Preparation method for producing vitamin K2 through high-density fermentation of bacillus natto

Abstract

The invention discloses a preparation method for producing vitamin K2 through high-density fermentation of bacillus natto, and belongs to the technical field of microbial fermention.The preparation method comprises the following steps that S1, strains are constructed and screened, and bacillus natto serves as a starting strain; s2, preparation of a seed solution and slant activation: inoculating a slant activation culture medium with the seed solution, and culturing to obtain a slant strain with stable activity; seed tank expanding culture: inoculating the slant strain into a seed tank expanding culture medium to obtain a high-activity and high-concentration seed solution; s3, high-density fermentation: inoculating the seed solution into the optimized fermentation culture medium according to the inoculum size of 10%-12% (v / v), adopting a two-stage pH-dissolved oxygen coordinated regulation strategy and regularly supplementing materials, and carrying out fermentation culture for 72-80 hours to obtain a fermentation solution with high biomass and high MK-7 yield; s4, extracting and purifying the product to obtain a vitamin K2 product. The method has the technical effects of high strain yield, high fermentation efficiency, simplicity and convenience in extraction and purification, high product purity and low cost.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation technology, and in particular relates to a method for preparing vitamin K2 by high-density fermentation of Bacillus natto. Background Technology

[0002] Vitamin K (MK) is a general term for a class of antihemorrhagic compounds containing a menadione structure, also known as clotting vitamin or antihemorrhagic vitamin. Vitamin K (MK) can be divided into two main categories: one is the fat-soluble compounds K1, K2, and K3, which can be extracted from animals and plants; the other is the water-soluble compound K4, which is artificially synthesized. Vitamin K2 (also known as menadione, MK-n) is a fat-soluble vitamin, with its core active ingredient being MK-7. It has important physiological functions such as promoting blood clotting, regulating bone metabolism, and preventing osteoporosis, and is widely used in medicine, health products, and food. Vitamin K2 is a metabolic product of intestinal bacteria. Small amounts of vitamin K2 are found in some fermented foods, with fermented natto being particularly rich in it. Currently, microbial fermentation is the mainstream method for preparing vitamin K2 due to its readily available raw materials, mild conditions, high product bioactivity, and good safety.

[0003] Bacillus natto is a food-grade safe strain that can naturally synthesize vitamin K2. However, the yield of wild-type strain MK-7 is extremely low, and its biomass is limited, making it difficult to meet the needs of industrial production. Existing technologies, such as strain mutagenesis, culture medium optimization, and fermentation condition control, still have many shortcomings: single mutagenic strains have poor stability and are not effective in increasing yield; unreasonable nutrient ratios in the fermentation medium and imprecise fermentation control lead to asynchronous cell growth and product synthesis; and the extraction and purification processes are cumbersome and prone to organic solvent residues. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention provides a method for preparing vitamin K2 by high-density fermentation of Bacillus natto. This method has the advantages of high strain yield, high fermentation efficiency, simple extraction and purification, high product purity, and low cost. It solves the problems of poor stability of single mutagenic strains, low yield of MK-7, limited cell biomass, imprecise fermentation control, cumbersome extraction and purification, and residual organic solvents in the prior art.

[0005] This invention is achieved through a method for preparing vitamin K2 by high-density fermentation of Bacillus natto, comprising the following steps: S1. Strain Construction and Screening Using Bacillus natto as the starting strain, a Phe- / Trp- dual auxotrophic strain with high vitamin K2 production (MK-7) was obtained by combining UV mutagenesis with diethyl sulfate mutagenesis and site-directed overexpression of the menA gene. The MK-7 yield of the strain was increased by more than 80% compared with the original starting strain and was preserved in glycerol tubes. S2, Seed Liquid Preparation Slant activation: The high-yield strains screened above were revived from the glycerol tubes, inoculated into slant activation medium, and cultured to obtain stable slant strains; Seed tank expansion culture: The slant culture is inoculated into the seed tank expansion culture medium, and cultured with aeration and stirring to obtain a highly active and high-concentration seed liquid; S3, High-density fermentation The above seed culture was inoculated into the optimized fermentation medium at an inoculation rate of 10%-12% (v / v). A two-stage pH-dissolved oxygen synergistic regulation strategy was adopted and the medium was fed regularly for fermentation culture. The fermentation cycle was 72-80 hours to obtain a fermentation broth with high biomass and high MK-7 yield. The optimized fermentation medium, by mass-volume ratio, comprises the following components: 5%-6% glycerol, 2%-3% soybean protein, 3%-4% shiitake mushroom residue, appropriate amounts of inorganic salts, L-tryptophan, and L-phenylalanine, with an initial pH of 7.0-7.2, and is sterilized at 120℃ for 20 min. S4. Product Extraction and Purification The fermentation broth was centrifuged to collect the cells, and the cells were extracted by ultrasonic disruption using a hexane-isopropanol mixed solvent. The extracted cells were then concentrated under reduced pressure, filtered through a nanofiltration membrane, and purified by silica gel column chromatography to obtain a vitamin K2 product with a purity ≥98%.

[0006] As a preferred embodiment of the present invention, in step S1, the combined mutagenesis is performed by ultraviolet irradiation combined with diethanol sulfate treatment, and the menA gene overexpression is performed by constructing a recombinant plasmid using the pBE-S vecto vector, which is then introduced into the mutagenized strain via electroporation.

[0007] This setting can significantly improve the mutation efficiency of the strain, enhance the genetic stability of the strain, break through the metabolic bottleneck of vitamin K2 synthesis, and greatly increase the yield of MK-7.

[0008] As a preferred embodiment of the present invention, in step S1, the starting strain of Bacillus natto is SD-3 or ND-1-A27, and the high-yield strain is stored in a glycerol tube at -80°C, resulting in high activation efficiency after revival.

[0009] This setting ensures that the starting strain is safe and reliable, and that the fermentation performance is stable, making it suitable for long-term preservation and continuous industrial production.

[0010] As a preferred embodiment of the present invention, in step S2, the slant activation culture medium contains glucose, peptone, NaCl, yeast extract and agar, and the culture temperature is 37°C, with continuous activation for 2-3 generations.

[0011] This setting allows the strain to quickly regain its activity, ensuring the purity and viability of the strain and providing a stable source of inoculum for subsequent propagation.

[0012] As a preferred embodiment of the present invention, in step S2, the seed tank expansion culture medium contains glucose, peptone and appropriate amount of inorganic salts, the culture temperature is 36-37℃, and the culture is carried out by aeration and stirring to obtain qualified seed liquid.

[0013] This setup allows for rapid cell proliferation, resulting in a high-concentration, highly viable seed culture, laying the foundation for high-density fermentation.

[0014] As a preferred embodiment of the present invention, in step S3, the two-stage pH-dissolved oxygen synergistic regulation strategy is specifically as follows: Stage 1 is the cell proliferation stage, where the pH is controlled at 7.0-7.2, dissolved oxygen is maintained at ≥20%, and high-density cell growth is promoted; Stage 2 is the product synthesis stage, where the pH is adjusted to 3.8-4.2, dissolved oxygen is maintained at 15%-20%, and MK-7 is induced to be synthesized in large quantities, with the fermentation endpoint yield of MK-7 ≥70mg / L.

[0015] This setting enables precise segmented control of cell growth and product synthesis, significantly improving biomass and synthesis efficiency, and solving the problem of imprecise fermentation control.

[0016] As a preferred embodiment of the present invention, in step S3, the periodic feeding includes a feeding solution containing glycerol, soybean protein, and amino acids to maintain fermentation stability.

[0017] This setting continuously replenishes the nutrients needed for cell growth and product synthesis, prevents premature cell aging, and ensures a stable and efficient fermentation process.

[0018] In a preferred embodiment of the present invention, step S4, the extraction and purification specifically comprises: Cell collection: Centrifuge the fermentation broth at 8000-9000 rpm and 4℃ for 8-12 min and collect the wet cells; Cell wall disruption extraction: Mix wet bacterial cells with a hexane-isopropanol mixed solvent, and extract by ultrasonic disruption at 40-45℃ and 200-300w for 25-35 minutes. Centrifuge to collect the organic phase. Repeat the extraction 2-3 times and combine the organic phase extracts. Concentration: The combined extracts were concentrated by vacuum distillation at 45-50℃ and 0.06-0.08MPa to remove organic solvents and obtain crude MK-7. Purification: The crude product was dissolved in a small amount of n-hexane, filtered through a nanofiltration membrane to remove impurities, and then subjected to silica gel column chromatography. The main elution peak was collected using a mixture of n-hexane and isopropanol as the eluent, concentrated under reduced pressure, and then dried under vacuum to obtain the product.

[0019] This setup improves cell disruption efficiency and product extraction efficiency, simplifies the purification process, reduces organic solvent residue, and significantly enhances product purity and recovery rate.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows: By combining compound mutagenesis with gene overexpression, a highly stable and high-yield strain was obtained, solving the problems of poor stability and low MK-7 yield associated with single-mutation strains. Optimizing the culture medium and implementing two-stage pH-dissolved oxygen synergistic regulation enabled high-density cell culture, addressing issues of low cell biomass and imprecise fermentation control. Employing a combined purification process of ultrasonic cell disruption, mixed solvent extraction, and nanofiltration-chromatography resolved problems such as cumbersome extraction and purification processes, low product purity and recovery rates, and the potential for organic solvent residue. The resulting vitamin K2 product is highly pure, safe, and reliable, possessing significant economic and application value. This invention's strain is highly efficient and stable, exhibits high-density fermentation efficiency, and its extraction and purification are simple and environmentally friendly, making it highly adaptable to industrial applications. Detailed Implementation

[0021] To further understand the invention's content, features, and effects, the following embodiments are provided and described in detail below.

[0022] The present invention will now be described in detail. Example 1

[0023] This invention provides a method for preparing vitamin K2 through high-density fermentation of Bacillus natto. The method includes the following steps: S1. Strain Construction and Screening Using Bacillus natto SD-3 as the starting strain, a combination of ultraviolet irradiation and diethanol sulfate treatment was used to complete the combined mutagenesis. The mutagenized bacterial suspension was serially diluted and spread on the screening medium. The MK-7 content was detected by HPLC, and strains with high MK-7 yield were screened. The menA gene was cloned into the pBE-S vecto vector to construct a recombinant plasmid, which was introduced into the mutagenized strain by electroporation. A high-yielding Phe- / Trp- dual auxotrophic strain was obtained, with its MK-7 yield increased by 90% compared with the original starting strain. The strain was stored in glycerol tubes at -80℃, and the activation efficiency after revival was 96%. S2, Seed Liquid Preparation Slant activation: The high-yield strains screened above were revived from glycerol tubes and inoculated into slant activation medium to obtain stable slant strains. The slant activation medium contained glucose, peptone, NaCl, yeast extract and agar. The culture temperature was 37℃, and the strains were activated for two generations. Seed tank expansion culture: The slant culture is inoculated into the seed tank expansion culture medium, which contains glucose, peptone and appropriate amount of inorganic salts. The culture temperature is 36℃ and the culture is carried out by aeration and stirring to obtain qualified seed liquid. S3, High-density fermentation The above seed culture was inoculated at a rate of 10% (v / v) into the optimized fermentation medium. The optimized fermentation medium, by mass-volume ratio, consisted of the following components: 5% glycerol, 2% soybean protein, 3% shiitake mushroom residue, appropriate amounts of inorganic salts, L-tryptophan, and L-phenylalanine, with an initial pH of 7.0, and sterilized at 120°C for 20 min. The culture was then placed in a fermenter. Stage 1 was the cell proliferation stage: pH was controlled at 7.0, dissolved oxygen was maintained at ≥20%, and high-density cell growth was promoted. Stage 2 was the product synthesis stage: pH was adjusted to 3.8, dissolved oxygen was maintained at 15%, and MK-7 synthesis was induced. A feed solution containing glycerol, soybean protein, and amino acids was added every 20 h. The fermentation cycle was 72 h, and the final MK-7 yield was 72 mg / L, yielding the fermentation broth. S4. Product Extraction and Purification Cell collection: The fermentation broth was centrifuged at 8000 rpm and 4℃ for 8 min, and the wet cells were collected; Cell wall disruption extraction: Wet bacterial cells were mixed with a hexane-isopropanol mixed solvent and ultrasonically disrupted at 40℃ and 200w for 25 min. The organic phase was collected by centrifugation. The extraction was repeated twice, and the organic phase extracts were combined. Concentration: The combined extracts were concentrated by vacuum distillation at 45℃ and 0.06MPa to remove organic solvents and obtain crude MK-7. Purification: The crude product was dissolved in a small amount of n-hexane, filtered through a nanofiltration membrane to remove impurities, and then subjected to silica gel column chromatography. The main elution peak was collected using a mixture of n-hexane and isopropanol as the eluent, concentrated under reduced pressure, and then dried under vacuum to obtain a vitamin K2 product with a purity of 98.5%. Example 2

[0024] A method for preparing vitamin K2 by high-density fermentation of Bacillus natto includes the following steps: S1. Strain Construction and Screening Using Bacillus natto ND-1-A27 as the starting strain, a combination of ultraviolet irradiation and diethanol sulfate treatment was used to complete the mutagenesis. The mutagenized bacterial suspension was serially diluted and spread on the screening medium. The MK-7 content was detected by HPLC, and strains with high MK-7 yield were screened. The menA gene was cloned into the pBE-S vecto vector to construct a recombinant plasmid, which was introduced into the mutagenized strain by electroporation. A high-yielding Phe- / Trp- dual auxotrophic strain was obtained, with its MK-7 yield increased by 95% compared with the original starting strain. The strain was stored in glycerol tubes at -80℃, and the activation efficiency after revival was 98%. S2, Seed Liquid Preparation Slant activation: The high-yield strains screened above were revived from glycerol tubes and inoculated into slant activation medium to obtain stable slant strains. The slant activation medium contained glucose, peptone, NaCl, yeast extract and agar. The culture temperature was 37℃, and the strains were continuously activated for 3 generations. Seed tank expansion culture: The slant culture is inoculated into the seed tank expansion culture medium, which contains glucose, peptone and appropriate amount of inorganic salts. The culture temperature is 37℃ and the culture is carried out by aeration and stirring to obtain qualified seed liquid. S3, High-density fermentation The above seed culture was inoculated at a rate of 12% (v / v) into the optimized fermentation medium. The optimized fermentation medium, by mass-volume ratio, consisted of the following components: 6% glycerol, 3% soybean protein, 4% shiitake mushroom residue, appropriate amounts of inorganic salts, L-tryptophan, and L-phenylalanine, with an initial pH of 7.2, and sterilized at 120°C for 20 min. The culture was then placed in a fermenter. Stage 1 was the cell proliferation stage: pH was controlled at 7.2, dissolved oxygen was maintained at ≥20%, and high-density cell growth was promoted. Stage 2 was the product synthesis stage: pH was adjusted to 4.2, dissolved oxygen was maintained at 20%, and MK-7 synthesis was induced. A feed solution containing glycerol, soybean protein, and amino acids was added every 20 h. The fermentation cycle was 80 h, and the final MK-7 yield was 85 mg / L, yielding the fermentation broth. S4. Product Extraction and Purification Cell collection: The fermentation broth was centrifuged at 9000 rpm and 4℃ for 12 min, and the wet cells were collected; Cell wall disruption extraction: Wet bacterial cells were mixed with a hexane-isopropanol mixed solvent and ultrasonically disrupted at 45℃ and 300w for 35 min. The organic phase was collected by centrifugation. The extraction was repeated 3 times and the organic phase extracts were combined. Concentration: The combined extracts were concentrated by vacuum distillation at 50℃ and 0.08MPa to remove organic solvents and obtain crude MK-7. Purification: The crude product was dissolved in a small amount of n-hexane, filtered through a nanofiltration membrane to remove impurities, and then subjected to silica gel column chromatography. The main elution peak was collected using a mixture of n-hexane and isopropanol as the eluent, concentrated under reduced pressure, and then dried under vacuum to obtain a vitamin K2 product with a purity of 99%.

[0025] This invention obtains highly stable and high-yield bacterial strains through combined mutagenesis and gene overexpression. By optimizing the culture medium and implementing two-stage pH-dissolved oxygen synergistic regulation, high-density bacterial culture is achieved. A combined purification process of ultrasonic cell disruption, mixed solvent extraction, and nanofiltration-chromatography is employed, simplifying the extraction and purification process and avoiding organic solvent residues. The resulting vitamin K2 product is highly pure, safe, and reliable, with significant economic and application value. The strains of this invention are highly efficient and stable, exhibit high fermentation efficiency at high densities, and the extraction and purification process is green, simple, and highly adaptable to industrial applications.

[0026] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0027] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing vitamin K2 by high-density fermentation of Bacillus natto, characterized in that, Includes the following steps: S1. Strain Construction and Screening Using Bacillus natto as the starting strain, a Phe- / Trp- dual auxotrophic strain with high vitamin K2 production (MK-7) was obtained by combining UV mutagenesis with diethyl sulfate mutagenesis and site-directed overexpression of the menA gene. The MK-7 yield of the strain was increased by more than 80% compared with the original starting strain and was preserved in glycerol tubes. S2, Seed Liquid Preparation Slant activation: The high-yield strains screened above were revived from the glycerol tubes, inoculated into slant activation medium, and cultured to obtain stable slant strains; Seed tank expansion culture: The slant culture is inoculated into the seed tank expansion culture medium, and cultured with aeration and stirring to obtain a highly active and high-concentration seed liquid; S3, High-density fermentation The above seed culture was inoculated into the optimized fermentation medium at an inoculation rate of 10%-12% (v / v). A two-stage pH-dissolved oxygen synergistic regulation strategy was adopted and the medium was fed regularly for fermentation culture. The fermentation cycle was 72-80 hours to obtain a fermentation broth with high biomass and high MK-7 yield. The optimized fermentation medium, by mass-volume ratio, comprises the following components: 5%-6% glycerol, 2%-3% soybean protein, 3%-4% shiitake mushroom residue, appropriate amounts of inorganic salts, L-tryptophan, and L-phenylalanine, with an initial pH of 7.0-7.2, and is sterilized at 120℃ for 20 min. S4. Product Extraction and Purification The fermentation broth was centrifuged to collect the cells, and the cells were extracted by ultrasonic disruption using a hexane-isopropanol mixed solvent. The extracted cells were then concentrated under reduced pressure, filtered through a nanofiltration membrane, and purified by silica gel column chromatography to obtain a vitamin K2 product with a purity ≥98%.

2. The method for preparing vitamin K2 by high-density fermentation of Bacillus natto as described in claim 1, characterized in that: In step S1, the combined mutagenesis is performed by ultraviolet irradiation combined with diethanol sulfate treatment, and the menA gene overexpression is performed by constructing a recombinant plasmid using the pBE-S vecto vector, which is then introduced into the mutagenized strain via electroporation.

3. The method for preparing vitamin K2 by high-density fermentation of Bacillus natto as described in claim 1, characterized in that: In step S1, the starting strain of Bacillus natto is SD-3 or ND-1-A27, and the high-yield strain is stored in glycerol tubes at -80℃, and has high activation efficiency after revival.

4. The method for preparing vitamin K2 by high-density fermentation of Bacillus natto as described in claim 1, characterized in that: In step S2, the slant activation medium contains glucose, peptone, NaCl, yeast extract and agar, and the culture temperature is 37℃, with continuous activation for 2-3 generations.

5. The method for preparing vitamin K2 by high-density fermentation of Bacillus natto as described in claim 1, characterized in that: In step S2, the seed tank expansion culture medium contains glucose, peptone and appropriate amount of inorganic salts, the culture temperature is 36-37℃, and the culture is carried out by aeration and stirring to obtain qualified seed liquid.

6. The method for preparing vitamin K2 by high-density fermentation of Bacillus natto as described in claim 1, characterized in that: In step S3, the two-stage pH-dissolved oxygen synergistic regulation strategy is as follows: Stage 1 is the cell proliferation stage, where the pH is controlled at 7.0-7.2 and the dissolved oxygen is maintained at ≥20%, promoting high-density cell growth; Stage 2 is the product synthesis stage, where the pH is adjusted to 3.8-4.2 and the dissolved oxygen is maintained at 15%-20%, inducing the large-scale synthesis of MK-7, with the fermentation endpoint yield of MK-7 ≥70mg / L.

7. The method for preparing vitamin K2 by high-density fermentation of Bacillus natto as described in claim 1, characterized in that: In step S3, the periodic feeding includes a feeding solution containing glycerol, soy protein, and amino acids to maintain fermentation stability.

8. The method for preparing vitamin K2 by high-density fermentation of Bacillus natto as described in claim 1, characterized in that: In step S4, the extraction and purification specifically involve: Cell collection: Centrifuge the fermentation broth at 8000-9000 rpm and 4℃ for 8-12 min and collect the wet cells; Cell wall disruption extraction: Mix wet bacterial cells with a hexane-isopropanol mixed solvent, and extract by ultrasonic disruption at 40-45℃ and 200-300w for 25-35 minutes. Centrifuge to collect the organic phase. Repeat the extraction 2-3 times and combine the organic phase extracts. Concentration: The combined extracts were concentrated by vacuum distillation at 45-50℃ and 0.06-0.08MPa to remove organic solvents and obtain crude MK-7. Purification: The crude product was dissolved in a small amount of n-hexane, filtered through a nanofiltration membrane to remove impurities, and then subjected to silica gel column chromatography. The main elution peak was collected using a mixture of n-hexane and isopropanol as the eluent, concentrated under reduced pressure, and then dried under vacuum to obtain the product.