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Bacillus CotA laccase improved the intestinal health, amino acid metabolism and hepatic metabolic capacity of Pekin ducks fed naturally contaminated AFB1 diet

Journal of Animal Science and Biotechnology volume 15, Article number: 138 (2024) Cite this article

Abstract

Background

Aflatoxin B1 (AFB1) is a prevalent contaminant in agricultural products, presenting significant risks to animal health. CotA laccase from Bacillus licheniformis has shown significant efficacy in degrading mycotoxins in vitro test. The efficacy of Bacillus CotA laccase in animals, however, remains to be confirmed. 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 purpose of this study was to evaluate the efficacy of Bacillus CotA laccase in alleviating AFB1 toxicosis of ducks.

Results

Bacillus CotA laccase alleviated AFB1-induced declines in growth performance of ducks accompanied by improved average daily gain (ADG) and lower feed/gain ratio (F/G). Bacillus CotA laccase ameliorated AFB1-induced gut barrier dysfunctions and inflammation testified by increasing the jejunal villi height/crypt depth ratio (VH/CD) and the mRNA expression of tight junction protein 1 (TJP1) and zonula occluden-1 (ZO-1) as well as decreasing the expression of inflammation-related genes in the jejunum of ducks. Amino acid metabolome showed that Bacillus CotA laccase ameliorated AFB1-induced amino acid metabolism disorders evidenced by increasing the level of glutamic acid in serum and upregulating the expression of amino acid transport related genes in jejunum of ducks. Bacillus CotA laccase ameliorated AFB1-induced liver injury testified by suppressing oxidative stress, inhibiting apoptosis, and downregulating the expression of hepatic metabolic enzyme related genes of ducks. Moreover, Bacillus CotA laccase degraded AFB1 in digestive tract of ducks, resulting in the reduced absorption level of AFB1 across intestinal epithelium testified by the decreased level of AFB1-DNA adduct in the liver, and the reduced content of AFB1 residues in liver and feces of ducks.

Conclusions

Bacillus CotA laccase effectively improved the growth performance, intestinal health, amino acid metabolism and hepatic aflatoxin metabolism of ducks fed AFB1 diets, highlighting its potential as an efficient and safe feed enzyme for AFB1 degradation in animal production.

Graphical Abstract

Background

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.

Materials and methods

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.

Table 1 Composition and nutrient levels of the basal diets
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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.

Results

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.

Table 2 Effect of Bacillus CotA laccase addition and AFB1 challenge on the growth performance of ducks
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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).

Fig. 1
figure 1

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

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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).

Fig. 2
figure 2

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)

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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).

Fig. 3
figure 3

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

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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.

Fig. 4
figure 4

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

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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).

Fig. 5
figure 5

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)

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Discussion

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.

Conclusion

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.

Availability of data and materials

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

Abbreviations

ADFI:

Average daily feed intake

ADG:

Average daily gain

AFB1 :

Aflatoxin B1

AFBO:

Aflatoxin B1-8,9-epoxide

ALT:

Alanine aminotransferase

AST:

Aspartate aminotransferase

Bak-1:

Bcl-2 antagonist/killer 1

Bcl-2:

B-cell lymphoma-2

BW:

Body weight

CAT:

Catalase

Caspase-1:

Cysteine-aspartic acid protease 1

Caspase-3:

Cysteine-aspartic acid protease 3

Caspase-9:

Cysteine-aspartic acid protease 9

CD:

Crypt depth

CLDN1:

Claudin 1

CP:

Crude protein

CYP1A1:

Cytochrome P450 family 1 subfamily A1

CYP1A4:

Cytochrome P450 family 1 subfamily A4

CYP2C9:

Cytochrome P450 family 2 subfamily C9

CYP2D17:

Cytochrome P450 family 2 subfamily D17

CYP3A8:

Cytochrome P450 family 3 subfamily A8

F/G:

Feed/gain ratio

GAPDH:

Glyceraldehyde 3-phosphate dehydrogenase

GST:

Glutathione S-transferase

H&E:

Haematoxylin and eosin

IFN-γ:

Interferon gamma

IL-8:

Interleukin 8

MDA:

Malondialdehyde

p53:

Tumor suppressor protein 53

SEM:

Standard error of the mean

SLC1A1:

Solute carrier family 1 member 1

SLC1A3:

Solute carrier family 1 member 3

SLC1A4:

Solute carrier family 1 member 4

SOD:

Superoxide dismutase

T-AOC:

Total antioxidant capacity

TJP1:

Tight junction protein 1

TNF-α:

Tumor necrosis factor alpha

VH:

Villi height

ZO-1:

Zonula occluden-1

ZO-2:

Zonula occluden-2

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Funding

This study was supported by the National Key Research and Development Program of China (2021YFC2103003), National Natural Science Foundation of China (31972604), Jinan Introductory Innovation Team Project (No. 202228037), and China Postdoctoral Science Foundation (2023M730998).

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Author notes

  1. Mingxin Ma and Qianqian Wang contributed equally to this work.

Authors and Affiliations

  1. State Key Laboratory of Animal Nutrition and Feeding, Poultry Nutrition and Feed Technology Innovation Team, College of Animal Science and Technology, China Agricultural University, No. 2. Yuanmingyuan West Road, Beijing, 100193, People’s Republic of China

    Mingxin Ma, Qianqian Wang, Yanrong Liu, Limeng Liu, Gaigai Wang, Shimeng Huang, Qiugang Ma, Cheng Ji & Lihong Zhao

  2. Poultry Institute, Shandong Academy of Agricultural Sciences, Jinan, 250100, China

    Guiming Li

  3. College of Animal Science and Technology, Henan Agricultural University, Zhengzhou, 450046, China

    Yongpeng Guo

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Contributions

The author’s contributions are as follows: LZ and MM conceived and designed the experiment. MM, QW, YL, GL, LL and GW were involved in the animal experiments, analysis, and data collection. MM and QW analyzed the data and drafted the original manuscript. QM, CJ, SH, YG and LZ made a revision of this manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Lihong Zhao.

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Ethics approval and consent to participate

All procedures mentioned in the present study were approved by the Laboratory Animal Welfare and Ethical Review Committee of China Agricultural University (approval No. AW41213202-1-3).

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Not applicable.

Competing interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Supplementary Information

Additional file 1

: Table S1 The determined levels of AFB1 in diets; Table S2 Sequences and product sizes of primers for target genes. 

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Ma, M., Wang, Q., Liu, Y. et al. Bacillus CotA laccase improved the intestinal health, amino acid metabolism and hepatic metabolic capacity of Pekin ducks fed naturally contaminated AFB1 diet. J Animal Sci Biotechnol 15, 138 (2024). https://doi.org/10.1186/s40104-024-01091-8

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Journal of Animal Science and Biotechnology

ISSN: 2049-1891