Bacillus natto and application thereof

Abstract

The invention provides bacillus natto and application, and belongs to the technical field of microorganisms. The bacillus natto disclosed by the invention is bacillus natto DG2-7 which is preserved in the China General Microbiological Culture Collection Center (CGMCC) and has the preservation number of CGMCC No.36482, and relates to application of the bacillus natto DG2-7 in improvement of intestinal functions and body metabolism. The strain can be used for preparing natto, natto powder, natto tablets and other preparations, has the effects of regulating constipation, diarrhea, flatulence, sour regurgitation and bad breath, can assist in reducing blood triglyceride and relieving gout, and is beneficial to relieving female dysmenorrhea. In a test, good intestinal regulation and metabolism improvement effects are shown, and the comprehensive function is superior to that of a commercially available probiotic product.

Description

Technical Field

[0001] This application belongs to the field of microbial technology, specifically relating to a strain of Bacillus natto and its application. Background Technology

[0002] Constipation and diarrhea are common functional gastrointestinal disorders in clinical practice. Constipation, in particular, is characterized by dry, hard stools, difficulty defecating, and reduced bowel movements. Currently, commonly used medications include: stimulant laxatives such as rhubarb and senna leaves, but their use should not exceed one week. Long-term use is not recommended for those with chronic constipation and may worsen intestinal problems, even potentially leading to colorectal cancer. Prokinetic drugs and gut microbiota regulators, such as mosapride, itopride, bifidobacteria triple live bacteria, and lactobacillus tablets, can promote gastrointestinal smooth muscle motility, alter the intestinal microecology, and relieve constipation and bloating. Laxatives, such as lactulose, polyethylene glycol, and magnesium sulfate, can change stool consistency, increase stool volume and water content, and promote rapid defecation.

[0003] However, existing products often have limited functionality and lack the ability to comprehensively improve accompanying metabolic problems (such as hyperlipidemia and gout) and microcirculatory disorders (such as dysmenorrhea).

[0004] Bacillus natto is a subspecies of Bacillus subtilis that produces various active substances such as nattokinase and elastase. Although natto bacteria resources exist in China, proprietary strains with high fermentation activity and proven multiple health benefits in humans (especially for non-traditional areas such as dysmenorrhea and gout) remain scarce. Summary of the Invention

[0005] In view of the shortcomings of existing products in the prior art, which have limited functions and lack comprehensive improvement capabilities for accompanying metabolic problems (such as hyperlipidemia and gout) and microcirculation disorders (such as dysmenorrhea), this application proposes a strain of Bacillus natto and its application. By starting from the source, a strain of Bacillus natto DG2-7 that can efficiently ferment soybeans and effectively promote digestion and metabolism has been screened and isolated. It has high bacterial activity, simple culture method, and can effectively help relieve constipation, diarrhea, and flatulence. It has broad prospects for industrial application.

[0006] This application discloses a strain of Bacillus subtilis natto. Bacillus subtilis subsp.natto It is preserved at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with a preservation date of November 6, 2025, and is classified and named as: Bacillus subtilis natto subsp. Bacillus subtilis subsp.natto The registration number is: CGMCC No. 36482.

[0007] Preferably, the Bacillus natto is Bacillus natto DG2-7.

[0008] In another aspect, this application proposes a Bacillus natto preparation comprising the cells, spores, or fermentation metabolites of Bacillus natto DG2-7 as described in claim 1 or 2.

[0009] Preferably, the product form of the preparation is any one of natto, natto powder, or natto tablets.

[0010] Preferably, the method for preparing natto includes primary strain activation, secondary strain activation, preparation of primary seed liquid, and inoculation of the primary seed liquid into soybean culture to obtain natto fermented by Bacillus natto DG2-7.

[0011] This application also proposes applications in products for improving intestinal function and metabolism.

[0012] Preferably, the improvement of gastrointestinal health is any one of regulating constipation and diarrhea, relieving bloating and acid reflux, or inhibiting bad breath.

[0013] Preferably, the regulation of body metabolism is either an aid in lowering blood triglycerides or an relief of gout.

[0014] Preferably, the application also includes relieving menstrual cramps in women.

[0015] Preferably, the soybeans are soybeans that have been sterilized and cooled under high temperature and high pressure.

[0016] The beneficial effects of the embodiments in this application are as follows: (1) High bacterial activity and strong fermentation ability: This bacterial strain exhibits excellent growth adaptability in soybean substrate, and the effective viable count in the fermentation product can reach 100%. The levels were significantly higher than those of conventional natto bacteria, laying the foundation for efficient production.

[0017] (2) Outstanding comprehensive regulatory ability: It can not only effectively regulate intestinal function in both directions (improve constipation, diarrhea, bloating, acid reflux and bad breath), but also assist in regulating the body's metabolism, such as helping to lower blood triglycerides, relieve gout, and extend to non-traditional areas such as relieving menstrual pain in women, thus realizing comprehensive intervention on gastrointestinal health and system metabolism.

[0018] (3) Diverse product forms and convenient application: Based on this strain, it can be prepared into a variety of dosage forms such as natto, natto powder, and natto tablets to meet different consumption scenarios and preferences. In particular, natto tablets are easy to carry and take in a quantitative manner, and the stability of live bacteria is well maintained through dry granulation and other processes. Detailed Implementation

[0019] The technical solutions of this application will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this application.

[0020] The raw materials and reagents used in the following examples are all commercially available.

[0021] Example 1

[0022] Preservation and culture of Bacillus natto LB agar medium preparation: 10g tryptone, 5g yeast extract, 5g sodium chloride, 15g agar, bring the volume to 1000mL with purified water, and adjust the pH to 6.8-7.2.

[0023] High-temperature treated rice straw was fermented with soybeans, and strains were isolated using LB agar plates prepared in step (1). Bacillus subtilis DG2-7 was obtained by streak plating. Bacillus subtilis DG2-7 was preserved at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, on November 6, 2025, and classified as: Bacillus subtilis natto subsp. Bacillus subtilis subsp.natto The registration number is: CGMCC No. 36482.

[0024] Select the bacterial strain and treat it with the above-mentioned Bacillus natto DG2-7 and Bacillus subtilis natto subsp. natto. Bacillus subtilis subsp.natto, CGMCC No. 36482 was cultured on a slant agar medium at 37°C for 28 hours.

[0025] Liquid culture of the strain: Take the slant culture from step (2) or take the Bacillus natto DG2-7 strain from the glycerol tube and inoculate it into the prepared shake flask LB medium, and culture it at 37℃ in a constant temperature shaker at 220r / min for 24h.

[0026] Inoculate the primary seed culture into the fermentation medium at an inoculation rate of 2%-20%, at a temperature of 38℃, a rotation speed of 200 rpm, an air flow rate of 0.2-3 vvm / min, and incubate for 28 h.

[0027] Viable bacteria determination: The viable bacteria count was determined using the method specified in GB 4789.2 "Food Microbiology Examination - Determination of Total Colony Count". The effective viable bacteria count reached (1.0-5.0)×10⁻⁶. 10The CFU / g was significantly higher than that of conventional Bacillus natto, which typically only reaches 10 under the same fermentation conditions. 8 -10 9 The strain exhibited excellent growth adaptability and metabolic activity in soybean substrate, and its high-density proliferation ability stemmed from its physiological characteristics of efficient carbon source utilization and tolerance to organic acid accumulation during fermentation.

[0028] Identification of strains: Morphological identification: Bacillus natto DG2-7 in the logarithmic growth phase was observed. The results are as follows: Colony characteristics: On solid culture medium, colonies are nearly round and opaque; the edges are irregular (uneven), and the surface is not smooth with slight wrinkles. Cell characteristics: Cells are rod-shaped and Gram-positive (G+); cell size is (0.6-1) μm × (2.0-2.5) μm; they possess lateral flagella and are highly motile. Spore characteristics: Produces endospores and is highly heat-resistant; spores are elliptical or columnar, located in the central or semi-central position of the cell; sporangia do not show obvious swelling. Physiological characteristics: This strain has a wide growth temperature range, with a minimum growth temperature of 5-20℃ and a maximum growth temperature of 45-55℃.

[0029] 16S rDNA sequence analysis: The 16S rDNA gene fragment of Bacillus natto strain DG2-7 was amplified using colony PCR technology. The amplified product was cloned and sequenced. The sequence is shown in SEQ ID NO:1. SEQ ID NO:1 (>16S-DG2-7.33813400):

[0030] By comparing the above 16S rDNA sequence with the microbial genome blast analysis in the NCBI database, Bacillus natto DG2-7 was identified as Bacillus natto, i.e., Bacillus subtilis subsp. natto.

[0031] Example 2

[0032] Natto preparation (1) First-time bacterial activation: Take the Bacillus natto DG2-7 bacterial strain from the glycerol tube and inoculate it into the prepared shake flask culture medium. Incubate at 36℃ for 14h. The shake flask culture medium consists of soybean water, glucose 2-30g / L, and pH 6.0-7.0.

[0033] (2) Secondary activation of the strain: Take an appropriate amount of the strain after the first activation and inoculate it into the prepared shake flask culture medium. Incubate at 38℃ in a shaking shaker for 16 hours. The shake flask culture medium consists of soybean water, glucose 2-30g / L, and pH 6.0-7.0.

[0034] (3) Preparation of primary seed: Inoculate the secondary activated strain into the primary seed culture medium at an inoculation amount of 1%-15%, at a temperature of 38℃ and a rotation speed of 200rpm for 12 hours to obtain the primary seed liquid; composition of primary seed culture medium: composition of shake flask culture medium: soybean water, glucose 2-30g / L, pH 6.0-7.0.

[0035] (4) Preparation of fermentation liquid and fermented natto: 5%-20% of the primary seed liquid was inoculated onto soybeans that had been sterilized and cooled by high temperature and high pressure. The temperature was 40℃, the rotation speed was 100rpm, the air volume was 0.2-3vvm / min, and the culture was carried out for 30h. Natto fermented by Bacillus natto DG2-7 was obtained.

[0036] Example 3

[0037] Natto powder preparation Pretreatment: Place the fermented natto from Example 2 on a sterile workbench, remove any small amount of clumping impurities produced during fermentation, and add a small amount of sterile purified water to make a uniform paste.

[0038] Low-temperature drying process: Spread the prepared natto evenly on a sterile drying tray (2-3cm thick), place it in a vacuum low-temperature drying oven, set the vacuum degree to -0.08~-0.10MPa, the drying temperature to 35~40℃, and the drying time to 18~24h. During this period, check the moisture content every 6h until the moisture content is below 5%.

[0039] Grinding and sieving: Place the dried natto into a sterile ultra-fine grinder, grind it, and then pass it through a 100-mesh sterile sieve to remove coarse particles that are not completely ground, ensuring that the natto powder has a uniform particle size.

[0040] Packaging and Storage: After grinding, the natto powder is cooled to room temperature (below 25℃) under sterile conditions. It is then packaged in sterile aluminum foil bags or vacuum-sealed bags, sealed, and stored in a cool, dry place with an effective viable bacteria count ≥1.0×10⁻⁶. 10 CFU / g.

[0041] Example 4

[0042] Natto flake preparation Ingredients: Natto powder from Example 3, maltodextrin, corn starch, yam powder, and silicon dioxide.

[0043] Mixing: Mix 15% natto powder, 40% maltodextrin, 25% corn starch, 15% yam powder, and 5% silica. Put all raw materials into a sterile three-dimensional mixer, set the speed to 100~150 r / min, and the mixing time to 15~20 min to ensure that the raw materials are mixed evenly. The mixing process is carried out in a sterile environment with relative humidity ≤40% and temperature ≤25℃ to avoid natto powder absorbing moisture and clumping.

[0044] Granulation: The evenly mixed raw materials are put into a sterile dry granulator, the granulation pressure is set to 5~8MPa, and after granulation, they are passed through a 20~30 mesh sterile sieve to obtain natto granules with uniform particle size, avoiding the inactivation of live bacteria caused by wet granulation.

[0045] Tableting: The prepared natto granules are fed into a sterile rotary tablet press. The tableting pressure is set to 8-12 kN, and the tablet weight is controlled at 0.3-0.5 g / tablet. A shallow concave die is used to ensure the tablets are formed completely, without cracks or sticking to the die. A sterile, low-humidity, and low-temperature environment is maintained throughout the process. The effective viable bacteria count is 9 × 10⁻⁶. 8 CFU / g.

[0046] Example 5

[0047] Natto flake preparation Ingredients: Natto powder from Example 3, maltodextrin, corn starch, rice flour, and silicon dioxide.

[0048] Mixing: Mix 15% natto powder, 40% maltodextrin, 25% corn starch, 15% rice flour, and 5% silica. Put all raw materials into a sterile three-dimensional mixer, set the speed to 100~150 r / min, and the mixing time to 15~20 min to ensure that the raw materials are mixed evenly. The mixing process is carried out in a sterile environment with relative humidity ≤40% and temperature ≤25℃ to avoid natto powder absorbing moisture and clumping.

[0049] Granulation: The evenly mixed raw materials are put into a sterile dry granulator, the granulation pressure is set to 5~8MPa, and after granulation, they are passed through a 20~30 mesh sterile sieve to obtain natto granules with uniform particle size, avoiding the inactivation of live bacteria caused by wet granulation.

[0050] Tableting: The prepared natto granules are fed into a sterile rotary tablet press. The tableting pressure is set to 8-12 kN, and the tablet weight is controlled at 0.3-0.5 g / tablet. A shallow concave die is used to ensure the tablets are formed completely, without cracks or sticking to the die. A sterile, low-humidity, and low-temperature environment is maintained throughout the process. The effective viable bacteria count is 9 × 10⁻⁶. 8 CFU / g.

[0051] Example 6

[0052] Natto flake preparation Ingredients: Natto powder from Example 3, maltodextrin, corn starch, potato starch, and silicon dioxide.

[0053] Mixing: Mix 15% natto powder, 40% maltodextrin, 25% corn starch, 15% potato starch, and 5% silica. Put all raw materials into a sterile three-dimensional mixer, set the speed to 100~150 r / min, and the mixing time to 15~20 min to ensure that the raw materials are mixed evenly. The mixing process is carried out in a sterile environment with relative humidity ≤40% and temperature ≤25℃ to avoid natto powder absorbing moisture and clumping.

[0054] Granulation: The evenly mixed raw materials are put into a sterile dry granulator, the granulation pressure is set to 5~8MPa, and after granulation, they are passed through a 20~30 mesh sterile sieve to obtain natto granules with uniform particle size, avoiding the inactivation of live bacteria caused by wet granulation.

[0055] Tableting: The prepared natto granules are fed into a sterile rotary tablet press. The tableting pressure is set to 8-12 kN, and the tablet weight is controlled at 0.3-0.5 g / tablet. A shallow concave die is used to ensure the tablets are formed completely, without cracks or sticking to the die. A sterile, low-humidity, and low-temperature environment is maintained throughout the process. The effective viable bacteria count is 9 × 10⁻⁶. 8 CFU / g.

[0056] Example 7

[0057] The regulatory effect of natto tablets on intestinal function Experimental Materials and Methods Test sample: Natto flakes prepared in Example 4.

[0058] Subject group: 166 volunteers with intestinal dysfunction (constipation, diarrhea, bloating, etc.) were selected.

[0059] Dosage: Take 2-4 tablets 1-2 times daily with warm water.

[0060] Evaluation criteria: Significant effect (related symptoms disappear, bowel movements become regular); Effective (related symptoms are significantly reduced, bowel movement frequency improves); Ineffective (no change in symptoms).

[0061] The statistical results are shown in Table 1: Table 1. Regulatory effects of natto tablets on intestinal function

[0062] (3) Typical case description: Case ND005 (Intractable Constipation): The patient, a 65-year-old female, suffered from chronic constipation and severe halitosis. After taking the preparation of this invention for half a month, her bowel movement cycle returned to once a day, and the bowel movement was smooth, and her halitosis completely disappeared.

[0063] Case ND093 (Antibiotic-associated diarrhea): A 74-year-old male volunteer suffered from dysbiosis-related diarrhea for over a month due to antibiotic use, with conventional medications proving ineffective. After taking the formulation of this invention for 3 days, the diarrhea stopped, and the stool characteristics returned to normal.

[0064] Example 8

[0065] Evaluation of the effect of natto tablets on regulating metabolism (uric acid / blood lipids) Test sample: Natto flakes prepared in Example 5.

[0066] For subjects with a history of hyperuricemia, hyperlipidemia, and gout, biochemical indicators and clinical signs were recorded before and after administration.

[0067] Experimental results Improvement in biochemical indicators: Subject ND146 (male, 47 years old) took the formulation of this invention (8 tablets daily) for one year. The comparison results are shown in Table 2.

[0068] Table 2: Comparison of biochemical indicators before and after ND146 administration in subjects

[0069] Gout relief: Subject ND123 (male, 69 years old) had a severe history of gout with a high frequency of attacks. After taking the preparation of this invention for 2 months, uric acid metabolism tended to stabilize, and no gout attacks occurred during the follow-up period. At the same time, the accompanying constipation symptoms disappeared.

[0070] Example 9

[0071] The auxiliary effects of natto tablets on endocrine regulation and sleep improvement Test sample: Natto flakes prepared in Example 6.

[0072] Endocrine and menstrual cycle regulation: Case ND159: A woman over 35 years old suffered from chronically irregular menstruation (intervals of up to 3 months) due to endocrine disorders, and traditional Chinese medicine treatment was ineffective. After taking the preparation of this invention for 3 months, her menstrual cycle returned to normal monthly starting in May 2025, and her weight returned to a normal range.

[0073] Case ND058: A 51-year-old female reported that after taking the product, not only was her lower abdominal distension relieved, but her menstrual cramps also significantly improved.

[0074] (3) Improvement in skin and sleep: Acne (pimples) improvement: Subjects ND044 (20 years old) and ND150 (39 years old) reported that after taking the preparation for 1 month to improve constipation, facial acne was significantly reduced and skin condition was significantly improved.

[0075] Sleep aid: Subjects ND001 and ND002 reported that after one week of use, their sleep depth increased, enabling them to "sleep through the night," and their sleep quality assessment was significantly improved.

[0076] Example 10

[0077] Comparative test of natto tablets and commercially available probiotic preparations Test sample: Natto flakes prepared in Example 4.

[0078] Case ND045: The patient suffered from irritable bowel syndrome (IBS) and had previously taken Bifidobacterium licheniformis preparations without significant improvement. After switching to the DG2-7 preparation of this application, the diarrhea symptoms were effectively controlled.

[0079] Case ND081: A customer purchased a commercially available probiotic containing 64 billion live bacteria to improve constipation, but the constipation worsened. After taking the formulation proposed in this application, daily bowel movements were achieved.

[0080] In summary, the Bacillus natto strain DG2-7 described in this application exhibits high fermentation activity and a high viable count. Its formulation not only effectively regulates intestinal function bidirectionally, improving constipation, diarrhea, and bloating, but also helps lower blood triglycerides, alleviate gout, and alleviate menstrual cramps in women, demonstrating comprehensive intestinal and metabolic regulatory capabilities. Its effects are superior to conventional probiotic products in experiments. Notably, no adverse reactions were reported by any subjects during continuous use.

[0081] Although embodiments of this application have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of this application. All equivalent changes and improvements made within the scope of this application shall still fall within the patent coverage of this application.

Claims

1. A strain of Bacillus natto, characterized in that, The Bacillus subtilis is Bacillus subtilis natto. Bacillus subtilis subsp.natto It is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 36482.

2. The Bacillus natto strain as described in claim 1, characterized in that, The Bacillus natto is Bacillus natto DG2-7.

3. A Bacillus natto preparation, characterized in that, The formulation comprises the cells, spores, or fermentation metabolites of Bacillus natto DG2-7 as described in claim 1 or 2.

4. The Bacillus natto preparation as described in claim 3, characterized in that, The product form of the preparation is any one of natto, natto powder, or natto tablets.

5. The Bacillus natto preparation as described in claim 4, characterized in that, The method for preparing natto includes primary strain activation, secondary strain activation, preparation of primary seed liquid, and inoculation of primary seed liquid into soybean culture to obtain natto fermented by Bacillus natto DG2-7.

6. The use of Bacillus natto as described in claim 1 or 2, or the Bacillus natto preparation as described in claim 3, in products for improving intestinal function and metabolism.

7. The application as described in claim 6, characterized in that, The improvement of gastrointestinal health refers to any one of the following: regulating constipation and diarrhea, relieving bloating and acid reflux, or suppressing bad breath.

8. The application as described in claim 6, characterized in that, The regulation of the body's metabolism is either an aid in lowering blood triglycerides or a relief of gout.

9. The application as described in claim 6, characterized in that, The application also includes relieving menstrual cramps in women.

10. The Bacillus natto preparation as described in claim 5, characterized in that, The soybeans mentioned are soybeans that have been sterilized and cooled under high temperature and high pressure.

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