Method for recovering MK-7 in crystallization mother liquor

Abstract

The invention discloses a method for recovering MK-7 in crystallization mother liquor, which comprises the following steps of: performing vacuum concentration on the crystallization mother liquor to remove a residual solvent, mixing and stirring the crystallization mother liquor and a solvent I until the crystallization mother liquor and the solvent I are completely dissolved, mixing a buffer solution and a solvent II, mixing and stirring the two mixed solutions to react, centrifuging, taking an upper organic phase, and refining through a reverse phase silica gel column to obtain the MK-7 in the crystallization mother liquor. Then adding the vitamin K2 crude product into a methanol solution to be heated and dissolved, slowly adding water until MK-7 is separated out, cooling to enable MK-7 to be continuously separated out, separating MK-7 crystals after separation is finished, and drying. According to the scheme, most impurities such as phospholipid impurities and pigments in the crystallization mother liquor are separated out by adopting the three-phase mixed liquor, a purified crude product of the vitamin K2 is obtained, then relatively pure MK-7 crystals are obtained through recrystallization by utilizing the solubility difference of MK-6 and NK-7 in methanol solutions with different concentrations and at different temperatures, the process is simple to operate, the production cost is reduced, and the method is suitable for industrial production. The detected purity of the separated and recovered MK-7 is 85%-90%, and the purity after recrystallization can reach 95%.

Description

Technical Field

[0001] The invention relates to the technical field of material extraction and recovery, and more particularly to a method for recovering MK-7 from a crystallization mother liquor. Background Art

[0002] Vitamin K2, also known as menaquinone, is abbreviated as MK-n, where M stands for menaquinone, K stands for vitamin K, and n stands for the number of isoprene side chain residues. MK-7 in vitamin K2 comes from synthesis and fermentation. The best source of fermented MK-7 is natto. MK-7 has high bioavailability, long half-life, and anti-osteoporosis activity. MK-7 can promote bone formation and inhibit bone resorption by stimulating osteoblast differentiation, regulating extracellular matrix mineralization of bone cells, upregulating bone marker gene expression, and inhibiting osteoclastogenesis, and has the effect of preventing and treating osteoporosis. A large number of research results have shown that the biological activity of MK-7 is better than other types of vitamin K2, which is caused by differences in enzyme affinity and different tissue distribution. MK-7 has better bioavailability and longer plasma half-life.

[0003] At present, the production method of vitamin K2 is mainly based on microbial fermentation. Patent CN116536220A provides a method for preparing Bacillus subtilis natto that can improve the fermentation level and fermentation yield of vitamin K2. Patent CN116283531 A discloses a vitamin K2 extraction process, which is to load the bacterial powder into a chromatography column and use an organic solvent for extraction. CN109824496 discloses a vitamin K2 extraction method, wherein the fermentation liquid is filtered and concentrated through a ceramic membrane, the bacterial cell is bombarded with a nitrogen ion beam to break the cell wall, the cell wall after the cell wall is broken, and then the supernatant is collected by centrifugation to obtain a vitamin K2 extract. In patent CN106631748, the extraction of vitamin K2 is to first obtain wet bacterial cells by centrifugal solid-liquid separation, the wet bacterial cells are broken by repeated freezing and thawing, and n-hexane is added for extraction. The obtained extract is purified and separated by a resin column and a silica gel column, and finally crystallized to obtain a pure K2 product. A small amount of other types of vitamin K2, such as MK-4, MK-6, etc., will be produced during the microbial fermentation process. The current purification process removes most of the other impurities and improves the purity of MK-7. However, during the extraction process, some fat-soluble impurities and MK-6 will enter the crystallization mother liquor along with the uncrystallized MK-7 and continue to be enriched in the mother liquor. Due to the excessive amount of impurities, it is difficult to further recover MK-7 in the mother liquor using traditional separation processes. This part of the crystallization mother liquor is generally treated as waste liquid, which not only increases the cost of environmental governance, but also wastes resources. Since MK-7 is relatively expensive, the recovery of MK-7 in the crystallization mother liquor still has great economic value. Summary of the invention

[0004] In view of this, the present invention provides a method for recovering MK-7 from a crystallization mother liquor. The method can solve the problem that MK-7 is difficult to recover from the crystallization mother liquor. The process is simple to operate and reduces production costs.

[0005] To achieve the above object, the present invention adopts the following technical solution:

[0006] A method for recovering MK-7 from a crystallization mother liquor comprises the following steps:

[0007] (1) vacuum concentrating the crystallization mother liquor to remove residual solvent;

[0008] (2) mixing the concentrated crystallization mother liquor with solvent 1 and stirring until completely dissolved to obtain solution A;

[0009] (3) mixing the buffer solution and solvent 2 to obtain solution B;

[0010] (4) Mix solution A and solution B, stir at 30-40°C for 1-2 hours, and then centrifuge to collect the upper clear organic phase;

[0011] (5) refining the organic phase collected in step (4) through a reverse phase silica gel column to obtain a crude vitamin K2 product;

[0012] (6) Add crude vitamin K2 to methanol, heat to 60°C to dissolve, slowly add water to continuously precipitate MK-7 from the solution until the final volume concentration of methanol is 85%, cool to 0-20°C, MK-7 continues to precipitate, and after precipitation is complete, separate the MK-7 crystals and dry them.

[0013] Preferably, the vacuum concentration temperature in step (1) is 65° C. and the vacuum degree is -0.09 MPa.

[0014] Preferably, the solvent 1 described in step (2) is one of n-hexane, ethyl acetate and butyl acetate.

[0015] Preferably, the amount of solvent added in step (2) is 4-6 times the weight of the crystallization mother liquor.

[0016] Preferably, the buffer in step (3) is a sodium carbonate-sodium bicarbonate system or a disodium hydrogen phosphate-sodium hydroxide system; and the second solvent is one of ethanol, methanol, and isopropanol.

[0017] Furthermore, the molar ratio of sodium carbonate to sodium bicarbonate in the sodium carbonate-sodium bicarbonate system is 1:1, and the molar ratio of disodium hydrogen phosphate to sodium hydroxide in the disodium hydrogen phosphate-sodium hydroxide system is 1:1.05.

[0018] Preferably, the weight of the buffer solution in step (3) is 1-2 times the weight of the crystallization mother liquor, and the weight of the second solvent is 6-8 times the weight of the crystallization mother liquor.

[0019] Preferably, the centrifugal speed in step (4) is 6000 rpm and the time is 10 min.

[0020] Preferably, the reverse phase silica gel column chromatography column in step (5) has a height-to-diameter ratio of 10:1, the column is eluted with ethyl acetate, and the fractions are collected, and the crude vitamin K2 is obtained after the solvent is evaporated.

[0021] Preferably, the weight of methanol in step (6) is 15-20 times that of the crude vitamin K2, and the drying is vacuum drying at a temperature of 45°C.

[0022] It can be seen from the above technical solution that, compared with the prior art, the present invention discloses a method for recovering MK-7 from a crystallization mother liquor, which has the following beneficial effects:

[0023] The scheme of the present invention adopts a three-phase mixed liquid to separate most of the impurities such as phospholipid impurities and pigments in the crystallization mother liquor to obtain a purified crude vitamin K2, and then utilizes the difference in solubility of MK-6 and NK-7 in methanol solutions of different concentrations and at different temperatures to obtain relatively pure MK-7 crystals through recrystallization, thereby solving the problem that MK-7 in the crystallization mother liquor is difficult to recover. The process is simple to operate and reduces production costs. The separated and recovered MK-7 has a detection purity of 85%-90%, and the purity can reach 95% after recrystallization. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0025] Figure 1 The solubility of MK-6 and MK-7 at different methanol ratios at 20°C;

[0026] Figure 2 is the solubility of MK-6 and MK-7 in 85% methanol at different temperatures;

[0027] Figure 3 is the solubility of MK-6 and MK-7 in 100% methanol at different temperatures;

[0028] Figure 4 The liquid phase spectrum of MK-6 and MK-7 in the crystallization mother liquor;

[0029] Figure 5 This is the crystal liquid phase spectrum after crystallization;

[0030] Figure 6 This is the liquid phase spectrum of the crystal after recrystallization. DETAILED DESCRIPTION

[0031] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] Example 1

[0033] (1) The crystallization mother liquor was concentrated under vacuum at 65°C and a vacuum degree of -0.09 MPa to remove the residual solvent;

[0034] (2) fully mixing the concentrated crystallization mother liquor with n-hexane in an amount of 4 to 6 times the weight of the crystallization mother liquor, stirring until completely dissolved, to obtain solution A;

[0035] (3) mixing a sodium carbonate-sodium bicarbonate system buffer solution (the molar ratio of sodium carbonate to sodium bicarbonate is 1:1) in an amount of 1-2 times the weight of the crystallization mother liquor with ethanol in an amount of 6-8 times the weight of the crystallization mother liquor to obtain a solution B;

[0036] (4) Solution A and Solution B were mixed, stirred and reacted at 30-40° C. for 1-2 h to obtain a reaction solution C. The reaction solution C was centrifuged at 6000 rpm for 10 min. After centrifugation, there were three layers, the top layer was a clear organic phase, obtaining solution D, the second layer was a dark-colored aqueous phase, and the bottom layer was a light-colored aqueous phase;

[0037] (5) Solution D was purified by reverse phase silica gel column with a height-to-diameter ratio of 10:1. The column was eluted with ethyl acetate and fractions were collected. The solvent was evaporated to obtain crude vitamin K2.

[0038] (6) Add crude vitamin K2 to 15-20 times its weight of methanol, heat at 60°C to dissolve, slowly add water, MK-7 continues to precipitate from the solution, add water to a final methanol volume concentration of 85%, cool to 0-20°C, MK-7 continues to precipitate,

[0039] After separating the MK-7 crystals, vacuum drying was performed at 45°C, and the purity was detected to be 85%-90%. The purity could reach 95% after recrystallization.

[0040] Experimental example

[0041] 1. Pre-set the constant temperature water bath temperature to 20°C, add 70%, 75%, 80%, 85%, 90%, 95%, 100% methanol solution to the constant temperature jacket reactor, then add sufficient MK-7 or MK-6, adjust the stirrer speed, stir vigorously for 2-3 hours, ensure that the system reaches solid-liquid equilibrium, stop stirring, keep the temperature constant, wait for the solid particles to settle completely, take the supernatant, and measure the concentration of MK-7 and MK-6. The solubility curves of MK-6 and MK-7 at different methanol ratios at 20°C are obtained, such as Figure 1 As shown, Figure 1 The results show that when the methanol concentration is 85%, the solubility difference between MK-6 and MK-7 is the largest.

[0042] 2. Pre-set the constant temperature water bath temperature to 60℃, add 85% methanol solution to the constant temperature jacket reactor, then add sufficient MK-7 or MK-6, adjust the stirrer speed, stir vigorously for 2-3h, ensure that the system reaches solid-liquid equilibrium, stop stirring, keep it at constant temperature, wait for the upper solution to clarify, take the supernatant, and measure the concentration of MK-7 and MK-6. Measure once every 10℃ between 0 and 60℃, and obtain the solubility curves of MK-6 and MK-7 in 85% methanol solution at different temperatures, such as Figure 2 As shown, Figure 2 It is shown that in the range of 0-20°C, the solubility of MK-6 and MK-7 in 85% methanol has a greater difference.

[0043] 3. Pre-set the constant temperature water bath temperature to 60℃, add 100% methanol solution to the constant temperature jacket reactor, then add sufficient MK-7 or MK-6, adjust the stirrer speed, stir vigorously for 2-3h, ensure that the system reaches solid-liquid equilibrium, stop stirring, keep it at constant temperature, wait for the upper solution to clarify, take the supernatant, and measure the concentration of MK-7 and MK-6. Measure once every 10℃ between 0 and 60℃ to obtain the solubility curves of MK-6 and MK-7 at different temperatures in 100% methanol solution, such as Figure 3 As shown, Figure 3 The results show that in 100% methanol solution, the solubility of MK-6 and MK-7 is roughly the same and difficult to separate.

[0044] In this specification, each embodiment is described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the embodiments can be referred to each other. For the device disclosed in the embodiment, since it corresponds to the method disclosed in the embodiment, the description is relatively simple, and the relevant parts can be referred to the method part.

[0045] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for recovering MK-7 from a crystallization mother liquor, characterized in that: The following steps are involved: (1) vacuum concentrating the crystallization mother liquor to remove residual solvent; (2) mixing the concentrated crystallization mother liquor with solvent 1 and stirring until completely dissolved to obtain solution A; (3) mixing the buffer solution and solvent 2 to obtain solution B; (4) Mix solution A and solution B, stir at 30-40°C for 1-2 hours, and then centrifuge to collect the upper clear organic phase; (5) refining the organic phase collected in step (4) through a reverse phase silica gel column to obtain a crude vitamin K2 product; (6) Add crude vitamin K2 to methanol, heat to 60°C to dissolve, slowly add water to continuously precipitate MK-7 from the solution until the final volume concentration of methanol is 85%, cool to 0-20°C, MK-7 continues to precipitate, and after precipitation is complete, separate the MK-7 crystals and dry them.

2. The method for recovering MK-7 from a crystallization mother liquor according to claim 1, characterized in that: The vacuum concentration temperature in step (1) is 65° C. and the vacuum degree is -0.09 MPa.

3. The method for recovering MK-7 from a crystallization mother liquor according to claim 1, characterized in that: The solvent 1 described in step (2) is one of n-hexane, ethyl acetate and butyl acetate.

4. The method for recovering MK-7 from a crystallization mother solution according to claim 1, characterized in that: The amount of solvent 1 added in step (2) is 4-6 times the weight of the crystallization mother liquor.

5. The method for recovering MK-7 from a crystallization mother liquor according to claim 1, characterized in that: The buffer solution in step (3) is a sodium carbonate-sodium bicarbonate system or a disodium hydrogen phosphate-sodium hydroxide system; and the second solvent is one of ethanol, methanol, and isopropanol.

6. The method for recovering MK-7 from a crystallization mother liquor according to claim 5, characterized in that: The molar ratio of sodium carbonate to sodium bicarbonate in the sodium carbonate-sodium bicarbonate system is 1:1, and the molar ratio of disodium hydrogen phosphate to sodium hydroxide in the disodium hydrogen phosphate-sodium hydroxide system is 1:1.

05.

7. The method for recovering MK-7 from a crystallization mother solution according to claim 1, characterized in that: The weight of the buffer solution in step (3) is 1-2 times the weight of the crystallization mother liquor, and the weight of the second solvent is 6-8 times the weight of the crystallization mother liquor.

8. The method for recovering MK-7 from a crystallization mother liquor according to claim 1, characterized in that: The centrifugal speed in step (4) is 6000 rpm and the time is 10 min.

9. The method for recovering MK-7 from a crystallization mother solution according to claim 1, characterized in that: The reverse phase silica gel column chromatography column described in step (5) has a height-to-diameter ratio of 10:

1. The column is eluted with ethyl acetate and the fractions are collected. The crude vitamin K2 is obtained after the solvent is evaporated.

10. The method for recovering MK-7 from a crystallization mother solution according to claim 1, characterized in that: In step (6), the weight of methanol is 15-20 times that of the crude vitamin K2, and the drying is vacuum drying at a temperature of 45°C.

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