Bacillus velezensis mitigates deoxynivalenol-induced intestinal inflammation and liver injury via modulating the gut microbiota

Abstract

Deoxynivalenol (DON), a prevalent mycotoxin in food and feed, induces gastrointestinal and liver damage. The potential probiotic Bacillus velezensis may mitigate DON toxicity, though its precise mechanisms remain unknown. Our study demonstrates that B. velezensis WMCC10514 effectively survives and degrades DON within simulated gastrointestinal fluid. Fluorescently labeled WMC10514-GFP colonized murine intestines and persisted in simulated intestinal fluid (SIF), confirming its colonization capacity. In vivo, WMCC10514 alleviated DON-induced anorexia, restored murine growth, and reduced liver injury. Furthermore, the strain elevated ZO-1 and Occludin expression, enhanced intestinal barrier integrity and reduced DON accumulation in host tissues. Integrated transcriptomic and microbiome analyses revealed that the strain suppressed TLR4/NF-κB pathway activation in the intestine and liver, increased Lactobacillus abundance, restored SCFAs level, and modulated liver energy metabolism. These findings elucidate B. velezensis’s role in mitigating mycotoxin toxicity through gut microbiota-driven regulation of the gut-liver axis.

Introduction

Deoxynivalenol (DON), a type B trichothecene mycotoxin produced by Fusarium graminearum, is a widespread contaminant of cereal crops globally¹. Grains are highly susceptible to DON contamination during harvesting, transportation, storage, and distribution². Owing to its chemical stability, DON persists in food and feed systems, posing significant safety risks. Dietary exposure to DON via contaminated food or feed threatens human and animal health by primarily targeting the gastrointestinal tract and liver—organs essential for nutrient absorption, detoxification, and immune regulation^3–5

Physical and chemical methods—such as adsorbents, alkalisation or ozone treatment—can prevent fungal growth or degrade DON during processing and storage, yet their industrial use is limited by low elimination efficiency, possible secondary pollution and sensory damage to the final product. Biological approaches, particularly those employing live microorganisms, offer a safer and more sustainable alternative^6,7. In vitro and in vivo studies demonstrate that Bacillus, Lactobacillus, Streptomyces, Devosia and Nocardioides reduce mycotoxin bioavailability and mitigate associated intestinal damage⁸. The underlying mechanisms were dissected into four complementary layers: (i) Adsorption and antagonism: high-affinity binding of the compound to bacterial surfaces, driven by non-covalent interactions with cell-wall teichoic acids and peptidoglycan layers^9,10; antagonize toxigenic pathogens by suppressing their growth, sporulation, and mycotoxin synthesis¹¹. (ii) Enzymatic transformation: secrete extracellular enzymes or intracellular enzymes, converting mycotoxins into less toxic metabolites¹². (iii) Gut-microbiota modulation: a selective increase in probiotic taxa (e.g., Lactobacillus, Bifidobacterium) and a concomitant reduction in opportunistic pathogens (e.g., Enterobacteriaceae)¹³. (iv) Immune regulation and intestinal protection: regulate immune response and reduce inflammation; microbial metabolites —particularly butyrate and indole-3-lactate—secreted by the restructured community activated the aryl-hydrocarbon receptor (AhR) and upregulated tight-junction proteins (ZO-1, occludin), thereby enhancing intestinal integrity^14,15. However, only a few studies have evaluated dedicated DON-degrading probiotics in animals, and their systemic detoxification mechanisms remain largely unexplored. Among these, Bacillus velezensis has emerged as a promising probiotic¹⁶. Studies indicate that B. velezensis produces and secretes digestive enzymes (protease, amylase, and lipase) alongside bioactive metabolites, enhancing its potential as a feed additive^17–19. Its benefits in poultry nutrition are well-documented, yet its ability to combat mycotoxin-induced systemic damage remains poorly understood²⁰.

Mounting evidence reveals that DON simultaneously injures both liver and intestine^21,22. Histopathological analyzes show swollen duodenal surfaces, jejunal ulceration, and ruptured ileal villi, together with hepatic mononuclear infiltration, centrilobular vein dilatation, and peri-portal necrosis²³. These findings underscore the importance of the gut–liver axis—a bidirectional communication network linking intestinal microbiota, epithelial barrier, and hepatic metabolism—in orchestrating systemic responses to DON challenge^24,25. At present, the specific mechanism by which probiotics can alleviate the toxicity of DON by regulating the gut-liver axis remains unclear. To address this gap, this study selected B. velezensis WMCC10514, which could efficiently degrade DON and was isolated from Wuliangye Daqu. Firstly, its survival rate under simulated gastrointestinal conditions and its ability to degrade DON in vitro were evaluated. Then, using a murine model, evaluate WMCC10514 ’s mitigation effects against DON toxicity in mice by analyzing growth performance, hematoxylin-eosin staining, histopathology, tissues DON residue and immune factors. Furthermore, integrated transcriptomic analysis of liver and jejunum tissues, coupled with intestinal microbiota profiling and short-chain fatty acid (SCFA) quantification, revealed how WMCC10514 modulates the gut-liver axis to counteract toxicity of DON. Our study elucidates the mechanism by which probiotics mitigate mycotoxin toxicity in mice and reveals novel insights into their role in modulating the gut-liver axis.

Results

WMCC10514 survives, colonizes, and degrades DON in simulated gastroenteric fluid

To assess the feasibility of oral administration in animals, we examined the survival of WMCC10514 in simulated gastroenteric fluid (SGF) of different pH (1–5) and simulated intestinal fluid (SIF) containing different bile salt concentrations (0.03–0.3%). As expected, strain survival was significantly reduced at low pH and high bile salt concentrations (p < 0.05). The survival rate was 46.46% at pH 3. When pH was 5, the highest survival rate was 89.78% (Fig. 1a). Similarly, the survival rate at 0.3% bile salt was 34.83%, and that at 0.03% bile salt was 80.01% (Fig. 1b). Meanwhile, after treatment at 37°C for 2 h, the degradation rate of WMCC10514 was 31.99% in LB medium, 23.44% in SGF, and 17.16% in SIF (Fig. 1c), confirming that the strain could maintain its DON degradation ability even under harsh gastrointestinal conditions.

Fig. 1. Stability of WMCC10514 in simulated gastric and intestinal fluids.

a pH tolerance. b Bile salt tolerance. c DON degradation. d Cell-surface hydrophobicity. e Auto-agglutination. f Biofilm. Data are expressed as mean ± SD and different letters (a–e) indicate significance (P < 0.05), n = 3.

Colonization efficiency and intestinal adaptation are key factors when evaluating strains for use in vivo. Cell-surface hydrophobicity (CSH) and autoagglutination capacity are commonly assessed^26–28. In this study, the CSH values of WMCC10514 for ethyl acetate, xylene and n-hexane were 41.49%, 32.50% and 22.64%, respectively (Fig. 1d). Auto-agglutination increased with incubation time, reaching 48.57% in the 24 h simulated gastric fluid, exceeding the recorded value in the SIF (Fig. 1e). The strain WMCC10514 demonstrates a robust capacity for biofilm formation in both SGF and SIF (Fig. 1f). In summary, strain WMCC10514 had good acid resistance, bile salt resistance, DON degradation ability and colonizing potential in animal intestine. These properties indicate that it can be used to reduce DON contamination in feed.

WMCC10514 alleviates anorexia and weight loss caused by DON

To investigate the potential of WMCC10514 as microbial feed additives for utilizing DON-contaminated grains, mice were employed to investigate the alleviating effect of WMCC10514 on DON-induced toxicity (Fig. 2a). After oral administration for 4 weeks, all groups’ mice exhibited increased body weight. However, the DON group showed significantly lower weight gain and food intake compared to other groups (p < 0.05). The SW and SN groups did not show significant differences in weight gain and food intake compared to the CK group (Fig. 2b, c). These results indicated that WMCC10514 could not affect mice growth and could alleviate the DON-induced suppress on mice growth. However, further investigation is needed to verify the action of WMCC10514.

Fig. 2. Strain WMCCC10514 alleviated the damage induced by DON in mice.

a Model for administration of strain WMCCC10514 and DON in mice. b Body weight gain (n = 8). c Average daily feed intake (n = 8). d, e Representative image of histopathological changes of liver and jejunum (The red box represents inflammatory cell infiltration;blue box represents punctate necrosis of cells; green box represents swelling and deformation of cells). f, g Representative image of immunohistochemical of ZO-1 and Occludin in the jejunum of mice. h-i Jejunum villus height and crypt depth in mice (n = 6). j, k Quantification of the integral optical density of ZO-1 and Occludin (n = 6). Data are expressed as mean ± SD and compared with one-way analysis of variance (ANOVA) after Tukey’s multiple comparisons test, ^*p < 0.05, ^**p < 0.01.

WMCC10514 improves liver and intestinal integrity exposed to DON

Analysis of organ indices (liver, kidney, spleen) revealed a significant increase in the liver index for the DON group compared to both the CK and SW groups (p < 0.05). However, no significant difference was found between the DON and SN groups. Furthermore, there was no significant difference in the SN group compared with the CK and SW groups (Supplementary Fig. 1a). Meanwhile, no significant differences were found in kidney and spleen indices among the experimental groups (Supplementary Fig. 1b, c). Furthermore, the intestine also represents a primary target organ for DON. To evaluate tissue-level effects, hematoxylin-eosin (HE) staining was conducted on both the liver and jejunum for histological assessment (Fig. 2d, e).

The liver tissues in the CK and SW groups had intact hepatocyte structures with no pathological changes, while the DON group exhibited severe hepatocyte necrosis, deformation, swelling, and pronounced inflammatory infiltration. Notably, the SN group showed significant improvement in hepatocyte morphology and reduced inflammation. Moreover, jejunal histological analysis (Fig. 2e, h, i) revealed that compared to the CK group, the villus height in the DON group was significantly reduced, and the crypt depth was significantly increased (p < 0.05), indicating jejunum damage. In contrast, the SN group exhibited increased significantly higher villus height and decreased significantly crypt depth compared to the DON group (p < 0.05), suggesting that WMCC10514 alleviates DON-induced jejunum damage. These results suggest that WMCC10514 could not change the histological obversion of liver and jejunum and could alleviate DON-induced damage of liver and jejunum.

IHC analysis was conducted to assess the distribution of tight junction proteins ZO-1 and Occludin in the jejunum (Fig. 2f, g, j, k). Compared to CK group, the expression levels of ZO-1 and Occludin in the DON group were significantly downregulated (p < 0.05). In contrast, the expression of ZO-1 and Occludin in the SN group was significantly upregulated compared to the DON group (p < 0.05). Thus, DON reduced the intestinal barrier permeability, and WMCC10514 could alleviate the DON-induced intestinal epithelial damage by upregulating tight junction proteins.

WMCC10514 reduced DON accumulation in different tissues and alleviates systemic inflammation in the mice

The contents of DON in the liver, fecal contents and serum of mice in each group were detected. The concentrations of DON in the liver and feces of the SN group were significantly lower than those of the DON group (Fig. 3a, b, p < 0.05). No detectable DON was observed in serum samples. These findings confirm that measurable concentrations of DON were present in both the liver and intestinal tract. Administration of WMCC10514 significantly reduced this accumulation.

Fig. 3. WMCC10514 reduced DON accumulation in different tissues and alleviates systemic inflammation in the mice.

a, b DON concentration in liver and fecal contents (n = 6). c–f Levels of TNF-α, IL-1β, IL-6 and IL-10 (n = 8). Data are expressed as mean ± SD and compared with one-way analysis of variance (ANOVA) after Tukey’s multiple comparisons test, ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001.

The effects of DON and WMCC10514 on immune factors in mice are shown in Fig. 3c–f. The results indicate that compared to the CK group, pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) were significantly elevated in the DON group (p < 0.001). No significant difference in pro-inflammatory cytokines was observed in the SW group. However, the SN group showed a significant reduction in pro-inflammatory cytokine levels compared to the DON group (p < 0.001). Furthermore, the anti-inflammatory cytokine IL-10 level in the DON group was significantly decreased compared to the CK group (p < 0.001), while IL-10 levels were significantly increased in the SN group compared to the DON group (p < 0.001). These results suggested that DON could induce immune response, and WMCC10514 suppress the immune response.

WMCC10514 alleviates liver inflammation induced by DON

Supplementary Fig. 2a shows that compared to the CK group, the SW group significantly upregulated 85 genes and downregulated 122 genes, with no disease-related genes identified. It further verified that WMCC10514 was safety to animals. The DON group significantly upregulated 124 genes and downregulated 42 genes, including notable upregulation of immune disease-related genes such as BCL6 and BTNL9 (Fig. 4a). Compared to the DON group, the SN group significantly upregulated 113 genes and downregulated 221 genes, including the significant downregulation of immune disease-related genes such as TUBB2A (Fig. 4b). These results were in accordance with the changes in immune factors, indicating that WMCC10514 could regulate the expression of genes related to immune response induced by DON.

Fig. 4. Different expression genes (DEGs) in liver and jejunum of mice after oral administration of DON and / or WMCC15014.

a–d Volcano map of DEGs in mice liver and jejunum. e–h KEGG enrichment of DEGs in the liver and jejunum of mice.

We performed GO functional enrichment analysis of the different expression genes (DEGs) in the liver. As shown in Supplementary Fig. 3a–d, the top 20 enriched GO terms for DEGs between CK and SW were mainly related to lipid metabolic processes and small biosynthetic molecule processes. Compared to the CK group, the top 20 GO terms in DON group were primarily related to muscle tissue development and muscle structure development, while the DEGs between DON and SN were mainly enriched in circulatory system development and system development. Further, KEGG analysis (Fig. 4e, f, and Supplementary Fig. 3i, j) was conducted to examine the top 20 signal pathways of DEGs in each group. The results showed that compared to the CK group, the differential genes in the SW group were enriched in pathways such as Circadian rhythm and Terpenoid backbone biosynthesis, which are not associated with diseases, indicating that WMCC10514 does not cause disease in mice. In contrast, the DEGs in the DON group were significantly enriched in disease-related pathways, including Inflammatory mediator regulation of TRP channels, Chemical carcinogenesis - DNA adducts, and PPAR signaling pathway, suggesting that DON exposure induces liver disease in mice. DEGs between the DON and SN groups were enriched in pathways related to Fluid shear stress and atherosclerosis, IL-17 signaling, and Transcriptional misregulation in cancer, with significant downregulation observed in the IL-17 signaling pathway, suggesting that WMCC10514 can downregulate genes involved in inflammation-related pathways, thereby mitigating the inflammatory response induced by DON. In this study, an important reason for alleviating the inflammation and immune response was WMCC10514 significantly reduced DON concentration in liver.

WMCC10514 alleviates jejunum inflammation induced by DON

The volcano plot (Fig. 4c, d, and Supplementary Fig. 2c, d) shows that compared to the CK group, the SW group significantly upregulated 353 genes and downregulated 423 genes, with no impact on disease-related genes in mice, indicating that WMCC10514 does not affect the health of mice jejunum. The DON group significantly upregulated 520 genes and downregulated 354 genes, with upregulation of immune disease-related genes such as REG4 and AFP. Compared to the DON group, the SN group upregulated 121 genes and downregulated 305 genes, with significant downregulation of immune disease-related genes such as MSR1 and TLR11.

GO enrichment analysis (Supplementary Fig. 3e–h) shows that the top 20 enriched GO terms for DEGs between CK and SW were primarily associated with extracellular region and extracellular space, while the top 20 enriched GO terms between CK and DON were mainly related to response to external stimulus and system development. The DEGs between DON and SN were primarily enriched in extracellular region and extracellular space. KEGG enrichment analysis (Fig. 4g, h, and Supplementary Fig. 3k, l) revealed that the DEGs in the CK and SW groups were enriched in pathways such as Cytokine-cytokine receptor interaction and Focal adhesion, with no involvement in disease-related pathways. Furthermore, the DEGs in these pathways showed both upregulation and downregulation, suggesting that WMCC10514 does not affect normal physiological activities or health in mice. In contrast, the DEGs between CK and DON were enriched in pathways like the Intestinal immune network for IgA production and Viral protein interaction with cytokine and cytokine receptors, indicating that DON exposure induces immune-related damage in mice. The DEGs between DON and SN were enriched in pathways related to Intestinal immune network for IgA production, Chemical carcinogenesis - DNA adducts, Viral protein interaction with cytokine and cytokine receptors, and Pathways in cancer, with significant downregulation observed in these pathways, suggesting that WMCC10514 can alleviate the intestinal immune-inflammatory response induced by DON.

WMCC10514 colonizes the intestine to regulate the composition of the gut microbiota

The GFP plasmid remained highly stable in strain WMCC10514, retaining 96% fluorescence after 30 generations, confirming its suitability for vivo tracking. In vivo and in vitro experiments revealed green fluorescence in fecal contents and SIF of SW-GFP group mice (Figs. 5a–c and 6a, b), demonstrating WMCC10514 has colonization capacity under both conditions. Principal coordinates analysis of fecal samples and SIF showed that the DON group significantly differed from the CK, SW, and SN groups (Supplementary Fig. 4a, b), while no significant differences were observed between the CK, SW, and SN groups. The diversity indices (Chao1, Simpson, Shannon) (Supplementary Fig. 4c, d) indicated that the control group maintained high diversity and richness. There were no significant differences in diversity indices between the experimental groups and the CK group, but the Shannon index in the DON group was significantly lower compared to the SW group. This suggests that DON reduces intestinal microbiota diversity, while WMCC10514 improves the composition of the microbiota. As expected, the relative abundance at the phylum level (Fig. 5d) showed that the dominant bacterial phyla across groups were Bacteroidetes, Firmicutes, and Proteobacteria. Compared to the CK group, the DON group exhibited an increase in Firmicutes and a decrease in Bacteroidetes and Proteobacteria, suggesting that DON disrupts the intestinal microbiota. Similar results were observed in SIF (Fig. 6c).

Fig. 5. Gut microbes of feces in mice.

a Direct observation of fecal contents in the SW-GFP group. b, c CK and SW-GFP fecal contents GFP were observed after culture. d, e Microbial community of feces in mice at phylum levels and genus levels. f–i Linear Discriminant Analysis Effect Size (LEFSe) between different treatment groups.

Fig. 6. Microbes of SIF.

a, b CK and SW-GFP of SIF. c, d Microbial community of SIF at phylum levels and genus levels. e–h CK, SW, DON and SN fecal microorganism’s co-occurrence network.

At the genus level (Fig. 5e), the CK and SW groups exhibited similar microbiota compositions, while the abundance of Lactobacillus decreased and Allobaculum increased in the DON group. In contrast, the SN group showed a higher abundance of Lactobacillus and lower abundance of Allobaculum compared to the DON group. In the SIF treatment (Fig. 6d), the abundance of Bacillus in the SW and SN groups increased following WMCC10514 inoculation. DON decreased the abundance of Parabacteroides and Bacteroides, but the SN group, with WMCC10514 supplementation, alleviated these changes. LEFSe analysis showed that DON exposure significantly altered the abundance of Clostridium, Rhodobacterales, Allobaculum, and Rhodobacteraceae (Fig. 5f). Notably, compared to the DON group, the SN group exhibited enrichment of Bacteroides, Photobacterium, Paraprevotella, and Streptococcus (Fig. 5h), which are associated with intestinal barrier stability. Therefore, WMCC10514 appears to restore the intestinal homeostasis disrupted by DON.

Microbial community relationships were analyzed through co-occurrence networks (Fig. 6e–h, and Supplementary Table 1). The CK and SW groups exhibited similar clustering patterns, demonstrating that WMCC10514 supplementation did not compromise microbial stability. In contrast, the DON group displayed distinct structural divergence, with significantly reduced clustering coefficients and average degrees. This suggests DON disrupted gut microbiota equilibrium and altered intermicrobial relationships. Conversely, the SN group demonstrated partial network recovery, marked by increased clustering coefficients and average degrees, indicating WMCC10514 mitigated DON-induced microdysbiosis.

Overall, these data confirm that DON destabilizes murine gut microbiota and diminishes beneficial bacterial populations. WMCC10514 colonize well and counteracts DON-induced microbial imbalance both in vivo and in vitro.

WMCC10514 restores the level of SCFAs and regulates the metabolism of the gut-liver axis

Analysis of SCFAs level in mice fecal contents (Fig. 7a–c) revealed that the DON group had significantly lower levels of acetic, butanoic, and valeric compared to the CK, SW, and SN groups (p < 0.05). Conversely, the SN group showed significantly higher SCFAs level than the DON group (p < 0.05). These results suggest that DON disrupts the metabolic processes in mice, leading to reduced SCFAs level. WMCC10514 helps restore these metabolic functions, thereby increasing SCFAs level.

Fig. 7. SCFAs metabolism.

a–c The concentrations of acetic acid, butanoic acid and valeric acid in CK, SW, DON, and SN treatments (n = 6). d Metabolism of SCFAs. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001.

As shown in Fig. 7d, WMCC10514 modulates key enzymes such as EC:2.7.2.7, EC:3.1.2.1 and EC:3.1.2.30 to regulate intestinal microbiota-derived SCFAs synthesis, including butanoic, acetic, and valeric acids. Following liver uptake of SCFAs via the portal vein, WMCC10514 further activates liver genes expression such as ACACA, CS and HMGCS2, directly enhancing critical metabolic pathways: TCA cycle activity, ketone body and fatty acid synthesis. This dual regulatory mechanism amplifies liver metabolic efficiency and energy output.

Potential mechanisms through which WMCC10514 alleviates DON toxicity

It was reported that TLR4/NF-κB signaling pathway played an important role in immune-inflammatory response induced by DON^29–31. Compared to the CK group, the expression of TLR4/NF-κB signaling pathway-related genes (TLR4, MYD88, NFKB1, CHUK) and pro-inflammatory cytokines (IL1A, IL1B) in liver and jejunum were significantly increased in the DON group (p < 0.01) (Supplementary Fig. 5). This increase was not observed in the CK or SW groups, suggesting that DON exposure activates the TLR4/NF-κB signaling pathway, inducing inflammation in the intestinal and liver tissues of mice. In contrast, the expressions of TLR4, MYD88, NFKB1, CHUK, IL1A, and IL1B in the SN group was significantly lower than in the DON group (p < 0.05), indicating that WMCC10514 inhibits the activation of TLR4/NF-κB signaling and the subsequent inflammatory signaling in the intestinal and liver.

To further investigate the relationship between gut microbes and the expression of TLR4/NF-κB-related genes, spearman correlation analysis was employed (Supplementary Fig. 6a). It indicated that the expression of TLR4/NF-κB-related genes in the intestinal was significantly negatively correlated with the abundance of Lactobacillus, Odoribacter, Oscillospira, Akkermansia, Bacteroides, Coprococcus and Parabacteroides. Moreover, the expression of genes in the TLR4/NF-κB pathway was significantly positively correlated with increased levels of Allobaculum, Prevotella, Ruminococcus and Helicobacter.

As shown in Supplementary Fig. 6b, acetic was negatively correlated with Prevotella and [Ruminococcus], and positively correlated with Oscillospira, Akkermansia, Coprococcus, and Parabacteroides. Butanoic was negatively correlated with Allobaculum, Helicobacter, and Ruminococcus, and positively correlated with Lactobacillus, Odoribacter, and Bacteroides. Valeric acid showed a negative correlation with Prevotella and [Ruminococcus], and a positive correlation with Oscillospira, Akkermansia, Coprococcus, and Parabacteroides.

Acetic and valeric acid were negatively correlated with the expression of IKBKB and IL1B in the liver, while butanoic was negatively correlated with CHUK, MYD88, NFKB1, IL1A, and TLR4 (Supplementary Fig. 6c). These results suggested that acetic, butanoic, and valeric acid regulated the community of gut microbes and inhibited the expression of genes related in TLR4/NF-κB signaling pathway.

Discussion

DON contamination poses a global threat to food and feed safety due to its persistent and recalcitrant nature²⁹. In food systems, this mycotoxin contaminates grains and derived products at high frequencies. Chronic dietary exposure compromises human immune and reproductive functions while elevating risks of carcinogenesis, teratogenicity, and mutagenicity. Within livestock production, DON-contaminated feed reduces growth performance through decreased appetite, impaired weight gain, and immunosuppression in animals^32–34. Furthermore, residual DON and its metabolites accumulate in meat, eggs, and dairy products, perpetuating exposure risks across the food chain³³. These multifaceted health impacts necessitate innovative strategies for DON detoxification. B. velezensis is an emerging probiotic. Studies demonstrate its efficacy as a feed additive for improving broiler growth metrics, including average daily weight gain and feeding conversion efficiency^20,35. Building on this, our study evaluates its capacity to mitigate DON contamination in mice models and regulatory mechanism on the combination of the intestine and liver was deeply investigated.

WMCC10514 retained 46.46% viability at pH 3 and 34.83% in 0.3% (w/v) bile salts, values that surpass the 26.06% (pH 1) and 22.26% (0.3% bile salts) reported for the aflatoxin-degrading B. licheniformis (Dong et al.)³⁶ and are comparable to the 53.1% (pH 2) and 39.0% (0.3% bile salts) reported for Leuconostoc mesenteroides LM187 (Zhang et al.)³⁷. Thus, WMCC10514 demonstrates acid and bile-salt tolerance within the range accepted for effective probiotic strains.

Existing studies demonstrate that DON impairs murine growth by inducing anorexia³⁸. Consistent with this, our data revealed a marked decrease in food intake and cumulative weight gain in DON-exposed mice. Treatment with WMCC10514 counteracted DON-induced anorexia and restored weight gain to baseline levels. Following oral ingestion, DON initially enters systemic circulation via plasma before undergoing rapid absorption and distribution to multiple organs, where it disrupts physiological functions and induces tissue damage^39,40. In this study, DON concentrations were quantified in serum, liver tissue, and fecal matter. No detectable DON was observed in serum across all groups. However, liver and fecal DON levels were identified in both DON-exposed and WMCC10514-treated mice, with significantly higher concentrations in the former group (p < 0.05). These findings confirm that WMCC10514 reduces systemic DON accumulation, thereby directly mitigating its toxicity.

Serum inflammatory cytokines serve as critical biomarkers for systemic inflammation⁴¹. DON exposure markedly elevated pro-inflammatory cytokine levels while suppressing anti-inflammatory factors. In contrast, SN-treated mice exhibited attenuated pro-inflammatory responses and enhanced anti-inflammatory activity. Collectively, these results demonstrate that DON is involved in the regulatory process of immune homeostasis imbalance. WMCC10514 appears to partially restore immune homeostasis through the modulation of key inflammatory factors. The precise molecular mechanism, however, requires further investigation.

The liver, the body’s primary metabolic and detoxification organ, represents a key target of DON toxicity⁴². In this study, DON exposure induced liver index increased significantly, hepatocellular structural damage and pronounced inflammatory cell infiltration. Transcriptomic profiling revealed that the DEGs were significantly enriched in inflammatory pathways within the DON-exposed group, notably through hyperactivation of the TLR4/NF-κB signaling cascade. WMCC10514 supplementation did not significantly restore liver indices, but it alleviated liver injury, suppressed inflammation, and inhibited TLR4/NF-κB pathway activation. These results indicated that WMCC10514 could alleviate DON-induced liver injury and inflammation to a certain extent.

The intestine, as the primary site of nutrient absorption, features a vast mucosal surface area with intricate villi and microvilli structures, rendering it directly exposed to ingested DON^43,44. Tight junction proteins are essential structural components of the intestinal epithelial barrier, responsible for maintaining its integrity and selective permeability⁴⁵. DON rapidly impairs barrier function by downregulating the expression of these proteins, a process frequently associated with the activation of inflammatory pathways such as NF-κB^46,47. Correspondingly, our study confirmed that DON exposure significantly reduced tight junction protein levels and concurrently activated the intestinal TLR4/NF-κB signaling pathway. Probiotic supplementation alleviates this damage and counteracts the resulting inflammation via immune pathway modulation. Specifically, treatment with strain WMCC10514 effectively upregulated tight junction protein expression, restored barrier integrity, and suppressed TLR4/NF-κB pathway activation. This concerted action fortified the mucosal architecture and diminished the inflammatory response.

Notably, the liver and intestinal structures of mice administered WMCC10514 alone were indistinguishable from those of the CK group. Transcriptomic analysis further supported this; compared to the CK group, no disease-associated genes were identified among the DEGs in the liver or jejunum of the SW group. While KEGG pathway analysis indicated enrichment in Drug Metabolism-Cytochrome P450 and Metabolism of Xenobiotics by Cytochrome P450 in these tissues, this specific enrichment of the CYP450 enzyme system likely represents an adaptive, host response to the newly introduced bacterium⁴⁸. Collectively, these findings demonstrate the safety of WMCC10514 and confirm B. velezensis as a safe probiotic candidate⁴⁹.

Compromised tight junction integrity elevates intestinal permeability, triggering inflammatory cascades and bacterial translocation that destabilize gut flora equilibrium⁵⁰. The intestinal microbiota critically regulates barrier function and overall gut homeostasis⁵¹. As demonstrated by prior studies, DON can alter the intestinal microbiota and reduce the diversity of intestinal microbiota^52,53. In our study, it was also found that DON exposure reduces microbial diversity, disrupts community balance, enriches pathogens, and depletes beneficial taxa—findings corroborated here in both in vivo and in vitro models. B. velezensis exhibits robust gastric survivability, resisting acid and bile stress to colonize the gastrointestinal tract effectively. Our data confirm that strain WMCC10514 establishes durable colonization in vivo and in vitro, counteracting DON-driven dysbiosis and restoring microbial symbiosis.

SCFAs are essential microbial metabolites, critically regulate intestinal health, immune modulation, and microbiota equilibrium^54,55. Research demonstrates their role in preserving intestinal barrier integrity and mucosal homeostasis, thereby preventing systemic translocation of toxins and inflammatory mediators to the liver via the gut-liver axis⁵⁶. In this study, DON exposure markedly decreased intestinal concentrations of acetic, butanoic, and valeric. Conversely, B. velezensis WMCC10514 elevated intestinal SCFAs level and enhanced liver SCFAs metabolism, modulating liver energy homeostasis. In addition, the intestinal ecological imbalance induced by DON also increases the level of Gram-negative bacteria (Prevotella), potentially elevating intestinal lipopolysaccharide (LPS) level⁵⁷. The LPS reaches the liver through the portal circulation, triggering the TLR4/NF-κB pathway and causing liver inflammation.

Correlation analysis revealed a negative association between jejunum TLR4/NF-κB gene expression and the abundance of specific gut microbiota, including Lactobacillus and Bacteroides. It was reported that the reduction of Lactobacillus induced the disruption of the microbiota balance^58,59 and the increase of intestinal permeability^60,61, which allowed harmful substances and pathogens to enter the bloodstream and trigger inflammation. Similarly, reduced Bacteroides levels correlate with heightened intestinal inflammation^62,63. Furthermore, shifts in microbial composition alter the production of SCFAs, metabolites critical for suppressing the TLR4/NF-κB pathway⁶⁴. These SCFAs travel via the portal vein to the liver, modulating liver TLR4/NF-κB expression.

Thus, the potential mechanisms were summary in Fig. 8. Oral ingestion of DON first reaches the mouse gut, increasing the expression of TLR4 genes and activating the TLR4/NF-κB signaling pathway. This process triggers gut inflammation and disrupts the intestinal barrier. The damage to the gut barrier further leads to dysbiosis, with DON significantly reducing beneficial bacteria (Lactobacillus) and increasing pathogenic bacteria (Allobaculum), which results in decreased SCFAs levels in the ceremonies and negative impacts liver metabolism. SCFAs reaching the liver through the gut-liver axis reduce and impair its metabolic functions, including its anti-inflammatory and regulatory abilities. Simultaneously, the damaged gut barrier allows unmetabolized DON and harmful substances to enter the liver via the bloodstream, re-activate inflammatory pathways in the liver and increase the expression of TLR4/NF-κB signaling, which induces liver inflammation. In addition, DON also increases the level of Gram-negative bacteria in the intestine, potentially increasing the LPS content in the intestine and further activating the TLR4/NF-κB signaling pathway in the liver (Fig. 8).

Fig. 8. Mechanism diagram.

Mechanism by which WMCC10514 alleviates DON-induced damage.

However, the supplementation of WMCC10514 effectively improves these conditions by reducing the DON concentration, regulating the community of gut microbes, increasing the concentrations of acetic, butanoic, and valeric acid, regulating the gut-liver axis and suppressing the expression of genes in TLR4/NF-κB signaling pathway (Fig. 8). In detail, WMCC10514 can significantly decrease the DON concentrations in mice, observing the reduction of the expression of TLR4 genes in the mouse jejunum, inhibiting TLR4/NF-κB signaling activation and alleviating intestinal inflammation while maintaining the integrity of the gut barrier. Moreover, WMCC10514 stabilizes the gut microbiota and increases SCFAs production. These changes help the liver take up more SCFAs from the intestine through the portal vein, enhance liver metabolic function and immune responses. With better protection of the intestinal barrier, the amount of unmetabolized DON and harmful substances entering the bloodstream is significantly reduced, further decreasing the expression of inflammatory genes in the liver and effectively mitigating liver inflammation. WMCC10514 also mitigated the DON-induced increase in intestinal Gram-negative bacteria. This reduction may decrease the subsequent translocation of LPS to the liver, thereby inhibiting the activation of the liver TLR4/NF-κB signaling pathway.

In summary, our findings demonstrate that B. velezensis WMCC10514 exhibits robust survival and DON-degrading activity in SGF and SIF. Oral administration of WMCC10514 restored murine growth performance and markedly reduced tissues DON accumulation. The strain effectively colonized the intestine in vivo and vitro, restructuring the gut microbiota, elevating SCFAs level, preserving intestinal barrier integrity, and attenuating inflammation. Furthermore, WMCC10514 modulated the gut microbiota to enhance liver SCFAs concentration, thereby regulating the gut-liver axis. This modulation suppressed liver pro-inflammatory genes expression, enhanced metabolic efficiency, mitigated DON-induced liver injury, and supplied energy substrates to peripheral tissues. WMCC10514 may also further attenuate liver inflammation by reducing liver LPS content, thereby suppressing activation of the TLR4/NF-κB signaling cascade. These findings provide foundational insights for employing probiotic additives in feed or food systems to neutralize DON toxicity.

Methods

Bacterial suspension preparation and DON

B. velezensis WMCC10514 was isolated from Wuliangye Daqu and stored in glycerol at −80°C. Strain WMCC10514 was cultured in LB broth with a 1% inoculum. After incubation, the suspension was centrifuged (6000 × g, 10 min), the supernatant discarded, and the pellet was retained. DON standard product is purchased from Shanghai Yuanye Biotechnology Co., LTD, purity ≥98%. Based on existing research and confirmed by our own preliminary experiments, we selected 2.4 mg/kg DON concentration for this study^38,65.

Study on pH and bile salt tolerance

To evaluate the pH tolerance of the strain, the amount of bacterial precipitate collected by centrifugation was suspended in buffer solutions with different pH values (1–5), and normal saline with pH 7.0 was used as the control (CK)⁶⁶ To evaluate bile salt tolerance, the amount of bacterial precipitate was suspended in solutions containing bile salts of different concentrations (0.03– 0.3%), and normal saline without bile salts was used as the control (CK)^67,68. After the above-mentioned bacterial suspension was incubated at 37°C for 2 h, a series of dilutions were carried out and spread on LB solid medium. Survival rates were calculated using Eqs. 1 and 2 after 24 h of incubation at 37°C.

$$\mathrm{Survival\; rate}(\%)=\frac{\lg \left(\mathrm{The\; number\; of\; viable\; bacteria\; in\; different\; pH\; solutions}\right)}{\lg (\mathrm{pH}=7\mathrm{Number\; of\; viable\; bacteria\; under\; normal\; saline})}\times 100\%$$ 1

$$\mathrm{Survival\; rate}(\%)=\frac{\lg \left(\mathrm{The\; number\; of\; viable\; bacteria\; under\; different\; bile\; salt\; solutions}\right)}{\lg \left(\mathrm{The\; number\; of\; viable\; bacteria\; under\; normal\; saline}\right)}\times 100\%$$ 2

Study on DON degradation

Strain WMCC10514 was inoculated (1% v/v) into three media: (1) LB + 100 µg/mL DON, (2) simulated gastric fluid (SGF) + 100 µg/mL DON and (3) SIF + 100 µg/mL DON. Cultures were incubated at 37°C with shaking (120 rpm) for 72 h. Following incubation, combine 5 mL of the culture medium with 15 mL of extract solution (acetonitrile: formic acid: water, 70:1:29, v/v/v). This mixture was vortexed for 5 min, then centrifuged at 6000 rpm for 5 min to promote sedimentation. Pass the resulting supernatant through a dedicated solid-phase purification column (PriboFast® MFC100, 3 mL). Finally, filter the eluate through a 0.22 μm membrane for HPLC analysis. The HPLC determination conditions were as follows: a Zorbax Eclipse Plus C18 column (4.6 × 250 mm, 5 μm) maintained at 35°C. An isocratic mobile phase of water: methanol (80:20, v/v) flowed at 0.6 mL/min. We injected 20 μL of sample and used UV detection at 220 nm over 15 min run time. DON degradation was calculated using Eq. 3:

$$\mathrm{DON\; Degradation}(\%)=\frac{\mathrm{DON\; initial\; concentration}-\mathrm{DON\; final\; concentration}}{\mathrm{DON\; initial\; concentration}}\times 100\%$$ 3

Study on cell-surface hydrophobicity (CSH)

Hydrophobicity was assessed using the method described by Zhao et al.⁶⁹. Strain WMCC10514 suspension was adjusted to an optical density (OD₆₀₀) of 1.0 ± 0.02 using sterile saline and recorded as A₀. A 4 mL aliquot of the adjusted suspension was mixed with 2 mL of solvent (ethyl acetate, xylene, or n-hexane), vortexed for 5 min, and incubated statically at 37 °C for 30 min. The aqueous phase was then aspirated, and the absorbance of the remaining phase was measured at OD₆₀₀ (A₁). Hydrophobicity was calculated using Eq. 4:

$$\mathrm{Hydrophobicity}(\%)=(1-{\mathrm{A}}_1/{\mathrm{A}}_0)\times 100\%$$ 4

Study on auto-agglutination

Auto-agglutination was evaluated using the method of Kuebutornye et al.⁷⁰. Bacterial pellets from cultures treated with LB medium (CK), SGF or SIF were collected separately. Each pellet was resuspended in saline and adjusted to an initial OD₆₀₀ of 1.0 ± 0.02, recorded as A₀. A 6 mL aliquot of each suspension was transferred to sterile tubes and incubated statically at room temperature for 4, 8, or 24 h. Supernatants were aspirated, and absorbance measured at OD₆₀₀ (A_t). Auto-agglutination was calculated using Eq. 5:

$$\mathrm{Auto}-\mathrm{agglutination}(\%)=(1-{\mathrm{A}}_{\mathrm{t}}/{\mathrm{A}}_0)\times 100\%$$ 5

Study on biofilm

Biofilm formation was quantified using the method of Fernández et al.⁷¹. Overnight cultures of strain WMCC10514 were inoculated into a 96-well polystyrene microplate (100 μL/well) and incubated at 37°C for 24 h. Unattached cells were removed by aspiration, and wells were washed twice with sterile saline. Biofilms were fixed with 100 μL methanol for 10 min, air-dried, and treated sequentially: (1) SGF for 1 h at 37°C, followed by washing with NaCl solution; (2) SIF for 1 h at 37°C, with two additional NaCl washes. Wells were stained with 100 μL crystal violet (20 min), unbound dye was aspirated, and excess stain removed by saline washing. Bound dye was solubilized with anhydrous ethanol, air-dried, and absorbance measured at OD₅₉₅ using a microplate reader. Triplicates were performed per group, with untreated wells serving as controls.

Animal experimental design

Male C57BL/6 mice (6 weeks old, average weight 20 ± 2 g) were obtained from Chengdu Dashuo Laboratory Animal Co., Ltd. The mice were housed under a 12 h light/dark cycle with free access to food and water for one week to acclimate to the environment, according to the guidelines of the Chinese Animal Care and Use Committee. Animal experimental procedures were approved by the Animal Care and Use Committee of Sichuan Normal University (NO. 2025LS0036). Following acclimation, the mice were randomly assigned to four groups (10 mice per group): the control group (CK), the strain WMCC10514 group (SW), the deoxynivalenol (DON) group, and the DON + strain WMCC10514 group (SN). Upon commencement of the experiment, the strain WMCC10514 group and the DON + strain WMCC10514 group received oral administration of a bacterial suspension of strain WMCC10514 at a concentration of 1 × 10⁹ CFU/mL, with a dosage of 0.1 mL per 10 g of body weight. The CK and DON groups administered an equal volume of sterile saline. In addition, the DON and DON + strain WMCC10514 groups were orally administered a DON solution at a dosage of 2.4 mg/kg body weight. All treatments were administered daily for 28 days.

During the experiment, body weight and food intake were recorded daily to calculate the average daily food intake (ADFI) and body weight gain. After the final treatment, the mice were fasted for 12 h before being weighed. They were then euthanized by cervical dislocation, and the liver, kidneys, and spleen were excised and weighed. The organs were rinsed with sterile saline, blotted dry, and weighed again to calculate their respective organ indices, which were determined by dividing the organ weight by the body weight.

Hematoxylin-eosin staining

Jejunum and liver tissues were fixed in formalin for 24 h, then embedded in paraffin for sectioning. The sections were stained with hematoxylin and eosin (HE) and observed under an optical microscope (Leica, Germany). Villus height and crypt depth in the jejunum were measured using ImageJ software.

Immunohistochemistry

Jejunum tissues were paraffin-embedded using a tissue processor and sectioned. Following deparaffinization and rehydration, antigen retrieval was performed, and nonspecific binding was blocked with serum. Sections were incubated overnight with rabbit anti-ZO-1 and anti-Occludin primary antibodies. After washing, secondary antibody incubation and PBS rinses were conducted, followed by DAB chromogenic development. Nuclei were counterstained, and sections were dehydrated, cleared, and mounted. Immunohistochemical images were acquired via light microscopy, and ImageJ software analyzed the average optical density to quantify protein expression levels.

Determination of DON content in tissues

Approximately 0.2 g of tissue was accurately weighed and homogenized in 500 μL of ice-cold PBS (0.01 M, pH 7.4), then add 1 mL of ethyl acetate. The mixture was vortexed for 5 min and centrifuged at 12,000 rpm for 5 min. Following centrifugation, the organic upper layer was carefully collected, and the extraction process was repeated twice with ethyl acetate. Then combined all organic supernatants into a clean vial and evaporated them to dryness at 40°C. The resulting residue was reconstituted in 1 mL of a methanol/water solution (15:85, v/v). Finally, all samples were vortexed for 1 min and filtered through a 0.22 µm membrane prior to HPLC analysis⁷². The HPLC analysis was performed according to the method described in Section “Study on DON degradation”.

ELISA

Blood was collected via retro-orbital puncture, allowed to clot at room temperature for 30 min, and centrifuged at 3000 × g for 20 min. The serum supernatant was transferred into sterile 1.5 mL microcentrifuge tubes for downstream analysis. The levels of TNF-α, IL-1β, IL-6, and IL-10 in the supernatant were tested with ELISA kits (Ruixin Biotechnology, Quanzhou, China) following the manufacturer’s instructions.

Transcriptome analysis

Liver and jejunum tissue samples were placed in 2 mL grinding tubes and homogenized using a grinding instrument at 60 Hz for 60 s. Total RNA was extracted using an RNA extraction kit (Thermo Fisher Scientific, USA). The degradation and contamination of RNA were assessed by agarose gel electrophoresis to ensure RNA purity, and the samples were used for RNA sequencing. Mixed RNA samples were used for RNA library construction and sequenced on NovaSeq 6000 platform (Illumina). Raw sequencing data were filtered to obtain high-quality sequences (Clean Data), which were then aligned to the reference genome of the species. Gene expression levels were calculated based on the alignment results. Differential expression analysis, enrichment analysis, and clustering analysis were performed on the samples.

16S rRNA sequencing

Genomic DNA was extracted from fecal bacteria using a DNA extraction kit (Omega Bio-Tek, Norcross, USA). The molecular size of the DNA was assessed by agarose gel electrophoresis, and the DNA concentration was quantified using a Nanodrop spectrophotometer. The V3-V4 hypervariable region of the bacterial 16S rRNA gene was amplified using universal primers 338F and 806R. The amplified samples were sequenced on the Illumina MiSeq platform to analyze changes in the gut microbiota community of mice.

SCFAs analysis

Freeze-dried and homogenized cecal content samples were accurately weighed 20 mg into 1.5 mL Eppendorf tubes and suspended in 1 mL of 0.5% (v/v) phosphoric acid. The mixture was vortexed for 10 min and ultrasonicated for 5 min. After centrifugation at 12,000 rpm for 10 min at 4°C, the supernatant was collected and extracted twice with an equal volume of ethyl acetate. The combined organic phases were filtered through a 0.22 μm filter membrane and transferred to an amber vial for GC-MS analysis. The GC operating conditions were as follows: helium carrier flow rate of 1.0 mL/min, with an inlet and detector temperature of 280°C. The temperature program began at 40°C for 2 min, then increased to 95°C at 40°C/min for 1 min, followed by a ramp to 140 °C at 5°C/min, and a final increase to 240°C at 40°C/min for 6 min. For MS detection, conditions included electron ionization at 70 eV, an ion source temperature of 230°C, and a scan range of m/z 40–20⁷³.

Construction and stability determination of WMCC10514-GFP strain

The plasmid pSU03-GFP was introduced into B. velezensis WMCC10514 by electroporation, yielding the fluorescently tagged strain WMCC10514-GFP. To evaluate plasmid stability, cultures were propagated for 30 serial passages in LB medium containing 50 μg mL⁻¹ kanamycin, followed by growth in LB at pH 2.0 supplemented with 0.3% (w/v) porcine bile salts for 24 h at 37°C and 220 rpm. After every passage, dilutions were plated on LB agar and incubated at 37°C for 24 h; 100 randomly selected colonies were examined for GFP fluorescence under a fluorescence microscope. Plasmid stability was calculated using Eq. 6:

$$\begin{array}{c}\mathrm{Plasmid}\mathrm{stability}\left(\%\right)\hfill \\ =\left(\mathrm{Fluorescent}\mathrm{colonies}/\mathrm{One}\mathrm{hundred}\mathrm{randomly}\mathrm{selected}\mathrm{colonies}\right)\times 100\hfill \end{array}$$ 6

Study on the in vivo and in vitro colonization

The WMCC10514-GFP strain was cultured in LB broth at 37°C with shaking (200 rpm) for 24 h. Cells were harvested by centrifugation at 8000 × g for 10 min, washed thrice with sterile phosphate-buffered saline (PBS, pH 7.4), and resuspended in 0.9% (w/v) NaCl to a final concentration of 1 × 10⁹ CFU/mL. For murine colonization assays, mice were randomly allocated into two groups: (1) control (CK) and (2) DON + WMCC10514-GFP (SW-GFP). The SW-GFP group received 0.1 mL of bacterial suspension (1 × 10⁹ CFU/mL) per 10 g body weight via injection, while the CK group received an equivalent volume of sterile saline. Additionally, the SW-GFP group was orally gavage with DON at 2.4 mg/kg body weight. Both treatments were administered daily for 28 days. Fecal samples were collected post-experiment for downstream analysis.

To simulate gastrointestinal conditions, 1 mL of 10% (w/v) CK fecal suspension, 1 mL of WMCC10514-GFP suspension (2 × 10⁹ CFU/mL), and 1 mL of 10 μg/mL DON were added to SIF. A blank control lacking the bacterial suspension was included. The mixture was incubated at 37°C with orbital shaking (200 rpm) for 48 h. Serial dilutions of the culture were prepared up to 10⁻⁶, and 100 μL of the 10⁻⁶ dilution was spread onto LB agar plates containing 50 μg/mL kanamycin to select drug-resistant colonies. Plates were incubated at 37°C for 24 h, and colonies were examined under a fluorescence microscope.

Data analysis

All experiments were conducted with at least three biological replicates. Data are presented as mean ± standard deviation (SD). Statistical analyses were performed using SPSS 16.0 software (SPSS, Chicago, IL, USA) to determine statistical significance. Comparisons among groups were conducted using one-way ANOVA, followed by Tukey’s post-hoc test for multiple comparisons.

Author contributions

X.Y.H.: Investigation, Formal Analysis, Visualization, Software, Writing - Original Draft; B.W.X.: Investigation, Visualization, Writing - Original Draft; Y.L., H.X.Q., and J.Y.L.: Conceptualization, Methodology, Investigation; J.Z., Y.X., D.Z., J.S., and J.Y.Z.: Conceptualization, Validation, Writing - Review & Editing, Supervision, Funding acquisition. All authors have read and approved the final manuscript for publication.

Data availability

The datasets analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests

The authors declare no competing interests.

Supplementary information

The online version contains supplementary material available at 10.1038/s41538-026-00707-9.