Bacillus subtilis, complex microbial inoculant and application thereof in degrading mycotoxin

Abstract

This invention discloses a strain of Bacillus subtilis, a compound bacterial agent, and its application in degrading mycotoxins, belonging to the fields of microbiology and fermentation engineering. This invention screened and obtained a strain of Bacillus subtilis with mycotoxin-degrading activity (…). Bacillus subtilis BLYC-1937 exhibits good degradation effects on single or combined contamination by T-2 toxin, vomitoxin, fumonisin B1, aflatoxin B1, and ochratoxin A. Furthermore, it demonstrates strong antagonistic effects against T-2 toxin-producing Fusarium oxysporum and ochratoxin A-producing Aspergillus ochraceus, thus reducing T-2 and ochratoxin A contamination at its source.

Description

Technical Field

[0001] This invention relates to the fields of microbiology and fermentation engineering, specifically to a strain of Bacillus subtilis, a compound microbial agent, and its application in the degradation of mycotoxins. Background Technology

[0002] Mycotoxins are naturally occurring toxic secondary metabolites produced by fungi, widely found in contaminated feed ingredients and compound feeds. They mainly include T-2 toxin, DON (vomiting toxin), aflatoxin B1 (AFB1), fumonisin B1 (FB1), and ochratoxin A (OTA). Among these, T-2 toxin is a highly toxic fungal toxin produced by Fusarium fungi, belonging to the trichothecene family of compounds. T-2 toxin can cause damage to tissues such as the intestines, liver, kidneys, and brain, with liver damage being the most typical symptom. In addition, T-2 toxin can also cause ataxia, anorexia, central nervous system damage, growth inhibition, bone deformities, and immune system destruction, posing a serious challenge to the livestock industry and human health.

[0003] The toxic epoxy groups and double bonds of T-2 toxin exhibit strong resistance to nucleophiles, making its chemical structure extremely stable. T-2 toxin retains its toxicity for 6-7 years at room temperature and its toxicity is difficult to reduce even under high pressure, ultraviolet light, or neutral or acidic environments. Therefore, detoxification of T-2 toxin is extremely difficult. Current detoxification methods mainly include physical, chemical, and biological methods. However, physical and chemical detoxification may alter feed palatability and reduce its nutritional and usability. In contrast, biological detoxification avoids the drawbacks of irritating odors and nutrient loss, and demonstrates good detoxification efficiency and specificity for T-2 toxin. Biological detoxification utilizes microorganisms or enzymes to convert fungal toxins into less toxic substances through biological metabolism. For example, studies have reported that *Eubacterium* partially degrades T-2 toxin into HT-2 and can convert T-2 toxin metabolites (HT-2, T-2 triol, T-2 tetraol, etc.) into their de-epoxylated forms, thus disrupting the chemical structure of the fungal toxin and achieving safe detoxification. Current research on T-2 toxin detoxification strains primarily focuses on *Lactococcus lactis* (…). Lactococcus lactis ), intertidal microbacteria ( Exiguobacteriumestuarine ), genus *Phyllostachys* ( Curtobacterium ), Eubacterium ( Eubacterium Bacteria such as ) and brewer's yeast ( Saccharomyces yeast Aspergillus niger ( Aspergillus niger While it can be used on fungi such as ), there are problems such as low degradation efficiency, unstable effect, and unclear degradation mechanism. The resources of strains that can efficiently degrade T-2 toxin are still relatively scarce.

[0004] In addition, there are synergistic toxic effects among different mycotoxins. Studies have found that the co-contamination rate of mycotoxins in feed ingredients and compound feed is 30.3%-88.9%, which makes the formulation of mycotoxin limit standards and pollution control more complicated. Summary of the Invention

[0005] To address the problems existing in the prior art, the purpose of this invention is to provide a strain of Bacillus subtilis, a compound bacterial agent, and its application in the degradation of mycotoxins.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a strain of Bacillus subtilis (… Bacillus subtilis BLYC-1937, this strain was deposited at the China Center for Type Culture Collection (CCTCC, Wuhan University, Wuhan, China) on July 28, 2025, with accession number: CCTCC NO: M 20251711.

[0007] The Bacillus subtilis of the present invention ( Bacillus subtilis Compared with existing reports of Bacillus subtilis, the main characteristics of BLYC-1937 are: (1) It can simultaneously degrade T-2 toxin, vomitoxin, fumonisin B1, aflatoxin B1 and ochratoxin A; and it can antagonize the growth of Fusarium oxysporum producing T-2 toxin and Aspergillus ochratoxin A producing ochratoxin A, thereby reducing the production of T-2 toxin and ochratoxin A at the source.

[0008] (2) It exhibits significant antibacterial activity against common pathogens in aquaculture, such as Escherichia coli, Staphylococcus aureus, Clostridium perfringens, Vibrio parahaemolyticus, Vibrio alginolyticus, Streptococcus pyogenes, Serratia marcescens, and Aeromonas hydrophila. Infection with these pathogens can exacerbate the harm caused by mycotoxins, resulting in a compounding of aquaculture risks. Strain BLYC-1937 not only has the ability to degrade various mycotoxins but also exhibits good antibacterial activity against the aforementioned pathogens, thus simultaneously addressing the challenges posed by both mycotoxins and pathogens.

[0009] (3) It has excellent fermentation performance and can obtain a high number of viable bacteria through short-time fermentation; it also has high temperature resistance, acid resistance and bile salt resistance, as well as the activity of producing neutral protease, α-amylase and cellulase.

[0010] A second aspect of the present invention provides a microbial agent containing the aforementioned Bacillus subtilis (Bacillus subtilis). Bacillus subtle BLYC-1937 and / or Bacillus subtilis ( Bacillus subtilis ) Metabolites of BLYC-1937.

[0011] Preferably, the bacterial agent contains Bacillus subtilis (B. subtilis). Bacillus subtilis BLYC-1937 exists in one or more of the following forms: cultured live bacteria, bacterial suspension, fermentation broth, and cell lysate.

[0012] Furthermore, the fermentation broth refers to the liquid produced after inoculating the microbial strain into a culture medium and culturing it for a period of time.

[0013] The bacterial suspension refers to the bacterial precipitate obtained by resuspending the precipitate after centrifugation of the fermentation broth.

[0014] The bacterial lysate refers to the bacterial suspension that has been broken up by ultrasonication and then the supernatant has been separated by centrifugation.

[0015] The Bacillus subtilis ( Bacillus subtilis The metabolites of BLYC-1937 mainly exist in the form of the fermentation supernatant of the strain. The fermentation supernatant refers to the clear liquid at the top after centrifugation of the fermentation broth, which contains abundant metabolites during the growth and reproduction of the strain.

[0016] In a third aspect, the present invention provides the above-mentioned Bacillus subtilis ( Bacillus subtilis BLYC-1937 or fungicides are used to degrade at least one of the following mycotoxins: (1)-(5) (1) T-2 toxin; (2) Vomiting toxins; (3) Fumonisin B1; (4) Aflatoxin B1; (5) Ochratoxin A.

[0017] In a fourth aspect, the present invention provides the above-mentioned Bacillus subtilis ( Bacillus subtilis The application of BLYC-1937 or the bacterial agent in the preparation of at least one of the following (1)-(3) antibacterial products: (1) Antibacterial products that inhibit T-2 toxin-producing Fusarium oxysporum; (2) Antimicrobial products that inhibit ochratoxin A-producing ochratoxin fungi; (3) Antibacterial products that inhibit Escherichia coli, Staphylococcus aureus, Clostridium perfringens, Vibrio parahaemolyticus, Vibrio alginolyticus, Streptococcus pyogenes, Serratia marcescens and / or Aeromonas hydrophila.

[0018] In a fifth aspect, the present invention provides a compound microbial agent, said compound microbial agent being composed of Bacillus subtilis (… Bacillus subtle It consists of BLYC-1937, Bacillus lateralis, and Aspergillus niger.

[0019] Preferably, the preservation number of the *Bacillus lateralis* is CCTCC NO: M2022932; and the preservation number of *Aspergillus niger* is CCTCC NO: M2015722.

[0020] Preferably, in the compound microbial agent, Bacillus subtilis (… Bacillus subtilis The ratio of viable counts of BLYC-1937, Bacillus lateralis, and Aspergillus niger was 1:1:1.

[0021] In a sixth aspect, the present invention provides the above-mentioned Bacillus subtilis ( Bacillus subtilis Application of BLYC-1937, microbial agents or compound microbial agents in the preparation of mycotoxin detoxification products.

[0022] A seventh aspect of the present invention provides a method for degrading mycotoxins, comprising the following steps: The above Bacillus subtilis ( Bacillus subtilis BLYC-1937, microbial agents or compound microbial agents are brought into contact with samples contaminated with mycotoxins to degrade the mycotoxins in the samples.

[0023] In the above method, the mycotoxin contamination is a single toxin contamination or a combination of multiple toxins, including T-2 toxin, vomitoxin, fumonisin B1, aflatoxin B1, and ochratoxin A.

[0024] In the above method, the sample contaminated with mycotoxins is grain, oil and / or feed contaminated with mycotoxins.

[0025] The beneficial effects of this invention are: (1) This invention screened and obtained a strain of Bacillus subtilis with activity in degrading mycotoxins ( ). Bacillus subtle BLYC-1937 exhibits good degradation effects against single or combined contamination by T-2 toxin, vomitoxin, fumonisin B1, aflatoxin B1, and ochratoxin A; it also shows significant antagonistic effects against Fusarium oxysporum producing T-2 toxin and Aspergillus ochratoxin A producing Aspergillus, thus reducing mycotoxin contamination at the source.

[0026] The Bacillus subtilis of the present invention ( Bacillus subtilis BLYC-1937 exhibits significant antibacterial activity against common pathogens in aquaculture, including Escherichia coli, Staphylococcus aureus, Clostridium perfringens, Vibrio parahaemolyticus, Vibrio alginolyticus, Streptococcus pyogenes, Serratia marcescens, and Aeromonas hydrophila. Mycotoxin contamination and bacterial pathogens pose a double threat to the aquaculture industry; the coexistence of these two pathogens greatly amplifies the harm to animal health, leading to more severe economic losses. However, the Bacillus subtilis strain of this invention… Bacillus subtilisBLYC-1937 has the dual function of degrading mycotoxins and inhibiting bacterial pathogens, and can simultaneously address the challenges of mycotoxins and pathogens.

[0027] (2) Based on Bacillus subtilis ( Bacillus subtilis BLYC-1937, this invention further develops a compound microbial agent that can significantly improve the prevention and control of combined contamination by T-2 toxin, vomitoxin, fumonisin B1, aflatoxin B1 and ochratoxin A. Attached Figure Description

[0028] Figure 1 The results of plate confrontation between strain BLYC-1937 and T-2 toxin-producing Fusarium oxysporum; the left figure shows T-2 toxin-producing Fusarium oxysporum, and the right figure shows T-2 toxin-producing Fusarium oxysporum + strain BLYC-1937.

[0029] Figure 2 Electron micrographs of the antagonistic effect of strain BLYC-1937 on Fusarium oxysporum producing T-2 toxin; the left image is the control group, and the right image is the antagonistic treatment group.

[0030] Figure 3 Plate confrontation results of strain BLYC-1937 and ochratoxin A-producing Aspergillus oryzae; where the left figure is ochratoxin A-producing Aspergillus oryzae and the right figure is ochratoxin A-producing Aspergillus oryzae + strain BLYC-1937.

[0031] Figure 4 Scanning electron microscopy image of the antagonistic effect of strain BLYC-1937 on ochratoxin A-producing ochratoxin A fungi; the left image is the control group, and the right image is the antagonistic treatment group.

[0032] Figure 5 Growth curve (left) and pH change (right) of strain BLYC-1937 fermentation in small tanks. Detailed implementation method: It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0033] As mentioned earlier, mycotoxins are widely present in contaminated feed ingredients and compound feeds, posing a serious challenge to the livestock industry and human health. Different mycotoxins also exhibit synergistic toxicity; when multiple mycotoxins are present in a co-contamination environment, the degradation effect of microbial strains with detoxification capabilities is reduced. Furthermore, the livestock farming process may also involve threats from various pathogens such as Escherichia coli, Staphylococcus aureus, Clostridium perfringens, Vibrio parahaemolyticus, Vibrio alginolyticus, Streptococcus pyogenes, Serratia marcescens, and Aeromonas hydrophila. The combined harm of these pathogens and mycotoxins further amplifies the risks in livestock farming.

[0034] In view of this, the present invention screened and obtained a strain of Bacillus subtilis with activity in degrading mycotoxins (… Bacillus subtle BLYC-1937, unlike existing reports of Bacillus subtilis that degrade mycotoxins, is a Bacillus subtilis strain of the present invention (BLYC-1937). Bacillus subtilis BLYC-1937 integrates multiple functions. On the one hand, it has a degradation effect on T-2 toxin, vomitoxin, fumonisin B1, aflatoxin B1, and ochratoxin A, and also has a significant antagonistic effect on Fusarium oxysporum producing T-2 toxin and Aspergillus ochratoxin A producing Aspergillus, which can reduce mycotoxin pollution at the source. On the other hand, it has significant antibacterial activity against a variety of pathogens such as Escherichia coli, Staphylococcus aureus, Clostridium perfringens, Vibrio parahaemolyticus, Vibrio alginolyticus, Streptococcus pyogenes, Serratia marcescens, and Aeromonas hydrophila, which can reduce the risk of combined harm from mycotoxins and pathogens.

[0035] Furthermore, to address the impact of combined contamination by multiple mycotoxins on the degradation efficiency of microbial strains, this invention utilizes Bacillus subtilis (… Bacillus subtilis Based on BLYC-1937, Bacillus subtilis ( Bacillus subtle BLYC-1937, when combined with Bacillus lateralis and Aspergillus niger, synergistically improves the degradation effect on multiple mycotoxin contamination.

[0036] To enable those skilled in the art to more clearly understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments. If specific experimental conditions are not specified in the embodiments, they are generally based on conventional conditions or conditions recommended by the reagent company; the reagents, consumables, etc. used in the following embodiments, unless otherwise specified, can be obtained commercially. Wherein: LB liquid medium: glucose 0.2%, peptone 1.0%, yeast extract 0.5%, NaCl 0.5%, pH 7.0, sterilized at 121°C for 30 min, where the percentages are mass percentages.

[0037] LB solid medium: Add 1.5% agar powder (by mass) to the above LB liquid medium.

[0038] NB liquid culture medium: 1.0% peptone, 0.3% beef extract, 0.5% sodium chloride, pH 7.2-7.4, sterilized at 121°C for 30 min, where the percentages are mass percentages.

[0039] NA solid medium: Add 0.7% / 1.5% agar powder (by mass) to the above NB liquid medium.

[0040] PDA liquid culture medium: 20% potato, 2% glucose, 0.5% peptone, 0.3% potassium dihydrogen phosphate, 0.15% magnesium sulfate, sterilized at 121°C for 30 min, where the percentages are by mass.

[0041] PDA solid medium: Add 1.5% agar powder (by mass) to the above PDA liquid medium.

[0042] Ferrous sulfite solid culture medium: 1.5% tryptone, 0.5% soybean peptone, 0.5% yeast extract, 0.1% sodium metabisulfite, 0.1% ferric ammonium citrate, 2% agar, pH 7.5–7.7, sterilized at 121°C for 15 min, where the percentages are by mass.

[0043] MRS liquid culture medium: 1% peptone, 0.5% beef extract, 2% glucose, 0.2% dipotassium hydrogen phosphate, 0.2% triammonium citrate, 0.02% magnesium sulfate, 0.0005% manganese sulfate, 0.1% Tween-80, pH 6.2, sterilized at 121°C for 30 min, where the percentages are mass percentages.

[0044] PBS buffer: Sodium chloride 0.8%, potassium chloride 0.02%, disodium hydrogen phosphate 0.142%, potassium dihydrogen phosphate 0.027%, pH 7.4, sterilized at 121°C for 30 min, where the percentages are mass percentages.

[0045] Physiological saline: 0.9% sodium chloride, sterilized at 121°C for 30 min, where the percentages are mass percentages.

[0046] 20L fermentation medium: glucose 5%, corn starch 5%, soybean meal 4%, corn steep liquor powder 2%, potassium dihydrogen phosphate 0.2%, magnesium sulfate 0.05%, manganese sulfate 0.1%, calcium chloride 0.3%, amylase 0.05%, and defoamer as needed; where the percentages are mass percentages.

[0047] T-2 toxin, vomitoxin, aflatoxin B1, fumonisin B1, ochratoxin A standards and corresponding toxin detection kits were purchased from Qingdao Pribang Biotechnology Co., Ltd. Porcine bile salts were purchased from Beijing Sorabo Technology Co., Ltd., and acetonitrile and other reagents were purchased from Shanghai Maclean Biotechnology Co., Ltd.

[0048] The pathogens used are: Escherichia coli BLCC8-0135 (ATCC25922), Salmonella BLCC8-0129, Staphylococcus aureus BLCC8-0138 (ATCC6538), Clostridium perfringens BLCC8-0136, Vibrio parahaemolyticus BLCC8-0110 (ATCC17802), Vibrio alginolyticus BLCC8-0109, Streptococcus pyogenes BLCC8-0141 (ATCC12344), and Serratia marcescens B. LCC8-0137 (ATCC14041), Aeromonas hydrophila BLCC8-0121 (CVCC4002), and Fusarium oxysporum BLCC6-0067, Fusarium oxysporum BLCC6-0069, Fusarium oxysporum BLCC6-0071, Aspergillus flavus BLCC6-0182, and Aspergillus ochraceus BLCC6-0076 are all from the strain resource bank of Shandong Baolai Lailai Biotechnology Co., Ltd. Fusarium novifluae and Fusarium moniliforme are currently commercially available pathogens.

[0049] Aspergillus niger ( Aspergillus niger The accession number of BLCC6-0009 is CCTCC NO: M2015722, and it is recorded in patent CN 105385609 B.

[0050] Lateral spores of Bacillus brevis ( Brevibacillus laterosporus The accession number of BLCC1-0170 is CCTCCNO: M2022932, and it is recorded in patent CN 115927058 B.

[0051] Example 1: Screening and preservation of T-2 toxin-degrading strains 1. Test method: The degradation effects of Bacillus subtilis Y-002, Y-009, Y-052, Y-105, Y-108, Y-124, Y-196, BLYC-1937 and Bacillus licheniformis Y-200, Y-203, Y-300, Y-311, Y-320, Y-324, preserved in the strain resource bank of Shandong Baolai Lailai Biotechnology Co., Ltd., on T-2 toxin were investigated.

[0052] 1.1 Preparation of Seed Fermentation Broth for Strains Using an inoculation loop, scrape one loopful of the test strain from the slant medium (LB solid medium) of the above-mentioned test strains, and inoculate them into LB liquid medium. Incubate at 37℃ and 180r / min for 16h to obtain the seed fermentation broth of the test strains.

[0053] 1.2 Evaluation of T-2 toxin degradation effect In the experimental group, 780 μL of LB liquid medium and 20 μL of the seed fermentation broth of the test strain were added, along with 200 μL of T-2 toxin standard working solution filtered through a 0.22 μm aqueous filter membrane (the final concentration of T-2 toxin after mixing was 1 μg / mL). The mixture was then incubated at 37℃ and 180 r / min for 48 h. A control group was also set up, consisting of 800 μL of LB liquid medium and 200 μL of T-2 standard working solution filtered through a 0.22 μm aqueous filter membrane (the final concentration of T-2 toxin after mixing was 1 μg / mL). The mixture was then incubated at 37℃ and 180 r / min for 48 h.

[0054] After the culture was completed, the solutions obtained above were centrifuged at 4000 r / min for 5 min, the supernatant was collected and stored at 4℃. The concentration of T-2 toxin in each group of samples was detected according to the instructions of the T-2 toxin detection kit, and the degradation rate of T-2 toxin by each test strain was calculated.

[0055] Degradation rate (%) = (T0 - T1) / T0 × 100%; T0 = ​​Toxin concentration in the control group T1 = Toxin concentration in the experimental group.

[0056] 1.3 Identification and Preservation of 16S rRNA in Strains The strain with the highest degradation rate of T-2 toxin was selected as the target strain, and its 16S rRNA gene sequence was amplified and sequenced. The sequencing results were compared with the NCBI database to determine its species name, and the target strain was biopreserved using a patented procedure.

[0057] 2. Test Results 2.1 Degradation effect of the tested strains on T-2 toxin The degradation effects of each tested strain on T-2 toxin are shown in Table 1.

[0058] Table 1: Degradation effect of the tested strains on T-2 toxin The results showed that strain BLYC-1937 had the best degradation effect on T-2 toxin, with a degradation rate of 93.70%, and it was selected as the target strain.

[0059] 2.2 Identification and Preservation of Target Strains The 16S rRNA gene sequence of strain BLYC-1937 is shown in SEQ ID NO.1. Comparative analysis showed that it had the highest homology with Bacillus subtilis. Combined with previous morphological and physiological biochemical identification results, strain BLYC-1937 was identified as Bacillus subtilis. Bacillus subtilis ).

[0060] The selected strain BLYC-1937 was deposited at the China Center for Type Culture Collection, and the deposit information is as follows: Reference biological material (strain): strain BLYC-1937; Suggested classification and nomenclature: Bacillus subtilis ( Bacillus subtilis ); Accession number: CCTCC NO: M20251711; Preservation date: July 28, 2025.

[0061] Example 2: Bacillus subtilis ( Bacillus subtilis Investigation of the degradation mechanism of T-2 toxin by BLYC-1937 1. Test Methods 1.1 Investigation of the Degradation Site Preparation of seed solution: Using an inoculation loop, scrape a loopful of bacterial cells from the slant medium (LB solid medium) for the preservation of strain BLYC-1937, inoculate it into LB liquid medium, and incubate at 37°C and 180 r / min for 16 h to obtain the seed culture of the strain.

[0062] Preparation of fermentation broth: The seed culture of strain BLYC-1937 was inoculated into LB liquid medium at a rate of 1% (v / v) and cultured at 37°C and 180 rpm for 24 h to obtain the fermentation broth of the strain.

[0063] Preparation of fermentation supernatant: Centrifuge the fermentation broth at 4000 r / min for 10 min, collect the supernatant, and prepare the fermentation supernatant.

[0064] Preparation of bacterial suspension: Centrifuge the fermentation broth at 4000 r / min for 10 min, collect the bacterial precipitate, and resuspend it in an equal volume of sterile PBS to prepare a bacterial suspension.

[0065] Preparation of cell lysate: The bacterial suspension was ultrasonically disrupted, centrifuged at 8000 r / min for 10 min, and the supernatant was collected to prepare the bacterial lysate.

[0066] Among them: fermentation broth was used to determine the degradation rate of T-2 toxin by bacterial culture; fermentation supernatant was used to determine the degradation rate of T-2 toxin by extracellular metabolites; bacterial suspension was used to determine the degradation rate of T-2 toxin by bacterial cells; and bacterial lysate was used to determine the degradation rate of T-2 toxin by intracellular metabolites.

[0067] The method for determining the degradation rate of T-2 toxin is the same as in Example 1.

[0068] 1.2 Investigation on antagonistic effect against T-2 toxin-producing Fusarium oxysporum 1.2.1 Screening of Fusarium oxysporum producing T-2 toxin Using Fusarium oxysporum BLCC6-0067, BLCC6-0069, and BLCC6-0071 as experimental subjects, the experimental strains were first activated with PDA solid slant, and then inoculated into 20 mL of PDA liquid medium. After fermentation at 28℃ and 180 r / min for 72 h, the fermentation broth was obtained.

[0069] The fermentation broth was centrifuged at 4000 r / min for 5 min, and the supernatant was collected. The content of T-2 toxin in the fermentation supernatant of Fusarium oxysporum was determined using a T-2 toxin detection kit.

[0070] 1.2.2 Investigation of antagonistic effects The antagonistic effect of strain BLYC-1937 on T-2 toxin-producing *Fusarium oxysporum* was investigated using the plate confrontation method. Six *Fusarium oxysporum* mycelial discs were taken using a 1.00 cm diameter punch and transferred to the center of new PDA medium plates. Three of the plates were inoculated with strain BLYC-1937 at four points around the center. Three other PDA medium plates inoculated only with *Fusarium oxysporum* mycelial discs served as controls. The plates were incubated at 28°C for 3 days. The diameter of *Fusarium oxysporum* colonies in the plates was measured using the cross-cross method, and the inhibition rate was calculated.

[0071] Inhibition rate (%) = (control colony diameter - test colony diameter) / control colony diameter × 100%.

[0072] Then, the T-2 toxin-producing Fusarium oxysporum under antagonistic effects was observed by electron microscopy. Specifically, a certain amount of 2.5% glutaraldehyde fixative was slowly and evenly added to the PDA culture dish after the above steps. The culture dish was then left to stand at room temperature for 24 hours. The fixative was then gently aspirated into the waste container using a dropper. After the fixative was dried, the edge of the Fusarium oxysporum in contact with the degrading bacteria in the culture medium was cut off with a scalpel for electron microscopy.

[0073] 2. Test Results 2.1 Results of investigation on the site of degradation The degradation rate of T-2 toxin by each active site of strain BLYC-1937 is shown in Table 2.

[0074] Table 2: Results of the investigation on the degradation sites (results are mean values, n=3) The results showed that the degradation of T-2 toxin by strain BLYC-1937 was mainly achieved through extracellular metabolites.

[0075] 2.2 Screening results of T-2 toxin-producing Fusarium oxysporum The screening results of Fusarium oxysporum producing T-2 toxin are shown in Table 3.

[0076] Table 3: Screening results of T-2 toxin-producing Fusarium oxysporum Note: "-" in the table indicates that the result was not measured.

[0077] The results showed that Fusarium oxysporum BLCC6-0071 could produce T-2 toxin at a concentration of 19.70 μg / mL, and this strain was subsequently used for further research.

[0078] 2.3 Antagonistic effect of strain BLYC-1937 against T-2 toxin-producing Fusarium oxysporum The results of the plate confrontation test between strain BLYC-1937 and T-2 toxin-producing Fusarium oxysporum are as follows: Figure 1 As shown in Table 4, the inhibition rate of strain BLYC-1937 against T-2 toxin-producing Fusarium oxysporum is shown in Table 4. Electron micrographs of T-2 toxin-producing Fusarium oxysporum under antagonistic conditions are shown in the figure. Figure 2 As shown.

[0079] Table 4: Inhibition rate (%) of strain BLYC-1937 against T-2 toxin-producing Fusarium oxysporum. The results showed that strain BLYC-1937 had a strong inhibitory effect on Fusarium oxysporum producing T-2 toxin; the mycelia of Fusarium oxysporum inhibited by the T-2 toxin degrading bacterium BLYC-1937 showed shrinkage and wrinkling, suggesting that its morphology and function were affected.

[0080] Example 3: Bacillus subtilis ( Bacillus subtilis Detoxification performance of BLYC-1937 against other mycotoxins 1. Test method: 1.1 Preparation of Seed Fermentation Broth for Strains Using an inoculation loop, scrape a loopful of bacterial cells from the slant medium (LB solid medium) for the preservation of strain BLYC-1937, inoculate it into LB liquid medium, and incubate at 37℃ and 180 r / min for 16 h to obtain the strain's seed fermentation broth.

[0081] 1.2 Degradation performance of vomitoxin The experimental group consisted of 780 μL of LB liquid medium, 20 μL of seed fermentation broth of strain BLYC-1937, and 200 μL of vomitoxin standard working solution filtered through a 0.22 μm aqueous filter (final concentration of vomitoxin after mixing was 1 μg / mL). The mixture was incubated at 37℃ and 180 rpm for 48 h. A control group was also included, consisting of 800 μL of LB liquid medium and 200 μL of vomitoxin standard working solution filtered through a 0.22 μm aqueous filter (final concentration of vomitoxin after mixing was 1 μg / mL). Each group was divided into three replicates. After fermentation, the culture medium was centrifuged at 4000 rpm for 5 min, and the supernatant was collected and stored at 4℃. The concentration of vomitoxin in each sample was measured according to the instructions of the vomitoxin detection kit, and the degradation rate was calculated.

[0082] 1.3 Degradation performance of fumonisin B1 The experimental group consisted of 780 μL of LB liquid medium, 20 μL of seed fermentation broth of strain BLYC-1937, and 200 μL of fumonisin B1 standard working solution (filtered through a 0.22 μm aqueous filter membrane, with a final concentration of fumonisin B1 of 1 μg / mL after mixing). The mixture was incubated at 37℃ and 180 rpm for 48 h. A control group was also included, consisting of 800 μL of LB liquid medium and 200 μL of fumonisin B1 standard working solution (filtered through a 0.22 μm aqueous filter membrane, with a final concentration of fumonisin B1 of 1 μg / mL after mixing). Each group was tested in triplicate. After fermentation, the culture medium was centrifuged at 4000 rpm for 5 min, and the supernatant was collected and stored at 4℃. The concentration of fumonisin B1 in each sample was determined according to the instructions of the fumonisin B1 detection kit, and the degradation rate was calculated.

[0083] 1.4 Degradation performance of aflatoxin B1 The experimental group consisted of 780 μL of LB liquid culture medium, 20 μL of seed fermentation broth of strain BLYC-1937, and 200 μL of aflatoxin B1 standard working solution filtered through a 0.22 μm aqueous membrane (final aflatoxin B1 concentration after mixing was 1 μg / mL). The mixture was incubated at 37℃ and 180 rpm for 48 h. A control group was also included, consisting of 800 μL of LB liquid culture medium and 200 μL of aflatoxin B1 standard working solution filtered through a 0.22 μm aqueous membrane (final aflatoxin B1 concentration after mixing was 1 μg / mL). Each group was tested in triplicate. After fermentation, the culture medium was centrifuged at 4000 rpm for 5 min, and the supernatant was collected and stored at 4℃. The aflatoxin B1 concentration in each sample was measured according to the aflatoxin B1 detection kit instructions, and the degradation rate was calculated.

[0084] 1.5 Degradation performance of ochratoxin A The experimental group consisted of 780 μL of LB liquid medium, 20 μL of seed fermentation broth of strain BLYC-1937, and 200 μL of ochratoxin A standard working solution (filtered through a 0.22 μm aqueous filter membrane, with a final concentration of ochratoxin A of 1 μg / mL after mixing). The mixture was incubated at 37℃ and 180 rpm for 48 h. A control group was also included, consisting of 800 μL of LB liquid medium and 200 μL of ochratoxin A standard working solution (filtered through a 0.22 μm aqueous filter membrane, with a final concentration of ochratoxin A of 1 μg / mL after mixing). Each group was tested in triplicate. After fermentation, the culture medium was centrifuged at 4000 rpm for 5 min, and the supernatant was collected and stored at 4℃. The concentration of ochratoxin A in each sample was measured according to the ochratoxin A detection kit instructions, and the degradation rate was calculated.

[0085] Toxin degradation rate = (T0-T1) / T0 × 100%; T0 = ​​concentrations of each toxin in the control group; T1 = concentration of each toxin in the experimental group.

[0086] 2. Test Results The degradation effects of strain BLYC-1937 on vomitoxin, fumonisin B1, aflatoxin B1 and ochratoxin A are shown in Tables 5, 6, 7 and 8, respectively.

[0087] Table 5: Effects on the degradation of vomitoxin (results are mean values, n=3) Table 6: Degradation effect on fumonisin B1 (Results are mean values, n=3) Table 7: Degradation effect on aflatoxin B1 (Results are mean values, n=3) Table 8: Degradation effect on ochratoxin A (Results are mean values, n=3) The results showed that strain BLYC-1937 had a certain ability to degrade vomitoxin, fumonisin B1, aflatoxin B1 and ochratoxin A.

[0088] Example 4: Bacillus subtilis ( Bacillus subtilis Investigation of the antagonistic properties of BLYC-1937 against other molds 1. Test method: Based on previous experiments, plate confrontation tests were conducted on molds producing corresponding mycotoxins (vomitoxin, fumonisin, aflatoxin B1, and ochratoxin A), using the same method as in Example 2. The antagonistic effects of strain BLYC-1937 on *Fusarium nivale* (vomitoxin-producing), *Fusarium moniliforme* (fumonisin B1-producing), *Aspergillus flavus* (aflatoxin B1-producing), and *Asteris ochratoxin A* (ochratoxin A-producing) were investigated.

[0089] 2. Test Results The inhibition rates of strain BLYC-1937 against other molds are shown in Table 9; the results of the plate confrontation test of strain BLYC-1937 against ochratoxin A-producing Aspergillus are shown in Table 9. Figure 3 As shown; electron micrographs of *Ochratoxin A* producing ochratoxin A under antagonistic effects. Figure 4 As shown.

[0090] Table 9: Inhibition rate (%) of BLYC-1937 against other molds (Results are mean values, n=3) Note: CK represents the mold growth control group, and T represents the group in which strain BLYC-1937 and the corresponding mold plate were grown in opposition.

[0091] The results showed that strain BLYC-1937 had no significant inhibitory effect on Fusarium nocturia, Fusarium moniliforme, and Aspergillus flavus, which produce vomitoxin, fumonisin B1, and aflatoxin B1, but had a good inhibitory effect on Aspergillus ochraceus, which produces ochratoxin A. The hyphae of Aspergillus ochraceus inhibited by strain BLYC-1937 showed shrinkage and wrinkling, suggesting that its morphology and function were affected.

[0092] Example 5: Bacillus subtilis ( Bacillus subtilis Performance evaluation of BLYC-1937 1. Experimental Methods 1.1 Growth performance assessment Using an inoculation loop, a loopful of bacterial cells was scraped from the BLYC-1937 culture slant medium (LB solid medium) and cultured at 37℃ and 180 rpm for 16 h to obtain a seed culture. The seed culture was then inoculated at a 1% (v / v) inoculation rate into a 20L fermentation tank for fermentation. The fermentation conditions were controlled as follows: rotation speed 200 rpm, air flow rate 16 L / min, and growth temperature 37℃. Samples were taken every 4 h to determine the viable cell count and pH. The total fermentation time was 40 h.

[0093] 1.2 Tolerance Performance Assessment 1.2.1 Preparation of Seed Fermentation Broth for Strains Using an inoculation loop, scrape a loopful of bacterial cells from the slant medium (LB solid medium) for the preservation of strain BLYC-1937, inoculate it into LB liquid medium, and incubate at 37℃ and 180 r / min for 16 h to obtain the strain's seed fermentation broth.

[0094] 1.2.2 Heat resistance test Take 100 mL of seed fermentation broth of strain BLYC-1937, centrifuge at 4000 r / min for 10 min, discard the supernatant, wash the cells with sterile PBS buffer, repeat this step twice, resuspend in 100 mL of sterile PBS buffer, mix well, and divide into 6 groups, with 3 replicates in each group and 5 mL of resuspension in each replicate; place the experimental groups in an 80℃ water bath for 15 min, 25 min, and 35 min, respectively, and place the control group (CK) in a 25℃ water bath for 15 min, 25 min, and 35 min, respectively. Then, use LB solid medium to determine the viable cell survival rate of each group.

[0095] Survival rate (%) = Number of viable bacteria in the experimental group / Number of viable bacteria in the control group × 100%.

[0096] 1.2.3 Acid resistance test Take 100 mL of seed fermentation broth of strain BLYC-1937, centrifuge at 4000 r / min for 10 min, discard the supernatant, wash the cells with sterile PBS buffer, repeat this step twice, then resuspend in 100 mL of sterile PBS buffer, mix well, and divide into 4 groups, with 3 replicates per group and 5 mL of resuspended solution per replicate; adjust the pH of the experimental groups to 2.5, 2.0, and 1.5, and place them together with the control group (natural pH) in a constant temperature incubator at 25℃ for 120 min. Then, use LB solid medium to determine the viable cell survival rate of each group. Viable cell survival rate (%) = viable cell count in experimental group / viable cell count in control group × 100%.

[0097] 1.2.4 Test of tolerance to bile salts In the experimental group, the seed fermentation broth of strain BLYC-1937 was inoculated at a 1% (v / v) in LB liquid medium supplemented with 0.3%, 0.4%, and 0.5% (w / v) porcine bile salts, respectively. In the control group, the seed fermentation broth of strain BLYC-1937 was inoculated at a 1% (v / v) in ordinary LB liquid medium. Each group was replicated three times. All groups were incubated at 37℃ and 180 rpm for 12 h to obtain the fermentation broth. Viable cell counts were performed using LB solid medium. Viability (%) = (Number of viable cells in experimental group / Number of viable cells in control group) × 100%.

[0098] 1.3 Enzyme production performance evaluation 1.3.1 Preparation of Seed Fermentation Broth for Strains Using an inoculation loop, scrape a loopful of bacterial cells from the slant medium (LB solid medium) for the preservation of strain BLYC-1937, inoculate it into LB liquid medium, and incubate at 37℃ and 180 r / min for 16 h to obtain the strain's seed fermentation broth.

[0099] 1.3.2 Performance evaluation of neutral protease production The seed fermentation broth of strain BLYC-1937 was inoculated into LB liquid medium at a 1% (v / v) inoculation rate. After fermentation at 37℃ and 180 r / min for 24 h, the neutral protease activity was determined. The neutral protease activity was determined according to the national standard GB / T28715—2012 "Determination of Acidic and Neutral Protease Activity in Feed Additives - Spectrophotometric Method". 1.3.3 Performance evaluation of α-amylase production The seed fermentation broth of strain BLYC-1937 was inoculated into LB liquid medium at a 1% (v / v) inoculation rate. After fermentation at 37℃ and 180 r / min for 24 h, the α-amylase activity was determined. The determination of α-amylase activity was performed according to the method for determining bacterial α-amylase activity in "Principles and Analytical Methods of Food Enzymology". 1.3.4 Performance evaluation of cellulase production The seed fermentation broth of strain BLYC-1937 was inoculated into LB liquid medium at a 1% (v / v) inoculum and fermented at 37℃ and 180 r / min for 24 h. Cellulase activity was then determined. The saccharification power method using sodium carboxymethyl cellulose (CMC) was employed to determine cellulase activity.

[0100] 1.4 Antibacterial performance evaluation 1.4.1 Preparation of fermentation supernatant from strain BLYC-1937 Using an inoculation loop, a loopful of bacterial cells was scraped from the slant agar (LB solid medium) of strain BLYC-1937 and inoculated into LB liquid medium. The culture was then incubated at 37°C and 180 rpm for 16 h to obtain the strain's seed fermentation broth. The seed fermentation broth was centrifuged at 4000 rpm for 10 min, and the supernatant was collected to prepare the fermentation supernatant.

[0101] 1.4.2 Preparation of Fermentation Broth for Pathogenic Bacteria Escherichia coli, Salmonella, Staphylococcus aureus, Vibrio parahaemolyticus, Vibrio alginolyticus, Streptococcus pyogenes, Serratia marcescens, and Aeromonas hydrophila were inoculated into NB medium (with 2% sodium chloride added to the Vibrio parahaemolyticus and Vibrio alginolyticus medium) and incubated overnight at 37°C and 180 rpm. The incubated pathogens were then diluted to 10⁻¹⁰ with sterile physiological saline. 7 CFU / mL, to form a pathogen dilution solution.

[0102] Clostridium perfringens was inoculated into thioglycolate liquid medium and incubated overnight at 45°C. The overnight Clostridium perfringens culture was then diluted to 10⁻⁶ with sterile physiological saline. 7 CFU / mL, to form a pathogen dilution solution.

[0103] 1.4.3 Antibacterial test The antibacterial test method refers to the method in GB / T 39101-2020 "Determination of Antibacterial Activity of Polypeptides - Inhibition Zone Method", and the Oxford cup and perforation method are used for the antibacterial test.

[0104] The Oxford cup method was used to test the antibacterial activity of *Escherichia coli*, *Salmonella*, *Staphylococcus aureus*, *Vibrio parahaemolyticus*, *Vibrio alginolyticus*, *Streptococcus pyogenes*, *Serratia marcescens*, and *Aeromonas hydrophila*. First, 10 mL of NA medium containing 1.5% agar was added to each petri dish. After cooling and solidification, 600 μL of the corresponding pathogen dilution was added, followed by 6 mL of NA medium containing 0.7% agar to achieve a pathogen concentration of 10-1. 6 CFU / mL (2% (m / v) sodium chloride added to 0.7% NA medium for Vibrio parahaemolyticus and Vibrio alginolyticus), shake quickly and mix well. After cooling and solidification, place in an Oxford cup (7.8 mm outer diameter), let stand for 30 min, and after the Oxford cup is stable, add 260 μL of fermentation supernatant of strain BLYC-1937, let stand for 30 min, and incubate at 37℃ for 12-16 h. Then measure the diameter of the inhibition zone.

[0105] The inhibition test of *Clostridium perfringens* was performed using the perforation method. First, 1.8 mL of *Clostridium perfringens* dilution was added to each petri dish, followed by 18 mL of ferrous sulfite medium to achieve a pathogen concentration of 10. 6CFU / mL, shake quickly. After cooling and solidification, punch holes using a 9mm outer diameter punch, add 90μL of BLYC-1937 fermentation supernatant, let stand for 30min, then incubate at 37℃ for 16h. Measure the diameter of the inhibition zone. *Clostridium perfringens* should be placed in an anaerobic culture bag for anaerobic culture.

[0106] For each pathogen, the plate was replicated 3 times, and the inhibition zone diameter was greater than 9 mm to indicate effective inhibition.

[0107] 2. Test Results 2.1 Results of Growth Performance Evaluation The fermentation growth and pH profiles of strain BLYC-1937 in a 20L tank are shown below. Figure 5 As shown, the results indicate that strain BLYC-1937 enters the logarithmic growth phase at 4 hours and reaches its peak viable count at 16 hours, exceeding 1×10⁻⁶. 10 It has a CFU / mL concentration and exhibits rapid growth and a high viable count.

[0108] 2.2 Results of the tolerance test The results of the investigation on the tolerance of strain BLYC-1937 to heat, acid and bile salts are shown in Tables 10, 11 and 12.

[0109] Table 10: Results of heat resistance test (results are mean values, n=3) Table 11: Results of acid resistance test (results are mean values, n=3) Table 12: Results of bile salt tolerance test (results are mean values, n=3) The results showed that strain BLYC-1937 exhibited good survival after being incubated in a water bath at 80℃ for 15 min, 25 min, and 35 min. It also showed good survival after being cultured at pH=2.5, 2.0, and 1.5 for 120 min, and good survival after being cultured at 0.3%, 0.4%, and 0.5% porcine bile salts for 12 h. Therefore, strain BLYC-1937 can be considered to have good heat resistance, acid resistance, and bile salt resistance.

[0110] 2.3 Results of enzyme activity assay The results of the investigation of the neutral protease, cellulase and α-amylase activities of strain BLYC-1937 are shown in Table 13.

[0111] Table 13: Results of enzyme activity study (results are mean values, n=3) The results showed that strain BLYC-1937 had a high ability to produce neutral protease, cellulase, and α-amylase.

[0112] 2.4 Results of Antibacterial Performance Evaluation The results of the antibacterial performance study of strain BLYC-1937 against different pathogens are shown in Table 14.

[0113] Table 14: Results of antibacterial performance evaluation (Results are mean values, n=3) Note: An inhibition zone larger than 9mm is considered an effective inhibition pore size; a larger pore size indicates stronger antibacterial ability. The results showed that strain BLYC-1937 exhibited good antibacterial activity against Escherichia coli, Staphylococcus aureus, Clostridium perfringens, Vibrio parahaemolyticus, Vibrio alginolyticus, Streptococcus pyogenes, Serratia marcescens, and Aeromonas hydrophila.

[0114] Escherichia coli, Staphylococcus aureus, Clostridium perfringens, Vibrio parahaemolyticus, Vibrio alginolyticus, Streptococcus pyogenes, Serratia marcescens, and Aeromonas hydrophila are all common pathogens in aquaculture. Infections with these pathogens can exacerbate the harm caused by mycotoxins, resulting in a compounding of aquaculture risks. Strain BLYC-1937 possesses the ability to degrade various mycotoxins while also exhibiting good antibacterial activity against the aforementioned pathogens. Therefore, strain BLYC-1937 can simultaneously address the challenges posed by both mycotoxins and pathogens.

[0115] Example 6: Bacillus subtilis ( Bacillus subtilis An investigation into the degradation performance of BLYC-1937 compound microbial agent against combined pollution by multiple mycotoxins. 1. Test Methods 1.1 Preparation of Seed Fermentation Broth for Strains Use an inoculation loop on Bacillus subtilis ( Bacillus subtilis A loopful of bacteria was scraped from the BLYC-1937 preservation slant and inoculated into LB liquid medium. The culture was carried out at 37℃ and 180r / min for 16h to obtain the seed fermentation broth of Bacillus subtilis.

[0116] Using an inoculation loop on Bacillus lateralis ( Brevibacillus laterosporus A loopful of bacterial cells was scraped from the BLCC1-0170 preservation slant and inoculated into LB liquid medium. The culture was carried out at 37℃ and 180r / min for 16h to obtain the seed fermentation broth of Bacillus laterosporus.

[0117] Use an inoculation loop to inoculate with Aspergillus niger ( Aspergillus nigerA loopful of bacteria was scraped from the BLCC6-0009 preservation slant and inoculated into seed culture medium (3% sucrose, 0.2% sodium nitrate, 0.1% dipotassium hydrogen phosphate, 0.05% potassium chloride, 0.05% magnesium sulfate, 0.001% ferrous sulfate, pH natural; all percentages by mass). The culture was carried out at 30℃ and 180r / min for 3 days to obtain the seed fermentation broth of Aspergillus niger.

[0118] The viable cell counts of the seed fermentation broths of Bacillus subtilis, Bacillus laterosporus, and Aspergillus niger were all adjusted to 10. 8 CFU / mL.

[0119] 1.2 Preparation of Compound Microbial Agent The seed fermentation broth of Bacillus subtilis after adjusting the viable count was mixed with the seed fermentation broth of Aspergillus niger at a volume ratio of 1:1 to obtain compound microbial agent A; The seed fermentation broth of Bacillus subtilis after adjusting the viable count was mixed with the seed fermentation broth of Bacillus laterosporus at a volume ratio of 1:1 to obtain compound microbial agent B; The seed fermentation broth of Bacillus subtilis, Aspergillus niger, and Bacillus lateralis after adjusting the viable cell count were mixed at a volume ratio of 1:1:1 to obtain compound microbial agent C.

[0120] 1.3 Bacillus subtilis ( Bacillus subtilis Degradation effect of BLYC-1937 and its combined strains on multiple mycotoxin contamination. Corn-soybean meal feed was selected as the experimental subject. The experiment was divided into experimental groups 1, 2, 3, and 4 (T1, T2, T3, and T4) and a control group (CK). 100g of feed from each experimental group and the control group was spread evenly on a plastic film. T-2 toxin, vomitoxin, fumonisin B1, aflatoxin B1, and ochratoxin A were dissolved in 1mL of solvent, with vomitoxin dissolved in sterile water and the others in acetonitrile. The dissolved toxins were then evenly sprayed onto the feed using a small spray bottle to achieve a final concentration of 100μg / kg. After standing for 2 hours, the seed fermentation broth of Bacillus subtilis (adjusted for viable count), compound microbial agent A, compound microbial agent B, and compound microbial agent C, respectively, were evenly sprayed onto the feed using a spray bottle in experimental groups T1 to T4. The same volume of microbial solution was sprayed onto each experimental group, and the total viable count in each experimental group's feed was guaranteed to reach 1×10⁻⁶. 6 For the control group, the corresponding volume of solvent (1 mL sterile pure water and 4 mL acetonitrile) was sprayed, and the toxin content was determined after standing for 24 hours.

[0121] Take 1g of feed from experimental groups 1-4 and the control group respectively into 99mL sterile physiological saline conical flasks, and shake at 25℃ and 180r / min for 0.5h. Then centrifuge at 4000r / min for 5min, collect the supernatant, and determine the concentrations of T-2 toxin, vomitoxin, fumonisin B1, aflatoxin B1, and ochratoxin A in each group using a toxin detection kit, and calculate the degradation rate of each toxin. The calculation method is the same as in Example 3.

[0122] Bacillus subtilis ( Bacillus subtilis The degradation performance of BLYC-1937 and its strains against multiple mycotoxin contaminations is shown in Table 15.

[0123] Table 15: Degradation effect of combined strains on multiple mycotoxin contamination The results showed that co-contamination with T-2 toxin, vomitoxin, fumonisin B1, aflatoxin B1, and ochratoxin A increased the difficulty of toxin degradation and weakened the degradation effect of strain BLYC-1937 on mycotoxins to some extent.

[0124] Combining Bacillus subtilis, Bacillus lateralis, and Aspergillus niger can improve the degradation effect on multiple mycotoxin contamination, especially in synergistically reducing the content of T-2 toxin, vomitoxin, fumonisin B1, and ochratoxin A in combined toxin contamination.

[0125] Example 7: Animal Experiment 1. Test method: Two hundred and seventy three-day-old broiler chicks were used as experimental animals. Before the experiment, the chicken house floor, cages, water troughs, and feed troughs were cleaned and subsequently disinfected by fumigation. During the experiment, the temperature in the chicken house was controlled at 30±5℃ and the relative humidity at 40%. Multi-layer cages were used for rearing, and the stocking density was controlled to ensure the broilers had free movement, feeding, and drinking. The cages, water troughs, and feed troughs were cleaned daily, and manure was removed to ensure a hygienic growing environment for the broilers.

[0126] First, the chickens were acclimatized to a basal diet (purchased from Shannong Agricultural and Animal Husbandry (Tai'an) Co., Ltd.). Then, 270 broilers were randomly divided into three groups, with six replicates per group and 15 chickens per replicate. The three groups were the control group (CK), the T-2 toxin group (T-2), and the T-2 toxin detoxification group (T-2+JF). All groups of broilers were fed free access to food and water. The experimental period was 21 days. All three groups were fed the same basal diet. The T-2 toxin group was exposed to the toxin in a manner similar to natural feeding, and the specific treatment method is as follows: CK group: fed purified water and basal diet.

[0127] T-2 group: fed with purified water, and T-2 toxin was evenly mixed into the basal diet to achieve a T-2 toxin concentration of 100 μg / kg.

[0128] T-2+JF group: fed purified water, with T-2 toxin and Bacillus subtilis BLYC-1937 bacterial powder evenly mixed into the basal diet, so that the concentration of T-2 toxin in the diet reached 100 μg / kg and the number of viable Bacillus subtilis BLYC-1937 in the feed reached 1×10⁻⁶. 6 CFU / g.

[0129] During the experiment, each group's diet was prepared and used immediately, while other feeding and management practices remained consistent. Daily feed intake and body weight were recorded, and the feed conversion ratio (FCR, FCR = feed intake / body weight gain) was calculated. Sampling was conducted on days 7, 14, and 21 after grouping. Chickens were fasting for 24 hours prior to slaughter. Five chickens were selected from each replicate group. Body weight and venous blood were collected from the selected chickens before euthanasia. At slaughter, the bursa of Fabricius, spleen, and liver were removed, connective tissue and blood were removed, and their weight was measured. The organ index (organ index = organ weight / body weight × 100%) was determined. The kidneys and liver were removed, cleaned with 4°C physiological saline, and then stored at -20°C.

[0130] After incubating venous blood at 37°C for 30 minutes, centrifuging at 4000 r / min for 10 minutes, the supernatant was collected, and various biochemical indicators (alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin (ALB), creatinine (CREA), uric acid (UA), and urea (UREA)) of the 21-day serum were detected using a blood biochemistry analyzer.

[0131] Take 0.2g of liver and kidney samples respectively into test tubes, add 1.9mL of 80% methanol, grind using a homogenizer, shake at 180r / min for 16h at room temperature, centrifuge at 4000r / min for 5min, and determine the T-2 toxin concentration of each sample using a T-2 toxin detection kit.

[0132] 2. Test Results 2.1 Results of growth performance test The results of the growth performance test are shown in Table 16.

[0133] Table 16: Results of Growth Performance Measurement Note: Different lowercase letters in the superscript of peer data indicate significant differences. P <0.05); Different capital letters in the shoulder labels of peer data indicate extremely significant differences ( P <0.01), the same applies below.

[0134] The body weight at 21 days and the weight gain from 0 to 21 days in the T-2 group were significantly lower than those in the T-2+JF group and the CK group. P <0.05), while the FCR at 0-21d was significantly higher than that in the T-2+JF poison group and the CK group ( P The concentration of Bacillus subtilis (BLYC-1937) was <0.05, indicating a significant negative impact of the toxin on the growth performance of broiler chickens. The addition of Bacillus subtilis BLYC-1937 mitigated this negative impact.

[0135] 2.2 Results of Organ Index Measurement The organ index measurement results for each treatment group are shown in Table 17.

[0136] Table 17: Results of Organ Index Measurement Table 17 shows that the spleen index of group T-2 increased at 7 days ( P <0.05), indicating the stimulation of the organ's immune function; at 21 days, the spleen index of the T-2 group was lower than that of the CK group, and the spleen index of the T-2+JF group recovered to be close to that of the CK group, indicating that the attack of T-2 toxin affected the normal function of the spleen and did not recover by the end of 21 days, while Bacillus subtilis BLYC-1937 promoted the recovery of spleen growth and development.

[0137] 2.3 Results of serum biochemical index measurement The serum biochemical parameters of each treatment group are shown in Table 18.

[0138] Table 18: Results of Serum Biochemical Indicators Measurement Table 18 shows that, after 21 days of feeding, compared with the CK group, the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine, uric acid, and urea in the T-2 group were significantly higher. P <0.05), indicating that T-2 toxin damaged the liver and kidneys of chickens; compared with the CK group, the T-2+JF group had increased levels of alanine aminotransferase, aspartate aminotransferase, creatinine, uric acid, and urea, but the differences were not statistically significant. P >0.05); compared with the CK group, the albumin index in the T-2 group was significantly lower ( P <0.05), although albumin levels in the T-2+JF group showed a decreasing trend, the difference was not significant. P >0.05), indicating that Bacillus subtilis BLYC-1937 can reduce the damage to the liver and kidneys of broilers caused by T-2 toxin to some extent.

[0139] 2.4 Results of T-2 toxin residue detection The results of T-2 toxin content detection in the tissues of each group are shown in Table 19.

[0140] Table 19: T-2 toxin content in tissues Note: ND indicates not detected. As shown in Table 19, compared with the CK group, the T-2 toxin level was significantly increased in the T-2 group ( P <0.05); Compared with the T-2 group, the addition of Bacillus subtilis BLYC-1937 reduced the content of T-2 toxin in the liver, and the difference was significant at 21 days. P <0.05), and significantly reduced kidney function at 14d and 21d ( P The T-2 toxin content in <0.05) indicates that the degrading bacteria have the effect of reducing T-2 toxin in organisms.

[0141] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A strain of Bacillus subtilis ( Bacillus subtilis BLYC-1937, with accession number CCTCC NO: M20251711.

2. A microbial agent, characterized in that, The microbial agent contains Bacillus subtilis as described in claim 1 ( Bacillus subtilis )BLYC-1937.

3. The Bacillus subtilis strain according to claim 1 ( Bacillus subtilis The application of BLYC-1937 or the microbial agent according to claim 2 in the degradation of at least one of the following mycotoxins (1)-(5): (1) T-2 toxin; (2) Vomiting toxins; (3) Fumonisin B1; (4) Aflatoxin B1; (5) Ochratoxin A.

4. The Bacillus subtilis strain according to claim 1 ( Bacillus subtilis The use of BLYC-1937 or the microbial agent according to claim 2 in the preparation of at least one antibacterial product as described in (1)-(3) below: (1) Antibacterial products that inhibit T-2 toxin-producing Fusarium oxysporum; (2) Antimicrobial products that inhibit ochratoxin A-producing ochratoxin fungi; (3) Antibacterial products that inhibit Escherichia coli, Staphylococcus aureus, Clostridium perfringens, Vibrio parahaemolyticus, Vibrio alginolyticus, Streptococcus pyogenes, Serratia marcescens and / or Aeromonas hydrophila.

5. A compound microbial agent, characterized in that, The compound microbial agent is composed of Bacillus subtilis as described in claim 1 (… Bacillus subtilis Composed of BLYC-1937, Bacillus lateralis, and Aspergillus niger; The preservation number of the *Bacillus lateralis* is CCTCC NO: M 2022932; the preservation number of *Aspergillus niger* is CCTCC NO: M 2015722.

6. The Bacillus subtilis strain according to claim 1 ( Bacillus subtilis The application of BLYC-1937, the microbial agent according to claim 2, or the compound microbial agent according to claim 5 in the preparation of mycotoxin detoxification products, is characterized in that... The mycotoxins are T-2 toxin, vomitoxin, fumonisin B1, aflatoxin B1 and / or ochratoxin A.

7. A method for degrading mycotoxins, characterized in that, Includes the following steps: The Bacillus subtilis (as described in claim 1) Bacillus subtilis BLYC-1937, the microbial agent of claim 2 or the compound microbial agent of claim 5, are brought into contact with a sample contaminated with mycotoxins to degrade the mycotoxins in the sample; The mycotoxin contamination is either a single toxin contamination or a combination of multiple toxins, including T-2 toxin, vomitoxin, fumonisin B1, aflatoxin B1, and ochratoxin A.

8. The method according to claim 7, characterized in that, The samples contaminated with mycotoxins are grains, oils, or feeds contaminated with mycotoxins.

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