Bacillus velezensis and fermentation production method, bacterial agent and application thereof

Abstract

This invention belongs to the field of microbial technology, specifically relating to a strain of Bacillus belye and its fermentation production method, inoculum, and applications. Experiments have demonstrated that the Bacillus belye SDU1 provided by this invention possesses broad tolerance to acidity and alkalinity, growth temperature, and high salt, and simultaneously exhibits the ability to produce high levels of γ-polyglutamic acid and lipopeptides. It can be applied to promising applications such as saline-alkali soil improvement, microbial oil displacement agents in high-salt-alkali oil reservoir environments, and can also be used to prepare biocontrol agents for inhibiting plant and animal diseases, pharmaceutical carriers, cosmetic moisturizers, food thickeners, and environmentally friendly preparations, thus possessing significant practical application value.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of Bacillus belye and its fermentation production method, inoculum, and application. Background Technology

[0002] In the processes of agricultural production, livestock breeding, and energy production, the widespread use of chemical products such as pesticides, fertilizers, and antibiotics, while solving some problems, has also led to ecological and environmental issues and human health problems, including the emergence of drug-resistant bacteria and environmental pollution. Therefore, promoting green, low-carbon production and sustainable development has become an industry consensus. Biotechnology solutions centered on microorganisms are considered an important pathway to achieving green and sustainable development in agriculture, animal husbandry, and industry.

[0003] Existing research indicates that Bacillus belyssus ( Bacillus velezensis This species can produce γ-polyglutamic acid (γ-PGA), lipopeptides, and enzyme preparations, and has wide applications in agricultural growth promotion and disease resistance, petroleum enhanced oil recovery, and alkane degradation. However, most of the reported strains currently available have only one function, or while possessing multiple functions, their effects are limited. They generally suffer from high production costs and poor environmental adaptability, making it difficult to meet the needs of large-scale production and diversified application scenarios. For example, existing strains often perform poorly in key properties such as γ-PGA yield, lipopeptide yield, salt tolerance, and antibacterial spectrum, limiting their promotional value in actual production. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a strain of *Bacillus belye*, its fermentation production method, inoculum, and applications. Specifically, experimental verification shows that this strain exhibits broad-spectrum environmental adaptability, can simultaneously produce high levels of γ-polyglutamic acid and lipopeptides, and not only shows significant inhibitory effects against both bacteria and fungi but also possesses excellent emulsifying properties. Based on these research findings, this invention is thus completed.

[0005] To achieve the above-mentioned technical objectives, the present invention relates to the following technical solutions: In a first aspect, the present invention provides a strain of Bacillus belye ( Bacillus velezensis SDU1, this strain was deposited on December 25, 2025 at the China General Microbiological Culture Collection Center (address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, China), with the biological accession number CGMCC No. 37196.

[0006] A second aspect of the present invention provides a fermentation production method for the above-mentioned Bacillus belye, the fermentation production method comprising: inoculating the Bacillus belye into a fermentation medium for fermentation culture.

[0007] A third aspect of the present invention provides a microbial agent containing the aforementioned Bacillus belye or its fermentation product or its metabolites.

[0008] A fourth aspect of the present invention provides the use of the above-described Bacillus belye SDU1 and / or the above-described inoculum in any one or more of the following (a)-(c): (a) Preparation of biocontrol agents to inhibit pathogens; (b) Fermentation production of γ-polyglutamic acid and lipopeptides; (c) Extraction of crude oil from oil fields.

[0009] In application (a), the pathogen can be bacteria or fungi. The bacteria include, but are not limited to, Staphylococcus aureus and Pseudomonas aeruginosa, and the fungi are Aspergillus niger.

[0010] In application (b), the lipopeptide substance includes surfactants.

[0011] In application (c), the application is specifically as a crude oil displacement agent for the extraction of crude oil in oil fields, wherein the application environment can be a saline-alkali environment.

[0012] This invention demonstrates through research that this strain has excellent environmental adaptability, effectively tolerating sodium chloride concentrations not exceeding 150 g / L. Furthermore, the growth of the strain is not significantly affected at pH levels of 5-9, and it can grow and reproduce in environments ranging from 20-50°C, which is highly beneficial for its practical application.

[0013] The beneficial technical effects of one or more of the above technical solutions are as follows: The *Bacillus vesiculosus* SDU1 strain provided by the above-mentioned technical solution exhibits excellent broad-spectrum environmental adaptability, tolerating high salinity, wide pH, and wide temperature conditions, making it particularly suitable for extreme environments such as saline-alkali land and high-salt oil reservoirs. Furthermore, *Bacillus vesiculosus* SDU1 is a multifunctional strain that produces a variety of bioactive substances, simultaneously producing high levels of γ-polyglutamic acid and lipopeptides, significantly reducing the production cost of multi-target products. Moreover, experiments have demonstrated that the fermentation supernatant of this strain not only has significant inhibitory effects on *Staphylococcus aureus*, *Pseudomonas aeruginosa*, and *Aspergillus niger*, but also possesses excellent emulsifying properties, making it a superior biocontrol agent and oil displacement agent.

[0014] In summary, the above technical solutions effectively broaden the application scope of Bacillus belyss in the field of biotechnology, providing high-quality strain resources for green agriculture, environmental governance and energy extraction, and have great application value and market development potential. Attached Figure Description

[0015] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0016] Figure 1 This is a transmission electron microscope image of Bacillus belye SDU1 in Example 1 of the present invention.

[0017] Figure 2 The growth curves of Bacillus belye SDU1 under different concentrations of sodium chloride in Example 2 of this invention are shown.

[0018] Figure 3 The growth curves of Bacillus belyssus SDU1 at different pH values ​​are shown in Example 2 of this invention.

[0019] Figure 4 The growth curves of Bacillus belyssus SDU1 at different temperatures are shown in Example 2 of this invention.

[0020] Figure 5 This is a photograph of the product obtained after γ-polyglutamic acid extraction in Example 3 of the present invention. Detailed Implementation

[0021] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0022] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0023] In a typical embodiment of the present invention, a strain of Bacillus belye is provided ( Bacillus velezensis SDU1, this strain was deposited on December 25, 2025 at the China General Microbiological Culture Collection Center (address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, China), with the biological accession number CGMCC No. 37196.

[0024] When this strain is cultured on solid plates, a viscous substance adheres to the plate surface and inverted, sticky droplets appear.

[0025] Bacillus belye SDU1 can utilize glucose, lactose, sucrose, glycerol, xylose, esculin, mannitol, salicin, and cellobiose. It exhibits motility, starch hydrolysis, nitrate reduction, and anaerobic growth, but cannot utilize maltose and propionate. Furthermore, it is oxidase-negative, catalase-positive, urease-positive, Simon's citrate-negative, and lipase-negative.

[0026] Bacillus belye SDU1 can tolerate sodium chloride concentrations up to 150 g / L and grows well in environments with pH values ​​of 5-9. The optimal temperature range for optimal growth is 20℃-50℃. Therefore, its salt and alkali tolerance and wide temperature adaptability make it valuable for applications in saline-alkali environments.

[0027] In another specific embodiment of the present invention, a fermentation production method for the above-mentioned Bacillus belye SDU1 is provided, the fermentation production method comprising: inoculating the Bacillus belye SDU1 into a fermentation medium for fermentation culture.

[0028] In this invention, no specific limitation is made to the fermentation production method; any conventional bacterial fermentation culture method can be used for cultivation.

[0029] In one specific embodiment of the present invention, the specific steps of the fermentation production method include: inoculating activated Bacillus belye SDU1 into a fermentation medium for aerobic fermentation.

[0030] The specific conditions for the aerobic fermentation are: 30-40℃ (preferably 37℃), ventilation volume 1-1.6 vvm, rotation speed 350-600 rpm, and tank pressure not higher than 0.05 MPa.

[0031] The fermentation medium can be a common bacterial culture medium. In one specific embodiment of the present invention, the fermentation medium formula is as follows: glucose 30-50 g / L, yeast extract 3-5 g / L, peptone 5-10 g / L, potassium nitrate 4-8 g / L, dipotassium hydrogen phosphate 3-5 g / L, potassium dihydrogen phosphate 1-2 g / L, magnesium sulfate heptahydrate 1-2 g / L, potassium chloride 2-3 g / L, sodium chloride 10-50 g / L, and calcium chloride dihydrate 0.05-0.1 g / L. Using the above fermentation production method helps to produce high yields of γ-polyglutamic acid and lipopeptides.

[0032] In another specific embodiment of the present invention, a microbial agent is provided, which contains the Bacillus belye SDU1 or its fermentation product or its metabolites.

[0033] In this invention, the term "fermentation product" is used to refer to fermentation products. The corresponding fermentation product can be a liquid obtained from the fermentation culture of *Bacillus belye* SDU1, and therefore can also be called fermentation broth; the liquid may contain bacteria (cells), but does not necessarily need to contain bacteria. The liquid preferably contains metabolites produced by *Bacillus belye* SDU1 of this invention. The fermentation product or metabolites include γ-polyglutamic acid and lipopeptides.

[0034] Furthermore, in embodiments of the present invention, the fermentation broth or culture medium containing bacterial cells is separated from the liquid by centrifugation, filtration, sedimentation, or other means known in the art. The liquid remaining after removing the bacterial cells is called the "supernatant," and in the present invention, the supernatant contains extracellular metabolites of Bacillus belyssus SDU1. In embodiments of the present invention, the bacterial agent may also contain this supernatant.

[0035] Furthermore, in embodiments of the present invention, the fermentation broth or culture medium containing bacterial cells is centrifuged, filtered, settled, or otherwise known in the art to separate the bacterial cells grown in the fermentation broth or culture medium from the liquid to obtain bacterial cells. The bacterial cells can be broken up to obtain bacterial fragments. The breaking method can be ultrasound (e.g., ice bath ultrasound to break up cells) or other methods known in the art. Alternatively, the bacterial fragments can be centrifuged to collect the supernatant, which is designated as the cell-free extract. In the present invention, the bacterial fragments or cell-free extract contain intracellular metabolites of Bacillus belyssus SDU1. In embodiments of the present invention, the bacterial agent may also contain the bacterial fragments or cell-free extract.

[0036] Furthermore, in embodiments of the present invention, for ease of storage and transportation, and to improve the survival rate of the bacterial strain, the bacterial agent may also be a solid, and more preferably a freeze-dried powder. That is, it is obtained by further freeze-drying the aforementioned Bacillus belye SDU1 or its fermentation product or its metabolites. The freeze-drying technology (including vacuum freeze-drying technology) can be carried out using conventional methods, and will not be elaborated further here.

[0037] In another specific embodiment of the present invention, the microbial agent may further include excipients acceptable to the microbial agent.

[0038] In another specific embodiment of the present invention, the excipients are selected from one or more of dispersants, wetting agents, disintegrants, binders, defoamers, antifreeze agents, thickeners, fillers, and solvents. The present invention does not impose any special restrictions on the sources of acceptable excipients for the bacterial agent; commercially available products are generally sufficient.

[0039] In another specific embodiment of the present invention, the above-mentioned Bacillus belye SDU1 and / or the above-mentioned bacterial agent are provided for use in any one or more of the following (a)-(c): (a) Preparation of biocontrol agents to inhibit pathogens; (b) Fermentation production of γ-polyglutamic acid and lipopeptides; (c) Extraction of crude oil from oil fields.

[0040] In application (a), the pathogen can be bacteria or fungi. The bacteria include, but are not limited to, Staphylococcus aureus and Pseudomonas aeruginosa, and the fungi are Aspergillus niger.

[0041] In application (b), the lipopeptide substances include surfactants. It should be noted that, due to the wide range of applications of γ-polyglutamic acid and lipopeptides, which have significant value in agriculture, medicine, daily chemicals, food, oil extraction, and environmental protection, this strain can be used as a soil conditioner for saline-alkali land, an antibacterial agent for animal and plant breeding, and an oil displacement agent in oil extraction. It can also be used to produce pharmaceutical carriers, cosmetic moisturizers, and food thickeners, etc., without specific limitations.

[0042] In application (c), the application is specifically as a crude oil displacement agent for the extraction of crude oil in oil fields, wherein the application environment can be a saline-alkali environment.

[0043] The following examples further illustrate the present invention, but do not constitute a limitation thereof. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0044] Soil samples were taken from saline-alkali soil in the sweet sorghum planting area of ​​Dongying, with a salt content of 0.8%.

[0045] The culture medium used in the examples is as follows: Enrichment medium: glucose 10 g / L, yeast extract 2 g / L, sodium chloride 100.0 g / L, potassium nitrate 5 g / L, magnesium sulfate heptahydrate 0.5 g / L, dipotassium hydrogen phosphate 1 g / L, ferrous sulfate heptahydrate 5 mg / L, pH = 6.0-7.5.

[0046] Initial screening culture medium: glucose 20 g / L, yeast extract 2 g / L, peptone 10 g / L, sodium chloride 100 g / L, potassium nitrate 2 g / L, dipotassium hydrogen phosphate 2 g / L, pH = 5.5-7.5.

[0047] Activation medium: yeast extract 5 g / L, peptone 10 g / L, sodium chloride 30 g / L, natural pH.

[0048] Fermentation medium: glucose 30-50 g / L, yeast extract 3-5 g / L, peptone 5-10 g / L, potassium nitrate 4-8 g / L, dipotassium hydrogen phosphate 3-5 g / L, potassium dihydrogen phosphate 1-2 g / L, magnesium sulfate heptahydrate 1-2 g / L, potassium chloride 2-3 g / L, sodium chloride 10-50 g / L, calcium chloride dihydrate 0.05-0.1 g / L, pH = 6.8-7.5.

[0049] All the above culture media were sterilized at 121℃ for 20 min.

[0050] Reagents and equipment not labeled with manufacturers or brands in the examples can all be found and substituted on the market.

[0051] Example 1: Isolation and identification of Bacillus belyssus SDU1 Two g of soil sample was placed in 10 mL of sterile physiological saline and shaken at 20°C and 200 rpm for 6 h. Then, 5 mL of the sample was transferred to 200 mL of sterile enrichment medium and incubated at 30-37°C and 150-200 rpm for 2-3 days. The sample was then inoculated again into the enrichment medium at an inoculation rate of approximately 5%, and this enrichment process was repeated three times. The resulting culture was then spread onto solid primary screening medium and incubated upside down at 37°C for 2 days. Large, plump, and viscous colonies were selected for streak plating. One strain was observed that, after 2 days of incubation, produced a viscous coating and formed inverted, sticky droplets on the plate surface. This strain was purified and transferred to activation medium, incubated at 37°C and 200 rpm for 18 h. Molecular biological identification was performed, and its 16S rDNA sequence is shown in SEQ ID NO.1.

[0052] After culturing on solid activation medium for 28 h, the cell morphology was observed using transmission electron microscopy. The strain was rod-shaped with flagella, and a layer of substance adhered to the cell surface after culture. Figure 1 ).

[0053] Physiological and biochemical tests were performed on this strain using HBI Bacillus biochemical identification strips and other biochemical identification tubes (Haibo Biotechnology Co., Ltd.). The results showed that it can utilize glucose, lactose, sucrose, glycerol, xylose, esculin, mannitol, salicin, and cellobiose, and possesses the ability to motility, starch hydrolysis, nitrate reduction, and anaerobic growth, but cannot utilize maltose and propionate. Furthermore, it was negative for oxidase, positive for catalase, positive for urease, negative for Simon's citrate, and negative for lipase. Based on the combined identification results, this strain is identified as *Bacillus belye* (…). Bacillus velezensis It was named Bacillus belyssus SDU1.

[0054] Example 2 Environmental adaptability of Bacillus belyssus SDU1 Activation media with sodium chloride concentrations of 30, 100, 120, and 150 g / L were prepared, and the strains were cultured at pH 7.0, 37°C, and 200 rpm. For acid-base tolerance experiments, the initial pH of the sterilized activation media was adjusted to 5.0, 7.0, and 9.0 using HCl or NaOH, and the strains were cultured at 37°C and 200 rpm. Activation media with pH 7.0 was prepared, and the strains were cultured at 20, 30, 37, and 50°C, respectively, at 200 rpm. The inoculum size for the above environmental tolerance experiments was 2% (OD). 600 = 1), periodically sample and measure the optical density value of the bacterial solution at 600 nm, and plot the growth curve.

[0055] The strain's tolerance to different concentrations of sodium chloride, such as Figure 2 As shown, when the sodium chloride concentration was 100 g / L, the cell density of the strain at the stationary phase was approximately 60% of that at 30 g / L sodium chloride; when the sodium chloride concentration was 150 g / L, the growth of the strain was significantly inhibited. Figure 2 Therefore, this strain can tolerate sodium chloride concentrations not exceeding 150 g / L. With increasing sodium chloride concentration, the biomass of the strain decreases and the lag phase of strain growth increases.

[0056] The growth of the strain was not significantly affected when the pH was between 5 and 9, except that the lag phase of the strain increased at pH = 9. Figure 3 This indicates that the Bacillus berberis SDU1 provided by this invention can tolerate saline-alkali environments, which is of great significance for the development and application of biotechnology in high-salt-alkali environments.

[0057] Growth curves of strains at different temperatures are as follows Figure 4 As shown, under 20℃ incubation conditions, the strain began to grow slowly after 6 hours of incubation, and the final growth rate was close to that under 30℃ and 37℃ incubation conditions. Although the strain cultured at 30℃ had the highest cell density, the final growth rate was consistent with that under 37℃ incubation conditions. The strain cultured at 50℃ reached the stationary phase more quickly, but the cell density was approximately 50% of that under 37℃ incubation conditions. Therefore, high temperatures severely affected the growth of this bacterium, while low temperatures increased the lag phase and the time to reach the stationary phase.

[0058] Example 3: Fermentation of Bacillus belye SDU1 to produce γ-polyglutamic acid Strain activation: Bacillus berleis SDU1 stored at -80℃ was spread on solid activation medium and incubated upside down at 37℃ for 18 h.

[0059] Seed culture: Three single colonies were transferred to liquid activation medium and cultured at 37°C and 180 rpm for 16 h.

[0060] Fermentation in a fermenter: In a 3 L fermenter, the seed culture was transferred to the liquid fermentation medium at an inoculum rate of 2% (v / v). The fermentation was carried out at 37°C with an aeration rate of 1-1.6 vvm, a rotation speed of 350-600 rpm, and a pressure not exceeding 0.05 MPa. Fermentation was continued for 42 h, with no pH control during the process. The fermentation medium consisted of 30 g / L glucose, 5 g / L yeast extract, 10 g / L peptone, 5 g / L potassium nitrate, 3 g / L dipotassium hydrogen phosphate, 1 g / L potassium dihydrogen phosphate, 1 g / L magnesium sulfate heptahydrate, 2 g / L potassium chloride, 30 g / L sodium chloride, 0.08 g / L calcium chloride dihydrate, and a pH of 7.2.

[0061] The concentration of γ-PGA in the fermentation broth was measured by taking 10 mL samples at 18 h, 24 h, 32 h and 42 h of culture.

[0062] The extraction method for γ-PGA from the fermentation broth was as follows: the fermentation broth was centrifuged at 10,000 rpm at room temperature for 3 min, the supernatant was collected, 4 volumes of pre-cooled methanol at 4℃ were added, and the mixture was shaken thoroughly and allowed to stand overnight at 4℃. The precipitate was then centrifuged at 10,000 rpm at room temperature for 5 min, dried at 55℃ for 30 min to obtain crude γ-PGA, weighed, and reconstituted with 50 mL of ultrapure water.

[0063] Qualitative analysis was performed according to NY / T 3039-2016, using an amino acid analyzer to determine the amino acid composition of the monomers. The results showed that glutamic acid accounted for 94.13% of the extracted fermentation product.

[0064] Quantitative detection was performed using ultraviolet spectrophotometry. Specifically, the γ-PGA standard solution was scanned within the wavelength range of 190-600 nm to determine the maximum absorption peak of γ-PGA, which was found to be at 209 nm. Using distilled water as a blank, the absorbance values ​​of 0, 10, 20, 40, 80, and 100 mg / L γ-PGA standard solutions were measured, and a standard curve for γ-PGA was plotted. The concentration of γ-PGA at different fermentation time points is shown in Table 1. After 32 h of fermentation, the yield of γ-PGA tended to stabilize, and the purity of γ-PGA in the crude product ranged from 97.21% to 99.80%.

[0065] Table 1. γ-PGA production at different time points

[0066] The culture medium composition was further optimized, and the concentration of γ-PGA was measured at 18 h, 32 h, and 42 h of culture. The results are shown in Table 2. Under the conditions of 50 g / L glucose, 5 g / L yeast extract, 10 g / L peptone, and 8 g / L potassium nitrate as the main components, the highest yield of γ-PGA reached 10.85 g / L. The crude γ-PGA product after fermentation was white (…). Figure 5 ).

[0067] Table 2. Yield of γ-PGA under different culture media and time points.

[0068] Fermentation media with different compositions are as follows: Fermentation medium 1: glucose 30 g / L, yeast extract 5 g / L, peptone 10 g / L, potassium nitrate 8 g / L, dipotassium hydrogen phosphate 3 g / L, potassium dihydrogen phosphate 1 g / L, magnesium sulfate heptahydrate 1 g / L, potassium chloride 2 g / L, sodium chloride 10 g / L, calcium chloride dihydrate 0.1 g / L, pH = 6.8-7.2.

[0069] Fermentation medium 2: glucose 50 g / L, yeast extract 3 g / L, peptone 8 g / L, potassium nitrate 8 g / L, dipotassium hydrogen phosphate 3 g / L, potassium dihydrogen phosphate 1 g / L, magnesium sulfate heptahydrate 1 g / L, potassium chloride 2 g / L, sodium chloride 10 g / L, calcium chloride dihydrate 0.1 g / L, pH = 6.8-7.2.

[0070] Fermentation medium 3: glucose 50 g / L, yeast extract 3 g / L, peptone 5 g / L, potassium nitrate 8 g / L, dipotassium hydrogen phosphate 3 g / L, potassium dihydrogen phosphate 1 g / L, magnesium sulfate heptahydrate 1 g / L, potassium chloride 2 g / L, sodium chloride 10 g / L, calcium chloride dihydrate 0.1 g / L, pH = 6.8-7.2.

[0071] Fermentation medium 4: glucose 50 g / L, yeast extract 5 g / L, peptone 10 g / L, potassium nitrate 8 g / L, dipotassium hydrogen phosphate 3.5 g / L, potassium dihydrogen phosphate 1.5 g / L, magnesium sulfate heptahydrate 1.5 g / L, potassium chloride 2 g / L, sodium chloride 10 g / L, calcium chloride dihydrate 0.1 g / L, pH = 6.8-7.2.

[0072] Example 4: Fermentation of Bacillus belyssus SDU1 to produce lipopeptides and its antibacterial effect The process of producing lipopeptides by fermentation of Bacillus belye SDU1 is the same as in Example 3 above. Fermentation is terminated after 32 hours of cultivation, and samples are collected.

[0073] The fermentation medium consisted of: 50 g / L glucose, 5 g / L yeast extract, 10 g / L peptone, 8 g / L potassium nitrate, 3.5 g / L dipotassium hydrogen phosphate, 1.5 g / L potassium dihydrogen phosphate, 1.5 g / L magnesium sulfate heptahydrate, 2 g / L potassium chloride, 10 g / L sodium chloride, 0.1 g / L calcium chloride dihydrate, and a pH of 6.8-7.2.

[0074] Lipopeptide extraction: The fermentation broth was centrifuged at 10,000 rpm and 4°C for 5 min. The supernatant was adjusted to pH 2.0 with 6 mol / L hydrochloric acid and allowed to precipitate overnight at 4°C. The precipitate was then collected by centrifugation at 10,000 rpm and 4°C for 10 min. It was extracted with methanol (1 g: 50 mL), and the crude extract was obtained by rotary evaporation, with a yield of approximately 3.65 g / L.

[0075] The method for quantitative determination of surfactant was as follows: After redissolving the crude extract in methanol, the surfactant content in the crude extract was determined using the following detection parameters: chromatographic column: BEH C18 column (2.1×100mm×1.7μm); column temperature: 30℃; flow rate: 0.3 mL / min; acquisition mode: ESI+; ion spray voltage: 4000 V; ion source temperature: 400℃; desiccator temperature: 300℃; desiccator flow rate: 10.0 L / h; fragmentation voltage: 35 V; collision energy: 5 V. Positive ion scanning mode was used for acquisition, and the quantitative ion concentration was 1035.0. A series of standard solutions of surfactant were prepared with methanol, and the peak intensity of the standards at different concentrations was measured. A standard curve was plotted, and the results showed that the surfactant yield in the crude extract was 1.12 g / L.

[0076] The Oxford cup method was used to evaluate the antibacterial effect of Bacillus belyssioides SDU1 fermentation supernatant against Staphylococcus aureus, Pseudomonas aeruginosa, and Aspergillus niger after 32 h of fermentation. Staphylococcus aureus and Pseudomonas aeruginosa were cultured on commercially available LB premixed medium at 37°C for 24 h. Aspergillus niger was cultured on commercially available PDA premixed medium at 28°C for 48 h.

[0077] As shown in Table 3, the fermentation supernatant of Bacillus vesiculosus SDU1 has a certain inhibitory effect on Staphylococcus aureus, Pseudomonas aeruginosa, and Aspergillus niger.

[0078] Table 3. Inhibitory effect of Bacillus belyssus SDU1 fermentation supernatant on pathogens.

[0079] Example 5: Emulsification performance of Bacillus belyssus SDU1 fermentation supernatant The fermentation process of Bacillus belyssus SDU1 was the same as in Example 3 above, and the fermentation medium and sampling time were the same as in Example 4.

[0080] The emulsifying activity index (E24) was used to evaluate the emulsifying properties of the fermentation supernatant. The fermentation broth was centrifuged at 8000 rpm for 2 min at room temperature. 2 mL of the fermentation supernatant was collected and 2 mL of liquid paraffin was added. The height of the upper organic phase layer was recorded. The mixture was vortexed for approximately 2 min and allowed to stand at room temperature for 24 h. The height of the white emulsion layer was recorded. A blank culture medium was used as a control group, and three replicates were set up. The average value of the results was taken. The E24 was calculated as: E24 = (height of emulsion layer / height of organic phase layer) × 100%.

[0081] The surface tension of the fermentation supernatant and the interfacial tension between the fermentation supernatant and liquid paraffin were measured using a video optical contact angle meter, with water as a control group.

[0082] The results are shown in Table 4. The E24 of the fermentation supernatant of Bacillus vesiculosus SDU1 was 100%, the surface tension was 25.88 mN / m, and the interfacial tension with liquid paraffin was 1.12 mN / m, indicating that it has the performance as a crude oil displacement agent. In particular, due to its tolerance to high salinity and alkalinity environment, it is helpful for the extraction of crude oil in high salinity and alkalinity oil fields.

[0083] Table 4. Evaluation of Emulsification Performance of Fermentation Supernatant

[0084] Therefore, through the above examples of environmental tolerance testing of Bacillus belyssus SDU1, fermentation production of γ-PGA and lipopeptides and their antibacterial effects and emulsification properties of fermentation supernatant, it is shown that the strain SDU1 provided by the present invention has the ability to tolerate salt and alkali and produce high levels of γ-PGA and lipopeptides. It can be applied to saline-alkali land management, microbial fertilizer production, preparation of biocontrol agents to inhibit plant and animal diseases, and can also be used as an oil displacement agent for high-salt and alkali oil reservoirs. It can also be used to produce pharmaceutical carriers, cosmetic moisturizers, food thickeners, environmentally friendly preparations, etc.

[0085] The nucleotide sequence information involved in this invention includes: 16S rDNA sequence of Bacillus belyssus SDU1

[0086] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A strain of Bacillus belye ( Bacillus velezensis SDU1, characterized in that, This strain was deposited at the China General Microbiological Culture Collection Center (CGMCC) on December 25, 2025, with the accession number CGMCC No. 37196.

2. The fermentation production method of Bacillus belyssus SDU1 according to claim 1, characterized in that, The fermentation production method includes: inoculating the Bacillus berberis SDU1 into a fermentation medium for fermentation culture.

3. The fermentation production method as described in claim 2, characterized in that, The fermentation culture is an aerobic fermentation culture, and the specific conditions for the aerobic fermentation culture are: 30-40℃, ventilation rate 1-1.6 vvm, rotation speed 350-600 rpm, and tank pressure not higher than 0.05 MPa.

4. The fermentation production method as described in claim 2, characterized in that, The fermentation medium has the following formula: glucose 30-50 g / L, yeast extract 3-5 g / L, peptone 5-10 g / L, potassium nitrate 4-8 g / L, dipotassium hydrogen phosphate 3-5 g / L, potassium dihydrogen phosphate 1-2 g / L, magnesium sulfate heptahydrate 1-2 g / L, potassium chloride 2-3 g / L, sodium chloride 10-50 g / L, and calcium chloride dihydrate 0.05-0.1 g / L.

5. A microbial agent, characterized in that, The bacterial agent contains Bacillus belyssus SDU1 as described in claim 1, or its fermentation product or its metabolites.

6. The microbial agent as described in claim 5, characterized in that, The fermentation product or the metabolite contains γ-polyglutamic acid and lipopeptides.

7. The use of Bacillus belyssus SDU1 according to claim 1 or the inoculum according to any one of claims 5-6 in any one or more of the following (a)-(c): (a) Preparation of biocontrol agents to inhibit pathogens; (b) Fermentation production of γ-polyglutamic acid and lipopeptides; (c) Extraction of crude oil from oil fields.

8. The application as described in claim 7, characterized in that, In application (a), the pathogen is bacteria and fungi; wherein the bacteria include Staphylococcus aureus and Pseudomonas aeruginosa, and the fungi are Aspergillus niger.

9. The application as described in claim 7, characterized in that, In application (b), the lipopeptide substance includes surfactants.

10. The application as described in claim 7, characterized in that, In application (c), the application environment is a saline-alkali environment.

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