TW202548006A - A strain of bacillus subtilis and use thereof in the co-production of nattokinase and vitamin k2 - Google Patents

Abstract

The present invention relates to the field of microbial technology, in particular to a strain ofBacillus subtilisand use thereof in the co-production of nattokinase and vitamin K2. The accession number of the strain is CCTCC NO: M 2024343. The strain achieves synchronous fermentation of nattokinase and vitamin K2, while increasing the nattokinase enzyme activity and the content of vitamin K2, and has a short fermentation cycle and low cost, which is suitable for large-scale production.

Description

Bacillus subtilis and its application in the co-production of nattokinase and vitamin K2

This invention relates to the field of microbial technology, and in particular to a strain of Bacillus subtilis and its application in the co-production of nattokinase and vitamin K2.

Nattokinase is a basic serine protease secreted by Bacillus subtilis, with a molecular weight of approximately 28 kDa. It is a single-chain protein structure composed of 275 amino acid residues. Its molecular weight is much smaller than that of urokinase, streptokinase, and recombinant tissue plasminogen activator, making it easier for the human body to absorb. It can produce a strong thrombolytic effect in vivo and has significant effects in the treatment and prevention of thrombosis. Compared with clinical urokinase, streptokinase, and other plasminogen activators, it has advantages such as a long half-life, good safety, strong specificity, and oral administration.

Vitamin K2 is an important fat-soluble clotting vitamin used to maintain the body's normal blood clotting function. In addition, as an electron transport carrier in respiratory metabolism, vitamin K2 also participates in many important metabolic reactions in the body. It has many important physiological functions such as preventing and treating osteoporosis, arterial calcification, cardiovascular diseases, tumors and Parkinson's disease. It has advantages such as high affinity and long half-life in the human body.

Bacillus subtilis is a Gram-positive bacterium that produces not only nattokinase and vitamin K2, but also various proteases, glucosamine, pyridine dicarboxylic acid, and bioactive peptides, among other physiologically active substances. When a thrombus forms in the human body, enzyme systems with serine protease activity work synergistically to efficiently degrade cross-linked fibrin that forms the thrombus backbone. The abundance of these proteases is crucial for efficient thrombolysis. Because different Bacillus species produce proteases with significant differences in their enzyme systems, selecting suitable strains and fermentation processes is critical to the effectiveness of nattokinase-related products.

The industry of producing nattokinase using Bacillus subtilis is currently in its developmental stage, facing two main challenges: low enzyme activity and high cost. Regarding enzyme activity, previous reports indicate that most enzymes have activities below 500 FU/mL, which, overall, lags behind the fermentation levels of nattokinase reported in Japanese literature. The nattokinase content, temperature tolerance, and acid tolerance vary significantly among different strains and fermentation processes, necessitating low-temperature freeze-drying for most strains, resulting in high production costs (due to high drying costs).

The main strain used for vitamin K2 production is *Bacillus subtilis*. Previous reports indicated that the fermentation cycle of these strains was long, and the vitamin K2 yield was not ideal. Vitamin K2 is distributed both intracellularly and extracellularly in the fermentation broth. The highest reported total content is found in patent CN116536220A, which discloses a method for preparing *Bacillus subtilis* strains for natto and a method for preparing vitamin K2, with a vitamin K content of 124 mg/L and a fermentation time of 120-140 hours. The long fermentation cycle increases the risks of contamination and unstable product content. Combined with the industry's low fermentation yield, this ultimately leads to consistently high vitamin K2 production costs.

In addition, in the method for co-producing vitamin K2 and nattokinase by fermentation using Bacillus natto in patent CN106701719A, both nattokinase and vitamin K2 are in the fermentation supernatant, requiring a complicated purification process to separate them. Furthermore, the fermentation level is low, resulting in insufficient production efficiency and failing to fundamentally solve the problem of generally high production costs of vitamin K2.

In view of this, the present invention provides a strain of Bacillus subtilis and its application in the co-production of nattokinase and vitamin K2. This strain achieves simultaneous fermentation of nattokinase and vitamin K2, produces a rich enzyme system of proteases, and has a short fermentation cycle. The fermentation broth is mainly used to produce nattokinase-related products, and the separated heavy phase (bacterial sludge) is mainly used to produce vitamin K2-related products.

This invention isolated a strain RB4 from the traditional Baijiu cellar mud of Yibin, Sichuan. The strain is numbered AMCC 11927 in the Angel Yeast Co., Ltd. strain bank. It was identified as Bacillus subtilis by morphology and 16S rDNA. It was deposited at the Food Industry Development Institute Foundation in 2025 with deposit number CCTCC NO: M 2024343 (and deposited at the China Center for Type Culture Collection on February 27, 2024, at Wuhan University, Wuhan, China, with deposit number CCTCC NO: M 2024343).

The present invention also provides microbial agents, including at least one of the Bacillus subtilis RB4 or its culture, metabolite and fermentation product.

The present invention also provides the use of the Bacillus subtilis RB4 or the microbial agent in at least one of the following: (I) in the preparation of nattokinase and/or vitamin K2 or products thereof; (II) in the preparation of fermented soybean, natto or intestinal probiotic agents; (III) in the fermentation preparation of biological feed.

In the above applications, the product includes at least one of natto, natto powder, nattokinase fermented products, and vitamin K2 fermented products.

This invention also provides a culture medium for fed-batch fermentation of Bacillus subtilis, comprising a fermentation bottom water culture medium and supplements; the fermentation bottom water culture medium comprises: corn flour, soybean meal, yeast peptone, L-serine, glycine, and inorganic salts; the inorganic salts comprise sodium salts, calcium salts, potassium salts, magnesium salts, zinc salts, and iron salts; the supplements comprise supplement 1, supplement 2, and supplement 3. The supplement 1 comprises water and a component selected from any of the following combinations: a combination of liquefied corn flour and L-serine, a combination of maltodextrin and L-serine, a combination of corn flour and L-serine, or a combination of glycerol and L-serine; the supplement 2 comprises water and soybean meal, or an aqueous solution of ammonium sulfate; the supplement 3 is ammonia.

In some embodiments, the inorganic salt includes sodium chloride, calcium chloride, potassium dihydrogen phosphate, magnesium sulfate, zinc sulfate, and ferrous sulfate.

In some preferred embodiments, the fermented hydroponic culture medium comprises water and the following components by weight/volume percentage: 1-4% corn flour, 0.2-1% soybean meal, 0.1-0.5% yeast peptone, 0.05-0.3% L-serine, 0.001-0.01% glycine, 0.1-1% sodium chloride, 0.1-0.4% calcium chloride, 0.05-0.2% potassium dihydrogen phosphate, 0.05-0.2% magnesium sulfate, and 0.002-0.01% sulfur. Zinc sulfate and 0.001-0.01% ferrous sulfate; in some specific embodiments, the fermentation substrate comprises water and the following components by weight and volume percentage: 2% corn flour, 0.5% soybean meal, 0.1% yeast peptone (Angel FP103), 0.1% L-serine, 0.005% glycine, 0.6% sodium chloride, 0.2% calcium chloride, 0.1% potassium dihydrogen phosphate, 0.1% magnesium sulfate, 0.01% zinc sulfate and 0.005% ferrous sulfate.

In some specific embodiments, the pH of the fermented hydroponic culture medium may be 6.0, 6.1, or 6.2.

In some preferred embodiments, supplement 1 comprises water and any combination of the following: a combination of 5-25% corn flour and 0.05-0.3% L-serine, a combination of 10-25% maltodextrin and 0.05-0.3% L-serine, or a combination of 10-25% glycerol and 0.05-0.3% L-serine, wherein the corn flour comprises liquefied corn flour and/or non-liquefied corn flour; supplement 2 is 5-10% soybean meal and the remainder water, or 1-3.5% ammonium sulfate and the remainder water; supplement 3 is 10-15% ammonia. In some specific embodiments, supplement 1 comprises water and any combination selected from the following: a combination of 10% non-liquefied corn flour and 0.3% L-serine, a combination of 25% liquefied corn flour and 0.3% L-serine, a combination of 25% maltodextrin and 0.3% L-serine, or a combination of 25% glycerol and 0.3% L-serine; supplement 2 is 10% soybean meal and the remainder water, or 3.5% ammonium sulfate and the remainder water; supplement 3 is 15% ammonia. In the above supplements, the percentage "%" for each component refers to mass volume percentage, representing the proportion of each component in the supplement, in g/ml.

In some embodiments, the culture medium described in this invention further includes a bacterial slant culture medium; the bacterial slant culture medium comprises: soluble starch, yeast extract, yeast peptone, sodium chloride, agar, and water, with a pH of 6.0-6.2. Preferably, the bacterial slant culture medium comprises 1-3% soluble starch, 0.5-3% yeast extract, 0.2-1% yeast peptone, 0.1-1% sodium chloride, 2-3% agar, and the balance being water, with a pH of 6.0-6.2; in some specific embodiments, the bacterial slant culture medium comprises 2% soluble starch, 1% yeast extract, 0.4% yeast peptone, 1% sodium chloride, 3% agar, and the balance being water, with a pH of 6.2.

In some embodiments, the culture medium described in this invention further includes a primary seed culture medium; the primary seed culture medium comprises soluble starch, yeast extract, yeast peptone, sodium chloride, potassium dihydrogen phosphate, magnesium sulfate, and water, with a pH of 6.0-6.2. Preferably, the primary seed culture medium comprises 1-3% soluble starch, 0.5-3% yeast extract, 0.2-1% yeast peptone, 0.1-1% sodium chloride, 0.05-0.2% potassium dihydrogen phosphate, 0.05-0.2% magnesium sulfate, and the balance being water. In some specific embodiments, the primary seed culture medium comprises 2% soluble starch, 1% yeast extract, 0.4% yeast peptone, 0.6% sodium chloride, 0.1% potassium dihydrogen phosphate, 0.1% magnesium sulfate, with the balance being water, and a pH of 6.2.

In some embodiments, the culture medium described in this invention further includes a secondary seed culture medium; the secondary seed culture medium comprises corn flour, soybean flour, yeast extract, L-serine, glycine, potassium dihydrogen phosphate, magnesium sulfate, and water, with a pH of 6.0-6.2. In some preferred embodiments, the secondary seed culture medium comprises 1-3% corn flour, 0.2-1% soybean flour, 0.1-0.5% yeast extract, 0.05-0.3% L-serine, 0.001-0.01% glycine, 0.05-0.2% potassium dihydrogen phosphate, and 0.05-0.2% magnesium sulfate, with the balance being water, and a pH of 6.0-6.2. In some specific embodiments, the secondary seed culture comprises the following components by weight and volume percentage: 2% corn flour, 1% soybean flour, 0.2% yeast extract, 0.1% L-serine, 0.005% glycine, 0.1% potassium dihydrogen phosphate, 0.1% magnesium sulfate, with the balance being water, and pH 6.2.

This invention also provides a fermentation method for Bacillus subtilis, comprising: performing fed-batch fermentation of Bacillus subtilis using the culture medium described in this invention to obtain a fermentation broth; the fed-batch fermentation includes: inoculating the Bacillus subtilis into the fermentation bottom water culture medium described in this invention, and adding the feed in a fed-batch manner. The specific timing of adding the feed is determined based on changes in dissolved oxygen and total sugar content. In a specific embodiment of this invention, the feed is added 1-2 hours after inoculation into the fermentation bottom water culture medium.

In some embodiments, prior to the fed-batch fermentation, a step of scaling up the *Bacillus subtilis* using the strain slant culture medium, primary seed culture medium, and/or secondary seed culture medium described in this invention is included. In some specific embodiments, prior to the fed-batch fermentation, the step further includes scaling up the *Bacillus subtilis* using the strain slant culture medium described in this invention, or using the strain slant culture medium and primary seed culture medium described in this invention, or using the strain slant culture medium, primary seed culture medium, and secondary seed culture medium described in this invention.

In some embodiments, the initial fermentation temperature of the fed-batch fermentation is 36-45℃, the fermentation temperature increases by 1-2℃ every 2-4 hours during fermentation, and the fermentation temperature is maintained at 46-52℃ for 12-16 hours for subsequent fermentation, with a total fermentation cycle of 34-40 hours. In some specific embodiments, the initial fermentation temperature of the fed-batch fermentation is 42℃, the fermentation temperature increases by 2℃ every 4 hours during fermentation, and the fermentation temperature is maintained at 50℃ for 16 hours for subsequent fermentation, with a total fermentation cycle of 36-40 hours.

In some implementation schemes, the feed 1 is fed in a pulsed manner to maintain dissolved oxygen levels fluctuating between 10-30% and total sugar content <30g/L during the fermentation process, or a dissolved oxygen-coupled feed is used to maintain dissolved oxygen levels at 30% and total sugar content at 10-20g/L during the fermentation process; the feed 2 is fed in a segmented, uniform rate; the feed 3 is used to control the fermentation pH to be constant at 6.0, or to control the fermentation pH to be >5.8. When the pH of the fermentation broth is lower than 5.8, ammonia is used to adjust the pH back to 6.5.

In this invention, there are no special restrictions on the specific source of the yeast peptone and yeast extract powder; ordinary commercially available products are acceptable. In a specific embodiment of this invention, the yeast peptone is Angel FP103, and the yeast extract powder is Angel FM888.

The present invention also provides a method for preparing nattokinase and/or vitamin K2, comprising the following steps: Step (1) performing flocculation separation and plate and frame filtration sequentially on the fermentation liquid obtained by the fermentation method of the present invention, and collecting the clear liquid and bacterial residue respectively; Step (2) filtration of the clear liquid through a membrane followed by concentration and drying to obtain a product rich in nattokinase; Step (3) drying the bacterial residue to obtain a product rich in vitamin K2.

After obtaining products rich in nattokinase and vitamin K2 according to the above preparation method of the present invention, pure nattokinase and pure vitamin K2 can be prepared separately using the separation and purification methods disclosed in the art.

In some implementation schemes, in step (1), flocculants and perlite are used to flocculate and separate the fermentation liquid. In some specific embodiments, the flocculant is a composite clarifying flocculant, which can be polyacrylamide, calcium chloride, etc., and any type commonly used in this field is acceptable; the amount of perlite added is 2% of the total mass of the fermentation liquid. In some specific embodiments, the flocculation and separation includes: first adding composite clarifying flocculants to the fermentation liquid until the concentration of composite clarifying flocculants is 1.0 g/L, stirring at high speed for 2 hours, and then adding 2.0 g/L of perlite.

In some implementation schemes, step (2) after concentration and before drying includes: mixing the concentrated liquid and auxiliary materials to obtain raw materials containing nattokinase, and the drying is spray drying; in step (3), the drying includes mixing and granulating the bacterial residue and auxiliary materials before drying, and the drying is fluidized bed drying.

This invention provides a strain of Bacillus subtilis and its application in the co-production of nattokinase and vitamin K2. This strain enables simultaneous fermentation of nattokinase and vitamin K2, produces a rich enzyme system of proteases, has a short fermentation cycle, and is low in cost. The fermentation supernatant is mainly used to produce nattokinase-related products, and the separated heavy phase (bacterial sludge) is mainly used to produce vitamin K2-related products. This invention has the following advantages.

1. Using the strain of this invention, the fermentation cycle is less than 40 hours. The nattokinase content in the fermentation liquid is nearly 35% higher than the highest enzyme activity reported in the industry, and it is rich in a variety of protease systems. The temperature and acid resistance of the nattokinase produced by this strain is significantly improved, and the spray drying enzyme activity recovery rate is nearly 80%, effectively solving the problems of low fermentation level of nattokinase and large loss of enzyme activity during spray drying in the industry.

2. The vitamin K2 content (heavy phase) in the by-product sludge after fermentation of nattokinase by the strain of this invention reaches more than 140 ug/g, which can be directly used as a raw material for vitamin K2 extraction. Due to the short fermentation cycle and high utilization value of the by-product, it effectively solves the problems of long fermentation cycle and high production cost of vitamin K2.

Definition of terms

The "natto" described in this invention is a soybean product made from soybeans fermented by Bacillus subtilis natto.

The "nattokinase" described in this invention is an alkaline serine protease extracted from natto, which has been reported in the prior art. The "nattokinase fermented product" used in this invention refers to a fermented product containing nattokinase.

The term "vitamin K2 fermented product" as used in this invention refers to a fermented product containing vitamin K2.

The "fermentation method of Bacillus subtilis" described in this invention also refers to a fermentation method using Bacillus subtilis. For example, Bacillus subtilis RB4 of this invention is used to ferment a substrate (such as corn flour or soybean meal) to obtain a fermented product.

The "feedback fermentation" described in this invention, also known as fed-batch fermentation or semi-continuous fermentation, refers to a fermentation technique in which a certain amount of material is added to the fermentation system in a certain way during the microbial batch fermentation process, but the fermentation liquid is not continuously released. The "feedback addition" method used in this invention refers to adding feed in a fed-batch fermentation manner, for example, in batch culture, feed is gradually added as fermentation progresses.

The "fermented bottom water culture medium" mentioned in this invention refers to the culture medium used when fermentation begins, and can also be called fermented base culture medium.

This invention discloses a strain of Bacillus subtilis and its application in the co-production of nattokinase and vitamin K2. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the same result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art will clearly be able to modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

This invention isolated multiple Bacillus strains from the traditional Baijiu (Chinese liquor) cellar mud in Yibin, Sichuan. After initial screening using cellulose agar plates and high-temperature acclimatization, one strain, RB4, was selected, capable of growing well at 48-50℃. Finally, studies were conducted on this strain to produce nattokinase, protease, and vitamin K2-related metabolites during liquid fermentation. The results showed that this strain exhibited outstanding performance in producing nattokinase and vitamin K2. Based on morphological and molecular biological (16S rDNA) analysis, it was identified as * Bacillus subtilis *. The 16S rDNA gene sequence of strain RB4 is as follows:

(SEQ ID NO: 1): ACTTCACCCCAATCATCTGTCCCACCTTCGGCGGCTGGCTCCTAAAAGGTTACCTCACCGACTTCGGGTGTTACAAACTCTCGTGGTTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCAGCTTCACGCAGTCGAGT TGCAGACTGCGATCCGAACTGAGAACAGATTTGTGGGATTGGCTTAACCTCGCGGTTTCGCTGCCCTTTGTTCTGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTGAAT GCTGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACTCTGCCCCCGAAGGGGACGTCCTATCTCTAGGATTGTCAGAGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACAT GCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTAAGGGGCGGAAACCCCCTAACACTTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTC GCTCCTCAGCGTCAGTTACAGACCAGAGAGTCGCCTTCGCCACTGGTGTTCCTCCACATCTCTACGCATTTCACCGCTACACGTGGAATTCCACTCTCCTCTTCTGCACTCAAGTTCCCCAGTTTCCAATGACCCTCCCCGGTTGAGCCGGGGGCTTTCACATCAGACTTAAGAAACCGCCTGC GAGCCCTTTACGCCCAATAATTCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCAAGGTACCGCCCTATTCGAACGGTACTTGTTCTTCCCTAACAACAGAGCTTTACGATCCGAAAACCTTCATCACTCACGCGGCGT TGCTCCGTCAGACTTTCGTCCATTGCGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTACGCATCGTCGCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATGCGCCGCGGGTCCATCTGTAAGTGGTAGCCG AAGCCACCTTTTATGTTTGAACCATGCGGTTCAAACAACCATCCGGTATTAGCCCCGGTTTCCCGGAGTTATCCCAGTCTTACAGGCAGGTTACCCACGTGTTACTCACCCGTCCGCCGCTAACATCAGGGAGCAAGTCCCCATCTGTCCGCTCGACTTGCATGTATTAGGCACGCCGCCAGCG.

Unless otherwise specified, the concentration % of each component in the embodiments of the present invention is a percentage by mass volume (in g/ml).

The invention will be further explained below with reference to practical examples:

Implementation Examples 1

Bacillus subtilis RB4 (accession number CCTCC NO: M 2024343) was cultured on slant, then in primary seed culture, and fermented in a 50L tank using fed-batch fermentation. The nattokinase activity and heavy-phase vitamin K2 content of the fermentation broth were tracked and detected. The levels of nattokinase and vitamin K2 produced are shown in Tables 1 and 2.

The specific manufacturing process includes the following:

(1) Slant culture: The above strains were inoculated into the strain slant culture medium. The slant culture temperature was 42℃ and the time was 24h.

The slant culture medium was formulated as follows: 2% soluble starch, 1% yeast extract (Angel FM888), 0.4% yeast peptone (Angel FP103), 1% sodium chloride, 3% agar, with the remainder being water, pH 6.2, and sterilized at 121℃ for 30 min.

(2) Primary seed culture: The strain cultured on the slant was inoculated into a 5L shake flask containing 1L of primary seed culture medium. The culture temperature was 42℃, the shaker speed was 220r/min, and the culture time was 20h to obtain the primary seed culture solution.

The primary seed culture medium is formulated as follows: 2% soluble starch, 1% yeast extract (Angel FM888), 0.4% yeast peptone (Angel FP103), 0.6% sodium chloride, 0.1% potassium dihydrogen phosphate, 0.1% magnesium sulfate, with the balance being water, pH 6.2, and sterilized at 121℃ for 30 min.

(3) Feed fermentation: 1.2L of primary seed culture medium was inoculated into a 50L tank containing 6.8L of fermentation bottom water culture medium for feed fermentation. The fermentation program controlled the pH at 6.0, the fermentation temperature at 42℃, the fermentation tank speed at 200-250r/min, and the aeration rate at 1:1~1.2:1 vvm. Feed (including feed 1, feed 2 and feed 3) was added after 1~2 hours of incubation.

The culture medium used in this process includes a fermented bottom water culture medium and supplements. The fermented bottom water culture medium consists of: 2% corn flour, 0.5% soybean meal, 0.1% yeast peptone (Angel FP103), 0.1% L-serine, 0.005% glycine, 0.6% sodium chloride, 0.2% calcium chloride, 0.1% potassium dihydrogen phosphate, 0.1% magnesium sulfate, 0.01% zinc sulfate, and 0.005% ferrous sulfate. Supplement 1 consists of: 25% liquefied corn flour and 0.3% L-serine; Supplement 2 consists of: 10% soybean meal; Supplement 3 consists of: 15% ammonia water (used to adjust pH, added as needed according to pH changes).

All raw materials used in this invention can be purchased from the market, and the same products from different brands can be substituted for each other.

The fed-batch fermentation process is as follows: the volume of feed 1 is 10L, and dissolved oxygen is added in conjunction with the feed. The dissolved oxygen is maintained at 30% during the fermentation process, and the total sugar content is maintained at 10-20g/L. The total volume of feed 2 is 6L, and it is added in a segmented uniform flow rate. The flow rate of feed 2 is 65% of that of feed 1.

Regularly sample and centrifuge to test the nattokinase activity in the fermentation broth. High-pressure homogenization and cell wall disruption testing are performed on the heavy phase to determine the vitamin K2 content. Nattokinase activity reference: DBS44/013-2019 Local Food Safety Standard - Determination of Nattokinase in Natto Powder - Ultraviolet Spectrophotometry. Vitamin K2 content reference: Appendix A.4 Vitamin K2 Detection Method of Announcement No. 8 of the National Health and Family Planning Commission, 2016.

Test Case 1 Comparison of fermentation results of different strains

Multiple Bacillus strains selected in the laboratory, including SD, YJ, RB1, RB2, MG1, MG3, SJ1, SJ2, BL1, and BL2, were used as research subjects. Bacillus subtilis was cultured on slant, in primary seed culture, in secondary seed culture, and fermented in a 50L tank using the following process. The nattokinase activity and heavy-phase vitamin K2 content of the fermentation broth were tracked and detected. The levels of nattokinase and vitamin K2 produced by different strains are shown in Tables 1 and 2.

The main strains of Comparative Examples 1-10 are different from those of Example 1, while the other processes remain unchanged.

Table 1 Comparison of Nattokinase and Fermentation Time among Different Strains Implementation Examples Nattokinase activity (FU/g) Acidic protease (U/g) Neutral protease (U/g) Basic protease (U/g) Optimal fermentation time (h) Implementation Example 1 (RB4) 1873 1106 4935 2848 38 Comparative Example 1 (SD) 1560 1413 2083 1971 47 Comparative Example 2 (YJ) 754 229 1321 890 53 Comparative Example 3 (RB1) 211 342 837 423 66 Comparative Example 4 (RB2) 1269 1211 1628 1363 60 Comparative Example 5 (MG1) 83 213 594 298 60 Comparative Example 6 (MG3) 950 335 3255 1554 48 Comparative Example 7 (SJ1) 671 201 2101 982 32 Comparative Example 8 (SJ2) 720 197 1898 1562 36 Comparative Example 9 (BL1) 39 302 761 338 68 Comparative Example 10 (BL2) 90 643 1032 775 72

Table 2. Comparison of Vitamin K2 and Fermentation Time in the Heavy Phase of Fermentation Broth from Different Strains Implementation Examples Vitamin K2 (ug/g) Optimal fermentation time (h) Implementation Example 1 (RB4) 152 96 Comparative Example 1 (SD) 41 72 Comparative Example 2 (YJ) 138 120 Comparative Example 3 (RB1) 103 96 Comparative Example 4 (RB2) 156 168 Comparative Example 5 (MG1) 18 60 Comparative Example 6 (MG3) 90 108 Comparative Example 7 (SJ1) 62 168 Comparative Example 8 (SJ2) 26 70 Comparative Example 9 (BL) 39 68 Comparative Example 10 (YZ1) 80 108

As shown in Tables 1 and 2, strain RB4 of this invention exhibits outstanding fermentation performance of nattokinase and is rich in vitamin K2. This strain RB4 was deposited in 2025 at the Food Industry Development Institute Foundation, with accession number CCTCC NO: M 2024343 (and subsequently deposited on February 27, 2024, at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with accession number CCTCC NO: M 2024343).

Implementation Examples 2

Unlike Example 1, Feed 1 is fed in a pulsed manner, maintaining dissolved oxygen levels fluctuating between 10-30% during the fermentation process, keeping the total sugar content <30g/L, and the fermentation time is 34-36 hours. Other processes remain unchanged.

Implementation Examples 3

Unlike Example 2, feed 1 consists of 25% maltodextrin and 0.3% L-serine. The other processes remain unchanged.

Comparison 11

Unlike Example 2, feed 1 consists of 10% non-liquefied corn flour and 0.3% L-serine. The other processes remain unchanged.

Comparison 12

Unlike Example 2, Feed 1 consists of 25% glucose and L-serine, which is added after being sterilized separately, while the rest of the process remains unchanged.

Comparison 13

Unlike Example 2, supplement 1 consists of 25% sucrose and 0.3% L-serine. The other processes remain unchanged.

Comparison 14

Unlike Example 2, supplement 1 consists of 25% glycerol and 0.3% L-serine. The other processes remain unchanged.

Comparison 15

Unlike Example 2, supplement 2 consists of 10% yeast protein powder. The rest of the process remains unchanged.

Comparison 16

Unlike Example 2, feed 2 consists of 3.5% ammonium sulfate. The other processes remain unchanged.

Test Case 2 Comparison of fermentation results with different supplements

The nattokinase activity and vitamin K2 content in fermentation broths obtained from fermentation with different feeds were detected, and the results are shown in Table 3:

Table 3. Effects of different supplements on nattokinase activity and vitamin K2 content. Group Nattokinase activity (FU/g) Vitamin K2 (ug/g) Implementation Example 2 2601 146 Implementation Example 3 2538 159 Comparative Example 11 1810 85 Comparative Example 12 629 161 Comparative Example 13 738 63 Comparative Example 14 1847 182 Comparative Example 15 198 79 Comparative Example 16 2337 142

Table 3 shows that the nattokinase activity and vitamin K2 content of strain RB4 fermented under different fermentation raw materials and fed-batch process parameters varied greatly. When liquefied corn starch or dextrin was used as the carbon source, soybean meal was used as the nitrogen source, and the process was controlled by pulse feeding, the nattokinase activity and vitamin K2 content of fermented broth were relatively ideal, with an average nattokinase activity >2500 FU/g and a vitamin K2 content >140 ug/g in the heavy phase.

Implementation Examples 4

Unlike Example 2, in Feed 3, the fermentation pH is controlled to be greater than 5.8. When the pH of the fermentation liquid is lower than 5.8, ammonia water is used to adjust the pH back to 6.5 for control. The initial fermentation temperature is 42°C. During the fermentation process, the fermentation temperature is increased by 2°C every 4 hours. After 16 hours of fermentation, the fermentation temperature is maintained at 50°C for subsequent fermentation. Other processes remain unchanged. Add 0.1% of compound clarifying flocculant to the fermentation broth, stir for 2 hours at 500 r/min, add 2% perlite, and perform plate and frame filtration. The filtered liquid is concentrated 5-6 times by filtration through an 8000 Dalton membrane, heated to 50℃, and 5% dextrin is added and stirred evenly. A spray drying tower is used with an inlet air temperature of 110-120℃ and the outlet temperature is maintained below 70℃ to obtain raw material rich in nattokinase.

Comparison 17

Unlike Example 4, the fermentation temperature was maintained at 42°C, the fermentation pH was controlled at 6.0, and other processes remained unchanged.

Comparison 18

Unlike Example 4, the fermentation temperature was maintained at 42°C, while other processes remained unchanged.

Comparison 19

Unlike Example 4, the fermentation pH was controlled at 6.0, while other processes remained unchanged.

Comparison 20

Unlike Example 4, the fermentation temperature was maintained at 37°C and the fermentation pH was controlled at 6.0, while other processes remained unchanged.

Comparison twenty one

Unlike Example 4, the fermentation temperature was maintained at 50°C and the fermentation pH was controlled at 6.0, while other processes remained unchanged.

Test Case 3 Comparison of fermentation results from different processes

Table 4. Comparison of nattokinase activity and spray-dried enzyme activity yields under different processing control methods. Implementation Examples Nattokinase activity (FU/g) Parfumase activity yield (%) Example 4 (Variable Temperature, pH Fluctuation) 2763 76 Comparative Example 17 (Constant Temperature, Constant pH) 2575 71 Comparative Example 18 (Constant Temperature, pH Fluctuation) 2598 75 Comparative Example 19 (Variable Temperature, Constant pH) 2309 83 Comparative Example 20 (low temperature, constant pH) 2335 66 Comparative Example 21 (High Temperature, Constant pH) 1821 87

As shown in Table 4, under the variable temperature and pH fluctuation fermentation process control system in Example 4, the nattokinase activity in the fermentation broth reached a maximum of 2763 FU/g, and the powder spraying enzyme activity yield was >75%. Compared with the constant temperature and constant pH fermentation control process, both the nattokinase activity and the powder spraying enzyme activity yield in the fermentation broth were significantly improved.

The above description is merely a preferred embodiment of the present invention and should not be construed as limiting the scope of the invention's rights. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications are also considered to be within the scope of protection of the present invention.

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Domestic Storage Information 1. Republic of China, Bioresource Conservation and Research Center, Food Industry Development Institute, 2025.XX.XX, CCTCC NO: M 2024343.

Information on overseas storage: 1. China, China Center for Type Culture Collection, 2024.02.27, CCTCC NO: M 2024343.

TW202548006A_114119787_SEQL.xml

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Claims (12)

A species of Bacillus subtilis is characterized by its registration number CCTCC NO: M 2024343. A microbial agent characterized in that it includes Bacillus subtilis as described in claim 1. The use of Bacillus subtilis as described in claim 1 or a microbial agent as described in claim 2 in at least one of the following: (I) in the preparation of nattokinase and/or vitamin K2 or products thereof; (II) in the preparation of fermented soybeans, natto or intestinal probiotics; (III) in the fermentation preparation of biological feed. The application as described in claim 3, wherein the product includes at least one of natto, natto powder, nattokinase fermented products, and vitamin K2 fermented products. A method for fermenting Bacillus subtilis , characterized in that it includes: fed-batch fermentation of Bacillus subtilis as described in claim 1 using a fermentation substrate and feed to obtain a fermentation broth; the fed-batch fermentation includes: adding the Bacillus subtilis... The culture medium is inoculated into the fermented hydroponic substrate, and the feed additive is added sequentially. The fermented hydroponic substrate comprises water and the following components by weight and volume percentage: 1-4% corn flour, 0.2-1% soybean meal, 0.1-0.5% yeast peptone, 0.05-0.3% L-serine, 0.001-0.01% glycine, 0.1-1% sodium salt, 0.1-0.4% calcium salt, 0.05-0.2% potassium salt, and 0.05% sodium sulfate. -0.2% magnesium salt, 0.002-0.01% zinc salt, and 0.001-0.01% iron salt; the supplement includes supplement 1, supplement 2, and supplement 3; supplement 1 includes water and a component selected from any of the following combinations: 5-25% corn flour and 0.05-0.3% L-serine, 10-25% maltodextrin and 0.05-0.3% L-serine, 10-25% glycerol and 0.05-0.3%... The combination of L-serine; the corn flour includes liquefied corn flour and/or non-liquefied corn flour; the supplement 2 is 5-10% soybean meal and the remainder water, or 1-3.5% ammonium sulfate and the remainder water; the supplement 3 is 10-15% ammonia water. The fermentation method as described in claim 5, wherein the sodium salt in the fermentation substrate comprises sodium chloride; the calcium salt comprises calcium chloride; the potassium salt comprises potassium dihydrogen phosphate; the magnesium salt comprises magnesium sulfate; the zinc salt comprises zinc sulfate; and the iron salt comprises ferrous sulfate. The fermentation method as described in claim 5 further includes, prior to the fed-batch fermentation, a step of scaling up the Bacillus subtilis using a strain slant culture medium, a primary seed medium, and/or a secondary seed medium. The fermentation method as described in claim 7, wherein the slant culture medium for the strain comprises: soluble starch, yeast extract, yeast peptone, sodium chloride, agar, and water, with a pH of 6.0 to 6.2; the primary seed culture medium comprises: soluble starch, yeast extract, yeast peptone, sodium chloride, potassium dihydrogen phosphate, magnesium sulfate, and water, with a pH of 6.0 to 6.2; and the secondary seed culture medium comprises: corn flour, soybean flour, yeast extract, L-serine, glycine, potassium dihydrogen phosphate, magnesium sulfate, and water, with a pH of 6.0 to 6.2. The fermentation method described in claim 5, wherein the initial fermentation temperature of the fed-batch fermentation is 36~45℃, the fermentation temperature increases by 1~2℃ every 2~4 hours during the fermentation process, and the fermentation temperature is maintained at 46~52℃ for subsequent fermentation after 12~16 hours, and the total fermentation cycle is 34~40 hours. As described in claim 5, the fermentation method wherein the feed 1 is pulsed fed to maintain dissolved oxygen levels fluctuating between 10-30% and total sugar content <30g/L, or dissolved oxygen-coupled feeding is used to maintain dissolved oxygen levels at 30% and total sugar content at 10-20g/L; the feed 2 is fed in a segmented, uniform manner; the feed 3 is used to control the fermentation pH to be constant at 6.0, or to control the fermentation pH to be >5.8. When the pH of the fermentation broth is lower than 5.8, ammonia is used to adjust the pH back to 6.5. A method for preparing a product containing nattokinase and/or vitamin K2, characterized in that it includes the following steps: step (1) sequentially performing flocculation separation and plate and frame filtration on the fermentation broth obtained by the fermentation method described in claim 5, and collecting the clear liquid and bacterial residue respectively; step (2) filtration of the clear liquid through a membrane followed by concentration and drying to obtain a product rich in nattokinase; and step (3) drying the bacterial residue to obtain a product rich in vitamin K2. The preparation method described in claim 11, wherein step (2), after concentration and before drying, further includes: mixing the concentrated liquid and excipients to obtain a raw material containing nattokinase; the drying is spray drying; and step (3), before drying, further includes mixing and granulating the bacterial residue and excipients, wherein the drying is fluidized bed drying.