Bacillus mojavensis capable of degrading zearalenone and application of bacillus mojavensis

Abstract

The invention relates to bacillus mojavensis capable of degrading zearalenone and application of the bacillus mojavensis. The bacillus mojavensis capable of degrading zearalenone is L-4, the bacillus mojavensis is classified and named as bacillus mojavensis, and the preservation number of the bacillus mojavensis is CGMCC (China General Microbiological Culture Collection Center) No. 29681.

Description

Technical Field

[0001] The invention relates to Bacillus mohaievii capable of degrading zearalenone and application thereof, and relates to Bacillus strains and application fields thereof. Background Art

[0002] Probiotics for animals are usually added to animal feed. When adding probiotics to animal feed, their tolerance to acid, alkali, gastrointestinal fluid, salt and bile salt must be ensured to ensure that they can enter the gastrointestinal tract smoothly and colonize to play a probiotic role (Kwak et al, 2016). Bacillus generally has good stress resistance, but reports vary. Xinna et al. (2022) found that Bacillus subtilis has strong alkali resistance, but poor acid resistance.

[0003] Probiotics have the ability to produce digestive enzymes and are sensitive to antibiotics (Kwak et al, 2016). The resistance of probiotics to antibiotics can be used to determine whether they can be used clinically. If the probiotics have drug-resistant genes, they may transfer drug-resistant genes to normal intestinal flora and pathogenic bacteria, thereby causing intestinal flora disorders and affecting the health of the animal body.

[0004] In recent years, people have paid more and more attention to the topic of probiotics as alternatives to antibiotics, and the development of probiotics and probiotic preparations is particularly important. When adding probiotics to food or animal feed, it is necessary to ensure their tolerance to acid, alkali (Liu Zhiyuan et al., 2020), gastrointestinal fluid (Kwak et al., 2016), salt and bile salts (Gutiérrez et al., 2021), so that they can successfully enter the gastrointestinal tract to colonize and exert their effects.

[0005] Probiotics should also be safe, that is, they should not be toxic to the host. When conducting mouse tests, attention should be paid to organ index, intestinal villus-crypt ratio and bacterial translocation. Therefore, organ index and bacterial translocation tests are important evaluation indicators for the safety of probiotics.

[0006] The intestinal flora of animals is closely related to the gastrointestinal health of animals. If the probiotics enter the intestines and cause the intestinal flora to become disordered, it will lead to the occurrence of digestive tract and non-digestive tract diseases in the body.

[0007] Therefore, qualified probiotics for animals should meet the requirements in terms of stress resistance, enzyme production performance, antibiotic sensitivity and safety.

[0008] Bacillus mohaivi B. mojavensis ) was originally isolated from desert soil and is most closely related to Bacillus amyloliquefaciens, Bacillus atrophaeus, and Bacillus subtilis (Roberts et al, 1994). B. mojavensis It has antagonistic effects on mycotoxin-producing Fusarium and can be used as a biocontrol probiotic for plant diseases caused by pathogenic fungi of various crops (Rath et al, 2018; Choi et al, 2011; Bacon et al, 2006). B. mojavensis There are no reports on its use as a zearalenone-detoxifying bacterium.

[0009] Studies have shown that Bacillus subtilis and Bacillus amyloliquefaciens are commonly used fungal toxin degrading bacteria (K. et al, 2010; Lee et al, 2017).

[0010] Microbial detoxification methods include adsorption and degradation. The adsorption method is related to the cell structure of bacteria or fungi. Studies have shown that Saccharomyces cerevisiae (Chlebicz et al, 2019) has a good adsorption effect on ZEN (zearalenone), and the adsorption capacity gradually increases with the increase of temperature. However, the adsorption method is not a truly effective detoxification method for ZEN. It is often difficult to metabolize or incompletely metabolize, causing secondary pollution. The biodegradation method can metabolize ZEN into low-toxic or non-toxic substances through intracellular or extracellular proteins secreted by microorganisms. It is a very feasible ZEN detoxification method. However, there is currently no method for using ZEN to detoxify. B. mojavensis Study on the degradation of zearalenone.

[0011] Therefore, a probiotic for animals that also has the function of degrading zearalenone is the technical problem to be solved by the present invention. Summary of the invention

[0012] In view of the above problems, the present invention provides a Bacillus mohaiwei capable of degrading zearalenone and its application. The Bacillus mohaiwei capable of degrading zearalenone in the present invention is L-4, which is classified and named Bacillus mohaiwei Bacillus mojavensis The strain deposit number is CGMCC No.29681; the depository is the General Microbiology Center of China Microorganism Culture Collection Administration; the deposit date is January 19, 2024; the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0013] The detoxification rate of L-4 strain for liquid culture medium containing 0.5-5.0 μg / mL zearalenone was not less than 83.42%. Under the conditions of 240 r / min and 37 ℃, Bacillus mohaiwei L-4 could quickly detoxify 0.5 μg / mL ZEN; under the conditions of 37-45 ℃, the detoxification effect of Bacillus mohaiwei L-4 on 0.5 μg / mL zearalenone reached more than 90%; the detoxification effect on 0.5 μg / mL zearalenone was best under the conditions of pH 7-11.

[0014] The optimal detoxification conditions for zearalenone by the L-4 strain are an initial concentration of 0.5-1.0 μg / mL, 37°C, 24h, and a pH of 7, with a detoxification rate of 100%. The peroxidase and phosphotransferase secreted by the L-4 strain degrade zearalenone into a low-toxic, low-concentration 15-OH-ZEN product. The degradation rate of 532.91 μg / kg of zearalenone by the L-4 fermentation broth in contaminated corn flour is 49.41%. The present invention also protects the use of the Bacillus mohaiweiensis that can degrade zearalenone as a probiotic for animals.

[0015] A probiotic for animals, comprising Bacillus mohaiwei L-4. The initial concentration of Bacillus mohaiwei L-4 is 0.5-1.0 μg / mL.

[0016] The probiotics for animals are prepared by mixing the fermentation supernatant of Bacillus mohaiwei L-4 with animal feed for use.

[0017] Compared with the prior art, the present invention has the following advantages: (1) The Bacillus mohaiwei L-4 of the present invention begins to enter the logarithmic growth phase after 3 hours of culture and enters the plateau phase after 18 hours; (2) Tolerance to acids, alkalis, salts, bile salts and gastrointestinal fluids; (3) L-4 has the ability to produce protease, cellulase and amylase, and the activities of the three enzymes produced are at a high level; (4) The L-4 strain is sensitive to 15 antibiotics including carbenicillin, piperacillin, kanamycin, ceftazidime, erythromycin, tetracycline, cefuroxime, cephalexin, minocycline, doxycycline, cefradine, cefazolin, ceftriaxone, and cefoperazone. It does not produce hemolytic enzymes, promotes weight gain and intestinal absorption in mice, and does not have the risk of translocation. In summary, the L-4 strain is resistant to acid, alkali, bile salts, high salt, artificial gastric juice and simulated intestinal juice. It can produce protease, cellulase and amylase, is sensitive to multiple antibiotics, is safe and can promote absorption. It can also change the composition of intestinal microorganisms and thus increase microbial abundance, making it suitable for use as an animal probiotic.

[0018] (6) The detoxification rate of Bacillus mohaiwei L-4 of the present invention for 0.5 and 1.0 μg / mL of ZEN is 100%, and the detoxification rate for 5.0 μg / mL of ZEN is 83.42%. Moreover, L-4 bacteria can detoxify 0.5 μg / mL of ZEN by 100% after being treated for 24 hours. The L-4 strain shows a very high detoxification effect on ZEN in neutral and alkaline culture media and simulated intestinal fluid, and also has a detoxification effect in artificial gastric juice, acidic and strongly alkaline culture media. The detoxification effect on ZEN is the best at 37°C, and 0.5 μg / mL of ZEN can be completely detoxified. Increasing or decreasing the temperature will lead to a decrease in its detoxification effect.

[0019] (7) The present invention co-cultured different components of L-4 fermentation broth with ZEN and found that the L-4 fermentation supernatant could completely detoxify 0.5 μg / mL ZEN. After adding SDS+proteinase K, the detoxification rate dropped to 18.31%. This result showed that the detoxification effect of L-4 on ZEN was mainly manifested in the degradation of its secreted extracellular proteins in the fermentation supernatant.

[0020] (8) The peroxidase and phosphotransferase secreted by the L-4 strain degraded ZEN into a low-toxic, low-concentration 15-OH-ZEN product. The present invention co-cultured the L-4 fermentation broth with ZEN-contaminated corn feed for 5 days and found that the concentration of ZEN in corn flour decreased from 532.91 μg / kg to 269.60 μg / kg, with a degradation rate of 49.41%.

[0021] In summary, the Bacillus mohaiwei L-4 of the present invention can be used as a probiotic for animals and can be mixed with animal feed to simultaneously degrade zearalenone, and has extremely high application value for ZEN detoxification of animal feed. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Shown is the colony morphology of Bacillus mohaiwei L-4 in Example 1; Figure 2 Shown is a Gram staining microscopic image of Bacillus mohaiwei L-4 in Example 1 (400×); Figure 3 Shown is the electrophoresis diagram of Bacillus mohaiwei L-416S rDNA in Example 1; Figure 4 Shown is the phylogenetic tree of the Bacillus mohaiwei L-416S rDNA gene sequence in Example 1; Figure 5 Shown is a circle diagram of the genome of Bacillus mohaiwei L-4 in Example 1; Figure 6 Shown is the COG annotation of protein-coding gene function in the genome; Figure 7 Shown is the KEGG metabolic signaling pathway annotation; Figure 8 Shown is an alignment of the ZEN-degrading enzyme sequence from B. subtilis with the protein sequence from B. mojavensis; Fig. 9 Shown are flagellar assembly genes in the annotation of the strain L-4 genome; Fig.10 Shown are bacterial chemotaxis genes in the genome annotation of strain L-4; Fig.11 Shown is the signal peptide sequence of efeB 3668; Fig.12 Shown is the transmembrane sequence of efeB 3668; Fig.13 Shown is the hydrophilicity of efeB 3668; Fig.14 Shown is the coiled-coil domain of efeB 3668; Fig.15 Shown is the active center of efeB 3668; Fig.16 Shown is the conserved domain of efeB 3668; Fig.17 Shown is the molecular docking model of ZEN binding to efeB 3668; Fig.18 The growth time curve of the Bacillus mohaiwei L-4 strain in Example 2 is shown, wherein the data are expressed as mean ± SD. Since SD < 0.01, no bar is shown in the figure; Fig.19 Shown is the tolerance test of L-4 strain under different conditions in Example 2; Among them, (A): survival rate of strains in different pH medium, (B): survival rate of strains in different bile salt concentration medium, (C): survival rate of strains in gastric and intestinal fluids, (D): survival rate of strains in 8% NaCl medium. Data are expressed as mean ± SD; because SD < 0.01, no bar is shown in the figure; Fig. 20 The photos of the enzyme production experiment of the Bacillus mohaiwei L-4 strain in Example 2 are shown, wherein (A): the activity circle of the strain on the cellulase enzyme production medium, (B): the activity circle of the strain on the protease enzyme production medium, (C): the activity circle of the strain on the amylase enzyme production medium; Fig.21The figure shows the mouse ileum sections (200×) in Example 2, wherein (A), (B), and (C) are the ileum loop sections of male mice in the control group, the test group, and the blank group, respectively; (D), (E), and (F) are the ileum loop sections of female mice in the control group, the test group, and the blank group, respectively; Fig. 22 The figure shows the mouse duodenum sections (200×) in Example 2, wherein (A), (B), and (C) are the circumferential sections of duodenum of male mice in the control group, the test group, and the blank group, respectively; (D), (E), and (F) are the circumferential sections of duodenum of female mice in the control group, the test group, and the blank group, respectively; Fig.23 The figure shows the α diversity analysis and β diversity analysis of cecal microbial diversity in Example 2, wherein (A) is the α diversity analysis of the distribution of cecal intestinal flora in each group at the species level, (B) is the difference test between cecal intestinal flora groups in each group at the species level, and (C) is the PCoa analysis of each group at the OTU level; Fig.24 The figure shows the detection of zearalenone by HPLC in Example 3, wherein (A) is the peak time of ZEN standard, and (B) is the ZEN standard curve; Fig.25 The figure shows the detoxification rate of zearalenone by eleven strains of bacteria in Example 3, wherein Ef: Enterococcus faecalis, L. r: Lactobacillus rhamnosus, L. d: Lactobacillus delbrueckii subsp. bulgaricus, B. m: Bacillus mohaivi, L. p: Lactobacillus plantarum, Lc. p: Lactobacillus paracasei, S. t: Streptococcus thermophilus, B. l: Bacillus licheniformis, B. c: Bacillus cereus, S. c: Saccharomyces cerevisiae, B. s: Bacillus subtilis; Fig.26 The figures show the detoxification effect of the L-4 strain of the present invention on zearalenone under different conditions in Example 4, wherein: (A): the detoxification effect of L-4 at different ZEN concentrations, (B): the detoxification effect of L-4 at different treatment times, (C): the detoxification effect of L-4 in different pH culture media and artificial gastric juice and simulated intestinal juice, (D): the detoxification effect of L-4 at different temperatures; Fig. 27 The figure shows the detoxification effect of different components of the L-4 strain of the present invention on zearalenone in Example 4; Fig.28 The figure shows the research diagram of the degradation products of zearalenone in Example 4, wherein: (A): ZEN degradation chromatogram, (B): degradation product mass spectrum, (C): degradation product secondary mass spectrum; Fig.29The diagrams are shown for screening of zearalenone-degrading enzymes in Example 4, wherein (A) is a homology diagram of pilon3668 and Bsdyp, and (B) is a homology diagram of pilon3330 and ZPH; Fig.30 The figure shows the detoxification effect of the L-4 strain of the present invention on ZEN-contaminated corn flour in Example 4. DETAILED DESCRIPTION

[0023] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, the embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0024] The Bacillus mohaiweiensis capable of degrading zearalenone in the present invention is L-4, which is classified and named as Bacillus mohaiweiensis Bacillus mojavensis The strain deposit number is CGMCC No.29681; the depository is the General Microbiology Center of China Microorganism Culture Collection Administration; the deposit date is January 19, 2024; the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0025] Example 1 Isolation and Identification of Bacillus mohairi L-4 Capable of Degrading Zearalenone Materials and methods 1.1 Bacterial morphological identification Use an inoculation loop to pick a loop of the strain and inoculate it into the corresponding agar medium. Place the culture dish in a 37 ℃ constant temperature incubator for 16 to 24 hours, then observe the shape and color of the bacterial colony, and use Gram stain to stain and perform a preliminary morphological identification under a microscope.

[0026] 1.2 Bacterial physiological and biochemical identification Use a sterile inoculation loop to pick up a single colony into a centrifuge tube filled with sterile water, mix the bacterial suspension thoroughly until it reaches a McFarland turbidity of 0.5, and add 100 μL per well to the pH 5.7, gelatin, VP, starch hydrolysis, nitrate reduction, citrate and 7% sodium chloride physiological and biochemical wells for physiological and biochemical properties tests.

[0027] Use a sterile inoculation loop to pick up a single colony, and use the zigzag streak method to streak in the anaerobic growth biochemical tube; use a sterile inoculation needle to pick up a single colony and vertically puncture it into the physiological and biochemical wells of D-mannitol, D-xylose, propionate, and L-arabinose to conduct physiological and biochemical characteristic tests. Use a pH meter to detect the pH value of the fermentation liquid to determine the acid production characteristics of the bacteria.

[0028] 1.3 16S rDNA sequencing PCR amplification was performed using bacterial universal primers (27F: 5′-AGAGTTTGATCMTGGCTCAG-3′; 1492R: 5′-GGTTACCTTGTTACGACTT-3′). The obtained PCR products were electrophoresed and observed and recorded using a gel imager. The obtained PCR products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. After obtaining the results, the sequence obtained was uploaded to GenBank and the accession number was obtained. The sequence was compared with BLAST on the NCBI website, and the strain sequence with the highest homology and the common model strain sequence were selected. The phylogenetic evolutionary tree was drawn using MEGA 5.10 software to further identify the strain.

[0029] 1.4 Whole-genome sequencing The genomic DNA of strain L-4 was extracted using a bacterial DNA kit (Omega, USA), and the DNA concentration and purity were determined by a Thermo Scientific NanoDrop 2000 microspectrophotometer. The genome of strain L-4 was sequenced using the PacBio Sequel II platform (Shanghai Yuanxin Biopharmaceutical Technology Co., Ltd., China). The filtered sub-reads were assembled using Canu v1.6 software, and the assembly results were corrected using the third-generation sub-reads by Racon v1.4.20 software. Gene prediction was performed using Prodigal v2.6.3 software and Island Path-DIMOB v1.0 (She et al., 2009). Functional annotation was performed by searching public databases (including Nr, KEGG, Pfam, and Swissport) using blastp with an E value of 10, and the genome circle map of strain L-4 was drawn using Circos software (Krzywinski et al., 2009).

[0030] 1.5 Data Processing The experimental data were statistically analyzed and plotted using Excel software.

[0031] 2 Results and analysis 2.1 Morphological characteristics The morphological characteristics of L-4 strain are shown in the attached figure of the specification. Figure 1 and Figure 2 The single colonies of L-4 strain grown on agar medium are large, opaque, slightly yellowish, with a rough surface and wrinkled edges (see the attached figure in the specification). Figure 1 After Gram staining, the L-4 strain was found to be thick and long rod-shaped, arranged singly or in multiples (see the attached figure in the specification). Figure 2 is a Gram-positive bacterium.

[0032] 2.2 Physiological and biochemical characteristics As shown in Table 1: L-4 bacteria grow well under pH 5.7, can decompose starch, reduce nitrate, VP test is positive, can utilize xylose, L-arabinose, etc., produce gelatinase, can produce acid, cannot ferment D-mannitol, and cannot utilize citrate and propionate. It does not grow under anaerobic conditions. Referring to the Bergey's Manual of Bacterial Identification, it was found that the physiological and biochemical characteristics of the L-4 strain are similar to those of Bacillus.

[0033]

[0034] 16S rDNA sequence analysis As shown in the attached figure of the instruction manual Figure 3 As shown, the molecular size of the electrophoresis product of the L-4 strain is about 1500 bp. The sequence of the sequencing product was submitted to GenBank and the sequence accession number was OR649301. The phylogenetic tree of 16S rDNA sequences was constructed by BLAST analysis and MEGA5.10 software (see the attached figure of the specification). Figure 4 ) found that the L-4 strain sequence was 99.65% homologous to Bacillus mojavensis strain D50 MZ165352.1 and was in the same branch.

[0035] 2.4 Whole genome sequencing results of L-4 strain The genome circle diagram is shown in the attached description Figure 5 As shown, the 2nd and 3rd circles from the outside to the inside are genes on the positive and negative chains of the genome, and the color differences represent different COG functional classifications. The 4th circle represents tRNA and rRNA, and the 5th circle represents GC content. The outward red part indicates that the GC content of this region is higher than the average GC content of the whole genome. The higher the peak value, the greater the difference with the average GC content. The inward blue part indicates that the GC content of this region is lower than the average GC content of the whole genome. The higher the peak value, the greater the difference with the average GC content.

[0036]

[0037] After morphological, physiological and biochemical characteristics and molecular biological identification, the L-4 strain was finally determined to be a strain of Bacillus mojavensis, and was named Bacillus mojavensis L-4.

[0038] 2.5 Degradation enzyme prediction and molecular docking DNAMAN software was used to align all protein sequences obtained by sequencing with known ZEN degradation protein sequences on NCBI, and the sequences with the highest homology were screened out.

[0039] The PSORT server was used to predict the cellular sublocalization of efeB 3668 (Yu et al., 2010), and the ProtScale server was used to predict the hydrophilicity of the target protein. The SignalP 6.0 and TMHMM 2.0 servers were used to predict whether the amino acid sequence of efeB 3668 contained a signal peptide and a transmembrane region structure, respectively (Almagro et al., 2019). The SOMPA server was used to predict the secondary structure of efeB 3668, and PROSITE and NCBI conserved domain search tools were used to predict and analyze the functional sites of efeB 3668. The SWISS-MODEL online server was used to perform protein tertiary structure homology modeling of efeB 3668 using 7pkx.1.A as a template, and the Ramachandran online server was used to evaluate the reliability of the constructed model.

[0040] The 2D structure of the small molecule ligand was obtained through the PubChem database (http: / / pubchem.ncbi.nlm.nih.gov / ), and the 2D structure was input into the Chem Office 20.0 software (CambridgeSoft, USA) to generate its 3D structure. Then, the RCSB PDB database (http: / / www.rcsb.org / ) was used to screen the crystal structure of the protein target with high resolution as the molecular docking receptor, and the protein was dehydrated and dephosphorylated using the PyMOL2.6.0 software (DeLano Scientific LLC, USA) (El-Hachem et al., 2017). The Molecular Operating Environment 2019 software (Chemical Computing Group Inc., Canada) was used to minimize the energy of the compound, pre-process the target protein and search for active pockets. Finally, MOE 2019 was run for molecular docking, and the number of operations was set to 50. The binding activity of the two was evaluated according to the size of the binding energy, and the results were visualized using PyMOL2.6.0 and Discovery studio2019 software (BIOVIA, USA).

[0041] 2.6 Data Statistics and Analysis The data were statistically analyzed using Microsoft Excel 2019 (Microsoft, USA) and SPSS 22.0 (IBM, USA). One-way ANOVA was used for comparison, and the least significant difference (LSD) test method was used for multiple comparisons of each mean. The results are expressed as "mean ± standard error", and P < 0.05 indicates a significant difference. MEGA 5.10 was used to draw the L-4 phylogenetic tree. The degradation products were analyzed by mass spectrometry using Qualitative Analysis B.05.00 software (Agilent Technologies, USA), the degradation enzyme sequences were predicted using DNAMAN software (Lynnon Biosoft, USA), and the graphics were drawn using GraphPad prism 9 software (GraphPad Software, USA).

[0042] 2.7 Whole genome analysis of strain L-4 2.7.1 Basic genome annotation analysis As shown in the attached figure of the instruction manual Figure 6 The total length of the strain L-4 gene sequence shown is 4,027,871 bp, the GC content is 43.78%, and there are 3,659 predicted protein-coding genes (CDS).

[0043] (1) COG Annotation The genome of bacterial strain H27 was subjected to functional classification and prediction of COG (Cluster of Protein Orthologous Groups) to infer the function of proteins (Tatusov et al., 2003). Figure 6 As shown, 3080 genes were annotated into 22 COG categories. The most abundant functional genes were "unknown function prediction (354 CDS)", "amino acid transport and metabolism (286 CDS)", "carbohydrate transport and metabolism (282 CDS)", "transcription (275 CDS)", "cell wall / membrane / envelope biogenesis (194 CDSs)" and "inorganic ion transport and metabolism (190 CDS)".

[0044] (2) KEGG metabolic pathway analysis The whole genome sequencing of L-4 strain is conducive to studying the degradation mechanism of ZEN. Figure 7The KEGG (Kyoto Encyclopedia of Genes and Genomes) annotation classification statistics of the strain L-4 genome are shown, and a total of 2,637 gene annotations were found. Most of the genes of strain L-4 are related to metabolism, with a total of 558 genes, including carbohydrate, amino acid, and lipid metabolism, among which amino acid and lipid metabolism (β-oxidation of fatty acids) may be related to the formation of hydroxyl groups in ZEN degradation products. In addition, strain L-4 has complete glycolysis, transmembrane transport, oxidative phosphorylation, tricarboxylic acid cycle, and pyruvate metabolic pathways. This indicates that L-4 has a strong metabolic conversion effect in the process of degrading ZEN.

[0045] 2.7.2 Analysis of degradation features in genome annotation (1) ZEN degradation-related genes Exploring the enzymes involved in the degradation process is of great significance for understanding the environmental transformation process of pollutants and the transformation and regulation of the degradation process. Figure 8 As shown, the ZEN-degrading enzyme sequence from Bacillus subtilis was aligned with the protein sequence from B. mojavensis. The 3659 protein sequences annotated to strain L-4 were aligned with the known ZEN-degrading enzyme sequences using DNAMAN software, and a protein sequence was found to have a homology of 95.91% with BsDyP of Bacillus subtilis.

[0046] (2) Flagella formation and chemotaxis Bacterial chemotaxis includes flagellar movement and signal transduction, as shown in the attached figure of the instruction manual Fig. 9 , 10 As shown, strain L-4 has perfect flagellar assembly and chemotaxis-related genes, which facilitates its tendency to move toward favorable conditions and increase the degradation and bioavailability of ZEN.

[0047] 2.7.3 Subcellular localization, transmembrane region, signal peptide, hydrophilicity and secondary structure of efeB 3668 PSORT predicted that efeB 3668 was mainly located in the cytoplasm, with the specific distribution as follows: cyto 22.5%, cyto_nucl 20%, nucl: 12.5%, mito 12.5%, plas 10%, extr 7.5%, cysk 7.5%, pero 5%, ER 2.5%. See the attached figure in the manual Fig.11 As shown, the amino acid sequence of efeB 3668 was input into the SignalP 6.0 server. There was a signal peptide sequence in efeB 3668, as shown in the attached figure of the specification. Fig.12 As shown, the TMHMM 2.0 server predicted that efeB 3668 has a transmembrane sequence, and it is speculated that it is a transport protein. Fig.13 As shown in the figure, ProtScale was used to predict the hydrophilicity of efeB 3668. The results showed that the hydrophobicity value of alanine at position 25 in the efeB 3668 polypeptide chain was 2.133, which was the most hydrophobic amino acid, and the hydrophilicity value of lysine at position 53 was -3.633, which was the most hydrophilic amino acid. The number of hydrophilic amino acid residues in the entire polypeptide chain was significantly greater than that of hydrophobic amino acid residues, and it was inferred that efeB 3668 was a hydrophilic protein.

[0048] As shown in the attached figure of the instruction manual Fig.14 As shown, using sopma software to predict efeB 3668, the α-helix (H) accounts for 29.33%, the extended link (E) accounts for 14.42%, the β-turn (T) accounts for 5.29%, and the random coil (C) accounts for 50.96%. Fig.15 As shown, ScanProsite software was used to predict that efeB 3668 contained two active centers, namely the TAT active site (amino acids 1-44) and the dyp_peroxidase active site (amino acids 63-408). Fig.16 As shown, NCBI analysis showed that efeB 3668 had two conserved domains, belonging to the TAT subfamily and the Dyp_perox superfamily domain.

[0049] Analysis of the protein using ProtParam software showed that the molecular weight was approximately 45 kD, the molecular formula was C2024H3192N556O618S15, the theoretical isoelectric point was 8.86 (basic protein), the number of negatively charged amino acid residues Asp+Glu was 48, the number of positively charged amino acid residues Arg+Lys was 54, the extinction coefficient of the protein was 6405, the instability coefficient was 34.09, and the fat coefficient was 70.67.

[0050] 2.7.4 efeB 3668 (tertiary structure) binding model with ZEN by molecular docking Using SWISS-MODEL and 7pkx.1.A as template, homology modeling of efeB 3668 was performed, with a sequence identity of 95.91% and a similarity of 0.90, and its function was predicted to be peroxidase.

[0051] The three-dimensional structure of efeB 3668 was obtained by homology modeling, and then the interaction between efeB3668 and ZEN was studied by molecular docking simulation. Fig.17As shown, the docking model shows that the binding energy of ZEN to efeB 3668 is -6.9557 kcal / mol, which has a good binding effect. ZEN interacts with efeB3668 mainly through hydrogen bond formation and hydrophobic force. According to the docking results, His326, Arg299, and Gly243 residues on the protein receptor form hydrogen bond interactions with zearalenone, and residues Ala330, Lys331, Val316, and Val327 form hydrophobic interactions with zearalenone. In addition, the Arg339 residue on the receptor forms an electrostatic interaction with zearalenone. These results indicate that ZEN is a suitable substrate for efeB 3668.

[0052] 3. Discussion The classification and identification of bacteria mainly include conventional identification and molecular biological identification (Garrity et al, 2004; Yu et al, 2010). Conventional identification is a common method for early identification of bacterial species by observing colony morphology, staining microscopy and physiological and biochemical characteristics (Garrity et al, 2004). Although this method has certain limitations, it can only identify bacterial genus. Molecular biological identification is faster, more reliable and more accurate than conventional identification, and has become the current classic bacterial identification method (Yu et al, 2010). The present invention uses colony morphology, bacterial characteristics, and physiological and biochemical identification to find that the colony morphology and physiological and biochemical characteristics of the L-4 strain are consistent with Bacillus; the 16S rDNA gene sequence is 99.65% homologous to Bacillus mohaiwei D50 (MZ165352.1), and phylogenetic evolution analysis shows that it is in the same branch as Bacillus mohaiwei. Therefore, based on the above results, the L-4 strain is determined to be Bacillus mohaiwei.

[0053] The whole genome circle map displays various information in one map, which can comprehensively display the genomic characteristics of the L-4 strain, such as the total number of genes, protein-coding genes, COG functional classification, and GC content, so that we can have a more comprehensive and intuitive understanding of the genomic characteristics of the strain (Krzywinski et al, 2009). This provides strong data support for the next step of research on the strain. The mechanism of ZEN degradation was analyzed in detail from the aspects of cell movement and protein degradation related gene prediction of strain L-4.

[0054] 4 Summary The present invention screened a strain L-4 from chicken intestine, and the identification results showed that it was a strain of Bacillus mohaiwei. L-4 has a strong metabolic conversion effect in the process of degrading ZEN. The strain L-4 has perfect flagella assembly and chemotaxis-related genes, which is conducive to its tendency to favorable conditions, increasing the degradation and bioavailability of ZEN. ZEN is a suitable efeB 3668 substrate. The above results provide a theoretical basis for the future application of Bacillus mohaiwei. Example

[0055] Probiotic properties and safety evaluation of L-4 strain 1 Materials and methods 1.1 Experimental materials 1.1.1 Reagents and consumables Lactobacillus liquid culture medium, Lactobacillus agar culture medium, and TSBA (5% sheep blood TSA plate) were purchased from Qingdao High-Tech Industrial Park Haibo Biotechnology Co., Ltd. Artificial gastric juice and simulated intestinal fluid were purchased from Beijing Coolaibo Technology Co., Ltd. Potassium dihydrogen phosphate, sodium hydroxide, and hydrochloric acid were purchased from Beijing Solebow Technology Co., Ltd. Twenty common antibiotic susceptibility tablets: ampicillin, oxacillin, carbenicillin, penicillin G, piperacillin, kanamycin, neomycin, ceftazidime, gentamicin, amikacin, erythromycin, tetracycline, and cefuroxime Cephalexin, minocycline, doxycycline, cephradine, cefazolin, ceftriaxone, and cefoperazone were purchased from Hangzhou Microbiological Reagent Co., Ltd., ox bile salt, yeast powder, ammonium chloride, casein, sodium carboxymethyl cellulose, soluble starch, peptone, sodium chloride, agar, ammonium sulfate, magnesium sulfate heptahydrate, etc. were purchased from Jinan Rannuo Trading Co., Ltd., mouse feed, wood shavings bedding, etc. were purchased from Qingdao Daren Fucheng Animal Husbandry Co., Ltd., and various types of centrifuge tubes, disposable culture dishes, inoculation loops, syringes, paraffin, etc. were purchased from Qingdao Aidewang Chemical Technology Co., Ltd.

[0056] 1.1.2 Experimental animals Female and male Kunming mice were purchased from Qingdao Daren Fucheng Animal Husbandry Co., Ltd.

[0057] 1.1.3 Test equipment

[0058] 1.2 Enzyme production medium Protease screening medium formula: 5 g casein, 3 g yeast powder, 10 g peptone, 5 g sodium chloride and 20 g agar were added to 1 L distilled water, pH 7.0, and autoclaved at 121 °C for 15-20 min. Amylase screening medium formula: 5 g yeast powder, 20 g soluble starch, 10 g peptone, 5 g sodium chloride and 20 g agar were added to 1 L distilled water, pH 7.0, and autoclaved at 121 °C for 15-20 min. Formula of cellulase screening medium: add 10 g sodium carboxymethyl cellulose, 4 g ammonium sulfate, 1 g peptone, 1 g potassium dihydrogen phosphate, 0.5 g magnesium sulfate heptahydrate and 20 g agar to 1 L distilled water, pH 7.0, and sterilize under high pressure at 121 °C for 15-20 min.

[0059] 1.4 Test methods 1.4.1 Growth curve of L-4 strain The preserved L-4 bacterial liquid was streaked on a lactobacillus solid culture medium plate to obtain a single colony of the L-4 strain, and a single colony was picked up and placed in a lactobacillus liquid culture medium, and the bacterial liquid was shaken to activate the fermented bacterial liquid at a speed of 160 r / min in a shaker at 37 ℃ to obtain a fermented bacterial liquid. The fermented bacterial liquid was added to the lactobacillus liquid culture medium at a ratio of 5% (V / V), and incubated at a speed of 160 r / min in a shaker at 37 ℃ for 30 h, with three replicates. During this period, samples were taken every 3 h, and the absorbance of the L-4 strain fermented bacterial liquid at 600 nm was measured by a UV spectrophotometer, and the growth curve of the L-4 strain within 30 h was drawn.

[0060] 1.4.2 L-4 strain tolerance test 1.4.2.1 Acid and alkaline medium tolerance test In order to explore the tolerance of L-4 strain to acidic and alkaline lactobacillus liquid culture medium, L-4 fermentation broth (1.5×109 cfu / mL) was added to liquid culture medium with pH values ​​of 2.5, 4, 9 and 11 at a ratio of 5% (V / V), and incubated in a 37 ℃ constant temperature shaker at 160 r / min. Three replicates were taken at 0, 1, 2 and 3 h, respectively. The samples were gradiently diluted and spread on the surface of lactobacillus solid culture medium, and then cultured in a 37 ℃ constant temperature incubator for 16 h. The plate colony count method was used to determine the concentration of the culture medium and the tolerance curve was drawn.

[0061] 1.4.2.2 Bile salt medium tolerance test In order to explore the tolerance of L-4 strain to bile salts, in this experiment, L-4 fermentation broth (1.5×109 cfu / mL) was added to Lactobacillus liquid culture medium containing 0.1%, 0.3% and 0.5% (w / v) of ox bile salt at a ratio of 5% (V / V), and then cultured in a 37 ℃ shaker at a speed of 160 r / min. Samples were taken at 0, 1, 2, and 3 h, respectively, with three replicates for each group. After sampling, the samples were gradiently diluted and spread on the surface of Lactobacillus solid culture medium, and then cultured in a 37 ℃ constant temperature incubator for 16 h. The plate colony count method was used to determine the concentration of the bacterial solution and draw a tolerance curve.

[0062] 1.4.2.3 Artificial gastric juice and simulated intestinal juice tolerance test In order to explore the tolerance of L-4 strain to artificial gastric juice and simulated intestinal juice, 4 mL of L-4 fermentation liquid was taken in a 10 mL sterile centrifuge tube and centrifuged at 3000 r / min for 20 min. The obtained precipitate was washed twice with sterile PBS, and then 4 mL of artificial gastric juice and simulated intestinal juice were added respectively. The above centrifuge tubes were placed in a 37 ℃ shaker and incubated at 160 r / min, and samples were taken at 0, 1, 2, and 3 h, respectively, with three replicates in each group. After sampling, the samples were gradient diluted and spread on the surface of the lactobacillus solid culture medium, placed in a 37 ℃ constant temperature incubator for 16 h, and the plate colony count method was used to determine the concentration of the bacterial liquid and draw a tolerance curve.

[0063] 1.4.3 Antibiotic sensitivity test of L-4 strain In order to explore the sensitivity of Bacillus mohaiwei L-4 to common antibiotics, this experiment referred to the Kirby-Bauer paper diffusion method to spread the fermentation liquid of L-4 strain on lactobacillus agar plates, and placed drug sensitivity tablets (2 tablets / plate): ampicillin, oxacillin, carbenicillin, penicillin G, piperacillin, kanamycin, neomycin, ceftazidime, gentamicin, amikacin, erythromycin, tetracycline, cefuroxime, cephalexin, minocycline, doxycycline, cephradine, cefazolin, ceftriaxone, and cefoperazone. The concentrations reached the standard content of antibiotics on the microbial drug sensitivity paper, and three replicates were set for each drug sensitivity tablet. All lactobacillus solid plates were placed in a 37 ℃ constant temperature incubator for 24 h, and then the inhibition zone diameter was measured and recorded using a fully automatic colony counter.

[0064] 1.4.4 Enzyme production performance test of L-4 strain In order to explore the enzyme production performance of Bacillus mohaiwei L-4, this experiment used an inoculation loop to pick up the L-4 single colony obtained from the drawing plate and inoculated it on the cellulase, protease and amylase screening plates respectively, and then placed the plate in a 37 ℃ constant temperature incubator for 36 h. After taking it out, Lu's iodine solution was added to cover the entire plate to observe whether a transparent circle appeared, and an automatic colony counter was used to measure the size of the colony and the transparent circle it produced. The average value was taken after three measurements, and the ratio of the diameter of the transparent circle to the diameter of the colony was used to evaluate the size of the enzyme production capacity.

[0065] 1.4.5 Safety test of L-4 strain 1.4.5.1 In vitro safety tests In order to explore the in vitro safety of the L-4 strain, this experiment will pick a loop of L-4 fermentation liquid and make three-zone lines on the sheep blood agar plate, and then place the agar plate in a 37 ℃ constant temperature incubator for 16 hours. Evaluation and classification are based on the dissolution of red blood cells in the culture medium around the colonies: green areas appear around the colonies (α-hemolysis), transparent areas appear around the colonies (β-hemolysis), and no changes around the colonies (γ-hemolysis, no hemolysis). If γ-hemolysis is detected in the medium, the strain is considered safe.

[0066] 1.4.5.2 In vivo safety study in mice To investigate the in vivo safety of the L-4 strain and its potential as a probiotic, Kunming mice (male and female, 20.0 ± 2.0 g) were used for acute and chronic toxicity evaluations. During the study, they had free access to distilled water and commercial mouse chow.

[0067] Acute toxicity test In order to study the acute toxicity of Bacillus mohaiwei L-4, 30 Kunming mice (half male and half female) were adaptively fed for three days and then divided into three groups (n = 10), half male and half female in each group. Blank group: one-time gavage of 0.5 mL distilled water / mouse, control group: one-time gavage of 0.5 mL Lactobacillus liquid culture medium / mouse, experimental group: one-time gavage of 0.5 mL L-4 strain fermentation liquid (1.5×109 cfu / mL) / mouse. During the whole experiment, standard feed and distilled water were fed every day, and the fecal status, toxicity signs and mortality were recorded. After seven days, the mice were uniformly killed on the eighth day.

[0068] Chronic toxicity test To investigate the chronic toxicity of Bacillus mohaiwei L-4, 60 Kunming mice (half male and half female) were adaptively fed for 1 week and then divided into three groups (n = 20), half male and half female in each group. The blank group (group K) was gavaged with 0.2 mL of distilled water / mouse every 2 days, the control group (group D) was gavaged with 0.2 mL of Lactobacillus liquid culture medium / mouse every 2 days, and the experimental group (group S) was gavaged with 0.2 mL of L-4 strain fermentation liquid (1.5×109 cfu / mL) / mouse every 2 days. During the entire experiment, the mice were fed with standard feed and distilled water every day, and the initial and final weights of the mice were recorded. The signs of toxicity, mortality, and fur and feces were recorded every 2 days for 28 days.

[0069] The mice were fasted at night on the 28th day. After measuring their body weight on the 29th day, they were uniformly killed for autopsy, and the organs were collected and weighed to calculate the organ index. The duodenum and ileum of four mice were randomly taken from each group. After rapid fixation with 4% paraformaldehyde for 48 hours, they were dehydrated, transparent, wax-impregnated, and paraffin-embedded. After that, 4 μm sections were made, and the sections were stained with hematoxylin-eosin (H&E). Finally, they were sealed with neutral resin and observed and photographed under a microscope. The pictures were analyzed with Image j software; the cecal contents of four mice in each group were taken into sterile enzyme-free centrifuge tubes and sent to Sangon Biotech (Shanghai) Co., Ltd. for analysis of cecal microbial diversity.

[0070] 1.4.5.3 Translocation test of Bacillus mohaiwei L-4 In order to explore whether Bacillus mohaiwei L-4 will translocate in mice, the heart, liver, spleen, lung, and kidney of each group of mice obtained in 1.4.5.2 were immersed in sterile saline. After the above organs were cut open with a high-temperature sterilized scalpel in the clean bench, the organ section was scraped with a sterilized inoculation loop and inoculated on the surface of the Lactobacillus solid culture medium plate. The plate was placed in a 37 ℃ constant temperature incubator for 24 hours, and the growth of colonies on the surface of the culture medium was observed. If colonies grow on the surface of the plate, it indicates that Bacillus mohaiwei L-4 has translocated in the mouse body, and if no colonies grow, it indicates that no translocation has occurred.

[0071] 1.5 Data Processing The experimental data were statistically analyzed and plotted using Excel, Image j and GraphPad prism9 software.

[0072] 2 Results and Analysis 2.1 Growth curve of strain L-4 The growth curve of Bacillus mohaiwei L-4 in the lactobacillus liquid culture medium was determined by measuring the absorbance of L-4 fermentation liquid at 600 nm on a UV spectrophotometer. Fig.18 As shown in the figure, Bacillus mohaiwei L-4 entered the logarithmic phase at 3 h, reached the stable phase at 9 h, and extended to 18 h. Therefore, the logarithmic phase data showed that the L-4 bacterial solution proliferated rapidly between 3 and 9 h.

[0073] 2.2 Results of the L-4 strain tolerance test In order to determine whether the L-4 strain can survive in the gastrointestinal tract of animals, the tolerance to different pH values ​​(2.5, 4, 9 and 11), tolerance to artificial gastrointestinal and simulated intestinal fluid, tolerance to 8% NaCl, and tolerance to 0.1%, 0.3% and 0.5% bile salts were tested. It was found that the L-4 strain has a certain tolerance to culture media and artificial gastric fluid at pH 2.5, pH 4, and pH 11, and has a strong tolerance to liquid culture media and simulated intestinal fluid at pH 9, see the attached figure of the instruction manual Fig.19 (A, C). In addition, the L-4 strain has a certain tolerance to bile salts and 8% NaCl, see the attached figure of the specification Fig.19 (B, D) This result further indicates that the L-4 strain has the potential to be an animal probiotic.

[0074] 2.3 Results of antibiotic sensitivity test of L-4 strain The property of being sensitive to antibiotics is a prerequisite for safe probiotic strains. In this experiment, the Kirby-Bauer method was used to determine the sensitivity of the L-4 strain to 20 common antibiotics. As can be seen from Table 4, Bacillus mohaiwei L-4 is completely sensitive to carbenicillin, piperacillin, kanamycin, ceftazidime, erythromycin, tetracycline, cefuroxime, cephalexin, minocycline, doxycycline, cephradine, cefazolin, ceftriaxone, and cefoperazone, and has a certain resistance to ampicillin, oxacillin, penicillin, neomycin, gentamicin, and amikacin.

[0075]

[0076] The results were evaluated according to the Standard for Antimicrobial Susceptibility Test by Paper Disk Method WS / T 125-1999. μg / pc represents a piece of paper about the unit dose of antibiotics, and "pc" represents a piece of antibiotic paper. S means sensitive, I means intermediate, and R means insensitive.

[0077] 2.4 Enzyme production performance of strain L-4 In order to understand the enzyme production characteristics of the L-4 strain, a single L-4 colony was picked from the lactobacillus agar medium plate and inoculated onto three enzyme production screening plates and cultured at 37°C for 36 h. Fig. 20 As shown, it was observed that strain L-4 had obvious transparent circles on the cellulase, protease and amylase plates. The transparent circles and diameters were 15.63, 30.27 and 19.70 mm, respectively. The diameters of the colonies were 4.85, 8.03 and 13.63 mm, respectively. The ratios were 3.22, 3.77 and 1.45, respectively. In this experiment, the activities of the three enzymes were all detected at a high level. This shows that the strain is suitable for use as a probiotic.

[0078] 2.5 Results of safety test on L-4 strain 2.5.1 Results of acute toxicity test During the 7 days of the experiment, the mice in the blank group, control group, and experimental group were in good mental state and no deaths occurred. The drinking water, food intake, and feces were all normal, with no signs of toxicity.

[0079] 2.5.2 Chronic toxicity test results During the 28 days of the experiment, no mice in the three groups showed signs of poisoning or death. The drinking water, food intake, and feces of the mice in the experimental group were normal, and no pathological changes were found in the internal organs during autopsy. Therefore, oral administration of Bacillus mohaiwei L-4 at a dose of up to 1.5×109 cfu / mL is safe and will not produce any adverse reactions.

[0080] At the end of the experiment, the weight gain of male mice in each group was 14.18-15.90 g, and the weight gain of female mice was 7.30-10.45 g. The average weight gain of male mice in the experimental group was 15.90 g, which was significantly higher than that in the other two groups (P < 0.05), as shown in Table 5; the average weight gain of female mice in the blank group was 10.45 g, the average weight gain of female mice in the experimental group was 9.9 g, and the average weight gain of female mice in the control group was 7.3 g. There were significant differences among the three groups (P < 0.05), as shown in Table 6.

[0081] After feeding mice with Bacillus mohaiwei L-4 for 28 days, as shown in Tables 7 and 8, there was no statistical difference in the organ indexes of male and female mice in the three groups (P>0.05), which indicated that the L-4 strain had no adverse effects on the mouse organs. It can be considered that the L-4 strain is safe and has the potential to be fed to animals as a probiotic.

[0082] Observation of the ileum and duodenum sections of the three groups of mice revealed that the chorionic villus ratio of the experimental group was significantly higher than that of the other two groups (P < 0.05), as shown in the accompanying figure. Fig.21 , Fig. 22 , as shown in Table 9. The larger the villus to intestinal villi ratio, the longer the intestinal villi are, the larger the absorption area is, and the more it can promote the absorption of nutrients.

[0083]

[0084] Cecal microbial diversity α diversity analysis and β diversity analysis are shown in the attached figure of the specification. Fig.23 As shown, ( Fig.23 Figures A, B, and C) are used to reflect the differences in microbial communities among different samples in the experiment. It can be seen from Figures A and B that there is no significant difference in the diversity of sample community distribution among the three groups at the species level; Figure C shows that there is a certain overlap between the three groups, indicating that there is a certain similarity in the composition of intestinal microbial communities in each group, and the abundance of cecal microorganisms in the experimental group of mice is richer than that in the other two groups.

[0085] 2.5.3 Translocation test results The bacterial translocation test showed that 28 days after mice were gavaged with B. mojavensis L-4 fermented bacteria, no colonies grew when fresh organ sections of mice in the three groups were inoculated in culture dishes. This indicated that the B. mojavensis L-4 strain did not translocate into the heart, liver, spleen, lung and kidney tissues of mice, indicating that the B. mojavensis L-4 strain did not have the risk of bacterial translocation.

[0086] 3 Discussion This study investigated the growth curve, stress resistance, enzyme production, antibiotic sensitivity and safety of B. mojavensis L-4. The results showed that: (1) The L-4 strain entered the logarithmic growth phase after 3 hours and the plateau phase after 18 hours. It was tolerant to acid, alkali, salt, bile salt and gastrointestinal fluid. (2) L-4 had the ability to produce protease, cellulase and amylase, and the activities of the three enzymes were at a high level. (3) The L-4 strain was sensitive to 15 antibiotics, did not produce hemolytic enzymes, promoted weight gain and intestinal absorption in mice, and had no risk of translocation.

[0087] When probiotics are added to animal feed, their tolerance to acid, alkali, gastrointestinal fluid, salt and bile salts must be ensured to ensure that they can enter the gastrointestinal tract smoothly and colonize to play a probiotic role (Kwak et al, 2016). Bacillus generally has good stress resistance, but reports vary. Xinna et al. (2022) found that Bacillus subtilis has strong alkali resistance but poor acid resistance; Wu Zhenchao (2020) found that Bacillus mojavensis LSG3-8 has a certain tolerance to bile salts, 8% high salt, acidic liquid culture medium and artificial gastric juice; Adıgüzel (2020) found that B. mojavensis TH309 showed good tolerance in the pH range of 5-11. Similarly, B. mojavensis L-4 in this article has good tolerance to salt, bile salts, simulated intestinal fluid and artificial gastric juice, and is a good probiotic for animals.

[0088] Probiotics have the ability to produce digestive enzymes and are sensitive to antibiotics (Kwak et al, 2016). The resistance of probiotics to antibiotics can be used to determine whether they can be used clinically. If the probiotic has drug-resistant genes, it may transfer drug-resistant genes to normal intestinal flora, pathogenic bacteria, etc., thereby causing intestinal flora disorders and affecting the health of the animal body. Studies have found that B. mojavensis L-4 exhibits a strong ability to produce proteases, cellulases and amylases. Through drug resistance tests, it was found that the B. mojavensis L-4 strain is sensitive to a variety of antibiotics such as carbenicillin, piperacillin, kanamycin, ceftazidime, erythromycin, tetracycline, cefuroxime, cephalexin, minocycline, doxycycline, cefradine, cefazolin, ceftriaxone, and cefoperazone.

[0089] When adding probiotics to food or animal feed, it is necessary to ensure their tolerance to acid, alkali (Liu Zhiyuan et al., 2020), gastrointestinal fluid (Kwak et al., 2016), salt and bile salt (Gutiérrez et al., 2021), so as to ensure that they can enter the gastrointestinal tract smoothly and colonize and play a role. The present study found that Bacillus mohaiwei L-4 is tolerant to artificial gastric juice, acidic liquid culture medium, bile salt and high salt, and simulated intestinal fluid and alkaline liquid culture medium can promote the growth of L-4 strain.

[0090] Probiotics should also be safe, that is, they should not be toxic to the host. When conducting mouse experiments, attention should be paid to the organ index, intestinal villus-crypt ratio and bacterial translocation. The organ index reflects the effect of B. mojavensis L-4 strain on the growth and development of mice (Singh et al, 2019); the larger the intestinal villus-crypt ratio, the longer the villi of the small intestine, which is more conducive to its absorption of nutrients; if the B. mojavensis L-4 strain is translocated in the animal body, it indicates that it can be transferred from the gastrointestinal tract to other organs, which may cause diseases in other organs (Feng Yuanyuan et al, 2017). Therefore, the organ index and bacterial translocation test are important evaluation indicators for the safety of probiotics. The present invention found that the B. mojavensis L-4 strain had no translocation phenomenon; the villus-crypt ratio of mice in the experimental group was significantly higher than that of the other two groups (P < 0.05), and the final weight of male mice in the experimental group at 28 days of age was significantly higher than that of the other two groups (P < 0.05); there were no significant differences in the organ indexes of the heart, liver, spleen, lung and kidney of mice in each group.

[0091] The intestinal flora of animals is closely related to the health of their gastrointestinal tract. If the probiotic enters the intestine and causes intestinal flora disorder, it will lead to the occurrence of digestive and non-digestive tract diseases. This experiment found that the α diversity of the cecal microbial community distribution of mice in the experimental group was higher than that of the other two groups, but not significantly; β diversity analysis found that the abundance of cecal microorganisms in the experimental group was higher than that of the other two groups at the genus level. This result shows that the B. mojavensis L-4 strain has a certain ability to improve the diversity of intestinal microorganisms in mice.

[0092] 4 Summary In this example, it was found that the B. mojavensis L-4 strain has the characteristics of tolerance to acid, alkali, bile salt, high salt and artificial gastric juice and simulated intestinal juice, can produce protease, cellulase and amylase, is sensitive to multiple antibiotics, is safe and promotes absorption, and can change the composition of intestinal microorganisms to increase microbial abundance, and has the potential to be used as an animal probiotic.

[0093] Example 3 Effects of different bacteria on the degradation of zearalenone 1 Materials and methods 1.1 Test materials 1.1.1 Test strains Enterococcus faecalis, Lactobacillus rhamnosus, Lactobacillus delbrueckii subsp.bulgaria, Bacillus mojavensis, Lactobacillus plantarum, Lactobacillus paracasei, Streptococcus thermophilus, Bacillus licheniformis, Bacillus cereus, Saccharomyces cerevisiae, and Bacillus subtilis were isolated and stored in the Laboratory of Animal Nutrition, Metabolism and Toxic Diseases of Qingdao Agricultural University.

[0094] 1.1.2 Test instruments see Table 10

[0095] 1.1.3 Reagents and consumables required for the experiment LB solid medium, LB liquid medium, YPD liquid medium, YPD solid medium, nutrient broth medium, nutrient broth agar medium, Lactobacillus liquid medium, Lactobacillus agar medium, Wahaha purified water, Gram stain, HBI Bacillus biochemical identification strips, nitrate reduction reagent, VP kit, Lugol's iodine solution, methanol (chromatographic grade), acetonitrile (chromatographic grade), zearalenone (ZEN) standard, etc. were purchased from Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd., and various specifications of centrifuge tubes, 1.5 mL liquid phase vials, 1 L Shu Niu bottles, 0.22 μm PTFE filter membranes, 1 mL sterile syringes, 75% alcohol, etc. were purchased from Qingdao Aidewang Chemical Technology Co., Ltd.

[0096] 1.2 Test methods 1.2.1 Establishment of the zearalenone detection method Liquid chromatography conditions: Use a C18 chromatographic column (150 mm×4.6 mm, 5 μm specification), set the injection volume to 20 μL, the flow rate to 0.8 mL / min, the detection wavelength to 274 nm, the column temperature to 30 °C, and the mobile phase is acetonitrile: water: methanol = 46:46:8 (V / V / V). After preparation, ultrasonication is required for 40 min.

[0097] The ZEN standard was dissolved in acetonitrile to prepare a 100.0 μg / mL standard stock solution, and the standard stock solution was diluted with acetonitrile to a 5.0 μg / mL mother solution.

[0098] The ZEN mother solution was diluted with the mobile phase to 0.05, 0.10, 0.50, 1.00, and 2.00 μg / mL standard solutions, and 0.5-1.0 mL was taken to filter through a 0.22 μm PTFE filter membrane and directly into a liquid phase vial. The peak area was detected by high performance liquid chromatography, and finally a standard curve was drawn according to the ZEN concentration. The liquid chromatography gradient elution program is shown in Table 11.

[0099]

[0100] 1.2.2 Detoxification effect of zearalenone In addition to the laboratory-preserved strains of Enterococcus faecalis, Lactobacillus rhamnosus, Lactobacillus delbrueckii subsp.bulgaria, Lactobacillus plantarum, Lactobacillus paracasei, Bacillus licheniformis, Bacillus cereus, and Bacillus subtilis, this experiment collected chicken feces, Yingchunle naked yogurt, and Nongda Impression original beer samples, picked them up with cotton swabs, put them in sterile saline, shook them well, and then streaked them on solid culture medium for multiple purification to obtain purified colonies.

[0101] The prepared Enterococcus faecalis, Lactobacillus rhamnosus, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus plantarum, Lactobacillus paracasei, Bacillus licheniformis, Bacillus cereus, Bacillus subtilis, Bacillus mohaiwei, and Streptococcus thermophilus fermentation broth was inoculated into the corresponding liquid culture medium at a ratio of 5% and cultured on a shaker at 160 rpm for 48 h. The prepared Saccharomyces cerevisiae fermentation broth was inoculated into the corresponding liquid culture medium at a ratio of 5% and cultured on a shaker at 160 rpm for 72 h. The initial concentration of ZEN was set to 0.5 μg / mL, and the tube with only toxin added was used as the control. 460 μL of the test group and control group samples were added to 2 mL sterile centrifuge tubes with acetonitrile and methanol in a ratio of 46:46:8, and mixed by vortexing for 10 min. The mixed sample was drawn with a 1 mL sterile syringe and filtered through a 0.22 μm PTFE filter membrane into a liquid phase vial. The residual ZEN in the culture medium was detected by HPLC. Three replicates were set for each group in this experiment, and the test results were averaged.

[0102] The HPLC conditions were the same as those in 1.2.1, and the HPLC gradient elution program was shown in Table 2. The formula for calculating the detoxification rate was (1-peak area of ​​the experimental group / peak area of ​​the control group) × 100%.

[0103] 1.3 Data processing The experimental data were statistically analyzed and plotted using Excel software.

[0104] 2 Results and analysis 2.1 Zearalenone detection method As shown in the attached figure of the instruction manual Fig.24 As shown: HPLC detection found that the peak time of ZEN standard was about 7.4 minutes, and there was a good linear relationship between the concentration and peak area of ​​ZEN, with the correlation coefficient R2 maintained above 0.999.

[0105] 2.2 Detoxification effect of zearalenone After isolating and identifying the microorganisms in the samples, we obtained a strain of Bacillus mohaiwei from chicken feces, a strain of Saccharomyces cerevisiae from beer, and a strain of Streptococcus thermophilus from yogurt, plus the original strains in the laboratory, a total of eleven probiotic strains. Using the co-culture method of probiotic strains and ZEN toxin, we initially screened out a strain of Bacillus mohaiwei with a ZEN detoxification rate higher than 80%, and two strains of Saccharomyces cerevisiae and Bacillus subtilis with a detoxification rate higher than 50%. The results are shown in the attached figure of the specification. Fig.25 Among them, the highest detoxification rate of the detoxified bacteria was 81.04%, named Bacillus mohaiwei L-4, followed by 61.39% and 56.32%, named Bacillus subtilis L-11 (B. s 11) and Saccharomyces cerevisiae YPD10 (S. c 10), respectively.

[0106] 3 Discussion Zearalenone is mainly caused by the massive production of Fusarium due to improper storage of feed and grain crops, which has a serious impact on the agricultural and animal husbandry economy (Han et al, 2022). Therefore, ZEN detoxification has attracted much attention in current research, among which the biodegradation method is favored due to its strong specificity, safety, high efficiency and low cost. Based on the determination of the zearalenone detection method, the present invention screened the ZEN detoxification bacteria and identified them by morphological, physiological and biochemical and molecular biological methods. The results showed that: (1) The ZEN method in liquid culture medium was feasible by high performance liquid chromatography, and there was a good linear relationship between ZEN concentration and peak area; (2) A ZEN detoxification bacteria L-4 strain was initially screened and its whole genome was sequenced, and it was confirmed to be Bacillus mohaiwei.

[0107] High performance liquid chromatography is one of the most commonly used methods for detecting mycotoxins. The present invention first determined a ZEN detection method for liquid culture medium with reference to the national standard for the detection of zearalenone (2012), and formulated detailed parameters. It was found that there was a good linear relationship between the ZEN concentration and the peak area, and the correlation coefficient R2 remained above 0.999. Subsequently, a ZEN and probiotic fermentation broth co-culture method was used for preliminary screening, and finally a strain L-4 with high efficiency in detoxification of ZEN was screened. After 48 hours of incubation, the strain had a detoxification rate of 81.04% for the lactobacillus liquid culture medium with a ZEN content of 0.5 μg / mL.

[0108] 4 Summary The present invention establishes a method for detecting the content of zearalenone in liquid culture medium using high performance liquid chromatography, and the results show that the ZEN peak area and concentration have a good correlation. The chicken intestine strain L-4 has a ZEN detoxification rate of 81.04% in the lactobacillus liquid culture medium, which provides a theoretical basis for the application of Bacillus mohaiwei in the ZEN detoxification field.

[0109] Example 4 Study on the degradation conditions, components and products of strain L-4 1 Materials and methods 1.1 Experimental materials 1.1.1 Reagents and consumables Chromatographically pure methanol and acetonitrile were purchased from Tianjin Komiou Chemical Reagent Co., Ltd., Lactobacillus liquid culture medium, Lactobacillus agar culture medium, Wahaha purified water, etc. were purchased from Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd., artificial gastric juice and simulated intestinal fluid were purchased from Beijing Coolabo Technology Co., Ltd., hydrochloric acid and sodium hydroxide were purchased from Beijing Solable Technology Co., Ltd., and various specifications of centrifuge tubes, 1.5 mL liquid phase vials, 1 L Shu Niu bottles, 0.22 μm PTFE filter membranes, 1 mL sterile syringes, 75% alcohol, sealing films, etc. were purchased from Qingdao Aidewang Chemical Technology Co., Ltd.

[0110] 1.1.2 Test instruments see Table 12

[0111] 1.2 Test methods 1.2.1 Conditions for detoxification of ZEN by L-4 strain 1.2.1.1 Effect of initial ZEN concentration on detoxification effect In order to explore the effect of ZEN initial concentration on detoxification effect, the present invention inoculated the preserved Bacillus mohaiwei L-4 bacterial liquid into the lactobacillus liquid culture medium at a ratio of 5%, and placed it in a shaker at 37 ℃ and 160 r / min for 18 h to obtain the fermented bacterial liquid. Experimental group: first add 3.8 mL of lactobacillus liquid culture medium to a 10 mL sterile centrifuge tube, and then add 200 μL of L-4 strain fermented bacterial liquid. The final concentration of ZEN in the culture medium was set to 0.5, 1.0, and 5.0 μg / mL, respectively, and a centrifuge tube containing 4 mL of lactobacillus liquid culture medium + 0.5 μg / mL ZEN was used as the control group; three replicates were set for each group, and the results were averaged. The above centrifuge tubes were placed in a 37 ℃ shaker at 240 r / min for 48 h and then taken out. The residual amount of ZEN in the liquid culture medium was detected by high performance liquid chromatography, and the detoxification rate was calculated. The detection and calculation methods were the same as 1.2.2 in Chapter 2.

[0112] 1.2.1.2 Effect of treatment time on detoxification effect In order to explore the effect of different treatment time on the detoxification effect of ZEN, the following treatments were performed in this experiment: experimental group: 3.8 mL of lactobacillus liquid culture medium was transferred to a 10 mL sterile centrifuge tube, and 200 μL of L-4 fermentation liquid was added to make the final concentration of ZEN in the culture medium 0.5 μg / mL, and the centrifuge tube containing 4 mL of lactobacillus liquid culture medium + 0.5 μg / mL ZEN was used as the blank control group; three replicates were performed in each group. Culture was carried out at 37 ℃ and 240 r / min, and samples were taken at 24, 36, 48, 60, and 72 h, and high performance liquid chromatography was used to detect the residual amount of ZEN in the liquid culture medium, and the detoxification rate was calculated. The detection and calculation methods were the same as those in Chapter 2, 1.2.2, and the results were averaged.

[0113] 1.2.1.3 Effect of pH value on detoxification effect In order to explore the effect of the pH value of the liquid culture medium on the detoxification effect of ZEN, this experiment was conducted as follows: experimental group: 3.8 mL of lactobacillus liquid culture medium with different pH values ​​(the pH of the culture medium was adjusted to 2.5, 4, 9 and 11 with hydrochloric acid and sodium hydroxide) and artificial gastric juice and simulated intestinal fluid were connected to a 10 mL sterile centrifuge tube, and 200 μL of L-4 fermentation liquid was added. The final concentration of ZEN in the liquid culture medium was set to 0.5 μg / mL; the liquid culture medium with different pH values ​​containing 0.5 μg / mL ZEN was used as the blank control group, and three replicates were used in each group. After culturing in a shaker at 37 ℃ and 240 r / min for 24 hours, samples were taken, and the residual amount of ZEN in artificial gastric juice, simulated intestinal juice and liquid culture medium was detected by high performance liquid chromatography, and the detoxification rate was calculated. The detection and calculation methods were the same as those in 1.2.2 of Chapter 2, and the results were averaged.

[0114] 1.2.1.4 Effect of temperature on detoxification effect In order to explore the effect of different treatment temperatures on the detoxification effect of ZEN, this experiment was carried out as follows: experimental group: 3.8 mL of lactobacillus liquid culture medium was transferred to a 10 mL sterile centrifuge tube, and 200 μL of L-4 fermentation liquid was added to make the final concentration of ZEN in the culture medium 0.5 μg / mL. The lactobacillus liquid culture medium containing only 0.5 μg / mL ZEN was used as the blank control group, and each group was repeated three times. The samples of the experimental group and the control group were placed in a shaker at 25 ℃, 30 ℃, 37 ℃, 42 ℃, and 45 ℃, respectively, and cultured at 240 r / min for 24 h. The samples were taken separately, and the residual amount of ZEN in different liquid culture media was detected by high performance liquid chromatography, and the detoxification rate was calculated. The detection and calculation methods were the same as 1.2.2 in Chapter 2, and the results were averaged.

[0115] 1.2.2 L-4 strain detoxifies ZEN components In order to explore the cellular components of Bacillus mohaiwei L-4 that play a major role in ZEN detoxification, the L-4 fermentation broth was treated as follows in this experiment to obtain fermentation supernatant, bacterial suspension, inactivated bacterial suspension, inactivated supernatant, intracellular fluid and bacterial crushing precipitate.

[0116] The fermentation supernatant can be obtained by centrifuging the fermentation broth at 8,000 r / min for 20 min. The L-4 live bacterial suspension can be obtained by resuspending the bacterial precipitate twice with PBS. The L-4 live bacterial suspension can be inactivated by placing the bacterial suspension in a high-pressure steam sterilizer at 121°C for 15 min to obtain the L-4 inactivated bacterial suspension. The fermentation supernatant can be kept in a constant temperature water bath at 100°C for 10 min to obtain the L-4 inactivated supernatant. The live bacterial suspension is subjected to ultrasonic disruption (360 W, ultrasound 9.9s, stop 9.9s, a total of 40 min) and then centrifuged to obtain the supernatant, which is the L-4 intracellular fluid. The L-4 live bacterial suspension is subjected to ultrasonic disruption and then centrifuged to obtain the precipitate, which is resuspended in PBS to obtain the L-4 bacterial disruption precipitate. ZEN was added to the above 6 components, and the initial concentration of ZEN was set to 0.5 μg / mL. PBS or Lactobacillus liquid culture medium with a ZEN content of 0.5 μg / mL was used as a control. Three replicates were set for each group, and the results were averaged. After the above 6 groups were cultured in a shaker at 37°C and 240 r / min for 72 h, the residual amount of ZEN in PBS and liquid culture medium was detected by high performance liquid chromatography. The detection and calculation methods were the same as 1.2.2 in Example 3, and the detoxification rate was calculated.

[0117] Proteinase K (1 mg / mL), SDS (final concentration of 1%) and proteinase K+SDS were added to the component with the highest detoxification rate obtained in the above test to set the final concentration of ZEN to 0.5 μg / mL. The lactobacillus liquid culture medium containing only 0.5 μg / mL ZEN was used as the control. Three replicates were added to each group. The above samples were placed in a 37 ℃ shaker at 240 r / min and cultured for 72 h. High performance liquid chromatography was used to detect the ZEN content and calculate the detoxification rate. The detection and calculation methods were the same as 1.2.2 in Chapter 2, and the results were averaged.

[0118] 1.2.3 ZEN detoxification products of L-4 strain After the preserved L-4 bacterial liquid was activated, it was added to the lactobacillus liquid culture medium at a ratio of 5%, and the final concentration of ZEN was set to 0.5 μg / mL. It was placed in a 37 ℃ shaker at 240 r / min and incubated for 20 h; the lactobacillus liquid culture medium, fermentation liquid, liquid culture medium and ZEN mother solution with only 0.5 μg / mL ZEN were used as controls, and three replicates were set for each group. After the end, 500 μL of the sample was taken into a 2 mL sterile centrifuge tube, an equal amount of methanol was added to terminate the reaction, and the mixture was vortexed for 5 min to mix, and then filtered through a 0.22μm PTFE filter into a liquid phase vial and sent to the Analysis and Testing Center of Qingdao Agricultural University for detection and analysis.

[0119] The chromatographic column was Agilent Poroshell 120 EC-C18 (2.1 mm×100 mm, 2.7 μm). The mobile phase was methanol in phase A and water (containing 0.1% formic acid and 5 mmol / L ammonium formate) in phase B. The gradient elution program was 0-0.5 min, 90%B, 10%A; 0.51-5 min, 90%-5%B, 10%-95%A; 5.01-8 min, 5%B, 95%A; 8.01-9 min, 90%B, 10%A. The flow rate was 0.2 mL / min, the column temperature was 30 °C, and 5 μL was injected.

[0120] Mass spectrometry detection conditions: electrospray ionization (ESI) source; negative ion mode scanning, ion source temperature: 350 °C; drying gas flow rate: 8 L / min; nebulizer gas pressure: 25 psi; capillary voltage: 3000 V; scanning range: m / z 50~1000; secondary mass spectrometry analysis setting induced collision dissociation energy: 20 eV.

[0121] 1.2.4 Analysis of ZEN-degrading enzymes by L-4 strain Log in to the NCBI website to download the known ZEN-degrading enzyme sequences, including the lactonase ZHD101 from Clonostachys rosea IFO7063, the phosphotransferase ZPH from Bacillus subtilis Y816, the CotA laccase from Bacillus licheniformis, the peroxidase BsDyP from Bacillus subtilis, the ZENC enzyme from Neurospora crassa and the ZLHY-6 enzyme from P. pastoris GSZ. Use DNAMAN software to perform multiple sequence alignment between all protein sequences obtained by third-generation sequencing and the above six protein sequences, and screen out the sequence of the protein with the highest homology.

[0122] 1.2.5 Detoxification test of ZEN-contaminated corn flour by strain L-4 10.0±0.01 g of finely ground corn flour was added to a 250 mL conical flask, sealed and sterilized in a high pressure steam autoclave at 121 ℃ for 15 min. 10 mL of sterile distilled water was added to the conical flask under a sterile environment and mixed well. ZEN standard was added to make the final concentration of 532.91 μg / kg. The preserved L-4 bacterial solution was activated and treated as follows: 1 mL of fermentation bacterial solution (1.5×109 cfu / mL) was added to the experimental group at a ratio of 10%, and 1 mL of lactobacillus liquid culture medium was inoculated to the control group. Each group was repeated three times. The conical flask was sealed with a sealing film and placed in a 37 ℃ constant temperature incubator for 5 days. Samples were taken at 1, 2, 3, 4, and 5 days, and the ZEN residue in the corn flour was detected by upconversion luminescence method. Its degradation rate was calculated and the results were averaged.

[0123] The formula for calculating the degradation rate = [(ZEN content in the control group - ZEN content in the experimental group) / ZEN content in the control group] × 100%.

[0124] 1.3 Data Processing The data obtained from the experiment were analyzed and plotted using Excel, Qualitative Analysis B.05.00, DNAMAN and GraphPadprism 9 software. Fig.26 shown.

[0125] 2 Results and Analysis 2.1 Detoxification of ZEN by Bacillus mohaiwei L-4 2.1.1 Detoxification results of different ZEN concentrations By studying the effect of different initial ZEN concentrations on the detoxification effect, it was found that the detoxification rate of L-4 strain in the liquid culture medium containing 0.5 and 1.0 μg / mL ZEN was 100%, and the detoxification rate in the liquid culture medium containing 5.0 μg / mL ZEN was 83.42% (e.g. Fig.14 A). It can be seen that Bacillus mohaiwei L-4 has a good detoxification effect on 0.5~5.0 μg / mL ZEN.

[0126] 2.1.2 ZEN detoxification results after different treatment times By studying the effect of different treatment times on the detoxification of ZEN, it was found that the L-4 strain could completely detoxify the liquid culture medium containing 0.5 μg / mL ZEN in 24 h. This result shows that at 240 r / min and 37 ℃, Bacillus mohaiwei L-4 can quickly detoxify 0.5 μg / mL ZEN (such as Fig.14 B).

[0127] 2.1.3 ZEN detoxification results at different pH values By studying the effect of different pH values ​​of liquid culture medium on the detoxification effect of ZEN, it was found that the L-4 strain had the best detoxification effect on liquid culture medium containing 0.5 μg / mL ZEN and simulated intestinal fluid under neutral and alkaline conditions (pH7, 9, 11), and the detoxification effect on liquid culture medium containing 0.5 μg / mL ZEN and artificial gastric fluid gradually weakened under acidic conditions (pH4, 2.5). This result shows that strong acid conditions will inhibit the growth of Bacillus mohaiwei L-4 and reduce its detoxification effect on ZEN (such as Fig.14 C).

[0128] 2.1.4 ZEN detoxification results at different temperatures By exploring the effect of different temperatures on the detoxification effect, it was found that the detoxification effect of the L-4 strain on the liquid culture medium containing 0.5 μg / mL ZEN can reach more than 90% at 37~45 ℃, and the temperature drop has a greater impact on the detoxification effect. At the same time, this also shows that on the basis of 37 ℃, the increase or decrease of temperature will weaken the detoxification effect of Bacillus mohaiwei L-4 on ZEN (such as Fig.14 D).

[0129] 2.2 Results of ZEN degradation by different components of L-4 strain Different components of L-4 fermentation broth were obtained by separation and extraction, and these components were incubated with 0.5 μg / mL ZEN respectively. It was found that the fermentation supernatant of L-4 strain had the best detoxification effect on ZEN, and the liquid culture medium containing 0.5 μg / mL ZEN could be completely detoxified within 72 hours. The detoxification effects of the remaining components on ZEN were all less than 25% (see the attached figure of the specification). Fig. 27 ). This result shows that the key to the detoxification of ZEN by L-4 strain lies in the extracellular substances it secretes, rather than the adsorption of cell membrane and the detoxification effect of intracellular substances.

[0130] After adding proteinase K, SDS and proteinase K+SDS to the fermentation supernatant of the component with the best detoxification effect, it was found that the detoxification rates of the three for ZEN decreased to 89.88%, 36.13% and 18.13% respectively (as shown in the attached figure of the specification) Fig. 27 This result indicates that the key to the detoxification of ZEN by the L-4 strain lies in the extracellular protein it secretes.

[0131] 2.3 Results of ZEN degradation by strain L-4 The UPLC-MS method was used to detect the ZEN residues and degradation product chromatograms in the experimental and control groups. It was found that the degradation product peak appeared at 6.12 min (see the attached figure in the manual). Fig.28A), the molecular weight of the substance was found to be 333.1000 and 379.1000 by mass spectrum analysis of the degradation product (see the attached figure of the specification). Fig.28 B), the two products were subjected to secondary mass spectrometry analysis, and the fragment ions were mainly m / z 333.1000 at a collision energy of 20 eV (as shown in the attached figure of the specification Fig.28 C), indicating that the degradation product of ZEN by L-4 strain is mainly a substance with a molecular weight of 333.1000, and it is inferred that it is 15-OH-ZEN, which is much less toxic than ZEN.

[0132] 2.4 Screening of ZEN-degrading enzymes After homology comparison, we found a protein sequence pilon3668 with 95.91% homology to the peroxidase Bsdyp produced by Bacillus subtilis and a protein sequence pilon3330 with 70.26% homology to the phosphotransferase ZPH produced by Bacillus subtilis Y816 (see the attached figure of the specification). Fig.29 (as shown in A and B).

[0133] 2.5 Detoxification effect of L-4 strain on contaminated corn flour By co-culturing ZEN and Bacillus mohaiwei L-4 fermentation liquid in corn flour to simulate the detoxification effect of L-4 strain on ZEN-contaminated corn flour, it was found that the detoxification rate of L-4 strain on ZEN-contaminated corn flour reached 49.41% (see the attached figure of the specification) Fig.30 After 5 days of cultivation, the concentration of ZEN in corn flour decreased from 532.91 μg / kg to 269.60 μg / kg.

[0134] 3 Discussion Bacillus mojavensis was originally isolated from desert soil and is most closely related to Bacillus amyloliquefaciens, Bacillus atrophaeus, and Bacillus subtilis (Roberts et al, 1994). Previous reports have shown that B. mojavensis has antagonistic effects on mycotoxin-producing Fusarium and can be used as a biocontrol probiotic for plant diseases caused by pathogenic fungi of various crops (Rath et al, 2018; Choi et al, 2011; Bacon et al, 2006). However, there have been no reports on the use of B. mojavensis as a zearalenone-detoxifying bacterium. The present invention uses the methods of co-culturing B. mojavensis L-4 with ZEN, UPLC-MS mass spectrometry analysis, and protein sequence homology comparison to conduct in-depth research on its detoxification conditions, detoxification components, detoxification products, degradation proteins, and its application in moldy feed. The results show that: (1) The optimal detoxification conditions for B. mojavensis L-4 to ZEN are when the initial concentration is 0.5-1.0 μg / mL, 37°C, 24 h, and pH is 7, and the detoxification rate is the highest, which is 100%; (2) The detoxification components of B. mojavensis L-4 to ZEN are mainly the extracellular proteins secreted by it; (3) The homology comparison results show that B. mojavensis L-4 can secrete two enzymes that have high homology with the ZEN degradation enzyme peroxidase Bsdyp and phosphotransferase ZPH, respectively. It is speculated that these two enzymes play a role in degrading ZEN. The degradation effect of these two enzymes on ZEN is manifested in that they can convert ZEN into low-concentration, low-toxic 15-OH-ZEN; (4) B. The degradation rate of ZEN in moldy feed by mojavensis L-4 bacterial solution was 49.41%.

[0135] Studies have shown that Bacillus subtilis and Bacillus amyloliquefaciens are commonly used fungal toxin degrading bacteria (K. et al, 2010; Lee et al, 2017). This experimental study found that B. mojavensis L-4 had a detoxification rate of 100% for 0.5 and 1.0 μg / mL ZEN, and a detoxification rate of 83.42% for 5.0 μg / mL ZEN. Moreover, L-4 bacteria could detoxify 0.5 μg / mL ZEN by 100% after 24 h of treatment. The L-4 strain showed a very high detoxification effect on ZEN in neutral and alkaline culture media and simulated intestinal fluid, and also had a detoxification effect in artificial gastric fluid, acidic and strongly alkaline culture media. The detoxification effect on ZEN was the best at 37 ℃, and 0.5 μg / mL ZEN could be completely detoxified. Increasing or decreasing the temperature would lead to a decrease in its detoxification effect.

[0136] Microbial detoxification methods include adsorption and degradation. The adsorption method is related to the cell structure of bacteria or fungi. Studies have shown that Saccharomyces cerevisiae (Chlebicz et al, 2019) has a good adsorption effect on ZEN, and the adsorption capacity gradually increases with the increase of temperature. However, the adsorption method is not a truly effective detoxification method for ZEN. It is often difficult to metabolize or incompletely metabolize, causing secondary pollution. The biodegradation method can metabolize ZEN into low-toxic or non-toxic substances through intracellular or extracellular proteins secreted by microorganisms. It is a very feasible ZEN detoxification method. In the present invention, different components of L-4 fermentation broth were co-cultured with ZEN, and it was found that L-4 fermentation supernatant could completely detoxify 0.5 μg / mL of ZEN. After adding SDS+proteinase K, the detoxification rate dropped to 18.31%. This result shows that the detoxification effect of L-4 on ZEN is mainly manifested in the degradation of its secreted extracellular proteins in the fermentation supernatant.

[0137] The degradation products of ZEN mainly include cis-ZEN, α-ZAL, β-ZAL, α-ZEL, β-ZEL, α-cis-ZEL, β-cis-ZEL, ZAN, 15-OH-ZEN, ZEN-14-sulfate and 17-β-estradiol (Sarah S et al, 2015). According to literature reports, ZEN can be degraded into 15-OH-ZEN by the peroxidase BsDyP secreted by Bacillus subtilis (Xing et al, 2021). The present invention uses UPLC-MS to detect and analyze the experimental group and the control group and compares them. It is found that the peak of its degradation product appears at 6.12 min. The product is subjected to mass spectrometry analysis and secondary mass spectrometry analysis to infer that it degrades ZEN into 15-OH-ZEN with a molecular weight of 333.1000, and its relative estrogenicity is one-fiftieth of that of ZEN (Sarah S et al, 2015).

[0138] ZEN is most frequently contaminated in corn (Wu Tingting et al., 2019). To explore the degradation effect of B. mojavensis L-4 on ZEN in corn, the present invention co-cultured L-4 fermentation broth with ZEN-contaminated corn feed for 5 days. It was found that the concentration of ZEN in corn flour decreased from 532.91 μg / kg to 269.60 μg / kg, and the degradation rate reached 49.41%.

[0139] 4 Summary The present invention determines that the optimal detoxification condition of Bacillus mojavensis L-4 for ZEN is that in a neutral liquid medium, when the initial concentration of ZEN is 0.5~1.0 μg / mL, it can be completely detoxified after 24 hours of co-incubation at 37°C; the present invention further verifies that the substance that the L-4 strain has a detoxification effect on ZEN is mainly the extracellular protein secreted by it; after identification of degradation products, it is found that the peroxidase and phosphotransferase secreted by the L-4 strain degrade ZEN into low-toxicity, low-concentration 15-OH-ZEN products; L-4 fermentation broth also shows a good ability to degrade ZEN in contaminated corn flour, and the degradation rate of 532.91 μg / kg of ZEN is 49.41%. The present invention analyzes the detoxification conditions, detoxification components, detoxification products, degradation proteins of B. mojavensis L-4 for ZEN and its application in moldy feed, and provides reliable experimental data for the application of B. mojavensis in the detoxification of ZEN in animal feed.

Claims

1. A Bacillus mohaiweii capable of degrading zearalenone, characterized in that: The Bacillus mohaiwei that can degrade zearalenone is L-4, and its classification name is Bacillus mohaiwei Bacillus mojavensis The strain collection number is CGMCC No.29681.

2. The Bacillus mohaiweiensis capable of degrading zearalenone according to claim 1, characterized in that: The detoxification rate of L-4 strain in liquid culture medium containing 0.5-5.0 μg / mL zearalenone was no less than 83.42%.

3. The Bacillus mohaievii capable of degrading zearalenone according to claim 1, characterized in that: At 240r / min and 37 ℃, Bacillus mohaiwei L-4 can quickly detoxify 0.5 μg / mL ZEN; at 37-45 ℃, the detoxification effect of Bacillus mohaiwei L-4 on 0.5 μg / mL zearalenone is more than 90%; the detoxification effect is best at pH 7-11 for 0.5 μg / mL zearalenone.

4. The Bacillus mohaiweiensis capable of degrading zearalenone according to claim 1, characterized in that: The optimal detoxification conditions for zearalenone by strain L-4 were as follows: initial concentration of 0.5-1.0 μg / mL, 37 ℃, 24 h, pH 7, and the detoxification rate was 100%.

5. The Bacillus mohaievii capable of degrading zearalenone according to claim 1, characterized in that: The peroxidase and phosphotransferase secreted by the L-4 strain degraded zearalenone into a low-toxic, low-concentration 15-OH-ZEN product.

6. The Bacillus mohaievii capable of degrading zearalenone according to claim 1, characterized in that: The degradation rate of L-4 fermentation broth on 532.91 μg / kg of zearalenone in contaminated corn flour was 49.41%.

7. Use of the Bacillus mohaiweii capable of degrading zearalenone as claimed in claim 1 as a probiotic for animals.

8. A probiotic for animals, characterized in that: Contains Bacillus mohaievii L-4.

9. The probiotic for animals according to claim 8, characterized in that: The initial concentration of Bacillus mohaiwei L-4 was 0.5~1.0 μg / mL.

10. The probiotic for animals according to claim 8, characterized in that: The fermentation supernatant of Bacillus mohaiwei L-4 is used and mixed with animal feed.

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