A type of Bacillus belye and its application

Abstract

This invention discloses a strain of *Bacillus belyssus* and its applications, relating to the field of microbial technology. The *Bacillus belyssus* strain has the preservation number CGMCC No. 25990. This strain exhibits strong reproductive capacity and acid / alkali resistance, as well as high protease activity and peptide conversion rate. This invention also provides a culture medium for culturing the above-mentioned strain, comprising soybean meal, rapeseed meal, peanut meal, wheat bran, zinc sulfate solution, sodium selenite solution, potassium dihydrogen phosphate solution, dimethyl hydrogen phosphate solution, tofu whey, and corn steep liquor. The fermented product after culture is dried and made into a powder, which can be added to feed or dissolved in water for direct feeding.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a Bacillus belye and its applications. Background Technology

[0002] Animal intestinal probiotics are live bacterial preparations containing beneficial microorganisms and their metabolites, developed based on the theory of intestinal microecology. They exert their effects by maintaining the balance of the intestinal microecology. They have multiple functions, including preventing and resisting intestinal diseases in animals, enhancing the body's immunity, promoting growth, and increasing weight. Moreover, they are pollution-free, residue-free, and do not induce drug resistance.

[0003] Currently, the main microorganisms used in microecological feed additives include: Bacillus, lactic acid bacteria, yeast, actinomycetes, and photosynthetic bacteria. Bacillus is an aerobic bacterium with a strong oxygen-depleting ability. It grows rapidly in the animal's intestines, creating strong competition with pathogenic bacteria and quickly establishing an anaerobic environment that favors the growth of lactic acid bacteria. Under certain conditions, it produces spores, is resistant to high temperatures and pressure, and exhibits high stability in the acidic environment of the intestines. It can lower the intestinal pH and ammonia concentration, and produce proteases and amylases, breaking down proteins into small peptides that facilitate nutrient absorption. Simultaneously, it produces a bacillusin, which promotes growth and enhances immunity.

[0004] Chinese Patent CN202011336925.5 discloses a Bacillus belysin microbial agent and its application. The invention uses Bacillus belysin TK2019 isolated from the rhizosphere soil of cotton fields in Group 44. The microbial agent prepared using Bacillus belysin TK2019 showed an inhibition rate of 94.37% against Verticillium dahliae in cotton. This indicates that Bacillus belysin TK2019 and its microbial agent provided by the invention have the advantages of high efficiency and broad bactericidal spectrum, and have broad value for expanding the application field of microbial strains.

[0005] Chinese patent CN2022116111951.3 discloses a probiotic microecological preparation, its preparation method, and its application. The microecological preparation of this invention contains Bacillus belye BzAH-1, whose fermentation metabolites have strong stress resistance and are characterized by high temperature resistance (treatment at 100℃ for 2 hours has no effect on antibacterial activity) and long duration (antibacterial rate is maintained above 93% within 8 hours). It can effectively inhibit the growth and reproduction of Escherichia coli, thereby maintaining the balance of the animal intestinal microbial community. This Bacillus belye microecological preparation can be applied to the prevention and control of pathogens in poultry farming, improve feed digestibility and absorption, thereby enhancing animal immunity and promoting growth.

[0006] Chinese patent CN201910011966.8 discloses a method for preparing Bacillus belyss DH82 and its antimicrobial protein, and its application. The preservation number of this bacterium is CGMCC No.16865. This invention discloses a method for preparing its antimicrobial protein using Bacillus belyss DH82, and the application of the antimicrobial protein in aquaculture feed additives. Its antimicrobial protein can effectively prevent and control diseases of aquatic animals caused by aquatic pathogens, and has good application prospects.

[0007] Currently, it remains promising to develop a strain of Bacillus belysus with strong reproductive capacity, acid and alkali resistance, high protease activity, and peptide conversion rate. Summary of the Invention

[0008] The purpose of this invention is to provide a Bacillus belyssus strain. Through screening of strains, the obtained Bacillus belyssus strain has strong reproductive and acid / alkali resistance capabilities, as well as high protease activity and peptide conversion rate.

[0009] To achieve the above-mentioned objectives, the technical solution of the present invention is as follows:

[0010] On the one hand, the present invention provides a Bacillus velezensis, which has the accession number CGMCC No.25990 and was deposited at the China General Microbiological Culture Collection Center on October 28, 2022.

[0011] In another aspect, the present invention provides a culture containing Bacillus belye.

[0012] Preferably, the culture is obtained by inoculating the above-mentioned Bacillus belye into a culture medium and then culturing it.

[0013] Preferably, the culture medium is selected from one or more of solid culture medium, liquid culture medium, and semi-fluid culture medium.

[0014] More preferably, the culture medium is a semi-fluid culture medium.

[0015] More preferably, the culture medium includes soybean meal, rapeseed meal, peanut meal, wheat bran, zinc sulfate solution, sodium selenite solution, potassium dihydrogen phosphate solution, dimethyl hydrogen phosphate solution, tofu whey, and corn steep liquor.

[0016] Preferably, the preparation of the culture medium includes the following steps:

[0017] (1) Crush soybean meal, rapeseed meal, peanut meal and wheat bran, weigh soybean meal, rapeseed meal, peanut meal and wheat bran according to weight, mix them evenly to obtain mixed powder.

[0018] (2) Prepare solutions of zinc sulfate, sodium selenite, potassium dihydrogen phosphate and dimethyl hydrogen phosphate respectively.

[0019] (3) Measure out the tofu yellow liquid, corn milk, zinc sulfate solution, sodium selenite solution, potassium dihydrogen phosphate solution, and dipotassium hydrogen phosphate solution, mix them evenly, and prepare a mixed solution.

[0020] (4) Pour the mixed solution into the mixed powder, stir evenly to make a semi-fluid culture medium, put it into a fermenter, sterilize, cool to room temperature, and set aside.

[0021] In some embodiments of the present invention, the preparation of the culture medium includes the following steps:

[0022] (1) Grind soybean meal, rapeseed meal, peanut meal and wheat bran to 40-100 mesh. Weigh 30-80g of soybean meal, 10-50g of rapeseed meal, 10-40g of peanut meal and 5-30g of wheat bran, mix them evenly and obtain mixed powder.

[0023] (2) Prepare solutions of zinc sulfate with a mass fraction of 10-30%, sodium selenite with a mass fraction of 10-30%, potassium dihydrogen phosphate with a mass fraction of 5-10%, and dimethyl hydrogen phosphate with a mass fraction of 5-10%.

[0024] (3) Measure 50-150mL of tofu yellow liquid, 10-50mL of corn liquor, 1-10mL of zinc sulfate solution, 1-10mL of sodium selenite solution, 1-10mL of potassium dihydrogen phosphate solution, 1-10mL of dipotassium hydrogen phosphate solution, and 100-1000mL of water, mix them evenly, and prepare a mixed solution.

[0025] (4) Pour the mixed solution into the mixed powder, stir evenly to make a semi-fluid culture medium, put it into a fermenter, sterilize at 105-121℃ for 10-30 minutes, cool to room temperature, and use it for later use.

[0026] Specifically, the mass fraction mentioned above refers to the ratio of the mass of the solute in the solution to the mass of the solution.

[0027] Preferably, the culture temperature is 32-37℃ and the culture time is 48-96 hours.

[0028] More preferably, the culture environment is a closed system with strong ventilation, and the fermentation method is stirring fermentation.

[0029] Preferably, the viable count of Bacillus belye in the culture is not less than 9 × 10⁻⁶. 10 CFU / g.

[0030] In another aspect, the present invention provides a microbial inoculant.

[0031] Preferably, the microbial agent includes the aforementioned Bacillus berberis or its culture.

[0032] Preferably, the microbial agent is a liquid preparation or a powder.

[0033] More preferably, the microbial agent is a powder.

[0034] In another aspect, the present invention provides the application of the above-mentioned microbial agents as additives in the preparation of feed.

[0035] In another aspect, the present invention provides a feed containing the aforementioned microbial agent.

[0036] The beneficial effects of this invention are as follows:

[0037] (1) The present invention obtained a strain of Bacillus belysin from nature. This strain of Bacillus belysin has strong reproductive ability and acid and alkali resistance, high protease activity and peptide conversion rate, can prevent and resist animal intestinal pathogens, can decompose plant protein into small peptide substances, and can produce bacillusin.

[0038] (2) The present invention also provides a culture medium for culturing the above-mentioned Bacillus belye, the culture medium comprising soybean meal, rapeseed meal, peanut meal, wheat bran, zinc sulfate solution, sodium selenite solution, potassium dihydrogen phosphate solution, dimethyl hydrogen phosphate solution, tofu yellow liquid and corn steep liquor, the cultured fermented product is dried into powder, which can be added to feed or dissolved in water for direct feeding.

[0039] Preservation Instructions

[0040] Chinese scientific name: Bacillus belye;

[0041] Latin name: Bacillus velezensis;

[0042] Strain number: MH-Bs-051;

[0043] Accession number: CGMCC No. 25990;

[0044] Deposit date: October 28, 2022;

[0045] Preservation institution: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Cultures;

[0046] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Detailed Implementation

[0047] To make the technical means, creative features, and achieved objectives and effects of this invention easier to understand, the invention is further illustrated below with specific embodiments. However, the following embodiments are merely preferred embodiments of this invention and not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments described herein without creative effort are all within the protection scope of this invention. Unless otherwise specified, the operating methods and equipment used in the following embodiments are conventional operating methods, and the materials and equipment used in each embodiment are the same.

[0048] Example 1

[0049] 1. Obtaining Bacillus belyssus strains

[0050] Samples of silage and potato starch wastewater were collected, and 586 superior strains were screened using the plate separation method. After repeated primary and secondary screening, a strain with fast growth and reproduction, strong acid and alkali resistance, high protease activity, and excellent performance was finally selected, namely Bacillus belyss MH-Bs-051.

[0051] The above-mentioned strain underwent morphological and physiological / biochemical identification, as well as molecular genetic identification. A BLAST comparison with the 16S rDNA submitted to GenBank confirmed its classification as belonging to the genus *Bacillus*. Phylogenetic tree construction using MEGA 6.0 software showed that this strain shared 99.9% homology with the 16S rDNA sequence of *Bacillus velezensis*, consistent with the physiological and biochemical results. Therefore, this strain was identified as *Bacillus velezensis*, with the strain number MH-Bs-051.

[0052] The strain MH-Bs-051 was sent to the Microbial Culture Collection Center of the Beijing Institute of Microbiology, Chinese Academy of Sciences for identification, and the identification results were completely consistent. This strain MH-Bs-051 has been deposited by the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 25990.

[0053] The detection of Bacillus belyssus MH-Bs-051 obtained by the present invention includes the following four aspects.

[0054] ①Growth rate measurement

[0055] Experimental methods: Beef extract peptone medium, pH 7.0±0.5, inoculum size 0.5%, culture temperature 36±0.5℃. Sampling began at the 8th hour and continued every two hours. Viable cell counts were performed using the plate count method, expressed as an exponential form (1.00×10⁻⁶). x(units / mL).

[0056] The experimental results are shown in the table below:

[0057] Table 1. Growth rate of Bacillus belyss MH-Bs-051

[0058]

[0059] Comparative Example 1: The viable count of Bacillus belyss JT3-2 (48 hours) was less than 2 × 10⁻⁶. 9 CFU / mL. (Reference: Study on high-density culture and preparation of inoculum of Bacillus belyssus JT3-2 and Lactobacillus plantarum FBL-3a).

[0060] Comparative Example 2: The highest viable count of Bacillus belyss J-4 (24.15 hours) was 3.52 × 10⁻⁶. 9 CFU / mL (Reference: Study on the biocontrol effect and growth-promoting effect of Bacillus berreatus J-4 on peach root rot).

[0061] Comparative Example 3: The viable count of Bacillus belyceta var. dY-6 (24 hours) was less than 1.5 × 10⁻⁶. 9 CFU / mL (Reference: Study on the antibacterial properties of Bacillus belyssus DY-6 and its effects on sea cucumber growth immunity and pond microbial community structure).

[0062] The above results indicate that the average viable count of Bacillus belyssus MH-Bs-051 in this application was 8.98 × 10⁻⁶ over 24 hours. 10 The CFU / g count was still higher than that of Comparative Example 1 after 48 hours of culture and Comparative Example 2 after 24.15 hours of culture, and also higher than that of Comparative Example 3, indicating that the Bacillus belyssus MH-Bs-051 of this application has a faster growth rate.

[0063] ②Acid and alkali resistance

[0064] Experimental methods: Beef extract peptone medium, inoculation amount 0.5%, culture temperature 36±0.5℃, samples were taken after 20 hours of culture, and the viable cell count was performed using the plate count method, expressed in exponential form (1.00×10⁻⁶). x (units / mL).

[0065] The experimental results are shown in the table below:

[0066] Table 2. Acid and alkali resistance of Bacillus belyss MH-Bs-051

[0067]

[0068] The survival rate of Bacillus belyssus MH-Bs-051 in this application was 95.1% at pH values ​​ranging from 7.5 to 4.5.

[0069] Comparative Example 4: The survival rate of Bacillus belyss HF-14109 was 88.89% when the pH changed from 7 to 5 (Reference: Isolation and screening of fish-derived enzyme-producing Bacillus belyss and its beneficial effects on carp).

[0070] The above results show that the pH decrease of Bacillus belyss MH-Bs-051 of the present invention is greater than that of Comparative Example 4, and the pH is lower than that of Comparative Example 4 (4.5 < 5), but its survival rate is still much higher than that of Comparative Example 4, indicating that Bacillus belyss of the present invention has better acid and alkali resistance.

[0071] ③ Protease activity assay

[0072] Experimental methods: Prepare soybean protein culture medium (weigh 15g soybean protein isolate, adjust the volume to 100mL), concentration 15.00% (g / L), pH 7.0±0.5, sterilize at 121℃, inoculum size 1.0%, shaker speed 150rpm, culture temperature 36±0.5℃, take samples at 20 hours of culture, and take samples every 8 hours. Determine the neutral protease activity (U / mL) using the Folin method (GB / T23527-2009).

[0073] The experimental results are shown in the table below:

[0074] Table 3. Protease activity of Bacillus belyssus MH-Bs-051

[0075] Enzyme activity (U) 20h 28h 36h 44h 52h 60h 1 1533.25 2863.45 3762.56 3743.36 3706.45 3688.66 2 1545.62 2864.65 3725.33 3782.15 3710.12 3697.23 3 1568.36 2798.66 3821.65 3746.22 3689.78 3686.45 average 1549.08 2842.25 3769.85 3757.24 3702.12 3690.78

[0076] Comparative Example 5: Bacillus belye LfF-1 (accession number: GDMCC No:60741), the method for determining its protease activity was the same as above.

[0077] Table 4. Protease activity of Bacillus belyssus LfF-1

[0078] Enzyme activity (U) 20h 28h 36h 44h 52h 60h Comparative Example 5 1514.42 2773.97 3679.00 3642.34 3601.53 3485.26

[0079] As can be seen from the results in Tables 3-4 above, compared with Bacillus belysinus of Comparative Example 5, Bacillus belysinus MH-Bs-051 of the present invention has higher neutral protease activity.

[0080] ④ Peptide conversion rate determination

[0081] Experimental methods: Prepare defatted soybean meal culture medium (weigh 15g defatted soybean meal, make up to 100mL), concentration 15.00% (g / L), pH 7.0±0.5, sterilize at 121℃, inoculum size 1.0%, shaker speed 150rpm, culture temperature 36±0.5℃, take samples at 36 hours, and take samples every 4 hours. Peptide conversion rate is determined according to the method of "Soybean Peptide Powder" GB / 22492-2008.

[0082] The experimental results are shown in the table below:

[0083] Table 5. Peptide conversion rate of Bacillus belyssus MH-Bs-051

[0084] Peptide conversion rate % 36h 40h 44h 48h 52h 1 78.8 89.6 95.3 94.7 95.1 2 78.6 88.7 94.5 95.2 95.5 3 78.2 90.5 94.6 95.6 95.4 average 78.5 89.6 94.8 95.2 95.3

[0085] Example 2: Preparation of Bacillus belye powder

[0086] Experimental steps:

[0087] 1. Grind soybean meal, rapeseed meal, peanut meal, and wheat bran to 80 mesh. Weigh 30.0g of soybean meal, 10.0g of rapeseed meal, 15.0g of peanut meal, and 10.0g of wheat bran, mix them evenly, and obtain a mixed powder.

[0088] 2. Prepare solutions of zinc sulfate, sodium selenite, potassium dihydrogen phosphate, and dimethyl hydrogen phosphate, respectively: ZnSO4 20.0%, Na2SeO3 20.0%, KH2PO4 10.0%, and K2HPO4 10.0%.

[0089] 3. Measure 50 mL of tofu whey, 10 mL of corn syrup, 1.0 mL of zinc sulfate solution, 1.0 mL of sodium selenite solution, 2.0 mL of potassium dihydrogen phosphate solution, 2.0 mL of dipotassium hydrogen phosphate solution, and 150 mL of water, mix them thoroughly to obtain a mixed solution.

[0090] 4. Pour the mixed solution into the mixed powder, stir well to make a semi-fluid culture medium, put it into a fermenter, sterilize at 121℃ for 20 minutes, cool to room temperature, and set aside.

[0091] 5. Dilute the above culture medium twice with sterile water, place it in an Erlenmeyer flask, inoculate the pre-cultured MH-Bs-051 test tube slant culture, place it in a constant temperature shaker at 35℃ and a rotation speed of 120 rpm for 24 hours to obtain a mature liquid seed.

[0092] 6. Inoculate the matured MH-Bs-051 liquid seed at 5% into the sterilized semi-fluid culture medium described above. Control the temperature at 35°C, introduce sterile air at a ventilation ratio of 1.0:1.5, turn on the stirrer, and stir at 80 rpm for 72 hours.

[0093] 7. After fermentation, place the fermented material in a dryer and dry it at 90℃ until the moisture content is ≤13.0%. Then pulverize it to make powder, which can be added to other animal and pet feeds or dissolved directly in water for feeding.

[0094] Example 3: Preparation of Bacillus belye powder

[0095] 1. Grind soybean meal, rapeseed meal, peanut meal, and wheat bran to 60 mesh. Weigh 40.0g of soybean meal, 10.0g of rapeseed meal, 20.0g of peanut meal, and 15.0g of wheat bran, mix them evenly, and obtain a mixed powder.

[0096] 2. Prepare solutions of zinc sulfate, sodium selenite, potassium dihydrogen phosphate, and dimethyl hydrogen phosphate, respectively: ZnSO4 25.0%, Na2SeO3 25.0%, KH2PO4 10.0%, and K2HPO4 10.0%.

[0097] 3. Measure 80mL of tofu whey, 15mL of corn syrup, 1.5mL of zinc sulfate solution, 1.5mL of sodium selenite solution, 2.5mL of potassium dihydrogen phosphate solution, 2.5mL of dipotassium hydrogen phosphate solution, and 200mL of water, mix them evenly to obtain a mixed solution.

[0098] 4. Pour the mixed solution into the mixed powder, stir well to make a semi-fluid culture medium, put it into a fermenter, sterilize at 121℃ for 20 minutes, cool to room temperature, and set aside.

[0099] 5. Dilute the above culture medium with sterile water by 1:1, place it in an Erlenmeyer flask, inoculate the MH-Bs-051 test tube slant culture, place it in a constant temperature shaker at 36℃ and a rotation speed of 120 rpm for 24 hours to obtain a mature liquid seed.

[0100] 6. Inoculate the matured MH-Bs-051 liquid seed at 6% into the sterilized semi-fluid culture medium described above. Maintain the temperature at 36°C, introduce sterile air at a ventilation ratio of 1.0:2.0, turn on the stirrer, and stir at 100 rpm for 72 hours.

[0101] 7. After fermentation, place the fermented material in a dryer and dry it at 95℃ until the moisture content is ≤13.0%. Then pulverize it to make powder, which can be added to other animal and pet feeds or dissolved directly in water for feeding.

[0102] Example 4: Preparation of Bacillus vesiculosus powder

[0103] 1. Grind soybean meal, rapeseed meal, peanut meal, and wheat bran to 100 mesh. Weigh 45.0g of soybean meal, 15.0g of rapeseed meal, 25.0g of peanut meal, and 15.0g of wheat bran, mix them evenly, and obtain a mixed powder.

[0104] 2. Prepare solutions of zinc sulfate, sodium selenite, potassium dihydrogen phosphate, and dimethyl hydrogen phosphate, respectively, with ZnSO4 30.0%, Na2SeO3 30.0%, KH2PO4 10.0%, and K2HPO4 10.0%.

[0105] 3. Measure 150mL of tofu whey, 15mL of corn whey, 3.0mL of zinc sulfate solution, 3.0mL of sodium selenite solution, 2.5mL of potassium dihydrogen phosphate solution, 2.0mL of dipotassium hydrogen phosphate solution, and 300mL of water, mix them evenly to obtain a mixed solution.

[0106] 4. Pour the mixed solution into the mixed powder, stir well to make a semi-fluid culture medium, put it into a fermenter, sterilize at 105℃ for 30 minutes, cool to room temperature, and set aside.

[0107] 5. Dilute the above culture medium with sterile water three times, place it in an Erlenmeyer flask, inoculate the MH-Bs-051 test tube slant culture, place it in a constant temperature shaker at 37℃ and a rotation speed of 110 rpm for 18 hours to obtain a mature liquid seed.

[0108] 6. Inoculate the matured MH-Bs-051 liquid seed at 5% into the sterilized semi-fluid culture medium described above. Control the temperature at 37°C, introduce sterile air at a ventilation ratio of 1.0:2.5, turn on the stirrer, and stir at 100 rpm for 60 hours.

[0109] 7. After fermentation, place the fermented material in a dryer and dry it at 95℃ until the moisture content is ≤13.0%. Then pulverize it to make powder, which can be added to other animal and pet feeds or dissolved directly in water for feeding.

[0110] Example 5: Preparation of Bacillus belye powder

[0111] 1. Grind soybean meal, rapeseed meal, peanut meal, and wheat bran to 80 mesh. Weigh 50.0g of soybean meal, 15.0g of rapeseed meal, 25.0g of peanut meal, and 20.0g of wheat bran, mix them evenly, and obtain a mixed powder.

[0112] 2. Prepare solutions of zinc sulfate, sodium selenite, potassium dihydrogen phosphate, and dimethyl hydrogen phosphate, respectively, with ZnSO4 30.0%, Na2SeO3 30.0%, KH2PO4 10.0%, and K2HPO4 10.0%.

[0113] 3. Measure 150mL of tofu whey, 10mL of corn syrup, 5.0mL of zinc sulfate solution, 5.0mL of sodium selenite solution, 3.0mL of potassium dihydrogen phosphate solution, 3.0mL of dipotassium hydrogen phosphate solution, and 350mL of water, mix them evenly to obtain a mixed solution.

[0114] 4. Pour the mixed solution into the mixed powder, stir well to make a semi-fluid culture medium, put it into a fermenter, sterilize at 121℃ for 30 minutes, cool to room temperature, and set aside.

[0115] 5. Dilute the above culture medium with sterile water three times, place it in an Erlenmeyer flask, inoculate the MH-Bs-051 test tube slant culture, place it in a constant temperature shaker at 37℃ and a rotation speed of 110 rpm for 20 hours to obtain a mature liquid seed.

[0116] 6. Inoculate the matured MH-Bs-051 liquid seed at 10% into the sterilized semi-fluid culture medium described above. Control the temperature at 37°C, introduce sterile air at a ventilation ratio of 1.0:2.5, turn on the stirrer, and stir at 100 rpm for 65 hours.

[0117] 7. After fermentation, place the fermented material in a dryer and dry it at 80℃ until the moisture content is ≤13.0%. Then pulverize it to make powder, which can be added to other animal and pet feeds or dissolved directly in water for feeding.

[0118] Example 6: Preparation of Bacillus belye powder

[0119] 1. Grind soybean meal, rapeseed meal, peanut meal, and wheat bran to 70 mesh. Weigh 60.0g of soybean meal, 10.0g of rapeseed meal, 25.0g of peanut meal, and 20.0g of wheat bran, mix them evenly, and obtain a mixed powder.

[0120] 2. Prepare solutions of zinc sulfate, sodium selenite, potassium dihydrogen phosphate, and dimethyl hydrogen phosphate, respectively, with ZnSO4 30.0%, Na2SeO3 30.0%, KH2PO4 10.0%, and K2HPO4 10.0%.

[0121] 3. Measure 120mL of tofu whey, 15mL of corn syrup, 6.0mL of zinc sulfate solution, 6.0mL of sodium selenite solution, 3.0mL of potassium dihydrogen phosphate solution, 3.0mL of dipotassium hydrogen phosphate solution, and 450mL of water, mix them evenly to obtain a mixed solution.

[0122] 4. Pour the mixed solution into the mixed powder, stir well to make a semi-fluid culture medium, put it into a fermenter, sterilize at 121℃ for 30 minutes, cool to room temperature, and set aside.

[0123] 5. Dilute the above culture medium with sterile water three times, place it in an Erlenmeyer flask, inoculate the MH-Bs-051 test tube slant culture, place it in a constant temperature shaker at 37℃ and a rotation speed of 110 rpm for 16 hours to obtain a mature liquid seed.

[0124] 6. Inoculate the matured MH-Bs-051 liquid seed at 8% into the sterilized semi-fluid culture medium described above. Maintain the temperature at 37°C, introduce sterile air at a ventilation ratio of 1.0:2.0, turn on the stirrer, and stir at 100 rpm for 72 hours.

[0125] 7. After fermentation, place the fermented material in a dryer and dry it at 85℃ until the moisture content is ≤13.0%. Then pulverize it to make powder, which can be added to other animal and pet feeds or dissolved directly in water for feeding.

[0126] 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 principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A strain of Bacillus velezensis, characterized in that, It is preserved in the China General Microbiological Culture Collection Center on October 28, 2022, and its preservation number is CGMCC No. 25990.

2. A culture comprising Bacillus velezensis, characterized in that, The bacillus velezensis of claim 1 is inoculated into a culture medium for culture.

3. The culture of claim 2, wherein, The culture medium comprises soybean meal, rapeseed meal, peanut meal, bran, zinc sulfate solution, sodium selenite solution, potassium dihydrogen phosphate solution, dimethyl hydrogen phosphate solution, tofu yellow slurry and corn slurry.

4. The culture of claim 3, wherein, The preparation of the culture medium comprises the following steps: (1) The soybean meal, rapeseed meal, peanut meal and bran are crushed, and 30-80g of soybean meal, 10-50g of rapeseed meal, 10-40g of peanut meal and 5-30g of bran are weighed and mixed to prepare a mixed powder; (2) 50-150mL of tofu yellow slurry, 10-50mL of corn slurry, 1-10mL of zinc sulfate solution, 1-10mL of sodium selenite solution, 1-10mL of potassium dihydrogen phosphate solution, 1-10mL of dimethyl hydrogen phosphate solution and 100-1000mL of water are measured and mixed to prepare a mixed solution; (3) Pour the mixed solution into the mixed powder, mix well to prepare a semi-liquid culture medium, and sterilize in a fermentation tank.

5. The culture of claim 4, wherein, The mass fraction of zinc sulfate is 10-30%, the mass fraction of sodium selenite is 10-30%, the mass fraction of potassium dihydrogen phosphate is 5-10%, and the mass fraction of dimethyl hydrogen phosphate solution is 5-10%.

6. The culture of claim 2, wherein, The culture temperature is 32-37℃, and the culture time is 48-96 hours.

7. A microbial inoculant, characterized in that, The microbial agent comprises the bacillus velezensis of claim 1 or the culture of any one of claims 2-6.

8. The microbial inoculant of claim 7, wherein, The microbial agent is a liquid preparation or a powder.

9. The microbial inoculant of claim 8, wherein, The microbial agent is a powder.

10. Use of the microbial agent of claim 7 as an additive in the preparation of feed.

11. A feed, characterized in that, The feed comprises the microbial agent of claim 7.

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