Bacillus subtilis, complex microbial inoculant and application of bacillus subtilis and complex microbial inoculant in degradation of mycotoxin

Abstract

The invention discloses bacillus subtilis, a complex microbial inoculant and application of the complex microbial inoculant to degradation of mycotoxin, and belongs to the field of microorganisms and fermentation engineering. According to the invention, a strain of bacillus subtilis BLYC-1937 with the activity of degrading mycotoxin is obtained through screening, and the bacillus subtilis BLYC-1937 has a good degradation effect on pollution of single toxin of T-2 toxin, vomitoxin, fumonisins B1, aflatoxin B1 and ochratoxin A or combined pollution of multiple toxins; the strain has good antagonism on fusarium oxysporum capable of producing T-2 toxin and aspergillus ochraceus capable of producing ochratoxin A, and pollution of T-2 toxin and ochratoxin A can be reduced from the source.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microorganisms and fermentation engineering, and particularly relates to a bacillus subtilis, a complex microbial agent and application of the bacillus subtilis and the complex microbial agent in degrading mycotoxins. BACKGROUND

[0002] Mycotoxins are natural toxic secondary metabolites produced by fungi, which widely exist in contaminated feed raw materials and compound feed, and mainly include T-2 toxin, vomitoxin (DON), aflatoxin B1 (AFB1), fumonisin B1 (FB1) and ochratoxin A (OTA) and the like. Among them, T-2 toxin is a strong toxic fungal toxin produced by Fusarium fungi, which belongs to the trichothecene family. T-2 toxin can cause damage to tissues such as the intestinal tract, liver, kidney and brain, and the most typical symptom is liver damage. In addition, T-2 toxin can also cause ataxia, anorexia, central nervous damage, growth inhibition, bone deformity, immune system damage and the like, which has brought serious challenges to the breeding industry and human health.

[0003] The toxic groups of T-2 toxin, epoxy group and double bond, have strong resistance to nucleophiles, making the chemical structure of T-2 toxin very stable. T-2 toxin remains toxic even after being placed at room temperature for 6-7 years, and it is also difficult to reduce its toxicity in high pressure, ultraviolet light, neutral or acidic environments. Therefore, it is extremely difficult to detoxify T-2 toxin. At present, the detoxification methods of T-2 toxin mainly include physical, chemical and biological detoxification methods, but physical detoxification and chemical detoxification may change the palatability of feed and reduce the nutritional value and use value of feed; in contrast, the use of biological detoxification method can avoid the disadvantages such as the generation of irritating odor and the loss of nutrients, and shows good detoxification effect and specificity to T-2 toxin. Biological detoxification method uses microorganisms or biological enzymes to convert fungal toxins into substances with lower toxicity through biological metabolism, such as the research report that Eubacterium makes T-2 toxin partially degrade into HT-2, and can convert the metabolic products (HT-2, T-2 triol, T-2 tetrol and the like) of T-2 toxin into its de-epoxy form, thereby destroying the chemical structure of fungal toxins and achieving the purpose of safety and detoxification. At present, the research on T-2 toxin detoxification strains is mainly focused on bacteria such as Lactococcus lactis ( Lactococcus lactis ), intertidal zone microbacterium ( Exiguobacterium aestuarii ), Brevibacterium ( Curtobacterium ), Eubacterium ( Eubacterium ) and fungi such as Saccharomyces cerevisiae ( Saccharomyces cerevisiae ), Aspergillus niger ( Aspergillus niger ), but there are problems such as low degradation efficiency, unstable effect and unclear degradation mechanism, and the resources of strains with high efficiency in degrading T-2 toxin are still scarce.

[0004] In addition, there is also a joint toxicity between different mycotoxins. Studies have found that the joint pollution rate of mycotoxins in feed raw materials and compound feed is 30.3%-88.9%, thereby causing the establishment of mycotoxin limitation standards and the prevention and control of pollution to be more complex. SUMMARY

[0005] In view of the problems in the prior art, the purpose of the present application is to provide a bacillus subtilis, a compound microbial agent and the application thereof in degrading mycotoxins.

[0006] To achieve the above-mentioned purpose, the present application adopts the following technical solutions: In a first aspect of the present application, a bacillus subtilis (BLYC-1937) is provided. Bacillus subtilis The bacillus subtilis (BLYC-1937) has been preserved in the China Center for Type Culture Collection (CCTCC) in Wuhan, China, on July 28, 2025, and the preservation number is CCTCC NO: M 20251711.

[0007] The bacillus subtilis (BLYC-1937) of the present application has the following main characteristics compared with the bacillus subtilis reported in the prior art: Bacillus subtilis (1) It can simultaneously degrade T-2 toxin, vomitoxin, fumonisin B1, aflatoxin B1 and ochratoxin A; and can antagonize the growth of T-2 toxin-producing fusarium oxysporum and ochratoxin A-producing aspergillus ochraceus, thereby reducing the production of T-2 toxin and ochratoxin A at the source.

[0008] (2) It has significant antibacterial activity against common pathogenic bacteria in the breeding process, such as escherichia coli, staphylococcus aureus, clostridium perfringens, vibrio parahaemolyticus, vibrio alginolyticus, streptococcus pyogenes, serratia marcescens and aeromonas hydrophila. Infection of these pathogenic bacteria can exacerbate the harm of mycotoxins and cause the superposition of breeding risks. Strain BLYC-1937 not only has the performance of degrading multiple mycotoxins, but also has good antibacterial activity against the above-mentioned pathogenic bacteria, and can simultaneously cope with the challenges of mycotoxins and pathogenic bacteria.

[0009] (3) It has excellent fermentation performance, can obtain high viable cell count through short-time fermentation, and has high-temperature resistance, acid resistance and bile salt resistance activity, as well as neutral protease, alpha-amylase and cellulase activity.

[0010] In a second aspect of the present application, a microbial agent is provided, which contains the above-mentioned bacillus subtilis (BLYC-1937) and / or the metabolites of bacillus subtilis (BLYC-1937). Bacillus subtilis In a second aspect of the present application, a microbial agent is provided, which contains the above-mentioned bacillus subtilis (BLYC-1937) and / or the metabolites of bacillus subtilis (BLYC-1937). Bacillus subtilis In a second aspect of the present application, a microbial agent is provided, which contains the above-mentioned bacillus subtilis (BLYC-1937) and / or the metabolites of bacillus subtilis (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 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 (… subtilis Bacillus subtilis 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 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 mycotoxin-contaminated sample is mycotoxin-contaminated grain, oil and / or feed.

[0025] The beneficial effects of this invention are: (1) This invention screened and obtained a strain of Bacillus subtilis with activity in degrading mycotoxins ( ). subtilis Bacillus subtilis 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 ( Figure 1 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 2 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 3 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 4 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 5 Electron micrographs 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] Bacillus Growth curve (left) and pH change (right) of strain BLYC-1937 fermentation in small tanks. Detailed Implementation 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 (… subtilis Bacillus subtilis 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 Based on BLYC-1937, Bacillus subtilis ( subtilis Aspergillus niger 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: 1.5% (mass percent) agar powder was added to the above LB liquid medium.

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

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

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

[0041] PDA solid medium: 1.5% (mass percent) agar powder was added to the above PDA liquid medium.

[0042] Iron subsulfate solid 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, wherein the percentages are mass percentages.

[0043] MRS liquid medium: 1% protein peptone, 0.5% beef extract, 2% glucose, 0.2% potassium phosphate dibasic, 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, wherein the percentages are mass percentages.

[0044] PBS buffer: 0.8% sodium chloride, 0.02% potassium chloride, 0.142% sodium phosphate dibasic, 0.027% potassium phosphate dibasic, pH 7.4, sterilized at 121°C for 30 min, wherein the percentages are mass percentages.

[0045] Normal saline: 0.9% sodium chloride, sterilized at 121°C for 30 min, wherein the percentage is a mass percentage.

[0046] 20L small tank fermentation medium: 5% glucose, 5% corn starch, 4% soybean meal, 2% corn syrup dry powder, 0.2% potassium dihydrogen phosphate, 0.05% magnesium sulfate, 0.1% manganese sulfate, 0.3% calcium chloride, 0.05% amylase, and an appropriate amount of defoaming agent; wherein 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 ( Brevibacillus laterosporus 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 ( Bacillus subtilis 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 inoculation ring to scrape each ring of the above-mentioned test strain on the slope culture medium (LB solid medium), inoculate in LB liquid medium respectively, 37°C, 180r / min culture for 16h, respectively to obtain the seed fermentation broth of the test strain.

[0053] 1.2 T-2 toxin degradation effect investigation The test group respectively takes LB liquid medium 780μL, seed fermentation broth 20μL of the test strain, adds 200μL T-2 toxin standard working solution filtered by 0.22μm water system filter film (the final concentration of T-2 toxin after mixing is 1μg / mL), 37°C, 180r / min culture for 48h; At the same time, set the system without adding strain seed fermentation broth as the control group, the control group takes LB liquid medium 800μL, adds 200μL T-2 standard working solution filtered by 0.22μm water system filter film (the final concentration of T-2 toxin after mixing is 1μg / mL), 37°C, 180r / min culture for 48h.

[0054] After culture, the obtained solution of each group is centrifuged at 4000r / min for 5min, the supernatant is taken, then stored in the refrigerator at 4°C, according to the T-2 toxin detection kit instruction, the T-2 toxin concentration in each group sample is detected, and the degradation rate of each test strain on T-2 toxin is calculated.

[0055] Degradation rate (%) = (T0-T1) / T0×100%; T0=the toxin concentration of the control group T1=the toxin concentration of the test group.

[0056] 1.3 16S rRNA identification and preservation of the strain The strain with the highest T-2 toxin degradation rate is selected as the target strain, and the 16S rRNA gene sequence of the target strain is amplified and sequenced. The sequencing result sequence is compared in the NCBI database to determine its species name, and the target strain is preserved according to the patent procedure.

[0057] 2、Test results 2.1 T-2 toxin degradation effect of the test strain The T-2 toxin degradation effect of each test strain is shown in Table 1.

[0058] Table 1: T-2 toxin degradation effect of the test strain The results show that the strain BLYC-1937 has the best T-2 toxin degradation effect, with a degradation rate of 93.70%, which is selected as the target strain.

[0059] 2.2 Identification and preservation of the target strain 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 ( Figure 1 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 2 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. Bacillus subtilis 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 inoculation ring to scrape a ring of bacteria on the strain BLYC-1937 preservation slope culture medium (LB solid medium), inoculate in LB liquid medium, 37℃, 180r / min culture for 16h, to obtain the strain seed fermentation liquor.

[0081] 1.2 Investigation of degradation performance on vomitoxin The test group takes LB liquid medium 780μL, strain BLYC-1937 seed fermentation liquor 20μL, adds 200μL filtered by 0.22μm water system filter vomitoxin standard working solution (after mixing, the final concentration of vomitoxin is 1μg / mL), 37℃, 180r / min culture for 48h; At the same time, set the system without adding strain seed fermentation liquor as the control group, the control group takes LB liquid medium 800μL, adds 200μL filtered by 0.22μm water system filter vomitoxin standard working solution (after mixing, the final concentration of vomitoxin is 1μg / mL). Each group sets 3 repeats, after fermentation, the culture liquid is centrifuged at 4000r / min for 5min, the supernatant is taken, then stored in the refrigerator at 4℃, according to the vomitoxin detection kit instruction, the vomitoxin concentration in each group sample is detected, and the degradation rate is calculated.

[0082] 1.3 Investigation of degradation performance on fumonisin B1 The test group takes LB liquid medium 780μL, strain BLYC-1937 seed fermentation liquor 20μL, adds 200μL filtered by 0.22μm water system filter fumonisin B1 standard working solution (after mixing, the final concentration of fumonisin B1 is 1μg / mL), 37℃, 180r / min culture for 48h; At the same time, set the system without adding strain seed fermentation liquor as the control group, the control group takes LB liquid medium 800μL, adds 200μL filtered by 0.22μm water system filter fumonisin B1 standard working solution (after mixing, the final concentration of fumonisin B1 is 1μg / mL). Each group sets 3 repeats, after fermentation, the culture liquid is centrifuged at 4000r / min for 5min, the supernatant is taken, then stored in the refrigerator at 4℃, according to the fumonisin B1 detection kit instruction, the fumonisin B1 concentration in each group sample is detected, and the degradation rate is calculated.

[0083] 1.4 Investigation of degradation performance on aflatoxin B1 The test group took LB liquid medium 780 μL, seed fermentation liquid 20 μL of strain BLYC-1937, added 200 μL of aflatoxin B1 standard working solution filtered by 0.22 μm water system filter (after mixing, the final concentration of aflatoxin B1 was 1 μg / mL), and was cultured at 37°C, 180 r / min for 48 h; at the same time, the system without adding strain seed fermentation liquid was set as the control group, the control group took LB liquid medium 800 μL, added 200 μL of aflatoxin B1 standard working solution filtered by 0.22 μm water system filter (after mixing, the final concentration of aflatoxin B1 was 1 μg / mL). Each group was set with 3 repeats, and the culture liquid of each group was centrifuged at 4000 r / min for 5 min after fermentation, the supernatant was taken, and then stored in a 4°C refrigerator. According to the aflatoxin B1 detection kit instruction, the aflatoxin B1 concentration in each group sample was detected, and the degradation rate was calculated.

[0084] 1.5 Degradation performance of ochratoxin A The test group took LB liquid medium 780 μL, seed fermentation liquid 20 μL of strain BLYC-1937, added 200 μL of aflatoxin B1 standard working solution filtered by 0.22 μm water system filter (after mixing, the final concentration of aflatoxin B1 was 1 μg / mL), and was cultured at 37°C, 180 r / min for 48 h; at the same time, the system without adding strain seed fermentation liquid was set as the control group, the control group took LB liquid medium 800 μL, added 200 μL of aflatoxin B1 standard working solution filtered by 0.22 μm water system filter (after mixing, the final concentration of aflatoxin B1 was 1 μg / mL). Each group was set with 3 repeats, and the culture liquid of each group was centrifuged at 4000 r / min for 5 min after fermentation, the supernatant was taken, and then stored in a 4°C refrigerator. According to the aflatoxin B1 detection kit instruction, the aflatoxin B1 concentration in each group sample was detected, and the degradation rate was calculated.

[0085] Toxin degradation rate = (T0-T1) / T0 x 100%; T0=the toxin concentration of the control group; T1=the toxin concentration of the test 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: Degradation effect on 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 ( Figure 3 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 4 As shown; electron micrographs of *Ochratoxin A* producing ochratoxin A under antagonistic effects. Bacillus subtilis 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 ( Figure 5 Performance evaluation of BLYC-1937 1. Experimental Methods 1.1 Growth performance investigation A loop of bacteria was scraped from the strain BLYC-1937 preservation slope medium (LB solid medium) using an inoculation loop, and was cultured at 37°C and 180 r / min for 16 h to obtain a seed solution. The seed solution was inoculated into a 20 L tank fermentation medium at an inoculation amount of 1% (v / v) for fermentation culture, and the fermentation conditions were controlled as follows: rotation speed 200 rpm, air flux 16 L / min, and growth temperature 37°C. Samples were taken every 4 h for determination of viable cell count and pH, and the total fermentation time was 40 h.

[0093] 1.2 Tolerance performance investigation 1.2.1 Preparation of strain seed fermentation solution A loop of bacteria was scraped from the strain BLYC-1937 preservation slope medium (LB solid medium) using an inoculation loop, and was inoculated into LB liquid medium, and was cultured at 37°C and 180 r / min for 16 h to obtain a strain seed fermentation solution.

[0094] 1.2.2 Investigation of heat tolerance 100 mL of the seed fermentation solution of the strain BLYC-1937 was centrifuged at 4000 r / min for 10 min, and the supernatant was discarded. The bacterial cells were washed with sterile PBS buffer, and the step was repeated twice. Then, 100 mL of sterile PBS buffer was used for resuspension, and the mixture was divided into 6 groups, each with 3 repeats, and each repeat was 5 mL of the resuspension. The test groups were placed in a water bath at 80°C for 15 min, 25 min, and 35 min, respectively, and the control group (CK) was placed in a water bath at 25°C for 15 min, 25 min, and 35 min, respectively. Then, LB solid medium was used to determine the survival rate of viable cells in each group.

[0095] Survival rate (%) = viable cell count of the test group / viable cell count of the control group x 100%.

[0096] 1.2.3 Investigation of acid tolerance 100 mL of the seed fermentation solution of the strain BLYC-1937 was centrifuged at 4000 r / min for 10 min, and the supernatant was discarded. The bacterial cells were washed with sterile PBS buffer, and the step was repeated twice. Then, 100 mL of sterile PBS buffer was used for resuspension, and the mixture was divided into 4 groups, each with 3 repeats, and each repeat was 5 mL of the resuspension. The pH of the test groups was adjusted to 2.5, 2.0, and 1.5, respectively, and the control group (natural pH) was placed in a constant temperature incubator at 25°C for 120 min. Then, LB solid medium was used to determine the survival rate of viable cells in each group. Survival rate (%) = viable cell count of the test group / viable cell count of the control group x 100%.

[0097] 1.2.4 Investigation of bile salt tolerance The seed fermentation liquor of strain BLYC-1937 was inoculated into LB liquid medium added with 0.3%, 0.4%, 0.5% (w / v) porcine bile salt at an inoculation amount of 1% (v / v), and the seed fermentation liquor of strain BLYC-1937 was inoculated into ordinary LB liquid medium at an inoculation amount of 1% (v / v) as a control group. Each group was cultured at 37°C and 180 r / min for 12 h, and the fermentation liquor was obtained. The viable count was determined using LB solid medium. Survival rate (%) = viable count of test group / viable count of control group x 100%.

[0098] 1.3 Enzyme production performance investigation 1.3.1 Preparation of seed fermentation liquor of strain A loop of bacteria was scraped from the strain BLYC-1937 preservation slant medium (LB solid medium) using an inoculation loop, inoculated into LB liquid medium, and cultured at 37°C and 180 r / min for 16 h to obtain the seed fermentation liquor of strain.

[0099] 1.3.2 Investigation of neutral protease production performance The seed fermentation liquor of strain BLYC-1937 was inoculated into LB liquid medium at an inoculation amount of 1% (v / v), and the neutral protease enzyme activity was determined after 24 h of fermentation at 37°C and 180 r / min. The neutral protease enzyme activity was determined according to GB / T 28715-2012 “Determination of Acid and Neutral Protease Activity in Feed Additives by Spectrophotometry”; 1.3.3 Investigation of α-amylase production performance The seed fermentation liquor of strain BLYC-1937 was inoculated into LB liquid medium at an inoculation amount of 1% (v / v), and the α-amylase enzyme activity was determined after 24 h of fermentation at 37°C and 180 r / min. The determination of α-amylase enzyme activity was performed according to the method for determining bacterial α-amylase activity in “Principles and Analysis Methods of Food Enzymology”; 1.3.4 Investigation of cellulase production performance The seed fermentation liquor of strain BLYC-1937 was inoculated into LB liquid medium at an inoculation amount of 1% (v / v), and the cellulase enzyme activity was determined after 24 h of fermentation at 37°C and 180 r / min. The determination of cellulase enzyme activity was performed using the carboxymethyl cellulose sodium (CMC) saccharification method.

[0100] 1.4 Investigation of bacteriostatic performance 1.4.1 Preparation of fermentation supernatant of strain BLYC-1937 A loop of bacteria was scraped from the strain BLYC-1937 preservation slope culture medium (LB solid culture medium) using a inoculation loop, inoculated into LB liquid culture medium, and cultured at 37°C and 180 r / min for 16 h to obtain a strain seed fermentation liquor. The seed fermentation liquor was centrifuged at 4000 r / min for 10 min, and the supernatant was collected to prepare the fermentation supernatant.

[0101] 1.4.2 Preparation of pathogenic bacteria fermentation liquor Escherichia coli, Salmonella, Staphylococcus aureus, Vibrio parahaemolyticus, Vibrio alginolyticus, Streptococcus pyogenes, Serratia marcescens, and Aeromonas hydrophila were inoculated into NB culture medium (2% sodium chloride was added to the culture medium for Vibrio parahaemolyticus and Vibrio alginolyticus), and cultured overnight at 37°C and 180 r / min. Then, the overnight cultured pathogenic bacteria were diluted to 10 7 CFU / mL with sterile normal saline to form a pathogenic bacteria diluent.

[0102] Clostridium perfringens was inoculated into a liquid thiosulfate culture medium and cultured overnight at 45°C. Then, the overnight cultured Clostridium perfringens was diluted to 10 7 CFU / mL with sterile normal saline to form a pathogenic bacteria diluent.

[0103] 1.4.3 Bacteriostatic test The bacteriostatic test method refers to the method in GB / T 39101-2020 “Determination of Polypeptide Antimicrobial Activity by Bacteriostatic Zone Method”, and the Oxford cup and punching method were used for the bacteriostatic test.

[0104] Escherichia coli, Salmonella, Staphylococcus aureus, Vibrio parahaemolyticus, Vibrio alginolyticus, Streptococcus pyogenes, Serratia marcescens, and Aeromonas hydrophila were subjected to the bacteriostatic test using the Oxford cup method. First, 10 mL of NA culture medium containing 1.5% agar was added to each culture dish, and after cooling and solidification, 600 μL of the corresponding pathogenic bacteria diluent was added, followed by the addition of 6 mL of NA culture medium containing 0.7% agar to make the pathogenic bacteria concentration 10 6 CFU / mL (2% (m / v) sodium chloride was added to the 0.7% NA culture medium for Vibrio parahaemolyticus and Vibrio alginolyticus), and quickly shaken. After cooling and solidification, the Oxford cup (outer diameter 7.8 mm) was placed, and after standing for 30 min, 260 μL of the fermentation supernatant of strain BLYC-1937 was added, and after standing for 30 min, it was placed in a 37°C incubator for 12-16 h, and then the diameter of the bacteriostatic circle was measured.

[0105] Clostridium perfringens was subjected to the bacteriostatic test using the punching method. First, 1.8 mL of Clostridium perfringens diluent was added to each culture dish, followed by the addition of 18 mL of ferrous sulfite culture medium to make the pathogenic bacteria concentration 10 6CFU / mL, shake quickly. After cooling and solidification, use the hole puncher (outer diameter 9 mm) to punch, add 90 μL of strain BLYC-1937 fermentation supernatant, stand for 30 min, put into a 37°C incubator, and after 16 h of culture, measure the diameter of the inhibition zone. Clostridium perfringens needs to be put into an anaerobic culture bag and cultured anaerobically.

[0106] Each pathogenic bacteria plate was repeated 3 times, and an inhibition zone with a diameter greater than 9 mm was considered effective.

[0107] 2. Test results 2.1 Growth performance test results The fermentation growth curve and pH curve of strain BLYC-1937 in a 20 L small tank are shown in Bacillus subtilis The results show that strain BLYC-1937 enters the logarithmic growth phase at 4 hours, and the viable cell count reaches a peak at 16 h, exceeding 1×10 10 CFU / mL, with fast growth and high viable cell count.

[0108] 2.2 Tolerance test results The tolerance of strain BLYC-1937 to heat, acid and bile salts is shown in Tables 10, 11 and 12.

[0109] Table 10: Heat tolerance test results (results are means, n=3) Table 11: Acid tolerance test results (results are means, n=3) Table 12: Bile salt tolerance test results (results are means, n=3) The results show that strain BLYC-1937 has good survival at 80°C water bath for 15 min, 25 min and 35 min, good survival at pH=2.5, 2.0 and 1.5 for 120 min, and good survival at 0.3%, 0.4% and 0.5% pig bile salt for 12 h, and it can be considered that strain BLYC-1937 has good heat tolerance, acid tolerance and bile salt tolerance.

[0110] 2.3 Enzyme activity test results The enzyme activity test results of strain BLYC-1937 producing neutral protease, cellulase and alpha-amylase are shown in Table 13.

[0111] Table 13: Enzyme activity test results (results are means, 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 ( Brevibacillus laterosporus 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 ( Aspergillus niger 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 ( Bacillus subtilisA 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 laterosporus 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] The feed of 1-4 test groups and control group was respectively taken 1 g in 99 mL sterile normal saline conical flask, 25 °C, 180 r / min, shaking for 0.5 h, then 4000 r / min, centrifuging for 5 min, taking supernatant, determining the concentration of T-2 toxin, vomitoxin, fumonisin B1, aflatoxin B1 and ochratoxin A in each group by toxin detection kit, and calculating the degradation rate of each toxin. The calculation method is the same as example 3.

[0122] Bacillus subtilis (BLYC-1937) and strain combination Bacillus subtilis The results of the degradation performance of the strain combination on the combined pollution of multiple mycotoxins are shown in Table 15.

[0123] Table 15: Degradation effect of strain combination on multiple mycotoxin pollution The results show that the combined pollution of T-2 toxin, vomitoxin, fumonisin B1, aflatoxin B1 and ochratoxin A increases the difficulty of toxin degradation, and to some extent, weakens the degradation effect of strain BLYC-1937 on mycotoxins.

[0124] The combination of Bacillus subtilis, Brevibacillus laterosporus and Aspergillus niger can improve the degradation effect on the combined pollution of multiple mycotoxins, especially can synergistically reduce the content of T-2 toxin, vomitoxin, fumonisin B1 and ochratoxin A in the combined toxin pollution.

[0125] Example 7: Animal experiment 1. Test method: 270 3-day-old white-feather broiler chickens were used as test animals. Before the test, the floor, cage, water tank and trough of the chicken house were cleaned, and then fumigated and disinfected. The temperature of the chicken house during the test was controlled at 30±5°C, and the relative humidity was 40%. Multi-layer cages were used for feeding, and the feeding density was controlled to ensure that the broilers could move, eat and drink freely. The cage, water tank and trough were cleaned every day, and the manure was cleaned to ensure that the growth environment of the broilers was clean and hygienic.

[0126] Firstly, the basic feed (purchased from Shannong Nongma (Taian) Co., Ltd.) was fed for domestication, and then 270 broilers were randomly divided into 3 groups, 6 replicates per group, 15 chickens per replicate, and the three groups were control group (CK), T-2 toxin group (T-2) and T-2 toxin detoxification group (T-2+JF). Each group of broilers was fed in a free-feeding and free-drinking manner. The test period was 21 days. The three groups were fed with the same basic feed, and the T-2 toxin group was simulated to eat naturally. The specific treatment method is as follows: CK group: feeding with pure water and basic feed.

[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 saline solution, 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 and weight gain of T-2 group from 0 to 21 d were significantly lower than those of T-2+JF group and CK group P <0.05), and the FCR from 0 to 21 d was significantly higher than that of T-2+JF group and CK group P <0.05), which indicated that the toxin had a significant negative impact on the growth performance of broilers, and the addition of Bacillus subtilis BLYC-1937 alleviated this negative impact.

[0135] 2.2 Determination results of organ index The determination results of organ index of each treatment group are shown in Table 17.

[0136] Table 17: Determination results of organ index As shown in Table 17, the spleen index of T-2 group increased at 7 d P <0.05), indicating that the immune function of the organ was stimulated; at 21 d, the spleen index of T-2 group was lower than that of CK group, and the spleen index of T-2+JF group recovered to close to that of CK group, indicating that the challenge of T-2 toxin affected the normal function of the spleen, and did not recover by the end of 21 d, while Bacillus subtilis BLYC-1937 promoted the recovery of the growth and development of the spleen.

[0137] 2.3 Determination results of serum biochemical indicators The determination results of serum biochemical indicators of each treatment group are shown in Table 18.

[0138] Table 18: Determination results of serum biochemical indicators As shown in Table 18, the serum biochemical indicators of chickens showed that, after being fed for 21 d, the alanine aminotransferase, aspartate aminotransferase, creatinine, uric acid and urea indicators of T-2 group were significantly higher than those of CK group P <0.05), indicating that the T-2 toxin damaged the liver and kidney of chickens; compared with CK group, the alanine aminotransferase, aspartate aminotransferase, creatinine, uric acid and urea indicators of T-2+JF group were increased, but the difference was not significant P >0.05); compared with CK group, the albumin indicator of T-2 group was significantly decreased P <0.05), and the albumin of T-2+JF group had a decreasing trend, but the difference was not significant P >0.05), indicating that Bacillus subtilis BLYC-1937 could reduce the damage of T-2 toxin challenge to the liver and kidney of broilers to a certain extent.

[0139] 2.4 Detection results of T-2 toxin residue The detection results of T-2 toxin content 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. An inoculant characterized in that, The bacterial agent contains the Bacillus subtilis (BLYC-1937) and / or the metabolic product of Bacillus subtilis (BLYC-1937) of claim 1. Bacillus subtilis The bacterial agent contains the Bacillus subtilis (BLYC-1937) and / or the metabolic product of Bacillus subtilis (BLYC-1937) of claim 1. Bacillus subtilis The bacterial agent contains the Bacillus subtilis (BLYC-1937) and / or the metabolic product of Bacillus subtilis (BLYC-1937) of claim 1.

3. The bacterial agent of claim 2, wherein The bacterial agent, wherein the Bacillus subtilis (BLYC-1937) exists in one or more of a form of a cultured live bacteria, a bacterial suspension, a fermentation broth, and a bacterial lysate. Bacillus subtilis The BLYC-1937 exists in one or more of a form of a cultured live bacteria, a bacterial suspension, a fermentation broth, and a bacterial lysate.

4. The Bacillus subtilis (BLYC-1937) or the microbial agent of claim 2 for use in degrading at least one mycotoxin selected from the group consisting of: (1) aflatoxins, (2) ochratoxins, (3) fumonisins, (4) zearalenone, and (5) patulin. Bacillus subtilis ) (1) T-2 toxin; (2) vomitoxin; (3) fumonisin B1; (4) aflatoxin B1; (5) ochratoxin A.

5. 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) bacteriostatic product for inhibiting T-2 toxin-producing Fusarium oxysporum; (2) bacteriostatic product for inhibiting ochratoxin A-producing Aspergillus ochraceus; (3) bacteriostatic product for inhibiting Escherichia coli, Staphylococcus aureus, Clostridium perfringens, Vibrio parahaemolyticus, Vibrio alginolyticus, Streptococcus pyogenes, Serratia marcescens and / or Aeromonas hydrophila.

6. A complex microbial agent, characterized in that, The complex microbial agent is composed of Bacillus subtilis (BLYC-1937), Brevibacillus parabrevis and Aspergillus niger. Bacillus subtilis ) BLYC-1937, Brevibacillus parabrevis and Aspergillus niger. Preferably, the Brevibacillus laterosporus has the preservation number CCTCC NO: M2022932, and the Aspergillus niger has the preservation number CCTCC NO: M2015722.

7. The Bacillus subtilis (BLYC-1937) of claim 1, the bacterial agent of claim 2 or the complex bacterial agent of claim 6 for use in the preparation of a mycotoxin detoxification product. Bacillus subtilis ) BLYC-1937, the bacterial agent of claim 2 or the complex bacterial agent of claim 6 for use in the preparation of a mycotoxin detoxification product.

8. A method of degrading mycotoxins, characterized by, The method comprises the following steps: The Bacillus subtilis of claim 1 Bacillus subtilis ) BLYC-1937, the microbial inoculant of claim 2, or the complex microbial inoculant of claim 6 is contacted with a sample contaminated with mycotoxins to degrade the mycotoxins in the sample.

9. The method of claim 8, wherein, The mycotoxin contamination is single toxin contamination or combined contamination of T-2 toxin, vomitoxin, fumonisin B1, aflatoxin B1 and ochratoxin A.

10. The method of claim 8, wherein, The sample of the mycotoxin contamination is mycotoxin-contaminated grain, oil and / or feed.

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