Ameliorating Effects of Bacillus subtilis ANSB060 on Growth Performance, Antioxidant Functions, and Aflatoxin Residues in Ducks Fed Diets Contaminated with Aflatoxins

Bacillus subtilis ANSB060 isolated from fish gut is very effective in detoxifying aflatoxins in feed and feed ingredients. The purpose of this research was to investigate the effects of B. subtilis ANSB060 on growth performance, body antioxidant functions, and aflatoxin residues in ducks fed moldy maize naturally contaminated with aflatoxins. A total of 1500 18-d-old male Cherry Valley ducks with similar body weight were randomly assigned to five treatments with six replicates of 50 ducks per repeat. The experiment design consisted of five dietary treatments labeled as C0 (basal diet containing 60% normal maize), M0 (basal diet containing 60% moldy maize contaminated with aflatoxins substituted for normal maize), M500, M1000, and M2000 (M0 +500, 1000 or 2000 g/t aflatoxin biodegradation preparation mainly consisted of B. subtilis ANSB060). The results showed that ducks fed 22.44 ± 2.46 μg/kg of AFB1 (M0) exhibited a decreasing tendency in average daily gain (ADG) and total superoxide dismutase (T-SOD) activity in serum, and T-SOD and glutathione peroxidase (GSH-Px) activities in the liver significantly decreased along with the appearance of AFB1 and AFM1 compared with those in Group C0. The supplementation of B. subtilis ANSB060 into aflatoxin-contaminated diets increased the ADG of ducks (p > 0.05), significantly improved antioxidant enzyme activities, and reduced aflatoxin accumulation in duck liver. In conclusion, Bacillus subtilis ANSB060 in diets showed an ameliorating effect to duck aflatoxicosis and may be a promising feed additive.


Introduction
Aflatoxins (AF) are secondary toxic metabolites produced by certain fungi belonging to the genus Aspergillus and can occur as natural contaminants of poultry food [1][2][3][4][5]. The principal aflatoxins commonly found in feedstuffs are aflatoxin B 1 (AFB 1 ), aflatoxin B 2 (AFB 2 ), aflatoxin G 1 (AFG 1 ), and aflatoxin G 2 (AFG 2 ) [6]. AFB 1 has the highest toxic potency and is classified as a Group I carcinogen by the International Agency for Research on Cancer (IARC, International Agency for Research on Cancer, 1993). After being ingested by animals, AFB 1 is converted to AFB 1 -8, 9-epoxide in

Growth Performance
The effects of B. subtilis ANSB060 on growth performance of Cherry Valley ducks fed moldy maize naturally contaminated with aflatoxins are presented in Table 1. There were no significant difference on ADG and ADFI among ducks fed basal diet without AFB 1 (C0), a moldy diet naturally contaminated with AFB 1 (M0), and diets (M500, M1000, and M2000) naturally contaminated with AFB 1 mixed with B. subtilis ANSB060 during the experimental phase (p > 0.05). However, birds fed a diet with 22.44 ± 2.46 µg/kg of AFB 1 (M0) had a relatively smaller ADG (p > 0.05) compared to those fed the basal diet without AFB 1 (C0), while ducks fed 22.44 ± 2.46 µg/kg of AFB 1 with B. subtilis ANSB060 exhibited relatively higher ADG (p > 0.05) compared to those in Group M0. The feed/gain ratio (F:G) of ducks was significantly affected by the dietary aflatoxins and B. subtilis ANSB060 during the experimental phase (p < 0.05). The F:G of birds in Group M1000 was markedly decreased compared with that in Group M0 (p < 0.05), implying that B. subtilis ANSB060 in diets might relieve the negative effects of aflatoxion on birds and improve the feed efficiency of ducks.

Serum Antioxidant Indices
The effects of B. subtilis ANSB060 on the serum antioxidant indices of ducks exposed to aflatoxins are shown in Table 2. The total superoxide dismutase (T-SOD) activity of the M0 group was decreased by the dietary aflatoxins compared to that in Group C0 during the experimental phase (p < 0.05). However, the addition of B. subtilis ANSB060 into moldy diets significantly improved the activity of serum T-SOD in the Group M500, M1000 and M2000 (p < 0.05), maintaining it in a normal status showed in Group C0. Though the glutathione peroxidase (GSH-Px) activity and malondiadehyde (MDA) content were not different among all groups (p > 0.05), Group M0 had the lowest GSH-Px activity and the highest MDA content, and the dietary B. subtilis ANSB060 could raise the activity of GSH-Px and decrease MDA concentration, keeping them to the normal level.

Liver Antioxidant Indices
The effects of B. subtilis ANSB060 on liver antioxidant indices of ducks exposed to aflatoxins are presented in Table 3. The result indicated that a low dose of aflatoxins in diets (Group M0) can decrease the activities of liver T-SOD and GSH-Px in ducks during the experimental period (p < 0.05); however, the presence of B. subtilis ANSB060 in some diets (Group M500, M1000 and M2000) exhibited a powerful capability of recovering T-SOD and GSH-Px activities in duck livers. The MDA level in liver was detected as the index of lipid peroxidation, and there were no significant effects of dietary aflatoxins and B. subtilis ANSB060 on MDA levels in duck livers in all groups (p > 0.05).

Aflatoxin Residues in Liver
The aflatoxin residues in the livers of ducks fed moldy diet naturally contaminated with 22.44 ± 2.46 µg/kg AFB 1 with or without B. subtilis ANSB060 are given in Table 4. Aflatoxins were not detected in the livers of ducks consuming a normal diet (Group C0). The AFB 1 and AFM 1 residues in Group M0 were the highest, being 0.12 and 0.10 ng/g, respectively. B. subtilis ANSB060 in diets could degrade aflatoxins into nontoxic products in the duck intestinal tract such that AFB 1 and AFM 1 residues in the liver were decreased. The AFB 1 residues was significantly decreased by 41.7%, 50.0%, and 58.3% respectively in Groups M500, M1000, and M2000 compared to those in Group M0 (p < 0.05). Similarly, the AFM 1 residues were significantly reduced by 40.0% in Groups M500, M1000, and M2000, respectively. The AFB 2 , AFG 1 , and AFG 2 residues in liver were not detected in all treatments. Table 4. The effects of B. subtilis ANSB060 on aflatoxin residues (ng/g) in livers from ducks fed moldy maize naturally contaminated with aflatoxins during the experiment.
Each value is a mean ± SD from 6 replicates of 1 duck each; a-c Means values with different superscripts in each column are significantly different (p < 0.05); * ND = Not detected.

Growth Performance
Aflatoxins are mycotoxins produced by Aspergillus spp. that contaminate animal feed and human food. Aflatoxins can lead to growth reduction, immunity suppression, and oxidative damage, along with residues in animal tissues [9,[32][33][34], and it has become a threat to human health. B. subtilis ANSB060 has shown a strong ability to detoxify aflatoxins in vitro; the degradation percentages of aflatoxins B 1 , M 1 , and G 1 in this strain are 81.5%, 60%, and 80.7%, respectively [25]; meanwhile, their protective effects on the performance and antioxidant function in layers [26] and the reduced accumulation of aflatoxin residues in livers of broilers [27] have also been verified.
In this study, ducks fed diets contaminated with 22.44 ± 2.46 µg/kg of AFB 1 show a tendency to decline in body weight, ADG, and feed efficiency compared with ones in the control group, illustrating a toxic effect of aflatoxins on the growth performance of ducks, which is consistent with previous research. A significant decrease in ADG and ADFI in ducks fed a diet containing 200 ppm AFB 1 for three weeks were observed in the research conducted by Cheng et al. (2001) [35]. Meanwhile, Fowler et al. (2015) reported a reduced body weight (BW) from the first week to the third week in broilers fed aflatoxin-contaminated diets, and this reduction grew greater along with the increase of aflatoxin concentrations in diets from 0 to 1800 µg/kg; the percentages of body weight reduction ranged from 8.8% to 24.5% during Week 2, and from 15.6% to 41.3% during Week 3. Additionally, a negative effect of aflatoxins on the cumulative F:G ratio of broiler chicks was also shown [36]. However, the deleterious effect on the ADG of ducks in our study was not significant during the trial phase. This may be due to lower concentration of aflatoxins and the inadequate duration of the experiment. Previous results indicate that a decrease in ADG was due to anorexia, reluctance, and an inhibition of protein synthesis and lipogenesis [37][38][39]. This growth inhibition in ducks suggests that diets naturally contaminated with aflatoxins at a low level (22.44 ± 2.46 µg/kg) appear to have a toxic effect on the growth performance of ducks in vivo. This result indicates that aflatoxin degradation preparation mainly consisting of B. subtilis ANSB060 has a great ability to ameliorate the toxic effects of aflatoxins on the growth performance of ducks. The basic mechanism seems to be one in which the spores of B. subtilis ANSB060 can survive and colonize in an animal's intestinal tract and secrete active substances to degrade aflatoxins in diets so that the adsorption of aflatoxins declines [27]. Some studies have suggested that microbial enzymes cleave the lactone ring of the AFB 1 molecule in vitro to lower its toxicity [40,41]. Nevertheless, the specific biotransformation mechanism of B. subtilis ANSB060 detoxifying aflatoxins in the animal's intestinal tract is unclear, and further studies should be performed with this strain [28].

Serum and Liver Antioxidant Indices
The inability to protect against reactive oxygen species (ROS) is the origin of oxidative stress. ROS are produced during the procedure of normal metabolism, and do much harm to the animal body. ROS mainly contain hydrogen peroxide (H 2 O 2 ), a superoxide anion radical (O 2 •− ), and a hydroxyl radical ( • OH). • OH is one of the most toxic oxygen-based radicals and wreaks havoc within cells, particularly with macromolecules. There is a dynamic equilibrium between oxidant and antioxidant systems and is vital for cell function, regulation, and adaptation to diverse growth conditions to remain healthy in animal bodies [42], and oxidant stress can be induced subsequently if the balance is broken down and the generation of ROS in a system exceed the system's ability to neutralize and eliminate them [43]. Oxidant stress can cause biological membrane lipid oxidation, denaturation of intracellular protein and enzymes, DNA damage, pain, and ultimately various diseases. Enzymes such as SOD and GSH-Px are crucial components of the antioxidant system and play a key role in removing ROS and relieving oxidative damage [42]. MDA is the main product of lipid peroxidation that can bring about protein damage and inactivation of membrane-bound enzymes [44]; thus, the level of MDA can be an important index reflecting the body's antioxidant ability [45]. One of our aims is to study the effects of adding B. subtilis ANSB060 to diets contaminated with a concentration of 22.44 ± 2.46 µg/kg aflatoxins on the antioxidant function in ducks. Our findings related to body antioxidant ability indicate that feeding diets contaminated with 22.44 ± 2.46 µg/kg aflatoxins can result in a significant decrease in T-SOD activity and an increase in the MDA level both in serum and the liver, while the activity of GSH-Px in duck livers is markedly decreased compared with that in the control group from Day 18 to Day 39. These results are in accordance with previous studies [46][47][48][49], demonstrating that lipid peroxidation took place in the ducks fed diet contaminated with aflatoxins.
While the activity of T-SOD is significantly increased and the MDA level is relatively decreased both in serum and liver, the activity of GSH-Px has a tendency to increase in serum but markedly increase in liver when ducks ingest aflatoxin-contaminated diets containing B. subtilis ANSB060 during the experiment compared with those fed no B. subtilis ANSB060 (M0). SOD and GSH-Px take part in lipid peroxidation by catalyzing the conversion of lipid hydroperoxide to hydroxy acids in the presence of GSH [50]. Liver is shown to be the major target organ for aflatoxins [51], and the increase in SOD and GSH-Px activities as well as the decrease in MDA level in serum and liver reflect that B. subtilis ANSB060 contributes to the inhibition of lipid peroxidation and relieve the harm produced by lipid peroxidation in ducks.
As a cause of liver degeneration and cancer, the elimination of these toxins from feedstuffs and foodstuffs bears great importance. In poultry, several nonnutritive adsorbents have been tested and have made some progress [46,52]; now, B. subtilis ANSB060 has been shown to have the capacity to inhibit lipid peroxidation, providing another biological method to alleviating oxidative damage to ducks caused by aflatoxins.

Aflatoxin Residues in Liver
The residue of aflatoxins and their metabolites in animal products have caused wide public concern. Liver is the main detoxifying organ that removes wastes and xenobiotics by metabolic conversion and biliary excretion [53]. Among the metabolites of AFB 1 , the toxicity of AFM 1 in animals seems to be comparable to or slightly less than that of AFB 1 [12]. In our study, low levels (0.12 and 0.10 ng/g) of AFB 1 and AFM 1 are retained in the livers of ducks fed the aflatoxin-contaminated diet for 21 days. Residue levels of AFB 1 (0.05 and 0.13 µg/kg) and AFM 1 (0.10 and 0.32 µg/kg) were also observed in the livers of broilers given diets contain 50 and 100 µg of AFB 1 /kg for 42 days [15]. AFB 1 residue was found in the livers of laying hens fed 2.5 mg/kg AFB 1 diet for four weeks [54]. Residue levels may be different because of the type of bird and diet, the concentrations of AFB 1 , and the duration, and enhanced tolerance to aflatoxins. In many countries, the maximum tolerance level of AFB 1 in human food products is 2 ng/g. AFB 1 residues in liver not only affect the performance and health of poultry, but also impair the health of the poultry product consumers as aflatoxins accumulate in edible parts of poultry, so it is necessary to control the quality of poultry products and analyze aflatoxin residues in different tissues of birds considering public health and safety. The results of our study show significantly decreased residue levels of AFB 1 -and AFM 1 -incorporated B. subtilis ANSB060 in ducks exposed to aflatoxins. The protective effects of B. subtilis ANSB060 from aflatoxins may be due to their specific biotransformation of aflatoxins in the intestinal tract, which leads to the reduction of aflatoxins absorbed by the intestinal tract and, consequently, a decrease in aflatoxin residues in the liver. However, the specific mechanism has not been tested, and more research should be done to determine this.

Conclusions
In conclusion, this study clearly indicates that aflatoxins in diets at a level of 22.44 µg/kg resulted in depressed growth performance and antioxidant capacity in liver and serum, as well as aflatoxin liver residues in ducks. Adding aflatoxin biodegradation preparation B. subtilis ANSB060 into moldy diets significantly recovers the growth performance of ducks and reduces the accumulation of aflatoxin residues in liver, effectively improving the antioxidant capacity to some extent. These results suggest that B. subtilis ANSB060 counteract the adverse effects of aflatoxins in animal diets, and thus is believed to be a very promising feed additive to detoxify aflatoxins in feed.

Ducks, Diets, and Management
One-day-old Cherry Valley ducks were obtained from a commercial hatchery and fed a commercial rearing diet for 17 days to adapt to the surroundings. On Day 18, 1500 male ducks were randomly assigned into 5 treatment groups with 6 replicates of 50 birds each. Five dietary treatment groups were composed as follows: Group C0 (the negative control), consisting of the basal diet with 60% normal maize; Group M0 (the positive control), containing 60% moldy maize taking the place of normal maize; and Groups M500, M1000, and M2000, containing an added 500 g/t, 1000 g/t and 2000 g/t of aflatoxin biodegradation preparation in the diet of Group M0, respectively. In this experiment, aflatoxin biodegradation preparation was mainly composed of industrially fermented and dried B. subtilis ANSB060 products via certain processing technology. The viable count of B. subtilis ANSB060 was more than 1 × 10 9 CFU/g in the aflatoxin biodegradation preparation. The composition of the basal diet was presented in Table 5. This study was approved by the Animal Care and Use Committee of the China Agricultural University (ethical approval code: CAU20131028-2; Date: 28 October 2013). All ducks were reared on floor in an environmentally controlled house equipped with central heating and temperature controllers. The size of barrier was 8 m × 2 m. Animals were cared for in accordance with the guidelines for the care and use of laboratory animals presented in the guide issued by the National Institute of Health and China's Ministry of Agriculture. Body weight (BW), average daily gain (ADG), average daily feed intake (ADFI), and feed/gain ratio (F:G) were measured on a replicate basis at the end of the experiment (39 day old).

Analysis of Dietary Mycotoxin
The contents of mycotoxins (AFB 1 , AFB 2 , AFG 1 , AFG 2 , deoxynivalenol, zearalenone, and ochratoxin A) in feed ingredients and formulated diets in the study were determined using high performance liquid chromatography (HPLC) according to appropriate methods [55]. After these five diets were prepared, the final contents of mycotoxins for each diet were determined. The concentration of AFB 1 , AFB 2 , AFG 1 , AFG 2 , and zearalenone of the diets fluctuated at 22.44 ± 2.46 µg/kg, 6.69 ± 1.32 µg/kg, 1.65 ± 0.65 µg/kg, 0.00 µg/kg, and 5.20 ± 0.68 µg/kg in Groups M0, M500, M1000, and M2000, respectively. Only AFB 1 was found in diet of Group C0, but the concentration of AFB 1 was less than 0.20 µg/kg in this group.

Sampling of Antioxidant Indices and Measurements
At 39 day of age, 12 ducks (2 birds for each replicate) close to the average weight of treatment were slaughtered by dislocation of the neck vertebrae and bleeding after fasting for 12 h. Blood samples were collected by puncture of wing vein; after being centrifuged at 1000× g at 4 • C for 10 min, the serum was separated and transferred into 1.5 mL plastic centrifuge tubes for analysis of antioxidant indices including superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) activity, and malondiadehyde (MDA) content.
The liver tissue samples were washed with ice-cold sterilized saline (0.85%), snap frozen in liquid nitrogen, and stored at −80 • C for further analysis of liver antioxidant indices. Liver tissue (1 g) was cut into small pieces and homogenized in ice-cold saline buffer (0.85%, pH = 7.4) (1:9, w/v) with an Ultra-Turrax (T8, IKA-labortechnik, Staufen, Germany) to form homogenates at a concentration of 0.1 g/mL for further analysis. Liver homogenates were centrifuged at 1000 g for 15 min at 4 • C, and the supernatants were collected. The supernatants were used for the assays of SOD and GSH-Px activity, and MDA content.
These parameters were determined with the clinical chemistry analyzer (Commercial Kit, Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer's recommended procedure.

Analysis of Aflatoxin Residues in Liver
Livers samples from the pectoralis major of the right side of the duck were excised and frozen at −20 • C to analyze the residues of the aflatoxins. For this analysis, liver samples of six birds from each treatment (one for each replicate) were selected. Analysis of AFB 1 , AFB 2 , AFG 1 , AFG 2 , and AFM 1 residues in the tissues was performed according to Fan et al. [27].
The ground defrosted liver sample (25 g), when 5 g of NaCl was added, was blended in 100 mL of methanol-water (80:20) for 3 min. Through a paper filter, an aliquot of 10 mL of filtrate was diluted with 40 mL of PBS/0.1% Tween-20 Wash Buffer and applied to an immunoaffinity column. Aflatoxins were eluted with 1.0 mL of methanol in a glass vial and dried near to dryness under a gentle stream of nitrogen and dissolved in a HPLC mobile phase. AFB 1 , AFB 2 , AFG 1 , and AFG 2 were determined by the HPLC system (Shimadzu LC-10 AT) equipped with reverse phase column (Diamonsil, C18, 5 µm, 15 cm × 4.6 cm ID) and post-column photochemical derivatization (AURA, New York, NY, USA) and fluorescence monitor (Shimadzu RF-20A), with excitation at 360 nm and emission at 440 nm, with methanol/water (45:55) as the mobile phase, at a flow rate of 1 mL/min. For the determination of AFM 1 , HPLC was performed with a fluorescence monitor at 365 nm for excitation and 425 nm for emission, and water/acetonitrile/methanol (68:24:8) as the mobile phase at a flow rate of 1 mL/min.

Statistical Analyses
Data were analyzed using the GLM procedure of SAS software (version 9; SAS Institute, Inc., Cary, NC, USA). Duncan's multiple range tests were used for multiple comparisons when the analysis indicated significant differences among treatments. All statements of statistical significance were based on probability (p < 0.05). All data were expressed as means ± SD.