A bacillus subtilis preparation, its preparation method and application

Abstract

The application discloses a bacillus subtilis preparation and a preparation method and application thereof, and belongs to the technical field of microbial inoculants. The bacillus subtilis has a preservation number of CGMCC No. 19440, a preservation date of March 2, 2020, and a taxonomic name of Bacillus subtilis. In view of the defects that live bacteria attenuate quickly and mechanical stability is poor when existing microbial inoculants are mixed with coated compound fertilizers, the application prepares a high-spore-rate fermentation liquor through a staged fermentation process, and uses sodium alginate-chitosan-sodium carboxymethyl cellulose-montmorillonite composite wall materials to prepare a microencapsulated microbial inoculant. The application has a live bacteria retention rate of more than 93% when mixed with mineral oil coated compound fertilizers and stored for 60 days, and a mixed damage rate of less than 5%, and can significantly improve soil fertility and increase crop yield by 22.7% to 37.5% when used in combination, and is suitable for industrialized production of compound microbial fertilizers.

Description

Technical Field

[0001] This invention belongs to the field of microbial inoculant technology, and particularly relates to a Bacillus subtilis preparation, its preparation method, and its application. Background Technology

[0002] As a core category of agricultural probiotics, the survival rate of Bacillus subtilis directly determines the synergistic effect of bacterial fertilizer. Currently, the application of Bacillus subtilis fermentation liquid is mainly through direct spraying and spray drying of bacterial powder. However, when combined with slow-release compound fertilizer (especially oil-coated compound fertilizer), it faces multiple stresses that lead to a sharp decline in the number of viable bacteria: (1) Complexity of oil film composition: The compound fertilizer oil film contains a variety of hydrophobic components such as mineral oil, sulfur, polymer resin, and asphalt. Its hydrophobic environment will damage the integrity of the bacterial cell membrane; (2) Chemical stress: The emulsifiers, organic solvents and pH fluctuations (5.0-9.0) in the oil film will inhibit spore germination and accelerate the death of vegetative cells. The viable bacteria of conventional spray powder preparations decreased by more than 90% in 30 days; (3) Mechanical damage: The shear force during the mixing process of compound fertilizer can easily cause the unprotected bacterial cells to rupture.

[0003] Existing microencapsulation technologies have significant drawbacks: ① They use a single wall material (mostly simple sodium alginate or chitosan), failing to address the hydrophobic and chemical stresses of the oil film; ② They neglect the diversity of compound fertilizer oil film components (such as sulfur coating, resin coating, etc.), resulting in poor compatibility; ③ They do not solve the problem of the mechanical stability of microcapsules in compound fertilizer mixing. Therefore, there is an urgent need to develop a composite microencapsulation formulation that combines hydrophobic barrier, chemical protection, and mechanical strength, clarifying its effect differences from direct spraying formulations, and meeting the needs of industrial applications. Summary of the Invention

[0004] To address the problems of poor viable bacterial stability, narrow compatibility, and insufficient mechanical strength of existing formulations in compound fertilizer oil film environments, this invention provides a Bacillus subtilis, with the preservation number CGMCC No. 19440.

[0005] The present invention also provides a method for preparing the above-mentioned Bacillus subtilis fermentation broth, comprising the following steps: (1) Activation of the strain: Bacillus subtilis was inoculated into a primary seed culture medium and cultured at 36-38℃ with shaking for 10-14 h; the primary seed culture medium consisted of: peptone 13-17 g / L, corn steep liquor 18-22 g / L, glucose 8-12 g / L, KH2PO4 0.8-1.2 g / L, MgSO4·7H2O 0.4-0.6 g / L, sodium glutamate 0.8-1.2 g / L, pH 6.5-7.5; (2) Secondary seed culture: The activated strain was inoculated into a secondary seed culture medium and cultured with shaking at 36-38℃ for 7-9 h; the secondary seed culture medium consisted of: peptone 13-17 g / L, corn steep liquor 18-22 g / L, glucose 13-17 g / L, KH2PO4 0.8-1.2 g / L, MgSO4·7H2O 0.4-0.6 g / L, sodium glutamate 1.8-2.2 g / L, pH 6.5-7.5; (3) Fermentation tank culture: The fermentation medium consists of 23-27 g / L corn flour, 16-20 g / L hydrolyzed soybean meal, 7-9 g / L glucose, 1-1.4 g / L KH2PO4, 0.5-0.7 g / L MgSO4·7H2O, and 2.2-2.8 g / L CaCO3, with a pH of 6.5-7.5; the fermentation is regulated in stages as follows: a. Adaptation period: 0-4h, 36-38℃, 180-220rpm stirring, aeration rate 0.4-0.6vvm; b. Logarithmic growth phase: 4-12h, 37-38℃, 260-300rpm stirring, aeration rate 0.8-1.0vvm, supplement with 8-12wt% glucose, residual sugar 1-2g / L; c. Stabilization period: 12-20h, 36-37℃, 230-280rpm stirring, aeration rate of 0.7-0.9vvm, and replenishment with 5wt% soybean meal hydrolysate; d. Spore formation period: 20-28h, 34-36℃, 180-220rpm stirring, aeration rate 0.4-0.6vvm, stop feeding, residual sugar ≤0.5g / L; e. Fermentation endpoint: Spore count ≥ 92%, viable cell count ≥ 1.1 × 10⁻⁶ 10 CFU / mL.

[0006] Preferably, the primary seed culture medium in step (1) is: 15 g / L peptone, 20 g / L corn steep liquor, 10 g / L glucose, 1 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, 1 g / L sodium glutamate, pH 7.0.

[0007] More preferably, the secondary seed culture medium in step (2) is: 15 g / L peptone, 20 g / L corn steep liquor, 15 g / L glucose, 1 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, 2 g / L sodium glutamate, pH 7.0.

[0008] More preferably, the fermentation medium in step (3) is composed of 25 g / L corn flour, 18 g / L hydrolyzed soybean meal, 8 g / L glucose, 1.2 g / L KH2PO4, 0.6 g / L MgSO4·7H2O, 2.5 g / L CaCO3, and pH 7.1.

[0009] The present invention also provides a fermentation broth prepared by any of the above-described preparation methods.

[0010] The present invention also provides a microencapsulated bacterial agent containing the above-mentioned Bacillus subtilis.

[0011] The present invention also provides a method for preparing the above-mentioned microencapsulated bacterial agent, comprising the following steps: (1) Core material preparation: The above fermentation broth is centrifuged to collect the bacterial sludge, and a protective agent compound system is added, with the addition amount being 45-55% of the bacterial sludge mass; the protective agent compound system is: sodium alginate 45wt%, chitosan 30wt%, CMC 15wt%, montmorillonite 10wt%; (2) Preparation of composite wall material solution: 45wt% sodium alginate, 30wt% chitosan, 15wt% CMC and 10wt% montmorillonite were stirred and dissolved at 60℃, pH was adjusted to 5.5, 4-6% of the total mass of the wall material was added to polyethylene glycol 6000, and water was added to dilute to a total solid content of 18% and a viscosity of 200 mPa·s to ensure uniform atomization; (3) Atomized co-crosslinking and in-situ curing: The core material obtained in step (1) and the composite wall material solution prepared in step (2) are thoroughly mixed at a volume ratio of 1:1-1:2. 0.3% glutaraldehyde is added to the mixture in advance. Using a dual-fluid nozzle, the mixture and 0.8 mol / L calcium chloride solution are atomized simultaneously at a volume ratio of 1:0.8 and crosslinked by collision in the drying tower. (4) Spray drying yields microencapsulated Bacillus preparations.

[0012] This invention also provides the application of the above-mentioned microencapsulated Bacillus preparation in the preparation of compound microbial fertilizers.

[0013] Preferably, the compound microbial fertilizer is a mixture of microencapsulated bacterial agent and coated compound fertilizer.

[0014] Compared with the prior art, the present invention has the following beneficial effects: Currently, when microbial agents are mixed with compound fertilizers, a large number of microorganisms die due to the chemical components and cannot play their corresponding role. The present invention realizes the microencapsulation of microorganisms and adopts the method of protecting microorganisms when mixed with compound fertilizers. The microorganisms will not die in large numbers and can play their role. At the same time, the microorganisms decompose the compound fertilizer, allowing its fertilizer effect to be fully exerted, improving soil fertility and promoting increased crop yield and income.

[0015] Biological preservation instructions for Bacillus subtilis BK5: Preservation institution: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Cultures; Accession number: CGMCC No. 19440; Date of deposit: March 2, 2020; Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; Taxonomic name: Bacillus subtilis. Detailed Implementation

[0016] The preparation method of the soybean meal hydrolysate used in the following examples is as follows: Take soybean meal powder, add water at a material-to-liquid mass ratio of 1:10 and stir evenly. Adjust the pH to about 7.0, add 2%~3% of the soybean meal powder mass of a mesophilic protease (i.e., Hupro protease, Wuhan Xinhua Yang Biotechnology Co., Ltd.), and stir at 45℃ for 2 hours for enzymatic hydrolysis. Then, raise the temperature to 95℃ and keep it at that temperature for 10 minutes, and dilute with water to prepare a 5wt% soybean meal hydrolysate. If you want to prepare hydrolyzed soybean meal powder, after the enzyme inactivation step, you need to add a concentration and spray drying (or other dehydration and powdering process) step to obtain solid powdered hydrolyzed soybean meal powder.

[0017] Example 1 1. Preparation of Bacillus subtilis fermentation broth (1) Activation of the strain: After Bacillus subtilis BK5 (preservation number CGMCC No. 19440) was revived, it was cultured on LB solid medium at 37℃ for 20h. Single colonies were picked and inoculated into primary seed medium (15g / L peptone, 20g / L corn steep liquor, 10g / L glucose, 1g / L KH2PO4, 0.5g / L MgSO4·7H2O, 1g / L sodium glutamate, pH 7.0) and cultured at 37℃ and 200rpm for 12h (OD200). 600 ≈1.2, viable count ≥10 8 (CFU / mL) (2) Secondary seed culture: Inoculate with secondary seed culture medium at a 3% inoculum (glucose increased to 15 g / L and sodium glutamate to 2 g / L based on the primary seed culture medium), and culture at 37℃ and 220 rpm for 8 h (OD 600 ≈2.5, spore rate ≤8%). (3) Fermentation tank culture: In a 50L fermenter, the fermentation medium consisted of 25g / L corn flour, 18g / L hydrolyzed soybean meal, 8g / L glucose, 1.2g / L KH2PO4, 0.6g / L MgSO4·7H2O, and 2.5g / L CaCO3, with a pH of 7.1; the pH was adjusted in stages. a. Adaptation period (0-4h): 37℃, stirring at 200rpm, aeration 1:0.5vvm; b. Logarithmic growth phase (4-12h): 37.5℃, stirring at 280rpm, aeration 1:0.9vvm, and addition of 10wt% glucose (controlling residual sugar in fermentation broth to 1-2g / L). c. Stabilization period (12-20h): 36.5℃, stirring at 250rpm, aeration 1:0.8vvm, add 5wt% soybean meal hydrolysate, total feed volume: 75~125mL / 50L tank; d. Spore formation period (20-28h): 35℃, stirring at 200rpm, aeration 1:0.5vvm, stop feeding (residual sugar ≤0.5g / L). e. Fermentation endpoint: Spore count ≥ 92%, viable cell count ≥ 1.1 × 10⁻⁶ 10 CFU / mL.

[0018] 2. Preparation of direct spray powder formulations The fermentation broth was filtered through a 0.45μm filter membrane (viable bacteria count ≥9.5×10⁻⁶). 9 CFU / mL), spray drying parameters: inlet temperature 125℃, outlet temperature 65℃, feed rate 15mL / min, atomization pressure 0.3MPa, finished product index: viable count ≥8.7×10⁻⁶ 10 CFU / g, moisture ≤5%, particle size 10-20μm.

[0019] 3. Preparation of microencapsulated formulations (1) Core material preparation: Centrifuge the fermentation broth at 8000 rpm for 20 min and collect the bacterial sludge (spore rate 95~98%). Add the protective agent compound system (sodium alginate 45% + chitosan 30% + CMC 15% + montmorillonite 10%, weight ratio), and the amount added is 50% of the bacterial sludge mass. (2) Preparation of composite wall material solution: 45wt% sodium alginate + 30wt% chitosan + 15wt% CMC + 10wt% montmorillonite, stir and dissolve at 60℃, adjust pH to 5.5, add 6000 polyethylene glycol (6% of the total mass of the wall material), dilute with water to 18% total solids and 200mPa·s viscosity to ensure uniform atomization.

[0020] (3) Atomized co-crosslinking and in-situ curing: The core material obtained in step (1) and the composite wall material solution prepared in step (2) are thoroughly mixed at a volume ratio of 1:1. 0.3wt% of glutaraldehyde is added to the mixture in advance. The mixture is atomized simultaneously with 0.8mol / L calcium chloride solution at a volume ratio of 1:0.8 using a dual-fluid nozzle and crosslinked by collision in the drying tower.

[0021] (4) Spray drying: control the inlet air temperature to 125℃, the outlet air temperature to 60℃, the spray pressure to 0.45MPa, the feed rate to 30mL / min, and the hot air velocity to 1.5m / s. After drying, the microencapsulated preparation of Bacillus spores is obtained.

[0022] 4. Scenario-based application verification (1) Comparison of indoor storage stability Table 1

[0023] Table 2

[0024] (2) Comparison of element content in soil between compound microbial functional fertilizer and control soil Select the same plot of land and divide it into 3 plots, each 4m². 2 The plots were labeled A, B, and C. Plot A was treated with a single compound fertilizer; plot B was treated with a mixture of a common formulation (the aforementioned powdered formulation) and compound fertilizer; and plot C was treated with a mixture of the microencapsulated formulation of this invention and compound fertilizer. The compound fertilizer (high-concentration potassium sulfate compound fertilizer) was applied at a conventional rate of 50 kg / mu. The formulation was diluted 30 times and sprayed evenly at a rate of 5 L / mu, with a 10-day interval between applications, for a total of 3 applications. After the 3 applications, 100 g soil samples were randomly selected from 5 points to measure soil elements. The compound microbial functional fertilizer effectively improved the soil and increased the soil element content. (Note: The compound fertilizer was from the same bag.) Table 3

[0025] (3) Comparison of effects of application on multiple crops Select a single, fertile, flat plot of land free from continuous cropping obstacles. A randomized block design was used, dividing the land into experimental units of 1 acre each, categorized by crop type. Three treatment groups were established: control group 1 (single compound fertilizer), control group 2 (ordinary formulation + compound fertilizer), and experimental group (the formulation of this invention + compound fertilizer). The compound fertilizer was applied at 50 kg / acre, and the formulation at 5 kg / acre, both applied simultaneously with the compound fertilizer before sowing. No further application was made during the crop growth period. Field management (sowing density, weeding, basic pest and disease control, etc.) was maintained consistently across all plots, and flood irrigation was used uniformly to ensure no other interfering factors besides fertilization.

[0026] Table 4

[0027] Note: The testing methods refer to NY / T 2321-2013, NY / T 1121-2021, and GB / T 17891-2021. The field trial adopted a randomized block design.

[0028] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A Bacillus subtilis strain, characterized in that, The Bacillus subtilis has the accession number CGMCC No. 19440.

2. A method for preparing the fermentation broth of Bacillus subtilis according to claim 1, characterized in that, Includes the following steps: (1) Activation of the strain: Bacillus subtilis was inoculated into a primary seed culture medium and cultured at 36-38℃ with shaking for 10-14 h; the primary seed culture medium consisted of: peptone 13-17 g / L, corn steep liquor 18-22 g / L, glucose 8-12 g / L, KH2PO4 0.8-1.2 g / L, MgSO4·7H2O 0.4-0.6 g / L, sodium glutamate 0.8-1.2 g / L, pH 6.5-7.5; (2) Secondary seed culture: The activated strain was inoculated into a secondary seed culture medium and cultured with shaking at 36-38℃ for 7-9 hours; the secondary seed culture medium consisted of: peptone 13-17 g / L, corn steep liquor 18-22 g / L, glucose 13-17 g / L, KH2PO4 0.8-1.2 g / L, MgSO4·7H2O 0.4-0.6 g / L, sodium glutamate 1.8-2.2 g / L, pH 6.5-7.5; (3) Fermentation tank culture: The fermentation medium consists of 23-27 g / L corn flour, 16-20 g / L hydrolyzed soybean meal, 7-9 g / L glucose, 1-1.4 g / L KH2PO4, 0.5-0.7 g / L MgSO4·7H2O, and 2.2-2.8 g / L CaCO3, with a pH of 6.5-7.5; the process is regulated in stages as follows: a. Adaptation period: 0-4h, 36-38℃, 180-220rpm stirring, aeration rate 0.4-0.6vvm; b. Logarithmic growth phase: 4-12h, 37-38℃, 260-300rpm stirring, aeration rate 0.8-1.0vvm, supplement with 8-12wt% glucose, residual sugar 1-2g / L; c. Stabilization period: 12-20h, 36-37℃, 230-280rpm stirring, aeration rate of 0.7-0.9vvm, and replenishment with 5wt% soybean meal hydrolysate; d. Spore formation period: 20-28h, 34-36℃, 180-220rpm stirring, aeration rate 0.4-0.6vvm, stop feeding, residual sugar ≤0.5g / L; e. Fermentation endpoint: Spore count ≥ 92%, viable cell count ≥ 1.1 × 10⁻⁶ 10 CFU / mL.

3. The preparation method according to claim 2, characterized in that, The primary seed culture medium in step (1) is: 15 g / L peptone, 20 g / L corn steep liquor, 10 g / L glucose, 1 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, 1 g / L sodium glutamate, pH 7.

0.

4. The preparation method according to claim 3, characterized in that, The secondary seed culture medium in step (2) is: 15 g / L peptone, 20 g / L corn steep liquor, 15 g / L glucose, 1 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, 2 g / L sodium glutamate, pH 7.

0.

5. The preparation method according to claim 4, characterized in that, In step (3), the fermentation medium consists of 25 g / L corn flour, 18 g / L hydrolyzed soybean meal, 8 g / L glucose, 1.2 g / L KH2PO4, 0.6 g / L MgSO4·7H2O, 2.5 g / L CaCO3, and pH 7.

1.

6. The fermentation broth prepared by the preparation method according to any one of claims 2-5.

7. A microencapsulated bacterial agent, characterized in that, The microencapsulated bacterial agent contains Bacillus subtilis as described in claim 1.

8. A method for preparing the microencapsulated bacterial agent according to claim 7, characterized in that, Includes the following steps: (1) Core material preparation: The fermentation broth described in claim 6 is centrifuged to collect bacterial sludge, and a protective agent compound system is added, with the addition amount being 45-55% of the bacterial sludge mass; the protective agent compound system is: sodium alginate 45wt%, chitosan 30wt%, CMC 15wt%, montmorillonite 10wt%; (2) Preparation of composite wall material solution: 45wt% sodium alginate, 30wt% chitosan, 15wt% CMC and 10wt% montmorillonite were stirred and dissolved at 60℃, pH was adjusted to 5.5, 4-6% of the total mass of the wall material was added to polyethylene glycol 6000, and water was added to dilute to a total solid content of 18% and a viscosity of 200 mPa·s to ensure uniform atomization; (3) Atomized co-crosslinking and in-situ curing: The core material obtained in step (1) and the composite wall material solution prepared in step (2) are thoroughly mixed at a volume ratio of 1:1-1:

2. 0.3% glutaraldehyde is added to the mixture. Using a dual-fluid nozzle, the mixture and 0.8 mol / L calcium chloride solution are atomized simultaneously at a volume ratio of 1:0.8 and crosslinked by collision in the drying tower. (4) Spray drying yields microencapsulated Bacillus preparations.

9. The application of the microencapsulated Bacillus preparation according to claim 7 in the preparation of compound microbial fertilizer.

10. The application according to claim 9, characterized in that, The compound microbial fertilizer is a mixture of microencapsulated bacterial agents and coated compound fertilizers.

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