*Article* **Isolation, Purification, and Characterization of a Laccase-Degrading Aflatoxin B1 from** *Bacillus amyloliquefaciens* **B10**

**Dongwei Xiong †, Jun Wen †, Gen Lu †, Tianxi Li and Miao Long \***

Key Laboratory of Livestock Infectious Diseases, Ministry of Education, College of Animal Science & Veterinary Medicine, Shenyang Agricultural University, Shenyang 110866, China; 2019220557@stu.syau.edu.cn (D.X.); 2020240593@stu.syau.edu.cn (J.W.); 2019220539@stu.syau.edu.cn (G.L.); 2020240619@stu.syau.edu.cn (T.L.)

**\*** Correspondence: longmiao@syau.edu.cn

† These authors contributed equally to this work.

**Abstract:** Aflatoxins, widely found in feed and foodstuffs, are potentially harmful to human and animal health because of their high toxicity. In this study, a strain of *Bacillus amyloliquefaciens* B10 with a strong ability to degrade aflatoxin B1 (AFB1) was screened; it could degrade 2.5 μg/mL of AFB1 within 96 h. The active substances of *Bacillus amyloliquefaciens* B10 for the degradation of AFB1 mainly existed in the culture supernatant. A new laccase with AFB1-degrading activity was separated by ammonium sulfate precipitation, diethylaminoethyl (DEAE) and gel filtration chromatography. The results of molecular docking showed that B10 laccase and aflatoxin had a high docking score. The coding sequence of the laccase was successfully amplified from cDNA by PCR and cloned into *E. coli*. The purified laccase could degrade 79.3% of AFB1 within 36 h. The optimum temperature for AFB1 degradation was 40 ◦C, and the optimum pH was 6.0–8.0. Notably, Mg2+ and dimethyl sulfoxide (DMSO) could enhance the AFB1-degrading activity of B10 laccase. Mutation of the three key metal combined sites of B10 laccase resulted in the loss of AFB1-degrading activity, indicating that these three metal combined sites of B10 laccase play an essential role in the catalytic degradation of AFB1.

**Keywords:** aflatoxin; *Bacillus amyloliquefaciens*; laccase; degradation; molecular docking; mutagenesis

**Key Contribution:** A novel laccase was isolated and purified, and some key sites for the catalytic degradation of aflatoxin were identified by targeted mutagenesis.

### **1. Introduction**

Aflatoxins are difuranocoumarin derivatives produced mainly by strains of *Aspergillus flavus* and *Aspergillus parasiticus*. They are produced during the growth and storage of crops and are chemically and thermally stable. Aflatoxin is highly hepatotoxic, nephrotoxic, acutely toxic, and immunotoxic, and belongs to a class of teratogenic, carcinogenic, and mutagenic compounds. In decreasing order of toxicity, the various metabolites are aflatoxin B1 (AFB1), AFM1, AFG1, AFB2, AFM2, and AFG2 [1–4]. Aflatoxins pose a significant risk to human health through the food chain [5].

It is essential to avoid AFB1 contamination and develop safe and effective detoxification methods to improve food safety. Traditional methods of AFB1 degradation include physical, chemical, and microbiological techniques [6]. Physical methods possess the disadvantage of being time consuming and less efficient in removing aflatoxin, while chemical methods lead to the loss of nutrients in food or feed [7]. In previous research focused on microbial degradation, AFB1 was degraded into nontoxic or less toxic metabolites by microorganisms or enzymes. The furan and lactone rings are the two key sites influencing the toxicity of AFB1 [8]. The existing studies on the microbial degradation of AFB1 also revolve around these two key sites. Some microorganisms are not probiotics, and their

**Citation:** Xiong, D.; Wen, J.; Lu, G.; Li, T.; Long, M. Isolation, Purification, and Characterization of a Laccase-Degrading Aflatoxin B1 from *Bacillus amyloliquefaciens* B10. *Toxins* **2022**, *14*, 250. https://doi.org/ 10.3390/toxins14040250

Received: 17 January 2022 Accepted: 29 March 2022 Published: 31 March 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

safety should be evaluated. Meanwhile, some microorganisms may inadvertently disrupt the nutritional properties of the product or even introduce other toxic substances that are harmful to the organism [9]. Enzymes are a promising choice, being ecofriendly and endowed with high substrate specificity and catalytic efficiency [10].

Enzymes identified to degrade aflatoxins include laccase, oxidoreductase, peroxidase, and manganese peroxidase [11]. Laccase is widely found in bacteria, fungi, insects, and higher plants [12]. Previous studies indicated that laccase can catalyse the oxidation of various compounds such as phenol, aniline, aromatic amines, ascorbic acid, and certain inorganic compounds, and can be coupled to the four-electron reduction of dioxygen to water [6,13–15]. The fungus laccase with high redox potential, isolated from the whiterot fungus *Cerrena unicolor* 6884, could efficiently degrade AFB1 to AFQ1 [16]. The CotA protein, as the best-known bacterial laccase, is predominantly located in the outer endospore layer of *Bacillus subtilis* and other *Bacillus* species and has a larger substrate binding pocket than other laccases [17,18]. Guo et al. cloned and expressed a novel CotA laccase from *Bacillus licheniformis* that converts AFB1 to AFQ1 and epi-AFQ1 [19]. Interestingly, Loi et al. also found that peroxidase converted AFB1 to AFQ1 [20]. Furthermore, many studies have verified that the toxicity of AFQ1 is one order of magnitude lower than that of AFB1 [21,22]. Typically, bacterial laccase exhibits higher thermal and alkaline stability than fungus laccase and other laccase [23]. Therefore, bacterial laccase is a promising biocatalyst to degrade AFB1 in feed and food.

In this study, a strain of *Bacillus amyloliquefaciens* B10 with efficient AFB1-degrading activity was isolated from 74 strains, and the active substance responsible for AFB1 degradation by this strain was localised. A new laccase was found to efficiently degrade AFB1. The laccase was expressed in *E. coli* and the degradation characteristics of recombinant laccase were determined. This study provides a theoretical basis for AFB1 degradation by laccase and promotes the development of the enzymatic degradation of AFB1 in feed and food.

#### **2. Results**

#### *2.1. Isolation and Identification of AFB1-Degrading Bacteria*

The initial screening of the 74 strains kept in the laboratory revealed that the strain labelled B10 grew relatively well in the medium containing different concentrations of AFB1. Strain B10 was white on LB (Luria-Bertani) medium, slightly elevated, with a rough surface and folded colonies; single colonies were round and 3–4 mm in diameter (Figure 1A). Gram staining was positive with bluntly rounded ends, and rod-shaped cells were seen (Figure 1B). The complete 16S rDNA gene of length 1462 bp and the complete *gyrB* gene of size 1186 bp were obtained through amplification and sequencing with 16S rDNA and *gyrB* universal primers. Phylogenetic analysis of the 16S rDNA gene showed that the homology of B10 with the *Bacillus amyloliquefaciens* strain GD4a was 98.8%. Phylogenetic analysis of the *gyrB* gene implied that B10 was 99.8% homologous to the *Bacillus amyloliquefaciens* strain JX014631.1 on the same branch (Figure 1E,F). This strain was identified as *Bacillus amyloliquefaciens* based on the biochemical response characteristics (Table 1), combining morphological and phylogenetic characteristics. The OD600 value of strain B10 was also measured using a UV spectrophotometer at 6 h intervals. The results indicated that the strain entered the log phase at 12–18 h of incubation (Figure 1C). In further AFB1 degradation tests, the efficiency of AFB1 degradation increased significantly with the increase in co-culture time; almost complete degradation of 2.5 μg/mL AFB1 was found after 96 h of co-culture (Figure 1D).

#### *2.2. Localisation of Degradation Active Substances*

The high-performance liquid chromatography (HPLC) results showed that the extracellular fluid of *B. amyloliquefaciens* B10 had the best degradation ability on AFB1, with a degradation rate of 72.9%. The other components of strain B10 caused a minor AFB1 degradation at a level far inferior to the extracellular fluid effect. Bacteria inactivated by

high temperature could degrade 15.2% of AFB1, probably due to the physical adsorption of the cell membrane of the B10 strain (Figure 2A).

**Figure 1.** Characteristics of strain B10 and efficiency of AFB1 degradation: (**A**) Morphology of strain B10 cultured on LB medium for 24 h. (**B**) Gram stain of strain B10. (**C**) Growth curve of strain B10. (**D**) Effect of co-culture time on the degradation efficiency of AFB1 (2.5 μg/mL). (**E**) Phylogenetic tree of the 16S rDNA gene sequence of strain B10 using the neighbour-joining method. (**F**) Phylogenetic tree of the *gyrB* gene sequence of strain B10 using the neighbour-joining method.


**Table 1.** Physiological and biochemical characteristics of strain B10.

**Figure 2.** Localization of active substances for aflatoxin B1 degradation by strain B10: (**A**) Evaluation on the degradation effect of AFB1 (2.5 μg/mL) by each component of strain B10. (**B**) Evaluation on the degradation effect of AFB1 (2.5 μg/mL) by the crude protein extract of culture supernatant with different concentrations of ammonium sulfate.

The crude protein of the supernatant from strain B10 was extracted with different concentrations of ammonium sulfate and dialysed for desalination. The degradation efficiency of AFB1 was 69.13% when the ammonium sulfate crude protein concentration was 80%. (Figure 2B). Therefore, we further determined that the AFB1-degrading substance might be a type of enzyme.

#### *2.3. Isolation of AFB1-Degrading Proteins*

The crude protein from the extracellular fluid was concentrated and initially purified using a DEAE ion-exchange chromatography column to obtain an elution curve with four component peaks (Figure 3D). The degradation of AFB1 by each component showed that components 3 and 4 exhibited vigorous AFB1 degradation activity after 36 h incubation, and component 4 had the highest degradation activity with regard to AFB1 with a rate of 73.4% (Figure 3A). Therefore, component 4 was further purified by gel filtration chromatography and seven component peaks were obtained (Figure 3E). Among the seven protein components, components 4-3 demonstrated the highest degradation activity to AFB1 with a rate of 87.3% (Figure 3B). After SDS-PAGE and Coomassie brilliant blue R-250 staining of

components 4-3, an obvious protein band of 35 kDa was observed. Components 4-3 were identified by protein mass spectrometry and matched to 60 proteins. The laccase component was contained in these 60 proteins by protein blast in NCBI. Therefore, we speculated that the proteins with AFB1 degradation activity in strain B10 probably contained laccase. However, the protein profile did not include a single protein, and whether all other proteins have AFB1-degrading activity warrants further research.

**Figure 3.** Isolation of AFB1 degrading enzyme from strain B10: (**A**) Degradation efficiency of AFB1 by each protein component precipitated by DEAE. (**B**) The efficiency of degradation of AFB1 by protein component precipitated by Superdex separation. (**C**) DEAE separation chromatogram (peaks 1 to 4 correspond to different protein fractions). (**D**) Superdex separation chromatogram (peaks 4-1 to 4-7 correspond to different protein fractions).

#### *2.4. Molecular Docking for Function Prediction*

Due to the excellent results achieved by AlphaFold2 in protein structure prediction [24], the predicted structure of a laccase from strain B10 was further refined and evaluated by Rasch plot, and 97% of the residues fell within the permissible interval (Figure 4A). This finding indicated that our predicted structure of B10 laccase could be used for molecular docking studies.

**Figure 4.** Molecular docking of aflatoxins with B10 laccase: (**A**) Ramachandran diagram of B10 laccase protein structure. (**B**) Models of the interaction of AFB1, AFB2, AFG1, AFG2, AFM1, and AFM2 with B10 laccase (grey, Zn2+; yellow, ligand).

The molecular docking of the six aflatoxins (AFB1, AFB2, AFG1, AFG2, AFM1, and AFM2) was performed using the B10 laccase prediction model, and each aflatoxin had a high docking score with B10 laccase (Table 2, Figure 4B). Therefore, it is important to further verify the aflatoxin degradation ability of B10 laccase. The differences in docking scores with B10 laccase were also minor as the chemical structures of the six aflatoxins were similar. The docking score results indicated that AFB2 had the most vital binding capacity for laccase. Both Lys-153 and Arg-268 in B10 laccase produced hydrogen-bonding interactions with each aflatoxin, and these two residues were most likely to be the key amino acids for binding the toxins. His-87 made hydrophobic interactions with each aflatoxin, while His-87 was one of the amino acids with a coordination bond with Zn2+. The O-1 site on the terminal furan ring of AFB1 that acted as an acceptor with O-5 on the five-membered ring of AFB2 formed two hydrogen bonds as an acceptor with the side chain of Arg-268 and with Tyr-191. AFM1 formed hydrogen bonds with Arg-268, His-259, and Tyr-191. AFM2 formed hydrogen bonds with Trp-152, Lys-153, and Tyr-191 (Table 2, Figure 4B). The results of the molecular docking analysis showed that B10 laccase had a stable binding mode with aflatoxin, which was predicted to play a role in the degradation of aflatoxin. Therefore, we continued to clone the B10 laccase gene and investigated its protein expression in order to verify the ability of B10 laccase to degrade aflatoxin.

**Table 2.** Molecular docking results.



**Table 2.** *Cont*.

#### *2.5. Cloning, Expression, and Purification of Laccase from Strain B10*

The amino acid sequence of B10 laccase obtained in Section 2.3 was matched by NCBI BLAST and had the highest similarity with laccase No. ASB53002.1. The cloning primers were designed according to the base sequence of the CDs region of ASB53002.1 laccase, and the B10 laccase gene was cloned from strain B10 (Figure 4A). The open reading frame of the B10 laccase gene was 837 bp (Figure 5A), encoded 278 amino acids, and was predicted to have a molecular weight of 30.9 kDa and an isoelectric point 5.84. The amino acid homology of laccase cloned from the B10 strain to *Bacillus amyloliquefaciens* 629 laccase was 99.64% (GenBank: KNX34508.1) [25], to *Bacillus velezensis* RC218 laccase was 99.28% (GenBank: KUP42711.1) [26], and to *Bacillus* 916 laccase was 99.22% (GenBank: AIW29756.1) (Figure 5D) [27].

The laccase gene was transformed into *E. coli* DH5α and verified by double digestion (Figure 5B) and expressed in *E. coli* BL21(DE3). The purified recombinant laccase was then purified by Ni Sepharose 6 Fast Flow affinity chromatographic packing, and the purified recombinant laccase demonstrated a more distinct, but single, band by SDS-PAGE gel electrophoresis. The apparent molecular weight of the band was approximately 35 kDa, which was close to the predicted molecular weight (Figure 5C).

#### *2.6. Efficiency of AFB1 Degradation by the Recombinant Laccase and the Effects of Different Conditions on AFB1 Degradation*

A series of experiments proved that the recombinant laccase of strain B10 could efficiently degrade AFB1. The rate of degradation of AFB1 gradually increased with the increase in co-culture time, reaching 79.3% after 36 h. Meanwhile, the degradation rate of AFB1 reached a plateau at 48 h (Figure 6A,F). The results with regard to the effects of temperature on the degradation of AFB1 by the recombinant laccase showed that the degradation rate of AFB1 was gradually increased with the increase in temperature from 20 to 40 ◦C at pH 7.2; the degradation rate of AFB1 reached 82.4% at 40 ◦C. The degradation rate of AFB1 decreased rapidly because of the high temperatures ranging from 70 to 90 ◦C. However, the degradation rate of AFB1 exceeded 65% between 30 and 60 ◦C (Figure 6B). pH value had a strong influence on the degradation of AFB1. At pH 6.5, the degradation rate of AFB1 was the highest, reaching 84.2%. Under acidic conditions, the degradation rate of AFB1 was lower; at pH 4.0, the degradation rate was only 7.1%. From pH 6.0 to pH 8.0, the degradation rate of AFB1 exceeded 60% (Figure 6D). As AFB1 was efficiently

degraded under strongly alkaline conditions, the effects of a pH greater than 9.0 on the degradation of AFB1 by the recombinant laccase were not considered in the present study.

**Figure 5.** Gene cloning, expression, purification, and identification of B10 laccase: (**A**) Agarose gel electrophoresis to isolate the B10 laccase gene (M, marker; 1, B10 laccase PCR product; C, blank control). (**B**) Agarose gel electrophoresis to validate the results of pET-28a/B10 laccase recombinant vector (M, marker; 1, pET-28a/B10 laccase recombinant vector double digestion; 2, pET-28a double digestion; 3, pET-28a; 4, B10 laccase gene; C, blank control). (**C**) SDS-PAGE analysis of purified recombinant B10 laccase (M, marker; 1, beads; 2, 300 mM imidazole; 3, 200 mM imidazole; 4, 150 mM imidazole; 5, 100 mM imidazole; 6, 50 mM imidazole; 7, 10 mM imidazole; 8, imidazole-free buffer). (**D**) Amino acid sequence alignment of B10 laccase with *Bacillus amyloliquefaciens* 629 laccase, *Bacillus velezensis* RC218 laccase, and *Bacillus* 916 laccase.

**Figure 6.** The efficiency of B10 laccase in degrading AFB1 and the effects of different conditions on the degradation: (**A**) Effect of co-culture time on the degradation of AFB1 by B10 laccase. (**B**) Effect of temperature on the degradation of AFB1 by B10 laccase. (**C**) Effect of common solutions on the degradation of AFB1 by B10 laccase (CK: In the control group, no substance was added). (**D**) Effect of pH on the degradation of AFB1 by B10 laccase. (**E**) Effect of metal ions on the degradation of AFB1 by B10 laccase (CK: In the control group, no metal ions was added). (**F**) Liquid chromatogram of AFB1 at 36 h co-culture (left, blank control; right, 36 h co-culture). \*\*: *p* < 0.05; \*\*\*: *p* < 0.001.

The results with regard to the effects of different metal ions on the degradation of AFB1 by the recombinant laccase indicated that Ca2+, Cu2+, Co2+, Fe3+, Mn2+, and Zn2+ significantly reduced the degradation activity of AFB1 by the recombinant laccase compared with the control group with an 80.3% degradation rate of AFB1 (*p* < 0.001), while Na+ and K+ slightly reduced the degradation rate of AFB1 (*p* < 0.05). The metal ion Ni2+ had no significant effect on the degradation activity of AFB1 (Figure 6E). It was also shown that the metal chelators EDTA (*p* < 0.05) and SDS (*p* < 0.001) significantly reduced the AFB1-degrading activity of the recombinant laccase (Figure 6C), and the AFB1-degrading efficiency was significantly increased with the addition of DMSO (*p* < 0.05).

#### *2.7. Site-Specific Mutagenesis of B10 Laccases*

The amino acid sequence of B10 laccase was matched to asb53002 by the NCBI protein database BLAST. The laccase domain protein YlmD score of 1 was 99.64%. After query, the annotation of the GenBank CDs region was directed to uniprotkb o31726. We found that B10 laccase contained three Zn2+ binding sites: namely, H-87, C-132, and H-149. Therefore, the roles of these three residues in laccase were assessed by site-specific mutagenesis. There were seven mutant proteins, single mutant H87, C132, and H149; double mutant H87/C132,

H87/H149, and C132/H149; and triple mutant H87/C132/H149. These proteins were obtained by the polymerase chain reaction (PCR) method to mutate these sites to alanine. The results showed that after expression and purification, single mutants H87, C132, and H149 could be analysed by measuring AFB1-degrading activity. The AFB1-degrading activity of the single and double mutants H87, C132, H149, H87/C132, H87/H149, and C132/H149 was significantly decreased compared with the wild type, while the AFB1-degrading activity was further weakened by the triple mutant H87/C132/H149. In contrast, the triple mutant H87/C132/H149 decreased the degradation rate of AFB1 to 6.7% (Figure 7).

**Figure 7.** Effects on AFB1 degradation activity of B10 laccase mutants. \*\*: *p* < 0.05; \*\*\*: *p* < 0.001.

#### **3. Discussion**

Aflatoxin is of widespread concern due to its high toxicity, with approximately 4.5 billion people worldwide chronically exposed to aflatoxin through contaminated food [28,29]. In addition, data show that the consumption of aflatoxin-contaminated food is expected to further increase in incidence due to the ongoing COVID-19 pandemic, which has made the management of food and feed more difficult and complex [30]. The detoxification of aflatoxins by microorganisms is a promising new technology with broad application prospects. The control of aflatoxin by microorganisms mainly includes the inhibition of aflatoxin production, the adsorption of aflatoxin, and the degradation of aflatoxin [11]. To date, strains that are highly efficient in degrading aflatoxins have been isolated from various environmental sample species, including fungi, bacteria, actinomycetes, and protozoa [31]. In this study, 74 strains were screened in the laboratory, and one strain (laboratory number B10), was found to be highly efficient in degrading AFB1. The strain was identified morphologically, characterized biochemically, and analysed in a phylogenetic tree as *Bacillus amyloliquefaciens*. The B10 strain was 99.8% homologous to the *Bacillus amyloliquefaciens* strain JX014631.1 on the same branch and 98.8% homologous to the *Bacillus amyloliquefaciens* strain GD4a. Xu et al. isolated a strain of *Bacillus shark ii* L7 from 43 strains of bacteria, which could degrade 92.1% of AFB1 at a final concentration of 100 μg/L for 72 h at 37 ◦C. Further experiments implied that the active substance degrading AFB1 of strain L7 was mainly present in the culture supernatant, which could degrade 77.9% of the AFB1 within 72 h [32]. This was similar to the results of this study, where the supernatant of strain B10 could degrade AFB1 by 72.9% at 24 h. In contrast, the degradation rate of AFB1 was lower in the high-temperature inactivation group, the intracellular fluid group, and the bacterial suspension group compared with the culture supernatant. The high-temperature inactivation group presented the degradation of AFB1 probably due to the physical adsorption capacity of the cell membrane of the B10 strain. The presence of active substances in culture supernatants for AFB1 degradation by *Bacillus* was also revealed in tests by Gayatri [33], Wang [34], and Shu [35]. In this study, we found that protein precipitated by an ammonium sulfate gradient had the highest AFB1-degrading efficiency, so the AFB1-degrading substance was likely to be an enzyme or several enzymes in the culture supernatant.

In this study, the protein precipitated by ammonium sulfate was further purified, and the active component was measured through separation by DEAE anion exchange chromatography and gel filtration chromatography. A novel laccase that can efficiently degrade AFB1 was identified by protein mass spectrometry. The laccase gene had an open reading frame of 837 bp, an apparent molecular weight of 35 kDa, and a predicted isoelectric point of 5.84, and encoded 278 amino acids. The laccase cloned from strain B10 showed 99.64% amino acid homology to *Bacillus amyloliquefaciens* 629 laccase [25], 99.28% to *Bacillus velezensis* RC218 laccase [26], and 99.22% to *Bacillus* 916 laccase [27]. Proteins with AFB1-degrading activity were previously isolated using similar methods from culture supernatants of *Aspergillus flavus* ANSM068 [36], and the edible fungus *Pleurotus ostreatus* [37].

Superior results in all previous applications were achieved when Alphafold2 was used to predict the structure of the B10 laccase protein, and the release of Deepmind's Alphafold2 software has ushered in a new revolution in high-quality three-dimensional (3D) protein structure prediction [38–40]. The predicted 3D model of B10 laccase was evaluated by structural refinement with a Rasch diagram, which allowed us to examine the model of the interaction of B10 and laccase with the substrate through molecular docking simulations without resolving the crystal structure. The active pocket of laccase appeared semi-open, with Zn2+ at the bottom of the pocket interacting with His-87, C-132, and H-149. This leads to speculation that Zn2+ may also be complexed with substrate or water molecules. In molecular docking, an implicit water model was used to approximate the solvent interaction, and a high degree of precision was employed to reveal the possible conformation of the ligand in the pocket. The docking results showed that both Lys-153 and Arg-268 could exert hydrogen bonding forces with aflatoxin, and therefore these two residues play an essential role in the binding of aflatoxin to B10 laccase. Notably, His-87 is known to play an essential role in catalysing the degradation of AFB1 by B10 laccase using a targeted mutation assay, and the docking results also indicated that His-87 can exert hydrophobic forces with aflatoxin, so His-87 is equally important in the binding of B10 laccase and substrate. The amino acid residues involved in hydrogen bonding were considered key residues for the interaction of laccase with specific ligands [41]. Lys-153 and Arg-268 will therefore be the focus of our future studies.

B10 laccase and aflatoxin have high docking scores. Therefore, we continue to verify the efficiency of B10 laccase in degrading aflatoxin. The efficiency of AFB1 degradation by B10 laccase was significantly different at different temperatures. The maximum degradation activity of AFB1 by this laccase reached 82.4% at 40 ◦C. Unlike the laccase identified by other researchers, the degradation activity of AFB1 decreased at temperatures ranging from 60 to 90 ◦C. The degradation rate of AFB1 by laccase isolated from *Stenotrophomonas* sp. CW117 was less than 60% at 40 ◦C but increased rapidly when treated at a high temperature for a short period; the degradation rate of AFB1 exceeded 84.6% at above 70 ◦C [31]. This may be due to evolutionary factors in the strain that led to the evolution of the laccase into a heat-resistant enzyme [42]. The highest AFB1-degradation activity of B10 laccase was achieved at pH 6.5, with a rate of degradation of 79.2%. The AFB1-degrading activity of this enzyme was deficient when pH was below 4.5, yielding a result more similar to that of Guo et al. [19]. The laccase isolated by Cai et al. exhibited low AFB1-degrading activity at pH 7–8 [31]. Since AFB1 was also efficiently degraded under strongly alkaline conditions, the effects of conditions above pH 9.0 on the degradation of AFB1 by recombinant laccase were not considered herein. Laccase activity could be increased at pH 9.8, but this condition was difficult to apply in food and feed detoxification [43]. In this study, we found that the addition of all metal ions exerted a negative effect on AFB1 degradation by B10 laccase, except for Mg2+, which enhanced the AFB1-degrading activity of B10 laccase. Notably, the addition of Cu2+ significantly reduced the AFB1-degradation activity of B10 laccase. This finding was consistent with results that laccase activity can be enhanced by 0.4–1 mM Cu2+ but was inhibited by high concentrations of Cu2+ [44]. In the present study, the organic solvent DMSO significantly improved the degradation activity of AFB1, in line with the

findings of Wu al. [45], which may be attributed to changes in enzymatic activity due to the altered microenvironment caused by the organic solvent [46].

The residues H87, C132, and H149 of B10 laccase are the three metal-binding sites of this enzyme. The metal-binding sites of laccase are associated with the catalytic degradation of AFB1. When supplemented with metal ion chaperones during the expression of laccase, there was an increase in the metal-ion content of laccase and an improvement in its specific activity [47]. Previous research showed that the ribose portion of such proteins was coordinated by the side chains of these three residues. One of the conserved triplets of B10 laccase was predicted to be the active site, with similar results to the YlmD protein [48,49]. In the presence or absence of excess metal ions, the conformation of the protein was found to be significantly altered, including the R-loop, MR helix, and metal site centre, which destabilized the structure of the protein [50]. Although this does not change the overall structure, it can significantly affect enzymatic activity [51,52]. The present study showed that these three residues are essential for the degradation of AFB1 by B10 laccase. When these residues were replaced by alanine, the AFB1-degrading activity of the mutants was significantly decreased compared with the wild type.

#### **4. Conclusions**

In this study, a strain of *Bacillus amyloliquefaciens* B10 with high AFB1-degradation capacity was obtained and the active component of AFB1 degradation was located in the culture supernatant of this strain. A new laccase with high AFB1 degradation activity was obtained by the crude extraction of culture supernatant proteins with ammonium sulfate, DEAE, and gel filtration chromatography. The molecular docking results showed higher scores. Meanwhile, it was speculated that Lys-153, Arg-268, and His-87 residues in this laccase played an essential role in the binding of aflatoxin to this laccase. The B10 laccase gene was cloned from the bacterial genome and was expressed in *E. coli*. The recombinant laccase showed the highest AFB1-degradation activity at 40 ◦C and pH 6.5. The mutation of three key metal sites of the laccase implied that the AFB1-degradation activity of the triple mutants was almost wholly lost. These results suggested that B10 laccase is a promising enzyme for aflatoxin degradation. Our laboratory will continue to explore B10 laccase to improve enzymatic activity and stability, with a view to prompting the application of this enzyme in practical production as soon as possible.

#### **5. Materials and Methods**

#### *5.1. Chemicals and Reagents*

Gram stain, TAE, nucleic acid dye, Komas Brilliant Blue R-250, and ammonium sulfate were purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China); the plasmid extraction kit, gel recovery kit, and PCR product recovery kit were purchased from Thermo Fisher Scientific (Waltham, MA, USA); DH5α and BL21 (DE3) receptor cells were sourced from TransGen Biotech (Beijing, China); point mutation kits from TransGen Biotech (Beijing, China) were used; point-mutation kits were purchased from Vazyme Biotech Co., Ltd. (Beijing, China); HiTrap Capto DEAE (5 mL), SuperdexTM 75 Increase 10/300 GL, and ÄKTA protein purification system were purchased from Cytiva (Washington, USA); and AFB1 was sourced from Sigma-Aldrich (Shanghai, China). All other reagents were at least of analytical purity.

#### *5.2. Isolation of AFB1-Degrading Strains*

Each of the 74 strains stored at −80 ◦C in the laboratory was inoculated into tubes containing 5 mL of LB liquid medium and incubated overnight at 37 ◦C on a shaker at 150 rpm. An inoculation loop was adopted to pick up a certain number of bacteria from the overnight culture of the strains to be tested on an LB agar gel plate for scribing. The strain was allowed to grow as a single colony for use, or the culture was purified if there were any stray colonies.

The purified strains to be tested were compared with the final concentration of 2.5 μg/mL AFB1 standard cultured in LB medium at 37 ◦C and 150 rpm for 24 h. The growth status of the strains in medium containing AFB1 was observed, and the strains that grew well were tested sequentially and at final concentrations of 5 and 10 μg/mL in AFB1 standard co-culture.

After initial screening, a laboratory strain labelled B10 grew well in AFB1 medium containing different concentrations of AFB1. All the components were incubated at 37 ◦C and 150 rpm for 12, 24, 48, and 96 h. The content of AFB1 was determined by high-performance liquid chromatography and ultraviolet (HPLC, UV) methods, and the degradation efficiency of strain B10 was calculated.

Chromatographic conditions: The chromatographic column was a C18 reverse adsorption column (4.6 mm × 250 mm, 5 μm), with an injection volume of 20 mL, a mobile phase of a 1:1 (*v*/*v*) water:methanol mixture, a flow rate of 1 mL/min, and a detection wavelength of 365 nm. The standard curve for the HPLC detection of AFB1 was *y* = 0.7735*x* + 0.1521, *R*<sup>2</sup> = 0.9975.

#### *5.3. Localisation of AFB1-Degrading Active Substances by Strain B10*

The degradation of active substances from the fermentation broth of strain B10 was localised in four fractions: 500 mL of fermentation broth was taken and centrifuged through a low-temperature high-speed centrifuge at 4 ◦C, 8000 rpm for 10 min. The separate of the supernatant of the fermentation broth was separated from the bacterial precipitation, and 3 mL was taken as the cell secretion group (extracellular fluid group). The bacterial sediment was resuspended with 20 mL of sterile PBS buffer; we centrifuged and poured some PBS and washed it thrice before resuspension in 20 mL of sterile PBS. Afterwards, 3 mL was taken as the live-cell group (bacterial suspension group). Of the bacterial suspension, 3 mL was placed in an autoclave at 121 ◦C for 30 min and allowed to cool before use (inactivation group). The remaining bacterial suspension was dosed with PMSF at a final concentration of 1%, crushed in a low-temperature ultrasonic cell crusher, then centrifuged at 4 ◦C, 13,000 rpm for 30 min. Each component was incubated with AFB1 standard at a final concentration of 2.5 μg/mL for 24 h in an incubator at 37 ◦C, and the degradation rate of each component against AFB1 was measured using HPLC.

#### *5.4. Crude Extraction of Protein from Culture Medium*

The B10 strain was inoculated in four vials of LB medium and incubated overnight at 37 ◦C. The supernatant was removed by high-speed low-temperature centrifugation, and ammonium sulfate was added to give final concentrations of 20, 40, 60, and 80%. After thorough mixing with a magnetic stirrer, the culture was kept at 4 ◦C overnight. The supernatant of each group was centrifuged at 13,000 rpm for 30 min at 4 ◦C. The supernatant was discarded, and the residue was dissolved in 10 mL of sterile PBS, transferred to a dialysis bag with a cut-off volume of 3.5 kDa, and dialysed in PBS buffer for 24 h at 4 ◦C.

One millilitre of crude protein solution of each component after dialysis was taken separately, filtered through a 0.22 μL needle filter, and incubated with 500 μL of AFB1 standard at a final concentration of 2.5 μg/mL for 24 h. The rate of degradation of each component was measured using HPLC.

#### *5.5. Isolation and Identification of AFB1-Degrading Proteins*

The crude extracted protein from the fermentation broth culture supernatant was concentrated and initially purified by passing through a DEAE ion-exchange column. The crude proteins were loaded onto a column pre-equilibrated with buffer (containing 20 mM/L Tris, 10 mM/L NaCl, pH 8.0) and eluted with a 0.01–2.0 M/L linear concentration gradient NaCl buffer at a flow rate of 1 mL/min. The eluate was collected separately according to the absorption peaks. Each component was incubated with 500 μL of AFB1 standard at a final concentration of 2.5 μg/mL for 24 h. The efficiency of each component in degrading AFB1 was obtained using HPLC.

The fraction with good degradation was purified by gel filtration chromatography. The solution to be measured was loaded onto a column pre-equilibrated with buffer (containing 20 mM/L Tris, 50 mM/L NaCl, pH 8.0) and eluted at a flow rate of 0.2 mL/min. The eluate was collected separately according to the absorption peaks. The degradation rate of each group was detected as described. The components with the best degradation effect were analysed by SDS-PAGE, and the proteins in the optimal components were identified by protein mass spectrometry.

#### *5.6. Structural Modelling of the Recombinant Laccase and Molecular Docking*

Through recombinant laccase amino acid sequencing using AlphaFold2 on the Beijing Supercomputing Cloud N22 partition with a full\_dbs preset [25], a total of five initial models and five models relaxed by the Amber relaxation procedure were generated, after which the highest-ranked conformation was selected for subsequent docking analysis based on the average plDDT ranking. The recombinant laccase's optimal structure was plotted in Python 3.8 using the RamachanDraw package for quality and poor contact assessment between residues.

As retrieved from AFB1 (PubChem CID: 186907), AFB2 (PubChem CID: 2724360), AFG1 (PubChem CID:133065469), AFG2 (PubChem CID: 2724362), AFM1 (PubChem CID: 15558498), and AFM2 (PubChem CID: 23318), six molecules were downloaded and saved in SDF format, semi-empirically optimized using MOPAC2016 (http://openmopac. net/, accessed on 15 November 2020) under the PM7 PRNT = 2 parameter. Optimized structures were prepared using the MGLTools 1.57 suite of prepare\_ligand.py which could be converted to pdbqt format to give the Gasteiger charge and retain the total hydrogen.

The prediction of the recombinant laccase Zn2+ binding site using the bioinformatics tool UniProt (EMBL-EBI, Cambridge, UK) was followed by use of the NCBI BLAST tool (National Center for Biotechnology Information, Bethesda, MD, USA). The PDB database was selected, the crystal structure of a purine nucleoside phosphorylase with 49.40% homology (PDB ID: 6T0Y) was retrieved, the two structures were fitted in pymol2.5, the template Zn2+ was retained, the amino acids in the Zn2+ binding site were adjusted, and the MGLTools 1.57 suite was used to create a recombinant laccase pdbqt file containing charge and H atoms.

Docking studies of the recombinant laccase were performed using watvina (https:// github.com/biocheming/watvina, accessed on 30 November 2021, Ximing Xu, Qingdao, China), a deeply optimized offshoot of vina, using the getbox plugin in pymol to generate docking boxes centred on the metal-binding site of the recombinant laccase. The docking box was centred at center\_x = 0.8, center\_y = −3.1, center\_z = −5.7; size\_x = 19.5, size\_y = 21.2, size\_z = 18.5, after which the molecule and recombinant laccase were docked using watvina, and the solvent interaction was approximated by using an implicit water model. The docking results were plotted using pymol.

#### *5.7. Gene Cloning, Protein Expression, and Purification of the Recombinant Laccase*

Strain B10 was cultured overnight in LB medium, and DNA was extracted from the strain using a DNA extraction kit. Amino acid sequence results obtained from protein mass spectrometry identification in 5.5 were aligned by NCBI BLAST. Cloning primers were designed based on the CDs region base sequences of the most similar proteins. The restriction enzymes BamH I and Xho I were selected, and the expression vector pET-28a was designed with fragment amplification primers as follows:

#### Lac-F: 5 CGGGATCCATGAATACATATCACCCGTTTAGTCTT3

#### Lac-R: 5 CCCTCGAGTTATGCCTCCTTCATTCCGATAAAG3 .

PCR reaction conditions consisted of denaturation at 98 ◦C for 10 s, annealing at 55 ◦C for 5 s, extension at 72 ◦C for 30 s, 34 cycles from denaturation to extension steps, and storage at 4 ◦C. PCR fragments were recovered using an agarose gel DNA extraction kit. The

double-cleaved B10 laccase gene fragment was inserted into the expression vector pET-28a, transformed into Trans5α chemically competent cells, coated onto LB medium containing 100 mg/mL kanamycin, and incubated overnight. Three positive clones were selected for sequencing, and the plasmids from the positive clones corresponding exactly to the B10 laccase gene were extracted and transformed into BL21 (DE3) chemically competent cells. Positive clones coated on LB medium containing 100 mg/mL kanamycin were inoculated in LB liquid medium. Expressed cells were collected by centrifugation and resuspended in sterile PBS, fragmented by ultrasonic cell disruptor, and purified by Ni Sepharose 6 FF affinity chromatography packing with His-tagged laccase in the supernatant. The purity of the recombinant laccase was determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and the protein concentration was detected by bicinchoninic acid (BCA) assay. BCA standard curve regression equation was *y* = 0.7618*x* + 0.1371.

#### *5.8. Efficiency of AFB1 Degradation by a Recombinant Laccase and Its Effect on AFB1-Degrading Activity under Different Conditions*

A recombinant laccase (20 μg/mL) was incubated with AFB1 standard at a final concentration of 2.5 μg/mL for 12, 24, 36, 48, 60, and 72 h at pH 7.2 and 37 ◦C. The samples were centrifuged in 1.5 mL centrifuge tubes at 13,000 rpm for 5 min at room temperature, and the supernatant was removed from the tubes. A total of 500 μL of the supernatant was transferred to a new 1.5 mL sterile centrifuge tube, and all mixed samples were withdrawn using a 1 mL syringe and filtered through a 0.22 μm organic phase needle filter. A total of 20 μL of the filtrate was injected into the HPLC detection system using a microsampling needle (the same treatment was applied to the control). The samples were assayed for AFB1 according to the AFB1 standard curve.

To evaluate the effects of different temperatures on the degradation of AFB1 by the recombinant laccase from strain B10, the recombinant laccase (20 μg/mL) and AFB1 standard at a final concentration of 2.5 μg/mL were dissolved in PBS buffer at pH 7.2 and incubated at 20, 30, 40, 50, 60, 70, and 80 ◦C for a total of 36 h.

To characterize the effects of different pH values on the degradation of AFB1 by recombinant laccase of strain B10, the recombinant laccase (20 μg/mL) and the AFB1 standard at a final concentration of 2.5 μg/mL were incubated at pH 4, 5, 6, 7, 8, 9, and 10 for 36 h at 37 ◦C.

To determine the effects of different metal ions on the degradation of AFB1 by the recombinant laccase from strain B10, the recombinant laccase (20 μg/mL) and 2.5 μg/mL of AFB1 standard were dissolved in PBS buffer at pH 7.2 and incubated for 36 h at 37 ◦C with 10 mM Na+, K+, Co2+, Fe3+, Mg2+, Cu2+, Ca2+, Mn2+, Zn2+, and Ni2+ for 36 h.

To estimate the effects of other conditions on the degradation of AFB1 by the recombinant laccase of strain B10, the recombinant laccase (20 μg/mL) and the AFB1 standard at a final concentration of 2.5 μg/mL were dissolved in PBS buffer at pH 7.2 and incubated for 36 h at 37 ◦C in 10 mM ethylenediamine tetraacetic acid (EDTA), sodium dodecyl sulfate (SDS), and dimethyl sulfoxide (DMSO), respectively.

The PBS buffer was incubated with AFB1 standard at a final concentration of 2.5 μg/mL for the same length of time as the control group. HPLC was used to assay all such samples, and the content of AFB1 was determined according to the AFB1 standard curve.

#### *5.9. Targeted Mutagenesis of the Recombinant Laccase*

The amino acid sequence of the recombinant laccase was blasted through the UniProt database and the PDB database to determine proteins with a high matching score for this laccase, and its metalloid sites were recorded as H87, C132, and H149. Targeted mutations were achieved by the Fast Mutagenesis kit according to the instructions supplied with the kit. Mutation primers were as follows:

#### H87-F: 5 CCAGACAgctGAAAACCGCGTCCGGCGCGTGA3

#### H87-R: 5 GGTTTTCagcTGTCTGGTCGGCGAACACCCAG3

#### C132-F: 5 TGTTTTGCGGACgctGTGCCCTTGTATTTTTATGACCCG3

#### C132-R: 5 ACagcGTCCGCAAAACAAAGGGCCAAAAAAAG3

#### H149-F: 5 TTATCGGCGCTGCCgctGCCGGATGGAAGGGGACG3

#### H149-R: 5 agcGGCAGCGCCGATAATGGATTTCACCGGGT3 .

The mutants were transformed into DH5α, and assays determined the correct sequence of the recombinant laccase mutant. The plasmid carrying the mutant was also transformed into *E. coli* BL21 receptor cells, and the purified mutant was expressed as described above, while its AFB1-degrading activity was determined.

**Author Contributions:** Methodology, M.L. and D.X.; software, D.X. and G.L.; validation, D.X., J.W. and T.L.; formal analysis, D.X. and J.W.; writing—original draft preparation, D.X.; writing—review and editing, M.L.; visualization, D.X. and G.L.; supervision, M.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the National Natural Science Foundation of China (Grant Nos. 31772809, 31972746, and 31872538) and through a Key Grant Project of Liaoning Provincial Department of Education (Grant No. LJKZ0632).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available in this article.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


### *Article* **The Antagonistic Effect of Glutamine on Zearalenone-Induced Apoptosis via PI3K/Akt Signaling Pathway in IPEC-J2 Cells**

**Tianhu Wang †, Jingjing Wang †, Tong Zhang, Aixin Gu, Jianping Li \* and Anshan Shan \***

Institute of Animal Nutrition, Northeast Agricultural University, Harbin 150030, China; wthqh123@163.com (T.W.); jf\_jing@163.com (J.W.); neauzt@outlook.com (T.Z.); aixingu@hotmail.com (A.G.) **\*** Correspondence: ljpneau@neau.edu.cn (J.L.); asshan@neau.edu.cn (A.S.); Tel.: +86-0451-5519-1439 (J.L.)

† These authors contributed equally to this work.

**Abstract:** Zearalenone (ZEN) is a non-steroidal estrogen mycotoxin produced by *Fusarium* fungi, which inevitably exists in human and animal food or feed. Previous studies indicated that apoptosis seems to be a key determinant of ZEN-induced toxicity. This experiment aimed to investigate the protective effects of Glutamine (Gln) on ZEN-induced cytotoxicity in IPEC-J2 cells. The experimental results showed that Gln was able to alleviate the decline of cell viability and reduce the production of reactive oxygen species and calcium (Ca2+) induced by ZEN. Meanwhile, the mRNA expression of antioxidant enzymes such as glutathione reductase, glutathione peroxidase, and catalase was up-regulated after Gln addition. Subsequently, Gln supplementation resulted in the nuclear fission and Bad-fluorescence distribution of apoptotic cells were weakened, and the mRNA expression and protein expression of pro-apoptotic genes and apoptotic rates were significantly reduced. Moreover, ZEN reduced the phosphorylation Akt, decreased the expression of Bcl-2, and increased the expression of Bax. Gln alleviated the above changes induced by ZEN and the antagonistic effects of Gln were disturbed by PI3K inhibitor (LY294002). To conclude, this study revealed that Gln exhibited significant protective effects on ZEN-induced apoptosis, and this effect may be attributed to the PI3K/Akt signaling pathway.

**Keywords:** zearalenone; glutamine; PI3K/Akt pathway; apoptosis; IPEC-J2 cells

**Key Contribution:** The addition of Gln (2 mM) alleviated the negative effects resulting from ZEN (160 μM) in IPEC-J2 cells. Gln (2 mM) exerts an antagonistic effect on apoptosis by activating PI3K/Akt signaling pathway.

#### **1. Introduction**

Zearalenone (ZEN), a mycotoxin produced by *Fusarium*, is one of the vital sources of food contamination. Its ubiquity in food and feed poses a threat to humans and animals health [1]. Studies have shown that after ingesting ZEN-contained foods, the toxic compound was absorbed through the gastrointestinal tract (GIT), metabolized, and distributed to different parts of the body [2]. Reports indicated that ZEN can induce hepatotoxicity, immunotoxicity, hematotoxicity, and genotoxicity, and lead to cell death by inducing oxidative stress, mitochondrial damage, and apoptosis [3–5]. Several toxicological models of ZEN's effects in the body and cells have been carried out in the past years. For instance, previous studies from this lab have shown that ZEN increased the levels of reactive oxygen species (ROS) and repressed the activity and expression of anti-oxidative enzymes in porcine kidney cells (PK15) or porcine intestinal epithelial cells (SIEC02), resulting in cell apoptosis [6,7]. However, little is known and it is worthy to further investigate ways to detoxify for ZEN-poisoned cells and organs.

Glutamine (Gln) is an α-amino acid and the most abundant free amino acid in the body [8]. As a precursor for nucleotide biosynthesis, Gln is one of the crucial substances for intestinal epithelial cell proliferation and integrity repair [9,10]. It was reported that

**Citation:** Wang, T.; Wang, J.; Zhang, T.; Gu, A.; Li, J.; Shan, A. The Antagonistic Effect of Glutamine on Zearalenone-Induced Apoptosis via PI3K/Akt Signaling Pathway in IPEC-J2 Cells. *Toxins* **2021**, *13*, 891. https://doi.org/10.3390/ toxins13120891

Received: 15 November 2021 Accepted: 10 December 2021 Published: 12 December 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

the dietary addition of Gln reduced weaning stress caused intestinal dysfunction by cell proliferation and increased expression of tight junction proteins in weaned pups [11,12]. Similarly, in vitro studies also indicated that Gln could promote the proliferation of intestinal porcine epithelial cell lines [13,14]. In addition, Gln was reported toxic protection effects on the intestinal damage and the intestinal epithelial cell apoptosis caused by clostridium *difficile* toxin-A in the rabbit model [15]. At present, the mechanism of Gln that protects cells from ZEN-induced apoptosis is rarely reported and needs further exploration.

The gastrointestinal tract is a multifunctional and complex organ [16]. It is not only an organ for digestion and absorption of nutrients, but the first barrier to protect animal health from ingested chemicals, food contaminants, and natural toxins. Furthermore, intestinal homeostasis depends on the diverse functions of intestinal epithelial cells [17,18]. Hence, porcine jejunal epithelial cells (IPEC-J2) were selected for the study.

It was hypothesized that Gln might protect the cells against ZEN-induced apoptotic, and it may work via PI3K/Akt signaling pathway. Therefore, the IPEC-J2 cell line was studied as a model to investigate the detoxification of Gln addition on ZEN-induced cells in this study.

#### **2. Results**

#### *2.1. Effects of ZEN and Gln on Cell Viability*

To determine the suitable concentration of Gln in subsequent experiments, the viability of the IPEC-J2 cells (Figure 1) was measured by the CCK-8 method at first. As shown in Figure 1, compared with the control group, exposure to 160 μM ZEN for 48 h, the cell viability was reduced significantly (*p* < 0.001). The addition of 2 mM Gln significantly increased the cell viability compared with the ZEN group (*p* < 0.001).

**Figure 1.** Effects of ZEN and Gln on the viability of the IPEC-J2 cells. Values are expressed as means ± SD of three independent experiments. \*\*\* *p* < 0.001 ZEN vs. control. ### *p* < 0.001 ZEN vs. ZEN + Gln 2.

#### *2.2. Effects of ZEN and Gln on the Activities of Enzymes*

When IPEC-J2 cells were exposed to ZEN and different concentrations of Gln for 48 h. As shown in Figure 2, the three enzyme activities (glutathione reductase (GR), glutathione peroxidase (GPx), and catalase (CAT)) decreased significantly upon exposure to ZEN compared with the control group (*p* < 0.05). Compared with the ZEN treatment, no differences were observed in the three enzyme activities at 0.5 mM Gln; however, the level of 1 and 2 mM Gln were showed significant increases (*p* < 0.05) in these three kinds of enzymes. Meanwhile, the concentration of 4 and 8 just observed improvements in one kind of enzyme (Gpx and CAT), respectively. Based on these data, a protective concentration of Gln (2 mM) was selected and incubated with ZEN for 48 h in subsequent experiments.

**Figure 2.** Effects of ZEN and Gln on the activities of the enzymes (GR, Gpx and CAT) in the IPEC-J2 cells. Values are expressed as means ± SD of three independent experiments. \* *p* < 0.05 and \*\*\* *p* < 0.001 ZEN vs. control. # *p* < 0.05, ## *p* < 0.01, and ### *p* < 0.001 ZEN vs. ZEN + Gln. (**A**) Effects of ZEN and Gln on the activity of GR; (**B**) Effects of ZEN and Gln on the activity of Gpx; (**C**) Effects of ZEN and Gln on the activity of CAT.

#### *2.3. Intracellular ROS Generation*

To determine changes in oxidative damage, IPEC-J2 cells were exposed to different drugs for 48 h. The ROS production results were obtained by the fluorescein assay (Figure 3). The figure showed that the level of ROS was significantly higher in the ZEN group than that in the control group (*p* < 0.001). Compared with the ZEN group, the intracellular ROS production was decreased significantly after Gln addition (*p* < 0.001).

**Figure 3.** Effects of ZEN and Gln on intracellular ROS production. Values are expressed as means ± SD of three independent experiments. \*\*\* *p* < 0.001 ZEN vs. control. ### *p* < 0.001 ZEN vs. ZEN + Gln.

#### *2.4. Intracellular Ca2+*

IPEC-J2 cells were incubated with Fluo-4 AM, bound to Ca2+ to produce strong fluorescence. As shown in Figure 4, compared with the control group, ZEN-induced levels of intracellular Ca2+ increased significantly (*p* < 0.001). Gln supplementation significantly reduced intracellular levels of Ca2+ in the ZEN-induced cells (*p* < 0.001).

**Figure 4.** Effects of ZEN and Gln on intracellular Ca2+ production. (**A**) Values are expressed as means <sup>±</sup> SD of three independent experiments. (**B**) Fluorescence microscopy observation of intracellular Ca2+ fluorescence intensity, Scale bar: 200 μm. \*\*\* *p* < 0.001 ZEN vs. control. ### *p* < 0.001 ZEN vs. ZEN + Gln.

#### *2.5. Immunofluorescence Staining of Cells*

The morphologic changes of apoptotic nuclei were observed by fluorescence microscopy with Hoechst-33258 staining (Figure 5). In control group cells, the nuclei displayed uniformly blue-stained with a smooth appearance. However, uneven nuclear staining, nuclear condensation, and fragmentation of nuclei were shown clearly in the ZEN group. In Comparison with the ZEN group, although Gln addition reduced nuclear shrinkage and rupture, pretreatment with LY294002 that did not reduce nuclear apoptosis.

**Figure 5.** Effects of ZEN, Gln, and LY294002 on the apoptotic nuclei (Hoechst 33258 staining). The red color frame in the figure indicates the obvious change area. The arrow represents a change in nuclear morphology. Scale bar: 200 μm.

#### *2.6. Apoptosis Rate in IPEC-J2 Cells*

The Annexin V/FITC/PI apoptosis kit was used to analyze different drugs effects on apoptosis of IPEC-J2 cells. As shown in Figure 6A, ZEN induced a significant increase in the number of early apoptotic cells (Q2), as well as in the number of late apoptotic cells (Q4) in IPEC-J2 cells. The total apoptotic cell proportion was increased by 56.8% (Figure 6B) compared with the control group. Compared with the ZEN group, Gln addition significantly reduced early apoptosis and late apoptosis, and the total apoptotic cell proportion (12.5%) was decreased by 44.3%. In addition, pretreatment with LY294002 significantly increased late apoptosis, and the total cell apoptotic rate (31.6%) was increased by 19.1% compared with the ZEN + Gln group. This result was consistent with the results of nuclear apoptosis staining.

**Figure 6.** (**A**) The apoptotic cells were determined by annexin V-FITC/PI staining using flow cytometry. The Q1, Q2, Q3, and Q4, respectively, represented dead cells, the late cells apoptosis, normal cells, and the early cells apoptosis. Apoptosis was the sum of early apoptosis and late apoptosis. (**B**) The percentage of IPEC-J2 cells apoptosis was shown in statistical analysis. Each value represents the mean ± SD of the three independent experiments. \*\*\* *p* < 0.001 ZEN vs. control, ### *p* < 0.001 ZEN vs. ZEN + Gln. ∧∧ *p* < 0.01 ZEN + Gln vs. ZEN + Gln + LY294002.

#### *2.7. The mRNA Expression of Apoptosis-Related Genes*

To further investigate the effects of these drugs on cell apoptotic, the mRNA expression of apoptosis-related genes was measured. As shown in Figure 7, compared with the control group, ZEN induced a significant increase in the mRNA expression levels of pro-apoptotic genes: Caspase-3, Caspase-9, Cytochrome c (Cyto-c), and Bad (*p* < 0.05). Conversely, the mRNA expression of anti-apoptosis genes (Bcl-xl and Bcl-2) was significantly reduced (*p* < 0.001). After Gln addition, the mRNA expression of five pro-apoptotic genes (Caspase-3, Caspase-9, Cyto-c, Bax and Bad) were significantly down-regulated (*p* < 0.001), anti-apoptotic genes (Bcl-xl and Bcl-2) were significantly up-regulated (*p* < 0.05). Compared with the ZEN + Gln group, pretreatment with LY294002, the mRNA expression of three pro-apoptotic genes (Caspase-3, Caspase-9, and Bax) were increased significantly (*p* < 0.05) and had no significant effect on anti-apoptotic genes.

**Figure 7.** Effects of ZEN, Gln and LY294002 on the apoptosis-related genes in the IPEC-J2 cells. Values are expressed as means ± SD of three independent experiments. \* *p* < 0.05, \*\* *p* < 0.01, and \*\*\* *p* < 0.001 ZEN vs. control. # *p* < 0.05 and ### *p* < 0.001 ZEN vs. ZEN + Gln. ∧ *p* < 0.05 ZEN + Gln vs. ZEN + Gln + LY294002. (**A**) Effects of ZEN, Gln and LY294002 on the mRNA expression of Caspase-3; (**B**) Effects of ZEN, Gln and LY294002 on the mRNA expression of Caspase-9; (**C**) Effects of ZEN, Gln and LY294002 on the mRNA expression of Cyto-c; (**D**) Effects of ZEN, Gln and LY294002 on the mRNA expression of Bax; (**E**) Effects of ZEN, Gln and LY294002 on the mRNA expression of Bcl-2; (**F**) Effects of ZEN, Gln and LY294002 on the mRNA expression of Bcl-xl; (**G**) Effects of ZEN, Gln and LY294002 on the mRNA expression of Bad.

#### *2.8. Immunofluorescence*

The Bad protein is involved in initiating apoptosis. Next, we investigated the expression of Bad by immunofluorescence. As displayed in Figure 8, in the control group, the fluorescence of Bad was very weak. While in the ZEN group, Bad fluorescence expression was strongly positive, distributed around the nucleus in a spotted manner and decreased number of IPEC-J2 cells. In the Gln + ZEN group, the immunostaining of Bad was relatively weaker than the ZEN group, and there was no distribution pattern of aggregated spots. Moreover, in the LY294002 pretreatment group, the immunostaining of Bad was relatively stronger than that in the ZEN + Gln group.

**Figure 8.** Expression of Bad in IPEC-J2 cells. Cells were stained with antibodies for Bad and detected by immunofluorescence after treatment. The images were collected by the nuclei showed blue fluorescence after counterstaining with DAPI. The red color frame in the figure indicates the obvious change area. Scale bar: 200 μm.

#### *2.9. Western Blotting*

To further verify that the PI3K/Akt signaling pathway is the mechanism by which Gln protects against ZEN-induced apoptosis, the related proteins of the PI3K/Akt signaling pathway and apoptosis-related proteins were measured by western blotting. As shown in Figure 9, there was no difference in the protein expression of Akt in the four treatment groups (Figure 9D). Compared with the control group, after ZEN-exposed, the protein expression of pro-apoptotic gene Bax and anti-apoptotic gene Bcl-2 were significantly increased (*p* < 0.01) and significantly decreased (*p* < 0.001), respectively. The addition of Gln significantly decreased the protein expression of Bax compared with the ZEN group (*p* < 0.05), conversely, the protein expression of Bcl-2 was significantly elevated (*p* < 0.05). Compared with the ZEN + Gln group, the pretreatment of LY294002 significantly decreased the protein expression of Bcl-2 (*p* < 0.01), and there was no significant change in Bax protein expression. Also, as shown in Figure 9E, after ZEN-exposed, a remarkable decrease in the protein expression of the p-Akt compared with the control group, the addition of Gln increased the protein expression of p-Akt but did not reach a significant level. In addition, pretreatment with LY294002 significantly reduced p-Akt protein expression.

**Figure 9.** Effects of ZEN, Gln and LY294002 on the PI3K/Akt pathway- and apoptosis-related genes in the IPEC-J2 cells. Values are expressed as means ± SD of three independent experiments. \*\* *p* < 0.01 and \*\*\* *p* < 0.001 ZEN vs. control. # *p* < 0.05 ZEN vs. ZEN + Gln. ∧ *p* < 0.05 and ∧∧ *p* < 0.01 ZEN + Gln vs. ZEN + Gln + LY294002. (**A**) Effects of ZEN, Gln and LY294002 on the protein expression of PI3K/Akt pathway; (**B**) Effects of ZEN, Gln and LY294002 on the protein expression of Bax; (**C**) Effects of ZEN, Gln and LY294002 on the protein expression of Bcl-2; (**D**) Effects of ZEN, Gln and LY294002 on the protein expression of Akt; (**E**) Effects of ZEN, Gln and LY294002 on the protein expression of p-Akt.

#### **3. Discussion**

ZEN is widely found in cereals and animal feed worldwide, which has a negative impact on human and animal health [4,19,20]. Intestinal epithelial cells are the first target of ZEN after ingestion of feed and foods contaminated with ZEN [21]. In vitro and in vivo studies found that oxidative damage was one of the crucial pathways by which ZEN induced cytotoxicity, resulting in cell apoptosis [22,23]. Oxidative damage is mainly caused by the mass production of ROS and free radicals [24]. Oxidative stress is caused by the excessive generation of ROS or the disruption of the oxidoreductase balance in the cell. It not only activated cell signaling but also induced apoptosis [25]. As described above, the results of this study found that ZEN exposure produced excess ROS (Figure 3), numerous uneven nuclear staining, nuclear fissures, mass cells apoptosis (Figure 6). To date, these antioxidant enzymes, comprising CAT, Gpx and GR, etc., are a source of protection against oxidative stress [26,27]. Overall, cells exposed to ZEN induced oxidative damage and reduced the intracellular antioxidant enzyme activities (Figure 2).

Gln, a major substrate utilized by intestinal cells, is not only a source of the main energy of the cell mitochondria, but it can eliminate some of the strong oxidants and protect cells from oxidative damage [28,29]. In gut physiology, the addition of Gln can promote enterocyte proliferation and protect against apoptosis under stress conditions [30]. Therefore, Gln was used to investigate the protective mechanism against ZEN-induced apoptosis in this study. It was observed that the addition of Gln increased cell survival (Figure 1), reduced nuclear shrinkage (Figure 5), and decreased apoptosis rate (Figure 6). The results showed Gln alleviated the apoptosis induced by ZEN. At the same time, the activities of antioxidant enzymes in the cells also increased, and the effect was the best when Gln concentration was 2 mM (Figure 2). Hence, 2 mM Gln was selected as the concentration for subsequent verification. Compared with 2mM Gln, the protective effect of Gln (4 and 8 mM) is weaker. The reason may be consistent with Curi's conclusion that although Gln supplementation can bring obvious benefits in many cases, the adverse effects of long-term use of high concentrations of glutamic acid might not be completely ruled out [31]. Further, the results of pretreatment with LY294002 that did not reduce nuclear apoptosis are consistent with earlier studies because it has cytostatic, but no cytotoxicity effects on cells [32–34].

ZEN exposure can induce IPEC-J2 cells apoptosis by mitochondrial damage [6,7,35]. The literature demonstrated that mitochondria-dependent apoptotic pathways involved a variety of events, such as the production of ROS, the release of Cyto-c in mitochondria, Bcl-2 family members, and activation of caspases-9 and caspases-3 [36]. We found that ZEN induced apoptosis via the mitochondrial pathway of IPEC-J2 cells. The expression of Cyto-c, caspases-9, caspases-3, and pro-apoptotic genes (Bax and Bad) were increased, while anti-apoptotic genes (Bcl-2 and Bcl-xl) were reduced (Figures 7 and 9B,C). In addition, mitochondria are a storage room for intracellular calcium [37]. Recently, with the in-depth discussion of the apoptotic process, it was suggested that intracellular Ca2+ and ROS surges were vital mediators of cell death [38,39]. The current study showed that ZEN exposure increased intracellular ROS and Ca2+ levels (Figures 3 and 4). Oppositely, the addition of Gln reduced the content of ROS and Ca2+ in cells (Figures 3 and 4). These results were consistent with the present study. Overall, Gln improved cell survival rate and protected cells from ZEN-induced mitochondrial apoptosis.

The PI3K/Akt pathway is an important regulator of cellular homeostasis in vivo [40,41]. In addition, it is a vital anti-apoptosis/proliferation signaling pathway that plays a key role in cellular functioning [42,43]. Recently, it was found that Gln increased the antioxidant capacity by activating PI3K/Akt signaling pathway in Parkinson's disease [44]. Phosphatidylinositol-3 kinases (PI3Ks) are a family of lipid kinases that regulate various metabolic activities in the cell [45]. Activated Akt is a downstream effector of PI3K, which can inhibit apoptosis by regulating multiple targets such as MPTP, ATPase, NF-κB, and the Bcl-2 family proteins [46,47]. We speculated that Gln could exert the effect of anti-apoptosis via PI3K/Akt signaling pathway and improve cell survival. The Bad protein is a downstream substrate of Akt, Akt-phosphorylate (p-Akt) can activate it [48]. In the presence of survival factors, the Bad protein is phosphorylated at two serine sites (Ser-112 and Ser-136) and sequestered in the form of inactive molecules in the cytosol, receiving the death signal, Bad dephosphorylates and interacts with Bcl-xl–Bcl-2 to form dimers that accumulate in mitochondria [49,50]. Therefore, to examine the position of Bad in the case of cell survival and death, immunofluorescence staining was used to observe the fluorescence change of the Bad gene. The results clearly showed that the strong accumulation of Bad gene fluorescence occurred in ZEN-induced cell apoptosis (Figure 8), the mRNA expression of Bad was increased simultaneously. However, there was no accumulation of Bad gene fluorescence occurred, but the mRNA expression of Bad was decreased (Figure 7G), and decreased cell apoptotic rate (Figure 6) when treated with Gln. Importantly, after pretreatment with inhibitor (LY294002) of the PI3K/Akt signaling pathway, the Bad gene strong accumulation of fluorescence occurred (Figure 8). This result suggested that Gln may play

an anti-apoptotic effect via the PI3K/Akt signaling pathway. The same result was found in primary liver cancer cells increases of Bad-expressing caused by Akt-knocked [43].

The literature suggested that the activation of the PI3K/Akt signaling pathway could suppress apoptosis [46]. Western blotting results in this study showed that Gln increased the expression of p-Akt protein and anti-apoptotic protein Bcl-2 (Figure 9C,E), these results were consistent with the activation of the PI3K/Akt pathway leading to increased expression of Bcl-2 [51]. Our results also showed that Gln treatment stimulated phosphorylation of Akt, and reduced the apoptosis rates (Figure 6), consistent with a previous study showing that growth factor receptor Akt activation prevented apoptosis [52]. Thus, the addition of Gln activated the PI3K/Akt signaling pathway and protected cells from ZEN-induced apoptosis. Meanwhile, pretreatment with LY294002 reduced p-Akt protein expression, suggesting that the anti-apoptotic pathway of PI3K/Akt was inhibited. This result was supported by an early study that LY294002 inhibited the PI3K/Akt signaling pathway and significantly enhanced bufalin-induced apoptosis [53]. As described above, these results suggested that Gln protected cells from ZEN-induced apoptosis, and activation of the PI3K/Akt signaling pathway was one of the factors.

#### **4. Conclusions**

In conclusion, ZEN exposure induced the excessive generation of ROS, increased intracellular Ca2+ concentration, induced oxidative damage, and activated the intrinsic apoptotic cascade reaction in IPEC-J2 cells. However, Gln addition increased the activities of intracellular antioxidant enzymes, increased the expression of anti-apoptotic genes and p-Akt, reduced the expression of pro-apoptotic genes and caspase cascade enzymes. Overall, these findings suggested that Gln antagonized ZEN-induced apoptosis, possibly via the PI3K/Akt signaling pathway in IPEC-J2 cells.

#### **5. Materials and Methods**

#### *5.1. Chemicals and Reagents*

The Zearalenone (ZEN) and Glutamine (Gln) were obtained from Sigma-Aldrich (St. Louis, MO, USA) and were dissolved in ethanol and deionized water to a stock solution of 100 mM and 200 mM, respectively. DMEM-F:12 cell culture medium was purchased from Thermo Fisher (Hyclone, Beijing, China). Fetal bovine serum (FBS) was supplied by Gibco-Life Technology (Grand Island, NY, USA). Trypsin/EDTA, Penicillin/streptomycin, the activity assay kits of Catalase (CAT), Glutathione reductase (GR), Glutathione peroxidase (Gpx), and Alkaline phosphatase (AP), ECL detection kit, the Annexin V-FITC/PI apoptosis detection kit, Hoechst-33258 Staining Kit, Reactive Oxygen Species Assay Kit, Fluo-4 AM and BCA Assay Kit were supplied by Beyotime Biotechnology (Nantong, China). Cell Counting Kit-8 (CCK-8) was supplied by Dojindo (Kumamoto, Japan). Phosphate buffered saline (PBS) was purchased from Biotopped (Beijing, China).

#### *5.2. Cell and Cell Culture*

IPEC-J2 cells were donated by China Agricultural University. The cells were cultured in a complete medium composed of DMEM-F:12 medium (Hyclone, Beijing, China), 10% FBS (GIBCO, Grand Island, NY, USA), and 1% penicillin and streptomycin (Beyotime Biotechnology, Nantong, China). Cells were cultured in an incubator at 37 ◦C, with a continual supply of 5% CO2.

#### *5.3. Cell Viability Assay*

Based on our previous findings (not yet published), we selected ZEN (160 μM) to infect IPEC-J2 cells. IPEC-J2 cells (0.8–1.0 × <sup>10</sup><sup>5</sup> cells/mL) were seeded in 96-well culture plates; culture medium was changed every 24 h. When cells became monolayer, cells were washed twice with PBS, then treated with ZEN (160 μM) and different concentrations of Gln (0.5, 1, 2, 4, and 8 mM) for 48 h. IPEC-J2 cells were washed three times with PBS after the cell culture medium was removed, then CCK-8 (10 μL) was added and incubated at 37 ◦C for

3 h. Cell viability was measured by absorbance on a microplate reader at 450 nm emission wavelength (Tecan Austria GmbH Untersbergatr, Austria). The microplate readers used in this study were from one manufacturer.

#### *5.4. Determination of IPEC-J2 Cellular the Activities of Enzymes*

IPEC-J2 cells (2.0–2.5 × 106 cells/mL) were grown in 6-well culture plates and treated with drugs as described in the previous section. The CAT, GR, and Gpx activities were determined according to the manufacturer's instructions.

#### *5.5. Detection of ROS Generation*

Changes in intracellular ROS was detected with dichlorofluorescein diacetate (DCFH-DA). IPEC-J2 cells (4.0–5.0 × <sup>10</sup><sup>5</sup> cells/mL) were grown in 24-well culture plates and treated with drugs as described in the previous section. After treatment, cells were washed thrice with PBS and incubated with 10 μM DCFH-DA at 37 ◦C for 20 min. Finally, cells were washed thrice with PBS and left a small amount of PBS. Intracellular production of ROS was measured by a microplate reader (Ex = 488 nm and Em = 525 nm). ROS production was expressed as a percentage of the control.

#### *5.6. Measurement of Intracellular Calcium (Ca2+) Levels*

Changes in intracellular Ca2+ were detected by using the intracellular Ca2+ indicator Fluo-4 AM. Fluo-4 AM, an acetyl methyl ester derivative of Fluo-4, can penetrate cell membranes. Upon entering the cell, Fluo-4 AM can be cleaved by intracellular esterase to form Fluo-4, which retain in the cell. After treatment, cells were incubated with Fluo-4 AM (1 μM) at 37 ◦C for 30 min. Intracellular Ca2+ was measured by microplate reader (Ex = 488 nm and Em = 520 nm). Ca2+ images were obtained using a fluorescence microscope (Life Technologies Crop Bothell, Bothell, WA, USA).

#### *5.7. Hoechst-33258 Staining*

After treatment, cells were washed 2–3 times with PBS and added 200 μL Hoechst-33258 at room temperature for 3–5 min in the dark. Lastly, aspirated Hoechst-33258 staining solution and washed with PBS 2–3 times, 3–5 min each time. The stained cells were visualized and photographed under a fluorescence microscope (Life Technologies Crop Bothell, Bothell, WA, USA).

#### *5.8. Apoptosis Detection*

According to the manufacturer's protocol, the apoptosis rate of IPEC-J2 cells was measured by the Annexin V-FITC/PI apoptosis detection kit. After treatment, the cells were rinsed 2–3 times using PBS, trypsinized, and collected. Next, cells were resuspended in 195 μL of binding buffer. Then, 5 μL of Annexin V- FITC and 10 μL of propidium iodide (PI) were added to the tubes and gently vortexed. Lastly, the cells were analyzed by flow cytometry (Becton, Dickinson and Company, Franklin Lakes, NJ, USA).

#### *5.9. Realtime PCR (RT-PCR) Assay*

TRIzol reagent (Invitrogen, Shanghai, China) was used to isolate Total RNA from the cells. The concentration of RNA (A260/A280 ratio) was measured by using a Nano Photometer P-Class (IMPLEN, München, Germany). Reverse transcription of 5 μL RNA was performed using the PrimeScript™ RT reagent kit and the concentration of total RNA was 300 ng μL−1. SYBR Green I RT-PCR kit (Takara, Dalian, China) was performed in a reaction volume of 10 μL using RT-PCR. Relative quantification of gene expression was calculated using the 2−ΔΔCt method and normalized to GAPDH in each sample. The gene-specific primers are shown in Table 1.


#### **Table 1.** Primers used for RT-PCR.

#### *5.10. Western Blotting Analyses*

The density of each protein was detected by BCA Assay kit. Total protein was loaded onto 6–15% SDS–PAGE gel electrophoresis, separated by electrophoresis, and then was transferred to PVDF membranes. The membranes were blocked with 5% BSA in TBST at room temperature for 2 h, and probed with the indicated primary antibodies: Akt (1:2000, Sangon Biotech, Shanghai, China), p-Akt (1:1000, Cell Signaling Technology, Danvers, MA, USA), Bax, and Bcl-2 (1:1000, Beyotime Institute of Biotechnology, Nantong, China) at 4 ◦C overnight. Then, the members were washed in TBST three times, incubated with goat antirabbit/mouse secondary antibodies (1:1000; Beyotime Institute of Biotechnology, Nantong, China) at room temperature for 2 h and visualized using ECL Plus detection system (P1010, Applygen, Beijing, China). The density of the bands was analyzed using Image J software (National Institutes of Health, Bethesda, Rockville, MD, USA) and normalized to GAPDH.

#### *5.11. Immunofluorescence Staining of Cells*

IPEC-J2 cells were seeded in a polylysine-coated confocal dish (2.0–2.5 × 106 cells/mL). After treatment, the cells were washed with PBS three times, fixed with 4% polyoxymethylene for 30 min, and 0.2% Triton X-100 for 10 min. Subsequently, 2% BSA-PBS was added dropwise and blocked for 60 min at 37 ◦C. Cells were stained with primary rabbit anti-Bad (1:1000, Abcam, Cambridge, UK) antibody for one night at 4 ◦C, followed by incubation with Alexa Fluor 555-conjugated anti-rabbit secondary antibody (1:200; Beyotime Institute of Biotechnology, Nantong, China) for 120 min at 37 ◦C. Cell nuclei were stained with DAPI (Beyotime Institute of Biotechnology, Nantong, China) for 30 s at room temperature in the dark. 100 μL of PBS was added dropwise and photographed under a fluorescence microscope (Life Technologies Crop Bothell, Bothell, WA, USA).

#### *5.12. Statistical Analyses*

Data of three independent experiments were expressed as means ± Standard Deviation (SD) and performed using GraphPad Prime 6.0 software (GraphPad Software, Inc, CA, USA). Statistical figures were analyzed using SPSS 19.0 software (SPSS Inc, Chicago, IL, USA). All the experimental data were analyzed for variance uniformity, then analyzed by a one-way ANOVA and groups were compared using LSD's test. A *p*-value less than 0.05 was considered to indicate statistical significance.

**Author Contributions:** Conceptualization, J.L.; methodology, T.W. and J.W.; software, A.G. and T.Z.; validation, T.W., J.W., and T.Z.; formal analysis, T.W. and J.W.; investigation, J.L.; data curation, T.W. and T.Z.; writing—original draft preparation, T.W. and J.W.; writing—review and editing, J.W.; supervision, J.L. and A.S.; project administration, J.L.; funding acquisition, J.L. and A.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the Natural Science Foundation of Heilongjiang Province of China (LC2018007) and the National Key R&D Program (2016YFD0501207).

**Data Availability Statement:** The data presented in this study are openly available in this article.

**Conflicts of Interest:** The authors have declared that no competing interest exist.

#### **References**


### *Article* **Comparison of Ameliorative Effects between Probiotic and Biodegradable** *Bacillus subtilis* **on Zearalenone Toxicosis in Gilts**

**Wenqiang Shen 1,†, Yaojun Liu 1,†, Xinyue Zhang 1, Xiong Zhang 1, Xiaoping Rong 1, Lihong Zhao 1, Cheng Ji 1, Yuanpei Lei 1, Fengjuan Li 2, Jing Chen <sup>2</sup> and Qiugang Ma 1,\***


**Abstract:** This study was conducted to compare the potential ameliorative effects between probiotic *Bacillus subtilis* and biodegradable *Bacillus subtilis* on zearalenone (ZEN) toxicosis in gilts. Thirty-six Landrace×Yorkshire gilts (average BW = 64 kg) were randomly divided into four groups: (1) Normal control diet group (NC) fed the basal diet containing few ZEN (17.5 μg/kg); (2) ZEN contaminated group (ZC) fed the contaminated diet containing an exceeded limit dose of ZEN (about 300 μg/kg); (3) Probiotic agent group (PB) fed the ZC diet with added 5 <sup>×</sup> <sup>10</sup><sup>9</sup> CFU/kg of probiotic *Bacillus subtilis* ANSB010; (4) Biodegradable agent group (DA) fed the ZC diet with added 5 <sup>×</sup> <sup>10</sup><sup>9</sup> CFU/kg of biodegradable *Bacillus subtilis* ANSB01G. Results showed that *Bacillus subtilis* ANSB010 and ANSB01G isolated from broiler intestinal chyme had similar inhibitory activities against common pathogenic bacteria. In addition, the feed conversion ratio and the vulva size in DA group were significantly lower than ZC group (*p* < 0.05). The levels of IgG, IgM, IL-2 and TNFα in the ZC group were significantly higher than PB and DA groups (*p* < 0.05). The levels of estradiol and prolactin in the ZC group was significantly higher than those of the NC and DA groups (*p* < 0.05). Additionally, the residual ZEN in the feces of the ZC and PB groups were higher than those of the NC and DA groups (*p* < 0.05). In summary, the ZEN-contaminated diet had a damaging impact on growth performance, plasma immune function and hormone secretion of gilts. Although probiotic and biodegradable *Bacillus subtilis* have similar antimicrobial capacities, only biodegradable *Bacillus subtilis* could eliminate these negative effects through its biodegradable property to ZEN.

**Keywords:** zearalenone; degradable *Bacillus subtilis*; probiotic *Bacillus subtilis*; gilts

**Key Contribution:** The alleviating effects of biodegradable *Bacillus subtilis* ANSB01G on the ZENpoisoned gilts were compared to probiotic *Bacillus subtilis* ANSB010, which was isolated from the same source and with similar bacteriostatic activity. Results showed that the improvement of ANSB01G on ZEN-poisoned gilts comes from its biodegradation activity to ZEN; not from its antibacterial activity.

#### **1. Introduction**

Zearalenone (ZEN), known as an F-2 mycotoxin, is a powerful estrogenic metabolite produced by certain species of *Fusarium* and *Erysipelas* spp. [1]. Several results showed that many feedstuffs for animals have been seriously contaminated with ZEN around the world [2,3], which could cause hyperestrogenism and fertility disorder in sows [4]. Moreover, a previous study found that exposure of post-weaning gilts to ZEN could increase the oxidative stress and had a negative impact on genital organs [5]. In addition,

**Citation:** Shen, W.; Liu, Y.; Zhang, X.; Zhang, X.; Rong, X.; Zhao, L.; Ji, C.; Lei, Y.; Li, F.; Chen, J.; et al. Comparison of Ameliorative Effects between Probiotic and Biodegradable *Bacillus subtilis* on Zearalenone Toxicosis in Gilts. *Toxins* **2021**, *13*, 882. https://doi.org/10.3390/ toxins13120882

Received: 16 November 2021 Accepted: 8 December 2021 Published: 10 December 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

recent study found that ZEN can interfere with immune mediators at the spleen level and induce an intense inflammatory response [6]. Therefore, it is critical to find the appropriate and effective detoxifying strategies to prevent contamination by ZEN in animal husbandry.

Numerous researches have reported that physical, chemical and biological methods can be used to counter mycotoxicosis [7–9]. However, most of these methods are impractical or potentially unsafe because of losses in the nutritional value, high equipment costs and formation of toxic residues or derivatives [10]. Biodegradation is eco-friendly and highly efficient in minimizing the harmfulness of mycotoxins in feeds [11,12]. Previous work from our laboratory reported that *Bacillus subtilis* ANSB01G, which has both a biodegradable effect against ZEN and probiotic activities against pathogenic bacteria, can alleviate toxicosis of ZEN in pre-pubertal female gilts [13,14]. However, the article did not clarify whether the reduced toxicity was due to its probiotic or biodegradable properties.

Therefore, the aim of this study was to investigate the effects of biodegradable and probiotic *Bacillus subtilis* on growth performance, serum biochemical indexes and hormone, serum antioxidant, immune indicators and mycotoxin residue in gilts exposed to ZEN for 25 d in vivo, as well as the inhibitory activity of common harmful bacteria in vitro.

#### **2. Results**

#### *2.1. Biochemical and Physiological Characteristics of Bacillus subtilis ANSB010 and ANSB01G*

The colony morphologies showed that the surfaces of *Bacillus subtilis* ANSB010 and ANSB01G colonies are rough, opaque and milky white (Figure 1A,B). Under the microscope, the cells were found to be short, thin rods, positive for Gram staining and capable of forming spores (Figure 1C,D). As shown in Table 1, the physiological and biochemical results revealed ANSB010 and ANSB01G had typical characteristics of *Bacillus* spp., such as growing well at 37 ◦C but not at 10 and 55 ◦C; and being able to utilize cellulose, glucose and maltose as the only carbon source. Moreover, a phylogenetic tree based on 16s rDNA sequences suggested that both ANSB010 and ANSB01G have a close evolutionary relationship to *Bacillus subtilis* (Figure 1E).

**Figure 1.** Colony characteristics of ANSB010 (**A**) and ANSB01G (**B**); cell (**left**) and spore (**right**) morphology of ANSB010 (**C**) and ANSB01G (**D**), scale bar, 100 μm; (**E**) the phylogenetic tree of *Bacillus subtilis* ANSB010 and ANSB01G, the GenBank accession numbers of sequences are shown in round brackets.


**Table 1.** Biochemical and physiological characteristics of *Bacillus subtilis* ANSB010 and ANSB01G.

<sup>1</sup> '+', '−' and 'w' mean positive, negative and weak response, respectively. <sup>2</sup> The results of ANSB01G after domestication were consistent with Lei et al. [13].

#### *2.2. Bacteriostatic and ZEN-Degrading Effects of ANSB010 and ANSB01G*

As shown in Figure 2 and Table 2, probiotic *Bacillus subtilis* ANSB010 and biodegradable *Bacillus subtilis* ANSB01G have a visible antibacterial effect on *Escherichia coli* (*E. coli*), *Salmonella choleraesuis* (*S. choleraesuis*) and *Staphylococcus aureus* (*S. aureus*) compared to the control group (Con) (*p* < 0.05, Table 2), while there was no significant difference in the antibacterial effect between ANSB010 and ANSB01G (*p* > 0.05, Table 2). Importantly, we noticed that ANSB01G could degrade 65.13%, 92.57% and 100.00% of ZEN in the fermentation broth at 6 h, 24 h and 48 h, respectively, but ANSB010 could not (*p* < 0.05, Table 3). Additionally, Supplementary Material Figures S1 and S2 show the representative chromatograms of degradation tests.

**Figure 2.** The antibacterial effects of probiotic *Bacillus subtilis* ANSB010 and biodegradable *Bacillus subtilis* ANSB01G on *E. coli* (**A**), *S. choleraesuis* (**B**) and *S. aureus* (C). *E. coli*, *Escherichia coli*; *S. choleraesuis*, *Salmonella choleraesuis*; *S. aureus*, Staphylococcus aureus. Con: MRS medium; ANSB010, probiotic *Bacillus subtilis* ANSB010; ANSB01G, biodegradable *Bacillus subtilis* ANSB01G.


**Table 2.** Antibacterial effects of *Bacillus subtilis* ANSB010 and ANSB01G.

Con: MRS medium; ANSB010, probiotic *Bacillus subtilis* ANSB010; ANSB01G, biodegradable *Bacillus subtilis* ANSB01G. Different superscript letters represent significant difference.

**Table 3.** The degradation rate (%) of ANSB010 and ANSB01G on zearalenone in fermentation medium.


ANSB010, probiotic *Bacillus subtilis* ANSB010; ANSB01G, biodegradable *Bacillus subtilis* ANSB01G. Different superscript letters represent significant difference.

#### *2.3. Growing Performance*

As shown in Table 4, no significant difference were observed for the initial weight, terminal weight and average daily gain (ADG) (*p* > 0.05), while there was a decreasing trend of average daily feed intake (ADFI) in probiotic *Bacillus subtilis* ANSB010 agent (PB) and biodegradable *Bacillus subtilis* ANSB01G agent (DA) groups (*p* = 0.10) compared to ZEN contaminated (ZC) group. Of the four groups, the ZC group had the highest feed conversion ratio (F/G) value (*p* < 0.05), and was 1.07- and 1.13-fold higher than the PB and DA group, respectively.



ADG, average daily gain; ADFI, average daily feed intake; F/G, feed conversion ratio; NC, normal control diet group; ZC, Zearalenone (ZEN)-contaminated group; PB, probiotic *Bacillus subtilis* group; DA, biodegradable *Bacillus subtilis* group. Different superscript letters represent significant difference.

#### *2.4. Vulva Size*

The effects of the four diets on the vulva size are shown in Table 5. The vaginal length and area of the DA group was significantly lower compared to the ZC group (*p* < 0.05), and there was no significant difference between the ZC and PB groups (*p* > 0.05). Interestingly, there was a decreasing trend of vaginal width among four groups (*p* = 0.10). The vaginal volume of the DA group was dramatically lower compared to all other groups (*p* < 0.05), and that of the PB group was significantly lower than that of the ZC group (*p* < 0.05), but no significant difference existed between the ZC and normal control diet (NC) groups, or PB and NC groups (*p* > 0.05). There was no significant difference in the vaginal height among the four groups (*p* > 0.05).

#### *2.5. Serum Biochemical Indicators, Antioxidant and Immunology Parameters*

There was no significant difference in the serum biochemical indicators (e.g., total protein (TP), albumin (ALB), alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatinine (CRE) and urea nitrogen (BUN)) among these groups (Figure 3A–G, *p* > 0.05), nor in the levels of glutathione peroxidase (GSH-Px) (Figure 4A, *p* > 0.05) and malondialdehyde (MDA) (Figure 4C, *p* > 0.05), which were

indicators of antioxidant activities. Interestingly, the level of superoxide dismutase (SOD) in the PB group was significantly lower than that of the ZC group (Figure 4B, *p* < 0.05).

**Table 5.** The values of vulva indexes in different treatment groups.


NC, normal control diet group; ZC, ZEN-contaminated group; PB, probiotic *Bacillus subtilis* group; DA, biodegradable *Bacillus subtilis* group. Different superscript letters represent significant difference.

**Figure 3.** Comparison of serum levels of biochemical indicators among different treatment groups. (**A**) TP, total protein; (**B**) ALB, albumin; (**C**) ALP, alkaline phosphatase; (**D**) AST, aspartate aminotransferase; (**E**) ALT, alanine aminotransferase; (**F**) CRE, creatinine; (**G**) BUN, urea nitrogen. NC, normal control diet group; ZC, ZEN-contaminated group; PB, probiotic *Bacillus subtilis* group; DA, biodegradable *Bacillus subtilis* group.

**Figure 4.** Comparison of serum levels of antioxidant parameters among different treatment groups. (**A**) GSH-Px, glutathione peroxidase; (**B**) SOD, superoxide dismutase; (**C**) MDA, malondialdehyde. NC, normal control diet group; ZC, ZENcontaminated group; PB, probiotic *Bacillus subtilis* group; DA, biodegradable *Bacillus subtilis* group. \*, *p* < 0.05; \*\*, *p* < 0.01.

As shown in Figure 5, the levels of immunoglobulin G (IgG) and IgM in the PB and DA groups were dramatically lower than that of the ZC group (Figure 5B,C, *p* < 0.05). However, there was no significant difference in the levels of IgA (Figure 5A, *p* < 0.05). In addition, the levels of pro-inflammatory factors (e.g., interleukin 2 (IL-2) and tumor necrosis factor-α (TNFα)) in the serum of the ZC group was significantly higher than these of other groups (Figure 5E,G, *p* < 0.05). Additionally, the DA group had the lowest level of IL-2 (Figure 5E, *p* < 0.05). However, other pro-inflammatory factors (e.g., IL-1β and IL-6) were not significantly different (Figure 5D,F, *p* > 0.05).

**Figure 5.** Comparison of serum levels of immune and inflammatory parameters in different treatment groups. (**A**–**C**) IgA, immunoglobulin A; IgG, immunoglobulin G; IgM, immunoglobulin M; (**D**–**G**) IL-1β, interleukin 1β; IL-2, interleukin 2; IL-6, interleukin 6; TNFα, tumor necrosis factor-α; NC, normal control diet group; ZC, ZEN-contaminated group; PB, probiotic *Bacillus subtilis* group; DA, biodegradable *Bacillus subtilis* group. \*, *p* < 0.05; \*\*\*, *p* < 0.001.

#### *2.6. Serum Hormone Parameters*

The effects of diet supplemented with ZEN or *Bacillus subtilis* on the serum hormone of gilts were also shown in Figure 6. The level of estradiol (E2) in the NC and DA groups were significantly lower than that in the ZC groups (Figure 6C, *p* < 0.05), while no significant difference in the levels of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were observed in each treatment group (Figure 6A,B, *p* > 0.05). In addition, the level of prolactin (PRL) in the ZC group was the highest among the four groups (Figure 6D, *p* < 0.05). There was no difference in the PRL levels of the PB and DA groups compared to the NC group (Figure 6D, *p* > 0.05).

**Figure 6.** Comparison of serum levels of hormone parameters in different treatment groups. (**A**–**D**) FSH, follicle-stimulating hormone; LH, luteinizing hormone; E2, estradiol; PRL, prolactin. NC, normal control diet group; ZC, ZEN-contaminated group; PB, probiotic *Bacillus subtilis* group; DA, biodegradable *Bacillus subtilis* group. \*, *p* < 0.05; \*\*, *p* < 0.01; \*\*\*, *p* < 0.001; \*\*\*\*, *p* < 0.0001.

#### *2.7. ZEN Residues*

As shown in Table 6, mildewed maize enormously increased the content of ZEN in feed and feces. The NC group had very low levels of ZEN in feed (*p* = 0.06) and fecal samples (*p* < 0.05). In contrast, the ZC group had the highest levels of ZEN in feed and fecal samples (*p* < 0.05). Surprisingly, the content of ZEN in feces of the DA group were dramatically lower than the ZC and PB groups (*p* < 0.05). Additionally, the ZEN content in feces of the PB group was nearly same to that of the ZC group (*p* > 0.05). Intriguingly, although the ratio of ZEN contents between feces to diet was not significantly among the four groups (*p* > 0.05), the value in the DA group was indeed half that of the remaining three groups. In this study, both ZEN and its metabolites (α-zearalanol (α-ZAL), β-zearalanol (β-ZAL), α-zearalenol (α-ZOL), β-zearalenol (β-ZOL) and zearalanone (ZAN)) were not found in serum samples.

**Table 6.** Content of ZEN in feed and fecal samples.


NC, normal control diet group; ZC, ZEN-contaminated group; PB, probiotic *Bacillus subtilis* group; DA, biodegradable *Bacillus subtilis* group. Different superscript letters represent significant difference.

#### **3. Discussion**

The recent years have witnessed growing interests in finding practical and effective methods to detoxify ZEN in contaminated cereals and feeds [15–17]. Previous studies had been focused on mycotoxin adsorbents used to control mycotoxins in animal feed [18]. However, these adsorbents would contribute to environmental pollution as they transfer mycotoxins to surrounding areas [19]. Biodegradation of mycotoxins was considered as an efficient and environmentally protective method for the treatment of contaminated diets in the livestock production [11]. Previous studies have shown that some strains of *Bacillus* spp. were able to prevent the toxicity of ZEN [13,14], while it is not well known whether these effects were due to their probiotic or degradative capacities. Previous reports from our laboratory suggested biodegradable *Bacillus subtilis* ANSB01G could degrade 84.58%, 83.04% and 66.34% of ZEN in naturally contaminated maize, swine complete feed and dried distillers' grains with solubles, respectively [13]. For these reasons, it is worth comparing the ameliorative effects between probiotic and biodegradable *Bacillus subtilis* in a ZEN-contaminated diet. The persuasion of the present study was ensured by the similarity of *Bacillus subtilis* ANSB010 and ANSB01G on the habitat source and the bacteriostatic activity against common pathogenic bacteria, including *E. coli, S. choleraesuis* and *S. aureus*.

It has been reported that the presence of ZEN reduced feed consumption, caused a subsequent growth depression, and increased susceptibility to diseases [20,21]. Although we did not observe significant difference in gilts weight in the present study, there is a decreasing trend in ADFI among these groups. In line with previous reports [22], our study showed that the gilts fed on diets containing ZEN significantly increased F/G. In our study, the contaminated diet contained about 300 μg/kg of ZEN, leading to an increase in ZEN levels in the feces. Our data revealed that biodegradable *Bacillus subtilis* ANSB01G alleviated the toxicity while significantly decreasing the F/G, but probiotic *Bacillus subtilis* ANSB010 did not.

Previous study has indicated that vulva swelling is the main clinical symptom of ZEN-induced toxicity in mammals [23,24], which has an adverse impact on the reproductive system and the breeding performance. Similarly, our data also indicated that ZEN significantly caused an increase in vulva size. Previous research found that the mycotoxin biodegradation agent composed of *Bacillus subtilis* ANSB01G and *Devosia* sp. ANSB714 can effectively reduce the estrogenic swelling of the vulva caused by ZEN in immature gilts [25].

In this study, we also discovered that only biodegradable *Bacillus subtilis* ANSB01G mediates vulva swelling caused by ZEN. Some available evidence also demonstrated the obvious adverse effects of ZEN on the secretion of these hormones and productivity of animals [26]. In addition, similar results were reflected by a fluctuation in hormone levels. Although there were no significant difference in the levels of FSH and LH, ZEN diets markedly increased the level of E2 in the gilts. It was well known that ZEN is a competitive substrate for endogenous estrogens, binding to estrogen receptors and thereby having a damaging effect on the function of gonads [27]. A recent long-term (48 d) study found that low doses of ZEN (20 μg/kg BW) induced changes in the concentrations of E2 levels in pre-pubertal gilts [28]. The addition of biodegradable *Bacillus subtilis* ANSB01G, in this study, restored serum E2 levels and modulated the function of gonads in gilts.

Van and his colleagues reported that ZEN ingestion partially induced oxidative stress in piglets, as it revealed an increased content of MDA and activity of SOD [29]. In addition, even low levels of ZEN (246 ug/kg) in the diet of gestation sows can lead to an increase in the level of serum MDA and cause cell apoptosis and moderate lesions of the liver, kidney, uterus, and ovary [30]. Importantly, our research showed that only biodegradable *Bacillus subtilis* ANSB01G reversed these increasing trends. However, no changes in the levels of antioxidant enzymes (e.g., GSH-Px and MDA) or SOD activity were observed in the serum of gilts. Moreover, we did not observe that diets treated with ZEN or both strains of *Bacillus subtilis* affected the serum biochemical indicators (e.g., ALB, AST, ALP and CRE). These data were inconsistent with the previous reports [31,32]. The difference in results might be attributed to the age difference of the animal model and different doses of ZEN contaminated. However, we noticed that ZEN increased IgG level in serum, but not in the levels of IgA and IgM. Gilts treated with biodegradable *Bacillus subtilis* ANSB01G and probiotic *Bacillus subtilis* ANSB010 decreased IgG levels in serum. IgG, IgM and IgA are the main components of immunoglobulins, of which the content of IgG is up to 70–75%. These results suggested that ZEN has antigenic activity which stimulated the immune system of gilts, and both biodegradable and probiotic *Bacillus subtilis* have the capacity to recover the stimulation caused by ZEN.

Moreover, it has been reported that ZEN could increase the synthesis and expression of pro-inflammatory factors through JNK signaling pathway activation [6]. Currently, few studies have focused on the effects of ZEN on the modulation of inflammation in gilts. In the present study, ZEN in the feed significantly increased the levels of TNFα and IL-2, and had no effects on IL-1β and IL-6 in serum. An increase in levels of TNFα, one of the most powerful pro-inflammation factors, might generate a risk of a more severe inflammatory response [33]. The inflammatory response in this trial was similar to previous results from our lab that showed an enormous increase in inflammatory cytokines (e.g., IL-2, IL-8 and IL-10) [25]. The elevation of cytokines caused by ZEN would impair the erythroid progenitor and red blood cells [34], which revealed a potential cancerotoxic effect of ZEN. Biodegradable *Bacillus subtilis* ANSB01G mitigated the acute inflammatory response, confirming the reliability of a biodegradation approach in myotoxin degradation. In comparison, the lack effect of probiotic *Bacillus subtilis* ANSB010 on the elevated inflammatory response demonstrated that probiotic *Bacillus subtilis* had no effects on alleviating the toxicity of ZEN. Therefore, we concluded that biodegradable *Bacillus subtilis* was more protective against ZEN toxicosis in gilts than the probiotic *Bacillus subtilis*. Taken together, this study provided further evidence that the specific strain of *Bacillus subtilis* ANSB01G can alleviate the toxicity of ZEN, mainly due to its biodegradable capacity.

#### **4. Conclusions**

This study demonstrated that a feeding diet contaminated with ZEN of 300 μg/kg had a damaging impact on the growth performance, plasma immune function and hormone secretion of gilts. Although probiotic *Bacillus subtilis* ANSB010 and biodegradable *Bacillus subtilis* ANSB01G have similar antimicrobial capacities and alleviate inflammatory responses, only biodegradable *Bacillus subtilis* ANSB010 could regulate estrogen levels,

relieve swelling of the vulva, and reduce the F/G and fecal ZEN residues. Hence, the biodegradable *Bacillus subtilis* ANSB01G used in this study is considered to have great and promising potential for biodegradation of mycotoxin in feed industrial applications.

#### **5. Materials and Methods**

#### *5.1. Source and Identification of Bacterial Strains*

The two strains of *Bacillus subtilis* used in this study were isolated from healthy broiler intestinal chyme and identified and characterized using a standard method described by Holt et al. [35]. Gram-staining was performed using the Gram staining kit (G1060, Solarbio, Beijing, China). Briefly, the bacteria were activated in Luria-Bertani (LB) medium at 37 ◦C for 12 h. Then, a 2.5 μL sample was stained with crystal violet for 1 min, mordanted with iodine solution 1 min, decolorized for 30 s, and counterstained with safranine for 1 min. Spore-staining was performed using the Spore stain kit (G1133, Solarbio, Beijing, China). First, a 5 μL sample, cultured in LB medium at 37 ◦C for 48 h, was stained in malachite green solution for 10 min and counterstained with safranine for 3 min. Finally, the staining results were observed using a microscope. Then, DNA was extracted from the bacterial isolates using the Bacterial Genomic DNA kit (Beijing Zoman Biotechnology Co., Ltd., Beijing, China) according to the manufacturer's instructions. PCR amplification of 16S rDNA was performed with the primers 27F (5 -AGAGTTTGATCMTGGCTCAG-3 ) and 1492R (5 -CGGTTACCTTGTTACGACTT-3 ), and PCR products were purified and sequenced by Sangon Biotech (Beijing, China). Probiotic and biodegradable *Bacillus subtilis* were identified as strains of *Bacillus subtilis* named ANSB010 and ANSB01G, respectively. It has been shown that ANSB01G could degrade ZEN in naturally contaminated maize with high efficiency [13]. The *Bacillus subtilis* ANSB01G in our experiment was a domesticated strain based on the wild bacteria obtained by Lei et al. [13]. The method of microbial domestication was as follows: after activation, *Bacillus subtilis* ANSB01G was induced and cultured in a series of MRS mediums with gradually increasing concentrations of ZEN [36]. The efficiency of *Bacillus subtilis* ANSB01G in degrading ZEN was further improved after several domestications. The 16 s rDNA sequences of ANSB010 and domesticated ANSB01G are shown in the Supplementary Materials. A phylogenetic tree was drawn with the neighbor-joining method of 1000 bootstrap replications within Mega 5.0.

#### *5.2. Antibacterial Activity and ZEN Degradation Tests*

Selected indicated bacteria *E. coli* (No.10003), *S. choleraesuis* (No.21493) and *S. aureus* (No.10384) were purchased from the China Center of Industrial Culture Collection (CICC, Beijing, China). *E. coli, S. choleraesuis* and *S. aureus* were inoculated in MRS medium and incubated in MRS medium at 180 rpm for 24 h at 37 ◦C. After diluting twice in a gradient, 200 μL of the bacterial solution was added to MRS medium and spread evenly, then z sterilized Oxford cup was placed on the medium. Then, 200 μL of the supernatant of the probiotic and biodegradable *Bacillus subtilis* solution was aspirated into an Oxford cup and the non-inoculated MRS medium was used as a control. After incubating at 37 ◦C for 24 h, the antibacterial circle diameter (cm) was measured. For the ZEN degradation test, ZEN solution (100 μL, 2 μg/mL) was added into the LB medium of ANSB010 and ANSB01G (900 <sup>μ</sup>L, 3.0 × <sup>10</sup><sup>8</sup> CFU/mL) and incubated with shaking for 6 h, 24 h, and 48 h at 37 ◦C in the dark, followed by measurement of ZEN levels using the HPLC method.

#### *5.3. Animals and Experimental Treatments*

The animal experiments were conducted according to the animal welfare requirements and approved by the Animal Protocol Review Committee of the China Agriculture University (Beijing, China).

Thirty-six healthy gilts (Landrace × Yorkshire, average body weight = 64 kg) were selected for the experiment. Then, these animals were randomly assigned to four treatments with nine replicates of one gilt per replicate for each group: (1) Normal control diet group (NC) fed the basal diet containing few ZEN (17.5 μg/kg diet) by controlling the quality of maize; (2) ZEN-contaminated group (ZC) fed the contaminated diet containing an exceeded limit dose of ZEN (about 300 μg/kg diet) by replacing normal maize in the basal diet with moldy maize; (3) Probiotic agent group (PB) fed the ZC diet with added 5 × <sup>10</sup><sup>9</sup> CFU/kg of probiotic *Bacillus subtilis* ANSB010; (4) Biodegradable agent group (DA) fed the ZC diet with added 5 × <sup>10</sup><sup>9</sup> CFU/kg of biodegradable *Bacillus subtilis* ANSB01G. The contaminated maize was purchased in 2018 from a small family farm in Henan Province, China. The *Bacillus subtilis* ANSB010 and ANSB01G were incubated in LB medium for 24 h at 37 ◦C, followed by drying at 65 ◦C, and then evenly mixed into the diets. Diets are formulated to meet or exceed nutrient requirements (Table 7) recommended by the National Research Council for replacement gilts (NRC, 2012). The experimental period lasts for 25 d. The contaminated diets were prepared by replacing corn in the control with the naturally contaminated maize. During the supplementation period, all piglets were individually housed in temperature-controlled stainless steel metabolism pens (25 ± 2 ◦C), allowing free access to drinking water. Animal care and experimental procedures were in accordance with the guidelines of the National Institutes of Health Guide and the China Ministry of Agriculture for the care and use of laboratory animals.


**Table 7.** Ingredients and compositions of the basal diet, as fed basis.

<sup>1</sup> The value is calculated. <sup>2</sup> Supplied the following per kilogram of diet: vitamin A, 5000 IU; vitamin D3, 900 IU; vitamin E, 40 IU; vitamin K, 2.5 mg; vitamin B1, 1.5 mg; vitamin B2, 6.4 mg; vitamin B6, 2.5 mg; vitamin B12, 0.025 mg; pantothenate, 20 mg; nicotinic acid, 30 mg; choline, 0.50 g; Fe 100 mg; Cu, 6 mg; Zn, 50 mg; Mn, 20 mg; Se, 0.30 mg; I, 0.24 mg.

#### *5.4. Growth Performances*

The initial body weight and terminal body weight were recorded. Moreover, ADG, ADFI, and F/G (ADFI/ADG) were calculated.

#### *5.5. Vulva Size Determination*

The vulva length (a), width (b) and height (h) were measured and recorded at 0, 12d and 24 d. The determination of vulva area and volume was performed and results were calculated according to the method previously described by Zhao and his colleagues [14]. The area and volume of vulva are approximately elliptical and conical, respectively. Therefore, the area of the vulva is in accordance with the formula: S = (π × a × b)/4; and the volume of the vulva is calculated with the equation: V = 1/3 × S × h.

#### *5.6. Serum Parametes*

Blood was collected from the marginal ear vein at the end of the experiment period. Then, the blood samples were centrifuged at 3000× *g* for 10 min to obtain serum for further analysis. The serum biochemical indicators including the TP, ALB, ALT, AST and ALP, CRE and BUN were measured with an automatic biochemical analyzer (Hitachi 7160, Hitachi High-Technologies Corporation, Tokyo, Japan). Immunoglobulin A (IgA), IgG and IgM

were also determined with an automatic biochemical analyzer (Hitachi 7160). In addition, interleukin 1β (IL-1β), IL-2, IL-6 and TNFα were measured by an enzyme-linked immune sorbent assay (ELISA) kit (YuanMu Biotechnology, Shanghai, China). Serum SOD, GSH-Px and MDA were detected according to the instructions of the manufacturer using microplate test kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The concentrations of serum E2, FSH, LH and PRL were determined by radioimmunoassay (RIA). The serum samples were treated with radioactive-125I according to the instructions of the RIA kit (Beijing Kemei Biotechnology Co., Ltd., Beijing, China). Then, a GC-1200 radio-immunity gamma-counter (KeDa Innovation Co., Ltd., Hefei, China) was used to measure hormone concentrations. For each RIA, the intra- and inter-assay coefficients of variation were less than 15% and less than 10%, respectively. Hormone concentration was determined according to each sample's level of radioactivity.

#### *5.7. Determination of ZEN in the Feed, Feces, Broth and Serum*

Two days before the end of the test, fresh fecal samples were taken to determine the content of ZEN in feces according to the description of Chinese certification GB/T 23504- 2009 and Lei et al. [13]. For feed and feces samples, 50 g ground samples were extracted by acetonitrile-water (70:30, *v/v*, 200 mL), followed by filtration with Whatman 4 filter paper. After dilution with PBS solution (PH = 7.40), the mixing solution were filtered through a micro-filter. A volume of 20 mL of suspension was passed through the immunoaffinity column (Femdetection FD-C21, Nanjing, China) at a flow rate of 1.0 mL/min under gravity. After washing the column with distilled water, the ZEN was subsequently eluted with 2 mL of methanol into a centrifuge tube for HPLC analysis. For liquid medium, samples (1 mL) were extracted with acetonitrile (9 mL) at 180 rpm for 2 h. The mixed samples were filtered using glass fibre filter paper, and then collected for subsequent HPLC analysis. ZEN and its metabolites in serum were analyzed using the method described by Duca et al. [37]. Briefly, serum (2 mL) was mixed with buffer ammonium acetate solution (8 mL). The mixed solution was incubated with glucuronidase/arylsulfatase (50 μL) at 37 ◦C for 15 h. After the samples were centrifuged at 5000 rpm for 10 min, the supernatant was passed through the immunoaffinity column. The column was then rinsed with 20 mL of ultrapure water. The analytes were then eluted with acetonitrile (2 mL). Subsequently, the solution was dried using a Speed Vac concentration system after which 200 μL mobile phase was added. For HPLC analysis, 20 μL sample solution was injected into the HPLC system. Separation was in a C18 column (4.6 mm × 150 mm, 5 μm; Thermo Fisher Scientific, Waltham, MA, USA) with mobile phase (water: acetonitrile, 50:50, *v/v*) at a flow rate of 1.0 mL/min. The analytes were detected by a fluorescence detector (Waters, Milford, MA, USA), excitation and emission wavelengths were 274 and 440 nm, respectively. The retention time was 7–8 min.

#### *5.8. Statistical Analysis*

Data were analyzed statistically using SAS 9.4 software (SAS Institute Inc., Cary, NC, USA) and were presented as mean ± SEM. The significance of difference between groups of gilts were analyzed by one-way ANOVA. A normality test (Shapiro–Wilk) was performed to determine normality before one-way ANOVA analysis. Differences were regarded as statistically significant at a probability of *p* < 0.05, and *p*-values < 0.10 were regard as a trend.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/toxins13120882/s1.

**Author Contributions:** W.S. and Y.L. (Yaojun Liu): conceptualization, conducted the animal experiments; methodology, writing—review and editing. X.Z. (Xinyue Zhang), X.Z. (Xiong Zhang), F.L. and J.C.: assisted with the experiments; X.R. and Y.L. (Yuanpei Lei): conducted the ZEN degradation test; L.Z. and C.J.: supervision; Q.M.: conceptualization, funding acquisition, writing—review and editing. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was funded by the National Natural Science Foundation of China (Grant No. 31772637, 31301981), and the Special Fund for Agro-scientific Research in the Public Interest (201403047).

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Beijing Muncipality on the Review of Welfare and Ethics of Laboratory Animals, and approved by the China Agricultural University Animal Care and Use Committee (Approval No. Aw72011202-1-6).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data are available from the first author.

**Acknowledgments:** The authors wish to thank the students who participated in our study and the workers of the Fengze Farm.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

