Bacillus subtilis and its use for increasing polyglutamic acid and / or organic acids

Abstract

The present application relates to the field of microbial technology, and more particularly to bacillus subtilis and its application in producing polyglutamic acid and / or organic acid. The present application provides a bacillus subtilis with a preservation number of CCTCC NO: M 20251821, which can produce polyglutamic acid (content reaching more than 6%) and organic acid (lactic acid and acetic acid content > 1%); a liquid microbial inoculant rich in polyglutamic acid can be prepared by using the strain, wherein the number of viable bacillus subtilis is more than 10 8 cfu / ml, the content of gamma-polyglutamic acid is more than 6%, the inoculant can produce a large amount of lactic acid and acetic acid in soil, has a significant effect of phosphorus and potassium release, and the rich polyglutamic acid can promote plant rooting, resist adversity and retain water, and improve crop yield.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and more particularly to Bacillus subtilis and its application in enhancing polyglutamic acid and / or organic acids. Background Technology

[0002] In agricultural production, soil quality is a core factor determining crop yield and quality, and the content of functional substances in the soil directly affects soil fertility, nutrient availability, and the crop growth environment. Organic acids, as important organic compounds widely present in soil, originate from soil organic matter decomposition, plant root secretions, and microbial metabolism. They can improve soil ecology by regulating soil physicochemical properties, activating insoluble nutrients, and promoting crop root absorption, thus providing favorable conditions for crop growth and positively impacting crop yield. γ-Polyglutamic acid, a natural biopolymer produced by microorganisms, possesses excellent water and fertilizer retention, nutrient chelation, and heavy metal passivation capabilities due to its abundant hydrophilic carboxyl groups in its molecular structure. It not only reduces soil nutrient leaching and volatilization and improves fertilizer utilization efficiency, but its degradation products can also directly promote plant growth as nutrients. Simultaneously, it can improve soil aggregate structure, enhance soil permeability and water retention, playing a crucial role in soil remediation and crop yield increase. The synergistic effect of organic acids and γ-polyglutamic acid can create a suitable soil environment for crop growth from multiple dimensions such as nutrient supply, water retention, and soil structure optimization. Therefore, increasing the content of these two types of substances in the soil is an important way to achieve soil improvement and crop yield increase.

[0003] Microbial inoculants, due to their environmental friendliness and long-lasting effects, have become an important means of improving soil quality in agricultural production. Bacillus subtilis, as a common functional strain, is widely used in the research and development of agricultural microbial agents because of its ability to produce active substances, inhibit harmful microorganisms, and adapt to complex environments. However, the field of microbial inoculants still faces many technical bottlenecks. Most strains suffer from single-function problems, weak environmental adaptability, and insufficient activity stability, making it difficult to continuously and efficiently produce sufficient organic acids under complex field conditions. While some strains screened at the laboratory level show potential, limitations such as imperfect functional validation systems and poor field adaptability often prevent them from achieving the expected results in practical applications, exhibiting insufficient remediation efficiency and stability. Furthermore, existing strains perform poorly in terms of soil colonization ability and compatibility with indigenous microbial communities, limiting their efficiency in producing functional substances and failing to fully realize their soil improvement and yield-increasing effects. Given the crucial role of organic acids and γ-polyglutamic acid in crop yield increase and the inadequacy of existing strains, screening for Bacillus subtilis strains that efficiently produce these two substances is the core direction for overcoming these technical bottlenecks. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide Bacillus subtilis and its application in improving polyglutamic acid and / or organic acids.

[0005] This invention provides Bacillus subtilis with accession number CCTCC NO: M 20251821 ( Bacillus subtle ).

[0006] This invention provides Bacillus subtilis that produces high levels of polyglutamic acid (greater than 6%) or organic acids (lactic acid, acetic acid, greater than 1%). Since Bacillus subtilis requires aerobic conditions to produce polyglutamic acid and anaerobic conditions to produce organic acids, creating a contradiction, this invention creatively prepares a microbial agent rich in polyglutamic acid through aerobic fermentation. After being applied to the soil, the microorganisms rely on the anaerobic environment to produce a large amount of organic acids. Simultaneously, the polyglutamic acid introduced into the agent promotes plant root growth and enhances stress resistance and water retention, resulting in a significant improvement in crop yield and quality. The newly generated organic acids in the soil have a significant effect on phosphorus and potassium solubilization, leading to a significant increase in phosphorus and potassium content in crops and soil remediation.

[0007] The present invention also provides a microbial agent, the raw materials for which include Bacillus subtilis as described above.

[0008] The viable count of Bacillus subtilis in the bacterial agent of this invention is not less than 10. 8 The cfu / ml concentration and the mass fraction of polyglutamic acid are not less than 6%.

[0009] More specifically, the viable count of Bacillus subtilis in the bacterial agent of this invention is 5.3 × 10⁻⁶. 8 cfu / ml ~6.6×10 8 CFU / ml; polyglutamic acid mass fraction is 6.2%~7.3%.

[0010] Furthermore, the present invention also provides a method for preparing the bacterial agent as described above, which includes fermenting the Bacillus subtilis as described above in a culture medium.

[0011] In this invention, a product rich in polyglutamic acid is obtained by fermentation. The culture medium includes water and the following components in mass fractions: glucose 60-80 g / L, sodium glutamate 60-80 g / L, yeast extract 10-20 g / L, potassium dihydrogen phosphate 2 g / L, magnesium sulfate heptahydrate 2 g / L, and pH 7.0.

[0012] In a specific embodiment, the culture medium includes water and the following components by mass fraction: glucose 80 g / L, sodium glutamate 80 g / L, yeast extract 20 g / L, potassium dihydrogen phosphate 2 g / L, magnesium sulfate heptahydrate 2 g / L, and pH 7.0.

[0013] In another specific embodiment, the culture medium comprises water and the following components by mass fraction: glucose 60 g / L, monosodium glutamate 60 g / L, yeast extract 10 g / L, potassium dihydrogen phosphate 2 g / L, magnesium sulfate heptahydrate 2 g / L, with the pH controlled at a constant 7.0.

[0014] In this invention, the fermentation conditions for obtaining a product rich in polyglutamic acid include: temperature 37°C, pressure 0.05 MPa, dissolved oxygen > 20%, continuous addition of glucose solution to control the reducing sugar concentration in the fermentation broth to be not less than 1.5%, continuous addition of ammonia water to adjust the pH value to 7.0, and culture time 50 h.

[0015] To increase the yield of polyglutamic acid and provide conditions for the subsequent production of organic acids, this invention optimizes the preparation parameters of the inoculant. By controlling dissolved oxygen to be above 20%, good proliferation and metabolic activity of the strain are maintained. Under the regulation of fed-batch feeding, a stable supply of carbon source is ensured, so that the polyglutamic acid content in the final fermentation product is stably above 6%. At the same time, sufficient active Bacillus subtilis is retained, providing a basis for the strain to produce organic acids after entering the soil.

[0016] In this invention, the pre-fermentation process further includes steps of slant activation, primary seed activation, and secondary seed activation; The slant seed culture medium is LB medium; The culture medium for primary and secondary seed activation was: glucose 20 g / L, sodium glutamate 5 g / L, yeast extract 3 g / L, potassium dihydrogen phosphate 1 g / L, magnesium sulfate 0.8 g / L, with an initial pH of 7.0.

[0017] In this invention, the slant activation condition is incubation at 37℃ for 18-24 hours. The primary seed activation condition includes incubation at 37℃ for 24 hours; the secondary seed activation condition includes incubation at 37℃ with shaking until the OD600 value is 5-7.

[0018] Furthermore, the present invention also provides Bacillus subtilis as described above, and the bacterial agent prepared by the aforementioned method can increase the level of organic acids in the soil. Moreover, the metabolites of Bacillus subtilis provided by the present invention can also promote plant growth, thereby increasing crop yield and improving crop quality.

[0019] Specifically, the Bacillus subtilis provided in this invention can ferment to obtain polyglutamic acid in a culture medium containing monosodium glutamate or glutamic acid. When this agent is applied to the soil, the polyglutamic acid effectively promotes the growth and development of plant roots, enhancing their ability to absorb water and nutrients. Simultaneously, in the anaerobic environment of the soil, Bacillus subtilis further metabolizes to produce large amounts of organic acids such as lactic acid and acetic acid. These organic acids can significantly lower the soil pH, especially for soils with high pH, ​​such as saline-alkali land, creating a suitable acid-base environment for crop growth. More importantly, organic acids can chelate or dissolve minerals such as phosphorus and potassium in the soil that are difficult for crops to absorb and utilize, converting them into soluble phosphates and potassium salts. This greatly increases the content of available phosphorus and potassium in the soil, achieving a significant effect of phosphorus and potassium solubilization, thus enabling crops to more fully absorb these key nutrients and providing a sufficient material basis for their growth, flowering, and fruiting. Furthermore, the metabolites of Bacillus subtilis in the microbial agent have a very complex composition. However, experiments have shown that the fermented product of the microbial agent described in this invention has a very viscous texture and good water retention properties. It can improve the soil's water holding capacity, reduce water loss, enhance the crop's resistance to drought conditions, and ensure that the crop can maintain a good growth state under different environmental conditions, thereby synergistically improving the yield and quality of crops.

[0020] As a feasible example, the organic acid is generated after the microbial agent is applied to the soil. In this invention, the organic acid includes lactic acid and acetic acid.

[0021] In this invention, the crop is a grass, a solanaceous plant, a cucurbitaceous plant, or a rutaceous plant.

[0022] In some embodiments, the grasses include rice, wheat, and corn; the solanaceae crops include peppers, tomatoes, and eggplants; the cucurbitaceae crops include cucumbers, watermelons, and cantaloupes; and the Rutaceae crops include citrus fruits, oranges, and pomelos.

[0023] In this invention, the applications include increasing the reducing sugar content, protein content, dry matter content, or nitrogen, phosphorus, and potassium levels in crop fruits, increasing crop fruit volume and / or weight, increasing plant height, promoting root growth and development, and increasing yield per unit area and / or average yield per plant.

[0024] Furthermore, the present invention also provides a method for crop cultivation, which includes applying the microbial agent as described above or the microbial agent prepared by the method described above to the soil.

[0025] In this invention, the amount applied is 1 kg per acre, diluted 250 times and applied by irrigation, once every 5-10 days.

[0026] The Bacillus subtilis strain with accession number CCTCC NO: M 20251821 provided by this invention can produce high levels of polyglutamic acid (content exceeding 6%) or organic acids (content exceeding 1%, mainly lactic acid or acetic acid); this strain can be used to prepare liquid microbial inoculants rich in polyglutamic acid, wherein the viable count of the above-mentioned Bacillus subtilis reaches 10. 8 The microbial agent, rich in polyglutamic acid, has a cfu / ml concentration of over 6% and a γ-polyglutamic acid content of over 6%. When applied to soil improvement, this agent produces a large amount of organic acids, significantly improving phosphorus and potassium solubility. Simultaneously, the abundant polyglutamic acid promotes plant root growth, enhances stress resistance and water retention, and ultimately increases crop yield.

[0027] Biological Preservation Instructions

[0028] Bacillus subtilis SD-1 ( Bacillus subtilis SD-1 was deposited on August 12, 2025, at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20251821. Attached Figure Description

[0029] Figure 1 Statistical analysis of chili pepper yield in the experimental and control groups is shown. Detailed Implementation

[0030] This invention provides Bacillus subtilis and its application in improving polyglutamic acid and / or organic acids. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0031] Unless otherwise defined in this invention, the scientific and technical terms associated with this invention shall have the meanings understood by one of ordinary skill in the art.

[0032] The terms “comprising,” “including,” and “having” are used interchangeably to indicate the inclusiveness of a scheme, meaning that the scheme may contain elements other than those listed. It should also be understood that the use of “comprising,” “including,” and “having” herein also provides for schemes “consisting of…”.

[0033] When used herein, the term “and / or” includes the meaning of “and,” “or,” and “all or any other combination of elements linked by the term.”

[0034] The term "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of single or multiple items.

[0035] It should be understood that in the various embodiments of this application, the order of the above processes does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0036] The numerical ranges and parameters involved in this invention have been presented as precisely as possible in the specific embodiments. However, any numerical value inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all numerical ranges or specific data used in this disclosure may have a reasonable deviation within a certain range, such as ±10%, ±5%, ±1%, or ±0.5%.

[0037] The test materials used in this invention are all common commercial products and can be purchased on the market.

[0038] The compound fertilizers involved in the embodiments include: compound fertilizers 15-5-25 and 15-15-15, where the three numbers represent the mass fractions of N, P, and K, respectively. Any compound fertilizer that meets the above requirements for the mass fractions of N, P, and K can be used in this embodiment.

[0039] The present invention will be further illustrated below with reference to the embodiments: Example 1: Screening of bacterial strains Twelve Bacillus subtilis strains isolated from Yichang's traditional food "douchi" (fermented black soybeans) were selected to screen for dominant bacteria that produce high levels of polyglutamic acid or organic acids. Since polyglutamic acid production requires a neutral pH, while organic acid production lowers the pH and inhibits polyglutamic acid synthesis, the strains were screened using two different culture media.

[0040] Screening of polyglutamic acid-producing strains: Twelve isolated Bacillus subtilis strains were cultured in shake flasks in a medium containing monosodium glutamate (MSG). The medium consisted of 60 g / L glucose, 60 g / L MSG, 2 g / L potassium dihydrogen phosphate, 2 g / L magnesium sulfate, and 5 g / L yeast extract. The pH was adjusted to 7 with sodium hydroxide solution, and the pH was kept constant at 7.0 with ammonia. The culture was carried out at 37℃ and 200 rpm for 24 h. The strain with the highest polyglutamic acid content was selected as the target strain.

[0041] Screening of organic acid-producing bacteria: Twelve isolated Bacillus subtilis strains were cultured in shake flasks. The culture medium consisted of 60 g / L glucose, 2 g / L potassium dihydrogen phosphate, 2 g / L magnesium sulfate, and 5 g / L yeast extract. The pH was adjusted to 7 with sodium hydroxide solution and allowed to decrease naturally. The culture was carried out at 37℃ and 200 rpm for 24 h. The strain with the lowest pH in the fermentation broth was selected as the target strain.

[0042] Conclusion: Strain 88-2 exhibited the strongest polyglutamic acid (PCA) production capacity, followed by 88-10. Strain 88-8 had the lowest fermentation pH, indicating the strongest acid production capacity, followed by 88-2, with 88-12 showing the weakest. Based on comprehensive analysis, it is planned to select strain 88-2 for application testing in PCA and organic acid production. The preservation number for strain 88-2 is CCTCC NO: M20251821.

[0043] Table 1. Production capacity of polyglutamic acid and organic acids by each strain

[0044] Example 2: Fermentation of polyglutamic acid by Bacillus subtilis

[0045] 2.1 Fermentation with 7% polyglutamic acid

[0046] Bacillus subtilis CCTCC NO: M 20251821 was activated, inoculated into LB slant seed medium, and incubated at 37℃ for 18-24 hours in a constant temperature incubator. Under aseptic conditions, the slant culture was inoculated into the primary seed culture medium and cultured at 37°C for 24 hours to obtain the primary seed liquid; The primary seed culture was inoculated into the secondary seed culture medium at an inoculation rate of 5%, and cultured at 37°C with shaking until the OD600 value reached 5-7 to obtain the secondary seed culture. The slant seed culture medium is LB medium; The components of the primary and secondary seed culture media were: glucose 20 g / L, sodium glutamate 5 g / L, yeast extract 3 g / L, potassium dihydrogen phosphate 1 g / L, magnesium sulfate 0.8 g / L, with the initial pH controlled at 7.0. The fermentation medium consisted of: 80 g / L glucose, 80 g / L sodium glutamate, 20 g / L yeast extract, 2 g / L potassium dihydrogen phosphate, and 2 g / L magnesium sulfate heptahydrate, with the pH maintained at a constant 7.0.

[0047] The secondary seed culture of Bacillus subtilis was transferred at an inoculum rate of 10% into the fermentation medium in a 50L fermenter for further cultivation. The culture was maintained at pH 7.0, temperature 37℃, and pressure 0.05MPa. Aeration was controlled by adjusting the airflow and stirring speed in conjunction with dissolved oxygen levels to maintain a dissolved oxygen content >20%. The reducing sugar concentration in the fermentation broth was maintained at no less than 1.5% by adding a 30% glucose solution. The pH was adjusted to 7.0 by adding ammonia. The culture time was 50 hours, yielding a microbial inoculum rich in γ-polyglutamic acid, with a viable Bacillus subtilis count of 5.3 × 10⁻⁶ cells / year. 8 CFU / ml, polyglutamic acid production reached 7.3%.

[0048] 2.2 Fermentation with 6% polyglutamic acid

[0049] Bacillus subtilis CCTCC NO: M 20251821 was activated, inoculated into LB slant seed medium, and incubated at 37℃ for 18-24 hours in a constant temperature incubator. Under aseptic conditions, the slant culture was inoculated into the primary seed culture medium and cultured at 37°C for 24 hours to obtain the primary seed liquid; The primary seed culture was inoculated into the secondary seed culture medium at an inoculation rate of 5%, and cultured at 37°C with shaking until the OD600 value reached 5-7 to obtain the secondary seed culture. The slant seed culture medium is LB medium; The components of the primary and secondary seed culture media were: glucose 20 g / L, sodium glutamate 5 g / L, yeast extract 3 g / L, potassium dihydrogen phosphate 1 g / L, magnesium sulfate 0.8 g / L, with the initial pH controlled at 7.0. The fermentation medium consisted of: 60 g / L glucose, 60 g / L sodium glutamate, 10 g / L yeast extract, 2 g / L potassium dihydrogen phosphate, and 2 g / L magnesium sulfate heptahydrate, with the pH maintained at a constant 7.0.

[0050] The secondary seed culture of Bacillus subtilis was transferred at a 5% inoculum to the fermentation medium in a 50L fermenter for further cultivation. The pH was maintained at 7.0, the temperature at 37℃, and the pressure at 0.05MPa. Aeration was controlled by adjusting the airflow and stirring speed in conjunction with dissolved oxygen levels to maintain a dissolved oxygen content >20%. The reducing sugar concentration in the fermentation broth was maintained at no less than 1.5% by adding a 30% glucose solution. The pH was adjusted to 7.0 by adding ammonia water. The cultivation time was 50 hours, yielding a microbial inoculum rich in γ-polyglutamic acid (labeled as organic water-soluble fertilizer). The viable Bacillus subtilis count was 6.6 × 10⁶. 8 The cfu / ml polyglutamic acid yield reached 6.2%.

[0051] Example 3: Fermentation of organic acids by Bacillus subtilis in a fermenter

[0052] 3.1 Fermentation with 1.3% organic acid content

[0053] Bacillus subtilis CCTCC NO: M 20251821 was activated, inoculated into LB slant seed medium, and incubated at 37℃ for 18-24 hours in a constant temperature incubator. Under aseptic conditions, the slant culture was inoculated into the primary seed culture medium and cultured at 37°C for 24 hours to obtain the primary seed liquid; The primary seed culture was inoculated into the secondary seed culture medium at an inoculation rate of 5%, and cultured at 37°C with shaking until the OD600 value reached 5-7 to obtain the secondary seed culture. The slant seed culture medium is LB medium; The components of both the primary and secondary seed culture media were: glucose 20 g / L, yeast extract 3 g / L, potassium dihydrogen phosphate 1 g / L, and magnesium sulfate 0.8 g / L, with the initial pH controlled at 7.0. The fermentation medium consisted of: 60 g / L glucose, 10 g / L yeast extract, 2 g / L potassium dihydrogen phosphate, and 2 g / L magnesium sulfate heptahydrate, with a natural pH.

[0054] The secondary seed culture of Bacillus subtilis was transferred to the fermentation medium in a 50L fermenter at an inoculation rate of 3% and cultured for a further period of time. The pH was 7.0, the temperature was 37℃, the pressure was 0.05MPa, and the dissolved oxygen was controlled by adjusting the air volume and stirring speed. The dissolved oxygen was controlled to be <10%. The concentration of reducing sugar in the fermentation broth was controlled to be no less than 1.5% by adding 30% glucose solution. The culture time was 50h, and the content of organic acids (lactic acid and acetic acid) reached 1.3%.

[0055] 3.2 Fermentation with 1.6% organic acid content

[0056] Bacillus subtilis CCTCC NO: M 20251821 was activated, inoculated into LB slant seed medium, and incubated at 37℃ for 48-72h in a constant temperature incubator. Under aseptic conditions, the slant culture was inoculated into the primary seed culture medium and cultured at 37°C for 24 hours to obtain the primary seed liquid; Inoculate the primary seed culture into the secondary seed culture medium at an inoculation rate of 6-8%, and culture at 37°C with shaking until the OD600 value is 5-7 to obtain the secondary seed culture. The slant seed culture medium is LB medium; The components of both the primary and secondary seed culture media were: glucose 20 g / L, yeast extract 3 g / L, potassium dihydrogen phosphate 1 g / L, and magnesium sulfate 0.8 g / L, with the initial pH controlled at 7.0. The fermentation medium consisted of: 80 g / L glucose, 20 g / L yeast extract, 2 g / L potassium dihydrogen phosphate, 2 g / L magnesium sulfate heptahydrate, and natural pH.

[0057] The secondary seed culture of Bacillus subtilis was transferred to the fermentation medium in a 50L fermenter at an inoculation rate of 8% and cultured for a further period of time. The pH was 7.0, the temperature was 37℃, the pressure was 0.05MPa, and the dissolved oxygen was controlled by adjusting the air volume and stirring speed. The dissolved oxygen was controlled to be <10%. The concentration of reducing sugar in the fermentation broth was controlled to be no less than 1.5% by adding 30% glucose solution. The culture time was 50h, and the content of organic acids (lactic acid and acetic acid) reached 1.6%.

[0058] Example 4: Production of organic acids in soil by liquid microbial agents

[0059] The reference for the detection method of organic acids in soil is "High Performance Liquid Chromatography for Simultaneous Determination of Multiple Organic Acids in Soil".

[0060] The soil pH test method refers to the national standard "Determination of Soil pH Value by Potential Method" (HJ 962-2018).

[0061] The detection methods for phosphorus and potassium in maize seeds refer to "NY / T 2017-2011 Determination of Nitrogen, Phosphorus and Potassium in Plants".

[0062] 4.1 pH test of saline-alkali soil

[0063] A sample of saline-alkali soil was taken from a certain location, and the pH was determined to be 8.18 using the national standard HJ 962-2018 method. The soil was then sieved and transferred into pot A, which was placed outdoors in a place with sunlight and ventilation.

[0064] The soil was sieved and thoroughly mixed with the liquid microbial inoculant in section 2.2 of Example 2 (the inoculant dosage was 2800 kg·hm²). -2 Then move it into pot B and place it outdoors in a place with sunlight and ventilation.

[0065] After a period of time, take a sample of the soil from the potted plant to test the pH. A The pH of the soil in pot A is given by pH. B The pH value is the soil pH in pot B of the potted plant.

[0066] Table 2. pH values ​​of soil in potted plants

[0067] Table 2 shows that the soil pH of the control group (potted plants A) without added microbial agent showed a slight upward trend over time, gradually increasing from an initial 8.18 to 8.25. This may be related to the ion changes in the saline-alkali soil during natural evaporation and concentration. In contrast, the soil pH of the treatment group (potted plants B) with added Bacillus subtilis liquid microbial agent showed a significant downward trend. In the first week, the soil pH of the treatment group had dropped to 7.72, 0.49 units lower than the control group. By the third week, the soil pH of the treatment group had further decreased to 7.55, a decrease of 0.63 units from the initial value, and remained lower than the pH of the control group at the same time point. This result indicates that the Bacillus subtilis agent described in this invention, after being applied to saline-alkali soil, can continuously produce organic acids through metabolic activity, effectively neutralizing alkaline substances in the soil, thereby significantly reducing the pH of the saline-alkali soil and improving the soil acid-base environment. As the duration of action increases, the alkalinity-reducing effect becomes more pronounced, demonstrating that the microbial agent has good environmental adaptability and continuous improvement capabilities, creating a more suitable slightly acidic to neutral soil environment for subsequent plant root growth.

[0068] 4.2 Organic acid production in soil by microbial agents

[0069] For the control group, 5.00 g of soil sample from a cornfield without microbial inoculant was placed in a 50 mL centrifuge tube. 10 mL of soil extract (50 mM Na₂SO₄ solution, pH adjusted to 2.65 with methanesulfonic acid) was added at a solid-liquid ratio of 1:2. The tube was then placed in a horizontal shaker and shaken at 150 rpm for 1 hour, followed by centrifugation at 5500 rpm for 15 minutes. The supernatant was filtered through a 0.22 μm filter to obtain the soil extract for organic acid analysis. High-performance liquid chromatography (HPLC) was used to detect the content of each organic acid in the soil extract, and the total organic acid (lactic acid, acetic acid) content in the soil was calculated. The organic acid content in the soil was calculated as follows: Soil organic acid content = Soil extract organic acid content × Extract volume × Dilution factor / Sample mass. The calculated organic acid content in the soil was 45 mg / kg.

[0070] Experimental group 1 applied the γ-polyglutamic acid-rich microbial agent obtained in 2.2 of Example 2 to the soil of the cornfield, at a dosage of 1 kg per mu diluted 250 times, and applied fertilizer once every 5-10 days. Soil samples were taken after 180 days. Weigh 5.00g of soil sample into a 50mL centrifuge tube, add 10mL of soil extract (50mM Na2SO4 solution, pH adjusted to 2.65 with methanesulfonic acid) at a solid-liquid ratio of 1:2, place in a horizontal shaker and shake at 150 rpm for 1 hour, then centrifuge at 5500 rpm for 15 min. Filter the supernatant through a 0.22μm filter membrane to obtain the soil extract for quantification of organic acids. Use high performance liquid chromatography (HPLC) to detect the content of each organic acid in the soil extract, and calculate the total organic acid (lactic acid, acetic acid) content in the soil. The organic acid content in the soil is calculated as: organic acid content in soil extract × extract volume × dilution factor / sample mass. The calculated organic acid content in the soil is 580 mg / kg.

[0071] Experimental group 2 applied the γ-polyglutamic acid-rich microbial agent obtained in 2.1 of Example 2 to the soil of the cornfield, at a dosage of 1 kg per mu diluted 250 times, and applied fertilizer once every 5-10 days. Soil samples were taken after 180 days. Weigh 5.00g of soil sample into a 50mL centrifuge tube, add 10mL of soil extract (50mM Na2SO4 solution, pH adjusted to 2.65 with methanesulfonic acid) at a solid-liquid ratio of 1:2, place in a horizontal shaker and shake at 150 rpm for 1 hour, then centrifuge at 5500 rpm for 15 min. Filter the supernatant through a 0.22μm filter membrane to obtain the soil extract for quantification of organic acids. Use high performance liquid chromatography (HPLC) to detect the content of each organic acid in the soil extract, and calculate the total organic acid (lactic acid, acetic acid) content in the soil. The organic acid content in the soil is calculated as: organic acid content in soil extract × extract volume × dilution factor / sample mass. The calculated organic acid content in the soil is 415 mg / kg.

[0072] Example 5: Application in maize cultivation

[0073] For the control group, at maturity, 12 uniformly growing corn plants from different regions were selected. The kernels were manually threshed, with an average kernel weight of 153g per plant. The samples were washed three times with deionized water, sterilized at 105℃ for 30 min, and dried at 75℃ to constant weight. The dry weight was then measured. The samples were ground and analyzed using the methods described in NY / T 2017-2011 "Determination of Nitrogen, Phosphorus, and Potassium in Plants". The average phosphorus content in the corn kernels was 2.5g / kg, and the potassium content was 4.1g / kg.

[0074] At the maturity stage of the experimental group of maize, 12 uniformly growing plants from different regions were selected. The kernels were manually threshed, and the average weight of the kernels per plant was 171.4 g. The samples were washed three times with deionized water, sterilized at 105 ℃ for 30 min, and dried at 75 ℃ to constant weight. The dry weight was then measured. The samples were ground and analyzed using the methods described in "NY / T 2017-2011 Determination of Nitrogen, Phosphorus, and Potassium in Plants". The average phosphorus content was 4.2 g / kg, and the average potassium content was 4.7 g / kg.

[0075] The above results indicate that, compared with the control group, the grain weight of individual maize plants in the experimental group increased significantly, by approximately 12.0%, demonstrating that the application of the Bacillus subtilis inoculant described in this invention can effectively promote maize growth and increase yield. Regarding mineral element content, the phosphorus content in the maize grains of the experimental group reached 4.2 g / kg, an increase of 68.0% compared to the control group (2.5 g / kg); the potassium content reached 4.7 g / kg, an increase of 14.6% compared to the control group (4.1 g / kg). This result confirms that the organic acids and polyglutamic acid produced by the inoculant's metabolism in the soil not only improve the soil's physical and chemical properties but also significantly enhance the soil's activation capacity for insoluble nutrients such as phosphorus and potassium, promoting the absorption and utilization of nutrients by crops.

[0076] Example 6: Application in eggplant cultivation

[0077] (1) Experimental location: at an eggplant plantation

[0078] (2) How to use: Experimental group: conventional fertilization (1.2 tons / mu of manure, 40 kg / mu of 15-15-15) + 10 kg / mu of organic water-soluble fertilizer prepared by microbial agent 2.2 in Example 2, 640 square meters, about 1360 plants; Control group: conventional fertilization (1.2 tons / mu of manure, 40 kg / mu of 15-15-15), 560 square meters, about 1200 plants.

[0079] (3) Results: In terms of appearance, the experimental group plants were thicker, with more abundant leaves and more developed root systems. Tests showed that the mature plant height of the experimental group was 116.32±7.66cm, while the mature plant height of the control group was 109.84±11.79cm.

[0080] The nutritional components of the fruit were tested. The following table shows the indicators for the control group, with the unit being mass fraction (%).

[0081] Table 3. Nutritional composition of eggplant fruit in the control group

[0082] The following table shows the indicators for the experimental group, with the unit being mass fraction (%).

[0083] Table 4 Nutritional composition of eggplant fruit in the experimental group

[0084] Table 5. Statistical analysis of nutritional components of eggplant fruit in the control and experimental groups.

[0085] The results showed that the organic water-soluble fertilizer prepared using the microbial agent in Example 2.2 could promote eggplant growth, increase plant height, promote more vigorous root growth, produce more thick roots, improve fruit nutrition indicators, and increase protein and dry matter accumulation, resulting in higher total nutrients.

[0086] Example 7: Application in chili pepper cultivation

[0087] (1) Experimental location: a chili pepper plantation

[0088] (2) How to use: Experimental group: conventional fertilization (1.2 tons / mu of manure, 40 kg / mu of 15-15-15) + 10 kg / mu of polyglutamic acid-containing organic water-soluble fertilizer prepared by the microbial agent in Example 2.2, 256 square meters, about 544 plants.

[0089] Control group: conventional fertilization (1.2 tons / mu of manure, 40 kg / mu of 15-15-15), 256 square meters, approximately 544 plants.

[0090] (3) Results: In terms of appearance, the experimental group plants were robust, had more abundant leaves, and more developed root systems. The yield statistics for the control and experimental groups are shown in the table below: Table 6. Pepper yield statistics for the experimental and control groups.

[0091] Table 7 Statistical analysis of pepper yield in experimental and control groups

[0092] The results are as follows Figure 1 The results showed that the average plant height of the second group was significantly higher than that of the first group (t = 3.005, df = 68, p < 0.01). A two-sample t-test showed a statistically significant difference between the two groups (p < 0.05). The mean difference was 4.36 cm, and the effect size Cohen's d ≈ 0.66 (moderately large).

[0093] The nutritional components of the fruits in the control group were tested, and the results are shown in the table below.

[0094] Table 8. Nutritional composition of pepper fruits in the control group

[0095] The nutritional components of the fruits from the experimental group were tested, and the results are shown in the table below.

[0096] Table 9 Nutritional components of pepper fruits in the experimental group

[0097] Table 10 Statistical analysis of nutritional components of pepper fruits in the control group and experimental group.

[0098] The results showed that the organic water-soluble fertilizer prepared using the microbial agent in Example 2.2 could promote the growth of chili peppers, increase plant height, improve crop yield, and improve fruit nutritional indicators. The chili peppers accumulated more protein and dry matter, and had more total nutrients.

[0099] Example 8: Application in cucumber cultivation

[0100] (1) Experimental location: a cucumber plantation

[0101] (2) How to use: Experimental group: conventional fertilization (1.2 tons / mu of manure, 40 kg / mu of 15-15-15) + 10 kg / mu of polyglutamic acid-containing organic water-soluble fertilizer prepared by the microbial agent in Example 2.2, 112 square meters, about 256 plants; Control group: conventional fertilization (1.2 tons / mu of manure, 40 kg / mu of 15-15-15), 112 square meters, approximately 256 plants.

[0102] (3) Results: In terms of appearance, the experimental group plants were robust, had more abundant leaves, and more developed root systems. The yield statistics for the control and experimental groups are shown in the table below: Table 11 Cucumber yield statistics for the experimental and control groups

[0103] Conclusion: Based on the yield results of three batches, it is shown that the organic water-soluble fertilizer prepared using the microbial agent 2.2 in Example 2 increased the yield by 6.67% compared with the fertilizer not used.

[0104] Example 9: Application in Citrus Cultivation

[0105] (1) Experimental location: a citrus orchard

[0106] (2) Crop: 4-5 year old citrus, variety: Ehime jelly orange

[0107] (3) How to use: Experimental group: 0.5 kg / plant of organic water-soluble fertilizer prepared by the microbial agent in Example 2.2 + 0.48 kg / plant of compound fertilizer 15-5-25, 10 mu, totaling 500 plants.

[0108] Control group: 0.48 kg / plant of compound fertilizer 15-5-25.

[0109] (4) Experimental results

[0110] The fruit quality is shown in the table below: Table 12 Citrus fruit quality of experimental and control groups

[0111] Conclusion: Using the microbial agent 2.2 in Example 2 to prepare organic water-soluble fertilizer can improve citrus yield and fruit quality, resulting in darker green and more resilient citrus leaves, higher sugar content, higher single fruit weight, and increased fruit diameter.

[0112] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Bacillus subtilis with accession number CCTCC NO: M 20251821 ( Bacillus subtilis ).

2. The Bacillus subtilis according to claim 1, characterized in that, After liquid fermentation, the Bacillus subtilis produces a fermentation product containing not less than 6% polyglutamic acid or not less than 1% organic acid.

3. A microbial agent, the raw materials for which are prepared include Bacillus subtilis as described in claim 1.

4. The microbial agent according to claim 3, characterized in that, The viable count of Bacillus subtilis in the bacterial agent is not less than 10. 8 The cfu / ml concentration and the mass fraction of polyglutamic acid are not less than 6%.

5. A method for preparing the bacterial agent according to claim 3 or 4, comprising fermenting the Bacillus subtilis according to claim 1 with a culture medium; wherein the culture medium comprises water and the following components in mass fractions: glucose 60-80 g / L, sodium glutamate 60-80 g / L, yeast extract 10-20 g / L, potassium dihydrogen phosphate 2 g / L, magnesium sulfate heptahydrate 2 g / L, and pH 7.

0.

6. The preparation method according to claim 5, characterized in that, The fermentation conditions include: temperature 37℃, pressure 0.05MPa, dissolved oxygen >20%, glucose solution added to control the reducing sugar concentration to be not less than 1.5%, ammonia added to adjust the pH to 7.0, and culture time 50h.

7. The preparation method according to claim 5 or 6, characterized in that, The process before fermentation also includes steps of slant activation, primary seed activation, and secondary seed activation. The slant seed culture medium is LB medium; The culture medium for primary and secondary seed activation was: glucose 20 g / L, sodium glutamate 5 g / L, yeast extract 3 g / L, potassium dihydrogen phosphate 1 g / L, magnesium sulfate 0.8 g / L, with an initial pH of 7.

0.

8. The application of Bacillus subtilis according to claim 1 or 2, the microbial agent according to claim 3 or 4, or the microbial agent prepared by the preparation method according to claim 5 or 6 in improving crop yield or crop quality; The crop is corn, and the effects of the Bacillus subtilis or inoculant include: Increase crop weight and improve P / K content; The crop is eggplant, and the effects of the Bacillus subtilis or inoculant include: increasing the plant height of eggplant and increasing the N / P / K content of eggplant; The crop is chili pepper, and the effects of the Bacillus subtilis or inoculant include: increasing the protein content of chili pepper and increasing the N / P / K content of the crop. The crop is cucumber, and the effects of the Bacillus subtilis or inoculant include: increasing cucumber yield; The crop is citrus, and the effects of the Bacillus subtilis or inoculant include: increasing the sugar content and / or weight of the citrus.

9. A method for planting a crop, characterized in that, This includes applying the microbial agent according to claim 3 or 4 to the soil, or the microbial agent prepared by the preparation method according to claim 5 or 6.

10. The method according to claim 9, characterized in that, The application rate is 1 kg per acre, diluted 250 times and applied by irrigation every 5-10 days.

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