High-salt-tolerant bacillus subtilis producing protease and nattokinase and application thereof

Abstract

The application provides a high-salt-tolerant Bacillus subtilis capable of producing protease and nattokinase and an application thereof, the Bacillus subtilis is named as Bacillus subtilis 3-1, is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO: M20252875, and the 16S rRNA nucleotide sequence is shown as SEQ ID NO: 1. The Bacillus subtilis of the application can be directly applied to a high-salt fermentation system due to excellent high-salt-tolerant performance, and solves the industrial problem that the activity of a conventional strain is inhibited; due to the protease production property, can be used for efficiently degrading raw material protein in the field of food fermentation, promoting flavor formation and shortening the production cycle; the nattokinase production property makes it have outstanding value in the field of functional food and preparation, and can be used for developing a fermented product with health care functions of dissolving thrombus, and improving the health added value of the product. The strain provides a core microbial resource for quality improvement and functional development of high-salt fermented food.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a high-salt-tolerant Bacillus subtilis that produces protease and nattokinase, and its applications. Background Technology

[0002] High-salt environments are a typical characteristic of many traditional fermented foods (such as fermented black beans, fermented bean curd, pickled products, etc.). High-salt conditions often inhibit the growth and metabolic activity of common microorganisms, leading to problems such as slow fermentation process and uneven product quality.

[0003] In fermented foods, proteases are key enzymes that hydrolyze proteins to produce peptides and amino acids, directly affecting the flavor, texture, and nutritional value of fermented foods. Nattokinase, a functional enzyme with significant fibrinolytic activity, shows great potential in supporting cardiovascular health; however, its traditional production relies on specific strains and processes used in natto fermentation, making stable production difficult in high-salt systems. Currently, functional microorganisms used in high-salt brewing often struggle to simultaneously achieve both salt tolerance and high enzyme production (especially simultaneous high production of protease and nattokinase), hindering the rapid improvement of the quality of high-salt fermented products and the development of functional products.

[0004] Therefore, screening and constructing new microbial resources with excellent salt tolerance, high protease activity and nattokinase production capabilities is of great significance for innovating traditional high-salt fermentation processes and developing a new generation of functional fermented foods. Summary of the Invention

[0005] To address the above issues, this invention provides a high-salt-tolerant Bacillus subtilis strain that produces proteases and nattokinase, along with its applications. This overcomes the problems of insufficient activity and low enzyme production efficiency of existing fermentation strains under high-salt conditions. This Bacillus subtilis can be used in the production of high-salt fermented foods, utilizing its salt tolerance to maintain fermentation stability and improving product texture and flavor through protease secretion. Simultaneously, the nattokinase it produces can provide a core ingredient for the development of related functional foods or pharmaceuticals, endowing products with potential health benefits such as improved blood circulation.

[0006] The technical solution of this invention is as follows:

[0007] A high-salt-tolerant Bacillus subtilis species that produces protease and nattokinase, characterized in that the Bacillus subtilis species is named Bacillus subtilis 3-1, which is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCCNO: M20252875 and deposit date of December 19, 2025; its 16S rRNA gene sequence is shown in SEQ ID NO:1.

[0008] This invention also provides the application of the aforementioned high-salt-tolerant and protease- and nattokinase-producing Bacillus subtilis 3-1, which is used in the production and processing of high-salt fermented foods to maintain the stability of the fermentation process and to prepare fermented foods, functional foods, health foods, or pharmaceuticals. The salt content of the salt-containing fermented foods is 6-10% NaCl.

[0009] The present invention also provides the application of the above-mentioned high-salt tolerant and protease- and nattokinase-producing Bacillus subtilis 3-1, which is used to prepare fermented products with protease activity, accelerate protein hydrolysis, and enrich the flavor of fermented products.

[0010] This invention also provides the application of the above-mentioned high-salt-tolerant and protease- and nattokinase-producing Bacillus subtilis 3-1, which is used to prepare functional foods, health foods or pharmaceuticals with nattokinase-producing activity.

[0011] Beneficial effects of this invention: 1. The present invention Bacillus subtilis 3-1 This strain was selected from fermented black soybeans, a traditional food from Guizhou, and has undergone long-term natural domestication, exhibiting good environmental adaptability. This strain simultaneously possesses three core functions: high salt tolerance, protease production, and nattokinase production. It can still grow and reproduce normally in a 10% NaCl culture medium, with a viable count reaching 103. 7 The strain exhibits a CFU / mL concentration, a protease activity of up to 33,966 U / mL, and the ability to produce nattokinase, with an activity of up to 63,169 IU / mL. Its overall functional performance surpasses that of most existing single-function strains.

[0012] 2. This strain can be used in the production of ordinary fermented foods to enhance the nutrition and flavor of the products; it can also be adapted to the processing of high-salt fermented foods to solve the problem of insufficient traditional high-salt fermentation strains; its characteristics of producing protease and nattokinase make it have important application potential in the field of functional foods and health products, which can meet the needs of different industries and has broad application prospects. Attached Figure Description

[0013] Figure 1 This is the invention Bacillus subtilis Growth curve of 3-1.

[0014] Figure 2 This is the invention Bacillus subtilis 3-1 Colony morphology plate diagram.

[0015] Figure 3 This is the invention Bacillus subtilis Microscopic image of bacterial cell morphology in 3-1 (1000X).

[0016] Figure 4 It is a strain Bacillus subtilis 3-1 Based on the results of 16S rRNA gene sequence alignment Paenibacillus polymyxa The Neighbor-Joining phylogenetic tree constructed using DSM 36 (AJ320493) as an outer branch.

[0017] Figure 5 The strains of the present invention Bacillus subtilis 3-1 (Identification number: 2025174) Heatmap of the whole genome of the strain calculated using OAT software.

[0018] Figure 6 This is the invention Bacillus subtilis Salt tolerance of 3-1.

[0019] Figure 7 This is the invention Bacillus subtilis 3-1 protein hydrolysis capacity.

[0020] Figure 8 This is the invention Bacillus subtilis 3-1's ability to produce nattokinase.

[0021] Figure 9 This is the invention Bacillus subtilis Drug resistance of 3-1. Detailed Implementation

[0022] The present invention will be better understood through specific examples below. However, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments.

[0023] Example 1 This embodiment describes a high-salt-tolerant Bacillus subtilis strain that produces proteases and nattokinase. The Bacillus subtilis strain is named... Bacillus subtilis 3-1, deposited at the China Center for Type Culture Collection, accession number CCTCC NO: M20252875, December 19, 2025; the aforementioned Bacillus subtilis The 16S rRNA nucleotide sequence of 3-1 is shown in SEQ ID NO:1.

[0024] This implementation Bacillus subtilis 3-1 Used in the production and processing of high-salt fermented foods to maintain the stability of the fermentation process and to prepare fermented foods, functional foods, health foods or medicines.

[0025] This implementation Bacillus subtilis 3-1 is used in fermented products with protease activity to accelerate protein hydrolysis and enrich the flavor of fermented products.

[0026] This implementation Bacillus subtilis3-1 is used to prepare functional foods, health foods, or medicines with nattokinase activity; the nattokinase activity is used to help improve the body's blood circulation function.

[0027] I. Sample Collection and Strain Isolation and Purification Fermented black soybean products were collected from Anshun, Guizhou Province. 10 g of the sample was placed in 90 mL of sterile water and treated in a 90℃ water bath for 15 min. The mixture was then shaken on a shaker for 30 min. 100 µL of the supernatant was spread onto LB agar plates in a clean bench and incubated in a 37℃ biochemical incubator for 24 h. Single colonies were picked for further purification and then stored on slant agar plates for later use.

[0028] II. Colony morphology identification, physiological and biochemical identification, and molecular biological identification (1) The strain was cultured on LB plates for 24 h, and a growth curve was plotted. Figure 1 And observe and record the colony morphology and color.

[0029] (2) Gram staining: pick up colonies from the plate, smear, fix, primary stain with crystal violet, mordant, decolorize, wash with water, counterstain with safranin, dry, and examine under a microscope.

[0030] The final Gram-positive strain was preserved and numbered 3-1 before proceeding to the next stage of the experiment.

[0031] The colony morphology of the strain was observed with the naked eye, and the results are as follows: Figure 2 The colonies of this strain were milky white and irregular in shape, and produced extracellular secretions. After Gram staining, the cell morphology was observed under an oil immersion optical microscope, and the results were as follows: Figure 3 It is a Gram-positive bacterium with spores.

[0032] (3) Physiological and biochemical characteristics Bacillus subtilis The physiological and biochemical characteristics of 3-1 are shown in Table 1-2.

[0033] Table 1. Physiological and biochemical characteristics of strain Bacillus subtilis 3-1 - enzyme activity and carbon source assimilation

[0034] +: Positive reaction; -: Negative reaction; Table 2. Physiological and biochemical characteristics of strain Bacillus subtilis 3-1 - Acid production using carbon sources

[0035] +: Positive reaction; -: Negative reaction; (4) Molecular biological characteristics strains Bacillus subtilis 3-1, 16S rRNA gene sequencing was performed, and the results were compared with those on NCBI. The identification results were as follows: Bacillus subtilis A 3-1 phylogenetic tree was constructed, and the results are as follows: Figure 4 As shown. The 16S rRNA nucleotide sequence is shown in SEQ ID NO:1.

[0036] (5) Strain identification report - Whole genome sequencing of microbial strain 3-1 Figure 5 This is a whole genome heatmap, showing strain 3-1 and... Bacillus subtilis The ANI value obtained by comparing the whole genome data of the (DSM10) type strain was 98.35%, which is greater than the threshold of 96% for ANI identification (this result was detected and provided by Wuhan Bena Technology Co., Ltd.).

[0037] III. Salt tolerance of the strain The bacteria to be tested were activated, and the bacterial concentration was adjusted to 1.0 × 10⁻⁶. 8 CFU / mL, inoculated at 3% (v / v) in LB liquid medium with NaCl contents of 0, 2%, 4%, 6%, 8%, and 10%, respectively, and incubated at 37°C for 24 h before counting viable cells. Figure 6 ) Strain 3-1 exhibits good tolerance to NaCl, growing well even in 10% NaCl conditions, with a viable count of 10-1. 7 CFU / mL.

[0038] IV. Protease Production Capacity of Strains (1) Initial screening of protease production capacity Bacillus subtilis 3-1 was activated and streaked onto an agar plate containing casein. After incubation, a clear zone could be observed. Figure 7 This indicates that the strain can effectively break down proteins.

[0039] (2) Rescreening of protease production capacity Preparation of crude enzyme solution: The strain was inoculated into LB medium and cultured in liquid at 37°C for 24 hours. Then, the fermentation broth was centrifuged at 8000 r / min for 10 minutes. The resulting supernatant was the crude enzyme solution.

[0040] The Folin-Ciocalteu method, as specified in the national standard GB / T23527.1-2023, was used to determine protease activity. One unit of protease activity (U / mL) is defined as the amount of enzyme required to catalyze the production of 1 μg of tyrosine from 1% casein solution per minute under standard assay conditions (40℃ constant temperature, pH 7.0 buffer system). 1 mL of crude enzyme solution was added and reacted at 40℃±0.2℃ for 2 min. 1 mL of casein solution was added and shaken well, then reacted at 40℃±0.2℃ for 10 min. 2 mL of trichloroacetic acid was added, shaken well, and allowed to stand for 10 min before filtration. 1 mL of the filtrate was taken, and 5 mL of sodium carbonate and 1 mL of Folin-Ciocalteu solution were added. The mixture was incubated at 40℃±0.2℃ for 20 min. Neutral protease was used as a positive control. The absorbance values ​​of the sample group and the control group were measured at 680 nm and converted to protease activity.

[0041] The enzyme activity of the crude enzyme solution was 33966 U / mL, while the enzyme activity of the positive control group was 15953 U / mL.

[0042] V. Ability of the strain to produce nattokinase (1) Initial screening of nattokinase production capacity Genomic DNA was extracted from the screened strains as PCR templates. Nattokinase gene primers (synthesized by Sangon Biotech) were synthesized, and their sequences are shown in Table 3. PCR was performed in a 25 μL system: 10×PCR Buffer 2.2 μL; dNTPs (2.5 mmol / L) 2.5 μL; ddH2O 17.5 μL; forward and reverse primers (10 μmol / L) 0.5 μL each; DNA template 1 μL; Taq enzyme 0.5 μL. The PCR program for the nattokinase gene was: 94 ℃ pre-denaturation for 3 min, 94 ℃ denaturation for 30 s, 52 ℃ annealing for 30 s, and 72 ℃ extension for 75 s, for a total of 30 cycles; sterile ddH2O was used as a blank control. The PCR products were identified by 1% agarose gel electrophoresis, showing the amplification of a distinct specific band approximately 1200 bp in size. Figure 8 (This is in line with expectations.)

[0043] The results showed that the strain produced nattokinase.

[0044] Table 3 Amplification Primer Sequences

[0045] (2) Rescreening of nattokinase production capacity After streak activation of the selected nattokinase-producing strains, a single colony was inoculated into LB liquid medium and cultured in shake flasks at 37°C for 24 hours. After the shaking period, the bacterial culture was sampled and centrifuged at 12000 rpm for 10 minutes at 4°C. The supernatant was used as the crude enzyme solution, and nattokinase activity was determined using the fibrin plate method. The nattokinase activity was calculated using the urokinase standard curve based on the size of the lysis zone of the strains. The crude enzyme activity was 63169 IU / mL.

[0046] VI. Drug resistance of strains (1) Drug sensitivity The bacteria to be tested were activated, and the bacterial concentration was adjusted to 1.0 × 10⁻⁶. 8 CFU / mL, take 100µL and spread evenly. After the plate surface is fully dry, use sterile forceps to pick up antibiotic discs containing erythromycin (15µg), tetracycline (30µg), chloramphenicol (30µg), and kanamycin (30µg). Gently press the discs onto the plate after spreading the bacterial culture, ensuring complete contact. The discs should be spaced at least 24mm apart and at least 15mm from the edge of the plate. Do not move them after placement. Incubate at 37℃ for 24 h. Figure 9 The diameter (H) of the clear zone that completely inhibits bacterial growth around each drug sensitivity tablet and the tablet diameter (C) were measured using vernier calipers. The H / C value was calculated, and the results are shown in Table 4.

[0047] Table 4. Susceptibility of strains to four antibiotics

[0048] The strain's antibiotic sensitivity is: chloramphenicol tetracycline Erythromycin Kanamycin. Given that this strain is sensitive to antibiotics and does not carry the risk of transferable resistance genes, it can be used as a strain with a natural safety advantage in the processing of fermented foods.

[0049] This invention isolates and screens the target strain from traditional high-salt fermented soybean foods; through colony morphology, physiological and biochemical characteristics, and 16S rRNA gene sequence analysis, it is identified as Bacillus subtilis (Bacillus subtilis). Bacillus subtilis By measuring its salt tolerance, protease and nattokinase content, and safety, it was confirmed that this strain possesses both high salt tolerance and enzyme production characteristics. Experimental verification shows that the *Bacillus subtilis* 3-1 described in this invention is a functional strain with application potential, especially demonstrating outstanding performance in tolerating high-salt environments and efficiently secreting proteases and nattokinase.

[0050] Specifically, this strain can grow normally in culture media with a NaCl concentration as high as 10%, and the viable cell count can still reach 10^6. 7The CFU / mL concentration exhibits excellent salt tolerance. In terms of fermentation performance, its protease activity reaches 33966 U / mL, effectively hydrolyzing proteins to generate flavor precursors. Furthermore, specific PCR amplification confirmed that it carries the complete nattokinase encoding gene, demonstrating its ability to produce nattokinase. Its nattokinase activity reaches 63169 IU / mL.

[0051] Based on the above characteristics, applying Bacillus subtilis 3-1 of this invention to the production of high-salt fermented foods can solve the technical bottlenecks of slow fermentation start-up, long cycle, and incomplete protein hydrolysis under high-salt conditions, thereby shortening the cycle and improving flavor and quality. Simultaneously, utilizing its nattokinase-producing properties, functional foods or related products with thrombolytic and cardiovascular health-promoting functions can be further developed.

[0052] The above embodiments are merely illustrative examples and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations; therefore, any obvious variations or modifications derived therefrom remain within the scope of protection of this invention.

Claims

1. A high-salt-tolerant Bacillus subtilis strain that produces protease and nattokinase, characterized in that, The Bacillus subtilis strain was named Bacillus subtilis 3-1 and is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCCNO: M20252875 on December 19, 2025.

2. The Bacillus subtilis strain tolerant to high salt and producing protease and nattokinase according to claim 1, characterized in that, The 16S rRNA gene sequence of Bacillus subtilis is shown in SEQ ID NO:

1.

3. The application of Bacillus subtilis according to any one of claims 1-2, characterized in that, The Bacillus subtilis is used in the preparation of salt-containing fermented foods.

4. The application of Bacillus subtilis according to claim 3, characterized in that, The salt content of the fermented food is 6-10% NaCl.

5. The application of Bacillus subtilis according to any one of claims 1-2, characterized in that, The Bacillus subtilis is used to prepare functional foods, health foods, or pharmaceuticals with protease activity.

6. The application of Bacillus subtilis according to any one of claims 1-2, characterized in that, The Bacillus subtilis is used to prepare functional foods, health foods, or pharmaceuticals with nattokinase activity.

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