Aflatoxins are noxious secondary metabolites that are produced by filamentous fungal species such as Aspergillus flavus and Aspergillus parasiticus, which mainly includes AFB₁, aflatoxin B₂ (AFB₂), aflatoxin G₁ (AFG₁), aflatoxin G₂ (AFG₂), aflatoxin M₁ (AFM₁) and aflatoxin M₂ (AFM₂). Among aflatoxins, AFB₁ is the most common, and also exhibits the highest toxicity, such as teratogenic, carcinogenic, and hepatotoxic toxicity [1,2,3]. Cereal crops are very susceptible to aflatoxins worldwide. The feed in China was universally found to be contaminated with AFB₁, which was detected in 81.9%–100% of feedstuff and complete feed collected from different regions of China with the average levels ranging from 1.2–27.4 μg/kg during 2018–2020 [4]. Ducks that consume feed contaminated with AFB₁ are at risk of poisoning, which can result in liver damage and immunotoxicity [5, 6]. The liver is the primary organ targeted by AFB₁. Within the liver, phase I metabolism of AFB₁ predominantly involves its conversion to AFB₁-8,9-epoxide (AFBO), facilitated by the cytochrome P450 (CYP450) enzyme and then gives rise to metabolites such as aflatoxin Q₁ (AFQ₁) and AFM₁ [7, 8]. Under phase II metabolism of AFB₁, it can be catalyzed by glutathione-S-transferase (GST) to form aflatoxin 8,9-dihydro-8-(S-glutathionyl)-9-hydroxy aflatoxin B₁ with lower toxicity [9]. Research has shown that AFB₁ impaired growth performance, disrupted liver metabolism, triggered liver inflammation, and resulted in liver conditions such as swelling, steatosis, and bleeding in ducks [10, 11]. Therefore, there is a need for an effective strategy to mitigate the toxicity of AFB₁ on ducks.

Previous studies summarized some approaches to detoxify AFB₁ from food and feed, including physical, chemical, and biological approaches. Heat treatment, ultraviolet irradiation, and adsorption treatment are examples of physical procedures [12, 13], while ozone treatment is an example of chemical method. Due to the high cost, low efficiency, loss of nutrients, and chemical residue in food and feed caused by physical and chemical methods, both approaches have not been proven worthy of thorough detoxification and widely applied in animal production [14].

Detoxification of AFB₁ by using microorganisms or enzymes can overcome the mentioned drawbacks and is considered an efficient, safe, and economical approach to detoxify AFB₁ from the contaminated feed [14]. Bacillus subtilis ANSB060 isolated from fish gut can degrade AFB₁, AFG₁, and AFM₁ in vitro, meanwhile this strain could resist unfavorable conditions within simulated gut environments [15]. The growth performance and meat quality of broilers were improved when the AFB₁ naturally moldy diet was added with Bacillus subtilis ANSB060 [16]. Moreover, the combined probiotics with aflatoxin B₁-degrading enzyme from Aspergillus oryzae could relieve the negative effect of AFB₁ on chicken’s production performance and nutrient metabolic rates, suggesting a promising future for the application of AFB₁-degrading enzymes [17, 18]. Presently, studies on AFB₁-degrading enzymes primarily focus on validating AFB₁ degradation in vitro, with limited in vivo experiments assessing the effectiveness and safety of AFB₁-degrading enzymes in animal production [19,20,21].

CotA laccase from Bacillus licheniformis ANSB821 identified by our laboratory is highly thermostable and can degrade 70% AFB₁ (2 µg/mL) within 30 min in vitro [22, 23], while the efficacy of Bacillus CotA laccase in animals remains to be confirmed. The current study aims to assess the AFB₁ detoxification ability of Bacillus CotA laccase in ducks exposed to diets contaminated with AFB₁.

Experimental animals and diets

Experimental procedures were approved by the Laboratory Animal Welfare and Ethical Review Committee of China Agricultural University (approval No. AW41213202-1-3). A total of 192 male Pekin ducklings were purchased from Beijing Golden Star Duck Co., Ltd. (Beijing, China) and randomly assigned to 4 treatments with 6 replicate cages of 8 ducks each. A 2 × 2 factorial design was used to investigate the effects of Bacillus CotA laccase level (0 or 1 U/kg), AFB₁ challenge (challenged or unchallenged) and their interactions on ducks. The 4 treatments were: (1) Control group (Control, basal diet); (2) CotA laccase group (CotA, basal diet with an additional 1 U/kg Bacillus CotA laccase); (3) AFB₁ group (AFB₁, moldy peanut meal taking the place of normal peanut meal); (4) AFB₁ and Bacillus CotA laccase group (AFB₁ + CotA, AFB₁ diet with an additional 1 U/kg Bacillus CotA laccase). CotA laccase from Bacillus licheniformis ANSB821 was expressed in Pichia pastoris GS115, and freeze-dried in a vacuum for 24 h and then incorporated into the feed. The final AFB₁ concentrations in the AFB₁ group and the AFB₁ + CotA group were set around 20 μg/kg, and the final AFB₁ concentrations in the Control group and the CotA group were below 10 μg/kg. The determined concentrations of AFB₁ in each of the four groups are presented in Table S1. Diets were pelleted in the KL-210 feed pellet extruder (Henan Qirun Machinery Equipment Co., Ltd., China). Ducks had ad libitum access to pellet feed and water, with continuous light. The experimental diets were formulated based on corn-soybean meal-peanut meal in accordance with the requirements of the National Research Council (NRC, 1994) [24]. Table 1 presents the composition and nutrients level of the basal diets.

Sample collection

On d 28, one duck from each replicate close to the average body weight was selected for sample collection. Polypropylene tubes were used to collect blood samples from the wing veins. By dislocating the neck vertebrae and bleeding from the carotid artery, ducks were slaughtered. Subsequently, liver tissues and jejunal samples between the endpoint of the duodenal loop and Meckel’s diverticulum were collected, flushed, snap-frozen in liquid nitrogen, and fixed with a 10% neutral buffered formalin solution for histological analysis. All tissues were kept at −80 °C. Feces were collected from each replicate using sterile sampling bags and kept at −20 °C.

Growth performance

On d 14 and 28, ducks were fed-deprived for 8 h to determine the body weight (BW). The average daily feed intake (ADFI), ADG, and F/G were calculated for d 1–14, 15–28 and 1–28, respectively. The data are presented as mean ± standard error of the mean (SEM) (n = 6).

Histopathology of liver and jejunum

Fixed liver and jejunum tissues were embedded in paraffin, and tissue rings were sliced into 5-μm thickness, deparaffinized in xylene, rehydrated, and mounted on glass slides [25, 26]. Sections were stained by haematoxylin and eosin (H&E). The slides were photographed on a Pannoramic MIDI digital slide scanner (3DHISTECH Ltd., Budapest, Hungary). Stained tissue sections were examined using CaseViewer V 2.43 (3DHISTECH Ltd., Budapest, Hungary).

Transcriptional analysis

Total RNA was extracted from the liver and jejunum samples, then reverse transcription was performed using commercial kits (RC112, R223-01; Vazyme Biotech Co., Ltd., Nanjing, China) according to the manufacturer’s instructions. Two-step quantitative real-time PCR was performed with Taq Pro Universal SYBR qPCR Master Mix (Q712-02; Vazyme Biotech Co., Ltd., Nanjing, China) on a Real-Time PCR Detection Systems (CFX Connect™, Bio-Rad, Hercules, California, USA). The relative levels of mRNA expression were calculated using the 2^−ΔΔCT method, which normalized to the reference mRNA level of GAPDH. The values of the control group were used as a calibrator. The primers used in this study are listed in Table S2.

Amino acid-targeted metabolome

Serum amino acids were analyzed by UHPLC-MS/MS. The UHPLC separation was performed by an Agilent 1290 Infinity II series UHPLC System (Agilent Technologies, Santa Clara, CA, USA). The assay development was performed on an Agilent 6460 triple quadrupole mass spectrometer) which was equipped with an AJS electrospray ionization (AJS-ESI) interface. The MRM data was analyzed using Agilent MassHunter Workstation Software (B.08.00).

Serum biochemical analysis

The activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT), catalase (CAT), superoxide dismutase (SOD), and the concentrations of total antioxidant capacity (T-AOC) and malondialdehyde (MDA) in serum were measured using commercial assay kits (C010-2-1, C009-2-1, A007-1-1, A001-3-2, A015-2-1, A003-1-2; Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions.

Determination of AFB₁ residues and AFB₁-DNA adduct levels

AFB₁ residues in liver and feces were extracted using the total aflatoxin immunoaffinity column (Clover Technology Group, Beijing, China) according to manufacturer's instructions. The extracted samples containing AFB₁ were measured by high-performance liquid chromatography (HPLC) [27]. In brief, sample containing AFB₁ was filtered using RC 0.22 μm filter and 20 μL of volume was injected into the HPLC injection system. AFB₁ detection was achieved using 360 and 440 nm as the wavelengths of excitation and emission, respectively. The mobile phase consisted of methanol–water (45:55, v/v), and the flow rate was 1 mL/min. The levels of AFB₁-DNA adduct in liver were measured by the Elisa kit (HB253-NC, Hengyuan Biological Institute, Shanghai, China) according to the manufacturer’s instructions.

Statistical analysis

The data was analyzed using GraphPad Prism V 8.0.1 (GraphPad Software, San Diego, California, USA). Two-way ANOVA was used to determine the main effects of Bacillus CotA laccase addition and AFB₁ challenge, and their interaction. Tukey’s multiple comparison was used to separate means when interactive effects were significant (P < 0.05). Results are presented as the mean ± SEM.

Bacillus CotA laccase alleviated AFB₁-induced declines in growth performance of ducks

The growth performance of ducks is presented in Table 2. Results showed that there were significant interactions of Bacillus CotA laccase addition and AFB₁ challenge on the BW at d 28, the ADG, and F/G of ducks during d 15–28, and d 1–28. AFB₁ challenge significantly decreased the BW at d 28 and the ADG of ducks during d 15–28 and d 1–28, while increased the F/G (P < 0.05) of ducks during d 15–28 and d 1–28 compared with those in the Control group. The BW at d 28 and the ADG of ducks during d 15–28 and d 1–28 were significantly improved and the F/G were reduced (P < 0.05) in the AFB₁ + CotA group compared with the AFB₁ group.

Bacillus CotA laccase ameliorated AFB₁-induced gut barrier dysfunctions and inflammation in ducks

H&E staining was utilized to observe the intestinal status of ducks in the four treatments. There was a significant interaction of Bacillus CotA laccase addition and AFB₁ challenge on the jejunal villi height of ducks. In contrast with the Control group, the jejunum of ducks in the AFB₁ group had severe pathological changes with the disappearance of villus architecture (Fig. 1A). The jejunal villi height in the AFB₁ group was significantly reduced compared to that in the Control group, while the AFB₁ + CotA group showed an observably higher villi height of jejunum compared with the AFB₁ group. No interacting effect was observed between Bacillus CotA laccase levels and AFB₁ challenge on jejunal crypt depth and VH/CD of ducks. AFB₁ challenge markedly increased crypt depth and decreased the VH/CD of jejunum, while dietary addition of Bacillus CotA laccase presented a decreased tendency on crypt depth (P = 0.0702) and significantly improved the VH/CD of jejunum (Fig. 1B–D).

Bacillus CotA laccase ameliorated AFB₁-induced gut barrier dysfunctions and inflammation in ducks. A H&E staining of jejunum in groups Control, CotA, AFB₁, and AFB₁ + CotA, scale bar = 100 μm; B Jejunal villi height; C Jejunal crypt depth; D Jejunal villi height/crypt depth; E–H The mRNA expression of TJP1, ZO-1, ZO-2, and CLDN1 in the jejunum of ducks; I–K The mRNA expression of IL-8, IFN-γ, and TNF-α in the jejunum of ducks. All data are presented as mean ± SEM (n = 6). ^a−cDifferent letters denote a significant difference (P < 0.05). ^*P < 0.05, ^**P < 0.01, P-value for the main effect of AFB₁

As to the mRNA expression of tight junction proteins, obvious interaction effects between Bacillus CotA laccase addition and AFB₁ challenge were observed in the mRNA expression of TJP1 and ZO-1 in the jejunum of ducks. AFB₁ challenge significantly decreased the mRNA expression of TJP1 and ZO-1 in the jejunum of ducks compared with the Control group, but these changes were markedly ameliorated in the AFB₁ + CotA group (Fig. 1E and F).

The mRNA expression of zonula occluden-2 (ZO-2) and claudin 1 (CLDN1) was obviously decreased in the AFB₁ treatment group compared to that in the group without AFB₁ treatment (Fig. 1G and H).

As to the mRNA expression of inflammatory cytokines, there was obvious interaction effect between Bacillus CotA laccase addition and AFB₁ challenge on the mRNA expression of interleukin 8 (IL-8), interferon gamma (IFN-γ), and tumor necrosis factor alpha (TNF-α) in the jejunum of ducks (Fig. 1I–K). The mRNA expression of IL-8, IFN-γ and TNF-α in the jejunum of ducks was observably increased in the AFB₁ group compared to the Control group, but these changes were significantly alleviated in the AFB₁ + CotA group.

In sum, these results indicated that Bacillus CotA laccase ameliorated AFB₁-induced gut barrier dysfunctions and inflammation in ducks.

Bacillus CotA laccase ameliorated AFB₁-induced amino acid metabolism disorders in ducks

The amino acid metabolome analysis was performed to evaluate the impact of Bacillus CotA laccase on serum amino acid metabolism of ducks exposed to AFB₁. Based on the OPLS-DA model (Fig. 2A), there was a clear separation in metabolites between the Control group and the AFB₁ group, indicating that AFB₁ treatment altered the serum metabolomics profile. And there was a clear separation of amino acid metabolites between the AFB₁ group and the AFB₁ + CotA group (Fig. 2B). A total of 24 amino acid metabolites were changed in the AFB₁ group, including 11 upregulated metabolites and 13 downregulated metabolites compared to those in the Control group (Fig. 2C). The AFB₁ + CotA group had 10 upregulated metabolites and 14 downregulated metabolites compared to the AFB₁ group (Fig. 2D). The heatmap showed the distinct expression patterns of 24 metabolites in the serum of ducks between the Control group and the AFB₁ group (Fig. 2E), as well as between the AFB₁ group and the AFB₁ + CotA group (Fig. 2F). Notably, compared with the Control group, glutamic acid level was lower in serum of ducks in the AFB₁ group, while the AFB₁ + CotA group reversed this change. KEGG classification analysis revealed that the biosynthesis of amino acids was the most enriched pathway among all the changed amino acid metabolite pathways in the AFB₁ and AFB₁ + CotA groups (Fig. 2G).

Bacillus CotA laccase ameliorated AFB₁-induced amino acid metabolism disorders in ducks. A and B The OPLS-DA score plot and VIP values of the model of Control vs. AFB₁ and AFB₁ vs. AFB₁ + CotA; C and D Volcano plots of amino acids in Control vs. AFB₁ and AFB₁ vs. AFB₁ + CotA groups, blue represents low content while red represents high content; E and F Heat maps of amino acids concentrations in serum samples. Columns represent the samples (Control vs. AFB₁ and AFB₁ vs. AFB₁ + CotA groups), and rows represent amino acids; G KEGG pathways enrichment analysis of AFB₁ vs. AFB₁ + CotA groups; H–J The mRNA expression of SLC1A1, SLC1A3, and SLC1A4 in jejunum of ducks. All data are presented as mean ± SEM (n = 6). ^a,bDifferent letters denote a significant difference (P < 0.05)

Additionally, we measured the mRNA expression of genes associated with glutamic acid transport in the jejunum of ducks. As shown in Fig. 2H–J, there was an obvious interaction effect between Bacillus CotA laccase addition and AFB₁ challenge on the mRNA expression of solute carrier family 1 member 1 (SLC1A1), solute carrier family 1 member 3 (SLC1A3) and solute carrier family 1 member 4 (SLC1A4) in the jejunum of ducks. AFB₁ exposure decreased the mRNA expression of SLC1A1, SLC1A3, and SLC1A4 in the jejunum of ducks compared to the Control group, but these changes were significantly alleviated in the AFB₁ + CotA group.

In sum, these results indicated that Bacillus CotA laccase ameliorated AFB₁-induced amino acid metabolism disorders in ducks.

Bacillus CotA laccase ameliorated AFB₁-induced liver injury in ducks

Histological analysis of liver was showed in Fig. 3A. In the AFB₁ group, liver cell displayed unclear line arrangement and inflammatory cell infiltration, these damages were disappeared in the AFB₁ + CotA group. To further investigate the status of liver injury, the serum activities of ALT and AST were measured. Results indicated that significant interactions were observed between Bacillus CotA laccase addition and AFB₁ challenge on the activities of AST and ALT in serum of ducks. The activities of AST and ALT in the serum were significantly higher in the AFB₁ group compared with those in the Control group, but these changes were significantly ameliorated in the AFB₁ + CotA group (Fig. 3B and C).

Bacillus CotA laccase ameliorated AFB₁-induced liver injury in ducks. A H&E staining of liver sections in groups Control, CotA, AFB₁, and AFB₁ + CotA, scale bars are 100 μm and 20 μm, respectively; B Serum AST activity; C Serum ALT activity; D Serum T-AOC content; E Serum CAT activity; F Serum SOD activity; G Serum MDA content; H–M The mRNA expression of p53, Caspase-1, Caspase-3, Caspase-9, Bak-1, and Bcl-2 in liver of ducks. All data are presented as mean ± SEM (n = 6). ^a−cDifferent letters denote a significant difference (P < 0.05). ^*P < 0.05, P-value for the main effect of AFB₁

The activities of antioxidant enzymes in the serum of ducks were determined to evaluate whether Bacillus CotA laccase could alleviate AFB₁-induced oxidative damage (Fig. 3D–G). There were significant interactions between Bacillus CotA laccase addition and AFB₁ challenge on the activities of CAT and SOD, and the concentrations of T-AOC and MDA in the serum of ducks. The lower activities of CAT and SOD, the lower concentration of T-AOC, and the higher concentration of MDA in the serum of ducks were observed in AFB₁ group compared with the Control group (P < 0.05). Bacillus CotA laccase supplementation in the AFB₁ diet reversed these changes compared with the AFB₁ group (P < 0.05).

It’s widely accepted that oxidative damage could cause cell apoptosis in the body, so the mRNA expression of apoptosis related genes in liver was measured to evaluate whether Bacillus CotA laccase could alleviate the apoptosis caused by dietary AFB₁. There were significant interactions between Bacillus CotA laccase addition and AFB₁ challenge on the mRNA expression of tumor suppressor protein 53 (p53), cysteine-aspartic acid protease 1 (Caspase-1), cysteine-aspartic acid protease 3 (Caspase-3), cysteine-aspartic acid protease 9 (Caspase-9) and Bcl-2 antagonist/killer 1 (Bak-1) in the liver of ducks. The mRNA expression of p53, Caspase-1, Caspase-3, Caspase-9, and Bak-1 in the liver of ducks in the AFB₁ group was significantly increased compared to those in the Control group. Conversely, dietary Bacillus CotA laccase supplementation remarkably reversed those changes caused by AFB₁ (Fig. 3 H–L). In addition, AFB₁ exposure decreased the mRNA expression of B-cell lymphoma-2 (Bcl-2) in the liver of ducks (P < 0.05) (Fig. 3M).

All the results revealed that Bacillus CotA laccase supplementation in the AFB₁ diet could ameliorate AFB₁-induced liver injury, oxidative damage, and cell apoptosis in ducks.

Bacillus CotA laccase neutralized hepatic metabolic enzyme changes induced by AFB₁ in ducks

The metabolic process of AFB₁ in the liver was conducted by the phase I enzyme cytochrome P450 (CYP450), which could metabolize AFB₁ to AFBO, then causing the toxicity to the body. There were significant interactions between Bacillus CotA laccase addition and AFB₁ challenge on the mRNA expression of CYP1A1, CYP1A4, CYP2D17, CYP2C9, and CYP3A8 in the liver of ducks (P < 0.05). AFB₁ challenge enhanced the mRNA expression of CYP1A1, CYP1A4, CYP2D17, CYP2C9, and CYP3A8 compared to the Control group (P < 0.05), while the mRNA expression of these genes was significantly downregulated in the AFB₁ + CotA group compared with the AFB₁ group (Fig. 4 A–E). In addition, AFB₁ challenge decreased the mRNA expression of phase II enzyme GST in the liver of ducks (P < 0.05; Fig. 4 F), and Bacillus CotA laccase addition alleviated this change. These results suggested that Bacillus CotA laccase ameliorated AFB₁-induced hepatic metabolic enzyme changes in ducks.

Bacillus CotA laccase neutralized hepatic metabolic enzyme changes in ducks induced by AFB₁. A–F The mRNA expression of CYP1A1, CYP1A4, CYP2D17, CYP2C9, CYP3A8, and GST in liver of ducks. All data are presented as mean ± SEM (n = 6). ^a−cDifferent letters denote a significant difference (P < 0.05). ^***P < 0.001, P-value for the main effect of AFB₁

Bacillus CotA laccase decreased AFB₁-induced AFB₁-DNA adduct formation in the liver and the contents of AFB₁ residues in the liver and feces of ducks

There were obvious interactions between Bacillus CotA laccase addition and AFB₁ challenge on the content of AFB₁-DNA adduct in the liver, and AFB₁ residues in the liver and feces of ducks. AFB₁ treatment significantly increased the content of AFB₁-DNA adduct in the liver of ducks, and the residues of AFB₁ in the liver and feces of ducks compared to those in the Control group. Whereas Bacillus CotA laccase supplementation in diet contaminated with AFB₁ reduced the content of AFB₁-DNA adduct in the liver of ducks, and the residues of AFB₁ in the liver and feces of ducks compared with the diet contaminated with AFB₁ without Bacillus CotA laccase (Fig. 5A–C).

Bacillus CotA laccase reduced AFB₁-induced AFB₁-DNA adduct in liver and AFB₁ residues in liver and feces of ducks. A AFB₁-DNA adduct in liver; B AFB₁ residues in liver (ND = not detected); C AFB₁ residues in feces. All data are presented as mean ± SEM (n = 6). ^a,bDifferent letters denote a significant difference (P < 0.05)

Long term consumption of AFB₁-contaminated feed by animals may result in the accumulation of AFB₁ in animal products, thereby presenting a substantial health hazard to human consumers [28]. Hence, finding an effective AFB₁ detoxification strategy and putting it into practical application is a crucial priority of the livestock industry. Enzymatic biotransformation is recognized as an efficacious and eco-friendly method for AFB₁ detoxification, because enzymes can efficiently degrade AFB₁ in the intestinal tract, then alleviate AFB₁-induced damage in animals [17]. However, it is currently unconfirmed whether dietary Bacillus CotA laccase supplementation can alleviate the toxicity induced by AFB₁ in ducks. In this study, AFB₁-contaminated diets induced numerous adverse effects on ducks such as intestinal barrier damage, inflammatory responses, amino acid metabolism disruption, abnormal CYP450 enzyme metabolism in the liver, and compromised growth performance. Nonetheless, dietary supplementation of Bacillus CotA laccase could effectively mitigate these adverse effects caused by AFB₁ in ducks.

Production performance serves as the primary indicator for assessing the health status of poultry. Research has demonstrated that dietary AFB₁ exposure adversely impacts the growth performance of animals, as evidenced by reductions in ADFI, ADG, and feed conversion ratio [29,30,31]. This study unequivocally emphasized the toxic effects of dietary AFB₁ at a concentration around 20 μg/kg on the growth performance of ducks, which was consistent with the previous research [32]. Nevertheless, this study proved that Bacillus CotA laccase effectively mitigated the toxicity induced by AFB₁ and improved the growth performance of ducks, highlighting the practical application potential of Bacillus CotA laccase in the poultry industry.

The integrity of the intestinal barrier could protect the host from various pathogens, bacterial metabolites, and toxins [33]. The intestinal barrier includes physical, immunologic, and microbial components. The physical barrier is the first barrier to resist various damage to intestine [34]. Further, villus height, crypt depth, and VH/CD are crucial indicators of intestinal integrity [35]. Disruption of the intestinal barrier may trigger inflammatory responses, thereby posing a significant threat to animal health [36]. In this research, Bacillus CotA laccase demonstrated a capacity to mitigate the jejunal barrier damage induced by AFB₁, as evidenced by improving the jejunal morphology, increasing the mRNA expression of tight junction proteins (TJP1 and ZO-1), and decreasing the mRNA expression of inflammatory cytokines (IL-8, IFN-γ, and TNF-α). This suggested that Bacillus CotA laccase alleviated the intestinal barrier damage and inflammation induced by AFB₁ in ducks.

Glutamic acid is crucial for the development of the intestinal mucosa, and plays an essential function in cellular metabolism, which benefits for the growth of young animals [37]. In this study, ducks exposed to AFB₁ had lower level of glutamic acid in serum compared to ducks in the Control group. This finding aligns with the previous research in dairy goats indicating that AFB₁ ingestion disrupts amino acid metabolism [38]. However, Bacillus CotA laccase ameliorated AFB₁-induced amino acid metabolism disorders testified by increasing the level of glutamic acid in the serum of ducks. Non-essential amino acids, such as glutamine, glutamate, and aspartate, are primarily metabolized in the intestine. Amino acid transporter carriers facilitate the transport of these amino acids from the intestinal lumen, across the parietal membrane, and into the intestinal epithelium [39]. In this study, Bacillus CotA laccase supplementation alleviated AFB₁-induced downregulation of mRNA expression of SLC1A1, SLC1A3, and SLC1A4 in the jejunum of ducks. Besides, the amino acid transport didn’t exhibit a significant difference between the CotA group and the Control group. This finding suggests that Bacillus CotA laccase does not influence the absorption of micronutrients in the intestinal tract of animals. However, previous studies have indicated that certain adsorbents may bind essential minerals and nutrients present in the feed during the AFB₁ detoxification process, potentially resulting in micronutrient deficiencies in animals [40].

Relevant studies have revealed that AFB₁ exposure could lead to liver injury, including vacuolar degeneration and increased ALT and AST activities in the serum [41, 42], which is consistent with this study. What’s more, dietary Bacillus CotA laccase addition ameliorated AFB₁ induced liver injury in ducks, which was proved by the decreased activities of ALT and AST in the liver and the serum. AFB₁ also could damage the antioxidant capacity in animal, including the reduction of antioxidant enzyme activities and the increase of MDA level [43]. Antioxidant enzymes such as CAT and SOD are widely acknowledged as key defenders in cells, protecting body against oxidative damage. MDA is an important biomarker for assessing lipid peroxidation [44]. In this study, increased serum MDA concentration and decreased serum T-AOC concentration, CAT and SOD activities were observed in the AFB₁ group, meanwhile the addition of Bacillus CotA laccase into the AFB₁ diet alleviated the reduction of antioxidant capacity induced by AFB₁.

Furthermore, the oxidative damage has the potential to cause cell apoptosis, which is associated with the activation of Caspase family [45, 46]. AFB₁ treatment increased the mRNA expression of p53, Caspase-1, Caspase-3, Caspase-9, and Bak-1, which is consistent with previous evidence that AFB₁ caused caspase-mediated apoptosis [47]. Notably, the addition of Bacillus CotA laccase into AFB₁ diet significantly reduced the mRNA expression of these genes in ducks compared to the AFB₁ diet. Thus, these findings suggested that Bacillus CotA laccase could mitigate AFB₁-induced oxidant damage and cell apoptosis testified by enhancing antioxidant enzyme activity and reducing apoptosis-related gene expression.

The process of AFB₁ metabolism mainly occurs in the liver, metabolizing AFB₁ to AFBO by CYP450 enzymes [14]. Moreover, AFBO bonds with biomacromolecules like DNA, resulting in the formation of AFB₁-DNA adduct [48]. AFB₁-DNA adduct represents promising biomarkers for evaluating AFB₁ exposure and AFBO production in animals [49]. In this study, the mRNA expression of CYP1A1, CYP1A4, CYP2D17, CYP2C9, and CYP3A8 was downregulated in the AFB₁ + CotA group compared to the AFB₁ group, indicating that the addition of Bacillus CotA laccase into diet mitigated the hepatotoxic effects of AFB₁. The decrease of AFB₁-DNA adduct content in the liver of ducks in the AFB₁ + CotA group further supported this finding. In the liver, AFB₁ also undergoes a phase II metabolism mediated by GST, metabolizing AFBO to metabolites with lower toxicity [9]. The mRNA expression of GST in the liver of ducks in the group with AFB₁ was significantly reduced. AFB₁ can induce the excessive production of lipid peroxidation in the body, reduce the activity of antioxidant enzymes in the liver, and ultimately compromise the total antioxidant capacity of the body. However, the addition of Bacillus CotA laccase into the AFB₁ diet significantly improved the mRNA expression of GST in the liver compared to the AFB₁ diet. These findings collectively indicated that Bacillus CotA laccase had the strong detoxification capability in intestinal tract of animal, and reduced the concentration of AFB₁ absorbed by enterocyte, which lead to the decreased levels of AFB₁-DNA adduct in the liver and the residues of AFB₁ in the liver and feces of ducks, thus maintaining the normal hepatic metabolism.

In summary, the current study firstly proved that Bacillus CotA laccase could alleviate AFB₁-induced liver and intestinal toxicity in ducks. Further studies need to be carried out to investigate whether Bacillus CotA laccase can effectively alleviate the toxicity of livestock and poultry fed with diets contaminated with multiple mycotoxins, and reduce the residues of mycotoxins in animal products.

Bacillus CotA laccase effectively improved the growth performance, intestinal health, amino acid metabolism and hepatic AFB₁ metabolism, reduced the content of AFB₁-DNA adduct in the liver and the residues of AFB₁ in the liver and feces of ducks fed naturally contaminated AFB₁ diet as it had the strong detoxification capability in intestinal tract of ducks, highlighting its potential as an efficient and safe feed enzyme for AFB₁ detoxification in the livestock and poultry production.