Bacillus subtilis, bacterial agent and preparation method thereof

Abstract

The application discloses Bacillus subtilis BS-56, a bacterial agent and a preparation method thereof. The preservation number of the Bacillus subtilis BS-56 is GDMCC No: 62324. The application further discloses a bacterial agent containing the Bacillus subtilis and a preparation method thereof. The fermentation production level of the Bacillus subtilis is increased by more than 40% compared with a starting strain, and the survival rate (recovery rate) of the Bacillus subtilis spores of the Bacillus subtilis bacterial agent product is still greater than or equal to 90% after being preserved for one year under commodity shelf conditions.

Description

Technical Field

[0001] This invention belongs to the field of microbiology, specifically relating to Bacillus subtilis, its inoculant, and its preparation method. Background Technology

[0002] The technology of preparing microbial agents and fertilizers using natural microorganisms is a green and environmentally friendly biological treatment technology that rapidly developed internationally in the mid-to-late 20th century. Through the integrated application of multiple disciplines, it utilizes mixed microbial communities in a specific environment to decompose multiphase organic matter, fermenting and maturing solid organic waste into humic acid. This technology is used for fertilizing and improving soil, and is economical, practical, and does not produce secondary pollution, thus attracting widespread attention. However, because the preparation of microbial agents and fertilizers seems simple, many individual farmers and small and micro-sized enterprises in China are currently mass-producing them. Generally, the microbial strains and raw materials are not screened or conditioned during the production process; they are directly piled up for fermentation. The result is a long fermentation cycle, low production efficiency, a large number of contaminating microorganisms (including harmful pathogens), unclear target beneficial bacteria, and low survival rates of target beneficial bacteria during use or on the shelf, seriously affecting product quality and user safety.

[0003] Bacillus subtilis is a common soil bacterium characterized by rapid and extensive reproduction and colonization within plants and soil, achieving antibacterial and disease-controlling effects on crops. Furthermore, it exhibits rapid growth, strong resistance to adverse conditions, and does not produce toxins, making it a non-pathogenic and safe microorganism. Therefore, targeted cultivation of bacteria that are resistant to adverse environments, readily form spores, and can thrive under suitable external conditions or with germination agents (such as L-alanine, Mn...) is crucial. 2+ Highly active Bacillus subtilis that rapidly germinates into reproductive cells (generally within a few minutes) in the presence of (MnSO4), surfactants (such as n-dodecylamine), and glucose, is essential for the development of modern and green agriculture. Summary of the Invention

[0004] To overcome the shortcomings of existing microbial agents and fertilizers, such as long fermentation cycles, low production efficiency, large numbers of contaminating bacteria (including harmful pathogens), unclear target beneficial bacteria, and low survival rates of the target beneficial bacteria during use or shelf life, this invention provides Bacillus subtilis, its inoculum, and its preparation method. This invention utilizes a pure-strain fermentation technique to proliferate the target beneficial bacteria, employing targeted selection pressure screening and fermentation. The selected Bacillus subtilis is not antagonized by the metabolites of Bacillus licheniformis or Paecilomyces lilacinus. When applied to the soil, it is more beneficial for killing harmful bacteria, increasing crop resistance, fixing nitrogen, promoting plant growth, improving crop quality, and achieving high yields. The Bacillus subtilis provided by this invention has a spore recovery survival rate of ≥90% after one year of preservation, demonstrating significant advantages, safe use, simple production process, ease of operation, energy saving, and emission reduction. It provides a highly efficient and environmentally friendly new type of microbial agent and fertilizer for the production of green and healthy agricultural products.

[0005] To address the aforementioned technical problems, the first aspect of this invention provides a Bacillus subtilis BS-56, the Bacillus subtilis BS-56 having the accession number GDMCC No:62324.

[0006] To solve the above-mentioned technical problems, the second aspect of the present invention provides a fermentation product obtained by fermentation using Bacillus subtilis BS-56 as described in the first aspect.

[0007] To address the aforementioned technical problems, the third aspect of this invention provides the application of Bacillus subtilis BS-56 as described in the first aspect and the fermentation products as described in the second aspect in microbial fertilizers.

[0008] To address the aforementioned technical problems, a fourth aspect of the present invention provides a microbial fertilizer comprising the aforementioned Bacillus subtilis BS-56 and / or the fermentation products described in the second aspect.

[0009] Optionally, the microbial fertilizer includes any one of microbial inoculants, compound microbial fertilizers, and bio-organic fertilizers.

[0010] Optionally, the content of Bacillus subtilis BS-56 in the bacterial agent is ≥100 billion CFU / g, for example, 2000 to 400 billion CFU / g.

[0011] To address the aforementioned technical problems, a fifth aspect of the present invention provides a method for preparing the microbial fertilizer as described in the fourth aspect of the present invention, the preparation method comprising the following steps:

[0012] The seed culture of Bacillus subtilis BS-56 was inoculated into a fermentation medium for fermentation culture to obtain a fermentation broth. The fermentation broth was then dried to obtain the microbial fertilizer.

[0013] Optionally, the culture medium used to prepare the seed solution comprises:

[0014]

[0015] Optionally, the culture medium used to prepare the seed solution has a pH of 6.5-7.5.

[0016] Optionally, the culture conditions for preparing the seed solution satisfy at least one of the following:

[0017] The rotation speed is 200-300 rpm;

[0018] The temperature is 30-37℃;

[0019] The culture period is 36-60 hours;

[0020] The ventilation rate is (0.8-1.2): 1v / vm.

[0021] Optionally, the fermentation medium comprises:

[0022]

[0023]

[0024] Optionally, the fermentation culture medium used has a pH of 6.5-7.5.

[0025] Optionally, the fermentation conditions satisfy at least one of the following:

[0026] The temperature is 32-36℃;

[0027] The cycle is 64-96 hours;

[0028] Reducing sugar content: 0.8-1.2%;

[0029] Dissolved oxygen 50-70%;

[0030] Control the pH to 7.0-7.7;

[0031] The inoculation rate is 5-15%.

[0032] To address the aforementioned technical problems, the sixth aspect of this invention provides the application of Bacillus subtilis BS-56 as described in the first aspect, the fermentation product as described in the second aspect, the microbial fertilizer as described in the fourth aspect, and the microbial fertilizer obtained according to the method described in the fifth aspect in the preparation of products that promote plant growth.

[0033] To address the aforementioned technical problems, the seventh aspect of this invention provides the application of Bacillus subtilis BS-56 as described in the first aspect, the fermentation product as described in the second aspect, the microbial fertilizer as described in the fourth aspect, and the microbial fertilizer obtained according to the method described in the fifth aspect in promoting plant growth.

[0034] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0035] The reagents and raw materials used in this invention are all commercially available.

[0036] The positive and progressive effects of this invention are as follows:

[0037] 1. Obtain a stable strain of Bacillus subtilis BS-56 that is unaffected by the fermentation metabolites of Bacillus subtilis, Bacillus licheniformis, and Paecilomyces lilacinus, and is resistant to high temperatures, acids, and alkalis. This strain is beneficial for forming a dominant symbiotic community with Bacillus licheniformis and Paecilomyces lilacinus, and has good application potential in the preparation of compound microbial agents. This will help to rapidly and directionally create a favorable microecological environment for plant growth, inhibiting and suppressing the growth and reproduction of pathogenic microorganisms, decomposing and degrading chemical substances in the soil, increasing soil aggregate structure, and increasing soil permeability. This favorable microecological environment will produce a large number of antibacterial and bactericidal substances, preventing soil-borne diseases, reducing plant diseases, promoting plant growth, being environmentally friendly, increasing yield, and reducing carbon emissions.

[0038] 2. The obtained Bacillus subtilis BS-56 strain has high production activity, and the number of colonies produced by deep fermentation is more than 40% higher than that produced by the control strain Bacillus subtilis CMCC63501.

[0039] 3. The obtained Bacillus subtilis produces products with a high spore recovery rate. Under shelf conditions, the spore survival rate (recovery rate) can reach 94% after one year of storage.

[0040] Information on the preservation of biological materials

[0041] The Bacillus subtilis BS-56 of this invention was deposited on March 25, 2022, at the Guangdong Provincial Microbial Culture Collection Center (Institute of Microbiology, Guangdong Academy of Sciences) (GDMCC). The deposit address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, 510070, China. The deposit number is GDMCC No:62324. The culture name is BS-56, and the classification name is Bacillus subtilis. Detailed Implementation

[0042] For the purposes of this invention, unless otherwise stated, the terms used in this application have the following meanings:

[0043] The term "submerged fermentation" refers to a fermentation method in which microorganisms are inoculated into a liquid fermentation medium, sterile air is continuously introduced, and the medium is stirred.

[0044] The term "microbial fertilizer" refers to products containing specific living microorganisms that are applied to agricultural production. Through the life activities of the microorganisms they contain, the supply of nutrients to plants is increased or plant growth is promoted, yields are increased, and the quality of agricultural products and the agricultural ecological environment are improved. Currently, microbial fertilizers include microbial inoculants (bacterial agents), compound microbial fertilizers, and bio-organic fertilizers.

[0045] Among them, microbial inoculants (bacterial agents) refer to live microbial products made by industrially producing and proliferating one or more target microorganisms, or by concentration, drying, or adsorption via a carrier, including:

[0046] Single-strain inoculant: A microbial inoculant made from a single microbial strain;

[0047] Compound microbial agents: microbial inoculants made from two or more non-antagonistic microbial strains;

[0048] Organic material composting microbial agent: a microbial inoculant that can accelerate the decomposition and composting of various organic materials (including crop straw, livestock and poultry manure, domestic waste and urban sludge, etc.);

[0049] Bioremediation agents: Microbial inoculants that can reduce the concentration of harmful substances in the environment, decrease their toxicity, or render them harmless through the growth and metabolic activities of microorganisms.

[0050] Compound microbial fertilizer refers to a live microbial product made by combining the target microorganisms with nutrients after industrial production and proliferation.

[0051] Bio-organic fertilizer refers to a live microbial product made by combining the target microorganisms with organic materials mainly derived from animal and plant residues (such as livestock and poultry manure, crop straw, etc.) that have undergone harmless treatment.

[0052] The term "CFU" (colony forming units) refers to each colony formed on an agar plate after incubation at a specific temperature and time.

[0053] In some embodiments, the bacterial agent provided in this application contains Bacillus subtilis BS-56 with a content of ≥100 billion CFU / g, preferably ≥150 billion CFU / g, for example, 2000-4000 billion CFU / g or 2300-4000 billion CFU / g.

[0054] In some embodiments, the method for preparing the microbial agent provided in this application includes the following steps:

[0055] The seed culture of Bacillus subtilis BS-56 was inoculated into a fermentation medium for fermentation culture to obtain a fermentation broth. The fermentation broth was then dried to obtain the bacterial agent.

[0056] Preferably, the viable count of the seed liquid is ≥3 billion CFU / ml; more preferably, the viable count is ≥3.5 billion CFU / ml, for example, 3.5-5 billion CFU / ml.

[0057] Preferably, the colony count of the fermentation broth is ≥15 billion CFU / ml; more preferably, the colony count is ≥20 billion CFU / ml, for example, 25-30 billion CFU / ml.

[0058] The following describes, in conjunction with the specific operating method of the present invention, a strain of Bacillus subtilis that is not antagonized by the fermentation metabolites of Bacillus licheniformis and Paecilomyces lilacinus and is resistant to high temperature, acid and alkali, as well as the industrial production and application of this strain. This further elaborates on the present invention. The provided examples are only for further elaboration of the present invention and do not constitute any limitation on the scope of the present invention.

[0059] Example 1: Preparation of Metabolite Concentrate Mixture

[0060] 1. Preparation of concentrated broth from the deep fermentation of Paecilomyces lilacinus:

[0061] Paecilomyces lilacinus CICC 40276 was submerged until the number of viable spores in the fermentation broth reached 3 billion CFU / ml. The fermentation broth was then filtered through a ceramic membrane, and the filtrate was concentrated 6 times. The concentrated fermentation broth was stored in a refrigerator at 4℃-6℃.

[0062] 2. Preparation of concentrated Bacillus licheniformis fermentation broth:

[0063] The number of viable spores in the deep fermentation broth of Bacillus licheniformis CICC 10037 was 12 billion CFU / ml. The fermentation broth was filtered through a ceramic membrane, the filtrate was concentrated 6 times, and the concentrated fermentation broth was stored in a refrigerator at 4℃-6℃.

[0064] 3. Preparation of concentrated Bacillus subtilis deep fermentation broth:

[0065] Bacillus subtilis CMCC 63501 was submerged until the number of viable spores in the fermentation broth reached 15 billion CFU / ml; the fermentation broth was filtered through a ceramic membrane, the filtrate was concentrated 6 times, and the fermentation concentrate was stored in a refrigerator at 4℃-6℃.

[0066] 4. Mix the three fermentation concentrates in equal proportions to obtain a mixture of fermentation concentrates rich in the fermentation metabolites of the three bacteria. Filter the mixture through a sterile filter under aseptic conditions to obtain a mixed fermentation concentrate.

[0067] Example 2: Selection of Dominant Bacillus subtilis Strains

[0068] 1. Using Bacillus subtilis CMCC 63501 as the starting strain, a dominant Bacillus subtilis strain that is not antagonized by the mixture of metabolite concentrates prepared in Example 1 and can grow normally under high-load pressure enrichment environment of fermentation metabolites was selected.

[0069] 1.1 Preparation of Bacillus subtilis CMCC 63501 slant culture: Bacillus subtilis CMCC 63501 was inoculated into slant culture and cultured at 35℃ for 36h.

[0070] 1.2 Preparation of Bacillus subtilis CMCC 63501 slant culture: Take 5 ml of the above-mentioned metabolite concentrate mixture, transfer it to the above-mentioned mature slant, scrape off the bacterial growth and colonies, and fully disperse the bacterial growth into single colonies;

[0071] 1.3 Preparation of thermostable bacterial culture: Take 1 ml of the above slant bacterial culture and dilute it with the metabolite concentrate mixture to a concentration of 10. -1 The dilution was determined and the sample was stored in a 70°C water bath for 30 minutes.

[0072] 1.4. Plate isolation and selection of strains:

[0073] Placed in a 70℃ constant temperature water bath for 10 -1Samples were taken from test tubes at 0, 5, 10, 15, 20, 25, and 30 minutes, and diluted with the metabolite concentrate mixture to a concentration of 10. -2 Up to 10 -9 Samples at dilution levels. Take samples at dilution levels of 10... -3 Up to 10 -9 Spread 1 ml of the sample onto a double-plate agar plate and incubate the plate at 35°C for 36 h.

[0074] The selection of temperature-resistant and antagonistic-resistant plate colonies is shown in Table 1:

[0075] Table 1. Selection of temperature-resistant and antagonistic plate colonies.

[0076]

[0077] 1.5 Selection of acid- and alkali-resistant plate colonies:

[0078] Select the above-mentioned antagonistic drugs that can withstand 70°C for 15 minutes (dilution 10). -7 ), 20 minutes (dilution 10) -5 ) and 25 minutes (dilution 10) -3 Three colonies of Bacillus subtilis (labeled T15, T20, and T25, respectively) were selected and isolated using acid-resistant plates (10g peptone, 3g beef extract, 5g sodium chloride, 10g soluble powder, 18g agar, 1000ml distilled water, pH adjusted to 4.5, sterilized at 121℃ for 30 minutes to prepare a double-disc isolation plate) and alkali-resistant plates (10g peptone, 3g beef extract, 5g sodium chloride, 10g soluble powder, 18g agar, 1000ml distilled water, pH adjusted to 9.5, sterilized at 121℃ for 30 minutes to prepare a double-disc isolation plate). One strain from each acid-resistant and alkali-resistant plate was selected and labeled NST15, NST20, and NST25, respectively.

[0079] 2. Strains preserved in sand tubes:

[0080] Three strains of Bacillus subtilis, NST15, NST20, and NST25, were prepared into Bacillus subtilis bacterial suspensions. These suspensions were then added to sand tubes, which were labeled with the corresponding bacterial number and time. The tubes were wrapped in kraft paper, placed in a desiccator, and then stored in a refrigerator at 4-6℃.

[0081] Example 3 Stability Test

[0082] 1. Take one sand tube from each of the three strains of Bacillus subtilis NST15, NST20 and NST25 that have been preserved in sand tubes for 60 days (with the starting strain Bacillus subtilis CMCC63501 as a control) and use them for shake-flask fermentation to verify their stability.

[0083] 1.1 Shake flask seed culture medium (wt%): soybean meal 3.0, soybean peptone 2.0, sodium chloride 0.3, glucose 2.0, corn starch 1.0, water balance, adjust pH to 7.2.

[0084] Shake flask seed culture conditions: In a 250ml Erlenmeyer flask, inoculate with a 25ml inoculated block, shake on a shaker at 200rpm, and incubate at 35±1℃ for 48h. The viable count of the liquid shake flask fermentation is about 4 billion CFU / ml.

[0085] 1.2 Shake flask fermentation medium (wt%): glucose 2.0, soybean peptone 1.5, Angel yeast powder 2.0, soybean meal 2.0, beef extract 2.0, corn starch 2.0, sodium chloride 0.3, potassium dihydrogen phosphate 0.2, magnesium sulfate 0.05, ammonium sulfate 0.15, manganese sulfate 0.03, water balance, adjust pH to 7.2.

[0086] The first group of shake flask fermentation culture conditions: 40 ml of shake flask fermentation culture medium and 5 ml of the metabolite concentrate mixture prepared in Example 1 were added to a 500 ml Erlenmeyer flask. The inoculum volume was 5 ml. The flask was shaken on a shaker at 200 rpm and cultured at 35 ± 1 °C for 84 h. There were 3 shake flasks in each group.

[0087] The second group of shake flask fermentation culture conditions: 45 ml of shake flask fermentation culture medium was added to a 500 ml Erlenmeyer flask, the inoculum volume was 5 ml, and the mixture was shaken on a shaker at 200 rpm and cultured at 35±1℃ for 84 h. There were 3 shake flasks in each group.

[0088] 2. The average data from each group of 3 shake-bottle verifications are as follows:

[0089] Table 2 Stability verification data

[0090]

[0091]

[0092] As shown in Table 2, after 60 days of storage in sand tubes, under conditions without the metabolite concentrate mixture, the colony counts of Bacillus subtilis NST15, NST20, and NST25, after fermentation, were 21.3 billion CFU / ml, 22.5 billion CFU / ml, and 24.8 billion CFU / ml, respectively, which were 23.84%, 30.81%, and 44.18% higher than the control group. This indicates that Bacillus subtilis NST15, NST20, and NST25 exhibited superior stability after 60 days of sand storage compared to the original strains. Furthermore, the metabolite concentrate mixture did not significantly inhibit the colony counts of NST15, NST20, and NST25, suggesting a significantly improved tolerance of NST15, NST20, and NST25 to the metabolite concentrate mixture. Among them, NST25 showed the best performance.

[0093] Example 4 Production Validation and Spray Drying

[0094] 1. Production Validation:

[0095] Take one sand tube each of Bacillus subtilis strains NST15, NST20, and NST25 that have undergone stability testing, and use the starting strain Bacillus subtilis CMCC 63501 as the control strain.

[0096] 1.1 Seed tank culture medium (wt%): soybean meal 3.0, soybean peptone 2.0, sodium chloride 0.3, glucose 2.0, corn starch 1.0, water balance, adjust pH to 7.2.

[0097] Seed culture conditions: rotation speed 220 rpm, aeration rate 1:1 v / vm, ​​culture at 35±1℃ for 48 h, seed culture viable count approximately 3 billion CFU / ml.

[0098] 1.2 Fermentation tank culture medium (wt%): glucose 2.0, soybean peptone 1.5, Angel yeast powder 2.0, soybean meal 2.0, beef extract 2.0, corn starch 2.0, sodium chloride 0.3, potassium dihydrogen phosphate 0.2, magnesium sulfate 0.05, ammonium sulfate 0.15, manganese sulfate 0.03, light calcium carbonate 0.3, water balance, adjust pH to 7.2.

[0099] Fermentation conditions in the fermenter: temperature 32℃, cycle 84h, with feed added from 15h to 70h to ensure reducing sugar content is 1.1% ± 0.2%, rotation speed 300rpm, aeration rate (1:1) v / vm, ​​dissolved oxygen in the culture medium controlled at 60 ± 5%, pH controlled at 7.5 ± 1.0, and inoculum size 10%.

[0100] 1.3 Fermentation Production Data:

[0101] Table 3 Fermentation Production Data

[0102] strains 63501 Reference NST15 NST20 NST25 Colony count (100 million CFU / ml) 181 235 241 256

[0103] As shown in Table 3, under the same culture conditions, the colony counts of Bacillus subtilis NST15, NST20, and NST25 were 23.5 billion CFU / ml, 24.1 billion CFU / ml, and 25.6 billion CFU / ml, respectively, which were 29.83%, 33.15%, and 41.43% higher than the control group. This indicates that under the same culture conditions, Bacillus subtilis NST15, NST20, and NST25 had higher fermentation rates than the starting strains and exhibited better production activity. Among them, NST25 showed the highest production activity.

[0104] 2. Spray drying:

[0105] 2.1 Add 0.2% manganese sulfate, 0.2% potassium sulfate, 0.3% magnesium sulfate, and 0.5% light calcium carbonate to the deep fermentation broth of each Bacillus subtilis strain, adjust the pH to 6.8-7.2, and stir well.

[0106] 2.2 Adjust the inlet temperature of the spray tower to 150±5℃ and the outlet temperature to 55±5℃. The Bacillus subtilis fermentation broth is preheated at 70±2℃ for 15-20 minutes before entering the spray tower.

[0107] 2.3. Mix each type of Bacillus subtilis spray product (bacterial agent) thoroughly, and measure the following data:

[0108] Table 4 Data on Bacillus subtilis spray products by strain number

[0109] strains 63501 Reference NST15 NST20 NST25 Colony count (100 million CFU / g) 1782 2452 2398 2531 Moisture (%) 3.5 3.4 3.6 3.3

[0110] As shown in Table 4, under the same preparation conditions, the colony counts of Bacillus subtilis NST15, NST20, and NST25 were 245.2 billion CFU / g, 239.8 billion CFU / g, and 253.1 billion CFU / g, respectively, which were 37.60%, 34.57%, and 42.03% higher than the control group. This indicates that under the same preparation conditions, compared with the starting strain, Bacillus subtilis NST15, NST20, and NST25 produced higher bacterial content, were more heat-resistant, and more stable during spray drying, with NST25 exhibiting the best stability.

[0111] Example 5: Product Shelf Stability Test

[0112] 1. All Bacillus subtilis products are packaged in aluminum-plastic vacuum packaging, 1000g per bag. They are stored under conditions of temperature ≤20℃, relative humidity ≤65%, and away from direct sunlight, and data testing is conducted every 3 months for a total of 12 months.

[0113] 2. The shelf stability data is as follows:

[0114] Table 5 Shelf stability data

[0115]

[0116] As shown in Table 5, under the same shelf-storage conditions, the survival rates (i.e., recovery rates) of Bacillus subtilis NST15, NST20, and NST25 were 90%, 91%, and 94%, respectively, which were 11%, 12%, and 15% higher than the control group. This indicates that under the same shelf-storage conditions, Bacillus subtilis NST15, NST20, and NST25 are more resistant to storage.

[0117] Based on the above data, the NST25 strain with the highest survival rate and best production performance was selected and named Bacillus subtilis BS-56. It was sent to the Guangdong Provincial Microbial Culture Collection Center (Institute of Microbiology, Guangdong Academy of Sciences) for preservation and is designated as Bacillus subtilis GDMCC No:62324.

[0118] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of the present invention. Therefore, any changes or modifications made in accordance with the claims and specification of the present invention should fall within the scope of the present invention.

Claims

1. A Bacillus subtilis BS-56 strain, characterized in that, The Bacillus subtilis BS-56 specimen has the accession number GDMCC No: 62324.

2. A fermentation product obtained by fermentation using Bacillus subtilis BS-56 as described in claim 1, wherein the fermentation product comprises Bacillus subtilis BS-56.

3. The application of Bacillus subtilis BS-56 as described in claim 1 and the fermentation product as described in claim 2 in microbial fertilizers.

4. A microbial fertilizer, characterized in that, The microbial fertilizer includes Bacillus subtilis BS-56 as described in claim 1 and / or the fermentation product as described in claim 2.

5. The microbial fertilizer as described in claim 4, characterized in that, The microbial fertilizer is a microbial agent, and the content of Bacillus subtilis BS-56 in the microbial agent is ≥100 billion CFU / g.

6. The microbial fertilizer as described in claim 5, characterized in that, The content of Bacillus subtilis BS-56 in the bacterial agent is 2000~4000 billion CFU / g.

7. A method for preparing the microbial fertilizer as described in any one of claims 4-6, characterized in that, The method includes the following steps: The seed culture of Bacillus subtilis BS-56 was inoculated into a fermentation medium for fermentation culture to obtain a fermentation broth. The fermentation broth was then dried to obtain the microbial fertilizer.

8. The method as described in claim 7, characterized in that, The fermentation medium comprises: Glucose 1.0-4.0 wt% Soy protein peptone 1.0-5.0 wt%; Angel Yeast Powder 1.0-4.0 wt% Soybean meal 1.0-4.0 wt% Beef extract 1.0-4.0 wt% Corn starch 1.0-4.0 wt% Sodium chloride 0.1-0.5 wt% Potassium dihydrogen phosphate 0.1-0.3 wt% Magnesium sulfate 0.01-0.06 wt% Ammonium sulfate 0.1-0.5 wt%; Manganese sulfate 0.01-0.05 wt% Light calcium carbonate 0.1-0.5 wt% The remainder is water.

9. The method as described in claim 7 or 8, characterized in that, The fermentation culture conditions satisfy at least one of the following: The temperature is 32-36℃; The culture period is 64-96 hours; Reducing sugar content: 0.8-1.2%; Dissolved oxygen 50-70%; Control the pH to 7.0-7.7; The vaccination rate is 5-15%.

10. The use of Bacillus subtilis BS-56 as described in claim 1, the fermentation product as described in claim 2, the microbial fertilizer as described in any one of claims 4-6, and the microbial fertilizer obtained by the method as described in any one of claims 7-9 in the preparation of products that promote plant growth.

11. The application of Bacillus subtilis BS-56 as described in claim 1, the fermentation product as described in claim 2, the microbial fertilizer as described in any one of claims 4-6, and the microbial fertilizer obtained by the method as described in any one of claims 7-9 in promoting plant growth.

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