Bacillus subtilis natto and application thereof in production of MK-7
By screening and breeding, Bacillus subtilis WD1, which produces high levels of MK-7, was obtained. Combined with optimized fermentation and purification processes, the problem of insufficient MK-7 yield in existing technologies was solved, and efficient and low-cost MK-7 production was achieved.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-31
- Publication Date
- 2026-04-10
AI Technical Summary
The existing production of MK-7 by Bacillus subtilis natto cannot meet market demand, and existing extraction and chemical synthesis methods suffer from low efficiency, high cost, or environmental pollution.
A high-yielding Bacillus subtilis WD1 strain for MK-7 was obtained by screening and UV mutagenesis breeding. Combined with optimized fermentation conditions and purification processes, including culture medium composition, culture temperature, pH value, and separation and purification steps, the yield and purity of MK-7 were improved.
It has achieved high-yield production of MK-7 with a short fermentation cycle, yield of 400~431mg/L, and purity of 98%~99.9%, meeting market demand and reducing production costs.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of microbial technology, and more specifically, relates to a Bacillus subtilis natto and its application in the production of MK-7. Background Technology
[0002] MK-7 (Menaquinone-7) is a fat-soluble vitamin and the most biologically active vitamin K2 in animals. It has a wide range of biological functions, such as improving hypoprothrombinemia, preventing and treating Parkinson's disease and osteoporosis, and other health and therapeutic effects.
[0003] MK-7 is widely found in nature and can be synthesized by various microorganisms. However, even in fermented foods rich in MK-7, such as fermented black soybeans and natto, the natural content of MK-7 is still extremely low. For example, 100g of natto contains only about 1mg of MK-7. Therefore, it is impossible to obtain enough MK-7 for health maintenance and disease treatment through diet alone; additional supplementation is required to meet therapeutic needs.
[0004] MK-7 is an internationally recognized third-generation health food for the prevention and treatment of osteoporosis. In 2016, my country officially approved Vitamin K2 as a nutritional fortification supplement and included it in the National Nutritional Fortification Catalogue. Its safety has been recognized by authoritative institutions such as the FDA, SFDA, the US Institute of Drug Research, and the Council of Europe. On December 13, 2019, the National Health Commission of China announced and confirmed Vitamin K2 as a new type of food fortification agent. Therefore, MK-7 has broad market prospects in the fields of medical pharmaceuticals and health care.
[0005] Currently, MK-7 on the market mainly comes from three sources: First, extraction from MK-7-rich substances. Natto is a major source of MK-7 through natural extraction. Although natto has a higher MK-7 content than other foods, its extremely low absolute MK-7 content leads to low extraction efficiency and high production costs, making this method far from meeting market demand. Second, MK-7 is obtained through chemical synthesis. Chemical synthesis can yield MK-7 with 99.9% high purity, but the process is complex and produces different cis isomers. The activity of cis isomer MK-7 is less than 10% of that of all-trans MK-7, and the process generates a large number of byproducts causing environmental pollution. Therefore, although the chemical synthesis of vitamin K2 has been industrialized, new chemical synthesis processes still need to be explored to overcome these drawbacks. Third, MK-7 is produced through fermentation microorganisms. Bacillus subtilis is the main strain for synthesizing MK-7. The use of Bacillus subtilis to produce MK-7 has a long history, and to date, Bacillus subtilis remains the strain with the highest MK-7 yield and is also the main strain for industrial microbial fermentation production of MK-7. However, the existing production of Bacillus subtilis MK-7 still cannot meet market demand.
[0006] Therefore, screening out a new strain that produces high levels of MK-7 is of great significance for achieving large-scale production of MK-7. Summary of the Invention
[0007] The purpose of this invention is to provide a strain of Bacillus subtilis natto and its application in the production of MK-7, so as to achieve high yield of MK-7 by screening out a new strain of Bacillus subtilis natto.
[0008] Therefore, the present invention provides the following technical solution.
[0009] The first aspect of the present invention provides a strain of Bacillus subtilisnatto, which was deposited on December 16, 2025 at the China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 36907.
[0010] In a preferred embodiment of the present invention, the Bacillus subtilis natto has the following microbiological characteristics: Gram-positive bacteria, rod-shaped, 0.5-0.7 μm wide and 1.7-4.8 μm long; on LB solid medium, the colonies are light yellow, nearly round, moist, opaque, and have neat edges.
[0011] In a preferred embodiment of the present invention, the Bacillus subtilis natto strain is a strain isolated from four different commercially available fresh natto sources. After isolation and purification, fermentation experiments were conducted to obtain the MK-7 producing strain. The MK-7 producing strain was then used as the starting strain. This strain underwent ultraviolet mutagenesis, and after 21 generations of ultraviolet mutagenesis, the yield of MK-7 increased 30-fold. This strain is designated WD1.
[0012] A second aspect of the present invention provides a microbial agent, including Bacillus subtilis natto as described above.
[0013] A third aspect of the invention provides the use of Bacillus subtilis natto or the aforementioned microbial agent in the production of MK-7 or its products.
[0014] In a preferred embodiment of the present invention, the product includes at least one of food, beverage, and MK-7 fermented product.
[0015] A fourth aspect of the present invention provides a method for producing MK-7 by fermentation using Bacillus subtilis natto as described above or the aforementioned microbial inoculant, the method comprising: The Bacillus subtilis natto was cultured in a culture medium to obtain a culture solution; MK-7 was obtained from the culture medium.
[0016] In a preferred embodiment of the present invention, the culture medium comprises: 6-60 g / L chickpea flour, 5-50 g / L glycerol, 3-30 g / L sucrose, 5-50 g / L glucose, 0.5-5 g / L sodium chloride, 0.1-1 g / L calcium chloride, 0.5-5 g / L magnesium sulfate, and 0.3-3 g / L dipotassium hydrogen phosphate.
[0017] In a preferred embodiment of the present invention, the culture conditions are as follows: the culture is carried out under aerobic conditions, the culture temperature is 35~50℃, the culture time is 24~48 h, the pH is 5.5~8.0, and the pressure is 0.03~0.08 MPa.
[0018] In a preferred embodiment of the present invention, obtaining MK-7 from the culture medium includes: S1: The fermentation cells are isolated from the culture medium and then subjected to a first drying process to obtain dried cells; S2: The dried bacterial cells are sequentially extracted, evaporated under reduced pressure, crystallized at low temperature, centrifuged, and dried again to obtain MK-7.
[0019] In a preferred embodiment of the present invention, in step S1, the separation is performed using one or more of tubular centrifuges, disc centrifuges, and ceramic membrane separation.
[0020] In a preferred embodiment of the present invention, in step S1, the separation is performed using ceramic membrane separation.
[0021] In a preferred embodiment of the present invention, in step S1, the first drying is performed by any one of freeze drying, spray drying, or vacuum drying.
[0022] In a preferred embodiment of the present invention, in step S1, the preferred first drying is performed by freeze drying.
[0023] In a preferred embodiment of the present invention, in step S2, the extraction is performed using an organic solvent, wherein the organic solvent is selected from any one of ethanol, methanol, n-hexane, isopropanol, and ethyl acetate.
[0024] In a preferred embodiment of the present invention, in step S2, the organic solvent is n-hexane.
[0025] In a preferred embodiment of the present invention, in step S2, the low-temperature crystallization condition is: a temperature of 2~8°C.
[0026] In a preferred embodiment of the present invention, in step S2, the low-temperature crystallization condition is: a temperature of 4°C.
[0027] In a preferred embodiment of the present invention, in step S2, the centrifugation is performed using a flatbed centrifuge.
[0028] In a preferred embodiment of the present invention, in step S2, the second drying is carried out by vacuum drying, wherein the vacuum drying temperature is 50~80℃ and the time is 20~40 h.
[0029] In a preferred embodiment of the present invention, in step S2, the second drying is carried out by vacuum drying, the vacuum drying temperature is 60°C and the time is 36 hours.
[0030] By employing the above technical solution, the present invention has at least the following advantages: This invention, through screening and breeding, yielded a novel *Bacillus subtilis natto* strain. This strain was deposited on December 10, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 36907 and classification name: *Bacillus subtilis*. The *Bacillus subtilis natto* WD1 strain of this invention can be used to produce MK-7, with a short production cycle of only 24-48 hours and a yield of up to 400-431 mg / L (mg / L refers to the ratio of MK-7 content to the total volume of the fermentation broth). After purification, the fermentation broth yields pure MK-7 with a purity of 98%-99.9%.
[0031] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the preferred embodiments of the present invention are described in detail below. Attached Figure Description
[0032] Figure 1 Comparison of MK-7 yields from different bacterial strains during fermentation; Figure 2 The colony morphology of Bacillus subtilis WD1 in natto on LB solid plates according to the present invention; Figure 3 The colony morphology of Bacillus subtilis WD1 in natto according to the present invention is shown under a microscope. Figure 4 The image shows a liquid chromatogram of MK-7 with a purity of 98.62% according to the present invention. Detailed Implementation
[0033] To make the technical means, creative features, achieved objectives, and effects of this invention readily understandable, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.
[0034] Unless otherwise specified, the percentage content mentioned in this invention refers to mass percentage for solid-liquid mixtures and solid-phase-solid mixtures, and volume percentage for liquid-phase-liquid mixtures.
[0035] Unless otherwise specified, all percentage concentrations mentioned in this invention refer to the final concentration. The final concentration refers to the proportion of the added component in the system after the addition of that component.
[0036] Unless otherwise specified, the temperature parameters in this invention can be either constant temperature processing or processing within a certain temperature range. The constant temperature processing allows temperature fluctuations within the precision range controlled by the instrument.
[0037] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.
[0038] The following embodiments involve and mention: The MK-7 high-performance liquid chromatography method is as follows: The chromatograph used was a Waters e2695 high-performance liquid chromatograph with an Agilent Poroshell 120 EC-C18 column (4.6 × 100 mm, 2.7 μm), a column temperature of 30℃, a detection wavelength of 268 nm, an injection volume of 10 μL, a mobile phase of anhydrous ethanol:water = 97:3, a flow rate of 0.7 mL / min, isocratic elution, and a MK-7 retention time of approximately 5.6 min.
[0039] Unless otherwise specified, the reagents mentioned in the following examples are all analytical grade reagents purchased domestically or internationally.
[0040] Example 1: Isolation and screening of Bacillus subtilis natto 1. Sample Source The strains used in this example were derived from four different commercially available natto products.
[0041] 2. Isolation and purification of strains Four different types of commercially available natto, each weighing 1 g, were placed in 5 mL of sterile water, crushed and diluted in a mortar, and then spread onto LB solid medium (formula: 5.0 g / L yeast powder, 10 g / L peptone, 10 g / L sodium chloride, 15 g / L agar powder, autoclaved at 121°C for 20 min) plates. The plates were incubated at 37°C for 24 h. Pale yellow colonies grew on the surface of the plates, exhibiting stringy behavior when picked up with an inoculation needle. These pale yellow colonies were then transferred to LB liquid medium (formula: 5.0 g / L yeast powder, 10 g / L peptone, 10 g / L sodium chloride, autoclaved at 121°C for 20 min) and incubated at 37°C for 24 h to obtain the fermentation broth. The obtained fermentation broth was then inoculated at a rate of 10% (v / v) into fermentation medium (50 mL in a 250 mL Erlenmeyer flask; the medium formulation was: 6 g / L chickpea flour (purchased from Qingdao Longhai Food Co., Ltd.), 5 g / L glycerol, 3 g / L sucrose, 5 g / L glucose, 0.5 g / L sodium chloride, 0.1 g / L calcium chloride, 0.5 g / L magnesium sulfate, and 0.3 g / L dipotassium hydrogen phosphate). The medium was incubated at 37℃ and 220 rpm for 48 h. After incubation, an equal volume of extraction buffer (n-hexane:isopropanol = 2:1, v / v) was added to the obtained fermentation broth for extraction. After separation, the upper layer was used for high-performance liquid chromatography (HPLC) analysis of MK-7 yield. Microorganisms corresponding to samples with the same retention time and higher peak area as the standard were selected to preliminarily screen for strains producing high levels of MK-7. The results are shown in Table 1.
[0042] Table 1. Yield of MK-7 strain isolated from four commercially available natto varieties. ; As can be seen from the results in Table 1, strain 4-13 produced the highest MK-7, and further UV mutagenesis breeding was carried out using strain 4-13 as the starting strain.
[0043] 3. Mutagenesis breeding of strains Further UV mutagenesis breeding was carried out using strain 4-13 as the starting strain. The specific procedure is as follows: (1) Preparation of bacterial suspension: ① Take the bacterial culture in the logarithmic growth phase After centrifugation and washing, the solution was resuspended in sterile physiological saline (0.85% NaCl). ② Adjust the bacterial concentration to ; ③ Add 0.1 mol / L PBS buffer (pH 7.0) to maintain DNA stability.
[0044] (2) Ultraviolet irradiation parameter settings: ① Use a 254 nm wavelength ultraviolet lamp, irradiate at a distance of 30 cm, and irradiate for 15 seconds; ② Immediately after irradiation, move the device to a dark environment to prevent photoreactivation and repair.
[0045] (3) Screening of high-yielding strains of Mk-7: ① Dilute the above-mentioned UV-irradiated strains and spread them on LB solid medium, and incubate at 37°C for 24 h; ② Twenty mutant strains were selected and inoculated into LB medium and cultured at 37°C with shaking for 8 h; ③ Transfer the inoculum at a rate of 5% (v / v) to the fermentation medium (6 g / L chickpea flour (purchased from Qingdao Longhai Food Co., Ltd.), 5 g / L glycerol, 3 g / L sucrose, 5 g / L glucose, 0.5 g / L sodium chloride, 0.1 g / L calcium chloride, 0.5 g / L magnesium sulfate, 0.3 g / L dipotassium hydrogen phosphate, sterilized at 115℃ for 20 minutes), and culture at 37℃ and 220 rpm for 48 h with shaking. ④ After the culture is completed, add an equal volume of extraction solution (n-hexane:isopropanol = 2:1, v / v) to the obtained fermentation broth for extraction. After separation, take the upper liquid for high performance liquid chromatography analysis of MK-7 yield, and screen the mutant strain with the highest MK-7 content for the next round of ultraviolet mutagenesis.
[0046] Steps (1) to (3) were repeated, and a total of 21 UV irradiation mutagenesis were performed. The results of the highest yield of MK-7 mutants after each batch of mutagenesis are shown in [the table below]. Figure 1 .
[0047] like Figure 1 As shown, the MK-7 yield of mutant strain T-5 was the highest after 21 generations of UV mutagenesis, reaching 425 mg / L.
[0048] 4. Strain identification 4.1 Colony characteristics like Figure 2 As shown, Bacillus subtilis T-5 colonies on LB solid medium are light yellow, nearly round, moist, opaque, and have neat edges.
[0049] 4.2 Microscopic morphology like Figure 3 As shown, Bacillus subtilis T-5 colony smear of natto: Gram-positive bacteria, rod-shaped, 0.5-0.7 μm wide and 1.7-4.8 μm long.
[0050] 4.3 16S rDNA Identification 16S rDNA sequencing results showed that the 16S rDNA of the mutant strain T-5 was similar to that of Bacillus subtilis NCBI 3610. TThe sequence similarity was ≥99.5%, and the gene sequence of strain T-5 16S rDNA is shown in SEQ ID NO. 1: SEQ ID NO. 1 Based on the above, the UV-mutated Bacillus subtilis T-5 was named Bacillus subtilis natto WD1 and deposited on December 16, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 36907. It was stored in a 50% glycerol solution at -80°C.
[0051] Example 2: Screening of optimal fermentation conditions for MK-7 production by Bacillus subtilis WD1 in natto 1. Determination of the optimal incubation temperature Seed culture: The *Bacillus subtilis* WD1 obtained in Example 1 was inoculated into a 500 mL Erlenmeyer flask containing 100 mL of seed culture medium. The seed culture medium consisted of: 5 g / L glycerol, 3 g / L sucrose, 5 g / L glucose, 0.5 g / L sodium chloride, 0.1 g / L calcium chloride, 0.5 g / L magnesium sulfate, and 0.3 g / L dipotassium hydrogen phosphate. The culture was incubated at 37°C and 220 rpm for 16 h to obtain the inoculum.
[0052] Fermentation Culture: Fermentation was carried out in a 5L tank. The inoculum to be inoculated was added to the fermentation medium at an inoculation rate of 10% (by volume) (the fermentation medium consisted of: 60 g / L chickpea flour (purchased from Qingdao Longhai Food Co., Ltd.), 50 g / L glycerol, 30 g / L sucrose, 50 g / L glucose, 5 g / L sodium chloride, 1 g / L calcium chloride, 5 g / L magnesium sulfate, 3 g / L dipotassium hydrogen phosphate, and water to a final volume of 2L; sterilized at 115℃ for 20 minutes). Fermentation was then carried out, with the pH controlled at 7.0, dissolved oxygen maintained above 30%, tank pressure controlled at 0.05 MPa, and temperatures set at 35℃, 40℃, 45℃, and 50℃ for a fermentation period of 48 hours to obtain the fermentation broth. After cultivation, an equal volume of extraction solution (n-hexane:isopropanol = 2:1, v / v) was added to the obtained fermentation broth for extraction. After separation, the upper layer was taken for high-performance liquid chromatography (HPLC) analysis to determine the yield of MK-7 at each cultivation temperature. The results are shown in Table 2 below. Table 2. MK-7 yield at different fermentation temperatures ; As shown in Table 2, WD1 had the highest MK-7 yield at 40℃ during fermentation, reaching 423.58 mg / L.
[0053] 2. Determination of the optimal fermentation pH Seed culture: The *Bacillus subtilis* WD1 obtained in Example 1 was inoculated into a 500 mL Erlenmeyer flask containing 100 mL of seed culture medium. The seed culture medium composition was: 5 g / L glycerol, 3 g / L sucrose, 5 g / L glucose, 0.5 g / L sodium chloride, 0.1 g / L calcium chloride, 0.5 g / L magnesium sulfate, and 0.3 g / L dipotassium hydrogen phosphate. The culture was incubated at 37°C and 220 rpm for 16 h to obtain the inoculum.
[0054] Fermentation Culture: Fermentation was carried out in a 5L tank. The inoculum to be inoculated was added to the fermentation medium at an inoculation rate of 10% (by volume) (the fermentation medium consisted of: 60 g / L chickpea flour (purchased from Qingdao Longhai Food Co., Ltd.), 50 g / L glycerol, 30 g / L sucrose, 50 g / L glucose, 5 g / L sodium chloride, 1 g / L calcium chloride, 5 g / L magnesium sulfate, 3 g / L dipotassium hydrogen phosphate, and water to a final volume of 2L; sterilized at 115℃ for 20 minutes). Fermentation was then carried out, with the pH controlled at 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0, dissolved oxygen maintained above 30%, tank pressure controlled at 0.05 MPa, temperature at 40℃, and fermentation cycle at 48 hours to obtain the fermentation broth. After cultivation, an equal volume of extraction buffer (n-hexane:isopropanol = 2:1, v / v) was added to the resulting fermentation broth for extraction. After separation, the upper layer was taken for high-performance liquid chromatography (HPLC) analysis to determine the yield of MK-7 at each cultivation pH. The results are shown in Table 3 below. Table 3 Fermentation pH of MK-7 ; As shown in Table 3, WD1 produced the highest MK-7 yield under pH 7.0 fermentation conditions, at 421.26 mg / L.
[0055] Based on the above results, the optimal fermentation conditions for the production of MK-7 by Bacillus subtilis WD1 in natto according to the present invention are: temperature 40℃ and pH 7.0.
[0056] Example 3: Fermentation comparison between WD1 and the original starting strain Seed culture: The *Bacillus subtilis* WD1 obtained in Example 1 and the original starting strain 4-13 were inoculated separately into 500 mL Erlenmeyer flasks containing 100 mL of seed culture medium. The seed culture medium consisted of: 5 g / L glycerol, 3 g / L sucrose, 5 g / L glucose, 0.5 g / L sodium chloride, 0.1 g / L calcium chloride, 0.5 g / L magnesium sulfate, and 0.3 g / L dipotassium hydrogen phosphate. The cultures were incubated at 37°C and 220 rpm for 16 h to obtain the inoculated bacterial strain.
[0057] Fermentation Culture: Fermentation was carried out in a 5L tank. The inoculum to be inoculated was added to the fermentation medium at an inoculation rate of 10% (v / v) (the fermentation medium consisted of: 60 g / L chickpea flour (purchased from Qingdao Longhai Food Co., Ltd.), 50 g / L glycerol, 30 g / L sucrose, 50 g / L glucose, 5 g / L sodium chloride, 1 g / L calcium chloride, 5 g / L magnesium sulfate, 3 g / L dipotassium hydrogen phosphate, and water to a final volume of 2L; sterilized at 115℃ for 20 minutes). Fermentation was then carried out, with the pH controlled at 7.0, dissolved oxygen above 30%, tank pressure at 0.05 MPa, temperature at 40℃, and a fermentation period of 48 h to obtain the fermentation broth. After fermentation, an equal volume of extraction solution (n-hexane:isopropanol = 2:1, v / v) was added to the obtained fermentation broth for extraction. After separation, the upper layer was taken for high-performance liquid chromatography (HPLC) analysis. The results are shown in Table 4 below. Table 4. Comparison of fermentation of Bacillus subtilis WD1 and 4-13 in natto. ; Note: - in the table, it indicates that MK-7 was not clearly detected.
[0058] As shown in Table 4, after 48 hours of fermentation with Bacillus subtilis WD1, the yield of MK-7 was 431.25 mg / L, while the yield of MK-7 from the starting strain 4-13 was only 13.59 mg / L. This indicates that the yield of MK-7 produced by fermentation of the mutant strain WD1 obtained through UV mutagenesis was significantly improved.
[0059] Example 4: Fermentation production and purification of MK-7 1. Fermentation production of MK-7 Seed culture: The *Bacillus subtilis* WD1 obtained in Example 1 was inoculated into a 500 mL Erlenmeyer flask containing 100 mL of seed culture medium. The seed culture medium composition was: 5 g / L glycerol, 3 g / L sucrose, 5 g / L glucose, 0.5 g / L sodium chloride, 0.1 g / L calcium chloride, 0.5 g / L magnesium sulfate, and 0.3 g / L dipotassium hydrogen phosphate. The culture was incubated at 37°C and 220 rpm for 16 h to obtain the inoculum.
[0060] Fermentation Culture: Fermentation was carried out in a 5L tank. The inoculum to be inoculated was added to the fermentation medium at an inoculation rate of 10% (v / v) (the fermentation medium composition was: 60 g / L chickpea flour (purchased from Qingdao Longhai Food Co., Ltd.), 50 g / L glycerol, 30 g / L sucrose, 50 g / L glucose, 5 g / L sodium chloride, 1 g / L calcium chloride, 5 g / L magnesium sulfate, 3 g / L dipotassium hydrogen phosphate, water added to a final volume of 2 L, sterilized at 115℃ for 20 minutes). Fermentation was then carried out, with the pH controlled at 7.0, dissolved oxygen above 30%, tank pressure controlled at 0.05 MPa, temperature at 40℃, and fermentation cycle at 48 h to obtain the fermentation broth. After the fermentation was completed, an equal volume of extraction solution (n-hexane:isopropanol = 2:1, v / v) was added to the obtained fermentation broth for extraction. After separation, the upper layer was taken for high-performance liquid chromatography (HPLC) analysis to determine the yield of MK-7.
[0061] The test results showed that the fermentation yield of MK-7 was 430 mg / L.
[0062] 2. Isolation and purification of MK-7 The fermentation broth was subjected to solid-liquid separation using a 100 nm ceramic membrane to obtain a concentrated bacterial cell solution. This concentrated solution was then freeze-dried (-20℃, -0.1 MPa) for 24 hours to obtain dried bacterial powder. Hexane was added to the bacterial powder at a weight ratio of 1:5 to extract the bacterial powder. After extraction, the organic phase was separated to obtain a crude extract containing MK-7. This crude extract was concentrated by vacuum evaporation to remove excess solvent. Vacuum evaporation was stopped when the liquid volume decreased to one-third of its original volume, yielding a concentrated solution. This concentrated solution was subjected to low-temperature crystallization at 4℃ for 16 hours, followed by centrifugation to recover the crystals, obtaining MK-7 crystals. The recovered MK-7 crystals were placed in a vacuum drying oven and dried at 60℃ for 36 hours to obtain MK-7 pure product with a purity of 98%. The liquid chromatogram is shown below. Figure 4 As shown.
[0063] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the methods and techniques disclosed above without departing from the scope of the present invention to create equivalent embodiments. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.
Claims
1. A strain of Bacillus subtilis natto, characterized in that, It was deposited on December 16, 2025, at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 36907.
2. A microbial inoculant, characterized in that, Includes Bacillus subtilis of Natto as described in claim 1.
3. The application of Bacillus subtilis natto according to claim 1 or the microbial agent according to claim 2 in the production of MK-7 or its products.
4. The application according to claim 3, characterized in that, The product includes at least one of food, beverage, and MK-7 fermented products.
5. The method for producing MK-7 by fermentation with Bacillus subtilis natto according to claim 1 or the microbial agent according to claim 2, characterized in that, The method includes: The Bacillus subtilis natto was cultured in a culture medium to obtain a culture solution; MK-7 was obtained from the culture medium.
6. The method according to claim 5, characterized in that, The culture medium consists of: 6-60 g / L chickpea flour, 5-50 g / L glycerol, 3-30 g / L sucrose, 5-50 g / L glucose, 0.5-5 g / L sodium chloride, 0.1-1 g / L calcium chloride, 0.5-5 g / L magnesium sulfate, and 0.3-3 g / L dipotassium hydrogen phosphate.
7. The method according to claim 5, characterized in that, The culture conditions are as follows: the culture is carried out under aerobic conditions, with a culture temperature of 35~50℃, a culture time of 24~48 h, a pH of 5.5~8.0, and a pressure of 0.03~0.08 MPa.
8. The method according to claim 5, characterized in that, Obtaining MK-7 from the culture medium includes: S1: The fermentation cells are isolated from the culture medium and then subjected to a first drying process to obtain dried cells; S2: The dried bacterial cells are sequentially extracted, evaporated under reduced pressure, crystallized at low temperature, centrifuged, and dried again to obtain MK-7.
9. The method according to claim 8, characterized in that, In step S1, the separation is performed using one or more of the following: tubular centrifuge, disc centrifuge, and ceramic membrane separation. The first drying process is carried out using any one of freeze drying, spray drying, or vacuum drying.
10. The method according to claim 8, characterized in that, In step S2, the extraction is performed using an organic solvent, wherein the organic solvent is selected from any one of ethanol, methanol, n-hexane, isopropanol, and ethyl acetate. The low-temperature crystallization conditions are: a temperature of 2~8℃; The centrifugation was carried out using a flatbed centrifuge. The second drying is carried out by vacuum drying, with a temperature of 50~80℃ and a time of 20~40 h.