Introduction

Mycotoxins are a chemically diverse group of toxic secondary metabolites produced by mycotoxigenic fungi, primarily belonging to Aspergillus, Fusarium, Penicillium, Claviceps, and Alternaria [1]. Of approximately 400 mycotoxins identified up to now, aflatoxin B1 (AFB1), zearalenone (ZEN), and deoxynivalenol (DON) have received particular concern because they cause enormous economic loses in animal husbandry and pose a significant threat to human health. Among aflatoxins, AFB1 is the most toxic and well known for its genotoxic, carcinogenic, teratogenic and immunosuppressive characteristics [2]. AFB1 has been classified as Group I human carcinogen by the International Agency for Research on Cancer (IARC) [3]. Both DON and ZEN are Fusarium mycotoxins. Animal exposure to DON is usually associated with gastroenteritis, with clinical signs including feed refusal, vomiting, diarrhea, and gastrointestinal hemorrhage [4]. Immune system is also a central target of DON. Notably, chronic dietary exposure to DON promotes the expression of cytokines, chemokines, and inflammatory genes with concurrent immune stimulation, whereas high-dose exposure induces leukocyte apoptosis and impairment of macrophage/neutrophil function with concomitant immune suppression [5]. ZEN is an estrogenic toxin that can competitively bind to estrogen receptors, consequently leading to precocious puberty, infertility, abortion, and other reproductive problems [6].

Mycotoxins are frequently found in food and feed, and the point of contamination can be in the field before harvest, postharvest, during processing, storage, and feeding despite the most strenuous efforts of prevention [7]. Commonly contaminated agricultural commodities are maize, wheat, barley, peanuts, forage, rice, fruits, and their processed products. On a global scale, it is estimated that about 25% of the world’s crops are affected by mycotoxins annually [8]. In China, a recent survey showed that AFB1, ZEN, and DON contaminated 0.6%, 10.8%, and 38.2% of complete pig feeds, respectively, between 2016 and 2017, at levels exceeding the Chinese regulatory limits [9]. Often multi-mycotoxins occur in a contaminated substrate since a certain strain of mold may produce more than one mycotoxin, or several species of fungi can be present simultaneously, producing different kinds of mycotoxins [10, 11]. As example, Streit et al. [12] reported the presence of 7 to 69 mycotoxins in all of 83 naturally contaminated samples of feed and feed raw materials. The co-exposure to the mycotoxin combinations may lead to enhanced toxicity by the possible additive or synergic effect [13, 14].

In response to the risk of exposure to mycotoxins from food and feeds, diverse remediation approaches have been proposed to mitigate mycotoxin contamination in agricultural commodities. These strategies are often categorized into physical, chemical, and biological principles. Physical removal of highly contaminated grains may be achieved by cooking, roasting, cleaning, and milling [15]. Another physical removal approach is the application of mycotoxin binders. Although the incorporation of mycotoxin binders is economically feasible and there are a variety of commercially available mycotoxin binders as feed additives, the efficacy of these adsorbents towards different classes of mycotoxins is variable, with most commercial adsorbents being poorly able to bind DON [16, 17]. Furthermore, some may have adverse nutritional effects related to unspecific binding of minerals and vitamins exist [18]. The chemical strategies include the use of acids, bases, hydrogen peroxide, and the use of ozonation [19]. However, the application of chemical methods is limited due to the potential toxicity of chemical residues and their negative effects on the palatability and nutritive quality of food and feed [17, 18]. In contrast, biological degradation approaches, which comprise microbial and enzymatic transformation of mycotoxins into nontoxic or less toxic metabolites, are generally more specific, efficient, and environmentally friendly [19]. In our previous works, we isolated three bacterial strains Bacillus subtilis ANSB060 [20], Bacillus subtilis ANSB01G [21], and Devosia sp. ANSB714 [22], which could degrade 81.5 % of AFB1, 85 % of ZEN, and 97.3 % of DON, respectively, in liquid culture. Besides, the addition of Bacillus subtilis ANSB060 to aflatoxins contaminated diet could reduce aflatoxins residues in the liver of broilers [23]. Bacillus subtilis ANSB01G was shown to be capable of effectively alleviating ZEN toxicosis in gilts [24], and DON-induced negative effects in pigs could be counteracted by dietary supplementation with Devosia sp. ANSB714 [25]. In the current study, we are aiming at evaluating the efficacy of mycotoxin biodegradation agent (combination of B. subtilis ANSB060, B. subtilis ANSB01G, and Devosia sp. ANSB714) in counteracting the adverse effects of naturally moldy diet contaminated with multiple mycotoxins on mice.

Materials and Methods

Chemicals and Microbial Feed Additive

Aflatoxins (AFB1, AFB2, AFG1, and AFG2), zearalenone (ZEN), deoxynivalenol (DON), and ochratoxin A (OTA) standards were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other reagents were of analytical grade or higher and purchased from Sinopharm Chemical Reagent Beijing Co., Ltd (Beijing, China). The mycotoxin biodegradation agent (MBA) is a multiple bacterial strain mix in powder obtained from Henan Trillion-Zhongyuan Bio-Sin Co., Ltd (Zhengzhou, China), which contains B. subtilis ANSB060 (5 × 108 CFU g-1), B. subtilis ANSB01G (7.5 × 108 CFU g-1), and Devosia sp. ANSB714 (7.5 × 108 CFU g-1).

Animals and Diets

BALB/c female mice (35 d) were purchased from Vital River Laboratory Animal Technology Co., Ltd. in Beijing. All the mice were housed in transparent polypropylene cages in animal care room artificially conditioned at temperature 24 ± 1 °C and humidity 50 ± 5 %, and on a 12-h light-dark cycle. After 1 week of acclimation, all the mice were divided into three groups (six cages per group and three mice per cage) as follows: basal diet (CON), multi-mycotoxins contaminated diet (MCD), and multi-mycotoxins contaminated diet plus MBA at dose of 1.0 g kg-1 of feed (MCD + MBA). The contents of mycotoxins including AFB1, AFB2, AFG1, AFG2, DON, ZEN, and OTA in feed ingredients and formulated diets were measured with high performance liquid chromatography (HPLC) as described by Li et al. [26]. Ingredient composition and nutrient contents of CON and MCD diet were shown in Table 1. All mice were fed ad libitum for 4 weeks. Body weight gain and feed intake were recorded in the experimental period.

Table 1 Ingredient composition and nutrient contents of control and moldy diet
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Sample Collection and Serum Biochemistry Analyses

At the end of the 28-day feeding trial, all mice were fasted overnight. Samples of blood were obtained from orbital venous plexus. After centrifugation at 2000× g for 15 min, serum was separated and stored at −80 °C for further analysis. Mice were euthanized by cervical dislocation. The liver, heart, spleen, uterus, and kidneys were dissected out immediately, rinsed in chilled saline solution, dried on a filter paper, and weighed separately to calculate the organ index. The liver and kidney samples were stored at −80 °C for further examination.

The levels of serum immunoglobulin A (IgA), immunoglobulin G (IgG), immunoglobulin M (IgM), total superoxide dismutase (T-SOD), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA) were determined using commercial enzyme-linked immunosorbent assay (ELISA) kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The measurements were performed following the manufacturer’s instructions. Interleukin-1β (IL-1β), interleukin-2 (IL-2), interleukin-6 (IL-6), and necrosis factor α (TNF-α) were measured by R-911 automatic radioimmunoassay counter (China University of Technology Industrial Co., Hefei, China).

Determination of Mycotoxin Residues in Tissues

Analysis of AFB1 residues in liver was carried out according to the method provided by Fan et al. [23] with some modifications. Briefly, the ground-defrosted liver samples (2.5 g) were added with 0.5 g of NaCl and blended in 10 mL of methanol/water (80/20, V/V) for 3 min on a vortex shaker. After centrifugation at 5000× g for 10 min, the supernatant was transferred and filtered through a paper filter. An aliquot of 3 mL of filtrate was diluted with 12 mL of PBS containing 0.1 % Tween-20 and subjected to an immune-affinity column. Following washes with 10 mL of PBS and 10 mL of double distilled water, respectively, AFB1 was eluted with 1 mL of methanol. The eluent was evaporated to dryness at 40 °C under a stream of nitrogen. The residues were dissolved in 100 μL of mobile phase before HPLC analysis. HPLC system was equipped with a post-column photochemical derivation and a fluorescence monitor. The wavelengths for excitation and emission were 360 nm and 440 nm, respectively. The separation of AFB1 was achieved by a Diamonsil® C18 reverse phase column (5 μm, 4.6 × 150 mm). The mobile phase was methanol-water (45:55) with the flow rate of 1 mL min-1, and the injection volume was 20 μL.

Zearalenone residues in the liver were analyzed using the method of Duca et al. [27] with a few modifications. A quantity of 2.5 g of ground-defrosted liver samples were blended in 10 mL of buffer solution of acetic acid-ammonium acetate (pH 4.8). The solution was incubated at 37 °C for 15 h with 80 μL of a solution of glucuronidase/arylsulfatase with pH 4.0 adjusted with glacial acetic acid. Subsequently, the mixture was extracted with 10 mL of acetonitrile and 200 μL of NaOH solution (1 M) while being stirred at 200 rpm for 60 min. After centrifugation at 5000× g for 10 min, the supernatant (10 mL) was collected and mixed with 40 mL of buffer phosphate solution, pH 7.4. Then, the solution was transferred and filtered through a glass fiber filter paper. The filtrate (40 mL) was loaded onto an immune-affinity column, followed by washes with 10 mL of PBS and 10 mL of double distilled water, respectively. The retained ZEN was eluted with 1 mL of methanol. And the eluent was evaporated to dryness at 40 °C under a stream of nitrogen. The residues were dissolved in 100 μL of mobile phase and subjected to HPLC analysis. HPLC was performed with a fluorescence monitor at 274 nm for excitation and 440 nm for emission and acetonitrile-methanol-water (46:46:8) as the mobile phase at the flow rate of 1 mL min-1. The separation of ZEN was achieved by a Diamonsil® C18 reverse phase column (5 μm, 4.6 × 150 mm).The injection volume was 20 μL.

DON residues in kidney were determined using the method described by Zhao et al. [22] with a slight modification. Specifically, 1 g of freeze-dried kidney samples were placed in 10 mL of eppendorf tube containing 5 mL of sodium acetate buffer (pH 5.5). Afterwards, 100 μL of β-glucuronidase (85, 000 U mL-1) was added in the tube. Following enzymatic hydrolysis at 37 °C for 12 h, the samples were extracted with a mixture of acetonitrile and water and then centrifuged at 5000× g for 30 min. An aliquot of 8 mL of PBS containing 0.1 % Tween-20 was added to 2 mL of the collected extract and vortex agitated for 30 s. After centrifugation at 5000× g for 10 min, the supernatant (8 mL) was passed through an immune-affinity column at a flow rate of 1 mL min-1 by gravity and subsequently washed with 10 mL of PBS and 10 mL of double distilled water, respectively. The eluent was evaporated to dryness at 40 °C under a stream of nitrogen. The residues were dissolved in 100 μL of mobile phase and applied to HPLC analysis. HPLC was performed with a UV detector set at 218 nm and methanol-water (10:90) as the mobile phase at the flow rate of 1 mL min-1. The separation of DON was achieved by a Diamonsil® C18 reverse phase column (5 μm, 4.6 × 150 mm). The injection volume was 20 μL.

Statistical Analysis

All data obtained in this experiment were analyzed using SAS software (Version 9; SAS Institute, Inc., Cary, NC, USA) by performing one-way analysis of variance (ANOVA), followed by Duncan’s multiple range test as a post hoc comparison. All statements of statistical significance were based on a probability of p < 0.05.

Results

Growth Performance and Relative Organ Weight

The effects of MBA on the body weight gain and feed intake of mice fed with multi-mycotoxins contaminated diet are shown in Fig 1. The average body weight gain of mice in the MCD group (0.18 g) were significantly decreased (p < 0.05), whereas no difference was observed between the CON and MCD + MBA groups (both 0.21 g). There was no difference on average daily feed intake of mice among the three treatments (p > 0.05). As shown in Table 2, there were no differences in the relative weight of the heart and kidney among the three groups (p > 0.05). However, mycotoxins contaminated diet resulted in higher relative weight of the liver, spleen, and uterus compared with the control diet (p < 0.05). Supplementing MBA into naturally moldy diet counteracted the effect of mycotoxins on the relative weight of visceral organs, resulting in no significant differences between the CON and MCD + MBA groups (p > 0.05).

Fig. 1
figure 1

Effects of MBA on average daily gain and average daily feed intake of mice fed with muli-mycotoxins contaminated diet. CON = control diet; MCD = naturally contaminated diet with aflatoxin B1, zearalenone, and deoxynivalenol; MCD + MBA = naturally contaminated diet with aflatoxin B1, zearalenone, and deoxynivalenol plus 1.0 g MBA kg-1 diet. Columns with different letters differ significantly (p < 0.05)

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Table 2 Effects of MBA on the relative organ weight of mice fed with muli-mycotoxins contaminated diet.
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Serum Immunoglobulins and Cytokines Concentrations

The effects of MBA on serum immunoglobulins and cytokines concentrations of mice fed with multi-mycotoxins contaminated diet are summarized in Table 3. The level of serum IgG content in MCD treatment was not different with that in CON treatment (p > 0.05). However, the serum IgA and IgM levels were significantly reduced (p < 0.05) in mice fed diet contaminated with mycotoxins when compared with mice fed control diet. The addition of MCD with mycotoxin biodegradation agent improved serum IgA and IgM levels, and the values in the MCD + MBA treatment were not significantly different with that in mice fed a basal diet (p > 0.05). Regarding the serum cytokines, the concentrations of IL-1β, IL-2, and IL-6 were significantly increased (p < 0.05) in the MCD group compared to the control, but no statistical difference in the content of serum TNF-α was found among the three treatments (p > 0.05). Adding mycotoxin biodegradation agent into multi-mycotoxins contaminated diet significantly decreased the serum IL-1β, IL-2, and IL-6 levels (p < 0.05) compared with that in mice fed with multi-mycotoxins contaminated diet. Besides, there were no significant differences on the levels of serum IL-1β and IL-2 between the MCD + MBA treatment and CON treatment (p > 0.05), while the value of serum IL-6 in MCD + MBA treatment was higher(p < 0.05) than that in CON treatment.

Table 3 Effects of MBA on the serum immunoglobulins and cytokines concentrations of mice of mice fed with muli-mycotoxins contaminated diet
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Serum Antioxidant Parameters

The effects of MBA on the serum antioxidant parameters of mice fed with multi-mycotoxins contaminated diet are shown in Fig 2. There was no significant difference in the level of serum MDA content among the three treatments (p > 0.05). Serum GSH-PX (754.31 U mL-1) and SOD (71.46 U mL-1) activities in MCD treatment were significantly lower (p < 0.05) than that (867.69 and 88.05 U mL-1) in CON, whereas no difference was found between the MCD + MBA and CON groups (p > 0.05).

Fig. 2
figure 2

Effects of MBA on the serum antioxidant parameters of mice fed with muli-mycotoxins contaminated diet. CON = control diet; MCD = naturally contaminated diet with aflatoxin B1, zearalenone, and deoxynivalenol; MCD + MBA = naturally contaminated diet with aflatoxin B1, zearalenone, and deoxynivalenol plus 1.0 g MBA kg-1 diet. Columns with different letters differ significantly (p < 0.05)

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Mycotoxin Residues in Tissues

Neither AFB1 nor ZEN was determined in liver samples of mice exposed to multi-mycotoxins in this study. DON residue in kidney samples of mice fed with contaminated diet was 10.35 ± 1.38 ng g-1. Moreover, when supplemented with MBA, there were no detectable DON residues in the kidneys of mice consuming moldy diet.

Discussion

In the last few years, academics have focused on the occurrence, toxicity, and detoxification of a single mycotoxin. However, more often multi-mycotoxins occur in a naturally contaminated substrate, which has been a great concern since the health risk from this multi-mycotoxins exposure is not well-known [13]. In China, co-contamination with AFB1, ZEN, and DON was commonly found in feed and feed ingredients [9, 25].The consumption of mycotoxins by animals has been shown to decrease growth performance, cause organ damage, disrupt immune function, and induce oxidative stress [28,29,30,31], resulting in huge economic losses for producers. Moreover, previous studies indicated that the combination of multi-mycotoxins in diet led to further adverse effects on animals than when consumed alone [32,33,34]. Due to the structural diversity of mycotoxins, the current available mycotoxin-detoxifying agents aiming at a specific mycotoxin hardly meet the need of protecting animals from the negative effects of multi-mycotoxins exposure. In this situation, a novel mycotoxin biodegradation agent MBA, which could simultaneously detoxify AFB1, ZEN, and DON, was developed and applied as feed additive to alleviate mycotoxicosis in mice.

The reduction of body weight gain is an important manifestation of mycotoxicosis in animals. Weaver et al. [34] showed that 4.8 mg kg-1 DON and 0.3 mg kg-1 ZEN in feed caused a 33 % reduction in average daily gain in pigs. In another report, piglets fed diet contaminated with Fusarium mycotoxins (1.1 mg kg-1 DON and 0.5 mg kg-1 ZEN in feed) had a 41 % decrease in average daily gain after the feeding trial for 25 days [29]. Additionally, Chaytor et al. [28] found that body weight gain of pigs was reduced by 16 % after exposure to 120 μg kg-1 AF and 600 μg kg-1 DON in feed for 33 days. Consistent with these reports, the body weight gain of mice fed the contaminated diet was decreased by 12 % after the feeding trial for 28 days. However, the presence of MBA in the multi-mycotoxins contaminated diet effectively counteracted the adverse effects of mycotoxins on body weight gain in mice. Mycotoxicosis in animals can also cause alterations in visceral organ weights, including the enlargement of the liver and spleen. In the present study, the relative weights of the liver, spleen, and uterus were significantly increased in mice fed multi-mycotoxins contaminated diet. As a result of incorporation of MBA in the contaminated diet, the relative weight of the liver, spleen, and uterus returned to the normal proportions observed in the CON treatment. The liver is the main site for mycotoxins metabolism and a major target organ for mycotoxicosis. In the report of Weaver et al. [30], pigs challenged with AF and DON (0.15 mg kg-1 and 1.1 mg kg-1 in feed, respectively) resulted in remarkable increase in relative liver weights. Another research indicated that relative uterus weight was significantly increased in prepubertal gilts following exposure to 3.9 mg kg-1 DON and 0.42 mg kg-1 ZEN in feed [35]. Conversely, Shi et al. [29] did not observe any significant change in relative organ weights of the liver, spleen, and reproductive organs in immature gilts exposed to ZEN and DON (0.6 mg kg-1 and 0.8 mg kg-1 in feed, respectively). In another study, Chaytor et al. [28] found that pigs fed diets containing a combined contamination of DON and AF did not result in alteration of relative weights of visceral organs. These contrasting results may be attributed to the variation in animal species and age, health condition, and levels of mycotoxins as well as exposure time.

There is a lack of information on the combined impacts of multiple mycotoxins on animal immune function. The decrease in serum IgA and IgM levels in mice co-administrated with multiple mycotoxins was contradictory to the previous reports showing that individual and combined treatment with AFB1 and DON had no effect on serum Ig subclasses production [28, 36, 37]. However, a recent report found a significant increase in serum IgA and IgG in gilts fed diet contaminated with ZEN and DON [29]. On the other hand, Yin et al. [38] reported that maternal exposure to high levels of ZEN (2.77 mg kg-1 in feed) decreased serum IgA and IgG levels in pregnant sows and their offspring. Thus, more detailed studies are needed to clarify the mode of action of individual and combined mycotoxins on the humoral immune system. Nevertheless, the present study suggested that the addition of MBA in the diet naturally contaminated with multiple mycotoxins effectively alleviated the mycotoxins-mediated impairment of serum immunoglobulins production. There is accumulating evidence suggesting that mycotoxins can induce inflammatory reaction. In the current study, up-regulations of serum pro-inflammatory cytokines IL-1β, IL-2, and IL-6 were observed in mice following combined dietary exposure to AFB1, ZEN, and DON. Previous investigation on mice also demonstrated that DON exposure led to elevation of serum IL-1β, IL-2, IL-6, and TNF-α [22]. Besides, Pistol et al. [39] reported that ZEN could induce the expression of pro-inflammatory cytokines (IL-1β, IL-6, IL-8, TNF-α, and IFN-γ) in the spleen of pigs. In addition, Li et al. [37] also found an increase of mRNA expression of pro-inflammatory cytokines (IL-6, TNF-α, and IFN-γ) in the spleen of broiler chickens fed AFB1 contaminated diet. These findings suggested that dietary exposure to mycotoxins triggered inflammatory responses, which may further induce cell apoptosis, immune dysfunction, and organ damage. In the present study, the addition of MBA in the contaminated diet effectively reduced inflammation, resulting in serum cytokines levels similar to those in mice in control treatment.

Researches have indicated that oxidative stress plays critical roles in the cytotoxic mechanism of mycotoxins. Oxidative stress can be defined as the misbalance between the generation of intracellular oxidant species and antioxidant capacity, which subsequently causes damage to all major classes of cellular macromolecules, such as DNA, proteins, and lipids. As the direct product of lipid peroxidation, MDA is often seen as an indicator of oxidative stress in the organism. SOD and GSH-Px are major components of the antioxidant system and participate in reactive oxygen species (ROS) scavenging. SOD are a class of enzymes that catalyze the dismutation of superoxide (O2−) into hydrogen peroxide (H2O2), representing an important antioxidant defense in all cells exposed to oxygen, while GSH-Px catalyzes the conversion of hydrogen peroxide into water and molecular oxygen. In the current study, serum SOD and GSH-Px activities showed significant reduction in mice treated with mycotoxins, whereas MDA content was not altered. Similarly, lipid peroxidation was not affected in pigs following combined dietary exposure to 4.8 mg kg-1 DON and 0.3 mg kg-1 ZEN in feed [34]. In addition, consumption of 1.1 mg kg-1 DON and 0.5 mg kg-1 ZEN in feed by piglets did not alter MDA level in the liver, but the hepatic SOD activity was significantly decreased [31]. In the report of Zhang et al. [40], ducks fed moldy maize naturally contaminated with AFB1 (22 μg kg-1) had reduced SOD and GSH-Px activities and increased MDA level in the liver. Taking literatures and the present results together, it can be speculated that SOD activity is a sensitive indicator for mycotoxins exposure, and the decrease of SOD activity may trigger lipid peroxidation, resulting in elevation of MDA content. The supplementation of contaminated diet with MBA promoted serum SOD and GSH-Px activities, which indicated that MBA could overcome the combined toxicity of AFB1, ZEN, and DON on antioxidant system, by means of detoxifying these mycotoxins in digestive tract.

Apart from a direct exposure of humans to mycotoxins through contaminated cereals, a potential exposure route might be from edible tissues and products of animals consuming mycotoxins. In the current study, there were no detectable AFB1 or ZEN residues in the liver of mice fed contaminated diet, while DON was retained in the kidneys at the concentration of 10.35 ± 1.38 ng g-1. It has been well documented that the kidney is the major tissue for DON retention, and the concentrations of DON decrease from kidney to serum and liver of pigs exposed to DON [25, 41, 42]. Consistent with our previous reports [22, 25], the addition of MBA containing Devosia sp. ANSB714 eliminated DON residues in kidneys of mice fed DON-contaminated diet, suggesting that Devosia sp. ANSB714 could reduce DON absorption in digestive tract.

In conclusion, the results presented in this study indicated that consumption of diet contaminated simultaneously with AFB1, ZEN, and DON led to depression of body weight gain, immune function, and antioxidant capacity of mice. The adding of mycotoxin biodegradation agent into multi-mycotoxin-contaminated diet could effectively alleviate mycotoxicosis in mice. Thus, MBA has a great application potential in livestock production.