Preparation method of high-purity MK-7

Abstract

The invention provides a preparation method of high-purity MK-7, which comprises the following steps: (1) putting bacillus subtilis natto dry powder into subcritical extraction equipment, and adding a mixed solvent for subcritical extraction; (2) after the extraction is completed, carrying out separation, and carrying out reduced pressure distillation to obtain a concentrated solution; (3) adding an organic solvent into the concentrated solution, stirring and dissolving, and cooling and crystallizing; (4) carrying out suction filtration separation, recrystallization and vacuum drying on the crystallized substance to obtain high-purity MK-7; in the step (1), the subcritical extraction temperature is 0-30 DEG C, and the extraction pressure is 0.5-3 MPa. The MK-7 prepared by the preparation method of the high-purity MK-7 provided by the invention has high yield and purity, the drying weight loss is obviously reduced, and the MK-7 has no fermentation peculiar smell.

Description

Technical Field

[0001] This invention belongs to the field of material extraction technology, specifically relating to a method for preparing high-purity MK-7. Background Technology

[0002] MK-7, or vitamin K2, is an important fat-soluble vitamin and one of the essential vitamins for the human body. In the human body, MK-7 is mainly synthesized by intestinal bacteria and is also found in some animal meat products and fermented products, such as animal liver, fermented dairy products, and cheese. As a cofactor of γ-glutamyl carboxylase, MK-7 can activate various vitamin K-dependent proteins, playing important physiological functions in promoting bone metabolism and preventing cardiovascular calcification. This has led to the widespread use of MK-7 in health products, pharmaceuticals, and food additives, making its industrial production particularly important.

[0003] Currently, the industrial production of MK-7 mainly relies on the fermentation of Bacillus subtilis natto. Existing methods for preparing MK-7 include ethanol extraction, column chromatography, and solvent extraction. Ethanol extraction uses Bacillus subtilis powder with a content of 300-400 mg / kg as raw material. Although it can extract MK-7, the extracted MK-7 content is low and contains many impurities. It requires more than three crystallizations to reach the basic purity requirements. In some cases, it needs to be combined with column chromatography, which is cumbersome and costly. In addition, the product has a strong fermented odor. Column chromatography uses a silica gel column and hexane-ethyl acetate or hexane-acetone as the eluent to prepare MK-7. However, it has problems such as small batch production, large amount of silica gel waste, large amount of eluent, and low purification efficiency. Column chromatography is only suitable for small-batch preparation. Solvent extraction uses solvents such as hexane or ethanol. However, it has problems such as high extraction temperature, usually requiring reflux, insufficient solvent selectivity, large product loss, high energy consumption for solvent recovery, and difficulty in achieving a product purity of 95%.

[0004] To address the problems existing in the current technology, developing a method for preparing high-purity MK-7 that is suitable for large-scale production, requires less solvent, has a lower extraction temperature, and produces high product yield and purity is of great significance for verifying the feasibility of industrial scale-up and reducing production costs. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing high-purity MK-7, so as to solve the problems of low purity, low yield, high cost, and high extraction temperature of the MK-7 obtained in the above-mentioned background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution: The present invention provides a method for preparing high-purity MK-7, comprising the following steps: (1) The dried powder of Bacillus subtilis natto was put into a subcritical extraction device and a mixed solvent was added for subcritical extraction; (2) After extraction, separation and vacuum distillation are performed to obtain a concentrated solution; (3) Add organic solvent to the concentrated solution, stir to dissolve, and then cool to crystallize; (4) The crystallized substance is separated by filtration, recrystallized and dried under vacuum to obtain high-purity MK-7; In step (1), the subcritical extraction temperature is 0-30℃ and the extraction pressure is 0.5-3MPa.

[0007] As a further improvement, the ratio of the mixed solvent and Bacillus subtilis dry powder in step (1) is 5:1-10:1 (mL / g).

[0008] As a further improvement, the moisture content of Bacillus subtilis natto dry powder is ≤5%, and the particle size is 100-300 mesh.

[0009] As a further improvement, the mixed solvent in step (1) is n-butane and ethanol.

[0010] As a further improvement, the mixed solvent in step (1) is n-butane and ethanol in a volume ratio of 8:2-9.5:0.5.

[0011] To ensure higher purity of the prepared MK-7, the ratio of the mixed solvent to the Bacillus subtilis powder was 7:1 mL / g, and the mixed solvent was n-butane and ethanol with a volume ratio of 9.2:0.8.

[0012] In order to ensure a high yield of MK-7 and relatively high purity, the ratio of the mixed solvent to the Bacillus subtilis powder is 6:1 mL / g. The mixed solvent is n-butane and ethanol with a volume ratio of 8:2. In step (4), when stirring and slurrying with ethanol, the slurrying is performed twice.

[0013] As a further improvement, the crystallization temperature in step (3) is 0-10℃.

[0014] As a further improvement, the vacuum drying temperature in step (4) is 20-40℃, and the drying weight loss is ≤0.5%.

[0015] As a further improvement, the recrystallization in step (4) is performed 1-2 times.

[0016] As a further improvement, the organic solvent in step (3) is an alcohol-based organic solvent.

[0017] As a further improvement, the extraction process in step (1) includes a stirring step with a stirring rate of 50-150 r / min.

[0018] This invention employs subcritical extraction, which is simple and controllable to operate, providing precise data support for industrial-scale production. At the same time, the extraction temperature is controlled at 0-30℃, which is much lower than the 40℃ or above of traditional solvent extraction, effectively reducing the oxidation and decomposition of MK-7. The yield is increased from ≤70% of existing methods to ≥80%, with minimal extraction loss. n-Butane has high selectivity for lipid-soluble MK-7, and cannot dissolve impurities such as inorganic matter and bacterial fragments. The initial purity of the extract is over 90%, and the purity is ≥96% after 1-2 recrystallizations. The product has no fermentation odor and is a uniform and loose powder. It solves the defects of stickiness and odor in products extracted by direct ethanol extraction. The product has high purity and excellent quality. After the raw materials are dried and concentrated, combined with the high solubility of subcritical extraction, the amount of solvent used is less than 1 / 10 of that used when directly extracted from fermentation liquid, which reduces solvent consumption and environmental pressure, and significantly reduces solvent usage. No multiple crystallizations or column chromatography are required; high purity can be achieved with just 1-2 recrystallizations, simplifying experimental steps, improving experimental efficiency, and making the purification process simple.

[0019] Compared with the prior art, the beneficial effects of the present invention are: The high-purity MK-7 preparation method provided by this invention produces MK-7 with high yield and purity, low drying loss, achieving a yield of over 72.9%, a purity of over 97.5%, and a drying loss of less than 0.2%, and without any fermentation odor. Detailed Implementation

[0020] The present invention will be described below with reference to specific embodiments. It should be noted that the following embodiments are examples of the present invention and are used only to illustrate the invention, not to limit it. Other combinations and various modifications within the scope of the present invention can be made without departing from its spirit or scope.

[0021] The Bacillus subtilis dry powder in Examples 1-10 and Comparative Examples 1-3 of this invention has a moisture content of 4% and a particle size of 100-200 mesh.

[0022] Example 1: (1) 10g of dried Bacillus subtilis powder was put into a 100mL subcritical extraction device and 60mL of mixed solvent (the mixed solvent was n-butane and ethanol with a volume ratio of 9:1) was added. The subcritical extraction was carried out at a temperature of 20℃ and a pressure of 1MPa for 1h. During the extraction process, the stirring speed was 100r / min. (2) After extraction is completed, separation is performed. After separating the residue and extract, the extract is subjected to vacuum distillation at 30°C to recover the n-butane in the extract, and a concentrated solution is obtained. (3) Add 5 mL of ethanol to the concentrate, stir to dissolve, cool to 3 °C, and crystallize at a constant temperature for 12 h; (4) The crystallized material is separated by filtration, the crystallized product is collected, and then the crystallized product is stirred and slurried twice with 5 mL of ethanol and dried under vacuum at 20°C for 12 h to obtain a bright yellow loose powder, namely high-purity MK-7. The purity of the high-purity MK-7 in Example 1 was found to be 98.8%, the loss on drying was 0.1%, and the yield was 82.0%.

[0023] Example 2: (1) 20g of dried Bacillus subtilis powder was put into a 100mL subcritical extraction device and 80mL of mixed solvent (the mixed solvent was n-butane and ethanol with a volume ratio of 8.5:1.5) was added. The subcritical extraction was carried out at a temperature of 15℃ and a pressure of 1.5MPa for 2h. During the extraction process, the stirring speed was 80r / min. (2) After extraction is completed, separation is performed. After separating the residue and extract, the extract is subjected to vacuum distillation at 30°C to recover the n-butane in the extract, and a concentrated solution is obtained. (3) Add 8 mL of ethanol to the concentrate, stir to dissolve, cool to 3 °C, and crystallize at a constant temperature for 16 h; (4) The crystallized material is separated by filtration, the crystallized product is collected, and then the crystallized product is stirred and slurried once with 10 mL of ethanol and dried under vacuum at 35°C for 16 h to obtain a bright yellow loose powder, namely high-purity MK-7. The purity of the high-purity MK-7 in Example 2 was found to be 97.5%, the loss on drying was 0.2%, and the yield was 88.3%.

[0024] Example 3: (1) 15g of dried Bacillus subtilis powder was put into a 100mL subcritical extraction device and 75mL of mixed solvent (the mixed solvent was n-butane and ethanol with a volume ratio of 9.5:0.5) was added. The subcritical extraction was carried out for 1.5h at a temperature of 25℃ and a pressure of 0.8MPa. During the extraction process, the stirring speed was 120r / min. (2) After extraction is completed, separation is performed. After separating the residue and extract, the extract is subjected to vacuum distillation at 30°C to recover the n-butane in the extract, and a concentrated solution is obtained. (3) Add 8 mL of ethanol to the concentrate, stir to dissolve, cool to 3°C, stir to dissolve, cool to 0°C, and then crystallize at a constant temperature for 10 h. (4) The crystallized material is separated by filtration, the crystallized product is collected, and then the crystallized product is stirred and slurried twice with 5 mL of ethanol and dried under vacuum at 25°C for 15 h to obtain a bright yellow loose powder, namely high-purity MK-7. The purity of the high-purity MK-7 in Example 3 was found to be 98.2%, the drying loss was 0.1%, and the yield was 77.7%.

[0025] Example 4: (1) 10g of dried Bacillus subtilis powder was put into a 100mL subcritical extraction device and 60mL of mixed solvent (the mixed solvent is n-butane and ethanol with a volume ratio of 8:2) was added. The subcritical extraction was carried out at a temperature of 30℃ and a pressure of 2MPa for 3h. The extracted material was then further extracted at a temperature of 30℃ and a pressure of 2MPa for 3h. During the extraction process, the stirring speed was 150r / min. (2) After extraction is completed, separation is performed. After separating the residue and extract, the extract is subjected to vacuum distillation at 30°C to recover the n-butane in the extract, and a concentrated solution is obtained. (3) Add 5 mL of ethanol to the concentrate, stir to dissolve, cool to 5 °C, and crystallize at a constant temperature for 18 h; (4) The crystallized material is separated by filtration, the crystallized product is collected, and then the crystallized product is stirred and slurried twice with 5 mL of ethanol and dried under vacuum at 25°C for 16 h to obtain a bright yellow loose powder, namely high-purity MK-7. The purity of the high-purity MK-7 in Example 4 was 98.2%, the drying loss was 0.2%, and the yield was 90.7%.

[0026] Example 5: (1) 10g of dried Bacillus subtilis powder was put into a 100mL subcritical extraction device and 70mL of mixed solvent (the mixed solvent was n-butane and ethanol with a volume ratio of 9.2:0.8) was added. The subcritical extraction was carried out at a temperature of 10℃ and a pressure of 0.5MPa for 1h. During the extraction process, the stirring speed was 50r / min. (2) After extraction is completed, separation is performed. After separating the residue and extract, the extract is subjected to vacuum distillation at 30°C to recover the n-butane in the extract, and a concentrated solution is obtained. (3) Add 2 mL of ethanol to the concentrate, stir to dissolve, cool to 0 °C, and crystallize at a constant temperature for 18 h; (4) The crystallized material is separated by filtration, the crystallized product is collected, and then the crystallized product is stirred and slurried once with 5 mL of ethanol and dried under vacuum at 30°C for 16 h to obtain a bright yellow loose powder, namely high-purity MK-7. The purity of the high-purity MK-7 in Example 5 was found to be 99.4%, the loss on drying was 0.1%, and the yield was 82.1%.

[0027] Example 6: (1) 10g of dried Bacillus subtilis powder was put into a 100mL subcritical extraction device and 60mL of mixed solvent (the mixed solvent is n-butane and ethanol with a volume ratio of 9:1) was added. The subcritical extraction was carried out for 1h at a temperature of 5℃ and a pressure of 1MPa. During the extraction process, the stirring speed was 100r / min. (2) After extraction is completed, separation is performed. After separating the residue and extract, the extract is subjected to vacuum distillation at 30°C to recover the n-butane in the extract, and a concentrated solution is obtained. (3) Add 5 mL of ethanol to the concentrate, stir to dissolve, cool to 3 °C, and crystallize at a constant temperature for 12 h; (4) The crystallized material is separated by filtration, the crystallized product is collected, and then the crystallized product is stirred and slurried twice with 5 mL of ethanol and dried under vacuum at 20°C for 12 h to obtain a bright yellow loose powder, namely high-purity MK-7. The purity of the high-purity MK-7 in Example 6 was found to be 98.8%, the loss on drying was 0.1%, and the yield was 80.6%.

[0028] Example 7: (1) 20g of dried Bacillus subtilis powder was put into a 100mL subcritical extraction device and 80mL of mixed solvent (the mixed solvent was n-butane and ethanol with a volume ratio of 8.8:1.2) was added. The subcritical extraction was carried out at a temperature of 15℃ and a pressure of 1.5MPa for 2h. During the extraction process, the stirring speed was 80r / min. (2) After extraction is completed, separation is performed. After separating the residue and extract, the extract is subjected to vacuum distillation at 30°C to recover the n-butane in the extract, and a concentrated solution is obtained. (3) Add 8 mL of ethanol to the concentrate, stir to dissolve, cool to 3 °C, and crystallize at a constant temperature for 16 h; (4) The crystallized material is separated by filtration, the crystallized product is collected, and then the crystallized product is stirred and slurried once with 10 mL of ethanol and dried under vacuum at 35°C for 16 h to obtain a bright yellow loose powder, namely high-purity MK-7. The purity of the high-purity MK-7 in Example 7 was found to be 98.5%, the loss on drying was 0.2%, and the yield was 84.0%.

[0029] Example 8: (1) 10g of dried Bacillus subtilis powder was put into a 100mL subcritical extraction device and 60mL of mixed solvent (the mixed solvent is n-butane and ethanol with a volume ratio of 8:2) was added. The subcritical extraction was carried out at a temperature of 30℃ and a pressure of 2MPa for 3h. The extracted material was then further extracted at a temperature of 30℃ and a pressure of 2MPa for 3h. During the extraction process, the stirring speed was 150r / min. (2) After extraction is completed, separation is performed. After separating the residue and extract, the extract is subjected to vacuum distillation at 30°C to recover the n-butane in the extract, and a concentrated solution is obtained. (3) Add 5 mL of ethanol to the concentrate, stir to dissolve, cool to 5 °C, and then crystallize at a constant temperature for 18 h; (4) The crystallized material is separated by filtration, the crystallized product is collected, and then the crystallized product is stirred and slurried with 5 mL of ethanol three times and vacuum dried at 25°C for 16 h to obtain a bright yellow loose powder, namely high-purity MK-7. The purity of the high-purity MK-7 in Example 8 was found to be 99.1%, the loss on drying was 0.2%, and the yield was 78.4%.

[0030] Example 9: (1) 15g of dried Bacillus subtilis powder was put into a 100mL subcritical extraction device and 75mL of mixed solvent (the mixed solvent was n-butane and ethanol with a volume ratio of 9.5:0.5) was added. The subcritical extraction was carried out for 1.5h at a temperature of 25℃ and a pressure of 3MPa. During the extraction process, the stirring speed was 120r / min. (2) After extraction is completed, separation is performed. After separating the residue and extract, the extract is subjected to vacuum distillation at 30°C to recover the n-butane in the extract, and a concentrated solution is obtained. (3) Add 8 mL of ethanol to the concentrate, stir to dissolve, cool to 3 °C, and then crystallize at a constant temperature for 10 h; (4) The crystallized material is separated by filtration, the crystallized product is collected, and then the crystallized product is stirred and slurried twice with 5 mL of ethanol and dried under vacuum at 25°C for 15 h to obtain a bright yellow loose powder, namely high-purity MK-7. The purity of the high-purity MK-7 in Example 9 was found to be 97.9%, the loss on drying was 0.1%, and the yield was 81.4%.

[0031] Example 10: (1) 10g of dried Bacillus subtilis powder was put into a 100mL subcritical extraction device and 70mL of mixed solvent (the mixed solvent was n-butane and ethanol with a volume ratio of 9.2:0.8) was added. The subcritical extraction was carried out at a temperature of 10℃ and a pressure of 0.5MPa for 1h. During the extraction process, the stirring speed was 50r / min. (2) After extraction is completed, separation is performed. After separating the residue and extract, the extract is subjected to vacuum distillation at 30°C to recover the n-butane in the extract, and a concentrated solution is obtained. (3) Add 2 mL of ethanol to the concentrate, stir to dissolve, cool to 0°C after stirring to dissolve, and then crystallize at a constant temperature for 10 h. (4) The crystallized material is separated by filtration, the crystallized product is collected, and then the crystallized product is stirred and slurried once with 5 mL of ethanol and dried under vacuum at 30°C for 16 h to obtain a bright yellow loose powder, namely high-purity MK-7. Testing showed that the purity of the high-purity MK-7 in Example 10 was 99.5%, the loss on drying was 0.1%, and the yield was 72.9%.

[0032] The high-purity MK-7 prepared in Examples 1 to 10 had no fermentation odor.

[0033] Comparative Example 1 (using ethanol extraction method): Take 10g of dried Bacillus subtilis powder, add 500mL of ethanol, stir and extract at 85℃ for 3h, filter while hot, recover the ethanol by vacuum distillation at 50℃, then concentrate the liquid after ethanol recovery to 5mL, cool the concentrate to 5℃ for 24h for crystallization, collect the product by suction filtration, the purity is 68.2%; The product collected by vacuum filtration was crystallized a second time with 50 mL of ethanol, and the purity of the product after filtration was 77.5%. The product was crystallized a third time with 50 mL of ethanol, and the purity of the product after filtration was 85.3%. The product was then crystallized a fourth time, and the purity of the product after the fourth crystallization was 88.3%, the loss on drying was 0.7%, and the yield was 48.9%.

[0034] The MK-7 prepared in Comparative Example 1 had a strong fermented odor, was sticky, and the concentrate also had a strong fermented odor.

[0035] Comparative Example 2 (using ethanol extraction and column chromatography purification): Take 10g of dried Bacillus subtilis powder, add 500mL of ethanol, stir and extract at 85℃ for 3h, filter while hot, recover the ethanol by vacuum distillation at 50℃, then concentrate the liquid after ethanol recovery to 5mL, cool the concentrate to 5℃ for 24h for crystallization, collect the product by suction filtration, the purity is 68.2%; The crystalline product was dissolved in 20 mL of n-hexane and loaded onto a silica gel column. The column was eluted with n-hexane and ethyl acetate in a volume ratio of 20:1, and the target fraction was collected. The target fraction was concentrated under reduced pressure, and 15 mL of ethanol was added for crystallization. After filtration, the product was dried at 40 °C to obtain a bright yellow powder, namely MK-7. The purity of MK-7 was 97.1%, the loss on drying was 0.2%, and the yield was 44.6%.

[0036] The MK-7 prepared in Comparative Example 2 had a slightly fermented odor, a sticky texture, and the concentrate had a strong fermented odor.

[0037] Comparative Example 3 (using hexane extraction and column chromatography): Take 10g of dried Bacillus subtilis powder (Natto), add 500mL of n-hexane, and extract by heating and reflux stirring in an oil bath (set to 100℃) for 2.5h; after filtration, concentrate the mother liquor to 2mL, load it onto a silica gel column (15mm×300mm), and elute with n-hexane and acetone at a volume ratio of 25:1, and collect the target fraction; after concentration, add 5mL of ethanol to crystallize, filter and dry to obtain a bright yellow powder product, namely MK-7, with a purity of 98.8%, a drying loss of 0.1%, and a yield of 29.6%.

[0038] The MK-7 prepared in Comparative Example 3 had a slightly noticeable off-odor.

[0039] The above embodiments are only for illustrating the technical concept and features of the present invention. Their purpose is to enable those skilled in the art to understand the content of the present invention and implement it. They should not be used to limit the scope of protection of the present invention. All technical solutions obtained by adopting equivalent substitutions or equivalent transformations should be covered within the scope of protection of the present invention.

Claims

1. A method for preparing high-purity MK-7, characterized in that: Includes the following steps: (1) The dried powder of Bacillus subtilis natto was put into a subcritical extraction device and a mixed solvent was added for subcritical extraction; (2) After extraction, separation and vacuum distillation are performed to obtain a concentrated solution; (3) Add organic solvent to the concentrated solution, stir to dissolve, and then cool to crystallize; (4) The crystallized substance is separated by filtration, recrystallized and dried under vacuum to obtain high-purity MK-7; In step (1), the subcritical extraction temperature is 0-30℃ and the extraction pressure is 0.5-3MPa.

2. The method for preparing high-purity MK-7 according to claim 1, characterized in that: The ratio of the mixed solvent and Bacillus subtilis dry powder in step (1) is 5:1-10:1 (mL / g).

3. The method for preparing high-purity MK-7 according to claim 1, characterized in that: The Bacillus subtilis powder has a moisture content of ≤5% and a particle size of 100-300 mesh.

4. The method for preparing high-purity MK-7 according to claim 1, characterized in that: The mixed solvent in step (1) is n-butane and ethanol.

5. The method for preparing high-purity MK-7 according to claim 4, characterized in that: The mixed solvent in step (1) is n-butane and ethanol in a volume ratio of 8:2-9.5:0.

5.

6. The method for preparing high-purity MK-7 according to claim 2, characterized in that: The crystallization temperature in step (3) is 0-10℃.

7. The method for preparing high-purity MK-7 according to claim 1, characterized in that: The vacuum drying temperature in step (4) is 20-40℃, and the drying weight loss is ≤0.5%.

8. The method for preparing high-purity MK-7 according to claim 1, characterized in that: The recrystallization process in step (4) is performed 1-2 times.

9. The method for preparing high-purity MK-7 according to claim 1, characterized in that: The organic solvent in step (3) is an alcohol-based organic solvent.

10. The method for preparing high-purity MK-7 according to claim 1, characterized in that: The extraction process in step (1) includes a stirring step, with a stirring rate of 50-150 r / min.