A Bacillus velezensis YE-1 antagonistic to Aspergillus flavus and its application

Abstract

The present invention discloses a Bacillus velezensis YE-1 antagonistic to Aspergillus flavus and its application, which relates to the field of microbial control. In the present invention, an Aspergillus flavus antagonist, namely Bacillus velezensis YE-1, is screened from lichen and has a deposit number of CCTCC NO: M 20221143. Through experimental verification, it is found that this Bacillus velezensis strain can antagonize Aspergillus flavus, and at the same time has good high temperature resistance, osmotic pressure resistance, artificial gastric juice resistance and artificial intestinal juice resistance. The Bacillus velezensis bacterial solution is gavaged to Kunming mice, and the results show that this strain is safe and reliable. Therefore, the Bacillus velezensis screened in the present invention can be applied to the prevention and control of Aspergillus flavus contamination during the storage of food, feed and grains.

Description

Technical Field

[0001] The present invention relates to the field of microbial control, and particularly to a Bacillus velezensis YE-1 that antagonizes Aspergillus flavus and its application. Background Art

[0002] Aspergillus flavus is a common saprophytic fungus of Deuteromycetes, which can contaminate food and feed at all stages of the food / feed chain. The Food and Agriculture Organization of the United States estimates that 25% of the global food supply is contaminated with Aspergillus flavus. Aspergillus flavus can produce aflatoxins in the later stage of growth. Aflatoxins are a group of compounds with similar chemical structures. The basic structure of aflatoxins is coumarin and a furan ring. The aflatoxins that have been isolated and identified include b1, b2, g1, g2, m1, m2, p1, q, h1, gm, b2a and toxic alcohol. Aflatoxins are highly toxic substances, and have been proven to have carcinogenic, teratogenic and mutagenic effects on the human body. They were classified as Group 1 carcinogens by the cancer research agency of the World Health Organization (WHO) as early as 1993. Not only because of their toxicity, but also because of their stability under many conditions, they have brought significant health risks and losses in international trade. Therefore, Aspergillus flavus contamination has always been an urgent problem to be solved in the agricultural market and the food industry.

[0003] The control measures for Aspergillus flavus mainly include physical control, chemical control and biological control. Physical methods include dehumidification, temperature control, deoxidation and ultraviolet irradiation, etc. However, physical methods have adverse effects on the nutritional components of grains and feeds, even cause damage, and consume a large amount of energy. Chemical methods mainly use chemical mold inhibitors to treat products, which are prone to produce by-products and residues of treatment agents, bringing potential safety hazards to physical health. A large number of studies have shown that many microorganisms can inhibit the growth of Aspergillus flavus, including bacteria, actinomycetes, yeasts, algae, etc. Biological control has received more and more attention due to its advantages such as good specificity, environmental friendliness, mild treatment conditions and easy industrialization.

[0004] The genus Bacillus has come into the public eye due to its high stress resistance and good environmental adaptability, and has been widely used as a biological control agent. Bacillus velezensis is a new type of biocontrol bacterium discovered by Spanish scholars in 2005. Screening new microbial strains and obtaining reliable in vivo and in vitro results are very important for expanding the germplasm resource library of probiotics. Although many scholars at home and abroad have studied the control of Aspergillus flavus by antagonistic bacteria and published papers, there are few studies on the control of Aspergillus flavus by Bacillus velezensis at present and there are certain deficiencies. Summary of the Invention

[0005] The object of the present invention is to provide a Bacillus velezensis YE-1 antagonistic to Aspergillus flavus and its application, so as to solve the problems existing in the above-mentioned prior art. This strain can efficiently antagonize Aspergillus flavus and has safety, and can be applied to food, feed and grain storage.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a Bacillus velezensis YE-1 antagonistic to Aspergillus flavus, and the preservation number of this strain is CCTCC NO: M 20221143, the preservation time is July 21, 2022, the preservation unit is China Center for Type Culture Collection, and the preservation address is Wuhan University, Wuhan, China.

[0008] The present invention also provides a fungicide antagonistic to Aspergillus flavus, which contains the above-mentioned Bacillus velezensis YE-1 or its metabolites.

[0009] Preferably, the active ingredients of the fungicide include the live cells of Bacillus velezensis, the solid culture of Bacillus velezensis or the liquid culture of Bacillus velezensis.

[0010] The present invention also provides the application of the above-mentioned Bacillus velezensis YE-1 or the above-mentioned fungicide, and the application includes any one of the following:

[0011] (1) Inhibiting the growth of Aspergillus flavus;

[0012] (2) Preventing or controlling the contamination of Aspergillus flavus in food, grains or feeds.

[0013] Preferably, the inhibition of the growth of Aspergillus flavus includes inhibiting the germination of Aspergillus flavus spores, inhibiting the elongation of Aspergillus flavus germ tubes and inhibiting the expansion of Aspergillus flavus hyphae.

[0014] Preferably, the grains include corn and peanuts.

[0015] The present invention also provides the application of the above-mentioned Bacillus velezensis YE-1 or the above-mentioned fungicide in the preparation of feed additives.

[0016] The present invention also provides a feed additive antagonistic to Aspergillus flavus, which contains the above-mentioned Bacillus velezensis YE-1 or its metabolites.

[0017] Preferably, the active ingredients of the feed additive are the live cells of Bacillus velezensis, the solid culture of Bacillus velezensis or the liquid culture of Bacillus velezensis.

[0018] The present invention discloses the following technical effects:

[0019] (1) A Bacillus velezensis YE-1 was screened in the present invention, which can antagonize Aspergillus flavus and has a rod-shaped cell morphology.

[0020] (2) Bacillus velezensis YE-1 has good stress resistance. The survival rate was 33.60% after treatment at 80 °C for 20 min; the survival rate was 19.75% after treatment with 7.5% sodium chloride for 2 h; the survival rate was 33.57% after treatment in artificial gastric juice for 3 h; the survival rate was 64.24% after treatment in artificial intestinal juice for 6 h; and it is sensitive to most antibiotics.

[0021] (3) Bacillus velezensis YE-1 can significantly inhibit the spore germination, germ tube elongation and hyphal expansion of Aspergillus flavus.

[0022] (4) Bacillus velezensis YE-1 can significantly eliminate or inhibit the contamination of Aspergillus flavus in peanuts and corn.

[0023] (5) Bacillus velezensis YE-1 can significantly eliminate or inhibit the contamination of Aspergillus flavus in complete feed.

[0024] (6) Bacillus velezensis YE-1 poses no safety hazards to Kunming mice. Biochemical indexes, hematological indexes and anatomical observations are normal. Description of the Drawings

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0026] Figure 1 shows the morphology of Bacillus velezensis YE-1; (A) shows the colonies of Bacillus velezensis YE-1 grown on LB medium at 37 °C for 1 day, which are milky white, oval, with wavy edges, low convexities, dull, opaque and viscous in texture; (B) shows the morphological diagram of Bacillus velezensis YE-1 under a thermal field scanning electron microscope at 3.00KX. The bacterium is rod-shaped and the scale bar is 3 μm;

[0027] Figure 2 shows the antibacterial effect of Bacillus velezensis YE-1 against Aspergillus flavus; (A) is the control group, and (B) is the experimental group;

[0028] Figure 3 Inhibitory effect of Bacillus velezensis YE-1 on spore germination, germ tube elongation, and hyphal expansion of Aspergillus flavus; (A) shows the inhibitory effect on spore germination, (B) shows the inhibitory effect on germ tube elongation, and (C) shows the inhibitory effect on hyphal expansion; a, b, c, and d in the figure indicate significant differences, p < 0.05;

[0029] Figure 4 Inhibitory effect of Bacillus velezensis YE-1 on Aspergillus flavus in corn and peanuts; (A) is the peanut control group; (B) is the peanut treatment group; (C) is the corn control group; (D) is the corn treatment group;

[0030] Figure 5 Inhibitory effect of Bacillus velezensis YE-1 on complete pig feed; (A) is the control group, (B) is the experimental group;

[0031] Figure 6 Detection results of the tolerance of Bacillus velezensis YE-1 to temperature, osmotic pressure, artificial gastric juice, and artificial intestinal juice; a is the tolerance result map at different temperatures, b is the tolerance result map at different osmotic pressures, c is the tolerance result map at different treatment times in artificial gastric juice, and d is the tolerance result map at different treatment times in artificial intestinal juice; * indicates significant difference, p < 0.05; ** indicates highly significant difference, p < 0.01;

[0032] Figure 7 Results of changes in body weight and food intake of each group of mice during 14 days of gavage with Bacillus velezensis YE-1; a is the result map of body weight changes in female mice, b is the result map of body weight changes in male mice, c is the result map of food intake changes in female mice, and d is the result map of body weight intake changes in male mice;

[0033] Figure 8 Result map of the pathological effects of gavage with Bacillus velezensis YE-1 bacterial solution on the liver and kidney tissues of Kunming mice. Detailed implementation manners

[0034] The various exemplary implementation manners of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0035] It should be understood that the terms used in the present invention are only for describing specific embodiments and are not intended to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0036] 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 invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0037] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of this application are merely exemplary.

[0038] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0039] Example 1 Isolation, screening and identification of a Bacillus velezensis YE-1 antagonistic to Aspergillus flavus

[0040] (1) Isolation of the antagonistic bacterium

[0041] Lichen was collected from Baiyin City, Gansu Province and placed in a sterilized 500 mL conical flask containing 300 mL of sterile water. It was shaken at 160 r / min for 15 min to obtain a suspension of bacteria, which was then aliquoted into 1.5 mL EP tubes and treated in a water bath at 85 °C for 20 min. 100 μL of the suspension of bacteria was spread on an LB agar medium and incubated overnight in an inverted position in an incubator at 37 °C. Single colonies were selected and cultured overnight in an LB broth medium at 37 °C and 160 r / min. The suspension of bacteria was mixed with 50% glycerol in equal proportions and stored in a glycerol preservation tube for use in a -80 °C refrigerator.

[0042] (2) Screening of the antagonistic bacterium

[0043] Preparation of Aspergillus flavus spore suspension. Using potato dextrose agar medium (PDA), the Aspergillus flavus strain was activated and cultured in an incubator at 25 °C for 10 days. Take the Aspergillus flavus mycelial cake and place it in a conical flask containing 30 mL of sterile water. Shake it evenly with a vortex mixer and filter it twice with sterile gauze to remove mycelia and impurities. Count the spores under an optical microscope using a hemocytometer and adjust the spore concentration to 10 6 cells / mL.

[0044] Take 100 μL of Aspergillus flavus spore solution and spread it on the PDA medium. Culture it in an incubator at 25 °C for 24 h. Use a 7 mm punch to punch out the Aspergillus flavus mycelial cake on the petri dish. Transfer the Aspergillus flavus mycelial cake to the center of a new PDA plate and culture it in an incubator at 25 °C for 24 h. Take 20 μL of the activated bacterial solution and streak it on both sides about 2 cm away from the bacterial cake. Culture it in an incubator at 25 °C for 48 h.

[0045] (3) Identification of antagonistic bacteria

[0046] Morphological identification: Describe the single colony state of the strain in the logarithmic growth phase with a stable colony size, mainly including the size, color, transparency, surface state of the colony, and edge state of the colony. Secondly, observe the shape of the bacteria on the colony by scanning electron microscopy. As Figure 1 shown, after Bacillus velezensis YE-1 grew on the LB medium at 37 °C for 1 day, it formed a milky white, oval, wavy edge, low concave, dull, opaque, and viscous colony. The morphological diagram of Bacillus velezensis YE-1 at 3.00KX in a thermal field scanning electron microscope. The bacterium is rod-shaped and the scale bar is 3 μm.

[0047] As Figure 2 shown, in the growth direction of Bacillus velezensis YE-1, the growth of Aspergillus flavus mycelia was significantly inhibited. Select the strain that inhibits the growth of Aspergillus flavus, extract the strain DNA, amplify the 16s rDNA, translation initiation factor IF-2 (infB), and topoisomerase (topA) genes, and send them to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0048] The primers, PCR reaction conditions, and amplified fragment sequences used in the above identification are as follows:

[0049] PCR amplification system:

[0050] Table 1 Amplification system

[0051] Component Name Addition Amount 2XHieff PCR Master Mix 12.5 μL Primer F (100 μM) 0.5 μL Primer R (100 μM) 0.5 μL DNA Template 0.6 μL <![CDATA[ddH2O]]> 10.9 μL

[0052] The PCR systems used for amplifying the 16s rDNA, infB, and topA gene sequences of the biocontrol bacterium Bacillus velezensis YE-1 strain are listed in Table 1.

[0053] Gene primers and PCR conditions:

[0054] Table 2 Amplification primers and PCR conditions

[0055]

[0056] The primers used for amplifying the 16s rDNA, infB, and topA gene sequences of Bacillus velezensis YE-1 strain, as well as the annealing temperature and extension time during PCR amplification, are listed in Table 2. The specific PCR conditions are as follows: pre-denaturation at 94°C for 5 min, [94°C for 30 s, annealing temperature for 30 s, 72°C for 1 extension time] × 30 cycles, 72°C for 5 min. After PCR, when the fragment size was verified to be correct by gel electrophoresis, the PCR product was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0057] The amplified nucleotide sequences are shown as SEQ ID NO: 1-3. The sequencing results were subjected to Blast alignment in the NCBI (National Center for Biotechnology Information, USA) database. The alignment result showed that this strain belongs to Bacillus velezensis, and the strain was numbered YE-1. This Bacillus velezensis YE-1 that antagonizes Aspergillus flavus was deposited at the China Center for Type Culture Collection on July 21, 2022. The deposit address is Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: M 20221143.

[0058] SEQ ID NO: 1 is as follows:

[0059] gaaagtggcgggcgtgcctaatacatgcaagtcgagcggacagatgggagcttgctccctgatgttagcggcggacgggtgagtaacacgtgggtaacctgcctgtaagactgggataactccgggaaaccggggctaataccggatggttgtctgaaccgcatggttcagacataaaaggtggcttcggctaccacttacagatggacccgcggcgcattagctagttggtgaggtaacggctcaccaaggcgacgatgcgtagccgacctgagagggtgatcggccacactgggactgagacacggcccagactcctacgggaggcagcagtagggaatcttccgcaatggacgaaagtctgacggagcaacgccgcgtgagtgatgaaggttttcggatcgtaaagctctgttgttagggaagaacaagtgccgttcaaatagggcggcaccttgacggtacctaaccagaaagccacggctaactacgtgccagcagccgcggtaatacgtaggtggcaagcgttgtccggaattattgggcgtaaagggctcgcaggcggtttcttaagtctgatgtgaaagcccccggctcaaccggggagggtcattggaaactggggaacttgagtgcagaagaggagagtggaattccacgtgtagcggtgaaatgcgtagagatgtggaggaacaccagtggcgaaggcgactctctggtctgtaactgacgctgaggagcgaaagcgtggggagcgaacaggattagataccctggtagtccacgccgtaaacgatgagtgctaagtgttagggggtttccgccccttagtgctgcagctaacgcattaagcactccgcctggggagtacggtcgcaagactgaaactcaaaggaattgacgggggcccgcacaagcggtggagcatgtggtttaattcgaagcaacgcgaagaaccttaccaggtcttgacatcctctgacaatcctagagataggacgtccccttcgggggcagagtgacaggtggtgcatggttgtcgtcagctcgtgtcgtgagatgttgggttaagtcccgcaacgagcgcaacccttgatcttagttgccagcattcagttgggcactctaaggtgactgccggtgacaaaccggaggaaggtggggatgacgtcaaatcatcatgccccttatgacctgggctacacacgtgctacaatggacagaacaaagggcagcgaaaccgcgaggttaagccaatcccacaaatctgttctcagttcggatcgcagtctgcaactcgactgcgtgaagctggaatcgctagtaatcgcggatcagcatgccgcggtgaatacgttcccgggccttgtacacaccgcccgtcacaccacgagagtttgtaacacccgaagtcggtgaggtaaccttttaggagccagccgccgaaggtgacaagaagg。

[0060] SEQ ID NO: 2 is as follows:

[0061]

[0062] SEQ ID NO: 3 is as follows:

[0063]

[0064] Example 2: Stress resistance detection of Bacillus velezensis YE-1

[0065] (1) Detection of temperature and osmotic pressure tolerance

[0066] Take 1 mL of the bacterial solution and place it in 1.5 mL EP tubes, which are respectively placed in a water bath at 40 °C, 50 °C, 60 °C, 70 °C, and 80 °C for 20 min. Dilute it to an appropriate concentration with physiological saline at a 10-fold gradient. Take 100 μL of the bacterial solution and spread it on the solidified PCA medium, then invert it and culture it in an incubator at 37 °C for 12 h. Inoculate the activated bacterial solution into 5 mL of LB broth medium with different sodium chloride concentrations (1%, 2%, 3%, 4%, 5%, 6%, 7.5%) and treat it for 2 h. After pouring the plate, culture it anaerobically at 37 °C for 12 h. Each experiment is repeated three times, and the plate counting method is used to count and calculate the survival rate of bacteria:

[0067]

[0068] In the formula: N0 is the number of viable bacteria in the control group (CFU / mL); N1 is the number of viable bacteria in the experimental group (CFU / mL).

[0069] As Figure 6 shown, the results show that the survival rate of Bacillus velezensis YE-1 is 33.60% after being treated in a water bath at 80 °C for 20 min, and the survival rate is 19.75% after being treated with 7.5% sodium chloride for 2 h.

[0070] (2) Detection of tolerance to artificial gastric juice and artificial intestinal juice

[0071] To prepare artificial gastric juice, potassium chloride (7 mmol / L), sodium bicarbonate (45 mmol / L), sodium chloride (125 mmol / L), and pepsin (3 g / L) (Shanghai Yuanye Bio-Technology Co., Ltd.) are prepared, and finally the pH is adjusted to 3.0 with hydrochloric acid, and then filtered and sterilized with a 0.22 μm hydrophilic microfiltration membrane. The twice-activated bacterial solution is centrifuged at 6000 r / min for 8 min. After removing the supernatant, add the same volume of artificial gastric juice and culture it at 37 °C. Take out the bacterial solution and pour the plate for counting at 1 h, 2 h, and 3 h. Similarly, to prepare artificial intestinal juice, trypsin (1 g / L) (Shanghai Yuanye Bio-Technology Co., Ltd.) and porcine bile salt (3 g / L) are added, the pH is adjusted to 7.5 with sodium hydroxide, and then filtered and sterilized with a 0.22 μm hydrophilic microfiltration membrane. Add the artificial intestinal juice to the above-prepared bacterial cell precipitate and culture it at 37 °C. Take out and pour the plate to calculate the survival rate at 2 h, 4 h, and 6 h respectively (the calculation formula is the same as above). Each experiment is repeated three times to evaluate the tolerance of the strain to artificial gastric juice and artificial intestinal juice.

[0072] As Figure 6 shown by the results: the survival rate after 3 h of treatment in artificial gastric juice was 33.57%, and the survival rate after 6 h of treatment in artificial intestinal juice was 64.24%.

[0073] (3) Antibiotic sensitivity test

[0074] Take 100 μL of Bacillus velezensis YE-1 with a cell concentration of 10 7 CFU / mL and spread it on the solidified LB agar medium. Then, evenly place the drug sensitivity test paper (Thermo Fisher Scientific) on the LB agar medium. After culturing in an incubator at 37 °C for 24 h, accurately measure the diameter of the inhibition zone of the drug sensitivity test paper with a vernier caliper. If there is no inhibition zone, record the diameter of the antibiotic paper. By comparing with the zone diameter provided in the antibacterial disc sensitivity performance standard, the results are expressed as resistance (R), intermediate sensitivity (I), and sensitivity (S).

[0075] Table 3 Sensitivity results of Bacillus velezensis YE-1 to different antibiotics

[0076]

[0077] As shown in Table 3, the drug sensitivity test results showed that Bacillus velezensis YE-1 was sensitive to most antibiotics, so it is safe to be used as a probiotic.

[0078] Example 3 Antagonistic effect of Bacillus velezensis YE-1 against Aspergillus flavus and its application

[0079] (1) Effect of the antagonistic bacterium on the spore germination and germ tube elongation of Aspergillus flavus

[0080] Inoculate Bacillus velezensis YE-1 into LB broth medium and culture it overnight at 37 °C. Add the bacterial solution to the PDA medium at an appropriate temperature and mix well to make the final bacterial solution concentration 10 7 CFU / mL, 10 6 CFU / mL, 10 5 CFU / mL, and pour plates. After the plates solidify, spread 100 μL of Aspergillus flavus spore solution on the plates. After culturing in an incubator at 25 °C for 11 h, detect the spore germination rate and germ tube length. Each replicate contains 9 parallels, and each parallel contains 900 spores. At the same time, set up PDA without any treatment and PDA with added LB broth as controls.

[0081] From Figure 3As can be seen from A-B, the fermentation broth of Bacillus velezensis YE-1 has a highly significant inhibitory effect on the spore germination and germ tube elongation of Aspergillus flavus. There was no significant difference in the spore germination rate and germ tube length between the CK group and the LB group (p>0.05), excluding the influence of the bacterial liquid solvent LB broth on the spore germination rate and germ tube elongation. There was a highly significant difference between the CK group and the 10 5 CFU / mL group (p<0.01). There was a highly significant difference in the spore germination rate between the 10 6 CFU / mL group and the 10 5 CFU / mL group (p<0.01). There was a highly significant difference in the spore germination rate and germ tube length between the 10 6 CFU / mL group and the 10 7 CFU / mL group (p<0.01). In summary, it can be concluded that Bacillus velezensis YE-1 has a highly significant inhibitory effect on the spore germination and germ tube elongation of Aspergillus flavus, and this inhibitory effect is significantly enhanced with the increase in the content of Bacillus velezensis YE-1.

[0082] (2) Inhibitory effect of the antagonistic bacterium on the mycelial expansion of Aspergillus flavus

[0083] Inoculate Bacillus velezensis YE-1 into LB broth medium and culture it overnight at 37°C. Add the bacterial liquid to the PDA medium at an appropriate temperature and mix well to make the final bacterial liquid concentration 10 7 CFU / mL, 10 6 CFU / mL, 10 5 CFU / mL, and pour plates. Take 100 μL of Aspergillus flavus spore liquid and spread it on the PDA medium, culture it in an incubator at 25°C for 24 h, and use a 7-mm punch to punch out Aspergillus flavus mycelial cakes on the culture dish. Transfer the Aspergillus flavus mycelial cakes to PDA plates containing different concentrations of biocontrol bacterial liquid and culture them upright in an incubator at 25°C. At the same time, set up PDA without any treatment and PDA with added LB broth as controls. Measure the sizes of the perpendicular diameters of the Aspergillus flavus mycelial cakes with a vernier caliper every 24 h until 96 h.

[0084] As can be seen from Figure 3 C, there was no significant difference in the mycelial diameter of Aspergillus flavus between the CK group and the LB group at 24 h, 48 h, 72 h, and 96 h (p>0.05), thus excluding the inhibitory effect of the solvent LB broth of the antagonistic bacterium on the growth of Aspergillus flavus mycelia. After 24 h of treatment, 10 7 CFU / mL, 10 6 CFU / mL, 10 5There were significant differences between the CFU / mL group and the CK group (p < 0.05), but there were no significant differences between the treatment groups of each bacterial solution (p > 0.05). At 48 h, 72 h, and 96 h of treatment, 10 7 CFU / mL, 10 6 CFU / mL, and 10 5 CFU / mL groups had significant differences from the CK group (p < 0.05), and there were significant differences between the 10 7 CFU / mL and 10 6 CFU / mL treatment groups and 10 5 CFU / mL (p < 0.05), but there was no significant difference between the 10 7 CFU / mL group and the 10 6 CFU / mL group (p > 0.05). In summary, it can be seen that the antagonistic bacteria have a significant inhibitory effect on the growth of Aspergillus flavus hyphae, but this inhibitory effect does not increase with the increase in the concentration of the antagonistic bacteria.

[0085] (3) Inhibitory effect of antagonistic bacteria on Aspergillus flavus disease of corn and peanut

[0086] Select corn and peanuts without damage and diseases, wash them with clean water, soak them in 1% sodium hypochlorite for 3 min, then rinse them with sterile water and dry them. Soak the treated corn and peanuts in Aspergillus flavus spore solution (10 6 cells / mL) for 1 min, dry them, place a moist filter paper on the culture dish, evenly place the corn or peanuts on the filter paper, and spray the antagonistic bacteria on the experimental group. Place them in an incubator at 30 °C for 6 d. Each replicate contains 6 replicates, each replicate contains 3 parallels, and each parallel contains 7 grains of corn or peanuts.

[0087] It can be Figure 4 seen that compared with the control group (only inoculated with Aspergillus flavus spore solution), the disease severity of corn and peanuts in the treatment group (inoculated with Aspergillus flavus spore solution and Bacillus velezensis YE-1 bacterial solution) was lighter.

[0088] Since corn and peanuts are the grains most vulnerable to Aspergillus flavus contamination, the experimental results obtained with corn and peanuts as materials can reflect the antagonistic effect of Bacillus velezensis YE-1 antagonistic bacteria during grain storage.

[0089] (4) Application of antagonistic bacteria to complete swine feed

[0090] Weigh 10 g of complete swine feed, inoculate 1 mL of bacterial fermentation broth in the experimental group and 1 mL of normal saline in the control group. After treatment at 60 °C for 5 h, spray 1 mL of 10 6 cells / mL Aspergillus flavus spore solution on both the control group and the experimental group, and culture them in an incubator at 25 °C for 6 d, taking pictures and recording every 2 d.

[0091] Depend on Figure 5 It can be seen that compared with the control group (inoculated only with Aspergillus flavus spore solution), the disease severity of pigs in the treatment group (inoculated with Aspergillus flavus spore solution and Bacillus velezensis YE-1 bacterial solution) was relatively mild.

[0092] Example 4 Safety Evaluation of Bacillus velezensis YE-1 Bacterial Liquid

[0093] In this example, Kunming mice were used as experimental animals, and the safety of Bacillus velezensis YE-1 was evaluated by oral gavage. The specific method is as follows:

[0094] 1. After activation culture of Bacillus velezensis YE-1, the activated bacterial solution was centrifuged at 6000r / min for 8min, and the lower bacterial precipitate was resuspended by adding physiological saline.

[0095] 2. Kunming mice (License No. SCXK(豫)2020-0005), weighing 20±2 g, were purchased from Henan Sikebes Biotechnology Co., Ltd.

[0096] 3. Purchase 64 healthy mice, half male and half female. After acclimating to the experimental environment for 5 days, randomly divide them into 8 groups, 8 mice in each group, one blank control group for each male and one blank control group for each female, and three experimental groups, namely, low-dose group (10 7 CFU / mouse), medium dose group (10 8 CFU / mouse), high-dose group (10 9 CFU / each).

[0097] 4. The administration route is oral gavage, and the gavage volume is 20mL / Kg. The mice need to fast for 6 hours before gavage with bacterial solution, and continue to fast for 2 hours after gavage, and continue gavage for 14 days. The mice are free to drink water during feeding.

[0098] 5. Detection indicators

[0099] (1) Clinical observations: All mice showed no signs of poisoning, including abnormal gait, lethargy, hair, eyes, skin, salivation, and local lesions.

[0100] (2) Mouse weight and food intake: The weight of each mouse was weighed and recorded every day before intragastric administration of bacterial solution. The food intake of the mouse from the time of intragastric administration to the start of the next fasting was calculated and recorded. There was no significant difference in the weight and food intake of mice in each group (p>0.05). Figure 7 .

[0101] (3) Biochemical and hematological indices: After 14 days of intragastric administration of the bacterial solution, the mice were fasted overnight. The next day, blood was collected by eye enucleation. After the whole blood of the mice was left standing in a 4°C refrigerator for 4 h, it was centrifuged at 3000 r / min for 15 min in a 4°C centrifuge. The upper-layer serum was used for the determination of biochemical indices with a commercially available kit (Nanjing Jiancheng Bioengineering Institute). Blood urea nitrogen (BUN) and creatinine (CRE) were used to evaluate renal function; direct bilirubin (DBIL) and total bilirubin (TBIL) were used to evaluate liver function; total cholesterol (T-CHO), glucose (GLU), and triglyceride (TG) were used to evaluate cholesterol, blood glucose, and blood lipid levels, respectively. The whole blood of each group of mice was stored in an EDTA (K2) microcollection tube and sent to Wuhan Sevier Biotechnology Co., Ltd. as soon as possible for hematological parameter detection.

[0102] Table 4 Biochemical results of mice in each group

[0103]

[0104] As can be seen from Table 4, there were no significant differences in the indices of mice among different concentrations of the bacterial solution (p > 0.05), indicating that there were no adverse effects on liver function, renal function, cholesterol, blood glucose, and blood lipid levels.

[0105] Table 5

[0106]

[0107]

[0108] Table 6

[0109]

[0110] Table 7

[0111]

[0112]

[0113] Note: “**” in the table indicates a significant difference, p < 0.01.

[0114] Tables 5 - 7 show the hematological results of mice in each group. As can be seen from the results, there was a highly significant difference in platelets (PLT) between the control group and the treatment group of mice (p < 0.01), but this index had no dose-dependent relationship. Combining with the results of the other 16 hematological indices, which showed no significant difference (p > 0.05) between the treatment group and the control group, comprehensive analysis showed that the highly significant change in PLT had no biological or toxicological significance. In summary, different concentrations of the bacterial solution had no adverse effects on the hematological indices of each group of mice.

[0115] (4) Mouse anatomy and pathology examination: The mice were sacrificed by cervical dislocation and dissected. No abnormal edema or congestion was found in the heart, lungs, intestines, stomach, liver, thymus, and other organs. The weights of the heart, liver, spleen, lungs, and kidneys of each mouse were measured, and the organ indices were calculated. After staining the liver and kidneys, histopathological observations were made. The liver and kidney tissue cells had clear structures and regular shapes. The results are shown in Figure 8 .

[0116] Table 8 Results of organ indices of mice in each group

[0117]

[0118] As can be seen from Table 8, there were no significant differences in the organ indices between the mouse treatment group and the control group (p > 0.05), indicating that Bacillus velezensis YE-1 had no adverse effects on the mouse organs.

[0119] The embodiments described above are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A strain of Bacillus velezensis YE-1 that antagonizes Aspergillus flavus, characterized in that, The preservation number of the Bacillus velezensis is CCTCC NO: M 20221143.

2. A fungicide against Aspergillus flavus, characterized in that, Containing the Bacillus velezensis YE-1 described in claim 1.

3. The microbial agent according to claim 2, characterized in that, The active ingredient of the microbial agent includes the live cells of the Bacillus velezensis, the solid culture of the Bacillus velezensis or the liquid culture of the Bacillus velezensis.

4. Use of Bacillus velezensis YE-1 according to claim 1 or the microbial agent according to any one of claims 2-3, characterized in that, The application includes any one of the following: (1) Inhibiting the growth of Aspergillus flavus; (2) Preventing or controlling the contamination of Aspergillus flavus in food, grains or feeds.

5. The application according to claim 4, wherein The inhibition of the growth of Aspergillus flavus includes inhibiting the germination of Aspergillus flavus spores, inhibiting the elongation of Aspergillus flavus germ tubes and inhibiting the expansion of Aspergillus flavus hyphae.

6. The application according to claim 4, wherein, The grains include corn and peanuts.

7. The application of the Bacillus velezensis YE-1 described in claim 1 or the microbial agent described in any one of claims 2-3 in the preparation of a feed additive.

8. A feed additive for antagonizing Aspergillus flavus, characterized in that, Containing the Bacillus velezensis YE-1 described in claim 1.

9. The feed additive according to claim 8, characterized in that, The active ingredient of the feed additive includes the live cells of the Bacillus velezensis, the solid culture of the Bacillus velezensis or the liquid culture of the Bacillus velezensis.

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