Method for purifying vitamin K2 (MK-7) from bacillus natto fermentation liquor

Abstract

The invention relates to the technical field of biological medicine, in particular to a method for purifying vitamin K2 (MK-7) from bacillus natto fermentation liquor. The method comprises the following steps: concentrating a fermentation stock solution, carrying out high-pressure homogenization on the concentrated fermentation stock solution and an organic solvent, extracting vitamin K2 in the fermentation stock solution into an organic phase through high-pressure homogenization, and separating to obtain an organic phase component; the preparation method comprises the following steps: washing an organic phase component with a saturated saline solution to remove impurities, further concentrating and crystallizing to obtain a vitamin K2 crude product, further purifying the vitamin K2 crude product through melt crystallization to obtain a vitamin K2 pure product, and the purity of the obtained vitamin K2 pure product is greater than 99.9%. The method disclosed by the invention is simple in process flow and low in equipment cost, can be used for preparing a high-purity vitamin K2 product, and is convenient to apply in actual production.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to a method for purifying vitamin K2 (MK-7) from natto bacillus fermentation broth. Background Art

[0002] Vitamin K2 is a series of compounds. The pure product is pale yellow crystals. It is heat-resistant but sensitive to light and alkali. Vitamin K2 has many physiological functions. It can prevent liver cirrhosis from progressing to liver cancer, promote the formation of prothrombin, accelerate blood coagulation, treat osteoporosis, act as a diuretic, strengthen the liver's detoxification function, and lower blood pressure. It has great medical value.

[0003] There are two main methods for obtaining vitamin K2: artificial synthesis and fermentation extraction. The fermentation method is mostly synthesized by Bacillus natto, and the vitamin K2 obtained is mainly trans, with high activity and strong stability. However, the regulation of the fermentation process is extremely difficult, and the separation steps are complicated, which makes it difficult to obtain the final product and the cost is high. In the existing process of extracting vitamin K2 from the fermentation broth to obtain a pure product, the general process is complicated and cumbersome, and the purity is low. Patent CN106631748A adopts a process of adsorption by a macroporous adsorption resin column, purification by a molecular weight exclusion chromatography column, separation by a reversed-phase silica gel column, and final recrystallization to obtain a pure product with a purity greater than or equal to 98.2%. Patent CN112321408A purifies the product by a continuous dynamic countercurrent extractor and a medium-pressure preparative chromatography and other equipment, and then obtains a pure product by recrystallization. Although the product purity can reach more than 99%, its equipment investment is expensive.

[0004] Melt crystallization technology utilizes the difference in freezing points between the components of the separated substance, controls the input and removal of heat, causes the separated components to crystallize out from the molten liquid, and achieves separation and purification of the target components through operations such as washing and sweating. It has the advantages of no use of solvents, low energy consumption, small equipment size, and the ability to obtain high-purity products. Therefore, based on the melt crystallization technology, the present invention proposes a method for purifying vitamin K2 (MK-7) from natto Bacillus subtilis fermentation broth to solve the problems in the prior art that the extraction process of vitamin K2 (MK-7) is complex, the product purity is low and the cost is high, and it is not conducive to industrial application. Summary of the invention

[0005] The purpose of the present invention is to provide a method for purifying vitamin K2 (MK-7) from natto bacillus fermentation broth, so as to solve the problems of complex process, low product purity and disadvantageous for industrial application of existing extraction methods.

[0006] To achieve the above object, the present invention adopts the following technical scheme: a method for purifying vitamin K2 (MK-7) from Bacillus natto fermentation broth, comprising the following steps:

[0007] S11, concentrating the fermentation stock solution, mixing it with an organic solvent, homogenizing it under high pressure, and separating the organic phase components;

[0008] S12, washing the organic phase components with saturated brine, filtering, concentrating, and crystallizing to obtain crude vitamin K2;

[0009] S13. The crude vitamin K2 product is melt-crystallized to obtain a high-purity vitamin K2 product.

[0010] Furthermore, in the S11, the volume of the concentrate after the fermentation liquid is concentrated is 5-20% of the volume of the fermentation liquid, and the concentration methods include single-effect evaporator, multiple-effect evaporator and membrane concentration; the volume of the organic solvent is 1-5 times the volume of the concentrate.

[0011] Furthermore, the organic solvent is immiscible with water and includes one or more of n-hexane, ethyl acetate and dichloromethane.

[0012] Furthermore, in the above-mentioned S11, the pressure of high-pressure homogenization is 20 to 80 MPa, and the number of high-pressure homogenization is 2 to 4 times.

[0013] Furthermore, in the above S11, the separation method includes static separation and centrifugal separation.

[0014] Furthermore, the separation adopts a three-phase disc separator with a rotation speed of 5000-7000rpm.

[0015] Furthermore, in the S12, the amount of saturated brine used is 5-15% of the volume of the organic phase component, and the product is washed with water 2-3 times; the filtration accuracy is not greater than 220nm; the concentration is carried out by reduced pressure concentration, and the volume is concentrated to 5-20% of the volume of the organic phase component; the crystallization endpoint temperature is 0-5°C, the cooling rate is 1-10°C / h, and the crystal is grown for 0.5-3h after the cooling is completed.

[0016] Further, in said S13, the melt crystallization comprises the following steps:

[0017] S21, heating the crude vitamin K2 to 56-65° C. and completely melting it;

[0018] S22, cooling to 50-53°C at a rate of 0.2-5°C / h, and then heating to 53-55°C at a temperature gradient of 0.1-5°C, collecting sweating liquid during the process, and keeping each gradient warm until sweating stops before continuing to heat up;

[0019] S23. Continue to raise the temperature to 56-65°C and completely melt to obtain pure vitamin K2. The purity of pure vitamin K2 is greater than 99.9%.

[0020] Furthermore, the mother liquor produced in the crystallization process in S12 and the sweating liquid produced in S22 are collected and comprehensively utilized; the organic solvent produced in the concentration process in S12 is returned to the S11 step for recycling.

[0021] Furthermore, in the above-mentioned S11-S13, each step is performed in a light-proof manner.

[0022] Beneficial effects of the present invention: The process flow of the method of the present invention is simple, the equipment cost is low, and the obtained product has high purity, which can reach more than 99.9%, and is convenient for industrial application. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is a process flow chart of the method of the present invention. DETAILED DESCRIPTION

[0024] The technical solution of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention.

[0025] like Figure 1 As shown, in the process of the present invention, the fermentation broth is concentrated and then subjected to high-pressure homogenization with an organic solvent. During the high-pressure homogenization, the organic solvent extracts vitamin K2 from the fermentation broth. On the one hand, the bacterial wall can be broken during the mixing process to fully release the vitamin K2 component in the cells; on the other hand, the organic solvent is dispersed into nano-scale particles through the strong shear force of the high-pressure homogenizer to fully extract the vitamin K2 component in the fermentation broth; the organic solvent is selected from solvents that are miscible with water, such as n-hexane, ethyl acetate and dichloromethane; after the high-pressure homogenization, the organic solvent is separated from the fermentation broth to obtain an organic phase component.

[0026] The organic phase components are washed with saturated brine to remove impurities. Water-soluble and polar impurity components in the organic phase can be removed by washing with water. Saturated brine can effectively avoid emulsification during the washing process and reduce organic phase loss. After impurities are removed, reduced pressure concentration is used for further concentration. Organic solvents are volatilized during the process. The volatilized organic solvents can be collected and returned to the high-pressure homogenization treatment for recycling. The concentrated solution obtained by concentration is cooled and crystallized, and then separated to obtain crude vitamin K2 and crystallization mother liquor.

[0027] The crude vitamin K2 is further purified by melt crystallization to obtain pure microbial K2, and sweat fluid is collected during the process. The collected sweat fluid and the aforementioned crystallization mother liquor can be comprehensively utilized.

[0028] The fermentation broth of each embodiment of the present invention comes from the vitamin K2 production workshop of Xintuoyang Bioengineering Co., Ltd., and the strain is B. natto (commercially available). The fermentation substrate is glycerol, soy peptone and yeast powder, the fermentation temperature is 36-38°C, the pH is 7±0.5, the ventilation volume is 0.5-1.0 vvm, and the fermentation time is 90-100 h.

[0029] Example 1

[0030] The fermentation tank was 6300L, and the vitamin K2 content was detected to be 209mg / L, with a total of 1316.7g of pure vitamin K2.

[0031] The fermentation broth was concentrated by a single-effect concentrator at a pressure of -0.09 MPa and a temperature of 55°C to a volume of about 400 L.

[0032] Take 600L of n-hexane, mix it with the fermentation concentrate and feed it into the homogenizer. The concentrate and n-hexane are fed at a feed rate of 1:1.5, and the homogenization pressure is 70MPa; repeat the homogenization 3 times.

[0033] After homogenization, the mixed liquid is separated by a three-phase disc separator to obtain an organic phase; the rotation speed of the three-phase disc separator is 6000-6200rpm; the organic phase is washed with saturated brine, 60L of which is used for 3 times; after washing, the organic phase is filtered through a 220nm filter element; the filtered organic phase is concentrated by reduced pressure to a residual volume of 50L, the concentration vacuum degree is -0.09MPa, and the concentration temperature is 50°C; the concentrated organic phase is cooled to 3°C at 5°C / h and kept warm for 30min; then filtered to obtain the crude vitamin K2; light must be avoided during the above operations.

[0034] The crude vitamin K2 is further purified by melt crystallization. The temperature control process is controlled by a high-precision constant temperature water bath. The crude vitamin K2 is heated to 60°C and kept warm until it is completely melted. The temperature is lowered to 53°C at 1°C / h and kept warm for 1h. The temperature is gradually increased by 0.2°C, and the sweating liquid is collected. Each gradient is kept warm until sweating stops and then the temperature is continued to increase. After the temperature is increased to 54.4°C, it is heated to 60°C to melt all the vitamin K2 crystals, and the pure vitamin K2 is collected. The operation is protected from light, and the sweating liquid is mixed into the next batch of cooling crystallization process for extraction again.

[0035] The vitamin K2 mother liquor concentrate produced by the above concentration and filtration and the sweat liquid produced by melt crystallization are collected and can be used for the preparation of low-content powder or oil.

[0036] 177g of pure vitamin K2 was collected, with a purity of 99.93%; 741g of vitamin K2 sweat solution was collected, with a purity of 92.62%. A total of 863.2g of pure vitamin K2 was collected, with a yield of 65.56%.

[0037] Example 2

[0038] The fermentation tank was 6150L, ​​and the vitamin K2 content was detected to be 216mg / L, with a total of 1328.4g of pure vitamin K2.

[0039] The fermentation broth was concentrated by a single-effect concentrator at a pressure of -0.09MPa and a temperature of 55°C to a volume of about 400L.

[0040] Take 800 L of ethyl acetate, mix it with the fermentation concentrate and feed it into the homogenizer. The concentrate and ethyl acetate are fed at a feed rate of 1:2, and the homogenization pressure is 75 MPa; repeat the homogenization 3 times.

[0041] After homogenization, the mixed liquid is separated by a three-phase disc separator to obtain an organic phase, and the rotation speed of the three-phase disc separator is 6000rpm; the organic phase is washed with saturated brine, 80L of which is used for 3 times; after washing, the organic phase is filtered through a 220nm filter element; the oil phase after filtration is concentrated by reduced pressure to a residual volume of 50L, the concentration vacuum degree is -0.098MPa, and the concentration temperature is 60°C; after concentration, the oil phase is cooled to 3°C at 5°C / h and kept warm for 30min; then filtered to obtain the crude vitamin K2; light must be avoided during the above operations.

[0042] The crude vitamin K2 is further purified by melt crystallization. The temperature control process is controlled by a high-precision constant temperature water bath. The crude vitamin K2 is heated to 60°C and kept warm until it is completely melted. The temperature is lowered to 53°C at 0.7°C / h, kept warm for 1h, and gradually heated up by 0.2°C. The sweat solution is collected. Each gradient is kept warm until sweating stops and then the temperature is continued to rise. After the temperature is raised to 54°C, it is raised to 60°C to melt all the vitamin K2 crystals, and the pure vitamin K2 is collected. The operation is protected from light, and the sweat solution is mixed into the next batch of cooling crystallization process for extraction again.

[0043] The vitamin K2 mother liquor concentrate produced by the above concentration and filtration and the sweating liquid produced by melt crystallization are collected and can be used for the preparation of low-content powder or oil.

[0044] 200g of pure vitamin K2 was collected, with a purity of 99.96%; 765g of vitamin K2 sweat solution was collected, with a purity of 91.58%. A total of 900.5g of pure vitamin K2 was collected, with a yield of 67.79%.

[0045] The present invention is not limited to the above-mentioned optimal implementation mode. Anyone can derive other various forms of products under the inspiration of the present invention. However, no matter what changes are made in the shape or structure, all technical solutions that are the same or similar to those of the present application fall within the protection scope of the present invention.

Claims

1. A method for purifying vitamin K2 (MK-7) from Bacillus natto fermentation broth, characterized in that: The following steps are involved: S11, concentrating the fermentation stock solution, mixing it with an organic solvent, homogenizing it under high pressure, and separating the organic phase components; S12, washing the organic phase components with saturated brine, filtering, concentrating, and crystallizing to obtain crude vitamin K2; S13. The crude vitamin K2 product is melt-crystallized to obtain a high-purity vitamin K2 product.

2. A method for purifying vitamin K2 (MK-7) from Bacillus natto fermentation broth according to claim 1, characterized in that: In the above S11, the volume of the concentrate after the fermentation liquid is concentrated is 5-20% of the volume of the fermentation liquid, and the concentration methods include single-effect evaporator, multiple-effect evaporator and membrane concentration; the volume of the organic solvent is 1-5 times the volume of the concentrate.

3. A method for purifying vitamin K2 (MK-7) from Bacillus natto fermentation broth according to claim 2, characterized in that: The organic solvent is immiscible with water and includes one or more of n-hexane, ethyl acetate and dichloromethane.

4. A method for purifying vitamin K2 (MK-7) from Bacillus natto fermentation broth according to claim 1, characterized in that: In the above-mentioned S11, the pressure of high-pressure homogenization is 20-80 MPa, and the number of high-pressure homogenization is 2-4 times.

5. A method for purifying vitamin K2 (MK-7) from Bacillus natto fermentation broth according to claim 1, characterized in that: In the above-mentioned S11, the separation method includes static separation and centrifugal separation.

6. A method for purifying vitamin K2 (MK-7) from Bacillus natto fermentation broth according to claim 5, characterized in that: The separation adopts a three-phase disc separator with a rotation speed of 5000-7000rpm.

7. A method for purifying vitamin K2 (MK-7) from Bacillus natto fermentation broth according to claim 1, characterized in that: In the S12, the amount of saturated salt water is 5-15% of the volume of the organic phase component, and the organic phase is washed 2-3 times; the filtration accuracy is not greater than 220nm; the concentration is concentrated under reduced pressure to 5-20% of the volume of the organic phase component; The terminal temperature of crystallization is 0-5°C, the cooling rate is 1-10°C / h, and the crystal is grown for 0.5-3h after the cooling is completed.

8. The method for purifying vitamin K2 (MK-7) from Bacillus natto fermentation broth according to claim 1, characterized in that: In the S13, the melt crystallization comprises the following steps: S21, heating the crude vitamin K2 to 56-65° C. and completely melting it; S22, cooling to 50-53°C at a rate of 0.2-5°C / h, and then heating to 53-55°C at a temperature gradient of 0.1-5°C, collecting sweating liquid during the process, and keeping each gradient warm until sweating stops before continuing to heat up; S23. Continue to raise the temperature to 56-65°C and completely melt to obtain pure vitamin K2. The purity of pure vitamin K2 is greater than 99.9%.

9. The method for purifying vitamin K2 (MK-7) from Bacillus natto fermentation broth according to any one of claims 1 to 8, characterized in that: The mother liquor produced in the crystallization process in S12 and the sweating liquid produced in S22 are collected and comprehensively utilized; the organic solvent produced in the concentration process in S12 is returned to the step S11 for recycling.

10. A method for purifying vitamin K2 (MK-7) from Bacillus natto fermentation broth according to claim 1, characterized in that: In the above-mentioned S11-S13, each step is performed in a light-proof manner.

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