectively mitigated intestinal injury induced by AFB1 through prevention of necrosis and exfoliation of intestinal villus epithelial cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). Meanwhile, administration of \u003cem\u003eB. subtilis\u003c/em\u003e ZJ-2019-1 could reduce mouse uterus damage induced by ZEN while preventing necrotic exfoliation of uterine cells caused by this toxin (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eL). Therefore, oral administration of \u003cem\u003eB. subtilis\u003c/em\u003e ZJ-2019-1 exhibits significant efficacy in preventing AFB1- and ZEN-induced liver, intestinal, and uterus damage in mice.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn this study, \u003cem\u003eB. subtilis\u003c/em\u003e ZJ-2019-1 effectively degraded AFB1 and ZEN in corn gluten meal by simulating the gastrointestinal environment. The toxicity of the degradation products was significantly reduced to that of the original mycotoxin. Moreover, \u003cem\u003eB. subtilis\u003c/em\u003e ZJ-2019-1 successfully restored the abnormal serum biochemical indexes caused by mycotoxins in mice, mitigated liver and intestine damage induced by AFB1, and alleviated liver and uterus injury caused by ZEN. The results demonstrate that the capability of \u003cem\u003eB. subtilis\u003c/em\u003e ZJ-2019-1 to effectively degrade AFB1 and ZEN simultaneously, both in vitro and in vivo, thereby highlighting its potential as a promising detoxification agent for animals.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCompeting interests\u003c/strong\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis work was funded by the Science and Technology Innovation Engineering Fund of Institute of Feed Research of Chinese Academy of Agricultural Sciences (CAAS-ASTIP-2023-IFR-14).\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eXiumin Wang and Jin Quanwang provided the design and concept of the experiment, and provided material support for the experiment. Jian Wenwu and Wei An jointly completed the experiment and wrote the main manuscript text. Zhenlong Wang helped to draw Figures 1-5, while Boquan Gao, Jiaxue Wang, Ya Zhao, and Yaping Guo assisted in the dissection and organ processing of experimental mice. Bing Han and Hui Tao made revisions to the article and finalized it.\u003c/p\u003e\n\u003ch2\u003eAcknowledgments\u003c/h2\u003e\n\u003cp\u003eWe want to thank all the scientists, without their dedicated work, this review would not have been possible. We would also like to thank the editor and the reviewers for their critical reading, thoughtful comments, and constructive suggestions. We also thank the Science and Technology Innovation Engineering Fund of Institute of Feed Research of Chinese Academy of Agricultural Sciences for funding the research work.\u003c/p\u003e\n\u003ch2\u003eData availability\u003c/h2\u003e\n\u003cp\u003eData supporting the findings of this study are available within the paper. Additional data can be provided by the corresponding author upon reasonable request.\u003c/p\u003e "},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAbid-Essefi S, Baudrimont I, Hassen W, Ouanes Z, Mobio TA, Anane R, Creppy EE, Bacha H (2003) DNA fragmentation, apoptosis and cell cycle arrest induced by zearalenone in cultured DOK, Vero and Caco-2 cells: prevention by Vitamin E. Toxicology 192:237\u0026ndash;248.\u003c/li\u003e\n \u003cli\u003eAn YN (2017) Mechanisms of aflatoxins b1-induced autophagy and extracellular traps in macrophages. Dissertation, Jilin University, Jilin.\u003c/li\u003e\n \u003cli\u003eAssun\u0026ccedil;\u0026atilde;o R, Viegas S (2020) Mycotoxin exposure and related diseases. Toxins 12(3):17.\u003c/li\u003e\n \u003cli\u003eAwuchi CG, Ondari EN, Ogbonna CU, Upadhyay AK, Baran K, Okpala COR, Korzeniowska M, Guin\u0026eacute; RPF (2021) Mycotoxins affecting animals, foods, humans, and plants: Types, occurrence, toxicities, action mechanisms, prevention, and detoxification strategies-a revisit. Foods 10(6):1279.\u003c/li\u003e\n \u003cli\u003eCai GD, Sun K, Xiang ZL, Wang L, Zhou H (2017) Effect of zearalenone on activation and proliferation of mouse T lymphocyte in vitro. Chin J Anim Vet Sci 48:1357\u0026ndash;1364.\u003c/li\u003e\n \u003cli\u003eCheng CL, Zhao XH, Jiang HM, Fang J, Tian Y (2018) Research progress of aflatoxin and its control technology. Food Ind 39:296\u0026ndash;300.\u003c/li\u003e\n \u003cli\u003eCheng XY (2022) Autophagy mediated by TRPML1 as a target for aflatoxins B1-induced injury and repair of porcine intestinal epithelial cells. Dissertation, Jiangxi Agricultural University, Jiangxi.\u003c/li\u003e\n \u003cli\u003eDeng ZF, Wang LJ, Wu XQ, Yang HH, Li TY (2023) Research progress on metabolism and molecular toxicity of zearalenone in animals. China Anim Husb Vet Med 50:5194\u0026ndash;5203.\u003c/li\u003e\n \u003cli\u003eEshelli M, Harvey L, Edrada-Ebel R, McNeil B (2015) Metabolomics of the bio-degradation process of aflatoxin B1 by actinomycetes at an initial pH of 6.0. Toxins 7(2):439\u0026ndash;456.\u003c/li\u003e\n \u003cli\u003eFang QA, Du M, Chen J, Liu T, Zheng Y, Liao Z, Zhong Q, Wang L, Fang X, Wang J (2020) Degradation and detoxification of aflatoxin B1 by tea-derived \u003cem\u003eAspergillus niger\u003c/em\u003e RAF106. Toxins 12(12):777.\u003c/li\u003e\n \u003cli\u003eFrisvad JC, Hubka V, Ezekiel CN, Hong SB, Nov\u0026aacute;kov\u0026aacute; A, Chen AJ, Arzanlou M, LarsenTO, Sklen\u0026aacute;ř F, Mahakarnchanakul W, Samson RA, Houbraken J (2019) Taxonomy of \u003cem\u003eAspergillus\u003c/em\u003e section \u003cem\u003eFlavi\u003c/em\u003e and their production of aflatoxins, ochratoxins and other mycotoxins. Stud Mycol 93:1\u0026ndash;63.\u003c/li\u003e\n \u003cli\u003eGuengerich FP, Johnson WW, Shimada T, Ueng YF, Yamazaki H, Langou\u0026euml;t S (1998) Activation and detoxication of aflatoxin B1. Mutat res 402:121\u0026ndash;128.\u003c/li\u003e\n \u003cli\u003eGuindon KA, Bedard LL, Massey TE (2007) Elevation of 8-hydroxydeoxyguanosine in DNA from isolated mouse lung cells following in vivo treatment with aflatoxin B(1). Toxicology 98(1):57\u0026ndash;62.\u003c/li\u003e\n \u003cli\u003eGuindon-Kezis KA, Mulder JE, Massey TE (2014). In vivo treatment with aflatoxin B1 increases DNA oxidation, base excision repair activity and 8-oxoguanine DNA glycosylase 1 levels in mouse lung. Toxicology 321:21\u0026ndash;26.\u003c/li\u003e\n \u003cli\u003eGuo Q, Zhou H, Hao J, Ye J, Cao Y, Liu X, Hu H (2021) Research on the removal effect and mechanism of tibet \u003cem\u003eRhodotorula mucilaginosa\u003c/em\u003e on zearalenone. Sci Technol Cereals Oils Foods 29:192\u0026ndash;197.\u003c/li\u003e\n \u003cli\u003eHamilton D (2022) Understanding mycotoxin-induced illness: Part 1. Altern Ther Health Med 28(5):8\u0026ndash;11.\u003c/li\u003e\n \u003cli\u003eHao S, Pan S, Hu J, Gan F, Huang K (2015) Aflatoxin B1 suppressed T-cell response to anti-pig-CD3 monoclonal antibody stimulation in primary porcine splenocytes: A role for the extracellular regulated protein kinase (ERK1/2) MAPK signaling pathway. J Agric Food Chem 63(26):6094\u0026ndash;6101.\u003c/li\u003e\n \u003cli\u003eHarkai P, Szab\u0026oacute; I, Cserh\u0026aacute;ti M, Csilla K, Risa A, Rad\u0026oacute; J, Bal\u0026aacute;zs A, Berta K, Kriszt B (2016) Biodegradation of aflatoxin-B1 and zearalenone by \u003cem\u003eStreptomyces\u003c/em\u003e sp. collection. Int Biodeterior Biodegrad 108:48\u0026ndash;56.\u003c/li\u003e\n \u003cli\u003eHe R (2021) Screening, identification and degradation characteristics of zearalenone-degrading bacteria. Dissertation, Henan University of Technology, Henan.\u003c/li\u003e\n \u003cli\u003eHussein HS, Brasel JM (2001) Toxicity, metabolism, and impact of mycotoxins on humans and animals. Toxicology 167:101\u0026ndash;134.\u003c/li\u003e\n \u003cli\u003eJaskiewicz K, Close PM, Thiel PG, Cole RJ (1988) Preliminary studies on toxic effects of cyclopiazonic acid alone and in combination with aflatoxin B1 in non-human primates. Toxicology 52:297\u0026ndash;307.\u003c/li\u003e\n \u003cli\u003eJi J (2017) Combined toxicity of deoxynivalenol and zearalenone on ANA-1 metabolome of Kunming mice and macrophages. Dissertation, Jiangnan University, Jiangsu.\u003c/li\u003e\n \u003cli\u003eJi Q (2020) The toxic effect of AFB1 on primary mouse peritoneal macrophages and the mechanism of iron homeostasis. Dissertation, Northeast Agricultural University, Heilongjiang.\u003c/li\u003e\n \u003cli\u003eKang JG, Li Y, Jiang GJ, Wang YB, Ma ZF (2022) Lycopene \u003cem\u003eCuscuta chinensis\u003c/em\u003e and \u003cem\u003eTaishan panshi\u003c/em\u003e powder can alleviate the reproductive toxicity of zearalenone in pregnant rats. Chin J Vet Sci 42:1895\u0026ndash;1901, 1908.\u003c/li\u003e\n \u003cli\u003eKumar V, Bahuguna A, Lee JS, Sood A, Han SS, Chun HS, Kim M (2023) Degradation mechanism of aflatoxin B1 and aflatoxin G1 by salt tolerant \u003cem\u003eBacillus albus\u003c/em\u003e YUN5 isolated from \u0026apos;doenjang\u0026apos;, a traditional Korean food. Food Res 165:112479.\u003c/li\u003e\n \u003cli\u003eLiu SS, Shi JB, Li Q, Cai GD, Zhou H, Gu JH, Yuan Y, Liu XZ, Liu PZ, Bian JC (2020) Effects of zearalenone on activation, proliferation and differentiation of mouse lymphocyte B in vitro. Chin J Vet Sci 50:1602\u0026ndash;1608.\u003c/li\u003e\n \u003cli\u003eMalir F, Pickova D, Toman J, Grosse Y, Ostry V (2023) Hazard characterisation for significant mycotoxins in food. Mycotoxin Res 39:81\u0026ndash;93.\u003c/li\u003e\n \u003cli\u003eMonika C, Philippe D (2019) Mycotoxin migration in moldy foods. Curr Opin Food Sci 29:88\u0026ndash;93.\u003c/li\u003e\n \u003cli\u003ePang JC (2021) Study on the application of \u003cem\u003eBacillus subtilis\u003c/em\u003e in the elimination of feed toxin. Fuzhou University.\u003c/li\u003e\n \u003cli\u003ePatr\u0026iacute;cia V, Guilherme LDS, Isadora ZE, Maria CD, Daiane H, Danielle MP, Luc\u0026eacute;lia H, Maria LS, Patricia VNF (2021) Effects of infestations of the storage mite \u003cem\u003eTyrophagus putrescentiae\u003c/em\u003e (Acaridae) on the presence of fungal species and mycotoxin production in stored products. J Stored Prod 94:101883.\u003c/li\u003e\n \u003cli\u003eRobens JF, Richard JL (1992) Aflatoxins in animal and hman health. Rev Environ Contam. Toxicology 127:69\u0026ndash;94.\u003c/li\u003e\n \u003cli\u003eRotimi OA, Onuzulu CD, Dewald AL, Ehlinger J, Adelani IB, Olasehinde OE, Rotimi SO, Goodrich JM (2021) Early life exposure to aflatoxin B1 in rats: Alterations in lipids, hormones, and DNA methylation among the offspring. IJERPH 18(2):589.\u003c/li\u003e\n \u003cli\u003eRuzs\u0026aacute;s C, Bir\u0026oacute;-Gosztonyi M, W\u0026ouml;ller L, Mess B (1979) Effect of the fungal toxin (zearalenone) on the reproductive system and fertility of male and female rats. Acta Biol Acad Sci Hung 30(4):335\u0026ndash;345.\u003c/li\u003e\n \u003cli\u003eShi AM, Wang Y, Ma WQ, Liu N, Zhou ZH (2023) Analysis of liver biochemical indexes and metabolism related factors in patients with non-homogeneous fatty liver. Chin J Hepatol 28:1346\u0026ndash;1350.\u003c/li\u003e\n \u003cli\u003eSong C, Fu CQ, Huangfu HP, Zhang AG, Wang YK, Shi DM, Wang JD, Peng W (2023) Effects of curcumin on zearalenone-induced oxidative stress and NLRP3 inflammasome activation in mice liver. Chin J Vet Sci 43:1898\u0026ndash;1904.\u003c/li\u003e\n \u003cli\u003eSu Y, Sun Y, Ju D, Chang S, Shi B, Shan A (2018) The detoxification effect of vitamin C on zearalenone toxicity in piglets. Ecotoxicol Environ Saf 158:284\u0026ndash;292.\u003c/li\u003e\n \u003cli\u003eSun XQ, Cui XX, Li W, Han MY, Ishfaq M (2016) Establishment and evaluation of subchronic liver injury model induced by aflatoxins B in AA broilers. China Poul 38:28\u0026ndash;32.\u003c/li\u003e\n \u003cli\u003eTakemura H, Shim JY, Sayama K, Tsubura A, Zhu BT, Shimoi K (2007) Characterization of the estrogenic activities of zearalenone and zeranol in vivo and in vitro. J Steroid Biochem Mol Biol 103:170\u0026ndash;177.\u003c/li\u003e\n \u003cli\u003eTan H, Hu Y, He J, Wu L, Liao F, Luo B, He Y, Zuo Z, Ren Z, Zhong Z, Peng G, Deng J (2014) Zearalenone degradation by two \u003cem\u003ePseudomonas\u003c/em\u003e strains from soil. Mycotoxin Res 30:191\u0026ndash;196.\u003c/li\u003e\n \u003cli\u003eTian SL, Li W (2017) Research progress and control of zearalenone toxin. Cereal Feed Ind 12:36\u0026ndash;40.\u003c/li\u003e\n \u003cli\u003eVance CK, King EH, Bowers SD, Ryan P, Walters K, Shappell NW (2019) Reproductive Performance of Mares Fed Dietary Zearalenone. Front Vet Sci 6:423.\u003c/li\u003e\n \u003cli\u003eVlata Z, Porichis F, Tzanakakis G, Tsatsakis A, Krambovitis E (2006) A study of zearalenone cytotoxicity on human peripheral blood mononuclear cells. Toxicol Lett 165:274\u0026ndash;281.\u003c/li\u003e\n \u003cli\u003eWan BY (2022) Effect of zearalenone on ovarian development in weaned piglets via SIRT1/PGC \u0026minus;\u0026thinsp;1\u0026alpha; pathway. Dissertation, Shandong Agricultural University, Shandong.\u003c/li\u003e\n \u003cli\u003eWang S, Fu W, Zhao X, Liu H, Zhou L, Li J, Cheng R, Wu X, Li X, Sun C (2022) Zearalenone disturbs the reproductive-immune axis in pigs: the role of gut microbial metabolites. Microbiome 10:234.\u003c/li\u003e\n \u003cli\u003eWang YR, Bai HY, Wang GY, Yang BH, Liao GZ (2018) Effect of feed supplemented with \u003cem\u003eBacillus coagulans\u003c/em\u003e and \u003cem\u003eBacillus subtilis\u003c/em\u003e on growth performance of growing pigs. Feed Res 9:1\u0026ndash;5.\u003c/li\u003e\n \u003cli\u003eWu H, Xian J, Liu D (2009) Preliminary study on a zearalenone elimination yeast. Sci Technol Food Ind 30:215\u0026ndash;216, 220.\u003c/li\u003e\n \u003cli\u003eWu JW, Wang ZL, An W, Gao BQ, Li CX, Han B, Tao H, Wang JQ, Wang XM, Li HR. (2024) \u003cem\u003eBacillus subtilis\u003c/em\u003e simultaneously detoxified aflatoxin B1 and zearalenone. Appl Sci 14:1589.\u003c/li\u003e\n \u003cli\u003eXu J, Wang H, Zhu Z, Ji F, Yin X, Hong Q, Shi J (2016) Isolation and characterization of \u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003e ZDS-1: Exploring the degradation of Zearalenone by \u003cem\u003eBacillus\u003c/em\u003e spp. Food Control 68:244\u0026ndash;250.\u003c/li\u003e\n \u003cli\u003eYan JC, Zheng YF, Zeng QL, Zhu HJ, Zhu XQ (2004) Effect of zearalenone on gap junctional communication. Toxicology 3:160\u0026ndash;162.\u003c/li\u003e\n \u003cli\u003eYang ML, Wu FY, Liu J.H, Liu YJ, Chen BJ (2019) Research progress on toxicity of zearalenone. Feed Res 42:74\u0026ndash;77.\u003c/li\u003e\n \u003cli\u003eYu ZL, Zhang LS, Wu DS (2005) Effects of zearalenone on proliferation and apoptosis of breast cancer cell line MCF-7. Chin J Prev Med 5:34\u0026ndash;37.\u003c/li\u003e\n \u003cli\u003eZhang M, Zhao J, Xu JR, Cao L, Ma SB (2021) Toxic effect of AFB1 on canine hepatocytes and protective effect of NAC. J JNWAFU 49:1\u0026ndash;10.\u003c/li\u003e\n \u003cli\u003eZhang Q, Xiong J, Zhao C, Wang Y, Zhang X (2016) Screening of zearalenone virus-free strain and preliminary study on virus-free mechanism. Sci Technol Cereals Oils Foods 24:76\u0026ndash;81.\u003c/li\u003e\n \u003cli\u003eZhao CM, Xie WT, Lin HY, Hu YX, Ma WA, Li Y, Chen ZB (2024) Protective effect of \u003cem\u003eInonotus obliquus\u003c/em\u003e extract on liver injury induced by aflatoxins B1 in mice. China Anim Husb Vet Med 51:864\u0026ndash;874.\u003c/li\u003e\n \u003cli\u003eZhou JC, Shi DH, Ji C (2020) Research progress on toxicity of zearalenone and deoxynivalenol to animals. Chin J Anim Nutr 32:2460\u0026ndash;2466.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"biodegradation, aflatoxin, zearalenone, Bacillus subtilis, gastrointestinal environment, mice","lastPublishedDoi":"10.21203/rs.3.rs-4590182/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4590182/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAflatoxin B1 (AFB1) and zearalenone (ZEN) are the most prevalent mycotoxins in production, posing a serious threat to the food and feed industry and resulting in a substantial economic burden. Therefore, finding a safe and efficient method for biodegradation of mycotoxins is of utmost important in addressing this issue. \u003cem\u003eBacillus subtilis\u003c/em\u003e ZJ-2019-1, capable of degrading AFB1 and ZEN, was isolated and cultured in our laboratory in vitro. In this study, we conducted detoxification tests on AFB1 and ZEN using \u003cem\u003eB. subtilis\u003c/em\u003e ZJ-2019-1 in gastrointestinal environment and in mice, respectively. Our findings demonstrate that \u003cem\u003eB. subtilis\u003c/em\u003e ZJ-2019-1 can effectively degrade both mycotoxins present in feed within gastrointestinal environment. Following degradation by \u003cem\u003eB. subtilis\u003c/em\u003e ZJ-2019-1, the toxicity of the AFB1 and ZEN product decreased compared to their original levels. Furthermore, \u003cem\u003eB. subtilis\u003c/em\u003e ZJ-2019-1 exhibited excellent detoxification effects on AFB1 and ZEN when tested in mice. These results indicate that \u003cem\u003eB. subtilis\u003c/em\u003e ZJ-2019-1 possesses the ability to degrade both AFB1 and ZEN, making it suitable for application in the food and feed industry as a means to reduce economic losses caused by mycotoxins.\u003c/p\u003e","manuscriptTitle":"Biodetoxification of both AFB1 and ZEN by Bacillus subtilis ZJ-2019-1 in gastrointestinal environment and in mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-08 07:30:41","doi":"10.21203/rs.3.rs-4590182/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0b2f41a0-dbd7-41c8-9fe9-e998ab2646a6","owner":[],"postedDate":"July 8th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-11-19T22:23:09+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-08 07:30:41","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4590182","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4590182","identity":"rs-4590182","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}