A strain of Bacillus licheniformis capable of inhibiting the growth of Aspergillus flavus and its application

Abstract

The invention discloses a strain of Bacillus licheniformis capable of inhibiting the growth of Aspergillus flavus and application thereof, and particularly relates to the technical field of microbial control. The invention screens an Aspergillus flavus antagonist from the rumen of a dairy cow, namely, Bacillus licheniformis NXU98, with a deposit number of CGMCC No. 27652. It is found through experimental verification that the Bacillus licheniformis strain can antagonize Aspergillus flavus and has strong high temperature resistance, acid and alkali resistance. In addition, the addition of Bacillus licheniformis to silage can effectively reduce the reproduction of aerobic bacteria. Therefore, the Bacillus licheniformis screened by the invention can be used to prevent Aspergillus flavus contamination in the process of silage fermentation.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial control, and specifically relates to a strain of Bacillus licheniformis capable of inhibiting the growth of Aspergillus flavus and application thereof. Background Art

[0002] As a common saprophytic fungus of the class of Aspergillus flavus in nature, it is very easy to contaminate crops, grains, etc. due to the influence of various factors such as temperature, humidity, and light, thus causing huge economic losses and food safety problems. Aflatoxin is a class of toxic secondary metabolites containing a bifuran ring and a naphthalene-oxazolidinone structure produced by Aspergillus flavus during its growth and metabolism. More than 20 forms of AF have been isolated and discovered. Among them, AFB1 is listed as a Class I carcinogen by the World Health Organization due to its extremely strong carcinogenicity, teratogenicity, mutagenicity, and liver damage.

[0003] At present, the prevention and control measures for aflatoxin mainly include physical control, chemical control and biological control. Physical control mainly includes ultraviolet irradiation, microwave treatment, heating, germ removal (for corn), rinsing, adsorbent method, etc., but it is easy to damage the nutritional components of grains or the detoxification efficiency is low. Chemical methods use strong acids, strong alkalis or strong oxidants to treat feed to destroy the aflatoxin structure in the feed, thereby removing aflatoxin from the feed, but it is very easy to produce by-products and reagent residues. Biological control uses the transformation of microorganisms or enzymes produced by microorganisms to degrade aflatoxin. Due to its specificity, mild treatment conditions, no toxic side effects, and no residue, it is one of the very important research directions for the removal of aflatoxin in the future.

[0004] Bacillus licheniformis is a Gram-positive thermophilic bacterium with superb resistance to high temperature, choline, and acid. In harsh environments, it often exists in the form of spores. As a new type of probiotic currently emerging, Bacillus licheniformis has great potential in mycotoxin biological control and green control. It can quickly seize oxygen in the internal environment, create an anaerobic environment, and provide a good living environment for beneficial bacteria such as Lactobacillus, Bifidobacterium, and Bacteroides. In different environments, the types and antibacterial effects of Bacillus licheniformis will change. The rumen, as a digestive organ unique to ruminants, contains a rich variety of microorganisms. Screening this species from the rumen can determine its biosafety while developing new antibiotics. Summary of the invention

[0005] In order to solve the problems raised in the above background technology, the present invention provides a strain of Bacillus licheniformis for inhibiting the growth of Aspergillus flavus and its application. Aiming at the current research and development status of biological control agents, the present application has the advantage of significant inhibitory effect on the growth of Aspergillus flavus and can be applied to feed fermentation.

[0006] To achieve the above-mentioned purpose, the present invention provides the following technical solution: a strain of Bacillus licheniformis NXU98, which has been deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on June 16, 2023, with the deposit number: CGMCC No.27652.

[0007] The present invention also provides a bacterial agent for inhibiting Aspergillus flavus, comprising the Bacillus licheniformis NXU98 or its metabolites.

[0008] Preferably, the active ingredient of the bacterial agent includes live Bacillus licheniformis cells, solid culture of Bacillus licheniformis or liquid culture of Bacillus licheniformis.

[0009] The present invention also provides an application of the Bacillus licheniformis or the bacterial agent, wherein the application includes any one of the following:

[0010] S1. Inhibit the growth of Aspergillus flavus;

[0011] S2. Prevent aflatoxin contamination during feed fermentation.

[0012] Preferably, the inhibiting the growth of Aspergillus flavus includes destroying the spores and strain structure of Aspergillus flavus.

[0013] Preferably, the feed comprises corn silage.

[0014] The present invention also provides the use of the Bacillus licheniformis NXU98 or the bacterial agent in the preparation of feed additives.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] 1. The present invention screened out a Bacillus licheniformis NXU98, which can inhibit the growth of Aspergillus flavus and has a rod-shaped cell morphology.

[0017] 2. The Bacillus licheniformis NXU98 of the present invention has strong stress resistance, and the survival rate is 34.13% after being treated at 90° C. for 20 minutes; and the survival rate is 9.69% at pH 9.0.

[0018] 3. The Bacillus licheniformis NXU98 of the present invention can significantly destroy the structure of the Aspergillus flavus strain.

[0019] 4. The Bacillus licheniformis NXU98 of the present invention can significantly inhibit aflatoxin contamination during silage fermentation. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is a schematic diagram of the common nutrient agar culture characteristics of Bacillus licheniformis NXU98 in the present invention;

[0021] Figure 2This is a schematic diagram of the Gram staining microscopic examination results of Bacillus licheniformis NXU98 in the present invention;

[0022] Figure 3 This is a schematic diagram of the analysis results of the 16S rRNA gene sequence of Bacillus licheniformis of the present invention;

[0023] Figure 4 This is a schematic diagram of the genetic evolution tree constructed based on the 16SrRNA gene sequence of Bacillus licheniformis in the present invention;

[0024] Figure 5 is a schematic diagram of the genome information of Bacillus licheniformis NXU98 of the present invention;

[0025] Figure 6 It is a schematic diagram of the survival rate of the Aspergillus flavus strain of Bacillus licheniformis NXU98 of the present invention under different temperature and pH conditions;

[0026] Figure 7 This is a schematic diagram of Example 3 of the present invention. The marks in the plate represent: BL-JT-1, NXU98 strain cells; BL-FJ-1, fermentation broth; BL-SQ-1, sterile supernatant;

[0027] Figure 8 The light microscopic images (LM) and transmission electron microscopic images (TEM) of the inhibitory effects of the Bacillus licheniformis NXU98 strain culture and fermentation supernatant on the morphology of the Aspergillus flavus strain are shown in the figures. DETAILED DESCRIPTION

[0028] The present invention is described in detail below with reference to the accompanying drawings and specific examples. The technical means are conventional means used by those skilled in the art, and the materials and reagents used can be obtained from commercial sources unless otherwise specified.

[0029] Example 1: Isolation, screening and identification of a strain of Bacillus licheniformis NXU98 that inhibits Aspergillus flavus

[0030] The present invention is a facultative anaerobic bacterium with strong heat resistance, acid resistance and antioxidant ability screened from the rumen of dairy cows. The surface of the bacterial colony is rough and wrinkled, and the edges are dirty white with irregular edges. After long-term cultivation, the edges of the bacterial colony are hair-like, the color deepens, and there is a viscous liquid on the surface of the bacterial colony. We have isolated, cultured and purified the strain to be tested, as well as stained microscopy, biochemical tests, molecular identification, genome sequencing and antibacterial tests.

[0031] 1. Isolation and identification of strains

[0032] 1. Isolation, culture and purification

[0033] 500 ml of enrichment medium was prepared in advance, and the enrichment medium, test tube rack and cleaned Henkel roller tube were sterilized under high pressure at 121° C. for 15 min and then placed on a sterile operating table;

[0034] On the day of sampling, a 500 ml plastic bottle was filled with CO2, and 500 ml of the collected rumen fluid of the fistula Holstein cow was placed in the plastic bottle, and the plastic bottle filled with rumen fluid was placed in an incubator and brought back to the laboratory at a constant temperature;

[0035] The enriched culture medium is dispensed into the Henkel roller tubes, and the enriched culture medium in each tube occupies 1 / 3 of the tube volume, the bottle cap and the bottle stopper are sealed, 1 ml of the rumen fluid is inoculated into the Henkel roller tube using a 1 ml sterile syringe, mixed and placed in a bacterial incubator, and cultured at 37° C.;

[0036] Prepare 500 ml of separation and screening culture medium, sterilize it under high pressure at 121°C for 15 minutes, place it on a sterile operating table, and when the temperature reaches 55°C-65°C, dispense it into disposable culture dishes, pouring 25 ml-35 ml into each culture dish, and wait until the temperature of the culture medium drops to room temperature and solidifies;

[0037] The LB agar medium was dispensed into 30 ml Henle tubes, and the rumen fluid was gradiently diluted (10-1, 10-3, 10-6, 10-9) with sterile water. The diluted rumen fluid was inoculated into the rolling tube using a 1 ml sterile syringe, and the tube was sealed with a sealing film. The tube was cultured in a bacterial incubator at 37°C. It was observed that dirty white colonies with rough and wrinkled surfaces and irregular edges grew. The colonies were purified by streaking on a plate. Figure 1 .

[0038] LB broth medium was purchased from Qingdao Haibo Biotechnology Co., Ltd.

[0039] 2. Gram staining microscopy

[0040] The dirty white colonies with rough and wrinkled surfaces and irregular edges were selected and placed on a glass slide. After Gram staining and observation under an oil microscope, it was found that the strain was a rod-shaped, single, paired or chain-shaped Gram-positive bacterium. The results are shown in Figure 2 .

[0041] 3. Physiological Tests

[0042] The purified cultured strain was cultured in a microbial incubator at 80°C for two hours, and the strain was inoculated into the purified culture medium again to determine its heat resistance.

[0043] Hi Bio-ID Sugar Fermentation Kit (HiCarbo'M Kit (KB009A / KB009B1 / KB009C)) was purchased. The kit contains 35 sugar fermentation tests, of which Box A and Box B each contain 12 sugar fermentation tests, and Box C contains 11 sugar fermentation tests and 1 control test. 50 μl of bacterial solution was inoculated into each well and cultured for about 5 hours in a bacterial incubator at 39°C for 48 hours. The color of the culture medium changes through microbial fermentation reactions. Preliminary identification was performed in combination with the Common Bacteria Manual and the Bergey's Bacteria Manual (as shown in Table 1).

[0044] Table 1 35 sugar fermentations of Bacillus licheniformis

[0045]

[0046] 4. Molecular Identification

[0047] 1) Bacterial DNA extraction

[0048] Take 1-5ml of purified bacterial culture solution, centrifuge at 10,000rpm (~11,500×g) for 1min, and discard the supernatant. Add 200μl buffer GA to the precipitate and shake until the bacteria are suspended. Add 20μl Proteinase K solution to the tube, mix well, add 220μl buffer GB, shake for 15s, let stand at 70℃ for 10min, and when the solution becomes clear, add 220μl anhydrous ethanol, shake thoroughly for 15s, then load into adsorption column CB3, centrifuge at 12,000rpm (~13,400×g) for 30s, discard the waste liquid, and place CB3 in a collection tube. Add 500μl buffer GD to adsorption column CB3, centrifuge at 12,000rpm (~13,400×g) for 30s, discard the waste liquid, and place CB3 in a collection tube. Add 600μl of rinse solution PW to the adsorption column CB3, centrifuge at 12,000rpm (~13,400×g) for 30s, discard the waste liquid, and place CB3 in a collection tube. After repeating the above steps, put the adsorption column CB3 back into the collection tube, centrifuge at 12,000rpm (~13,400×g) for 2min, discard the waste liquid, and place the adsorption column CB3 at room temperature for several minutes to completely dry the residual rinse solution in the adsorption material. Transfer the adsorption column CB3 to a clean centrifuge tube, add 50-200μl of elution buffer TE to the middle part of the adsorption membrane, place it at room temperature for 2-5min, and centrifuge it at 12,000rpm (~13,400×g) for 2min. The supernatant obtained is the DNA template.

[0049] 2) PCR amplification and sequencing analysis of 16S rRNA:

[0050] PCR amplification of 16S rDNA was performed using primers 27F and 1492R. Primer sequences: 27F: AGAGTTTGATCMTGGCTCAG, 1492R: TACGGTTACCTTGTTACGACTT. Reaction conditions: 95°C pre-denaturation for 3 min; 95°C denaturation for 15 s, 60°C annealing for 15 s, 72°C extension for 15 s, for a total of 35 cycles; 72°C extension for 10 min.

[0051] The PCR amplified product was detected using 1% agarose gel, and the gel recovery kit was used for recovery and purification. The bacteria were submitted to Shanghai Sangon Biotechnology Co., Ltd. for high-throughput sequencing. The 16S rDNA sequence was compared with the ribosome database http: / / rdp.cme.msu.edu / indexjsp, and the bacteria were identified again at the species, kingdom, phylum, class, order, family, genus, and species classification levels. It was found that the 16S ribosomal RNA gene of Bacillus licheniformis CCMMB933 and 810 strains was 99% similar, and the 16S ribosomal RNA gene of Bacillus licheniformis CCMMB928, 925, and 885 strains was 100% similar ( Figure 3 ), confirming that the tested bacterium is Bacillus licheniformis. The genetic evolution tree constructed based on the 16S rRNA gene sequence of the tested bacterium (seq1) is shown in Figure 4 .

[0052] 5. Genome Sequencing

[0053] 1) DNA purification and detection

[0054] The concentration of genomic DNA was detected using TBS380 or Nanodrop2500 to ensure that the DNA quality for subsequent experiments was high enough (no degradation, OD260 / 280=1.8-2.0, and the total amount was not less than 10 μg).

[0055] 2) Single-molecule sequencing library construction

[0056] The G-tubes method is used to process genomic DNA into 8-10k fragments; the ends are filled in and connected to circular single strands: the two ends of the single strand are connected to the double-stranded positive and negative strands respectively, to obtain a dumbbell-like ("horse ring") structure, called SMRTBell.

[0057] 3) Single-molecule sequencing

[0058] The single-stranded loop of the library is annealed and bound to the polymerase at the bottom of the fixed ZMW (zero-mode waveguides). Once the binding is completed, it can be sequenced on the machine.

[0059] The Circos software was used to draw a genome circle map. The outermost circle of the circle map is the symbol of the genome size; the second and third circles are the CDS on the positive and negative chains, and different colors indicate the functional classification of different COGs of CDS; the fourth circle is rRNA and tRNA; the fifth circle is the GC content, the outward red part indicates that the GC content of this region is higher than the average GC content of the whole genome, and the higher the peak value, the greater the difference with the average GC content; the inward blue part indicates that the GC content of this region is lower than the average GC content of the whole genome, and the higher the peak value, the greater the difference with the average GC content; the innermost circle is the GC-Skew value, and the specific algorithm is GC / G+C, which can assist in judging the leading chain and the lagging chain. Generally, the leading chain GC skew>0, and the lagging chain GC skew<0. It can also assist in judging the replication start point (minimum cumulative offset) and end point (maximum cumulative offset), which is especially important for circular genomes ( Figure 5 ).

[0060] Example 2: Stress resistance of a strain of Bacillus licheniformis NXU98 that inhibits Aspergillus flavus

[0061] 10mL of seed solution was placed in a water bath at 30, 50, 70 and 90℃ for 20min, diluted to the appropriate concentration with physiological saline in a 10-fold gradient, and 100μL of bacterial solution was applied to the solidified LB broth medium, inverted and cultured in a 37℃ incubator for 12h. The activated bacterial solution was inoculated into 5mL of LB broth medium with different pH (3.0, 5.0, 7.0, 9.0, 11.0) for 2h, inverted and cultured at 37℃ for 12h. The activated bacterial solution was inoculated into 5mL of different pH (3.0, 5.0, 7.0, 9.0, 11.0) for 2h, inverted and cultured at 37℃ for 12h. Each group of experiments was repeated three times, and the plate count method was used to count and calculate the survival rate of bacteria.

[0062]

[0063] Where: N0 represents the number of viable bacteria in the control group (CFU / mL); N1 represents the number of viable bacteria in the experimental group (CFU / mL). Figure 6 As shown, the results showed that the survival rate of Bacillus licheniformis NXU98 was 34.13% when treated in a 90°C water bath for 20 minutes, and the survival rate was 9.69% when treated at pH 9.0 for 2 hours.

[0064] Example 3: Antibacterial effect of a strain of Bacillus licheniformis NXU98 that inhibits Aspergillus flavus

[0065] 1. Preparation of NXU98 fermentation broth

[0066] The NXU98 strain was inoculated into a 50 mL enzyme-free sterile centrifuge tube containing 30 mL LB medium, and cultured at 37° C. and 140 rpm for 24 h to obtain the fermentation broth of the NXU98 strain.

[0067] 2. Preparation of NXU98 strain

[0068] The NXU98 strain was inoculated into a 50 mL enzyme-free sterile centrifuge tube containing 30 mL LB medium, and cultured at 37°C and 140 rpm for 24 h. The fermentation broth was centrifuged at 4°C and 4000×g for 1 h. The precipitate obtained by centrifugation was the bacterial cell.

[0069] 3. Preparation of NXU98 Sterile Filtrate

[0070] The NXU98 strain was inoculated into a 50 mL enzyme-free sterile centrifuge tube containing 30 mL LB medium, cultured at 37°C, 140 rpm, and shaken for 24 h. The culture was centrifuged at 4°C, 10,000 × g for 15 min. The supernatant was filtered with a 0.22 um filter membrane and centrifuged again at 4°C overnight to obtain a sterile filtrate of the NXU98 strain.

[0071] 4. Determination of antibacterial effect by confrontation plate method

[0072] A 7mm diameter Aspergillus flavus was inoculated in the center of each PDA plate containing different fermentation broths, sterile filtrates and bacterial cells. The PDA plate with sterile liquid culture medium was used as a blank control and placed in a 30°C biochemical incubator for dark culture. Each treatment was repeated 5 times. When the pathogenic fungal colony grew to 2 / 3 of the control plate, the colony diameter was measured. The results showed that the antibacterial effect of bacterial cells and fermentation supernatant was significantly higher than that of culture fluid ( Figure 7 ).

[0073] 5. Observation of the ultrastructure of fungi using light microscopy and transmission electron microscopy of NXU98 strain

[0074] The fungi treated with bacterial cells, fermentation broth and fermentation supernatant were fixed with 3% glutaraldehyde and then used for subsequent experiments.

[0075] 4.1 Reagent preparation

[0076] 1% osmium acid mother solution: 1g osmium tetroxide ampoule is thoroughly cleaned and wrapped with filter paper. After smashing the ampoule, it is immediately placed in the mother solution bottle, and 50ml of pure water is poured in. The bottle mouth is sealed and mixed, and it is moved to a 4℃ refrigerator and sealed and stored away from light. Before use, take an equal volume of osmium acid mother solution and 0.2M PBS (PH≈7.4) and mix them to obtain 1% osmium acid working solution.

[0077] 3% glutaraldehyde fixative: Take 120ml of 25% glutaraldehyde, add 2.97g of NaH2PO4·2H2O and 29g of Na2HPO4·12H2O, make up to 1000ml with UP water, stir until completely dissolved, and mix well.

[0078] Dehydration reagent: Take anhydrous acetone and dilute it with purified water to make 30%, 50%, 70%, 80%, 90% and 95% acetone solutions.

[0079] Epon-812 embedding medium: Accurately measure 45 ml of Epon-812, 30 ml of DDSA and 25 ml of MNA and mix them. Stir continuously for 30 minutes. While stirring, add 2 ml of DMP-30 dropwise. Continue stirring for 10 minutes and divide into portions for later use.

[0080] Table 2 Reagents required for the test

[0081] name batch number Specification Manufacturer Sodium dihydrogen phosphate 1010580101700 500g / bottle Fuchen (Tianjin) Chemical Reagent Co., Ltd. Disodium hydrogen phosphate 1010590101700 500g / bottle Fuchen (Tianjin) Chemical Reagent Co., Ltd. Glutaraldehyde (AR grade) 20230828 100ml / bottle Sinopharm Chemical Reagent Co., Ltd. Hungry acid GP18456 1g / stick Beijing Zhongjing Scientific Instrument Technology Co., Ltd. Acetone (AR grade) - 500ml / bottle Xilong Scientific Co., Ltd. Uranyl acetate GS02624 25g / bottle Beijing Zhongjing Scientific Instrument Technology Co., Ltd. Lead citrate GA10701-1 100g / bottle Beijing Zhongjing Scientific Instrument Technology Co., Ltd.

[0082] 4.2 Test methods

[0083] Fixation: Samples were pre-fixed with 3% glutaraldehyde (customer fixation) and re-fixed with 1% osmium tetroxide.

[0084] Dehydration: Dehydration with acetone step by step, the concentration gradient of the dehydrating agent is 30% → 50% → 70% → 80% → 90% → 95% → 100% (100% concentration was changed 3 times).

[0085] Infiltration: Dehydrating agent and Epon-812 embedding medium were infiltrated in the ratio of 3:1, 1:1, and 1:3 respectively.

[0086] Embedding: Epon-812 pure embedding medium.

[0087] Ultrathin sections: Use an ultrathin microtome to prepare ultrathin sections of about 60 to 90 nm, spread the sections, and then scoop them onto a copper mesh.

[0088] Staining: First stain with uranyl acetate for 10 to 15 minutes, then stain with lead citrate for 1 to 2 minutes, stain at room temperature, and finally examine under a microscope.

[0089] Table 3 Test instruments

[0090] name model Manufacturer Pure water and ultrapure water system UPR-II-15TNZ Sichuan Youpu Ultrapure Technology Co., Ltd. High Precision Electronic Balance BN-200 Produced by Taiwan Julin Sakura Enterprise Desktop high-speed microcentrifuge D3024 Dalong Xingchuang Experimental Instruments (Beijing) Co., Ltd. Biological Microscope BA210Digital Motic Industrial Group Co., Ltd. Ultrathin slicer UC7 LEICA Tissue dehydrator EMTP LEICA Transmission electron microscopy JEM-1400FLASH JEOL

[0091] 4.3 Image acquisition

[0092] The JEM-1400FLASH transmission electron microscope produced by JEOL was used to collect images of the copper mesh. Each copper mesh was first observed under low magnification for the entire structure, and the area to be observed was selected to collect images and observe the specific structure (see Figure 8 )

[0093] Example 4: Application of a strain of Bacillus licheniformis NXU98 in the fermentation process of corn silage. Corn silage at the waxy stage was selected, crushed to 1.5-2.0 cm, mixed evenly, and a 20L plastic bucket was used as the silage container. The control group did not add Bacillus licheniformis, and the three experimental groups were inoculated with different concentrations of Bacillus licheniformis (0.8×10 6 , 1.6×10 6 and 3.2×10 6 CFU / gDM, manual compaction (550±30kg / m 3 ), and the fermentation was kept at controlled room temperature (20±1°C). Samples were taken on the 3rd, 7th, 14th, 28th, and 56th days of fermentation for aflatoxin determination (Table 2).

[0094] Weigh 2g±0.05g of crushed sample into a 50ml centrifuge tube, add 10ml of sample extract, shake for 5min, and centrifuge at 4000r / min at room temperature for 10min; take 0.5ml of supernatant, add 0.5ml of deionized water, mix well; take 50μL for analysis.

[0095] Percent absorbance calculation: The percent absorbance of the standard solution or sample is equal to the average value of the percent absorbance of the standard solution or sample (dual wells) divided by the absorbance value of the first standard solution (0 ppb), multiplied by 100%, that is

[0096]

[0097] A represents the average absorbance value of the standard solution or sample solution, and A0 represents the average absorbance value of the 0 ppb standard solution

[0098] Drawing and calculation of standard curve: Draw a semi-logarithmic curve of the standard solution with the percent absorbance of the standard solution as the ordinate and the logarithm of the corresponding concentration (ppb) of the standard solution as the abscissa. Substitute the percent absorbance of the sample into the standard curve, read the corresponding concentration of the sample from the standard curve, and multiply it by the corresponding dilution factor to obtain the actual concentration of the analyte in the sample.

[0099] Table 4 Changes of mycotoxins during whole-plant corn silage fermentation

[0100]

[0101] As shown in Table 4, the addition of different concentrations of Bacillus licheniformis had an inhibitory effect on AFB1 during silage fermentation, and the inhibitory ability was strongest when the addition amount was 3.2×106 CFU / g DM.

[0102] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A strain of Bacillus licheniformis NXU98, characterized in that: The Bacillus licheniformis NXU98 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on June 16, 2023, with the deposit number: CGMCC No.27652.

2. A bacterial agent for inhibiting Aspergillus flavus, characterized in that: The method comprises the Bacillus licheniformis NXU98 according to claim 1.

3. The bacterial agent according to claim 2, characterized in that: The active ingredients of the bacterial agent include living bacteria of Bacillus licheniformis, solid culture of Bacillus licheniformis or liquid culture of Bacillus licheniformis.

4. The use of the Bacillus licheniformis according to claim 1 or the bacterial agent according to any one of claims 2 to 3, characterized in that: The application includes any of the following: S1. Inhibit the growth of Aspergillus flavus and do not treat diseases; S2. Prevent aflatoxin contamination during feed fermentation.

5. The use according to claim 4, characterized in that: The feed is silage corn.

6. Use of the Bacillus licheniformis NXU98 according to claim 1 or the bacterial agent according to any one of claims 2 to 3 in the preparation of feed additives.

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