*Article* **Transcriptomics Reveals the Effect of Thymol on the Growth and Toxin Production of** *Fusarium graminearum*

**Lian-Qun Wang 1,2, Kun-Tan Wu 1, Ping Yang 1, Fang Hou 2, Shahid Ali Rajput 3, De-Sheng Qi 1,\* and Shuai Wang 1,\***


**Abstract:** *Fusarium graminearum* is a harmful pathogen causing head blight in cereals such as wheat and barley, and thymol has been proven to inhibit the growth of many pathogens. This study aims to explore the fungistatic effect of thymol on *F. graminearum* and its mechanism. Different concentrations of thymol were used to treat *F. graminearum*. The results showed that the EC50 concentration of thymol against *F. graminearum* was 40 μg/mL. Compared with the control group, 40 μg/mL of thymol reduced the production of Deoxynivalenol (DON) and 3-Ac-DON by 70.1% and 78.2%, respectively. Our results indicate that thymol can effectively inhibit the growth and toxin production of *F. graminearum* and cause an extensive transcriptome response. Transcriptome identified 16,727 non-redundant unigenes and 1653 unigenes that COG did not annotate. The correlation coefficients between samples were all >0.941. When FC was 2.0 times, a total of 3230 differential unigenes were identified, of which 1223 were up-regulated, and 2007 were down-regulated. Through the transcriptome, we confirmed that the expression of many genes involved in *F. graminearum* growth and synthesis of DON and other secondary metabolites were also changed. The gluconeogenesis/glycolysis pathway may be a potential and important way for thymol to affect the growth of *F. graminearum* hyphae and the production of DON simultaneously.

**Keywords:** thymol; *Fusarium graminearum*; deoxynivalenol; mycelial growth; toxin production; gluconeogenesis/glycolysis

**Key Contribution:** This study revealed the genetic mechanisms for the responses of mycelial growth and toxin production of *Fusarium graminearum* to thymol.

### **1. Introduction**

Crops and food are often contaminated by molds and mycotoxins [1]. *Fusarium* head blight (FHB) is the most common disease of wheat in the world [2]. Some species of *Fusarium* can cause FHB, like *F. graminearum*, *F. culmorum*, and *F. avenaceum* [3]. The infection starts from the flowering period and progresses to the harvest period, causing the crops to be unharvested. A large amount of feed and food are rendered unfit for consumption due to mycotoxins contamination around the globe [4]. Finished feeds have high contamination rates and are often co-contaminated with multiple mycotoxins [5]. Common mycotoxins include aflatoxin B1 (AFB1) [6,7] and zearalenone (ZEN) [8], but deoxynivalenol (DON) and its acetyl-derivatives (3-Ac-DON and 15-Ac-DON) produced by *F. graminearum* are particularly concerning [9]. DON often contaminates wheat, barley, and oats. It can affect animal growth, immunity, and intestinal barrier function. In severe

**Citation:** Wang, L.-Q.; Wu, K.-T.; Yang, P.; Hou, F.; Rajput, S.A.; Qi, D.-S.; Wang, S. Transcriptomics Reveals the Effect of Thymol on the Growth and Toxin Production of *Fusarium graminearum*. *Toxins* **2022**, *14*, 142. https://doi.org/10.3390/ toxins14020142

Received: 28 December 2021 Accepted: 11 February 2022 Published: 15 February 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/).

cases, it can induce vomiting, refusal to feed, and gastrointestinal bleeding in pigs [10]. Therefore, great attention is paid to controlling mycotoxins in feed to reduce economic losses. For compound feed for pigs, China's Hygienical standard for feeds (GB 13078-2017) stipulates that the maximum limit is 1 mg/kg, and in the United States, it is 5 mg/kg. Due to the urgency of controlling FHB and the toxicity of trichothecenes, *F. graminearum* is listed as one of the top 10 fungal plant pathogens. Benzimidazole (such as carbendazim) and triazole (such as tebuconazole) have been widely used to control contamination by *F. graminearum* [11]. However, chemical fungicides are toxic and induce *F. graminearum* to synthesize DON [11]. Its residues on crops pose potential risks to environmental, animal, and human health. Therefore, people are paying more and more attention to finding biological antifungal agents alternative to synthetic pesticides. Researchers are particularly interested in essential oils (EO) extracted from plants, among natural fungistatic products. Due to its great potential to inhibit pathogens and their medicinal properties, it is considered one of the most promising biological antifungal agents.

EO is considered to be a secondary metabolite of aromatic flowers and plants. Because of their antiviral, fungistatic, and insecticidal properties in plants, they have been included in the category of natural preservatives [12]. Thymol (2-isopropyl-5-methylphenol) is a natural phenolic monoterpene compound, mainly found in *thymus Vulgaris*, *orange peel*, and *origanum heracleoticum* [13]. Thymol also has an excellent inhibitory effect on other toxinproducing molds, such as *Aspergillus flavus* [14], *Rhizoctonia solani*, *Pyricularia oryzae* [15], *Rhizopus stolonifera* [16], and *Fusarium solani* [17]. At the same time, it can also reduce the production of DON [18] and ZEN [19], so using thymol to inhibit *F. graminearum* has good potential and application prospects.

Some physical adsorbents can adsorb trichothecenes [20], but the effect is still not satisfactory. Adding thymol to feed can inhibit the mycelial growth of *F. graminearum* and reduce the production of trichothecenes from the source. It will be more conducive to the control of mycotoxins in the feed. Thymol is natural and degradable, and it also has the advantage of enhancing the antioxidant capacity of animals. Therefore, this experiment aims to study the effect of thymol on the growth and toxin production of *F. graminearum*. At present, there are also some reports that thymol inhibits the growth of *F. graminearum*, such as accumulating reactive oxygen species (ROS), destroying the integrity of cell walls and cell membranes [21], through ergosterol biosynthesis [22], and that it can block the overproduction of ROS [23]. However, there is a lack of research on other approaches. The fungistatic mechanism of EO is usually not a single pathway. Many studies have reported that EO can also change the membrane potential and destroy the integrity of cell membranes [24], inhibit DNA repair and transcription processes [25], or form chimeras with DNA and other pathways [26,27]. Therefore, we also adopted transcriptome sequencing technology (RNA-Seq), a high-throughput and high-resolution tool widely used to study fungi [28,29]. It can provide a comprehensive view of the *F. graminearum* transcriptome to comprehensively understand and explore other mechanisms by which thymol inhibits the growth of *F. graminearum*.

#### **2. Results**

#### *2.1. The Effect of Thymol on the Growth of F. graminearum Hypha*

First, we added different concentrations of thymol to the medium to determine its inhibitory effect on *F. graminearum* (Figure 1). The results show that thymol had a good fungistatic effect, and the 10 μg/mL thymol treatment group significantly reduced the colony diameter on the fourth day (Figures 1C and 2B). The inhibitory effect of thymol on the growth of *F. graminearum* had an obvious dose effect and time effect (Figures 1 and 2A). At the same time, the inhibition rate of different concentrations of thymol on mycelial growth was calculated after the fourth day of culture (Figure 2B). The mycelial growth inhibition rate (MGIR) of *F. graminearum* reached 100%, and the thymol concentration was 160 μg/mL. The EC50 and EC90 calculated by the regression equation were 40.15 μg/mL and 139.12 μg/mL, respectively.

**Figure 1.** The effect of thymol on the growth of *F. graminearum* hyphae. (**A**) Control group; (**B**) 5 μg/mL thymol treatment group; (**C**) 10 μg/mL thymol treatment group; (**D**) 20 μg/mL thymol treatment group; (**E**) 40 μg/mL thymol treatment group; (**F**) 80 μg/mL thymol treatment group; (**G**) 160 μg/mL thymol treatment group.

**Figure 2.** Thymol inhibits the growth of *F. graminearum* and DON synthesis. (**A**) Effect of different concentrations of thymol on the mycelial diameter; (**B**) Inhibition rate of different concentrations of thymol on the growth of *F. graminearum*; (**C**) The effect of thymol on the synthesis of DON and 3-Ac-DON at 40 μg/mL (EC50) or 139 μg/mL(EC90) concentrations. a,b Columns with different lowercase letters indicated significant differences between the compared groups (*p* < 0.05).

#### *2.2. The Effect of Thymol on DON Production by F. graminearum*

According to the previous growth inhibition test results, 40 μg/mL or 139 μg/mL of thymol was used to treat *F. graminearum* to evaluate its effect on DON. The results are shown in Figure 2C. The contents of DON and 3-Ac-DON in the control group were 78.0 ± 10.8 mg/g and 1160 ± 130.5 mg/g, respectively. The DON and 3-Ac-DON in the EC50 thymol treatment group were 23.3 ± 7.5 mg/g and 255.0 ± 209.3, respectively. Compared with the control group, the DON and 3-Ac-DON of the thymol treatment group decreased by 70.1% and 78.2%, respectively. The DON and 3-Ac-DON of the EC90 thymol treatment group were 22.3 ± 8.0 mg/g and 166.4.0 ± 91.6 mg/g, respectively, which decreased by 71.4% and 85.7%, respectively. The results show that 40 μg/mL thymol could significantly inhibit the production of DON and 3-Ac-DON by *F. graminearum*.

#### *2.3. The Effect of Thymol on the Transcriptome of F. graminearum*

To evaluate the quality of RNA-Seq data, we conducted some quality control analyses. Illumina sequencing produced 47,244,179 reads (control group) and 49,121,259 reads (thymol group). Strict data cleaning and quality inspection of the Illumina platform sequencing results, the error rate, GC percentage, and Q20 percentage were 0.02%, 52.2%, and 98.3%, respectively. Using Trinity to assemble all clean data de novo, a total of 16,727 non-redundant unigenes were identified, and the proportion of the expected length of the sequence to the total BUSCO score was 95.9%. The Mapped ratio between the sequencing data and the assembly results was 89.27%, indicating that this study's assembled data were high quality.

To better understand the functions of these non-redundant unigenes, all unigenes were compared with the NCBI non-redundant protein database sequences, and the e-value threshold was 10−5. The comparison analysis showed that a total of 12,597 unigenes matched the known proteins in the NR database. The matching percentages of *F. graminearum*, *Fusarium pseudograminearum*, and *Fusarium culmorum* were 73.05%, 5.61%, and 4.22%, respectively. All predicted unigenes were classified by functional annotation and classification through the Gene Ontology (GO) and Cluster of Orthologous Groups of Proteins (COG) database (Figure 3). The Top3 of the Biological Process (BP) were Cellular process, Metabolic process, and Localization; the Top3 of the Cellular Component (CC) were Cell part, Membrane part, and Organelle; the Top3 of the Molecular Function (MF) were Catalytic activity, Binding, and Transcription regulator activity. COG annotated 281, 230, and 356 unigenes with known functional classifications on Cellular processes and signaling, Information storage and processing, and Metabolism, respectively. In addition, 1653 (65.6%) unigenes were annotated by COG as Function unknown, and many unigenes were not matched to the database. These unigenes have the potential to be translated into functional proteins. This study's RNA-seq data helps enrich the annotations of the unigenes group of *F. graminearum*. The qRT-PCR results of the candidate genes were compared with the corresponding RNA-seq data, and the results were the same (Figure S1 and Table S1), which confirmed th e accuracy of the expression profile based on the RNA-seq data.

RSEM quantitatively analyzed the expression level of the unigenes, and the quantitative index was TPM. The overall distribution diagram of the expression level is shown in Figure 4D. At the same time, the correlation of the expression levels between samples was analyzed, and the heat map results are shown in Figure 4C. The correlation coefficient between samples was >0.986 and >0.941 in the control and treatment groups, respectively. The results of the PCA are shown in Figure 4A. PC1 was 53.02%, and PC2 was 15.94%, indicating a high similarity between samples. These results suggest that the correlation between biological replicates was high and that the experimental design was reasonable. On this basis, the unigenes with a differential expression caused by thymol treatment were further screened out (Figure 4B), and *p*-adjust was <0.05. When Fold change (FC) = 1.5, a total of 4417 differential unigenes were identified, of which 1989 were up-regulated, and 2428 were down-regulated. When FC was 2.0 times, a total of 3230 differential unigenes were identified, of which 1223 were up-regulated, and 2007 were down-regulated. When FC was 3.0 times, a total of 1944 differential unigenes were identified, of which 529 were up-regulated and 1415 were down-regulated. When FC was 6.0 times, 884 differential unigenes were identified, of which 165 were up-regulated, and 719 were down-regulated.

First, we used a functional enrichment analysis to understand the differentially expressed unigenes' functional pathways. The functional enrichment analysis of GO is shown in Figure 5A. The Top3 of BP were Catalytic activity, Oxidoreductase activity, and Cofactor binding. The Top3 of CC were Membrane part, Intrinsic component of membrane, and Integral component of membrane; the Top3 of MF were tRNA aminoacylation, Amino acid activation, and Polysaccharide catabolic process. The functional enrichment analysis of the Kyoto Encyclopedia of Genes and Genomes (KEGG) can be seen in Figure 5B. The Top3 of MF were Ribosome, Protein processing in endoplasmic reticulum, and Glycolysis/Gluconeogenesis.

**Figure 3.** Function annotation analysis. (**A**–**C**) GO function annotation analysis; (**D**) COG function annotation analysis. Red markers represent annotations related to energy metabolism processes.

**Figure 4.** Analysis of the relationship between samples. (**A**) PCA analysis between samples; (**B**) Percentage of up-regulated/down-regulated unigenes identified by different multiples of difference; (**C**) Heat map of correlation analysis between samples; (**D**) Distribution of expression levels. CK is the control group, and Treat is the thymol treatment group.

**Figure 5.** Functional enrichment analysis (**A**) GO function enrichment analysis; (**B**) KEGG function enrichment analysis.

#### *2.4. Unigenes Related to Mycelial Growth and DON Production*

The differentially expressed unigenes were functionally annotated and summarized in each NR, EggNOG, GO, KEGG, and Swiss-Prot database, and then the unigenes of interest were examined. Many unigenes related to mycelial growth (Table 1), DON synthesis (Table 2), secondary metabolites (Supplementary Materials Table S2), and glycolysis process (Supplementary Materials Table S3) were identified.

**Table 1.** Unigenes related to mycelial growth.


**Table 2.** Unigenes related to the synthesis of DON.


#### **3. Discussion**

*3.1. The Effect of Thymol on the Growth of Mycelium*

The fungistatic activity of thymol against pathogenic microorganisms has been widely reported [17,30]. For example, the EC50 of *Staphylococcus aureus*, *Staphylococcus luteus*, *Escherichia coli*, and *Bacillus cereus* is 27.64–128.58 μM [31]. Studies have found that seven kinds of thymol have suitable antifungal activities, one of which has an activity similar to the commercial fungicide thiabendazole [32]. Thymol inhibits the growth of *Fusarium oxysporum* with an EC50 of 80 μg/mL [21], and *Allium tuberosum R*. inhibits the growth of *Fusarium oxysporum* with an EC50 of 400 mg/mL [33]. In contrast, thymol has a better fungistatic effect. Thymol shows extensive antifungal activity against various isolates

of *F. graminearum*, inhibiting the production of conidia, the growth of hyphae [20], and the production of DON [34]. By detecting the sensitivity of 59 *F. graminearum* strains to thymol, the EC50 values of thymol of these strains were 22.53–51.76 μg/mL, and the average value was 26.3 μg/mL [11]. In this study, the EC50 of thymol against *F. graminearum* was 40 μg/mL, compared with the EC50 of eugenol and its derivatives of 395.7–1163.9 μM, indicating that the sensitivity of *F. graminearum* to eugenol is lower than thymol [35]. Thymol can also be used in combination, such as cinnamaldehyde and carvacrol, as a synergist to enhance antifungal activity [36] and reduce the production of trichothecenes by 95–99% [24]. These studies have shown that thymol can very effectively inhibit the growth of *F. graminearum*, and it is a very potent plant and antifungal agent.

COG annotation results annotated many transcripts related to fungal growth, such as "Cell cycle control, Cell division, Chromosome partitioning" (16 unigenes) and "Cell wall/membrane/envelope biogenesis" (25 unigenes). From the Top10 of KEGG functional enrichment analysis, many transcripts were significantly enriched in "Protein processing in endoplasmic reticulum" (31 unigenes), and "Cell cycle" (23 unigenes) related to fungal growth was also screened (Figure 5B). Based on the functional annotations of related databases and previous research reports, we screened out unigenes associated with the effect of thymol on mycelial growth (Table 1). From the table, we can see that many related to cell growth processes, such as cycle control, the translation process, and ribosome and cell wall/membrane; cell division and replication processes, such as classification, chromosomes, and transcription processes; and material metabolism processes, such as carbohydrates, coenzymes, and lipids. The expression of unigenes during transportation and metabolism changed significantly.

Interestingly, COG annotated many unigenes related to "Ribosome" (66 unigenes). DON can interact with the peptidyl-transferase region of the 60 S ribosomal subunit to induce "ribosomal stress toxicity" [10]. It suggests that thymol may alleviate toxicity by alleviating the ribosomal stress caused by DON to animals [37]. However, the mechanism of action of thymol on the ribosomes of *F. graminearum* itself is still unclear, and further research is needed. The results show that thymol inhibits the growth of *F. graminearum* by affecting the expression of related unigenes in the various processes of mycelial growth.

#### *3.2. The Effect of Thymol on DON Production by F. graminearum*

During the process of *F. graminearum* infecting plants, it can produce a variety of secondary metabolites, and one of the most concerning products is DON [38]. Therefore, we also summarized the table where thymol affects mycelial DON (Table 2) and the synthesis of secondary metabolites (Supplementary Materials Table S2). *Tri* gene refers to a gene cluster related to the biosynthetic pathway of trichothecenes. *Tri1*, *Tri4*, *Tri13*, and *Tri11* are the more important CYPs in fungi. *Tri4* encodes a multifunctional oxygenase that converts trichodiene to isotrichotriol [39]. *Tri1* and *Tri11* encode 3-acetyltrich-othecen C-8 hydroxylase and isotrichodermin C-15 hydroxylase, respectively. *Tri13*, as the 3-acetyl trichothecenes C-4 hydroxylase, is responsible for the hydroxylation of C-4 [40]. *Tri4* is involved in the synthesis of the trichothecenes framework [34]. Compared with plants and animals, few fungal CYPs have been thoroughly studied for their functions. They may be the key enzymes fungi use to metabolize phenolic compounds and aromatic hydrocarbon compounds [41]. *Tri5* is the first gene in DON biosynthesis. *Tri6* and *Tri10* are unigenes that regulate the synthesis of DON [42]. Fusarium's self-protection mechanism pumps the toxin out of the cell through *Tri12* and reduces the toxicity of intermediates in the biosynthesis of trichothecenes through *Tri101* [42]. Thymol can reduce the expression of *Tri5* [43] and inhibit the function of the toxin efflux pump, thereby enhancing the sensitivity of the fungus to tetracycline and benzalkonium chloride [44]. This is consistent with our results; 15 unigenes related to the fungal trichothecene efflux pump, such as *Tri10*, *Tri12*, *FUS6*, *FUB11*, and *ROQT,* were significantly down-regulated after thymol treatment. Studies have found that plant essential oils reduce the production of DON, 3-Ac-DON, and 15-Ac-DON by 96.33–100% [18], consistent with our results. The results indicate that thymol may inhibit

the expression of unigenes clusters related to the trichothecene biosynthetic pathway and inhibit the Fungal trichothecene efflux pump, thereby inhibiting the synthesis of DON.

#### *3.3. The Effect of Thymol on Glycolysis in F. graminearum*

In addition to the synthesis of DON, thymol also affects the synthesis of many secondary metabolites [12]. For example, histone acetyltransferases, such as *Elp3*, *Sas3*, and *Gcn5*, are related to the regulatory effect induced by DON [45–47]. Earlier studies reported that thymol might cause cell membrane damage by inducing lipid peroxidation and inhibiting ergosterol biosynthesis, thereby inhibiting the growth of pathogenic fungus [22]. Interestingly, we found that thymol may also inhibit toxins' growth and production by inhibiting the fungus's glycolysis process through a further analysis of transcriptomics data. Supplementary Materials Table S3 found that the expression levels of many unigenes related to carbohydrates and protein methylases, acetylases, oxidoreductases, and hydrolases have undergone significant changes. *ADH* is responsible for catalyzing the last methanol synthesis step [48]. *ALDOC* participates in the aldol condensation reaction in glycolysis [49]. *NAGA* encodes α-N-acetylgalactosaminidase, which is mainly involved in regulating the metabolism of glycoproteins and glycolipids in lysosomes [50]. *PME* catalyzes the hydrolysis of pectin with pectinic acid and methanol. *DAK1* catalyzes the production of dihydroxyacetone phosphate and enters the glycolysis pathway. *ENOA* is the gene encoding enolase, the metallocenes responsible for catalyzing the ninth step of glycolysis, converting 2-phosphoglycerate to phosphoenolpyruvate. *CHI1* catalyzes the hydrolysis of chitin to N-acetylglucosamine. As a key enzyme in the glycolysis process, the phosphoglycerate kinase encoded by *PGK* can catalyze ATP production.

It shows that the synthesis of secondary metabolites is closely associated with gluconeogenesis/glycolysis. Cinnamaldehyde can regulate intracellular glucose metabolism through α-enolase [51]. *Chuzhou chrysanthemum* can inhibit the growth of *E. coli* through the hexose monophosphate pathway [12,52]. It proves that EO can hinder the growth of a fungus by affecting energy metabolism. Glycolysis is an important metabolic process of *F. graminearum*, so we infer that thymol should also be able to exert fungistatic effects through glycometabolism and energy utilization pathways (Supplementary Materials Table S3). The COG annotation results (Figure 3D) annotate that many unigenes are related to energy metabolism processes, such as "Coenzyme transport and metabolism" (33 unigenes), "Secondary metabolites biosynthesis, transport and catabolism" (33 unigenes), "Energy Production and conversion and Lipid transport and metabolism" (49 unigenes), "Amino acid transport and metabolism" (62 unigenes), and "Carbohydrate transport and metabolism" (89 unigenes). From the Top10 of the KEGG functional enrichment analysis, many unigenes were also screened to be significantly enriched in pathways related to the energy metabolism process (Figure 5B), such as "Starch and sucrose metabolism" (16 unigenes), "Thermogenesis" (18 unigenes), and "Glycolysis/Gluconeogenesis" (23 unigenes).

Studies have found that EO does not completely inhibit the production of AFB1 by inhibiting the growth of fungi. It may also interfere with the process of carbohydrate decomposition and metabolism, resulting in an insufficient supply of acetyl-CoA, thereby reducing the ability of fungi to produce aflatoxin [53]. This is because acetyl-CoA is a key component in the glycolysis process and a crucial substrate and raw material in the production of DON. Thymol can inhibit the expression of acetyl-CoA carboxylase and fatty acid synthase [54] and affect the utilization of farnesyl pyrophosphate FPP (the precursor of DON synthesis) [43]. Acetyl-CoA and Tri5 work together. Many studies have shown that thymol can affect many intermediate products in the tricarboxylic acid (TCA) cycle [17], such as citrate, fumarate, succinate, and α-ketoglutarate [21,55]. Thymol mediates its bactericidal activity against Staphylococcus aureus by targeting aldehyde-ketoreductase to consume NADPH [56]. On Caenorhabditis elegans, thymol accelerates glucose metabolism by regulating multiple targets in the glycolytic pathway and participates in the degradation of fatty acids [57]. In summary, thymol can affect the energy homeostasis in cells [58]. It may interfere with the glycolysis process and the formation of the DON toxin via acetyl-CoA or

other common substances. This is a new idea to study the effect of EO on fungi. Acetyl-CoA is used as the raw material for DON synthesis. If the acetyl-CoA produced by glycolysis is not enough to supply its DON synthesis, it will eventually reduce the production of DON by *F. graminearum*. As far as we know, this is the first article combining thymol on the growth inhibition and toxin production of *F. graminearum* and RNA-Seq to understand the effect of thymol on *F. graminearum* fully.

#### **4. Conclusions**

Thymol can effectively inhibit the growth of *F. graminearum* and the production of DON. These results prove that after thymol treatment, many genes related to growth, DON, and the secondary metabolite synthesis process of *F. graminearum* undergo significant changes, which ultimately affect the growth and toxin production of *F. graminearum*. The study has enriched the data about thymol's influence on the genes of *F. graminearum* from the transcriptomics level. In addition, since the acetyl-CoA produced by the gluconeogenesis/glycolysis process can simultaneously participate in growth and toxin production, we believe that gluconeogenesis/glycolysis can be a breakthrough point for future research on the regulation of other plant essential oils in *F. graminearum*.

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

#### *5.1. Fungal Strain, Media and Culture Condition*

The *F. graminearum* strain W3008 was kindly provided by the College of Plant Science and Technology of Huazhong Agricultural University, China [59]. The strain was routinely cultured at 25 ◦C on potato dextrose agar (PDA, Hopebio, Qingdao, China) plates and was preserved in 20% disinfected glycerol at −80 ◦C for long-term storage [60]. Thymol (HPLC grade standard, purity > 98%, B21153, Shanghai yuanye Bio-Technology Co., Ltd. Shanghai, China) was dissolved in acetone into a 100 mg/mL stock solution, protected from light, and stored at 4 ◦C.

#### *5.2. Determination of the Sensitivity of Mycelial Growth to Thymol*

According to the results of our previous experiments, thymol was diluted by a certain multiple and then added to the PDA medium. The control group only added an equal volume of acetone (0.5%); the final concentrations of the thymol treatment group were 0, 5, 10, 20, 40, 80, and 160 μg/mL, and the acetone concentration in all groups was 5 μL/mL (0.5% of acetone used). A 6 mm diameter bacterial cake was taken from the edge of a 3-day-old colony with a sterile puncher, placed in the culture medium's center, and cultured for 4 days. The colony diameter was measured by the cross method every 24 h to evaluate the sensitivity of mycelial growth to thymol. The experiment was repeated 3 times, with 3 repetitions for each concentration. The percentage of mycelial radial growth inhibition on the 4th day of the culture after inoculation was calculated by the MGIR formula, MGIR (%) = [(C−N)/(C–6)] × 100. Where C and N are the average diameter values of the control and treatment groups, respectively. The thymol concentration of 50% (EC50) and 90% (EC90) of mycelial growth inhibition rate were calculated by the regression equation (See Supplementary Material "regression equation" and Figure S2).

#### *5.3. Changes in DON and 3-Ac-DON*

The preparation method of the conidia can be seen the supplementary material "Conidiation Assays" [46]. We added 1 mL of the spore suspension (5 × 105 CFU/mL) to a flask containing 100 mL of GYEP (glucose yeast extract peptone) medium, and we incubated it with shaking (180 r/min) at 25 ◦C for 24 h [60]. Thymol was then added to the culture, and the same amount of acetone (0.5%) was added to the control culture. The final concentration of the thymol treatment group was 40 μg/mL (this concentration was close to the EC50) or 139 μg/mL (this concentration was close to the EC90). The acetone concentration in each culture was 5 μL/mL (0.5% of acetone used), and each treatment had 3 replicates. After 7 days of continuous cultivation, the mycelium was collected and dried at 60 ◦C for 3 h [61].

The filtrate was used for DON and 3-Ac-DON quantification. DON production in vitro was expressed as a ratio of DON content to dry mycelia weight (μg/g) [62]. The extraction and purification methods (see Supplementary Material "Changes in DON and 3-Ac-DON" for details) of DON and 3-Ac-DON were improved from Stroka [63], and the quantitative method was based on Diao [64]. The pure products of DON and 3-Ac-DON were from FERMENTEK, with a purity of >99.6%.

#### *5.4. Transcriptome Analysis*

A total of 100 mL of GYEP medium containing 1 mL of conidia suspension (5 × <sup>10</sup><sup>5</sup> conidia/mL) was incubated with shaking (180 rpm/min) at 25 ◦C; after 24 h, thymol was added to the culture; the concentration of the thymol treatment group was 40 μg/mL, and the amount of acetone (0.5%) in groups control and treatment was same. Then, we continued incubating for 24 h, filtered, and collected hyphae [60]. The quality of RNA was evaluated after total RNA isolation using TRIzol reagent (Invitrogen, Shanghai, China). After the mRNA was isolated and fragmented, the library was sequenced using IlluminaHiSeqTM2000. Quality control of the original reading was performed, and we removed the linker and other low-quality base sequences to obtain the clean data reading.

Trinity was used to assemble all samples from scratch. TransRate and CD-HIT were optimized and filtered to remove common errors and redundancies. Then, BUSCO was used for assembly evaluation, and finally, the clean reads of each sample were compared with the reference sequence obtained by the Trinity assembly to obtain the mapping result of each sample. Unigene was the longest transcript in the transcript cluster, and unigenes were used for functional database annotation analyses (NR, Swiss-prot, Pfam, COG, GO, and KEGG). The RPKM method was used to calculate the expression value of unigenes (Reads Perkbper Millionread). RSEM compared the quality-controlled sequencing data with the assembled transcriptome sequence through comparison software, such as the bowtie, and then it estimated the expression abundance of unigenes/transcripts based on the comparison results. The quantitative expression index was TPM for homogenization so that the total expression in the sample was consistent for a more intuitive comparison. We used DESeq2 to analyze the differences between groups based on the quantitative expression results. The screening threshold was |log2FC| ≥ 1.585 and *p*-adjust < 0.05 to obtain unigenes with a differential expression between the two groups. Finally, the functional enrichment analysis of GO and KEGG was performed on the differentially expressed unigenes.

#### *5.5. qRT-PCR*

The total RNA was extracted from the sample as described above for real-time quantitative qRT-PCR analysis. Reverse transcription was performed using ABScriptIII Reverse Transcriptase kit (RK20408, ABclonal Technology Co., Ltd. Wuhan, China) with gDNA Eraser. Then, cDNA and 2×Universal SYBR Green Fast qPCR Mix reagent (RK21203, ABclonal Technology Co., Ltd.) were added to the 384 plates, respectively. We used a CFX384 real-time PCR system (Bio-Rad, Hercules, CA, USA) to complete qRT-PCR detection. The PCR program was as follows: 95 ◦C for 1 min, 40 cycles of 95 ◦C for 10 s, 60 ◦C for 5 s, and 72 ◦C for 10 s. The melting curve analysis was performed between 60 ◦C and 95 ◦C. The primers were synthesized by TSINGKE (Beijing, China). The qRT-PCR experiment was repeated 3 times, and each sample was repeated 3 times for analysis. *EF1α* was used as the reference gene for normalized expression data, and the relative gene expression level was calculated based on 2−ΔΔCt. Detailed information about gene-specific primers and alignment results are listed in the Supplementary Materials.

#### *5.6. Statistical Analysis*

The data were shown as mean ± standard deviation, comparing colony diameter, inhibition rate, DON, and 3-Ac-DON concentration. The values were analyzed by a oneway analysis of variance (ANOVA), followed by Duncan's multiple range test, and different

letters indicated significant differences at *p* < 0.05, compared with different concentrations of thymol. All analyses were performed with the GraphPad Prism 8.0 software (GraphPad Software, Inc., San Diego, CA, USA).

#### *5.7. Availability of Supporting Data*

The original Illumina sequencing dataset was submitted to the NCBI Sequence Read Archive with the accession number PRJNA792342. CK1-CK3 is the control group, and Treat1-Treat3 is the 40 μg/mL thymol treatment group.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/toxins14020142/s1, Table S1: Primer sequence of quantitative fluorescence PCR; Table S2: Unigenes related to the synthesis of secondary metabolites; Table S3: Unigenes related to glycolysis; Figure S1: Comparison of gene expression (*Tri5*, *Tri6*, *Tri8*, *Tri14*, *Tri101*, *LEU1*, *6PGD1*, *ERG6*) levels based on RNA-seq and qRT-PCR; Figure S2: Sensitivity regression equation.

**Author Contributions:** Supervision and funding acquisition, D.-S.Q. and S.W.; investigation and writing—original draft preparation, L.-Q.W. and K.-T.W.; writing—review and editing, L.-Q.W., K.-T.W., P.Y., and F.H.; visualization and formal analysis, P.Y., F.H., and S.A.R. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the National Key Research and Development Program of China (no. 2016YFD0501207).

**Institutional Review Board:** Not applicable.

**Data Availability Statement:** Not applicable.

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

#### **References**


### *Article* **A Newly Isolated** *Alcaligenes faecalis* **ANSA176 with the Capability of Alleviating Immune Injury and Inflammation through Efficiently Degrading Ochratoxin A**

**Rui Zheng †, Hanrui Qing †, Qiugang Ma \*, Xueting Huo, Shimeng Huang, Lihong Zhao, Jianyun Zhang and Cheng Ji**

> State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing 100193, China

**\*** Correspondence: maqiugang@cau.edu.cn

† These authors contributed equally to this work.

**Abstract:** Ochratoxin A (OTA) is one of the most prevalent mycotoxins that threatens food and feed safety. Biodegradation of OTA has gained much attention. In this study, an *Alcaligenes faecalis* strain named ANSA176, with a strong OTA-detoxifying ability, was isolated from donkey intestinal chyme and characterized. The strain ANSA176 could degrade 97.43% of 1 mg/mL OTA into OTα within 12 h, at 37 ◦C. The optimal levels for bacterial growth were 22–37 ◦C and pH 6.0–9.0. The effects of ANSA176 on laying hens with an OTA-contaminated diet were further investigated. A total of 36 laying hens were assigned to three dietary treatments: control group, OTA (250 μg/kg) group, and OTA + ANSA176 (6.2 <sup>×</sup> <sup>10</sup><sup>8</sup> CFU/kg diet) group. The results showed that OTA decreased the average daily feed intake (ADFI) and egg weight (EW); meanwhile, it increased serum alanine aminopeptidase (AAP), leucine aminopeptidase (LAP), β2-microglobulin (β2-MG), immunoglobulin G (IgG), tumor necrosis factor-α (TNF-α), and glutathione reductase (GR). However, the ANSA176 supplementation inhibited or attenuated the OTA-induced damages. Taken together, OTA-degrading strain *A. faecalis* ANSA176 was able to alleviate the immune injury and inflammation induced by OTA.

**Keywords:** ochratoxin A; biodegradation; *Alcaligenes faecalis* ANSA176; immune injury; inflammation; layers

**Key Contribution:** We isolated an *A. faecalis* strain ANSA176 from donkey intestinal chyme which showed strong OTA degradation ability. In addition, ANSA176 could alleviate OTA-induced immune response and inflammation, indicating that ANSA176 might be used as a potential bioproduct for improving animal health and food safety.

#### **1. Introduction**

Ochratoxin A (OTA) is one of the most prevalent mycotoxins produced by fungi of the genus *Aspergillus* and *Penicillium* [1]. The natural contamination of OTA widely occurs in plant- and animal-derived food commodities, including cereal, fruit, coffee, meat, milk, and other agricultural food and feed [2,3]. Studies have indicated that OTA possesses carcinogenic, teratogenic, genotoxic, and immunotoxic properties [2,4,5]. The International Agency for Research on Cancer (IARC) has classified OTA as a probable carcinogen for humans (group 2B) [6].

In animals, OTA has a broad range of harmful effects on livestock health and productivity, resulting in food-safety incidents and economic losses [7]. Due to the high plasma-protein binding affinity and low metabolism rate of OTA, its bioaccumulation and carryover effects in edible tissues and animal-derived products further endanger human health [2,8]. For poultry's complete feed, the European Commission Recommendation 2006/576/EC limits the maximum tolerable level for OTA to 100 μg/kg [9]. Residues of

**Citation:** Zheng, R.; Qing, H.; Ma, Q.; Huo, X.; Huang, S.; Zhao, L.; Zhang, J.; Ji, C. A Newly Isolated *Alcaligenes faecalis* ANSA176 with the Capability of Alleviating Immune Injury and Inflammation through Efficiently Degrading Ochratoxin A. *Toxins* **2022**, *14*, 569. https://doi.org/10.3390/ toxins14080569

Received: 21 July 2022 Accepted: 17 August 2022 Published: 20 August 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/).

OTA are detectable in the liver, kidney, and eggs in OTA-challenged laying hens [10]. Feeding OTA contaminated diets to poultry typically causes decreased production performance, altered serum biochemical profiles, and impaired immune response [10,11]. The detrimental effects of OTA on the poultry industry have been indicated to be time- and dose-dependent, with the liver and kidney being the main target organs [4,12]. Exposure of 0.5–3 mg/kg OTA to poultry typically results in renal lesions, followed by decreases in feed consumption, grow rate, feed-conversion efficiency, egg production, and egg quality [7,12,13]. The effects of 1–5 mg/kg OTA on poultry serum biochemistry include decreased total protein (TP), albumin (ALB), and globulin levels and increased alkaline phosphatase (ALP) and gamma glutamine transpeptidase (GGT) levels [12]. Studies have been carried out for decades to elucidate the critical role of oxidative stress in OTA-induced toxicity [14]. The investigation of oxidative stress or antioxidant potential in OTA-fed (0–6.4 mg/kg) broiler chicks reveals a significant dose-dependent decrease in the superoxide dismutase (SOD), glutathione peroxidase (GPx), and total antioxidant status (TAS) in plasma and tissues [15].

To date, numerous physical, chemical, and biological methods have been proposed to eliminate OTA [16]. Among them, biodetoxification of OTA is a highly promising approach due to its specific, efficient, and environmentally friendly advantages [3]. Biological detoxification methods could be classified into adsorption and degradation. Many yeasts have been reported to remove OTA via high adsorption, in which cell viability is not a prerequisite and the most important influence factor is cell wall components [3,17]. *Lactobacillus rhamnosus* GG has also been found to decrease OTA during the first 15 h of culture growth [18]. However, the amount of toxin elimination could be partially reversed, indicating both a limitation and risk. The degradation of OTA to ochratoxin α (OTα: less toxic or almost nontoxic) and L-β-phenylalanine (Phe) by breaking the amide bond is considered the most important biodegradation mechanism [3,16]. A good deal of microorganisms have been identified as being able to degrade OTA, including fungi [19–21], bacteria [22,23], and yeast [24]. For example, *Aspergillus niger* M00120 isolated from the soil has the strong ability to detoxify 99% of OTA in 2 days [25]. Furthermore, the product has been identified as OTα and assessed for cytotoxic response, indicating that it does not induce cellular oxidative damage [25]. As for the bacteria, the strain *Acinetobacter* sp. Neg1 that was isolated from the OTA-contaminated vineyard is capable of converting 91% of OTA into OTα in six days, at 24 ◦C [26]. The biodegradation production has been confirmed as OTα by liquid chromatography with high-resolution mass spectrometer (LC–HRMS) [26]. However, the degradation efficiencies of some microbes might be limited, and those isolated from the gastrointestinal tract of healthy animal might be worth investigating. Notably, the application of microorganism to both the food and feed industries must be cautious of its safety.

The objective of this study was to obtain a high-efficient OTA-degrading strain, elucidate the detoxification mechanism, and examine its protective efficacy in OTA-fed poultry. The central hypothesis is that supplementation of this novel OTA biodegrading strain could alleviate the OTA-induced immune injury and inflammation in laying hens.

#### **2. Results**

#### *2.1. Isolation and Characterization of OTA-Degrading Strain*

Colonies grown on the Luria-Bertani (LB) agar plate were screened from the donkey intestinal chyme. One pure strain, ANSA176, exhibiting the highest OTA removal ability was isolated and stored in our laboratory (Figure 1). The degradation test showed that ANSA176 was able to degrade 97.43% of OTA and produce OTα within 12 h (Figure 2), indicating the cleavage of the amide bond in OTA. The constructed phylogenetic tree (Figure 3) was consistent with the phylogeny of some *Alcaligenes faecalis* (*A. faecalis* ANSA176). Considering the microscopic observations, biochemical characteristics (Table 1), and the 16S rRNA gene sequence, the strain used in this study was confirmed and named *A. Faecalis* ANSA176.


**Table 1.** Biochemical and physiological characteristics of *A. faecalis* ANSA176.

"+" and "−" represent the positive and negative response, respectively.

**Figure 2.** *Cont*.

**Figure 3.** Phylogenetic tree of the isolated ANSA176 and related taxa.

Furthermore, we determined the growth of ANSA176 by OD600 measurement. As shown in Figure 4, the optimal temperatures for growth were observed to be between 22 and 37 ◦C (OD600 = 1.73–1.87). The lower (17 ◦C) and upper (42 ◦C) temperatures limited growth to some extent. In addition, the approximate pH levels for growth were 6.0–9.0 (OD600 = 1.54–1.79) at 37 ◦C incubation. At pH 10.0, the growth was reduced. Bacterial species subjected to the tested pH ranging from 2.5 to 5.0 were unable to grow.

**Figure 4.** Growth characteristics of ANSA176. (**a**) Growth at different temperatures during a 24 h period. (**b**) Growth at different pH during a 24 h period. Data are presented as means ± SEM. a–d The different letters mean significant difference (*p* < 0.05).

#### *2.2. Protection of A. faecalis ANSA176 against OTA-Induced Damages in Laying Hens* 2.2.1. Production Performance in Laying Hens

In Figure 5a, we can see that the average daily feed intake (ADFI) of the OTA-fed group (2.02%, *p* < 0.05) and the OTA + ANSA176 group (6.75%, *p* < 0.05) was significantly lower than that of the control group. In the OTA + ANSA176 group, the ADFI was even lower (4.83%, *p* < 0.05) than that of the OTA-fed group. However, no statistically significant differences (*p* > 0.10) were found in the feed/egg ratio (FER, Figure 5b) when compared the OTA-fed group (an increase of 3.63%) or the OTA + ANSA176 group (a decrease of 5.16%) to the control group. Moreover, the FER was significantly decreased (8.48%, *p* < 0.05) in the OTA + ANSA176 group when compared to the OTA-fed group. During the experiment period, feeding layers with OTA at the concentration of 250 μg/kg decreased the egg production ratio (EPR; see Figure 5c; 4.61%, *p* > 0.10) and average daily egg mass (EM; see Figure 5d; 5.44%, *p* > 0.10) when compared with the control. Meanwhile, the supplementation of ANSA176 to the OTA-fed group ameliorated the negative effects by increasing EPR and EM at 0.94% (*p* > 0.10) and 2.46% (*p* > 0.10), respectively.

The effects OTA and ANSA176 on egg weight (EW), shell percentage, yolk percentage, and albumen percentages were significantly different (*p* < 0.05) during the experimental period, while there were no statistically significant differences (*p* > 0.10) in shell color, shell thickness, shell strength, Haugh unit, and yolk color (Table 2). As shown in Table 2, the EW in the OTA group was significantly lower than the control and OTA + ANSA176 groups (*p* < 0.05). The higher shell and yolk proportions and lower albumen proportion were found in the OTA group compared to those of the control group (*p* < 0.05). Meanwhile, the supplementation of ANSA176 in the OTA-containing diet ameliorated these changes. In addition, residues of OTA an OTα were not detected (limit of detection = 0.1 μg/kg) in the eggs of all groups.

**Table 2.** Effect of OTA and ANSA176 on the egg quality of layer hens.


<sup>1</sup> Pooled standard error of the mean. a,b Means with different letters within a row mean significant difference (*p* < 0.05).

**Figure 5.** Effects of OTA and ANSA176 on production performance of layers. (**a**) Average daily feed intake. (**b**) Feed/egg ratio. (**c**) Egg production ratio. (**d**) Average daily egg mass. Data are presented as means ± SEM. The differences are defined as \* *p* < 0.05.

#### 2.2.2. Kidney and Liver Damage Related Parameters in Laying Hens

The administration of OTA resulted in mild pathomorphological changes in kidney and liver. Histopathological examination revealed that OTA caused kidney tubulonephrosis with degeneration of the epithelial cells in proximal tubules and glomerulonephrosis with enlarged glomeruli and swollen capillary endothelial cells (Figure 6a). In addition, OTA induced inflammatory infiltration and hyaline degeneration in the liver, which were ameliorated in the OTA + ANSA176 group (Figure 6b).

**Figure 6.** Effects of OTA and ANSA176 on kidney and liver damage related parameters in layers. (**a**,**b**) Histopathological examination of kidney (**a1**, control; **a2**, OTA; **a3**, OTA + ANSA176) and liver (**b1**, control; **b2**, OTA; **b3**, OTA + ANSA176) tissues by H&E staining. Magnification times, 400 × (scale bars = 100 μm). Lesions such as epithelial cells degeneration (green arrow), inflammatory infiltration (yellow arrow), and hyaline degeneration (blue arrow) are shown with arrows. (**c**–**i**) Serum biochemical parameters. Data are presented as means ± SEM. The differences are defined as # 0.05 ≤ *p* < 0.10 and \* *p* < 0.05.

As shown in Figure 6c,d, the serum alanine aminopeptidase (AAP) (*p* < 0.05) and leucine aminopeptidase (LAP) (*p* = 0.05) levels increased in the OTA group compared to the control group, whereas the supplementation of ANSA176 obviously decreased (*p* < 0.05) them. The concentration of serum alanine aminotransferase (ALT) was significantly higher (*p* < 0.05) in the OTA + ANSA176 group than in the control and OTA groups. No differences (*p* > 0.10) were observed in phosphoenolpyruvate carboxykinase (PEPCK), creatinine (Cr), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) among the three groups (Figure 6e,f,h,i).

#### 2.2.3. Immune and Inflammatory Response in Laying Hens

The immune and inflammatory response of OTA and ANSA176-fed layers are presented in Figure 7. In the serum, the levels of β2-microglobulin (β2-MG) (*p* < 0.05), immunoglobulin G (IgG) (*p* = 0.06), and tumor necrosis factor-α (TNF-α) (*p* = 0.05) after OTA feeding showed increases compared with the control group. Meanwhile, the concentrations of IgG (*p* < 0.05), lysozyme (LZM) (*p* < 0.05), interleukin-2 (IL-2) (*p* < 0.05), and TNF-α (*p* = 0.09) in the OTA + ANSA176 groups were lower than the OTA-fed group. There were no obvious differences (*p* > 0.10) in total protein (TP), albumin (ALB), IgA, IgM, and IL-10 among all groups.

**Figure 7.** Effects of OTA and ANSA176 on immune and inflammatory response of layers. (**a**–**j**) Levels of TP, ALB, β2-MG, IgA, IgG, IgM, LZM, IL-2, IL-10, and TNF-α. Data are presented as means ± SEM. The differences are defined as # 0.05 ≤ *p* < 0.10 and \* *p* < 0.05.

2.2.4. Oxidative Stress and Antioxidant Status in Laying Hens

Figure 8 exhibits the oxidative stress and antioxidant status in laying hens caused by the OTA and ANSA176 diet. The serum total antioxidant capacity (T-AOC) and superoxide dismutase (SOD) levels were significantly higher (*p* < 0.05) in the OTA + ANSA176 group compared with other groups. The glutathione reductase (GR) level was significantly higher (*p* < 0.05) in the OTA-fed group than in the control group. The levels of malonaldehyde (MDA), total glutathione (T-GSH), and glutathione peroxidase (GSH-Px) differed nonsignificantly (*p* > 0.10) among all groups.

#### **3. Discussion**

Ochratoxin A is a toxic secondary fungal metabolite that widely contaminates agriculture products, thereby threatening animal and human health. Microorganisms with an OTA-biodegradation property have great administration prospects in the food and feed industries due to their advantages in high specificity and effectivity [3]. In this study, we isolated and identified the *A. faecalis* ANSA176 strain from donkey intestinal chyme and found that it is capable of degrading 97.43% of OTA to OTα within 12 h in vitro. In addition, ANSA176 showed optimal growth at 22–37 ◦C and pH 6.0–9.0. Taken together, these results indicated a potential application of ANSA176 for the OTA biodegradation use

due to its easy culture and high degradation efficiency. Microorganisms with a confirmed ability to transform OTA into OTα have been previously studied [3,27]. It is noteworthy that an *A. faecalis* ASAGF 0D-1 strain isolated from soil and a novel N-acyl-L-amino acid amidohydrolase cloned from *A. faecalis* DSM 16503 have been reported to degrade OTA into OTα [23,28]. The biodegradation of OTA into barely or non-toxic metabolites (OTα and Phe) by microorganisms and their intracellular or extracellular enzymes is a preferred method to eliminate the toxic and carcinogenic potential. In many cases, biodegradation entailed the use of enzymes, including crude and purified ones that were able to cleave OTA [3,16]. In recent years, carboxypeptidases from *Bacillus amyloliquefaciens* ASAG1 [29], *Acinetobacter* sp. neg1 ITEM 17016 [30], and *Bacillus subtilis* ANSB168 [31] have been cloned and expressed to hydrolyze OTA. Except for the well-studied carboxypeptidase, several other enzymes were also indicated to biodegrade OTA, including protease A [32], amidohydrolase [33], lipase A [34], and hydrolase [35]. However, the degradation efficiencies of some microbes might be limited. Isolating strains from the gastrointestinal tract of healthy animals might be worth investigating. Our assay may help shed some light on investigating highly efficient enzymes. Further purification and identification of the degradation enzymes involved in OTA degradation by the strain ANSA176 is still ongoing.

The exposure of OTA could cause safety issues and enormous economic losses. Meanwhile, the efficacy and melioration effects of microorganism on OTA challenged poultry are still limited [3]. A study of varying levels of OTA showed that 2 mg/kg OTA decreased EPR and increased FER, while 4 mg/kg OTA decreased shell thickness [36]. Denli et al. found that the 2 mg/kg OTA-contaminated diet significantly decreased EM compared to the control [11]. Moreover, OTA at a level of 0.25 mg/kg did not influence the number of daily eggs produced, but 1 mg/kg of OTA reduced that, indicating the dose-dependent effect [13]. In order to provide a theoretical basis for the practical application of *A. faecalis* ANSA176, we further analyzed its alleviation effects on the laying performance of OTA-fed hens. In this experiment, decreases in ADFI and EW were recorded in the OTA group, which were in line with other studies [11,36]. Notably, administration of ANSA176 obviously ameliorated OTA-induced effects on layer's EM and FER. In addition, the OTA changed shell, yolk, and albumen percentages were altered back toward the control's by ANSA176. The unexpected ADFI decline in the ANSA176 supplementation group could be due to the palatability change. Although little is known on the palatability effects of bacteria supplements in feed diet, it has been accepted that microbial fermentation could change palatability and consumption [37,38]. In addition, a pair-feeding model indicated that the adverse effects of mycotoxin on growth performance, antioxidative status, and inflammation reaction were more severe compared to the merely FI reduction [39].

The challenge of OTA has been shown to result in nephrotoxic, hepatotoxic, and immunotoxic issues [14]. Several biochemical indicators in the serum can be used to reflect the tissue damage and inflammatory status in animals. Tubular enzymes AAP and LAP were markers of nephron injury [40]. The high β2-MG level was considered a clinical marker for nephropathy, and its excretion was related to OTA in Balkan endemic nephropathy (BEN) [41]. Our results showed increases in serum AAP, LAP, and β2-MG concentrations in OTA-fed layers, indicating OTA-induced damage. In comparison, biodegradable ANSA176 significantly reduced AAP and LAP, revealing a mitigating effect. Antibody responses are initiated via immune cells and lead to the production of immunoglobulins. It has been reported that OTA-contaminated diet significantly increased IgA, IgG, and IgM, suggesting an immune response against mycotoxin [31]. Cytokines such as IL-2 and TNF-α were involved in systemic inflammation and immunity [42,43]. Al-Anati et al. demonstrated that OTA released TNF-α in a dose- and time-dependent fashion via the classical inflammatory signaling cascade [44]. In the current study, increases were observed in serum β2-MG, IgG, and TNF-α concentrations in layers exposed to dietary OTA, indicating the OTA induced immune response and inflammation. However, the supplementation of ANSA176 can reduce levels of IgG, LZM, IL-2, and TNF-α to present protective effects.

Oxidative stress and antioxidant potential are sensitive indicators reflecting the imbalance between systemic reactive oxygen species (ROS) and the biological ability to detoxify intermediates or repair damage [14,15]. Nowadays, the various detrimental effects of OTA have been associated with the toxin itself, as well as the influence of ROS and oxidative stress [14]. Both in vitro and in vivo studies have suggested that the OTA-induced oxidative stress and reduced antioxidant ability may be implicated in the renal toxicity and carcinogenicity [14,39,45]. Different antioxidant enzymes and total antioxidant status (TAS) reflected the disturbances in animals [15]. Considering that OTA enhances the production of free radicals, the activity of antioxidant enzymes such as SOD may be affected. A previous study showed that feeding OTA to broiler chicks resulted in a dose-dependent reduction of TAS and SOD in plasma and almost all the tissues [15]. Although limited information was available on the OTA-induced oxidative stress in poultry, studies on rats have been carried out [14]. The enzymatic antioxidant SOD was significantly decreased in the kidney of OTA-challenged rat, while the administration of alleviators restored it [46,47]. In addition, pretreatment of antioxidant inhibited OTA-induced ROS overproduction and SOD reduction [48]. Furthermore, the injection of antioxidants SOD and catalase provided protection against OTA in rats [49]. In the present study, levels of T-AOC and SOD were higher in the ANSA176-treated group, suggesting an ameliorative effect of oxidative stress inside layers. It also supported the hypothesis that the modulation of oxidative stress might be due to the change in antioxidant enzyme, which has been proposed before [15,50].

#### **4. Conclusions**

In summary, our findings showed that the OTA-biodegrading strain *A. faecalis* ANSA176 could be used as a potential bioproduct to alleviate OTA-induced kidney and liver damage, inflammation, and oxidative stress. The application of the ANSA176 in grain or feed needs to be further developed, especially in resolving the consumption decline. Studies on the identified OTA degradation enzymes from ANSA176 also have a promising future.

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

#### *5.1. Isolation and Identification of the OTA-Degrading Strain*

#### 5.1.1. Culture Media and Reagents

The LB broth containing 10 g/L peptone, 5 g/L yeast extract, and 10 g/L NaCl was used for bacteria growth. All the reagents used in this study were purchased from Sigma-Aldrich (St Louis, MO, USA), unless otherwise indicated. The OTα standard was purchased from Biopure, Romer Labs (Tulln, Austria).

#### 5.1.2. Isolation of Microorganisms with OTA Degradation Ability

Bacterial strains were isolated from the intestine of animals. About 0.05 g of intestinal content was added to 5 mL of phosphate saline buffer and mixed by vortex. The solutions were further diluted and cross-streaked in Petri dishes containing LB agar to generate individual colonies of bacteria. Then the colonies obtained were individually transferred to LB broth and incubated on a rotary shaker (*n* = 200 rpm) at 37 ◦C for 24 h. The cultures were obtained through two successive inoculations (inoculum size at 2%, *v*/*v*). An aliquot of 980 μL of the culture was mixed with 20 μL of 50 mg/mL OTA standard solution. Sterile LB medium was used as a control. The inoculated mixtures were incubated at 37 ◦C at 200 rpm for 24 h. An equal volume of methanol (1 mL) was added at the end of incubation.

#### 5.1.3. Determination of OTA and OTα by Using HPLC

After incubation, cells of microbes were removed by centrifugation at 12,000 rpm for 4 min. The supernatants were filtered through filters with a 0.22 μm pore size (Millipore, Temecula, CA, USA) and analyzed by using high-performance liquid chromatography (HPLC). The HPLC was performed on an instrument (RF-20A, SHIMADZU Corporation, Kyoto, Japan) that contained a fluorescence detector set at 333 nm excitation and 477 nm emission wavelengths. The analysis was carried out with a reversed-phase Agilent Eclipse

Plus C18 column (4.6 × 150 mm, 5 μm) at an injection volume of 20 μL, and the mobile phase was water/acetonitrile/acetic acid (99:99:2, *v*/*v*/*v*) at a flow rate of 1.0 mL/min. During the HPLC analysis of OTA with fluorescence detection, the peak of OTα production was also clearly observed.

#### 5.1.4. Identification of the OTA-Degrading Strain

The morphological observation of the strain named ANSA176 with high OTA-degrading ability was carried out. To identify the bacterial strain, the 16S rRNA gene sequence was amplified by using primer set 27f-1492r. The inspection and appraisal report were given by the Institute of Microbiology (Chinese Academy of Sciences, Beijing, China). The strain was preserved in 20% glycerol and stored at −20 ◦C. Bacteria were revived and grown in LB broth at 37 ◦C for 24 h, with shaking (*n* = 200 rpm), for experiment.

#### 5.1.5. Characterization of *A. faecalis* ANSA176 Growth with Different Temperature and pH Levels

In order to determine the growth of ANSA176 under different conditions, a range of temperature (17, 22, 27, 32, 37, and 42 ◦C) and pH (2.5, 3, 4, 5, 6, 7, 8, 9, and 10) levels were investigated during a period of 24 h. Optical densities (ODs) were measured and recorded at 600 nm. Subsequently, the number of bacteria was calculated from the equation of plate counts against OD and expressed as log10 CFU/mL.

#### *5.2. Animal Trial in Layer Hens*

#### 5.2.1. Dietary Treatments of Animal Trial

According to Qing et al. [31], an *Aspergillus ochraceus* (CGMCC 3.4412) strain was used to produce OTA by artificial infection of sterile maize for 21 days, at 25–28 ◦C, and then the maize was dried and smashed. The concentration of OTA in maize powder was measured by HPLC, which was later added into the basal diet at 18% to meet the predicted concentration and verified by HPLC (predicted = 250 μg/kg; measured = 247.8 μg/kg). This dose was intended to observe the adverse effect of OTA contamination and the alleviation effect of *A. faecalis* ANSA176 supplementation.

The broth of strain *A. faecalis* ANSA176 was transformed into a freeze-dried powder. The number of bacteria in the powder was later determined as 3.1 × 108 CFU/g. Then it was added to the contaminated basal diet up to an overdose of 2 kg/T feed to ensure the effectiveness of degradation.

#### 5.2.2. Design of Animal Experiments

All procedures were reviewed and approved by the Laboratory Animal Welfare and Animal Experimental Ethical Committee of China Agricultural University (No. AW 13301202-1-7). The trial strictly complied with the standard operating procedures for experimental animals of the Ministry of Science and Technology (Beijing, China), and every effort was made to minimize suffering.

A total of 36 Jingfen No.1 layers (26 weeks of age) were randomly allocated to three feeding treatments and 12 replicate pens per treatment (separately feeding): (1) basal diet group (A) fed the basal diet without OTA contamination; (2) OTA-contaminated group (B) fed the contaminated diet containing exceeded limit dose of OTA (about 250 μg/kg); and (3) biodegradable agent group (C) fed the OTA contaminated diet added 2 kg *A. faecalis* ANSA176 freeze-dried culture per ton (6.2 × 108 CFU/kg diet of ANSA176). For the nutritive values and feeding procedures, we referred to the NY/T 33-2004 (China) and recommendations for Jingfen No.1 layers (Huadu yukou, Beijing, China). The composition and nutrient levels of the basal and contaminated diets are shown in Appendix A Table A1. The feeding trial lasted 70 days. On the final day of the feeding trial, blood samples were collected from each layer via the wing vein. After that, layers were euthanized for tissue collection, following the sodium pentobarbital injection (0.4 mL/kg BW; Sile Biological Technology Co., Ltd., Guangzhou, China).

#### 5.2.3. Laying Performance and Egg Quality

The laying performance was recorded and calculated. At the end of the experiment, 30 eggs were randomly selected from each treatment group to assess the egg quality, as previously described [51]. Shell color was determined with a QCR color reflectometer (QCR SPA, TSS, York, England). Thickness and strength were tested by the eggshell tester (ESTG-01, Orka Technology Ltd., Ramat Hasharon, Israel). Haugh unit and yolk color were measured by a multifunctional egg quality tester (EA-01, Orka Technology Ltd., Ramat Hasharon, Israel). Then the yolk was separated with a separator and weighed. The relative shell, yolk, and albumen proportions were calculated.

#### 5.2.4. Residues of OTA in Eggs

Residues of OTA and OTα in eggs were determined weekly during the animal trial, as previously conducted [31]. Briefly, homogenized sample was weighed and added into an acetonitrile/water solution (60:40, *v*/*v*). The mixture was shaken, followed by filtering to get the supernatant. The cleanup step was performed by passing the extracted sample through the immunoaffinity column (OchraTestWB, VICAM, Watertown, MA, USA) at a rate of 1 or 2 drops per second, under gentle vacuum pressure. Then the column was washed with 10 mL of water–methanol (90:10, *v*/*v*) and dried under nitrogen gas (N2) for 5 min. Finally, OTA was eluted by 2 mL of methanol for HPLC analysis. The HPLC parameter settings were as described above. The determined limit of detection (LOD) for OTA and OTα was0.1 μg/kg (based on a signal–noise ratio of 3).

#### 5.2.5. Blood Sampling and Serum Biochemical Analysis

Blood samples were centrifuged at 3000 rpm for 15 min at room temperature, and then serum samples were stored at −20 ◦C until analyzed. Serum contents of AST, ALT, ALP, PEPCK, Cr, TP, ALB, LZM, T-AOC, SOD, and GR were measured by using diagnostic kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), according to the manufacturer's instructions. Serum activities of LAP, AAP, MDA, T-GSH, GSH-Px, globulins (β2-MG, IgA, IgG, and IgM), and cytokines (TNF-α, IL-2, and IL-10) were determined by ELISA kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), following the manufacturer's instructions.

#### 5.2.6. Liver and Kidney Histological Assessment

Part of the liver and kidney samples were fixed in 10% neutral-buffered formalin solution and embedded in paraffin. Then these sections were stained with hematoxylin and eosin for histopathological examination, as previously described [52].

#### *5.3. Statistical Analysis*

The experimental data were analyzed through one-way analysis of variance (ANOVA), using the Statistical Analysis System software (SAS) package version 9.4. Tukey multiple comparison analysis was performed to determine significance of differences among treatment means. Differences were considered significant if the *p*-value was < 0.05 and demonstrated a trend if the *p*-value was < 0.10. The GraphPad Prism 9 software was used to generate graphs.

**Author Contributions:** R.Z., methodology, formal analysis and data curation during screening and identification of *Alcaligenes faecalis* ANSA176, and writing—original draft preparation; H.Q., methodology, formal analysis and data curation during feeding trial evaluation, and writing—review and editing; Q.M., conceptualization, writing—review and editing, project administration, and funding acquisition; L.Z., C.J. and J.Z., validation and supervision; X.H. and S.H., writing—review and editing. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Taishan Leading Industry Talents—Agricultural Science of Shandong Province (LJNY202022), Beijing Municipal Science Foundation (No. 6172017), Anhui Province Key Laboratory of Livestock and Poultry Product Safety Engineering (No. XM2004), and China Agricultural Research System program (CARS-40-K08).

**Institutional Review Board Statement:** The study was conducted according to the guidelines for experimental animals of the Ministry of Science and Technology (Beijing, China) and approved by the Laboratory Animal Welfare and Animal Experimental Ethical Committee of China Agricultural University (No. AW 13301202-1-7, 25 November 2019).

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Acknowledgments:** We thank all the researchers at our laboratory for their help with sample collection.

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

#### **Appendix A**


**Table A1.** Composition and nutrient level of basal and contaminated diet.

<sup>1</sup> When the contaminated corn's supplemental level was 18%, the total OTA content in the diet was 247.8 μg/kg. <sup>2</sup> Provided per kilogram of diet: 6.8 mg of Cu, 66 mg of Fe, 83 mg of Zn, 80 mg of Mn, 1 mg of I, and 0.3 mg of Se. <sup>3</sup> Provided per kilogram of diet: 11,700 IU of vitamin A, 3600 IU of vitamin D3, 21 IU of vitamin E, 4.2 mg of vitamin K3, 3 mg of vitamin B1, 10.2 mg of vitamin B2, 0.9 mg of folic acid, 15 mg of calcium pantothenate, 45 mg of niacin, 5.4 mg of vitamin B6, 24 μg of vitamin B12, and 150 μg of biotin. <sup>4</sup> All nutrient levels were calculated.

#### **References**

