Applications of a single strain of Bacillus subtilis, and its simultaneous production of nattokinase and vitamin K2.

Abstract

To provide simultaneous production of Nattokinase and vitamin K2 by a specific Bacillus subtilis, and application thereof.SOLUTION: Provided are a Bacillus subtilis specified by the accession number CCTCC NO:M 2024343, or a microbial agent containing the Bacillus subtilis, and use of the Bacillus subtilis or the microbial agent in production of at least one of the following (I) to (III): (I) production of Nattokinase and / or vitamin K2, or a product thereof; (II) production of fermented black beans, Natto or probiotic; and (III) production of biological feed by fermentation.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and in particular to a strain of Bacillus subtilis and the use thereof in the simultaneous production of nattokinase and vitamin K2.

Background Art

[0002] Nattokinase is an alkaline serine protease secreted by Bacillus subtilis. It has a molecular weight of approximately 28 kDa and a single-chain protein structure composed of 275 amino acid residues. Its molecular weight is far smaller than that of urokinase, streptokinase and recombinant tissue-type plasminogen activator, so it is easily absorbed by the human body, can produce a strong thrombolytic effect in vivo, and is highly effective in the treatment and prevention of thrombosis. Compared with plasminogen products such as urokinase and streptokinase clinically used at present, nattokinase has the advantages of longer half-life, higher safety, stronger specificity and oral availability.

[0003] Vitamin K2 is an important fat-soluble coagulation vitamin for maintaining normal blood coagulation ability in the body. In addition, as an electron transfer carrier in respiratory metabolism, vitamin K2 also participates in many important metabolic reactions in the body, has many important physiological functions such as prevention and treatment of osteoporosis, arterial calcification, cardiovascular diseases, tumors and Parkinson's disease, and has the advantages of high affinity in the human body and long half-life.

[0004] Bacillus subtilis is a Gram-positive bacterium capable of producing not only nattokinase and vitamin K2, but also various physiologically active substances such as various proteases, glucoamylase, dipicolinic acid, and active peptides. When a thrombus forms in the human body, enzyme systems with serine protease activity exert a synergistic effect, efficiently degrading the cross-linked fibrin that constitutes the thrombus's skeleton. The abundance of related protease enzyme systems is extremely important for efficient thrombolysis. Since protease enzyme systems from different Bacillus species differ significantly, appropriate selection of strains and fermentation processes is considered crucial for the effectiveness of nattokinase-related products.

[0005] Currently, the nattokinase production industry using Bacillus subtilis is developing, but it faces two main problems: decreased enzyme activity and rising costs. Regarding enzyme activity, the enzyme activity reported in prior art is mostly less than 500 FU / mL, and overall, there is a certain difference compared to the fermentation levels of nattokinase reported in Japanese literature. In addition, the content, heat resistance, and acid resistance of nattokinase produced by different strains and fermentation processes vary greatly, so drying by low-temperature freeze-drying is necessary when producing nattokinase from most strains, resulting in high production costs (drying costs).

[0006] Bacillus subtilis is the main strain used for vitamin K2 production, but the strains reported in prior art had long fermentation periods and insufficient vitamin K2 yields. Vitamin K2 in the fermentation liquid is distributed both intracellularly and extracellularly, and Japanese Patent Publication No. CN116536220A, titled "Method for culturing Bacillus subtilis for natto and method for producing vitamin K2," describes a vitamin K content of 124 mg / L with a fermentation time of 120-140 hours, which is the highest amount reported to date. However, the long fermentation cycle increases the risk of contamination during production and instability of the product content, and also reduces the fermentation production volume in the industry, thus increasing the cost of vitamin K2 production.

[0007] Furthermore, in patent CN106701719A, titled "Method for simultaneous production of vitamin K2 and nattokinase by fermentation of natto bacteria," both nattokinase and vitamin K2 are present in the fermentation supernatant, requiring a complicated purification process to separate them. Moreover, the fermentation level is low, resulting in insufficient production efficiency and failing to fundamentally solve the problem of the generally high production cost of vitamin K2. [Overview of the project] [Problems that the invention aims to solve]

[0008] The present invention has been made in view of the above circumstances and provides a strain of Bacillus subtilis and its application in the simultaneous production of nattokinase and vitamin K2. With this strain, simultaneous fermentation of nattokinase and vitamin K2 can be achieved, the produced protease enzyme system is rich, the fermentation period is short, the fermentation liquid is mainly used for the production of nattokinase-related products, and the isolated bilayer (bacterial sludge) is mainly used for the production of vitamin K2-related products. [Means for solving the problem]

[0009] In this invention, one strain, RB4, was isolated from traditional baijiu cellar clay in Yibin City, Sichuan Province. This strain was numbered AMCC 11927 at the microbial resource center of Angel Yeast Co., Ltd., identified as Bacillus subtilis based on morphology and 16S rDNA, and deposited on February 27, 2024, at the China Center for Typical Cultures (CCTCC) of Wuhan University, Wuhan City, China, with deposit number CCTCC NO:M 2024343.

[0010] The present invention further provides a microbial agent comprising at least one selected from the group consisting of Bacillus subtilis RB4 or its culture, metabolites, and fermentation products.

[0011] The present invention further provides the use of Bacillus subtilis RB4 or the microbial agent in the production of at least one of the following: (I) the production of nattokinase and / or vitamin K2 or products thereof; (II) the production of fermented soybeans, natto, or probiotics; and (III) the production of biological feed by fermentation.

[0012] In the aforementioned use, the product comprises at least one selected from the group consisting of natto, natto powder, nattokinase fermented product, and vitamin K2 fermented product.

[0013] The present invention further provides a culture medium for fed-batch fermentation of Bacillus subtilis, comprising a fermented bottom water culture medium and a supplement. Herein, the fermented bottom water culture medium comprises corn flour, soybean meal flour, yeast peptone, L-serine, glycine and inorganic salts. The inorganic salts include sodium salts, calcium salts, potassium salts, magnesium salts, zinc salts, and iron salts. The aforementioned supplement comprises Supplement 1, which contains one combination of components selected from the group consisting of a combination of liquefied corn flour and L-serine, a combination of maltodextrin and L-serine, a combination of corn flour and L-serine, and a combination of glycerol and L-serine, and water; Supplement 2, which contains water and soybean meal powder or is an aqueous solution of ammonium sulfate; and Supplement 3, which is aqueous ammonia.

[0014] In some embodiments, the inorganic salt includes sodium chloride, calcium chloride, potassium dihydrogen phosphate, magnesium sulfate, zinc sulfate, and ferrous sulfate.

[0015] In some preferred embodiments, the fermentation bottom water medium comprises, by mass volume percentage, 1-4% corn flour, 0.2-1% soybean meal flour, 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, 0.005-0.2% magnesium sulfate, 0.002-0.01% zinc sulfate, and 0.001-0.01% ferrous sulfate, and water. In some specific embodiments, the fermentation bottom water medium comprises, by mass volume percent, 2% corn flour, 0.5% soybean meal flour, 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, along with water.

[0016] In some specific embodiments, the pH of the fermentation substrate is specifically 6.0, 6.1, or 6.2.

[0017] In some preferred embodiments, supplement 1 comprises any one combination of components selected from the group consisting of 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, and water, where the corn flour is liquefied corn flour and / or non-liquefied corn flour; supplement 2 comprises 5-10% soybean meal flour and the remainder water, or 1-3.5% ammonium sulfate and the remainder water; and supplement 3 comprises 10-15% aqueous ammonia. In some specific embodiments, Supplement 1 contains one combination of components selected from the group consisting of 10% unliquefied corn flour and 0.3% L-serine, 25% liquefied corn flour and 0.3% L-serine, 25% maltodextrin and 0.3% L-serine, and 25% glycerol and 0.3% L-serine, along with water; Supplement 2 contains 10% soybean meal flour and the remainder water, or 3.5% ammonium sulfate and the remainder water; and Supplement 3 contains 15% aqueous ammonia. The percentage "%" for each component in the supplements is a mass-volume percentage, representing the proportion of each component to the supplement (unit: g / ml).

[0018] In some embodiments, the culture medium of the present invention further comprises a strain slant medium. The strain slant medium contains soluble starch, powdered yeast extract, yeast peptone, sodium chloride, agar, and water, and has a pH of 6.0 to 6.2. Preferably, the strain slant medium contains 1-3% soluble starch, 0.5-3% powdered yeast extract, 0.2-1% yeast peptone, 0.1-1% sodium chloride, and 2-3% agar, with the remainder being water, and has a pH of 6.0 to 6.2. In some specific embodiments, the strain slant medium contains 2% soluble starch, 1% powdered yeast extract, 0.4% yeast peptone, 1% sodium chloride, and 3% agar, with the remainder being water, and has a pH of 6.2.

[0019] In some embodiments, the culture medium of the present invention further comprises a primary seed medium. The primary seed medium comprises soluble starch, powdered yeast extract, yeast peptone, sodium chloride, potassium dihydrogen phosphate, magnesium sulfate, and water, and has a pH of 6.0 to 6.2. Preferably, the primary seed medium contains 1-3% soluble starch, 0.5-3% powdered 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, with the remainder being water. In some specific embodiments, the primary seed medium contains 2% soluble starch, 1% powdered yeast extract, 0.4% yeast peptone, 0.6% sodium chloride, 0.1% potassium dihydrogen phosphate, 0.1% magnesium sulfate, with the remainder being water, and has a pH of 6.2.

[0020] In some embodiments, the culture medium of the present invention further comprises a secondary seed medium. The secondary seed medium comprises corn flour, soybean flour, powdered yeast extract, L-serine, glycine, potassium dihydrogen phosphate, magnesium sulfate, and water, and has a pH of 6.0 to 6.2. In some preferred embodiments, the secondary seed medium comprises 1-3% corn flour, 0.2-1% soybean flour, 0.1-0.5% powdered yeast extract, 0.05-0.3% L-serine, 0.001-0.01% glycine, 0.05-0.2% potassium dihydrogen phosphate, 0.05-0.2% magnesium sulfate, with the remainder being water, and has a pH of 6.0 to 6.2. In some specific embodiments, the secondary seed medium contains, by mass volume percentage, 2% corn flour, 1% soy flour, 0.2% powdered yeast extract, 0.1% L-serine, 0.005% glycine, 0.1% potassium dihydrogen phosphate, and 0.1% magnesium sulfate, with the remainder being water and a pH of 6.2.

[0021] According to the present invention, a method for fermentation using Bacillus subtilis is further provided, which includes the step of obtaining a fermented liquid by feeding-batch fermentation of Bacillus subtilis as described in the present invention using the culture medium described in the present invention. However, the fed-batch fermentation includes inoculating the Bacillus subtilis into the fermentation bottom water medium described in the present invention and adding the supplement by feeding it in. The specific timing of adding the supplement is determined according to the changes in dissolved oxygen and total sugar content, but in specific embodiments of the present invention, the supplement is added 1 to 2 hours after inoculation into the fermentation bottom water medium.

[0022] In some embodiments, prior to the fed-batch fermentation, the process further includes a step of culturing the Bacillus subtilis using the strain slant medium, primary seed medium and / or secondary seed medium described in the present invention. In some specific embodiments, prior to the fed-batch fermentation, the process further includes a step of culturing the Bacillus subtilis using the strain slant medium described in the present invention, or using the strain slant medium and primary seed medium described in the present invention, or using the strain slant medium, primary seed medium and secondary seed medium described in the present invention.

[0023] In some embodiments, the initial fermentation temperature for the fed-batch fermentation is set to 36-45°C, and the fermentation temperature is increased by 1-2°C every 2-4 hours during fermentation, and subsequent fermentation is carried out for 12-16 hours while maintaining the fermentation temperature at 46-52°C, resulting in a total fermentation cycle of 34-40 hours. In some specific embodiments, the initial fermentation temperature for the fed-batch fermentation is set to 42°C, and the fermentation temperature is increased by 2°C every 4 hours during fermentation, and subsequent fermentation is carried out for 16 hours while maintaining the fermentation temperature at 50°C, resulting in a total fermentation time of 36-40 hours.

[0024] In some embodiments, supplement 1 is added by pulsed feed so that the dissolved oxygen fluctuation during fermentation is maintained at 10-30% and the total sugar content is maintained at less than 30 g / L, or by dissolved oxygen-linked feed so that the dissolved oxygen is maintained at 30% and the total sugar content is maintained at 10-20 g / L; supplement 2 is added at a constant rate by stepped feed; supplement 3 is used to fix the pH of the fermentation liquid at 6.0 or to control the pH of the fermentation liquid to be above 5.8, and if the pH of the fermentation liquid is below 5.8, ammonia water is used to adjust the pH back to 6.5.

[0025] In the present invention, there are no particular limitations on the specific sources of yeast peptone and powdered yeast extract, and any common commercially available product may be used. In a specific embodiment of the present invention, the yeast peptone is Angel FP103, and the powdered yeast extract is Angel FM888.

[0026] According to the present invention, there is further provided a method for producing nattokinase and / or vitamin K2, which comprises the following steps. (1) A step of sequentially subjecting the fermentation broth obtained by the fermentation method described in the present invention to coagulation separation and plate-and-frame filter press filtration, and separately collecting the clear liquid and the bacterial residue; (2) A step of subjecting the clear liquid to membrane filtration, followed by concentration and drying to obtain a product rich in nattokinase; (3) A step of drying the bacterial residue to obtain a product rich in vitamin K2.

[0027] After obtaining products respectively rich in nattokinase and vitamin K2 by the production method of the present invention, pure nattokinase products and pure vitamin K2 products can also be obtained respectively by separation and purification methods disclosed in the art.

[0028] In some embodiments, in step (1), flocculant and perlite are used to perform coagulation separation on the fermentation broth. In some specific embodiments, the flocculant is a composite clarification flocculant, which may specifically be polyacrylamide, calcium chloride, or the like, and any conventional type in the art is acceptable. The addition amount of the perlite is 2% based on the total amount of the fermentation broth. In some specific embodiments, the coagulation separation step comprises: first adding the composite clarification flocculant to the fermentation broth until the concentration of the composite clarification flocculant reaches 1.0 g / L, stirring at high speed for 2 hours, and then adding 2.0 g / L of perlite.

[0029] In some embodiments, in step (2), after the concentration and before the drying, the method further comprises a step of mixing the concentrated solution obtained from the concentration with an adjuvant to obtain a raw material containing nattokinase, wherein the drying is spray drying, Step (3) further includes a step of mixing the microbial residue and the auxiliary agent and granulating them before drying, wherein the drying is performed by fluidized bed drying. [Effects of the Invention]

[0030] This invention provides Bacillus subtilis and its application in the simultaneous production of nattokinase and vitamin K2. This strain enables the simultaneous fermentation of nattokinase and vitamin K2, produces a rich protease enzyme system, has a short fermentation period, and the fermentation liquid is mainly used for the production of nattokinase-related products, while the isolated bilayer (bacterial sludge) is mainly used for the production of vitamin K2-related products. This invention offers the following advantages:

[0031] 1. Using the strain of the present invention, the fermentation period is less than 40 hours, the nattokinase content in the fermentation liquid is approximately 35% higher than the highest enzyme activity reported in the industry, and it is rich in various protease enzyme systems. Producing nattokinase from this strain significantly improves the heat resistance and acid resistance of nattokinase, and the yield of enzyme activity by spray drying reaches approximately 80%, effectively solving the problems in the industry such as a decrease in the fermentation level of nattokinase and a large loss of enzyme activity due to spray drying.

[0032] 2. When fermentation is carried out using the bacterial strain of the present invention, the bacterial sludge (biphase), which is a byproduct after nattokinase isolation, contains more than 140 μg / g of vitamin K2, and can be directly used as a raw material for vitamin K2 extraction. Since the fermentation period is short and the utility of the byproduct is high, the problems of long fermentation periods for vitamin K2 and increased production costs are effectively solved.

[0033] Term definition The "natto" described in this application is a soybean product prepared by fermenting soybeans with a subspecies of Bacillus subtilis, specifically Bacillus natto. The "nattokinase" relating to this application is known as a type of alkaline serine protease extracted from natto (fermented soybeans). The "nattokinase fermented product" used in this application refers to a fermented product containing nattokinase.

[0034] As used in this application, "vitamin K2 fermented product" refers to a fermented product containing vitamin K2. The "fermentation method using Bacillus subtilis" as described in this application refers to a method of fermentation using Bacillus subtilis. For example, a fermented product is obtained by fermenting a substrate (e.g., corn flour, soybean meal flour) using Bacillus subtilis RB4 of the present invention.

[0035] The "fed-batch fermentation" described in this application is also called supplement-fed batch fermentation or semi-continuous fermentation. It is a fermentation technique in which certain material raw materials are added to the fermentation system by a specific means during microbial-fed batch fermentation, but the fermentation liquid is not continuously released to the outside. The "fed-batch addition" method used in this application is the addition of supplements according to the method of fed-batch fermentation. For example, during batch culture, supplements are added gradually in several portions as fermentation progresses. The "fermented bottom water medium" as used in this application is a medium used at the start of fermentation, and is also called a fermentation base medium.

[0036] Description of the deposit of biological materials This specimen, Bacillus subtilis AMCC 11927, was deposited on February 27, 2024, at the China Center for Typical Cultures of Bacteria, Wuhan University, Wuhan City, China, with deposit number CCTCC NO:M 2024343. [Modes for carrying out the invention]

[0037] This invention discloses the application of Bacillus subtilis, as well as its co-production of nattokinase and vitamin K2. Those skilled in the art can realize this invention by referring to the contents of this specification and appropriately improving the process parameters. In particular, it should be noted that all similar substitutions and modifications are obvious to those skilled in the art and are all considered to be included in this invention. While the methods and applications of this invention have been described through preferred examples, those involved can implement and apply the technology of this invention by modifying, appropriately changing, and combining the methods and applications described herein without departing from the content, spirit, and scope of this invention.

[0038] In this invention, multiple strains of Bacillus bacteria were isolated from traditional baijiu cellar mud in Yibin City, Sichuan Province. Initial sorting using fibrin plates and subsequent temperature acclimatization were performed sequentially to select one strain, RB4, which could grow well in an environment of 48-50°C. The production of nattokinase, protease, and vitamin K2-related metabolites by liquid fermentation of this strain was then studied, and it was found that this strain had excellent production performance for nattokinase and vitamin K2. Combining this with morphological and molecular biological (16S rDNA) results, it was identified as Bacillus subtilis. The 16S rDNA gene sequence of the RB4 strain is as follows.

[0039] (Sequence 1):

[0040] Unless otherwise specified, the concentration percentages of each component in the examples of the present invention are given as mass volume percentages (unit: g / ml). The present invention will be further described below with reference to the examples. [Examples]

[0041] Example 1 Bacillus subtilis RB4 (deposit number: CCTCC NO: M 2024343) was subjected to slant culture, primary seed culture, and flow-boil fermentation in a 50L tank. The enzymatic activity of nattokinase and the content of biphase vitamin K2 in the fermentation liquid were tracked and detected. The production levels of nattokinase and vitamin K2 are shown in Tables 1 and 2 below.

[0042] The specific steps include the following: (1) Slant culture: The above strains were inoculated into strain slant culture medium and cultured slant at 42°C for 24 hours. The slant medium formulation consisted of 2% soluble starch, 1% powdered yeast extract (Angel FM888), 0.4% yeast peptone (Angel FP103), 1% sodium chloride, and 3% agar, with the remainder being water. The pH was 6.2, and the medium was sterilized at 121°C for 30 minutes.

[0043] (2) Primary culture: The bacterial species cultured on a slant was inoculated into a 5 L shaking flask containing 1 L of primary culture medium, and cultured under the conditions of a culture temperature of 42°C, a shaking speed of 220 r / min, and a culture time of 20 hours to obtain a primary culture solution. The primary culture medium formulation consisted of 2% soluble starch, 1% powdered yeast extract (Angel FM888), 0.4% yeast peptone (Angel FP103), 0.6% sodium chloride, 0.1% potassium dihydrogen phosphate, and 0.1% magnesium sulfate, with the remainder being water. The pH was 6.2, and the medium was sterilized at 121°C for 30 minutes.

[0044] (3) Fed-batch fermentation: 1.2 L of primary seed culture was inoculated into a 50 L tank containing 6.8 L of fermentation bottom water medium, and fed-batch fermentation was performed. During the fermentation period, the pH was controlled to 6.0, the fermentation temperature was 42°C, the fermentation tank rotation speed was 200-250 r / min, and the aeration rate was 1:1-1.2:1 vvm. After culturing for 1-2 hours, supplements (supplement 1, supplement 2, and supplement 3) were added.

[0045] In this process, fermented bottom water culture medium and supplements were used as culture media. The fermented bottom water culture medium consisted of 2% corn flour, 0.5% soybean meal flour, 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 consisted of 25% liquefied corn flour and 0.3% L-serine. Supplement 2 was 10% soybean meal flour. Supplement 3 was 15% ammonia water (used for pH adjustment and added as needed depending on pH changes).

[0046] All raw materials used in this invention are commercially available, and similar products from different brands and manufacturers may be used interchangeably.

[0047] Fed-boil fermentation process: Supplement 1 was added at a volume of 10 L by a dissolved oxygen-linked fed-boil method to maintain a dissolved oxygen level of 30% and a total sugar content of 10-20 g / L during the fermentation period. Supplement 2 was added at a constant rate by a stepped fed-boil method at a total volume of 6 L. However, the flow rate of supplement 2 was 65% of that of supplement 1.

[0048] Samples were taken periodically and centrifuged. The fermentation supernatant was analyzed for nattokinase activity, and the resulting distillate was subjected to high-pressure homogenization and cell wall disruption, after which the vitamin K2 content was detected. Nattokinase activity was measured according to the measurement method for nattokinase in natto powder - ultraviolet spectroscopy, as specified in DBS44 / 013-2019 Local Food Safety Standard. Vitamin K2 content was measured according to Appendix A.4 "Method for detecting vitamin K2" of Announcement No. 8 of the National Health and Family Planning Commission of 2016. Test Example 1: Comparison of fermentation results using different bacterial strains

[0049] Several Bacillus strains selected in the laboratory, including SD, YJ, RB1, RB2, MG1, MG3, SJ1, SJ2, BL1, and BL2, were used as research subjects. Following the procedures below, slant culture, primary culture, secondary culture, and flow-boiled fermentation in 50L tanks were performed on Bacillus subtilis, and nattokinase activity and biphase vitamin K2 content in the fermentation liquid were tracked and detected. The production levels of nattokinase and vitamin K2 for different strains are shown in Tables 1 and 2 below.

[0050] Comparative Examples 1 to 10 were carried out in the same manner as in Example 1, except that different main bacterial strains were used.

[0051] [Table 1]

[0052] [Table 2]

[0053] Tables 1 and 2 show that the nattokinase fermented with the RB4 strain of the present invention exhibits excellent performance and is rich in vitamin K2. This RB4 strain was deposited on February 27, 2024, at the China Center for Typical Cultures of Wuhan University in Wuhan City, China, with deposit number CCTCC NO:M 2024343.

[0054] Example 2 Supplement 1 was added using a pulsed feed method, and the dissolved oxygen level was maintained at a rate of 10-30% during the fermentation period, with the total sugar content being <30 g / L. The fermentation time was set to 34-36 hours. All other steps were carried out in the same manner as in Example 1.

[0055] Example 3 Except for using 25% maltodextrin and 0.3% L-serine in supplement 1, the other steps were carried out in the same manner as in Example 2.

[0056] Ratio Proportionality 11 Except for using 10% unliquefied corn flour and 0.3% L-serine as supplement 1, the other steps were carried out in the same manner as in Example 2.

[0057] Ratio Proportionality 12 The procedure was carried out in the same manner as in Example 2, except that Supplement 1 was separately sterilized as 25% glucose and L-serine before being added.

[0058] Ratio Proportionality 13 Except for using 25% sucrose and 0.3% L-serine in supplement 1, the other steps were carried out in the same manner as in Example 2.

[0059] Ratio Proportionality 14 Except for the fact that Supplement 1 consisted of 25% glycerol and 0.3% L-serine, the other steps were carried out in the same manner as in Example 2.

[0060] Ratio Proportionality 15 Except for using 10% yeast protein powder as supplement 2, the other steps were carried out in the same manner as in Example 2. Ratio Proportionality 16 Except for using 3.5% ammonium sulfate as supplement 2, the other steps were carried out in the same manner as in Example 2.

[0061] Test Example 2: Comparison of Fermentation Results Using Different Supplements Nattokinase activity and vitamin K2 content were detected in fermented liquids obtained by fermenting different supplements, and the results are shown in Table 3 below.

[0062] [Table 3]

[0063] As is clear from Table 3, the nattokinase activity and vitamin K2 content obtained from fermentation of the RB4 strain differed significantly under different fermentation raw material and feed-in-feed process parameters. When fermentation was carried out using liquefied corn starch or dextrin as the carbon source, soybean meal powder as the nitrogen source, and the supplement's pulsed feed-in-feed process, the nattokinase activity and vitamin K2 content were ideal, with an average nattokinase activity in the fermentation liquid exceeding 2500 FU / g and a vitamin K2 content in the biphase exceeding 140 ug / g.

[0064] Example 4 The procedure was the same as in Example 2. However, the pH of the fermentation liquid was controlled to be above 5.8 with supplement 3, and if the pH of the fermentation liquid fell below 5.8, ammonia water was used to adjust the pH back to 6.5. The initial fermentation temperature was set to 42°C, and the fermentation temperature was increased by 2°C every 4 hours during fermentation. After 16 hours of fermentation, the temperature was maintained at 50°C for subsequent fermentation. 0.1% of a compound clarifying flocculant was added to the fermentation liquid in the tank, stirred for 2 hours under conditions of 500 r / min, 2% perlite was added, and plate frame filter press filtration was performed. The supernatant obtained from the filtration was filtered through an 8000 Dalton membrane to concentrate it 5 to 6 times, the temperature was raised to 50°C, 5% dextrin was added and stirred well, and the suction temperature was set to 110 to 120°C and the discharge temperature to be maintained below 70°C using a spray drying tower to obtain a raw material rich in nattokinase.

[0065] Ratio Proportionality 17 Except for maintaining the fermentation temperature at 42°C and controlling the fermentation pH to 6.0, the other steps were carried out in the same manner as in Example 4.

[0066] Ratio Proportionality 18 Except for maintaining the fermentation temperature at 42°C, the other steps were carried out in the same manner as in Example 4.

[0067] Ratio Proportionality 19 Except for controlling the fermentation pH to 6.0, the other steps were carried out in the same manner as in Example 4.

[0068] Ratio 20 Except for maintaining the fermentation temperature at 37°C and controlling the fermentation pH to 6.0, the other steps were carried out in the same manner as in Example 4.

[0069] Ratio Proportional Relations 21 Except for maintaining the fermentation temperature at 50°C and controlling the fermentation pH to 6.0, the other steps were carried out in the same manner as in Example 4. Test Example 3: Comparison of Fermentation Results Using Different Processes

[0070] [Table 4]

[0071] As is clear from the data in Table 4, in Example 4, which used a fermentation process control system with temperature and pH fluctuations, the nattokinase activity in the fermentation liquid reached a maximum of 2763 FU / g, and the enzyme activity yield by spraying exceeded 75%. Compared to a fermentation control process with constant temperature and pH, the nattokinase activity in the fermentation liquid and the powder injection enzyme activity yield were significantly increased.

[0072] The above are merely preferred embodiments of the present invention and do not limit the scope of the claims. Those skilled in the art can make several improvements and modifications without departing from the principles of the present invention, and it should be noted that these improvements and modifications are also considered to be within the scope of protection of the present invention.

Claims

1. Bacillus subtilis, characterized by its deposit number CCTCC NO: M 2024343.

2. A microbial agent characterized by containing Bacillus subtilis as described in claim 1.

3. (i) Manufacturing of nattokinase and / or vitamin K2, or products thereof; (II) Production of fermented soybeans, natto, or probiotics; (III) Production of biological feed by fermentation; The use of Bacillus subtilis according to claim 1 or the microbial agent according to claim 2 in at least one of the above manufacturing methods (I) to (III).

4. The use according to claim 3, characterized in that the product comprises at least one selected from the group consisting of natto, natto powder, nattokinase fermented product, and vitamin K2 fermented product.

5. A method for fermentation using Bacillus subtilis, comprising the step of obtaining a fermented liquid by flow-batch fermentation of Bacillus subtilis as described in claim 1 using a fermented bottom water culture medium and a supplement, The aforementioned fed-batch fermentation includes inoculating the Bacillus subtilis culture medium into a fermentation substrate and adding the supplement via a fed-batch method. The fermented bottom water culture medium comprises, by mass volume percentage, 1-4% corn flour, 0.2-1% soybean meal flour, 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, 0.05-0.2% magnesium salt, 0.002-0.01% zinc salt, and 0.001-0.01% iron salt, and water. The aforementioned supplement comprises a supplement 1 containing water and any one combination of components selected from the group consisting of 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, and a combination of 10-25% glycerol and 0.05-0.3% L-serine (provided that the corn flour includes liquefied corn flour and / or non-liquefied corn flour), Supplement 2 consists of 5-10% soybean meal powder and the remaining amount of water, or 1-3.5% ammonium sulfate and the remaining amount of water, A fermentation method characterized by comprising supplement 3, which is 10-15% aqueous ammonia.

6. The fermentation method according to claim 5, characterized in that the fermentation bottom water medium contains sodium chloride as the sodium salt, calcium chloride as the calcium salt, potassium dihydrogen phosphate as the potassium salt, magnesium sulfate as the magnesium salt, zinc sulfate as the zinc salt, and ferrous sulfate as the iron salt.

7. The fermentation method according to claim 5, further comprising the step of culturing the Bacillus subtilis using a strain slant medium, a primary seed medium and / or a secondary seed medium, prior to the aforementioned flow-batch fermentation.

8. The aforementioned strain slant medium contains soluble starch, powdered yeast extract, yeast peptone, sodium chloride, agar, and water, and has a pH of 6.0 to 6.

2. The primary seed culture medium contains soluble starch, powdered yeast extract, yeast peptone, sodium chloride, potassium dihydrogen phosphate, magnesium sulfate, and water, and has a pH of 6.0 to 6.

2. The fermentation method according to claim 7, characterized in that the secondary seed culture medium contains corn flour, soy flour, powdered yeast extract, L-serine, glycine, potassium dihydrogen phosphate, magnesium sulfate, and water, and has a pH of 6.0 to 6.

2.

9. The fermentation method according to claim 5, characterized in that the feed-bottle fermentation is characterized by setting the initial fermentation temperature to 36 to 45°C, raising the fermentation temperature by 1 to 2°C every 2 to 4 hours during fermentation, fermenting for 12 to 16 hours, maintaining the fermentation temperature at 46 to 52°C while performing subsequent fermentation, and having a total fermentation cycle of 34 to 40 hours.

10. Supplement 1 is added by pulsed feed so that the dissolved oxygen level remains between 10% and 30% during the fermentation period and the total sugar content remains below 30 g / L, or by dissolved oxygen-linked feed so that the dissolved oxygen level remains at 30% during the fermentation period and the total sugar content remains between 10% and 20 g / L. Supplement 2 is added at a constant rate by a stepwise flow method. The method according to 5, characterized in that supplement 3 is used to fix the pH of the fermentation liquid at 6.0 or to control the pH of the fermentation liquid to be above 5.8, and if the pH of the fermentation liquid falls below 5.8, ammonia water is used to adjust the pH back to 6.

5.

11. A method for producing a product containing nattokinase and / or vitamin K2, (1) A step of sequentially subjecting the fermentation liquid obtained by the fermentation method described in claim 5 to coagulation separation and plate frame filter press filtration, and recovering the clear liquid and microbial residue separately, (2) The process of filtering the liquid through a membrane, concentrating it, drying it, and obtaining a product rich in nattokinase, (3) A manufacturing method characterized by comprising the step of drying the bacterial residue to obtain a product rich in vitamin K2.

12. In step (2), after concentration and before drying, the process further includes mixing the concentrated liquid obtained from the concentration with an auxiliary agent to obtain a raw material containing nattokinase, wherein the drying is spray drying. The manufacturing method according to claim 11, further comprising a step of mixing the microbial residue and the auxiliary agent and granulating them before drying, wherein the drying is fluidized bed drying.

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