*Article* **Four PQQ-Dependent Alcohol Dehydrogenases Responsible for the Oxidative Detoxification of Deoxynivalenol in a Novel Bacterium** *Ketogulonicigenium vulgare* **D3\_3 Originated from the Feces of** *Tenebrio molitor* **Larvae**

**Yang Wang 1, Donglei Zhao 2, Wei Zhang 1, Songshan Wang 1, Yu Wu 1, Songxue Wang 1, Yongtan Yang <sup>1</sup> and Baoyuan Guo 1,\***


**Abstract:** Deoxynivalenol (DON) is frequently detected in cereals and cereal-based products and has a negative impact on human and animal health. In this study, an unprecedented DON-degrading bacterial isolate D3\_3 was isolated from a sample of *Tenebrio molitor* larva feces. A 16S rRNA-based phylogenetic analysis and genome-based average nucleotide identity comparison clearly revealed that strain D3\_3 belonged to the species *Ketogulonicigenium vulgare*. This isolate D3\_3 could efficiently degrade 50 mg/L of DON under a broad range of conditions, such as pHs of 7.0–9.0 and temperatures of 18–30 ◦C, as well as during aerobic or anaerobic cultivation. 3-keto-DON was identified as the sole and finished DON metabolite using mass spectrometry. In vitro toxicity tests revealed that 3-keto-DON had lower cytotoxicity to human gastric epithelial cells and higher phytotoxicity to *Lemna minor* than its parent mycotoxin DON. Additionally, four genes encoding pyrroloquinoline quinone (PQQ)-dependent alcohol dehydrogenases in the genome of isolate D3\_3 were identified as being responsible for the DON oxidation reaction. Overall, as a highly potent DON-degrading microbe, a member of the genus *Ketogulonicigenium* is reported for the first time in this study. The discovery of this DON-degrading isolate D3\_3 and its four dehydrogenases will allow microbial strains and enzyme resources to become available for the future development of DON-detoxifying agents for food and animal feed.

**Keywords:** DON biodegradation; 3-keto-DON; toxicity; *Ketogulonicigenium vulgare*; PQQ-dependent alcohol dehydrogenases

**Key Contribution:** A novel DON-oxidizing strain, *Ketogulonicigenium vulgare* D3\_3, was isolated from yellow mealworm feces, with oxidizing ability under either aerobic or anaerobic conditions. The oxidation product, 3-keto-DON, exhibited a higher phytotoxicity compared to its parent DON. Furthermore, all four PQQ-dependent alcohol dehydrogenases in the DON degrader were found to possess DON-oxidizing activities.

### **1. Introduction**

Deoxynivalenol (DON) is a natural toxin that is produced as a secondary metabolite by certain species of phytopathogenic fungi belonging to the genus *Fusarium* [1]. Its high prevalence and widespread occurrence in small cereal grains and their derivatives have been extensively documented in the literature [2,3]. The presence of DON in the food supply chain constitutes a notable risk to food safety and public health. A range of adverse health effects, including gastrointestinal disturbances, dermatological manifestations, and immunosuppression, have been associated with varying doses and exposure durations to

**Citation:** Wang, Y.; Zhao, D.; Zhang, W.; Wang, S.; Wu, Y.; Wang, S.; Yang, Y.; Guo, B. Four PQQ-Dependent Alcohol Dehydrogenases Responsible for the Oxidative Detoxification of Deoxynivalenol in a Novel Bacterium *Ketogulonicigenium vulgare* D3\_3 Originated from the Feces of *Tenebrio molitor* Larvae. *Toxins* **2023**, *15*, 367. https://doi.org/10.3390/ toxins15060367

Received: 8 May 2023 Revised: 25 May 2023 Accepted: 26 May 2023 Published: 30 May 2023

**Copyright:** © 2023 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/).

DON [4–7]. Given DON's chemical and thermal stability, salvaging DON-contaminated cereal grains and avoiding the health risks associated with DON is technically challenging. Over recent decades, extensive efforts have been made to seek cost-effective and efficacious DON mitigation strategies. For instance, a range of physicochemical detoxification approaches, such as ultraviolet irradiation [8], argon plasma exposure [9], ozonation treatment [10,11], sorting and separation [12], washing [13,14], and thermal treatment [15] have been explored. However, each of these DON-mitigation methods possesses inherent limitations regarding environmental impacts, nutritional quality, and potential generation of toxic byproducts. The biodegradation method, involving enzymatic transformation of mycotoxins into less toxic or non-toxic products via selected microorganisms, is characterized by mild reaction conditions and a relatively high specificity. Therefore, it was viewed as a promising alternative strategy to reduce DON contamination in agricultural products.

The main toxicity determinants in the molecular structure of DON are the three hydroxyl moieties located at C3, C7, and C15, as well as the epoxy ring connecting C12 and C13 [7,16], with C3-OH and the epoxy moiety as the primary targets for biodegradation. The oxidation of C3-OH can yield 3-keto-DON, which can further undergo epimerization to form 3-epi-DON. Furthermore, de-epoxidation of the epoxy moiety can generate deepoxydeoxynivalenol (DOM-1). These three metabolites have demonstrated a lower toxicity than DON in vitro [17–19], and many species of microorganisms have been found to possess these metabolic capabilities. For instance, several soil-borne bacterial strains, including *Nocardioides* sp. WSN05-2 [20], *Devosia mutans* 17-2-E-8 [21], *Paradevosia shaoguanensis* DDB001 [22], and *Nocardioides* sp. ZHH-013 [23], can produce 3-epi-DON. The two-step enzymatic reaction mechanism converting DON to 3-epi-DON was first elucidated in *D. mutans* 17-2-E-8. The process involves a PQQ-dependent alcohol dehydrogenase (DepA) oxidizing DON to 3-keto-DON, followed by an NADPH dependent aldo-keto reductase (DepB) stereospecifically reducing the intermediate 3-keto-DON to 3-epi-DON [21,24]. Although DON can also be converted to 3-keto-DON by *Agrobacterium* E3-39 [17], *D. insulae* A16 [25], and *Pelagibacterium halotolerans* ANSP101 [26], these strains cannot further convert 3-keto-DON to 3-epi-DON, likely due to the absence of a functional enzyme such as DepB.

Despite numerous successful isolations of DON-degrading microorganisms from various sources, there have been no reports of such isolations from the intestines or feces of insects. As a kind of edible insect, *Tenebrio molitor* larvae (yellow mealworm) are an alternative protein source for food and feed, due to their low production costs and good nutritional characteristics [27]. They can be readily raised on cereal bran or flour; however, these diets may be contaminated with mycotoxins, particularly DON. Long-term feeding of mycotoxin-contaminated diets could thus create favorable conditions for the acclimation of mycotoxin-degrading microorganisms in yellow mealworm. Several investigations have explored the impacts of DON on the biological parameters of insect larvae, as well as the profiles of DON metabolism and excretion [28–30]. For example, Van Broekhoven et al. [30] found that DON and its metabolites generally do not remain in the bodies of insect larvae fed diets contaminated with mycotoxins, but can be detected in larval feces, implying that yellow mealworms and/or their gut microbiota may have the potential ability to metabolize DON. On the other hand, microbial resources capable of degrading DON under both anaerobic and aerobic conditions are limited, posing a challenge for the effective mitigation of DON contamination in various application scenarios. In this regard, there is an urgent need to screen for novel DON-degrading strains from diverse sources, to enrich the repository of DON-degrading strains. This can help to address specific application demands in different scenarios, such as agricultural production and animal feed.

In the current study, a novel microbial DON degrader D3\_3 was isolated from a sample of yellow mealworm feces using conventional enrichment and isolation procedures. Genome-based ANI analysis and 16S rRNA-based phylogenetic tree analysis consistently revealed its taxonomic position. The impact of various conditions on the strain's DONdegrading activity was also investigated. Subsequently, the chemical structure of the DON metabolite was determined via mass spectrometry, and its animal cell cytotoxicity and

phytotoxicity were evaluated using human gastric epithelial cells (GES-1) and duckweed (*Lemna minor*), respectively. Finally, four PQQ-dependent alcohol dehydrogenases (ADHs) in strain D3\_3 were identified as being responsible for the catalysis of the oxidation of DON to 3-keto-DON.

#### **2. Results**

#### *2.1. A Potent DON-Oxidizing Strain Ketogulonicigenium Vulgare D3\_3 Isolated from Yellow Mealworm Feces*

As demonstrated in Figure 1A, four successive subcultures of yellow mealworm excreta in MMFS (mineral medium supplemented fecal supernatant) liquid resulted in a 20.9% decrease in 50 mg/L DON at 30 ◦C and 220 rpm in a shaking incubator after 5 d. However, no DON reduction was observed for the sample of PYM that underwent the same enrichment procedure. Thus, the mixed culture with DON-degrading activity was spread on MMFS agar plates after serial dilution. After growing at 30 ◦C for 7 d, 40 colonies were examined for their ability to remove DON. This resulted in the isolation and selection of the DON-degrading strain D3\_3. As demonstrated in Figure 1B, a pure culture of this isolate D3\_3 grown in MMFS (initial pH 7) was capable of removing 50 mg/L of DON after 12 h of incubation. Furthermore, a peak in the MMFS+DON+D3\_3 sample had the same retention time (Rt = 10.7 min) as the 3-keto-DON standard, which was not detected when D3\_3 was incubated in a MMFS medium without DON (MMFS+D3\_3), implying that it was a DON metabolite and possibly 3-keto-DON.

**Figure 1.** DON degradation profiles of the enrichment cultures and the isolate D3\_3' pure culture, as well as determination of the chemical structure of DON metabolite. (**A**) DON residual rate in MMFS and PYM media containing 50 mg/L of DON with or without insect fecal slurry after four instances of serial subcultivation. (**B**) LC profiles of DON degradation by the isolate D3\_3 in MMFS medium containing 50 mg/L of DON. (**C**) MS profiles of DON, 3-keto-DON, and the putative metabolite 3-keto-DON generated by *K. vulgare* strain D3\_3.

The chemical structure of possible 3-keto-DON (Rt = 5.75 min in UPLC-Q-TOF-MS analysis) was further ascertained via MS analysis. Figure 1C reveals that there were several peaks at *m*/*z* 339.1057, 329.0755, 293.1013, and 263.0904 in the negative mode, which were

assigned to [M+HCOO]−, [M+Cl]−, [M-H]−, and [M-CH2O-H]−, respectively. These data were consistent with those of the 3-keto-DON standard. Furthermore, the MS data of DON (Rt = 3.84 min) showed that a number of peaks, such as *m*/*z* 341.1222 ([M+HCOO]−), 331.0919 ([M+Cl]−), 295.1154 ([M-H]−), and 265.1069 ([M-CH2O-H]−), were larger by 2 in comparison to the corresponding ions of the DON metabolites; such an increase was equal to the atomic weight of two hydrogen atoms. These findings conclusively determined that the DON metabolite produced by *K. vulgare* D3\_3 was indeed the oxidation product of DON, namely 3-keto-DON.

The 16S rRNA gene sequence of strain D3\_3 determined in this study comprised 1322 nt (GenBank accession no. OQ102971), exhibiting the highest sequence similarities with that of *Ketogulonicigenium vulgare* DSM 4025T (99.85%) and *Ketogulonicigenium robustum* X6LT (99.47%), respectively. Furthermore, phylogenetic analysis based on a 16S rRNA gene sequence also indicated that strain D3\_3 was positioned within the genus *Ketogulonicigenium* and formed a phylogenetic clade with *K*. *vulgare* DSM 4025<sup>T</sup> and *K*. *robustum* X6LT, suggesting that it belonged to the genus *Ketogulonicigenium* (Figure 2A). However, these results could not definitively confirm which species strain D3\_3 belonged to. To further clarify its taxonomic status, its genome sequence (obtained from the genome sequencing analysis) and several other genome sequences of *Ketogulonicigenium* available in the NCBI Genome database were subjected to pairwise average nucleotide identity (ANI) calculations using the FastANI algorithm [31]. As shown in Figure 2B, the heatmap indicated that the strain D3\_3 shared an ANI of 98.33%, 98.36%, 98.40%, 98.43%, 98.44%, and 81.69% with *K*. *vulgare* Y25 (GCF\_000164885), *K*. *vulgare* SKV (GCF\_001693655), *K*. *vulgare* SPU B805 (GCF\_001855295), *K*. *vulgare* Hbe602 (GCF\_001399515), *K*. *vulgare* WSH-001 (GCF\_000223375), and *K*. *robustum* SPU B003 (GCF\_002117445), respectively. According to the classification criteria of >95% intra-species and <83% inter-species ANI values [31], the DON-degrading strain D3\_3 was definitively identified as *K*. *vulgare.*

**Figure 2.** Taxonomic identification of DON-degrading strain D3\_3. (**A**) The 16S rRNA-based phylogenetic tree of strain D3\_3 reconstructed using the maximum likelihood method. The numbers near the nodes on the phylogenetic tree indicate that bootstrap values greater than 50%, while the content in parentheses are the GenBank accession numbers of the 16S rRNA sequences. Scale bar: 2 nucleotide substitutions per 100 positions. T: type strain, TP: use of patent strain as type strain. (**B**) ANI heatmap of seven *Ketogulonicigenium* strains. Heatmap generated based on ANI matrix obtained from *Ketogulonicigenium* genomes' average nucleotide identity (ANI) values, ranging from low (blue) to high (orange).

#### *2.2. Effects of Different Growth Factors on the DON-Degrading Activity of Strain D3\_3*

As shown in Figure 3A, residual DON was not detected after 12 h in samples with an initial pH of 7, 8, and 9, meanwhile the DON residual rate was not significantly different for the pH 5 and 6 groups compared to the control. After 72 h, the DON residual rate of pH 6 decreased to 96.7 ± 0.6%, exhibiting a significant contrast with the control and the 12 h rate for the same group (*p* < 0.05). However, no such change was observed for the pH 5 group. Conclusively, strain D3\_3 can degrade DON with a pH range of between 6 and 9, with greater activity between pH 7 and 9 than at pH 6, but no activity at pH 5.

**Figure 3.** The impacts of different factors on the DON-degrading activity of strain D3\_3 in the MMFS medium containing 50 mg/L of DON at incubation times of 12 and 72 h. (**A**) pH, (**B**) temperature, (**C**) carbon source, and (**D**) cultivation method. Significant differences (*p* < 0.05) in the degradation rates between the two groups, as determined via ordinary one-way ANOVA with a use of Tukey's multiple comparisons test, are indicated by the different letters (a–e) above the columns. "Control" stands for the MMFS medium containing 50 mg/L of DON, while "no addition" stands for the DON-added and inoculated MMFS medium.

As illustrated in Figure 3B, the 12 h DON residual rates for the samples at 4, 18, 30, 37, and 42 ◦C were 96.2 ± 0.8, 55.6 ± 0.3, 0, 97.6 ± 0.9, and 98.0 ± 0.6%, respectively. After 72 h, the DON residual rates at 4 and 18 ◦C decreased significantly to 75.2 ± 1.7 and 0% (*p* < 0.0001), respectively, while the rates at 37 and 42 ◦C remained statistically similar to their 12 h counterparts. Overall, strain D3\_3 exhibited DON-degradation activity at temperatures ranging from 4 to 30 ◦C, with an optimal temperature of 30 ◦C. No DONdegradation activity was observed at 37 ◦C or higher, which is logically understandable

considering that the strain does not grow at temperatures above 37 ◦C. Notably, even at 4 ◦C, D3\_3 still displayed a 24.8 ± 1.7% DON-degradation rate after 72 h.

As shown in Figure 3C, the addition of glucose, mannitol, and sucrose significantly enhanced the DON residual rate at 12 h (*p* < 0.05), with glucose having the greatest effect, followed by mannitol and sucrose. Fructose, sorbitol, and trehalose had no significant effect on the DON residual rate. After 72 h, the DON residual rate for samples with glucose, mannitol, and sucrose declined significantly compared to the respective 12 h treatments (*p* < 0.001). Overall, the D3\_3 strain's ability to degrade DON was inhibited by glucose, mannitol, and sucrose, but not by fructose, sorbitol, and trehalose.

As demonstrated in Figure 3D, the 12 h DON residual rates for samples under static, shaking, and anaerobic conditions were 0, 0, and 61.9 ± 2.9%, respectively. Despite extending the incubation duration to 72 h, the anaerobic DON residual rate remained virtually unchanged (62.1 ± 2.0%). These findings indicate that strain D3\_3 possesses the capacity to degrade DON under both anaerobic and aerobic conditions, with the latter being more favorable for DON degradation. This expands this strain's potential application scenarios, such as being adapted to be used in agricultural practice in aerobic condition, in addition to being used as a feed additive administered to the anaerobic gastrointestinal tract of animals.

#### *2.3. Cytotoxicity and Phytotoxicity of 3-Keto-DON*

#### 2.3.1. Effect of 3-Keto-DON on the Viability of GES-1 Cells

To gain a better understanding of the toxicity of 3-keto-DON, GES-1 was used as an in vitro model to investigate its toxicity. The results, as illustrated in Figure 4A,B, showed that both DON and 3-keto-DON reduced the viability of GES-1 cells in a dose-dependent manner, with GES-1 being more sensitive to DON than 3-keto-DON at the same treatment dose. The calculated IC50 values for DON and 3-keto-DON against GES-1 cells were 2.66 and 29.70 mg/L, respectively, indicating an 11.1-fold decrease in the toxicity of 3-keto-DON against GES-1 cells compared to the parent mycotoxin DON.

#### 2.3.2. Effect of 3-Keto-DON on L. Minor

As shown in Figure 4C, 0.5 mg/L of DON standard significantly decreased the number of fronds by 41.4% when compared to the control (13.3 ± 2.3 vs. 22.7 ± 6.1 fronds/well), while the area of fronds was lowered by 49.4% (0.43 ± 0.07 vs. 0.85 ± 0.24 cm2/well). Duckweed development was entirely repressed by DON at 1 mg/L or above, with no increase in both the area and number of fronds after 7 d of exposure.

As demonstrated in Figure 4D, 0.5 mg/L of the 3-keto-DON standard resulted in a considerable decrease of 72.2% in the number of fronds (6.3 ± 1.1 compared to 22.7 ± 6.1 fronds/well in the control) and a 77.7% decrease in area (0.19 ± 0.04 compared to 0.85 ± 0.24 cm2/well in the control), both of which were statistically significant (*<sup>p</sup>* < 0.05). When compared to 0.5 mg/L of DON standard, the number and area of fronds were reduced by 52.6% (6.3 ± 1.1 vs. 13.3 ± 2.3 fronds/well) and 55.8% (0.19 ± 0.04 vs. 0.43 ± 0.07 cm2/well), respectively, with statistical significance (*p* < 0.05). Furthermore, it was observed that only a small number of duckweed fronds were bleached when exposed to 3-keto-DON at 0.5 mg/L. In comparison, all of the fronds became bleached when exposed to 1 and 2 mg/L of the compound. However, the phenomenon of fronds becoming bleached did not occur at DON concentrations of 0.5 mg/L or higher.

Prior to the phytotoxicity experiment, UPLC analysis of MMSF+D3\_3+DON revealed that DON had been completely converted to 3-keto-DON at a concentration of around 50 mg/L. Moreover, 100-fold, 50-fold, and 25-fold dilutions of the sample yielded 0.5, 1, and 2 mg/L of 3-keto-DON, respectively. Exposure of duckweed to the 100-fold dilution resulted in a 76.7% reduction in number of fronds (5.3 ± 2.3 vs. 22.7 ± 6.1 fronds/well in the control) and a 76.5% reduction in the area of fronds (0.2 ± 0.09 vs. 0.85 ± 0.24 cm2/well in the control), while 50-fold and 25-fold dilutions completely inhibited growth. No significant differences in growth inhibition were observed between the 100-fold dilution of MMSF+D3\_3+DON and 0.5 mg/L of the 3-keto-DON standard, or between the 50-fold and

25-fold dilutions and 1 and 2 mg/L of the 3-keto-DON standard, respectively (*p* > 0.05). According to these results, we can conclude that the phytotoxicity of 3-keto-DON seems to be greater than that of the same amount of DON.

**Figure 4.** Toxicity evaluation of DON and 3-keto-DON. Effect of different concentrations of DON (**A**) and 3-keto-DON (**B**) on the viabilities of GES-1 cells. Phytotoxicity assessment of DON, 3-keto-DON, and cell-free supernatant of DON-degradation culture (CFS) on the number (**C**) and area (**D**) of duckweed fronds. The use of different lowercase letters (a–f) indicates that there are significant differences (*p* < 0.05) between the treatments being compared. Values that share the same letter are not significantly different.

#### *2.4. Four PQQ-Dependent Alcohol Dehydrogenases Responsible for DON Transformation*

To gain a greater understanding of *K. vulgare* strain D3\_3 and screen its DON-oxidizing genes, its genome was sequenced, assembled, and then annotated. The analysis of D3\_3's genome revealed that it comprised one circular chromosome and three plasmids, with a total length of 3,293,003 bp and a GC content of 61.36%. PGAP annotation predicted 3236 putative protein-coding genes, 5 rRNA operons, and 60 tRNAs (Table S1).

Currently, there are two different types of sequence-known enzymes that can oxidize the hydroxyl at C3 of DON into a keto group, namely an aldo-keto reductase AKR18A1 from *Sphingomonas* sp. S3-4 and two pyrroloquinoline quinone (PQQ)-dependent dehydrogenases DepA and QDAH from *D. mutans* 17-2-E-8 and *Devosia* sp. D6\_9 [24,32,33]. Since DepA and QDAH share the same amino acid sequences, only DepA and AKR18A1 were used as query protein sequences to search for potential genes involved in DON oxidation against the resulting genome database. Based on BLASTp search outcomes, the genome contained fourteen potential genes for DON oxidation, comprising eight ADH-encoding genes and six AKR-encoding genes. The protein sequences encoded by these genes displayed amino acid sequence similarities to DepA and AKR18A1, ranging 24.8–57.8% and

28.2–34.7%, respectively (Tables S2 and S3). Furthermore, six of the eight dehydrogenaseencoding genes were located on a 2.8 Mb bacterial chromosome, whereas the other two were separately found on two 0.22 Mb megaplasmids of pP1 and pP2 (Table S2). Five of the six aldo-keto reductase-encoding genes were located on the bacterial chromosome, and the other one was found on megaplasmid pP1 (Table S3).

The signal peptide sequences were predicted and then deleted prior to heterogeneously expressing candidate enzymes. As predicted using SignalP 5.0, seven of the eight dehydrogenases possessed a signal sequence ranging from 21 to 24 amino acids in length, whereas all aldo/keto reductases did not possess it (Tables S2 and S3). Cloning, expression, and confirmation of enzymatic activity were performed on the fourteen candidate genes. As shown in Figure 5A, four of the eight ADHs, designated *Kv*ADH1, *Kv*ADH2, *Kv*ADH3, and *Kv*ADH4, yielded a single band at a molecular mass of 61.7, 61.2, 61.3, and 61.6 kDa, respectively. *Kv*ADH1, *Kv*ADH2, *Kv*ADH3, and *Kv*ADH4 showed 57.76%, 55.14%, 54.87%, and 55.86% amino acid similarity with DepA or QDDH, respectively; the amino acid sequence identities between the four ADHs obtained in this study ranged from 80.7 to 86.5% (Table S4). The four purified *Kv*ADHs exhibited catalytic activity toward DON in the presence of the cofactors PQQ and CaCl2, as well as the artificial electron acceptor PMS (Figure 5B), but no activity was observed without PQQ being present. It is therefore suggested that PQQ is necessary for maintaining the enzymatic function of the four dehydrogenases. Additionally, none of the six AKRs had such enzymatic activity in the presence of NADH or NADPH. These results clearly demonstrated that the oxidation of DON to 3-keto-DON in the *K. vulgare* strain D3\_3 was caused by the PQQ-dependent alcohol dehydrogenase rather than the aldo-keto reductase.

**Figure 5.** Molecular weight characterization and enzymatic activity confirmation of four recombinant PQQ-dependent alcohol dehydrogenases. (**A**) SDS-PAGE analysis of four Ni-affinity purified recombinant ADHs. M: molecular weight markers (10–180 KD); Lane 1: control; Lane 2: *Kv*ADH1; Lane 3: *Kv*ADH2; Lane 4: *Kv*ADH3; Lane 5: *Kv*ADH4. (**B**) LC profiles of 3-keto-DON produced via in vitro DON oxidation using four ADHs with PQQ, Ca2+, and PMS present.

#### **3. Discussion**

Many species of DON-degrading microbes have so far been successfully isolated from a variety of sources, such as wheat leaf, wheat head, soil, lake water, seawater, various animal intestines, bovine rumen, and human milk [20,22,34–44]. Nevertheless, there have been no reports regarding the isolation of a DON-degrading strain from yellow mealworm. Herein, we successfully isolated a DON-degrading strain *K. vulgare* D3\_3 from a single insect feces sample. Additionally, three DON-degrading *Devosia* strains were effectively screened from five other yellow mealworm feces samples collected from different geographical areas [45]. These results exemplify that yellow mealworm feces are a highly efficient source for the isolation of mycotoxin-degrading microorganisms.

Furthermore, this is the first report on a member of the genus *Ketogulonicigenium* as a potent DON-degrading microorganism. *K. vulgare*, the type species of the genus *Ketogulonicigenium*, was first isolated from soil and taxonomically characterized in 2001 [46]. It is currently used in the microbial production of a key intermediate for the industrial synthesis of vitamin C, namely 2-keto-L-gulonic acid [47]. Due to its unique nature and lack of many amino acid biosynthesis pathways, *K. vulgare* grew poorly, even on nutrient-rich media [48]. The isolate D3\_3 also exhibited this poor growth phenotype, with a maximum optical density

(OD600) of 0.13 (4 × 106 CFU/mL) in MMFS medium and only 0.25 in nutrient-rich TSB medium after 72 h incubation. However, it exhibited a highly efficient DON-degradation rate of 104.1 μg/h/per 107 cells, exceeding the degradation rates reported in earlier studies for *D. mutans* 17-2-E-8, *P. shaoguanens* is DDB001, and *Devosia* sp. D6-9, which had degradation rates of 0.3, 6.8, and 15.0 μg/h/per 107 cells, respectively [22,33,37]. In addition, the versatile DONdegrading capabilities of the facultative anaerobic strain D3\_3 under aerobic or anaerobic conditions greatly expand its potential application scenarios.

3-keto-DON is a common intermediate or end metabolite in microbial DON degradation. Several strains of bacteria, including *Agrobacterium-Rhizobium* E3-39, *D. insulae* strain A16, and *P. halotolerans* ANSP101 can directly convert DON to 3-keto-DON [17,25,26]. Other strains, including *D. mutans* 17-2-E-8, *Devosia* sp. D6-9, *Lactobacillus rhamnosus* SHA113, *Nocardioides* sp. WSN05-2, *Nocardioides* sp. ZHH-013, and *Sphingomonas* sp. S3-4, could first convert DON to 3-keto-DON, which was then stereospecifically reduced to 3-epi-DON [23,32,33,37,42,43]. The absence of 3-epi-DON in the MMFS+DON+D3\_3 sample indicated that *K. vulgare* D3\_3 was incapable of reducing 3-keto-DON to 3-epi-DON, likely due to a lack of the responsible enzyme.

The bacterium *K. vulgare* D3\_3 could transform DON to 3-keto-DON. However, before considering its potential use, it is essential to evaluate the toxicity of its transformation product. Currently, there is a scarcity of toxicity data for 3-keto-DON, with only two studies assessing its in vitro cytotoxicity on human colon cancer cells (Caco-2) and mouse spleen lymphocytes [17,49], and data on cytotoxicity against GES-1 cells are also lacking. In this study, the calculated IC50 values for DON and 3-keto-DON against GES-1 cells were 2.66 and 29.70 mg/L, respectively. However, in a previously described MTT (3-[4,5 dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) bioassay assessing Caco-2 cell viability, the IC50 values for 3-keto-DON and DON were 1.24 mg/L and 0.409 mg/L, respectively [49]. It is considered that the discrepancy in IC50 values between the two studies mainly resulted from different cell lines (GES-1 vs. Caco-2) and assay methodologies (CKK-8 vs. MTT). In addition, taking into account the fact that the IC50 value (2.66 mg/L) of DON in this study is analogous to the IC50 value (2.99 mg/L) published in earlier studies [50] using the same cell lines and detection methods, the validity of the IC50 values of DON and 3-keto-DON in the current study should be verified. In summary, our study provides initial evidence that 3-keto-DON is less toxic to GES-1 compared to its parent mycotoxin DON, similarly to other cell lines.

PQQ-dependent alcohol dehydrogenases represent a class of oxidoreductases that utilize the cofactor PQQ to catalyze the oxidation of a variety of substrates, including alcohols and sugars [51–53]. Based on their tertiary structures, PQQ-dependent ADHs can be categorized into two families: six-bladed "propeller fold" and eight-bladed "propeller fold" [54]. The latter can be further classified into three types based on their location and structural characteristics: type I (periplasmic, dimeric), type II (periplasmic, monomeric, with a heme c group), and type III (membrane-bound, dimeric or trimeric, with multiple heme c groups) [55]. The crystal structures of many different types of PQQ-dependent ADHs have been determined, providing structural foundations for understanding the substrate–cofactor–enzyme binding and electron transfer mechanisms. Recently, the crystal structures of the first identified PQQ-dependent DON dehydrogenase, DepA (PDB:7WMD), and its complex with PQQ (PDB:7WMK), were determined, suggesting that DepA belongs to the Type I PQQ-dependent ADH [56]. These crystal structures, along with biochemical evidence, confirm the interactions between DepA, PQQ, and DON, and reveal a unique tyrosine residue crucial for substrate selection. In this study, four PQQ-dependent alcohol dehydrogenases (*Kv*ADHs) with DON oxidation activity in the genome of strain D3\_3 were identified. These four enzymes exhibit high amino acid homology (54.87–57.76%) with DepA and 80.7–86.5% homology with each other. This suggests that the four *Kv*ADHs and DepA may share a similar catalytic mechanism, but with some differences in their amino acid sequences that could indicate differences in substrate specificity or other functional properties. Further research is required to elucidate the structure and function of the four

*Kv*ADHs, including studies which involve the crystallization and determination of their three-dimensional structures, as well as observations of their kinetic properties with DON and other alcohol substrates. In addition, some bacteria, such as *Pseudomonas*, methanotrophic, and methylotrophic bacteria, generally express different categories and even multiple PQQ-ADHs of the same type, demonstrating the importance of these enzymes for the metabolism of various alcohol substrates [52,57,58]. Within D3\_3, there are eight putative PQQ-dependent dehydrogenases whose functional redundancy may enhance the adaptability of microorganisms in maintaining critical functions under fluctuating environmental conditions and diverse microbial communities.

Despite challenges such as low bacterial cell yields, high-cost cofactor PQQ requirements, and the weak robustness of wild enzymes, optimizing the cultivation conditions to improve bacterial cell yield and engineering the enzyme to enhance its robustness could help overcome these limitations and maximize the potential of the isolate and enzyme for industrial applications.

#### **4. Conclusions**

In this study, a novel DON-degrading bacterial strain, *K. vulgare* D3\_3, was isolated from yellow mealworm feces and was found to completely degrade 50 mg/L of DON within 12 h under optimal conditions of pH 7.0–9.0, 30 ◦C, and anaerobic cultivation. The metabolite 3-keto-DON displayed lower cytotoxicity against GES-1 cells than its parent mycotoxin DON, but greater phytotoxicity toward duckweed. Additionally, four genes encoding PQQ-dependent dehydrogenases in the genome of isolate D3\_3 were identified as being responsible for catalyzing the oxidation of DON. These findings suggest that the strain and enzyme have potential to be developed as detoxification agents to address DON contamination in food and animal feed, despite certain challenges, such as the low yields of bacterial cells, high-cost cofactor PQQ, and weak robustness of the wild-type enzyme.

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

#### *5.1. Chemicals and Reagents*

Standard DON and 3-keto-DON, both with a purity of 98%, were bought from Pribolab Pte. Ltd. (Qingdao, China) and TripleBond Corporation (Guelph, ON, Canada), respectively. The methanol and acetonitrile for ultra-performance liquid chromatography (UPLC) and UPLC coupled to quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) analysis were of chromatographic grade and were purchased from Fisher Scientific International Inc. (Pittsburgh, PA, USA). All other chemicals and reagents were analytical-grade and obtained from various commercial sources.

### *5.2. Enrichment, Isolation, and Identification of DON-Degrading Microorganisms*

#### 5.2.1. Yellow Mealworm Feces Collection and Processing

Approximately 1 kg of yellow mealworms were bought from a pet supply market located in the Daxing District of Beijing. Upon arrival at the laboratory, they were placed in a pre-sterilized plastic container and fasted for 48 h, to empty their gastrointestinal tract of feed and produce an abundance of fecal pellets. These pellets were sifted from the yellow mealworms using a 40-mesh sieve and harvested with a sterile sampling bag. Then, 25 g of fresh feces was weighed and immersed in 500 mL of aseptic phosphate buffer (50 mM, pH 7) in a 2 L sterile Erlenmeyer flask. The mixture was homogenized via shaking for 30 min at 220 rpm and 30 ◦C, to make a 5% (*w*/*v*) fecal slurry. A small portion of this slurry served as the inoculum for subsequent enrichment culture experiments. The remaining slurry was centrifuged and subsequently filter sterilized. The resulting supernatant, namely the cell-free yellow mealworm fecal supernatant (FS), was stored at −20 ◦C for further use.

#### 5.2.2. Enrichment and Isolation Procedures

For the enrichment and isolation of DON-degrading microorganisms, two different media, namely MMFS and PYM, were employed. The MMFS medium comprised

Na2HPO4·12H2O (4.03 g/L), KH2PO4 (1 g/L), KNO3 (0.59 g/L), (NH4)2SO4 (0.5 g/L), MgSO4·7H2O (0.5 g/L), CaCl2 (0.02 g/L), FS (10:90, *v*/*v*), and 1× trace metal mixture [59]; while the PYM medium contained peptone (10 g/L), yeast extract (2 g/L), and MgSO4·7H2O (1 g/L). For the enrichment procedure, 5 mL of each medium pre-spiked with 50ppm DON was inoculated with and without 500 μL fecal slurry as the treatment samples and control samples, respectively. These cultures were continuously shaken at 220 rpm under 30 ◦C for 5 d. After the initial incubation, two cultures were subcultured four times in the respective fresh media at an inoculum ratio of 1:100. Residual DON in the final enrichment cultures was assessed via UPLC, to ascertain DON-degrading capabilities. The positive culture was diluted and plated, and randomly picked single colonies were then cultivated individually in DON-containing liquid media for 3 d before being analyzed for residual DON via UPLC to obtain the DON-degrading microbial strain.

#### 5.2.3. 16S rRNA-Based Phylogenetic Analysis of DON-Degrading Strain

A 16S rRNA-based phylogenetic analysis was conducted to determine the taxonomic position of the DON-degrading strain. Briefly, a partial 16S rRNA gene fragment was amplified via PCR using universal primers 27F (AGAGTTTGATYMTGGCTCAG) and 1492R (CGGYTACCTTGTTACGACTT). Then, the amplified product was sequenced and compared with sequences in the 16S ribosomal RNA database to obtain evolutionarily closely related 16S rRNA gene sequences using the BLASTn program. Finally, a phylogenetic tree was built using the maximum likelihood method with MEGA 11.0 software [60].

#### *5.3. Effects of Various Factors on the DON-Degrading Activity of Strain D3\_3*

In a degradation system consisting of 2 mL of MMFS medium, 50 mg/L DON, and <sup>8</sup> × 105 CFU/mL of initial cell concentration (OD = 0.025), we investigated the effects on degradation activity as a result of four factors: incubation temperature (4, 18, 30, 37, and 42 ◦C); initial pH (5.0, 6.0, 7.0, 8.0, and 9.0); addition of 2% (*w*/*v*) sugar and sugar alcohol (glucose, fructose, mannitiol, sorbitol, sucrose, and trehalose); and cultivation conditions (shaking at 220 rpm, static, and anaerobic). During the investigation of the impact of a single factor, the other parameters were maintained constant at 30 ◦C, pH = 7.0, no sugar alcohol addition, and shaking at 220 rpm. A control group was established using MMFS with DON but without inoculum. Each treatment was conducted in triplicate and its residual DON was analyzed using the UPLC technique at 12 and 72 h. The DON residual rate (%) was calculated by measuring the peak area of DON (UV absorption at 220 nm) and using the following equation:

$$\text{DON residual rate} = \text{Assample/Ac control} \times 100\% \tag{1}$$

where Asample and Acontorl are the values of the sample and control, respectively.

#### *5.4. DON and Its Metabolite Analysis*

#### 5.4.1. DON Detection Using UPLC Technique

A Thermo Scientific Dionex UltiMate 3000 system comprising a quaternary RS pump, a column oven, an autosampler, a diode array detector, and a system controller software Chromeleon 7.2 was used for UPLC analysis. First, 2 μL of sample was injected into an Acquity BEH C18 column (1.7 μm, 100 mm × 2.1 mm) and analyzed under the conditions of 40 ◦C, 0.2 mL/min flow rate, and a 220 nm detection wavelength. The elution gradients of solvent A (water) and solvent B (acetonitrile) were used as follows: 0–6 min, gradient 5% to 25% B; 6–12 min, isocratic 25% B; 12–13 min gradient 25% to 5% B; 13–18 min, isocratic 5% B.

#### 5.4.2. Analysis of DON Degradation Metabolite Using the UPLC-Q-TOF-MS Method

The DON metabolite was analyzed using a Waters Xevo G2-S quadrupole time-offlight mass spectrometer with an electrospray ionization source (negative ion mode), cou-

pled to a Waters Acquity UPLC system. Liquid chromatography was performed similarly to the DON detection method 5.4.1, with the exception of using a different gradient elution program with solvent A (water containing 5 mM ammonium formate) and solvent B (methanol) as follows: 0–1 min, isocratic 10% B; 1–19 min, gradient 10–90% B; 19–24 min, isocratic 90% B; 24–25 min, gradient 90–10% B; 25-30 min, isocratic 10% B. The ionization source conditions were set as follows: 2 kV capillary voltage; 450 ◦C desolvation temperature; 800 L/h desolvation gas (N2); 120 ◦C source temperature; and 50 L/h cone gas (N2).

#### *5.5. Toxicity Assay of DON and Its Metabolite*

#### 5.5.1. In Vitro Cytotoxicity Assay Using GES-1

The in vitro toxicity of DON and its oxidation product (3-keto-DON) were investigated using GES-1 cells and a CKK-8 test. GES-1 cells were bought from Cobioer Biosciences Co., Ltd. (Nanjing, China), and maintained under the cultivation conditions reported by Yang et al. [50]. First, 10,000 cells/well were plated into 96-well plates and allowed to proliferate to 80% confluence before cells were collected and exposed to 3-keto-DON (0.5, 1, 3, 6, 12, 24, and 48 mg/L) and DON standard (0.5, 1, 2, 4, 8, 10, and 20 mg/L) for 24 h. Following that, the spent medium was exchanged with a fresh one containing 1 mg/mL of CCK-8, which was then incubated for 4 h. Finally, cell viability was determined using an ELISA reader to measure the optical density at 450 nm. The percentage inhibition compared to control-treated cells was calculated for each compound concentration. All analyses were performed in triplicate. Statistical differences were determined using one-way ANOVA, with *p* < 0.05 considered to be statistically significant. IC50 values were calculated using Dr Fit software version 1.042 [61] with default setting parameters.

#### 5.5.2. Phytotoxicity Assay Using *Lemna minor*

To evaluate the phytotoxicity of 3-keto-DON, the growth of *Lemna minor* (common duckweed) was examined after exposure to test samples. Duckweed was collected from a pond in Daxing District, Beijing, China, disinfected, and acclimated to experimental conditions according to the method described by Megateli et al. [62]. Four duckweed fronds were aseptically inoculated into each well of 24-well plates preloaded with 2 mL of media supplemented with different test samples, including working concentrations of 100-fold, 50-fold, and 25-fold dilutions of MMFS+D3\_3+DON (derived from cell-free culture supernatant of strain D3\_3 grown for 3 d in MMFS medium supplemented with 50 mg/L of DON); 0.5, 1, and 2 mg/L of DON and 3-keto-DON standard solution; and no supplement. All treatments were performed in triplicate. After 7 d of incubation, the number of fronds was counted with a microscope. Each well was photographed, and the frond area was calculated using ImageJ software version 1.53p.

#### *5.6. Identification of DON-Oxidizing Enzyme in Strain D3\_3*

5.6.1. Sequencing, Assembly and Annotation of D3\_3' Genome, as well as Scouting Potential Genes Involved in DON Oxidation

Genome sequencing was carried out at Biomaker Technology Inc. (Beijing, China) using a combination of the Nanopore PromethION 48 system and the Illumina NovaSeq 6000 platform, and technical details on sequencing, assembly, and annotation can be found in our previous report [63].

To screen the candidate enzymes responsible for DON degradation, the two protein sequences of DepA (GenBank accession no. KFL25551.1) and AKR18A1 (GenBank accession no. ASY03293.1), which have been reported to have the catalytic function of oxidizing DON into 3-keto-DON, were individually used as queries against the genome sequence of D3\_3 for homology search using BLASTp with an E-value of 10−<sup>6</sup> using TBtools version 1.098769 [64]. Based on the results of the homology search, superfamily classifications for these candidate proteins were predicted using Superfamily 2.0 [65,66], and their signal peptides were predicted with SignalP-5.0 [67], prior to recombinant protein expression. Multiple sequence alignment of all protein sequences was performed using Clustal Omega software version 1.2.2 [68].

#### 5.6.2. Cloning, Expression, and Activity Assay for Potential DON-Oxidizing Enzymes

To verify whether the candidate enzymes suggested by the homology search results really had the function of degrading DON, we purified fourteen candidate enzymes heterologously overexpressed in *E. coli* and tested their DNA degradation function. For the DNA cloning experiment, the fourteen candidate genes encoding mature enzymes and linearized expression vector pET28a were amplified via PCR using Q5 High-Fidelity DNA Polymerase (NEB), and the detailed information of the PCR primers and cycling conditions are listed in Table S5. The fourteen DNA inserts were then individually cloned into linearized vector pET28a via the T5 exonuclease-dependent DNA assembly (TEDA) method [69], and the resulting ligation reaction mixtures were individually transformed into the expression host *E.coli* BL21(DE3)pLysS for expression as N-terminal 6-His-tag fusion proteins. Recombinant proteins were expressed in an autoinduction medium at 18 ◦C, according to the previously reported method [59], and purified using PureCube Ni-NTA Agarose (Cube Biotech), according to the manufacturer's instructions. Purified recombinant protein was assessed using SDS-PAGE and Coomassie Brilliant Blue R-250 staining. Regarding the enzymatic assay for alcohol dehydrogenase, 20 μL of each of the purified recombinant proteins was added to 180 <sup>μ</sup>L of a reaction system containing 1mM Ca2+, 100 <sup>μ</sup>M PQQ·Na2, 400 μM phenazine methosulfate (PMS), and 50 mg/L DON in 50 mM Tris-HCl buffer (pH 8). Aldo-keto reductase (AKR) activity was determined according to the method reported by He et al. [32]. After 1 h, enzyme-catalyzed reactions were stopped by adding 200 μL of methanol to the reaction systems followed by UPLC analysis.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/toxins15060367/s1, Table S1: Genomic characteristics of *Ketogulonicigenium vulgare* D3\_3; Table S2: Eight candidate genes for oxidation of C3-OH group of DON; Table S3: Six candidate genes for oxidation of C3-OH group of DON; Table S4: Percent identity matrix of amino acid sequences created with the Clustal Omega program; Table S5: DNA primers and PCR conditions.

**Author Contributions:** Conceptualization, Y.W. (Yang Wang) and B.G.; methodology, Y.W. (Yang Wang), D.Z., W.Z., and S.W. (Songshan Wang); formal analysis, Y.W. (Yang Wang), W.Z., S.W. (Songshan Wang), and Y.W. (Yu Wu); investigation, Y.W. (Yang Wang); writing—original draft preparation, Y.W. (Yang Wang); writing—review and editing, Y.W. (Yang Wang), D.Z., and B.G.; supervision, S.W. (Songxue Wang), Y.Y., and B.G.; funding acquisition, Y.W. (Yang Wang) and B.G. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by National Natural Science Foundation of China (Grant No. 31972605) and Key Research and Development Program of Ningxia, China (Grant No. 2022BBF03031).

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

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The full genomic sequence of *K. vulgare* D3\_3 has been deposited in NCBI/GenBank under BioProject number (PRJNA875257) with the GenBank accession numbers (CP103997, CP103998, CP103999, and CP104000) and BioSample number (SAMN30609498). The 16S rRNA gene was deposited under the GenBank accession number OQ102971. Additionally, the data supporting the findings of this work are available within the paper and its Supplementary Information.

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

#### **References**


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### *Article* **Dietary Catalase Supplementation Alleviates Deoxynivalenol-Induced Oxidative Stress and Gut Microbiota Dysbiosis in Broiler Chickens**

**Weiwei Wang, Jingqiang Zhu, Qingyun Cao, Changming Zhang, Zemin Dong, Dingyuan Feng, Hui Ye \* and Jianjun Zuo \***

> Guangdong Provincial Key Laboratory of Animal Nutrition Control, College of Animal Science, South China Agricultural University, Guangzhou 510642, China

**\*** Correspondence: magicsmall@scau.edu.cn (H.Y.); zuoj@scau.edu.cn (J.Z.)

**Abstract:** Catalase (CAT) can eliminate oxygen radicals, but it is unclear whether exogenous CAT can protect chickens against deoxynivalenol (DON)-induced oxidative stress. This study aimed to investigate the effects of supplemental CAT on antioxidant property and gut microbiota in DON-exposed broilers. A total of 144 one-day-old Lingnan yellow-feathered male broilers were randomly divided into three groups (six replicates/group): control, DON group, and DON + CAT (DONC) group. The control and DON group received a diet without and with DON contamination, respectively, while the DONC group received a DON-contaminated diet with 200 U/kg CAT added. Parameter analysis was performed on d 21. The results showed that DON-induced liver enlargement (*p* < 0.05) was blocked by CAT addition, which also normalized the increases (*p* < 0.05) in hepatic oxidative metabolites contents and caspase-9 expression. Additionally, CAT addition increased (*p* < 0.05) the jejunal CAT and GSH-Px activities coupled with T-AOC in DON-exposed broilers, as well as the normalized DON-induced reductions (*p* < 0.05) of jejunal villus height (VH) and its ratio for crypt depth. There was a difference (*p* < 0.05) in gut microbiota among groups. The DON group was enriched (*p* < 0.05) with some harmful bacteria (e.g., *Proteobacteria*, *Gammaproteobacteria*, *Enterobacteriales*, *Enterobacteriaceae*, and *Escherichia*/*Shigella*) that elicited negative correlations (*p* < 0.05) with jejunal CAT activity, and VH. DONC group was differentially enriched (*p* < 0.05) with certain beneficial bacteria (e.g., *Acidobacteriota*, *Anaerofustis*, and *Anaerotruncus*) that could benefit intestinal antioxidation and morphology. In conclusion, supplemental CAT alleviates DON-induced oxidative stress and intestinal damage in broilers, which can be associated with its ability to improve gut microbiota, aside from its direct oxygen radical-scavenging activity.

**Keywords:** antioxidant property; catalase; deoxynivalenol; gut microbiota; intestinal health

**Key Contribution:** Supplemental 200 U/kg catalase is beneficial in attenuating deoxynivalenolinduced oxidative stress and intestinal damage in broilers.

### **1. Introduction**

Deoxynivalenol (DON), also known as vomitoxin, is a secondary metabolite of *Fusarium graminearum*. As one of the most severe mycotoxins prevalent in multifarious crops, especially grains [1,2], DON can elicit serious detriments to animal growth and health when diets are prepared with DON-contaminated grains [3,4]. Animals exposed to DON may exhibit acute or chronic poisoning, as manifested by the structural and functional injuries of multiple organs (e.g., liver, kidney, and intestine), accompanied with a series of clinical symptoms, such as vomiting, anorexia, diarrhea, and intestinal bleeding [5,6]. Despite the lesser susceptibility to DON exposure than other monogastric animals (e.g., pigs), poultry exposed to DON can still display metabolic abnormalities and health disorders [5,6].

**Citation:** Wang, W.; Zhu, J.; Cao, Q.; Zhang, C.; Dong, Z.; Feng, D.; Ye, H.; Zuo, J. Dietary Catalase Supplementation Alleviates Deoxynivalenol-Induced Oxidative Stress and Gut Microbiota Dysbiosis in Broiler Chickens. *Toxins* **2022**, *14*, 830. https://doi.org/10.3390/ toxins14120830

Received: 20 September 2022 Accepted: 25 November 2022 Published: 28 November 2022

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

**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/).

One of the toxicological mechanisms of DON for animals is to cause mitochondrial dysfunction, leading to increased generation of reactive oxygen radicals, concurrent with decreased production of antioxidants in the cells [6,7], which can break the redox balance inside the body [8]. Once the accumulation of oxygen radicals exceeds their elimination, there can be oxidative stress responses within the body with a peroxidation and destruction of certain biomacromolecules, especially DNA and proteins [8], subsequently impairing animal growth and health [6,8]. Thereby, suppression of the inducive oxidative stress is a potential strategy to attenuate the toxic effects of DON in chickens.

Antioxidant enzymes play crucial roles in contributing to the scavenging actions of antioxidant system on various free radicals generated in the body [9]. Among the antioxidant enzymes, catalase (CAT) is a key member capable of catalyzing the decomposition of hydrogen peroxide (H2O2), which is a kind of reactive oxygen radicals and may trigger oxidative stress of the body when it exceeds the physiologic concentration [10]. More importantly, CAT can prevent reactions of H2O2 with oxygen under the action of iron chelates from producing more toxic hydroxyl radicals (·OH) and some other radicals [11]. Additionally, certain hydrogen donors, such as methanol and ethanol, may also be scavenged during the catalytic reaction of CAT, which can benefit the mitigation of oxidative stress [11,12]. Previous reports have shown that dietary CAT addition resulted in an enhancement of antioxidant property, with simultaneous improvements of growth and health performance in both broilers and pigs [13,14]. Moreover, CAT addition was reported to alleviate oxidative stress-induced intestinal and hepatic damages in pigs [15–17]. However, few studies are available concerning the potentially beneficial roles of CAT in alleviating the detriments of DON to chickens.

Gut microbiota elicit profound roles in mediating the impacts of dietary treatments on chicken growth and health [18]. There is evidence that dietary mycotoxin, including DON contamination-induced toxicity and oxidative damages in animals, were at least partially realized by the inducive disturbance of gut microbiota [19–21]. Growing studies implied that CAT addition had an ability to enhance antioxidant capacity, as well as improve intestinal and hepatic health, through associating with an optimization of gut microbial composition in both broilers [14] and pigs [16,22]. Nevertheless, the protective effects of CAT on the antioxidant property and gut microbiota in chickens exposed to DON remain unclear. Accordingly, the present study aimed to investigate the potential roles of supplemental CAT in alleviating DON-induced oxidative stress and gut microbiota dysbiosis in broiler chickens.

#### **2. Results**

#### *2.1. Growth Performance and Organ Indexes*

As shown in Table 1, there were no differences (*p* > 0.10) in the initial body weight (IBW) and final body weight (FBW), average daily gain (ADG), and average daily feed intake (ADFI) coupled with feed conversion ratio (FCR) among groups. However, the DON group showed a decreasing trend (*p* = 0.093) of average daily feed intake (ADFI), relative to the control or DONC groups. Regarding the organ indexes (Table 2), the indexes of spleen and bursa of Fabricius showed no differences (*p* > 0.10) among groups. The liver index and kidney index in the DONC group were lower (*p <* 0.05) and tended to be higher (*p =* 0.078), respectively, than those of the DON group, but similar to (*p* > 0.10) the control group.

**Table 1.** Effect of catalase on growth performance <sup>1</sup> of broilers exposed to deoxynivalenol (DON).


**Table 1.** *Cont.*


<sup>1</sup> IBW, initial body weight; FBW, final body weight; ADG, average daily gain; ADFI, average daily feed intake; FCR, feed conversion ratio. <sup>2</sup> Broilers in control and DON group were fed a basal diet without and with DON contamination, respectively, while those in DONC group were fed a DON-contaminated basal diet supplemented with 200 U/kg catalase.



a,b Values within a row with unlike superscript letters differ significantly (*p* < 0.05). <sup>1</sup> Organ indexes were calculated as the ratio of organ weight (g) to body weight (kg). <sup>2</sup> Broilers in control and DON group were fed a basal diet without and with DON contamination, respectively, while those in DONC group were fed a DON-contaminated basal diet supplemented with 200 U/kg catalase.

#### *2.2. Oxidative Status of the Intestine and Liver*

As shown in Table 3, the contents of oxidative metabolites, including reactive oxide species (ROS), superoxide anion (O2 −), hydroxyl radical (OH), 8-hydroxy-2 -deoxyguanosine (8-OHdG), and malondialdehyde (MDA) in the jejunum, did not differ (*p* > 0.05) among groups. However, the DON group showed increases (*p <* 0.05) in hepatic ROS, 8-OHdG, and MDA contents, relative to the control group. Supplemental CAT normalized hepatic ROS and 8-OHdG contents in DON-exposed birds to levels similar (*p* > 0.05) to the control group, and it abolished (*p <* 0.05) the DON-induced increase in hepatic MDA content. Regarding the antioxidant indicators, jejunal CAT and GSH-Px activities, along with hepatic T-AOC, were detected to be lower (*p* < 0.05) in the DON group versus control group. However, supplementing CAT to DON-exposed birds attenuated (*p <* 0.05) the decreased activities of jejunal CAT and GSH-Px and increased (*p <* 0.05) jejunal T-AOC.

**Table 3.** Effect of catalase on antioxidant parameters <sup>1</sup> of broilers exposed to DON.



**Table 3.** *Cont.*

a,b,c Values within a row with unlike superscript letters differ significantly (*p* < 0.05). <sup>1</sup> ROS, reactive oxygen species (ng/mL); O2 −, superoxide anion (nmol/mL); OH, hydroxyl radical (ng/mL); 8-OHdG, 8-hydroxy-2 deoxyguanosine (ng/mL); MDA, malondialdehyde (nmol/mL); CAT, catalase (U/mL); GSH-Px, glutathione peroxidase (U/mL); T-AOC, total antioxidant capacity (U/mL). <sup>2</sup> Broilers in control and DON group were fed a basal diet without and with DON contamination, respectively, while those in DONC group were fed a DON-contaminated basal diet supplemented with 200 U/kg catalase.

#### *2.3. Relative mRNA Expression of Antioxidation- and Apoptosis-Related Genes*

As exhibited in Figure 1, the DON group displayed increases (*p* < 0.05) in the relative expression levels of nuclear factor erythroid-2 related factor (Nrf2) and Bcl-2 associated X protein (Bax) in the jejunum, together with heme oxygenase 1 (HO-1) and Caspase-9 in the liver, as compared with the control group. Supplementing CAT to DON-exposed birds did not change (*p* > 0.05) the relative expression of jejunal Nrf2 and Bax or hepatic HO-1. However, DON-exposed birds supplemented with CAT supported a similar (*p* > 0.05) expression of hepatic Caspase-9, relative to the control group.

**Figure 1.** Effect of catalase on the relative mRNA expression of antioxidation-related genes (Nrf2 and HO-1) and apoptosis-related genes (Bcl-2, Bax and Caspase-9) in the jejunum (**A**) and liver (**B**) of broilers exposed to DON. Broilers in control and DON groups were fed a basal diet without and with DON contamination, respectively, while those in DONC group were fed a DON-contaminated basal diet supplemented with 200 U/kg catalase. a,b Values with unlike superscript letters differ significantly (*p* < 0.05). Nrf2, nuclear factor erythroid-2 related factor; HO-1, heme oxygenase 1; Bcl-2, B-cell lymphoma-2; Bax, Bcl-2 associated X protein.

#### *2.4. Intestinal Morphological Structure*

As presented in Figure 2A, jejunal villi in the control group were straight with a completed structure, while the DON group had a destruction of jejunal morphological structure, as evidenced by the breakage, shedding, and shortening of villi. However, the above phenomena were alleviated when birds were supplemented with CAT. Concretely, the broilers in the DON group had reduced (*p <* 0.05) villus height (VH) and villus height to crypt depth ratio (VCR), rather than crypt depth (CD) of the jejunum, compared with those in control group (Figure 2B–D). Nevertheless, the DON-induced reduction of jejunal VCR

was reversed (*p <* 0.05) by CAT addition, which also rendered jejunal VH of DON-exposed birds, comparable (*p* > 0.05) to that in the control birds.

**Figure 2.** Effect of catalase on jejunal morphological structure of broilers exposed to DON. (**A**) Illustration (magnification 200×) of jejunal morphology of broilers from different groups. (**B**) Jejunal villus height of broilers from different groups. (**C**) Jejunal crypt depth of broilers from different groups. (**D**) Villus height to crypt depth ratio of the jejunum of broilers from different groups. a,b Values with unlike superscript letters differ significantly (*p* < 0.05). Broilers in control and DON groups were fed a basal diet without and with DON contamination, respectively, while those in DONC group were fed a DON-contaminated basal diet supplemented with 200 U/kg catalase.

#### *2.5. Gut Microbiota*

#### 2.5.1. Diversity of Gut Microbiota

No difference (*p* > 0.05) was noted in the α-diversity indexes of gut microbiota among the groups (Figure S1). β-Diversity analysis manifested a difference (*p* < 0.05) in the similarity of gut microbiota among the groups (Figure 3A). This could be visualized by partial least squares discriminant analysis (PLS-DA) plot (Figure 3B), which revealed a distinct separation of microbiota among the groups.

#### 2.5.2. Gut Microbial Composition

As shown in Figure S2, the predominant phylum of broiler gut was *Firmicutes*, followed by *Proteobacteria*. Within *Firmicutes*, the majority belonged to the classes *Clostridia* and *Bacilli*, while the majority within *Proteobacteria* were *Gammaproteobacteria*. Orders level analysis showed that the gut of the control group was mainly occupied by *Oscillospirales* and *Lachnospirales*, while the DON and DONC groups were dominated by *Oscillospirales*, *Lachnospirales*, and *Enterobacteriales*. At the family level, the major members in control group were *Lachnospiraceae* and *Ruminococcaceae*, while those in the DON and DONC groups were *Lachnospiraceae*, *Ruminococcaceae*, and *Enterobacteriaceae*. The dominating genera in the control group were *Faecalibacterium* and the unclassified *Lachnospiraceae*, while those in DON group were *Escherichia*/*Shigella* and the unclassified *Lachnospiraceae*. In comparison, DONC group was dominated by *Escherichia*/*Shigella* and *Ruminococcus torques group*.

**Figure 3.** β-Diversity of broiler gut microbiota. (**A**) ANOSIM analysis (similarity analysis); (**B**) partial least squares discriminant analysis (PLS-DA). Broilers in control and DON groups were fed a basal diet without and with DON contamination, respectively, while those in DONC group were fed a DON-contaminated basal diet supplemented with 200 U/kg catalase.

#### 2.5.3. Bacterial Richness among Groups

Bacterial richness (*p* < 0.05, linear discriminant analysis score (LDA) > 2.0) was identified by LDA combined effect size measurements (LEfSe) analysis. As illustrated in Figure 4, certain bacterial members, such as phylum Firmicutes, class Clostridia, orders Oscillospirales and Peptococcales, and families Ruminococcaceae and Peptococcaceae, together with genera Faecalibacterium, Flavonifractor, and Paludicola, were detected to be enriched in control group. Strikingly, the phylum Proteobacteria, class Gammaproteobacteria, order Enterobacteriales, family Enterobacteriaceae, and genus Escherichia/Shigella were enriched in the DON group. In comparison, the DONC group was differentially enriched with phylum Acidobacteriota, order Eubacteriales, and family Anaerofustaceae, along with genera Anaerofustis and Anaerotruncus.

**Figure 4.** Linear discriminant analysis (LDA) combined effect size measurements (LEfSe) analysis of bacterial richness (*p* < 0.05, LDA > 3.0) in gut microbiota of broilers. Broilers in control and DON groups were fed a basal diet without and with DON contamination, respectively, while those in DONC group were fed a DON-contaminated basal diet supplemented with 200 U/kg catalase.

#### 2.5.4. Correlations of Gut Microbiota with Other Intestinal Parameters

Spearman's correlation analysis was used to identify associations between gut microbiota and other intestinal parameters among groups. As shown in Figure S3, there were no correlations between gut microbiota and intestinal gene expression. However, the abundance of phylum Firmicutes showed a positive correlation (*p* < 0.05) with jejunal CAT activity and VH (Figure 5), whereas a contrasting pattern was found for the phylum Proteobacteria and its affiliate members, including class Gammaproteobacteria, order Enterobacteriales, family Enterobacteriaceae, and genus Escherichia/Shigella. The abundance of phylum Acidobacteriota had a positive correlation (*p* < 0.05) with jejunal T-AOC. The class Clostridia, order Peptococcales, and genus Flavonifractor were positively correlated (*p* < 0.05) with jejunal CAT activity, and the order Oscillospirales was positively correlated (*p* < 0.05) with jejunal CAT activity and VCR. Additionally, the genus Faecalibacterium elicited a positive correlation (*p* < 0.05) with jejunal VH.

**Figure 5.** *Cont*.

**Figure 5.** Correlation analysis of gut microbiota (**A**) at phyum level; (**B**) at class level; (**C**) at order level; (**D**) at family level; (**E**) at genus level) with intestinal antioxidant property and morphology in broilers. VH, villus height; CAT, catalase; VCR, villus height to crypt depth ratio; GSH-Px, glutathione peroxidase; T-AOC, total antioxidant capacity. The red and blue panes represent positive and negative correlations, respectively. Color intensity means the Spearman's r-value of correlations in each panel. The asterisks indicate significant correlations (\* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001). Broilers in control and DON groups were fed a basal diet without and with DON contamination, respectively, while those in DONC group were fed a DON-contaminated basal diet supplemented with 200 U/kg catalase.

#### **3. Discussion**

The presence of a control + CAT group is not necessary for this study, based on the following two reasons: (1) this study was aimed to evaluate the efficacy of CAT in alleviating DON-induced detriments to broilers; (2) the beneficial effects of CAT addition on the growth and antioxidant capacity of both broilers and pigs under normal status had been confirmed in either our previous experiments or other studies [13,14].

DON contamination of feedstuffs, especially grains, has been an increasingly common problem, as it can cause growth retardation in animals [1,5]. There is evidence that poultry are far less sensitive to DON, relative to other monogastric animals (e.g., pigs), based on a previous study in which poultry showed no clinical response to a DON level of less than 20 mg/kg DON in diets, whereas 1–2 mg/kg DON caused toxicity in pigs [5]. Similarly, Dersjant-Li et al. [23] reported that dietary DON level below 16 mg/kg elicited marginal impact on broiler growth. Azizi et al. [24] recorded little impairment except for a reduction of feed intake during the early stage of growth of broilers when fortifying DON in diet at 10 mg/kg. In this study, dietary DON fortification at a subclinical level (an estimate of 7 mg/kg) caused little compromise of broiler growth performance except for a decreasing trend of ADFI. Supplemental CAT failed to improve growth performance of broilers exposed to DON, which did not agree with the study of Tang et al. [14] who found increases in weight gain, feed intake and feed efficiency of non-challenged broilers in response to CAT addition. The discrepancy might be due to the difference in raising condition of broilers.

Organ index namely the relative organ weight is a common indicator of health status in chickens. The effect of DON contamination of diets on broiler organ index revealed in previous studies is quite conflicting. For example, broilers fed a diet containing 5 mg/kg DON was reported to have negligible change in organ index [25], whereas it was also manifested

to increase spleen index [26]. When fortifying DON at 10 mg/kg in diet, broilers were found to show an increase in jejunum index and a reduction of the index of bursa of Fabricius in the study of Awad et al. [27] and Wu et al. [28], respectively, with no changes in the indexes of other organs. The highly variable outcomes in these studies might be associated with the exposure time of DON and broiler breeds. In this study, we recorded an increase in the liver index, with a decreasing trend of kidney index in the broilers exposed to DON at a subclinical dosage (an estimate of 7 mg/kg). The increase in liver index might be ascribed to the fact that the increased free radicals induced by DON exposure perturbed protein synthesis via attack on ribosome and subsequently triggered a compensatory swelling of liver (the main site of protein synthesis) [8,29], while the decreasing trend of kidney index was presumably due to the inductive atrophy of kidney [30]. At present, no published study was available regarding the effects of CAT on organ index in animals. Herein, we noted that supplemental CAT abolished the increase in liver index and tended to reverse the decreasing trend of the kidney index of DON-exposed broilers. It was possible that the elimination of free radicals upon CAT addition resulted in less injuries of liver and kidney [22,31], thereby attenuating liver swelling and kidney atrophy.

The resultant redox imbalance of broiler organs, such as the intestine and liver, resulting from DON exposure have been well-established [5,32]. Analogously, this study manifested that broilers exposed to DON exhibited a disturbance of redox status, as exhibited by the increased contents of hepatic oxidative metabolites (ROS, MDA, and 8-OHdG), as well as impaired antioxidant property (decreases in hepatic T-AOC with jejunal CAT and GSH-Px activities). It is well-established that ROS represent the major kinds of free radicals with the ability to attack the unsaturated fatty acids in cell membrane, which cause lipid peroxidation chain reaction and, finally, generate a large amount of MDA [33]. The 8-OHdG, a biomarker of DNA oxidative damage, is also produced by ROS attacking the carbon atom of guanine base in DNA [34]. CAT and GSH-Px can prevent from redox imbalance by specifically scavenging hydrogen peroxide and lipid hydroperoxides, respectively [10]. In this study, the increased production of oxidative metabolites, together with impaired antioxidant property, due to DON exposure, was deduced to cause oxidative stress and the biomacromolecular damage of broilers [5,7]. However, supplementing CAT to DON-exposed birds normalized the hepatic ROS and 8-OHdG contents, alleviated the increase in hepatic MDA content with decreases in jejunal CAT and GSH-Px activities, and increased jejunal T-AOC. These results highlighted that exogenous CAT was beneficial for attenuating DON-induced oxidative stress in broilers. Likewise, some previous studies reported that dietary CAT addition improved redox status by enhancing the activities of antioxidant enzymes (SOD, CAT, and GSH-Px) and lowering oxidative metabolite MDA in the liver and intestine of both broilers [14] and pigs [16].

In order to cope with the tissue damage induced by free radicals, the body has evolved a complex mechanism responding to oxidative stress, among which Keapl/Nrf2-ARE is the most important endogenous pathway regulating the redox status inside the body [34]. Keap1, a cytosol binding protein of Nrf2, can bind to Nrf2 to prevent it from entering the nucleus under normal status, thereby avoiding the increase of cell sensitivity to stressors [34]. However, the overproduction of free radicals may activate Nrf2, which is then released from Keap1, enters the nucleus, and interacts with the antioxidant response element (ARE), subsequently promoting the expression of a series of downstream antioxidants and eliminating excess free radicals [35]. HO-1 is a crucial antioxidant enzyme with a binding site of a promoter similar to that of ARE, thus being targeted by Nrf2 [34]. It was emphasized that DON exposure-induced oxidative stress, which could be mediated by the Nrf2/HO-1 pathway [35]. Similarly, the current study revealed an upregulation of jejunal Nrf2 and hepatic HO-1 expression in broilers, due to DON exposure, which could be a feedback response of the host defense mechanism to the inducive oxidative stress. Strikingly, CAT addition did not affect either Nrf2 or HO-1 expression in DON-exposed broilers, demonstrating that exogenous CAT probably moderated DON-induced oxidative stress by directly eliminating radical accumulation and subsequently reducing the

consumption of antioxidant enzymes, instead of promoting the expression of antioxidant enzyme genes via the Nrf2/HO-1 pathway. DON-induced oxidative stress was known to closely involve in cell apoptosis [8], as supported by the findings that DON exposure increased the expression of several key pro-apoptosis genes (e.g., Bax and Caspase family proteins) and anti-apoptosis gene Bcl-2, as well as their ratio (Bax/Bcl-2) in the intestine and liver [36,37]. Herein, we observed that broilers exposed to DON displayed an increased expression of jejunal Bax and hepatic caspase-9 (a critical mediator of cell apoptosis), highlighting an initiation of cell apoptosis in the jejunum and liver of broilers, in response to DON contamination in diet. When supplemented with CAT, the increased expression of hepatic caspase-9 in DON-exposed broilers was normalized, demonstrating a potential of CAT addition to mitigate DON-induced hepatic cell apoptosis in broilers to some extent. This was similar to some previous studies in which CAT addition decreased the mRNA expression of Bax and its ratio to Bcl-2 expression in the liver, as well as the mRNA and protein levels of hepatic and intestinal caspase family proteins (caspase-3 and -9) in pigs confronted with an oxidative stress induced by lipopolysaccharide [16,22].

Oxidative stress in broilers resulting from dietary DON contamination has been documented to trigger a destruction of the intestinal structure [24,28]. As expected, the present study showed that DON exposure led to an impairment of the jejunal morphology, as evidenced by the breakage, shedding, and incompleteness of the villi, together with reductions in VH and VCR. These were likely connected with the detected redox imbalance of jejunum. However, supplementing CAT to DON-exposed broilers normalized jejunal VH and VCR and might, in turn, favor maintenance of intestinal absorption and barrier function. This benefit was speculated to be responsible by the observed protective effect of CAT addition on jejunal antioxidant enzyme activities in DON-exposed broilers, because it has evidenced that exogenous CAT ameliorated intestinal morphological structure, probably through associating with the simultaneous increase in intestinal antioxidant capacity in both broilers [14] and pigs [16].

Gut microbiota are well-known for exerting essential roles in regulating host growth and health. Increasing studies verified that DON exposure-induced oxidative damage was involved in the gut microbiota disturbance of animals [18,28]. On the other hand, CAT addition was reported to promote intestinal and hepatic health via an association with improvement of gut microbiota in animals [14,16,22]. Similarly, the PLS-DA plot in this study disclosed a distinct shift of gut microbiota of broilers following DON exposure; however, this shift was alleviated by CAT addition. Bacterial richness analysis supported changes in gut microbial composition among groups. Thereinto, the control group was enriched with certain beneficial bacteria, such as *Firmicutes*, *Clostridia*, *Oscillospirales*, *Peptococcales*, *Ruminococcaceae*, *Faecalibacterium*, and *Flavonifractor*. In general, *Firmicutes* and *Clostridia* are the predominating commensal bacteria in animal gut and encompass plentiful potentially beneficial bacteria, therefore benefiting intestinal health of host [38,39]. *Oscillospirales*, *Peptococcales*, *Faecalibacterium*, *Ruminococcaceae*, and *Flavonifractor* were characterized as producers of butyric acid [40–42], which has a strong ability to enhance the antioxidant properties of broilers [43]. In the current study, the abundances of *Firmicutes*, *Clostridia*, *Oscillospirales*, *Peptococcales*, *Faecalibacterium*, and *Flavonifractor* were positively correlated with intestinal CAT activity and/or VH, suggesting that the enrichments of these bacterial members in gut could be conducive to intestinal redox homeostasis of broilers in control group. Comparatively, several potentially pathogenic or harmful bacterial members, including *Proteobacteria*, *Gammaproteobacteria*, *Enterobacteriales*, *Enterobacteriaceae*, and *Escherichia*/*Shigella* were enriched in the DON group. *Proteobacteria* includes a mass of typical pathogens, such as *Salmonella*, *Shigella*, *Klebsiella*, and pathogenic *Escherichia coli* that can generate considerable toxins, thereby serving as a momemtous indicator of gut microbiota disturbance and health disorders of animals [44,45]. It has been documented that the expansions of *Proteobacteria*, *Gammaproteobacteria*, *Enterobacteriales*, *Enterobacteriaceae*, and *Escherichia*/*Shigella* in the gut could cause accumulation of lipopolysaccharide, rendering the occurrence of oxidative stress in animals [19,46–49]. Herein, we detected

negative correlations of the abundances of *Proteobacteria*, *Gammaproteobacteria*, *Enterobacteriales*, *Enterobacteriaceae*, and *Escherichia*/*Shigella* with both intestinal CAT activity and VH, demonstrating that the enrichments of these bacteria in the DON group conduced the simultaneous intestinal oxidative damage of broilers. In contrast to the DON group, the DONC group was differentially enriched with several potentially beneficial bacteria, such as the phylum *Acidobacteriota*, along with the genera *Anaerofustis* and *Anaerotruncus*. It was indicated that *Acidobacteriota* might favor the suppression of intestinal oxidative stress, due to its connection with intestinal anti-inflammation and the antioxidation of animals [50,51]. Likewise, this study disclosed a positive correlation of the abundance of *Acidobacteriota* with intestinal T-AOC of broilers. *Anaerofustis* and *Anaerotruncus* in the gut were indicated to prompt fiber digestion and the production of short-chain fatty acids that might allow for improvements of the intestinal antioxidant properties and morphological structures of animals [28,52–54]. Accordingly, the intestinal enrichments of *Acidobacteriota*, *Anaerofustis*, and *Anaerotruncus* due to CAT addition were probably associated with the observed alleviation of the intestinal oxidative damage of broilers exposed to DON. This was similar to a previous study in which dietary CAT addition improved the intestinal antioxidant capacity and gut microbial composition in broilers [14].

#### **4. Conclusions**

Supplemental CAT had a capacity to attenuate oxidative stress and intestinal injury of broilers exposed to DON. It is possible that the improved gut microbial composition (reflected by the enrichments of several beneficial bacteria) following CAT addition contributed to the observed protection against DON-induced oxidative damage in broilers. Our findings provided a strategy for limiting the detriments of dietary DON contamination to poultry.

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

#### *5.1. Animals and Experimental Design*

The experimental animal protocols for the present study were approved by the Animal Care and Use Committee of the South China Agricultural University. A total of 144 oneday-old Lingnan yellow-feathered male broilers were randomly assigned to 3 groups, with 6 replicates per group and 8 birds per replicate. The initial body weight of birds was similar across replicates. Broilers fed a basal diet were considered the control group, and the other two groups received a DON-contaminated basal diet added with 0 (DON group) or 200 U/kg CAT (DONC group). The content of DON (1.3 mg/kg) in basal diet was estimated using an AgraQuant® DON enzyme-linked immunosorbent assay (ELISA) kit (Romer Labs, Getzersdorf, Austria) with a Multiskan SkyHigh Microplate Reader (Thermo Fisher Scientific, Waltham, MA, USA). Dietary DON fortification was performed by supplementing DON-enriching rice to basal diet at the expense of corn. The final content of DON in diet was estimated at 7 mg/kg, which could be viewed as a subclinical dosage, as it was less than the clinical dosage (greater than 16 mg/kg) estimated in a previous study [23], but exceeded the maximum allowable dietary level (3 mg/kg), according to the Chinese Hygienical Standard for Feeds (GB13078-2017). CAT preparation (the theoretical value of enzyme activity was 200 U/g) was obtained from Vetland Bio-Technology Co., Ltd. (Shenyang, China). The actual activity of CAT in this supplement was determined to be 197 U/g. The supplemental CAT level was selected based on our preliminary experiment. Birds were housed in two-tier cages in an environmentally controlled room, in which the lighting program was 16 h per day, and room temperature was kept around 34 ◦C during the first three days and then gradually decreased to 24 ◦C on d 21. Birds had free access to the mash feed and water. The composition of basal diet is exhibited in Table S1. At 21 day (d) of age, birds were randomly selected from each replicate (6 birds/group) for determination of growth performance and sample collection. After sacrifice of these birds, visceral organs, including the liver, spleen, kidney, and bursa of Fabricius and intestines were separated. The midpoint of jejunal section was removed and cleaved into two segments, one of which

was fixed in 4% paraformaldehyde solution, while the other one was quick-froze by liquid nitrogen and reserved at −80 ◦C. Moreover, cecal digesta was collected for sequencing analysis of gut microbiota.

#### *5.2. Fabrication of DON-Enriching Rices*

DON-enriching rices were fabricated according to the following procedures: (1) the *Fusarium graminearum* strain PH-1, kindly provided by Prof. Chenglan Liu (College of Plant Protection, South China Agricultural University), was revived, the resulting hyphae were harvested and aerobically plated on potato dextrose agar medium at 25 ◦C for 7 d; (2) fresh rices were placed into conical flasks and soaked with pure water overnight, followed by autoclaved sterilization; (3) the plating medium containing *Fusarium graminearum* hyphae was split into smaller portions and evenly scattered on the sterile rice, followed by static culture at 25 ◦C for 3 d with a subsequent shake culture at 25 ◦C for 18 d under aerobic condition. The contaminated rice were then collected, dried at 50 ◦C, and smashed, and the contents of DON (117.12 mg/kg) and its acetylated derivatives 3-acetyl-DON (2.39 mg/kg) and 15-acetyl-DON (8.03 mg/kg) in the powder were determined using an AgraQuant® DON ELISA kit (Romer Labs, Getzersdorf, Austria) and a liquid chromatography/mass spectrometry system (LCMS-8060, Shimadzu, Kyoto, Japan), respectively. The contents of some other mycotoxins (Table S2) were also quantified using the corresponding ELISA kits (Romer Labs, Getzersdorf, Austria).

#### *5.3. Determination of Growth Performance and Organ Indexes*

Body weight and feed intake were recorded for each replicate on d 21 for calculating the average body weight (ABW) of broilers at 21 d of age, along with average daily gain (ADG), average daily feed intake (ADFI), and feed conversion ratio (FCR) during 1–21 d of age. The collected organs, including liver, spleen, kidney, and bursa of Fabricius were weighed for determination of organ indexes, as calculated by the ratio of organ weight (g) to body weight (kg).

#### *5.4. Measurement of Oxidative Status*

The liver and jejunum samples were separately homogenized with 1:9 (*w*/*v*) cold saline, followed by centrifugation in a high-speed refrigerated centrifuge (6380R, Eppendorf, Hamburg, Germany) at 6000 rpm for 10 min at 4 ◦C to obtain the supernatants. The levels of oxidative metabolites, including reactive oxygen species (ROS), superoxide anion (O2 *−***)**, hydroxyl radical (OH), 8-hydroxy-2 -deoxyguanosine (8-OHdG), and malondialdehyde (MDA), as well as the antioxidant indices, including the total antioxidant capacity (T-AOC), the levels of glutathione (GSH) coupled with the activities of CAT, total superoxide dismutase (T-SOD), and glutathione peroxidase (GSH-Px) in the supernatants were measured by micromethods using the corresponding kits provided by Meimian Bioengineering Institute (Nanjing, China) under the manufacturer's instructions.

#### *5.5. Measurement of Gene Expression*

Total RNA from the jejunum and liver samples was isolated and purified using the FastPure Cell/Tissue Total RNA Isolation Kit (Vazyme Biotech. Co., Ltd., Nanjing, China) under the corresponding instructions. The concentration of isolated RNA was measured with a NanoDrop-2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). RNA purity was estimated by detecting the absorbance ratio at 260:280 nm. RNA integrity was verified via detection of the 18S and 28S bands in 1% agarose gel electrophoresis. Thereafter, RNA samples were reverse transcribed to cDNA samples by using the HiScript II qRT SuperMix (Vazyme Biotech. Co., Ltd., Nanjing, China). Real-time PCR for examining gene expression was implemented in a CFX96Touch Real-Time PCR system (Bio-Rad Laboratories, Hercules, CA, USA) using the 2×Taq Master Mix (Vazyme Biotech. Co., Ltd., Nanjing, China). Reduced glyceraldehyde-phosphate dehydrogenase (GAPDH) acted as a reference gene. Information for the primers of GAPDH and target genes, including

nuclear factor erythroid-2 related factor (Nrf2), heme oxygenase 1 (HO-1), B-cell lymphoma-2 (Bcl-2), Bcl-2 associated X protein (Bax), and caspase-9, are displayed in Table 4. The relative mRNA expressions of genes were calculated using the 2−ΔΔCt method [55].

**Table 4.** Sequences for real-time PCR primers.


<sup>1</sup> *GAPDH*: reduced glyceraldehyde-phosphate dehydrogenase, *Nrf2*: nuclear factor erythroid 2-related factor 2, *HO-1*: heme oxygenase 1, *Bax*: B-cell lymphoma-2, *Bcl-2:* Bcl-2 associated X protein. <sup>2</sup> F, forward; R, reverse.

#### *5.6. Analysis of Intestinal Morphological Structure*

The fixed jejunal tissues were embedded in paraffin and stained with hematoxylineosin to obtain cross-sections. The intact and representative villi selected from each section were used for determining intestinal morphological structure with a light microscope. Villus height (VH) and crypt depth (CD) were defined as the height from villous tip to villus-crypt joint, respectively, based on which villus height to crypt depth ratio (VCR) was then calculated.

#### *5.7. High-Throughput Sequencing of Gut Microbiota*

Bacterial genomic DNA was extracted from cecal digesta using NucleoSpin® DNA Stool kit (Macherey-Nagel company, Düren, Germany). The concentration and quality of extracted DNA were checked using Nanodrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA) and gel electrophoresis. Bacterial 16S rDNA sequences spanning the variable regions (V3-V4) were amplified using primers 338F (5 -ACT CCT ACG GGA GGC AGC A-3 ) and 806R (5 -GGA CTA CHV GGG TWT CTA AT-3 ). The amplified products were paired-end sequenced on an Illumina Novaseq platform (Illumina, San Diego, CA, USA) at Biomarker BioTech. Inc. (Beijing, China). The effective reads were clustered into operational taxonomic units and classified at various taxonomic levels based on a 97% sequence similarity. Bacterial α-diversity was analyzed using the QIIME2 software, and bacterial β-diversity was assessed by the partial least squares discriminant analysis (PLS-DA). The differences in bacterial abundances among groups were detected using the linear discriminant analysis (LDA) combined effect size measurements (LEfSe) analysis. Spearman correlation analysis was used for detecting the correlations between bacterial composition and other parameters.

#### *5.8. Statistical Analysis*

Data are expressed as the mean ± standard deviation and analyzed by one-way ANOVA using the general linear model procedure of SPSS 20.0. Differences among groups were examined using Duncan's multiple comparisons. Statistical significance was set at *p* < 0.05, and 0.05 ≤ *p* < 0.10 was thought as a tendency towards significance.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/toxins14120830/s1. Tables S1 and S2 and Figures S1–S3. Table S1: Composition of the basal diet (air-dry basis). Table S2: The contents of some mycotoxins aside from deoxynivalenol and its acetylated precursors in DON-enriching rice powder. Figure S1: Alphadiversity analysis of gut microbiota among groups. (A) Shannon index; (B) Simpson index; (C) Chao1 index; (D) ACE index. Control, birds were fed a basal diet; DON, birds were fed a basal diet contaminated with DON; DONC, birds were fed a DON-contaminated basal diet supplemented with 200 U/kg CAT. Figure S2: Gut microbial composition of broilers at phylum (A), class (B), order (C), family (D), and genus (E) levels. Control, birds were fed a basal diet; DON, birds were fed a basal diet contaminated with DON; DONC, birds were fed a DON-contaminated basal diet supplemented with 200 U/kg CAT. Figure S3: Correlation analysis between gut microbiota (A) at phyum level; (B) at class level; (C) at order level; (D) at family level; (E) at genus level) and intestinal gene expression in broilers. Bax, Bcl-2 associated X protein; Nrf2, nuclear factor erythroid-2 related factor. The red and blue panes represent positive and negative correlations, respectively. Color intensity means the Spearman's r-value of correlations in each panel. The asterisks indicate significant correlations (\* *p* < 0.05; \*\* *p* < 0.01). Control, birds were fed a basal diet; DON, birds were fed a basal diet contaminated with DON; DONC, birds were fed a DON-contaminated basal diet supplemented with 200 U/kg CAT.

**Author Contributions:** W.W. contributed to the conceptualization of this work and wrote the manuscript. J.Z. (Jingqiang Zhu) conducted the investigation. Q.C. contributed to parameter determination. C.Z. assisted with bioinformatic analysis. Z.D. contributed to data curation. D.F. administrated this project. H.Y. supervised this work. J.Z. (Jianjun Zuo) revised the manuscript and obtained the funding. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was financially supported by the Science and Technology Planning Project of Guangzhou, the National Natural Science Foundation of China (No. 32102584) and the Modern Feed Industry Innovation Team Project of Guangdong Province (No. 2021KJ115).

**Institutional Review Board Statement:** This study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Animal Care and Use Committee of the South China Agricultural University (No. SCAU20210612, date of approval 12 June 2021). The welfare of all chickens was guaranteed in accordance with the national standard Laboratory Animal Requirements of Environment and Housing Facilities (GB 14925–2001).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The datasets used in this study are available from the corresponding author upon reasonable request.

**Acknowledgments:** We gratefully thank Yiliang Chen, Zheng Fan, Chong Ling, and Shuming Zhang (South China Agricultural University) for their assistance with sample collection.

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

#### **References**

