A high-vitamin K2-producing Bacillus subtilis KV-2 strain and its application

Abstract

This invention relates to the field of microbial technology, and in particular to a high-vitamin K2-producing Bacillus subtilis strain KV-2 and its applications. This invention utilizes directed mutagenesis and precise screening techniques to breed a high-vitamin K2 (MK-7)-producing Bacillus subtilis strain—Bacillus subtilis (KV-2). Bacillus subtilis KV-2, with accession number CGMCC No.38728; Bacillus subtilis KV-2 was fermented under the final optimized conditions of this invention, and the yield of MK-7 reached 831 mg / L after 154 h of fermentation.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a high-vitamin K2-producing Bacillus subtilis KV-2 strain and its applications. Background Technology

[0002] Vitamin K2 (menaquinone compounds, MK-n) is a class of fat-soluble vitamins with important physiological activities. Among them, MK-7 has become a core active ingredient in areas such as bone health maintenance, cardiovascular disease prevention, and anticoagulation due to its strongest biological activity, long half-life, and safety and non-toxicity, resulting in continuously rising market demand. Currently, the production of vitamin K2 mainly relies on two pathways: chemical synthesis and microbial fermentation. Chemical synthesis has inherent drawbacks such as limited precursor raw materials, numerous byproducts, low product activity (mainly cis structure), and serious environmental pollution, making it difficult to meet the quality requirements of high-end foods, health products, and pharmaceuticals. On the other hand, microbial fermentation, due to its natural all-trans structure, excellent biocompatibility, and environmental friendliness, has become the mainstream technology for the industrial production of MK-7.

[0003] In the selection of strains for microbial fermentation, Bacillus subtilis (Bacillus subtilis) is preferred. Bacillus subtilis natto Bacillus subtilis is a natural high-yield host for MK-7, possessing irreplaceable advantages compared to other strains. Firstly, as the core functional bacterium in the traditional fermented food "natto," its metabolic pathway is naturally adapted to the biosynthesis of MK-7, enabling highly selective synthesis of MK-7 (accounting for over 96% of total vitamin K2), rather than the mixed production of multiple MK-n subtypes, significantly reducing the cost and difficulty of subsequent separation and purification. Secondly, Bacillus subtilis possesses Generally Recognized As Safe (GRAS) status, allowing its fermentation products to be directly applied in the food, health product, and feed industries, effectively circumventing safety regulatory barriers imposed by engineered bacteria.

[0004] Although Bacillus subtilis is the preferred strain for producing MK-7, the fermentation performance of wild-type strains has limitations, restricting their industrial application. Therefore, selecting high-yielding, stable Bacillus subtilis strains suitable for industrial fermentation through efficient mutagenesis breeding technology combined with targeted screening strategies has become key to overcoming the bottlenecks in MK-7 fermentation production technology. Among mutagenesis breeding techniques, ambient pressure room temperature plasma (ARTP) mutagenesis technology has significant advantages over traditional mutagenesis techniques: using high-purity helium as the working gas, it generates plasma rich in high-energy chemically active particles at ambient temperature and pressure, which can cause precise and diverse DNA damage to the strain genome; the strain's SOS high-tolerance repair mechanism generates abundant gene mutation sites, including point mutations, fragment deletions, and insertions, significantly increasing the probability of positive mutations. Compared to the uniform mutation of ultraviolet mutagenesis and the toxicity and randomness of chemical mutagenesis, ARTP mutagenesis has the characteristics of high mutation efficiency, broad mutation spectrum, less damage to bacterial cells, and good genetic stability, and has been widely used in industrial microbial breeding.

[0005] To further screen for high-yielding mutants that overcome feedback inhibition in the MK-7 biosynthetic pathway, resistance screening strategies based on structural analogs have become the core approach for targeted breeding. Diphenylamine (DPA) and 1-hydroxy-2-naphthoic acid (HNA), as structural analogs of key precursors in the MK-7 biosynthetic pathway, can competitively inhibit the activity of key enzymes in MK-7 synthesis, thereby providing feedback inhibition to cell growth and product synthesis. Only mutants that exhibit structural mutations in key enzymes, thus overcoming feedback inhibition, can grow normally in selection media containing HNA or DPA, accompanied by a significant increase in MK-7 synthesis capacity.

[0006] In summary, the high-yield breeding method of Bacillus subtilis based on ARTP mutagenesis and resistance screening using structural analogs (HNA / DPA), combined with fermentation process optimization, has significant economic and social value for overcoming technical barriers in vitamin K2 production, reducing production costs, and promoting the independent development of my country's vitamin K2 industry. Summary of the Invention

[0007] The purpose of this invention is to provide a high-vitamin K2-producing Bacillus subtilis KV-2 strain and its applications, in order to solve the problems existing in the prior art.

[0008] To achieve the above objectives, the present invention provides the following solution: This invention provides a high-vitamin K2-producing Bacillus subtilis strain (B. subtilis). Bacillus subtilis The Bacillus subtilis KV-2 has the accession number CGMCC No. 38728.

[0009] This invention provides the application of the above-mentioned Bacillus subtilis KV-2 in the preparation of products for the production of vitamin K2.

[0010] The present invention provides a product for producing vitamin K2, the product comprising the above-mentioned Bacillus subtilis KV-2.

[0011] This invention provides the application of the above-mentioned Bacillus subtilis KV-2 or the product described herein in the production of vitamin K2.

[0012] The present invention provides a method for producing vitamin K2, the method comprising the step of fermenting and culturing Bacillus subtilis KV-2 as described above.

[0013] Optionally, the fermentation process may also include steps of feeding glucose solution and yeast extract.

[0014] Optionally, the fermentation culture is carried out at a stirring speed of 300 rpm, an aeration rate of 1 vvm, a tank pressure of 0.03 MPa, a temperature of 37°C, and a dissolved oxygen level controlled between 15% and 20%.

[0015] Optionally, when the glucose in the culture medium used for fermentation is depleted, a glucose solution is added; the initial flow rate is 4 mL / (L·h), and the glucose addition rate is increased by 2 mL / (L·h) every 4 hours, while controlling the residual sugar concentration to be below 0.5 g / L. The glucose concentration in the glucose solution is 500 g / L.

[0016] Optionally, during the fermentation process, the feed rate of the yeast extract is controlled to keep the oxygen consumption rate between 25-30 mmol / L / h; the feed time of the yeast extract is 70 h of fermentation.

[0017] Optionally, the culture medium used for the fermentation culture includes the following components at the following concentrations: Glucose 55 g / L, sucrose 50 g / L, potassium dihydrogen phosphate 0.25 g / L, yeast extract 30.21 g / L, corn steep liquor powder 21.55 g / L, soybean peptone 30 g / L, methionine 0.65 g / L, and 5-methyltryptophan 20.97 mg / L.

[0018] The present invention discloses the following technical effects: 1. This invention utilizes directed mutagenesis and precise screening techniques to breed a high-yield vitamin K2 (MK-7) Bacillus subtilis strain—Bacillus subtilis (… Bacillus subtilis KV-2, with accession number CGMCC No. 38728; 2. This invention optimizes and provides a high-efficiency fermentation medium formulation for Bacillus subtilis KV-2, providing nutritional support for the efficient synthesis of vitamin K2; 3. This invention establishes a control scheme based on the tail gas OUR (oxygen consumption rate) parameter to achieve precise control of nitrogen source feeding during Bacillus subtilis fermentation, thereby improving fermentation stability and product synthesis efficiency; 4. When Bacillus subtilis KV-2 was fermented under the conditions optimized in this invention, the yield of MK-7 reached 831 mg / L after 154 h of fermentation. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 To obtain the production content of MK-7 (MK7) in batches of the mutagenized strain; Figure 2 This is a graph showing the genetic stability results of the mutagenic strain; Figure 3 3D model diagrams of significant factors; where (A) is the response surface diagram of 5-methyltryptophan (5-MT) and yeast extract powder; (B) is the response surface diagram of 5-MT and corn steep liquor powder; (C) is the response surface diagram of corn steep liquor powder and yeast extract powder. Figure 4 Cell growth (A) and product synthesis (B) at different temperatures; Figure 5 The oxygen uptake rate (A) and product synthesis (B) under nitrogen source feeding are guided by OUR. Detailed Implementation

[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0022] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included within the scope of this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0023] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0024] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0025] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0026] Example 1: Screening of Vitamin K2-producing strains The inventors obtained a total of 15 soil samples, including fermented black soybeans and the surrounding environment of fermentation, from Shandong, Jiangsu, and Sichuan.

[0027] 1. Sample pretreatment Weigh 5g of sample, add 30mL of sterile physiological saline, shake for 30min to disperse the bacterial cells, then place in an 80℃ water bath for 15-20min to eliminate non-spore-forming bacteria by utilizing the heat resistance of spores. Dilute the treated bacterial solution to 10... -3 -10 -8 This is used for subsequent plate screening.

[0028] 2. Casein plate initial screening 2.1 Preparation of casein plates The formula consists of 5 g / L glucose, 2 g / L yeast extract, 1 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate heptahydrate, 10 g / L casein peptone, 0.75 g / L cymoxanil, and 15 g / L agar powder. The pH is adjusted to 7-7.2 with sodium hydroxide.

[0029] 2.2 Screening Using the QPix 400 series microbial cloning screening system, the above samples were plated and subjected to hydrolysis zone screening. A hydrolysis zone diameter to colony diameter ratio greater than 0.5 was used as the screening criterion to select strains with high proteolytic enzyme activity. Based on previous experience, although highly efficient secretory proteases and nattokinase are not directly related to high vitamin K2 production, they are strongly correlated with it. Highly efficient expression of these two enzymes also indicates better metabolic activity and nutrient utilization efficiency in the strains. Statistical analysis showed that a total of 109 suitable strains were screened from 15 samples in this embodiment.

[0030] 3. Fibrin plate rescreening 3.1 Preparation of fibrin plates Take 40 mL of 1.5 g / L fibrin solution, keep it in a constant temperature water bath at 50 °C while stirring, add 40 mL of 1.5 wt% agarose solution and 2 mL of 10 U / mL thrombin, mix well, then pour into a culture dish and cool to solidify.

[0031] 3.2 Screening Fermentation medium: glucose 55g / L, sucrose 50g / L, potassium dihydrogen phosphate 1g / L, yeast extract 15g / L, soybean peptone 35g / L, pH adjusted to 7-7.2 with sodium hydroxide; One hundred and nine qualified bacterial strains were inoculated into 30 mL / 250 mL fermentation medium via 200 μL glycerol tubes and cultured at 37 °C and 200 rpm for 72 h. 15 μL of the supernatant from the fermentation broth was dropped onto a fibrin plate and incubated at 37 °C for 24 h. The lysis zone was then measured.

[0032] After fibrin plate screening, 23 strains with fibrin activity were obtained, namely SC-1-25, SC-1-20, SC-1-94, SC-3-12, SC-3-41, SC-5-30, SC-5-32, SC-5-55, SC-7-64, SC-7-99, JS-1-9, JS-2-22, JS-2-37, JS-2-49, JS-2-38, JS-4-41, JS-4-64, JS-5-28, JS-5-40, SD-1-78, SD-2-66, SD-5-87 and SD-7-54.

[0033] 4. Determination through shake-flask fermentation Detection method: Agilent TC-C18(2) 250 × 4.6 mm column, mobile phase: anhydrous ethanol:water = 97:3 (V / V). Instrument parameters: column temperature 30℃, injection volume 10 μL, flow rate 1 mL / min, detection wavelength 268 nm.

[0034] Product extraction: Accurately pipette 1 mL of fermentation broth into a 50 mL centrifuge tube, add 10 mL of isopropanol, wrap with aluminum foil, sonicate in the dark for 15 min, centrifuge for 10 min at 6000 rpm, and filter through a 0.22 μm organic phase filter membrane into a liquid chromatography vial with an inner liner for later use.

[0035] The results of shake-flask fermentation of vitamin K2 by the 23 strains obtained above are shown in Table 1. The results showed that strain SC-7-99 had the highest yield, reaching 45.1 mg / L, and this strain was selected as the strain for subsequent mutagenesis screening.

[0036] Table 1 Results of strain screening Example 2: Molecular biological identification of bacterial strains The China Industrial Microbial Culture Collection Center (CICC) was commissioned to conduct polyphasic taxonomic identification of strain SC-7-99, which produces vitamin K2, provided by this invention. The identification was based on the "Methods for Polyphasic Identification and Detection of Bacteria" and the "Methods for Identification and Detection of Bacterial gyrB Housekeeping Gene," and the identification methods included microscopic observation of colony morphology, determination of physiological and biochemical characteristics, and analysis of 16S rDNA. gyrB The results of gene sequencing and phylogenetic analysis for the householder gene are as follows: Morphological characteristics: When strain SC-7-99 was cultured in TSA medium at 37℃ for 18 hours, the colonies were light yellow and nearly round, with a moist and opaque surface and neat edges; under microscopic observation, the cells were Gram-positive rod-shaped, with a cell size of 0.5~0.7μm×2.4~5.2μm, and the cells were arranged singly or in pairs. Physiological and biochemical characteristics: Using the API 50 CH carbon source utilization kit, this strain can utilize carbon sources such as glycerol, L-arabinose, D-glucose, D-fructose, D-mannose, inositol, mannitol, sorbitol, N-acetylglucosamine, amygdalin, aesculin, salicin, cellobiose, maltose, sucrose, trehalose, and inulin, but cannot utilize carbon sources such as erythritol, various types of xylose, lactose, melibiose, starch, and gluconate. Molecular biological identification: The 16S rDNA sequence of strain SC-7-99 was determined and... gyrB Housekeeping gene sequences were obtained and compared with the NCBI standard type strain database; the 16S rDNA sequence was consistent with... Bacillus subtilis Closely related species share a similarity of over 99.86%. gyrB Housekeeping gene sequence and Bacillus subtilis type strain Bacillus subtilis The similarity to BCRC 10255T was 100.00%; a phylogenetic tree was constructed using the neighbor-joining method, and the target strain clustered with the Bacillus subtilis type strain in the same evolutionary branch. Based on the comprehensive morphological, physiological, biochemical, and dual-gene molecular phylogenetic analysis results, the taxonomic position of this strain was determined to be Bacillus subtilis (…). Bacillus subtilis This strain possesses highly efficient fermentation properties for synthesizing vitamin K2 (MK-7).

[0037] Example 3: Targeted Mutagenesis Screening The vitamin K2-producing Bacillus subtilis SC-7-99 obtained through screening was mutagenized using ambient pressure room temperature plasma (ARTP) technology. Subsequently, the mutagenized bacterial solution was plated on dual resistance screening plates of 1-hydroxy-2-naphthoic acid (HNA) and diphenylamine (DPA) to selectively enrich mutant strains that relieved the feedback inhibition of the MK-7 synthesis pathway. Finally, the QPix 400 series microbial cloning screening system was used to achieve automated selection of target clones. Combined with shake flask screening, the screening efficiency and enrichment ratio of high-yield strains were greatly improved.

[0038] 1. Culture medium preparation Fermentation medium: glucose 55 g / L, sucrose 50 g / L, yeast extract 15 g / L and soybean peptone 35 g / L, pH adjusted to 7-7.2 with sodium hydroxide.

[0039] Screening medium: glucose 10 g / L, yeast extract 0.05 g / L, (NH4)2SO4 5 g / L, KH2PO4 1.0 g / L, MgSO4·7H2O 0.5 g / L and agar 18 g / L, with pH adjusted to 7.2-7.4 using sodium hydroxide.

[0040] 2. ARTP mutagenesis Pick a loopful of colonies from an LB agar plate and inoculate it into a 250 mL Erlenmeyer flask containing 25 mL of LB medium. Incubate at 37°C and 200 rpm until mid-logarithmic growth (approximately 5-7 hours). Centrifuge the fermentation broth to remove the supernatant and resuspend in physiological saline. OD values ​​are then calculated. 600 Approximately 1, the bacterial suspension was evenly spread onto a slide and aseptically transferred to an ARTP mutagenesis apparatus for mutagenesis. The instrument power was set to 100W, and the aeration rate to 10L / min for 35 seconds. After sample processing, the metal slide was transferred to sterile physiological saline, shaken, and the resulting mutagenic bacterial suspension was serially diluted.

[0041] 3. Screening with dual resistance plates The bacterial suspension was spread on screening solid plates containing 60 mg / L HNA and 30 mg / L DPA and incubated overnight at 37°C.

[0042] After three rounds of mutagenesis and resistance screening, a total of 21 single colonies were obtained in this example. For ease of collection and recording, they are ordered according to sequence numbers 1-21#, and the original strain is named 0#.

[0043] 4. Shake flask and screen again The selected bacterial strains were inoculated (200 μL each) into 250 mL Erlenmeyer flasks containing 30 mL of culture medium via glycerol tubes and cultured at 37°C and 200 rpm for 72 h. The vitamin K2 concentration in the fermentation broth was then investigated, and the results are shown below. Figure 1 The results showed that the vitamin K2 concentration was highest in the fermentation broth of strains #6 and #16.

[0044] 5. Stability of the mutagenized strain In this embodiment, strains 6# and 16#, as well as the original strain 0#, were further cultured for 10 generations to examine their stability. The results are as follows: Figure 2 As shown, the #6 mutagen strain exhibited good genetic stability within 10 generations. #6 was subsequently used as the strain for process optimization in the fermenter. In this embodiment, the mutagen strain was named *Bacillus subtilis* (…). Bacillus subtilisKV-2 was deposited on May 18, 2026, at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 38728.

[0045] Example 4: Optimization of Basal Culture Medium 1. Single-factor key factor optimization experimental design Fermentation medium: glucose, sucrose, potassium dihydrogen phosphate, soybean peptone, yeast extract, corn steep liquor powder, 5-methyltryptophan, and methionine. The concentrations of glucose and sucrose were fixed at 55 g / L and 50 g / L, respectively, with the remaining components as variables. A Plackett-Burman experimental design was used to screen the key influencing components of the culture medium during MK-7 synthesis. The experimental matrix was constructed using Design-Expert software, and the design levels of each factor are shown in Table 2.

[0046] Bacillus subtilis KV-2 was cultured using different culture media listed in Table 3. Statistical analysis was used to clarify the effect of each culture medium component on the response value (MK-7 yield). The results are summarized in Tables 3 and 4. The results showed that yeast extract powder, corn steep liquor powder, and 5-methyltryptophan... P The values ​​are all less than 0.05, indicating they are significant influencing factors in the synthesis of MK-7. Overall, the model... P A value of 0.0082 (<0.05) indicates that the regression model is highly significant, has a good fit within the regression region studied, and exhibits high model reliability; the correlation coefficient R0 2 =0.9335, indicating that the model has an excellent fit to the experimental data; the corrected coefficient of determination R0 adj The coefficient of variation (χ²) of 0.8536 indicates that the regression model can explain 85.36% of the experimental data variability, demonstrating strong explanatory power. The coefficient of variation of 5.3% is relatively low, validating the reliability and accuracy of the Plackett-Burman experiment. The precision of 11.23 (significantly greater than the critical value of 4) indicates that the model has sufficient signal strength to effectively distinguish between signal and noise. Through multiple regression fitting, the regression equations (coded equations) for MK-7 yield on each culture medium component were obtained: Y=98.88-0.2158A-3.78B+5.76C+8.75D+5.8E+1.66F.

[0047] A climbing experiment was conducted on the three significant factors mentioned above. Corn steep liquor powder was the most significant factor, so the climbing unit step size was 5. The step size for corn steep liquor powder was 5, the step size for yeast powder was 3, and the step size for 5-methyltryptophan was 2.5 (Table 5).

[0048] Table 2. Factor levels and codes in the Plackett-Burman experimental design Table 3 Results of shake-flask fermentation based on the Plackett-Burman experimental design Table 4. Analysis of Variance for the Plackett-Burman Experimental Design Table 5. Experimental Design and Results for the Steepest Climb (2) Determining the optimal values ​​of significant influencing factors using the Box-Behnken method A Box-Behnken experimental design was employed. Based on the experimental results in Table 5, the process of optimizing three key significant factors affecting MK-7 synthesis—yeast extract powder, corn steep liquor powder, and 5-methyltryptophan—was refined. Using MK-7 yield as the response index, the influence of each factor and its interactions on product synthesis was systematically investigated. Each experiment was conducted in triplicate to ensure data accuracy and reliability. The level settings, experimental schemes, and results for each factor are shown in Table 5, and the results of the analysis of variance are shown in Table 6.

[0049] The experimental data were fitted using Design-Expert software through multiple quadratic regression to establish the regression equation: MK-7 yield = 269.36 + 0.025A - 5.58B - 3.25C + 8.35AB - 2.4AC - 1.8BC – 15.36A² - 9.16B² - 2.01C²; where A represents 5-methyltryptophan, B represents yeast extract powder, and C represents corn steep liquor powder; A, B, and C are the coefficients of the linear terms of each factor, A², B², and C² are the coefficients of the quadratic terms, and AB, AC, and BC are the coefficients of the interaction terms. Response surface plots and contour plots were drawn based on the regression equation to analyze the influence trend and surface characteristics of each factor on MK-7 yield. The results are as follows: Figure 3 As shown in the figure. The results show that the peak centers of the three sets of response surface contour lines all fall within the factor level range set in this experiment, indicating that there are optimal process conditions within the factor level range designed in this invention.

[0050] Through numerical optimization analysis of the above three significant factors, the optimal process parameters were determined to be: yeast extract powder 30.21 g / L, corn steep liquor powder 21.55 g / L, and 5-methyltryptophan 20.97 mg / L.

[0051] The optimized MK-7 fermentation medium composition is as follows: glucose 55 g / L, sucrose 50 g / L, potassium dihydrogen phosphate 0.25 g / L, yeast extract 30.21 g / L, corn steep liquor powder 21.55 g / L, soybean peptone 30 g / L, methionine 0.65 g / L, and 5-methyltryptophan 20.97 mg / L.

[0052] The shake-flask fermentation verification showed that under the above-mentioned optimal culture medium conditions, the actual yield of MK-7 reached 281.4 mg / L, which was basically consistent with the model prediction of 271.62 mg / L. This indicates that the quadratic regression model has a good fit and high prediction accuracy, and can provide reliable data support and theoretical basis for the subsequent process scale-up and further optimization research of Bacillus subtilis fermentation to produce MK-7.

[0053] Table 6 Box-Behnken Design and Results Table 7 Analysis of Variance Table Example 5 Temperature optimization during fermentation Seed culture medium: 10 g / L yeast extract, 5 g / L peptone, 10 g / L sodium chloride, pH adjusted to 7-7.2 with sodium hydroxide solution, sterilized at 121℃ for 20 min before use.

[0054] The optimized MK-7 fermentation medium composition is as follows: glucose 55 g / L, sucrose 50 g / L, potassium dihydrogen phosphate 0.25 g / L, yeast extract 30.21 g / L, corn steep liquor powder 21.55 g / L, soybean peptone 30 g / L, methionine 0.65 g / L and 5-methyltryptophan 20.97 mg / L.

[0055] Seed culture: Take one loopful of Bacillus subtilis KV-2 and inoculate it into a 250 mL Erlenmeyer flask containing 30 mL of culture medium. Incubate at 37.0℃ and 220 rpm for 13-16 h. OD 600 It is around 3.9.

[0056] The specific operation for fermenter process control is as follows: Take 50 mL of seed culture and inoculate it into a 5 L fermenter containing 3 L of optimized MK-7 fermentation medium, and start the fermentation program. The initial fermentation conditions are set as follows: stirring speed 300 rpm, aeration rate 1 vvm, and tank pressure maintained at 0.03 MPa. During fermentation, the dissolved oxygen concentration is monitored in real time. When the dissolved oxygen value is lower than 15%, the dissolved oxygen level is stabilized within the range of 15%-20% by gradually increasing the stirring speed to ensure the dissolved oxygen requirements for cell growth and MK-7 synthesis. In the early stages of fermentation, when the initial carbon source (glucose) in the culture medium is depleted, the dissolved oxygen concentration in the fermentation system will rapidly increase. At this point, a sugar replenishment program is initiated: first, a glucose solution with a mass concentration of 500 g / L is added at a rate of 4 mL / (L·h). During the process, the sugar replenishment rate is increased by 2 mL / (L·h) every 4 hours, i.e., after 4 hours, the sugar replenishment rate is increased to 6 mL / (L·h), and after another 4 hours, the sugar replenishment rate is increased to 8 mL / (L·h), and so on, with a maximum sugar replenishment rate of 12 mL / (L·h). The residual sugar is controlled to be below 0.5 g / L throughout the process. pH control is maintained throughout the entire fermentation process. For the first 100 hours of fermentation, ammonia is used to adjust the pH. After 100 hours, a 2 mol / L sodium hydroxide solution is used for pH adjustment. The pH of the fermentation system is strictly controlled between 7.0 and 7.5 throughout the process to provide a stable acid-base environment for the growth of Bacillus subtilis and the synthesis of MK-7. During fermentation, the temperature was controlled at 37℃ throughout the entire process, 40℃ throughout the entire process, 37℃ during the growth phase and 40℃ during the stationary phase, and vice versa. Specific temperature controls are shown in Table 8. The OD of the fermentation broth was investigated at different fermentation times. 600 Values ​​and MK-7 production yields are shown in Table 8 and Figure 4 As shown in the figure. The results indicate that under controlled temperature conditions throughout the process, the highest synthesis yield of the target product, reaching 557 mg / L, was achieved at 37℃. While increasing the temperature can accelerate the product synthesis rate to some extent, it is not conducive to the continuous accumulation of the product and can easily lead to a decrease in the overall synthesis level. Considering both product synthesis efficiency and accumulation stability, the optimal fermentation temperature was determined to be 37℃.

[0057] Table 8. Results of the effect of different temperatures on vitamin K2 production by fermentation. Example 6: Nitrogen Source Feeding Control Based on Oxygen Consumption Rate (OUR) Physiological Parameter Feedback Seed culture medium: 10 g / L yeast extract, 5 g / L peptone, 10 g / L sodium chloride, pH adjusted to 7-7.2 with sodium hydroxide solution, sterilized at 121℃ for 20 min before use.

[0058] The optimized MK-7 fermentation medium composition is as follows: glucose 55 g / L, sucrose 50 g / L, potassium dihydrogen phosphate 0.25 g / L, yeast extract 30.21 g / L, corn steep liquor powder 21.55 g / L, soybean peptone 30 g / L, methionine 0.65 g / L and 5-methyltryptophan 20.97 mg / L.

[0059] Seed culture: Inoculate one loopful of bacteria into seed culture medium in a 50mL / 250mL seed shake flask and incubate at 37℃ and 220 rpm for 13-16 hours. OD 600 Around 3.9.

[0060] Yeast extract replenishment: 15g yeast extract to a final volume of 500mL.

[0061] The specific operation of the fermenter process control is as follows: Take 50 mL of seed culture and inoculate it into a 5 L fermenter containing 3 L of optimized MK-7 fermentation medium, and start the fermentation program. The initial fermentation conditions are set as follows: culture temperature 37℃, stirring speed 300 rpm, aeration rate 1 vvm, and tank pressure maintained at 0.03 MPa. During fermentation, the dissolved oxygen concentration is monitored in real time. When the dissolved oxygen value is lower than 15%, the dissolved oxygen level is stabilized within the range of 15%-20% by gradually increasing the stirring speed to ensure the dissolved oxygen requirements for cell growth and MK-7 synthesis. In the early stage of fermentation, when the carbon source (glucose) in the initial medium is exhausted, the dissolved oxygen concentration in the fermentation system will rapidly rise. At this time, the sugar replenishment program is started: first, a glucose solution with a mass concentration of 500 g / L is added at a rate of 4 mL / (L·h), and the sugar replenishment rate is increased by 2 mL / (L·h) every 4 hours during the process, while controlling the residual sugar to be below 0.5 g / L. pH control was maintained throughout the entire fermentation process. For the first 100 hours of fermentation, ammonia was used to adjust the pH. After 100 hours, a 2 mol / L sodium hydroxide solution was used for pH adjustment. The pH of the fermentation system was strictly controlled between 7.0 and 7.5 throughout the process, providing a stable acid-base environment for the growth of Bacillus subtilis and the synthesis of MK-7. After 70 hours of fermentation, yeast extract (concentration 30 g / L) was added as a significant factor. The addition of yeast extract was guided by online tail gas chromatography-mass spectrometry, and the OUR (Oxidative Range) was controlled at 20-25 mmol / L / h, 25-30 mmol / L / h, or above 30 mmol / L / h. A group without added yeast extract (i.e., without added nitrogen source) was also included. Specifically, the OUR parameters were generated by real-time monitoring of tail gas data using mass spectrometry. While maintaining constant sugar addition, yeast extract solutions were added at concentrations of 0.5-1 mL / (L·h), 1.5-2.5 mL / (L·h), and 4-6 mL / (L·h) to control the three OUR levels. The results are as follows: Figure 5As shown, by using OUR to guide the nitrogen source feeding of yeast extract, the OUR was controlled at 25-30 mmol / L / h, and the yield reached 831 mg / L after 154 h of fermentation.

[0062] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A strain of Bacillus subtilis that produces high levels of vitamin K2 ( Bacillus subtilis KV-2, characterized in that, The Bacillus subtilis KV-2 has the accession number CGMCC No. 38728.

2. The use of Bacillus subtilis KV-2 as described in claim 1 in the preparation of products for the production of vitamin K2.

3. A product for producing vitamin K2, characterized in that, The product includes Bacillus subtilis KV-2 as described in claim 1.

4. The use of Bacillus subtilis KV-2 as described in claim 1 or the product as described in claim 3 in the production of vitamin K2.

5. A method for producing vitamin K2, characterized in that, The method includes the step of fermenting and culturing Bacillus subtilis KV-2 as described in claim 1.

6. The method according to claim 5, characterized in that, The fermentation process also includes steps such as feeding glucose solution and yeast extract.

7. The method according to claim 5, characterized in that, The fermentation culture was carried out at a stirring speed of 300 rpm, an aeration rate of 1 vvm, a tank pressure of 0.03 MPa, a temperature of 37°C, and a dissolved oxygen level controlled between 15% and 20%.

8. The method according to claim 6, characterized in that, When the glucose in the culture medium used for fermentation is depleted, glucose solution is added; the initial flow rate is 4 mL / (L·h), and the glucose addition rate is increased by 2 mL / (L·h) every 4 hours, while controlling the residual sugar concentration to be below 0.5 g / L; The glucose concentration in the glucose solution is 500 g / L.

9. The method according to claim 6, characterized in that, During the fermentation process, the feed rate of the yeast extract was controlled to keep the oxygen consumption rate between 25-30 mmol / L / h; the feed time of the yeast extract was 70 h of fermentation.

10. The method according to claim 5, characterized in that, The culture medium used in the fermentation culture includes the following components at the following concentrations: Glucose 55 g / L, sucrose 50 g / L, potassium dihydrogen phosphate 0.25 g / L, yeast extract 30.21 g / L, corn steep liquor powder 21.55 g / L, soybean peptone 30 g / L, methionine 0.65 g / L, and 5-methyltryptophan 20.97 mg / L.

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