1. The preparation method of vitamin K2 is characterized by comprising the following steps:

(1) adding a composite flocculant into a fermentation liquid of bacillus natto, then carrying out solid-liquid separation, and collecting liquid as a liquid to be extracted for later use;

(2) concentrating the extract to be extracted to obtain concentrated solution for later use;

(3) freeze-drying the concentrated solution to obtain freeze-dried powder containing a vitamin K2 crude product for later use;

(4) dissolving the freeze-dried powder in acetone, adding water as a precipitator after the freeze-dried powder is completely dissolved, precipitating vitamin K2, separating out the precipitate, removing acetone in the residual liquid, and recovering, wherein the residual liquid is mother liquor for later use;

(5) and (4) merging the mother liquor with the concentrated solution prepared in the next step (3) to realize the recycling of the mother liquor.

2. The method for preparing vitamin K2 according to claim 1, wherein in step (1), the culture medium of Bacillus natto mainly comprises the following components: glucose 2g/L, peptone 20g/L, ammonium sulfate 0.5%, K₂HPO₄ 2g/L、KH₂PO₄ 1g/、CaCl₂ 0.1g/L、MgSO₄ 1g/L。

3. The method for preparing vitamin K2 according to claim 2, wherein the fermentation conditions of Bacillus natto in step (1) are: the temperature is 37 ℃, the initial pH value is 7.5-8.0, and the inoculation amount is 5-7.5%.

4. The method for preparing vitamin K2 according to claim 1, wherein in step (1), the composite flocculant is one of polyaluminium chloride, polyferric chloride and alum, and 2Na₂CO₃·3H₂O₂Or 2K₂CO₃·3H₂O₂The resulting mixture;

preferably, in the step (1), the concentration of the composite flocculant in the fermentation liquid is 45-60 mg/L.

5. The preparation method of vitamin K2, according to claim 4, wherein the mass ratio of any one of polyaluminium chloride, polyferric chloride and alum to sodium peroxycarbonate or potassium peroxycarbonate is 2.5-8: 1.

6. the method for preparing vitamin K2 according to claim 1, wherein in step (2), the extract is concentrated under reduced pressure to 45-60% of the original volume at a concentration temperature of 55-75 ℃.

7. The process for preparing vitamin K2, according to claim 1, wherein in step (3), the lyophilization process is: and rapidly freezing the concentrated solution to the temperature of minus 25-35 ℃ in a freeze dryer, then vacuumizing to the temperature of 0.1-0.15 mbra, and sublimating water in the concentrated solution to obtain the freeze-dried powder containing the vitamin K2 crude product.

8. The preparation method of vitamin K2, according to claim 1, wherein in step (4), the mass-to-volume ratio of the lyophilized powder to acetone is 1 g: 6-10 ml.

9. The method for preparing vitamin K2, according to claim 1, wherein in step (4), the precipitating agent comprises distilled water or purified water, preferably, the volume of the precipitating agent added is 2.5-4 times that of acetone.

10. The process for the preparation of vitamin K2 according to any one of claims 1 to 9, wherein in step (4), the recovery of acetone from the remaining liquid is achieved by distillation, preferably at a temperature above the boiling point of acetone and below the boiling point of water, more preferably at a temperature of 60 to 65 ℃.

Background

Vitamin K2(vitamin K2, VK2) is a generic name for a series of terpene side-chain compounds containing a 2-methyl-1, 4-naphthoquinone core and a C3 site with a variable number of isoprene structural units. Vitamin K2 is mostly present in fermented foods such as natto, which is a bean product fermented from soybeans and is rich in various nutrient elements, especially vitamin K2. Vitamin K2 has the effect of facilitating carboxylation of primary osteocalcin secreted by osteoblasts to become active osteocalcin, thereby promoting the deposition of calcium ions in the blood into the bone. Vitamin K2 can produce bone protein, and can be used together with calcium to produce bone substance, to increase bone density and prevent fracture. In addition, vitamin K2 also has the effect of preventing liver cirrhosis from progressing to liver cancer.

With the continuous development of technology, many methods for extracting vitamin K2 from fermentation broth of microorganisms are available, for example, a method for obtaining vitamin K2 by centrifuging the fermentation broth, collecting supernatant, and performing liquid chromatography. For example, after removing solids such as cells from the fermentation broth, vitamin K2 is purified and extracted by using macroporous resin, reverse phase silica gel, or the like. However, these methods have undesirable extraction effects, such as low extraction rate and low purity, or complicated process, high cost, and low production efficiency. In addition, the treatment of the mother liquor remaining after extraction also becomes an important problem in the production of vitamin K2.

Disclosure of Invention

Aiming at the problems, the invention provides the preparation method of the vitamin K2, which not only can effectively improve the purification purity of the vitamin K2, but also can realize the effective utilization of the mother liquor after the vitamin K2 is extracted. In order to achieve the purpose, the invention discloses the following technical scheme:

a preparation method of vitamin K2 comprises the following steps:

(1) adding a composite flocculant into the fermentation liquor of the bacillus natto, then carrying out solid-liquid separation, and collecting the liquid as the liquid to be extracted for later use.

(2) And concentrating the extract to be extracted to obtain concentrated solution for later use.

(3) And freeze-drying the concentrated solution to obtain freeze-dried powder containing a vitamin K2 crude product for later use.

(4) Dissolving the freeze-dried powder in acetone, adding water as a precipitator after the freeze-dried powder is completely dissolved to precipitate vitamin K2, separating out the precipitate, removing the acetone in the residual liquid, and recovering the residual liquid which is mother liquor for later use.

(5) And (4) merging the mother liquor with the concentrated solution prepared in the next step (3) to realize the recycling of the mother liquor.

Further, in the step (1), the culture medium of the bacillus natto mainly comprises the following components: glucose 2g/L, peptone 20g/L, ammonium sulfate 0.5%, K₂HPO₄ 2g/L、KH₂PO₄ 1g/、CaCl₂ 0.1g/L、MgSO₄ 1g/L。

Further, in the step (1), the fermentation conditions of the bacillus natto are as follows: the temperature is 37 ℃, the initial pH value is 7.5-8.0, and the inoculation amount is 5-7.5%.

Further, in the step (1), the composite flocculant is 2Na and any one of polyaluminium chloride, polyferric chloride and alum₂CO₃·3H₂O₂Or 2K₂CO₃·3H₂O₂The resulting mixture. Optionally, the mass ratio of any one of polyaluminium chloride, polyferric chloride and alum to sodium peroxycarbonate or potassium peroxycarbonate is 2.5-8: 1.

further, in the step (1), the concentration of the composite flocculant in the fermentation liquor is 45-60 mg/L.

Further, in the step (2), the liquid to be extracted is concentrated under reduced pressure to 45-60% of the original volume, wherein the concentration temperature is 55-75 ℃, so that the concentration of vitamin K2 in the supernatant is increased.

Further, in the step (3), the lyophilization method is as follows: and rapidly freezing the concentrated solution to the temperature of minus 25-35 ℃ in a freeze dryer, then vacuumizing to the temperature of 0.1-0.15 mbra, and sublimating water in the concentrated solution to obtain the freeze-dried powder containing the vitamin K2 crude product.

Further, in the step (4), the mass-to-volume ratio (m/v) of the freeze-dried powder to the acetone is 1 g: 6-10 ml.

Further, in the step (4), the precipitating agent comprises distilled water or purified water, and pure water is used as the precipitating agent as much as possible to reduce impurities introduced in the vitamin K2.

Further, in the step (4), the addition volume of the precipitating agent is 2.5-4 times of the volume of acetone, and the acetone is dissolved in the precipitating agent through strong dissolution and affinity action between the precipitating agent and the acetone, so that the vitamin K2 insoluble in the precipitating agent is separated out, and the purification is realized.

Further, in the step (4), the acetone in the residual liquid is recovered by distillation, preferably, the distillation temperature is higher than the boiling point of acetone and lower than the boiling point of water, for example, between 60 and 65 ℃.

Compared with the prior art, the invention has the following beneficial effects:

(1) the invention realizes the purification and separation of vitamin K2 by utilizing the dissolution characteristic of the vitamin K2 in an organic solvent, not only effectively improves the purity of vitamin K2 in a finished product, but also is simpler and more efficient compared with the existing purification methods. The reason for this is that: vitamin K2 is easily dissolved in acetone but not dissolved in water, so the invention firstly dissolves the freeze-dried powder containing vitamin K2 crude product in acetone, then water is added as a precipitating agent to force vitamin K2 to separate out, because water and acetone can be dissolved together strongly, therefore, the water molecules can capture the acetone molecules more strongly and dissolve the acetone molecules in more water than the water with the same volume of the sowing function, acetone is changed into a solute in the precipitator from a solvent serving as vitamin K2, and vitamin K2 cannot be dissolved into water along with the acetone, so that, in the process, the vitamin K2 is separated out from the acetone, and other parts of substances in the freeze-dried powder containing the vitamin K2 crude product have better water solubility and can be dissolved in a precipitator together with the acetone, thereby realizing the extraction of the vitamin K2 and improving the purity of the vitamin K2 in the extracted product.

(2) The method for preparing the freeze-dried powder from the concentrated solution and the method for combining the concentrated solution and the concentrated solution solve the problem of recycling the concentrated solution, the concentrated solution contains part of unextracted vitamin K2, and the vitamin K2 in the concentrated solution is extracted again after being combined with the concentrated solution, so that the problem that the vitamin K2 in the concentrated solution is wasted due to the fact that the vitamin K2 cannot be effectively recycled can be solved.

(3) The invention adopts flocculating agent to carry out flocculation precipitation on thalli in fermentation liquor, wherein 2Na is contained in the thalli₂CO₃·3H₂O₂Or 2K₂CO₃·3H₂O₂The linear oxygen generated after being dissolved in the fermentation liquor has strong oxidation effect, and can change the surface charge and the space structure of the bacillus natto, so that the balance of the bacillus natto in the fermentation liquor is damaged, the sedimentation of the bacillus natto is realized, and compared with the traditional centrifugal seam method, the method not only realizes the removal of the bacillus natto in the fermentation liquor, but also is beneficial to reducing the breakage of the bacillus natto, and the problem that the purification difficulty of vitamin K2 is increased due to the outflow of other metabolites in the broken bacillus natto is solved.

Detailed Description

It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. The invention will now be further illustrated by specific examples.

Example 1

A preparation method of vitamin K2 comprises the following steps:

(1) inoculating a bacillus natto culture medium into a fermentation tank as follows: glucose 2g/L, peptone 20g/L, ammonium sulfate 0.5%, K₂HPO₄ 2g/L、KH₂PO₄ 1g/、CaCl₂ 0.1g/L、MgSO₄1 g/L. Then inoculating bacillus natto into the culture medium, wherein the fermentation conditions are as follows: the fermentation temperature is 37 ℃, the initial pH value is 8.0, the inoculation amount is 6%, and the fermentation time is five days.

(2) Adding composite flocculant (polyaluminium chloride and 2 Na) into the fermentation liquor after the fermentation is finished according to the proportion that the concentration of the composite flocculant in the fermentation liquor is 50mg/L₂CO₃·3H₂O₂According to the mass ratio of 5: 1) and stirring, standing, and after the bacillus natto thalli are settled, extracting supernatant liquid by using a tube with a filtering function to obtain the extract for later use.

(3) And (3) concentrating the extract to 45% of the original volume at 60 ℃ under reduced pressure to obtain a concentrated solution, then placing the concentrated solution in a freeze dryer, rapidly cooling to-25 ℃, vacuumizing to 0.15mbra, and sublimating water in the concentrated solution to obtain the freeze-dried powder containing the crude vitamin K2.

(4) According to the mass volume ratio of 1g of freeze-dried powder to acetone: and (3) dissolving the freeze-dried powder in acetone according to the proportion of 8ml, adding purified water serving as a precipitator (the adding volume of the purified water is 3 times of the volume of the acetone) after the freeze-dried powder is completely dissolved, centrifuging to separate out precipitates after no precipitate is separated out, and drying in vacuum to obtain the vitamin K2 product.

(5) Distilling the residual liquid separated from the precipitate in the step (4) at 60 ℃, and recovering the distilled acetone, wherein the residual liquid is mother liquid which is reserved to be combined with the concentrated solution prepared in the next step (3), so that the recovery and the utilization of the mother liquid are realized.

Example 2

A preparation method of vitamin K2 comprises the following steps:

(1) inoculating a bacillus natto culture medium into a fermentation tank as follows: glucose 2g/L, peptone 20g/L, ammonium sulfate 0.5%, K₂HPO₄ 2g/L、KH₂PO₄ 1g/、CaCl₂ 0.1g/L、MgSO₄1 g/L. Then inoculating bacillus natto into the culture medium, wherein the fermentation conditions are as follows: the fermentation temperature was 37 ℃, the initial pH 7.5, the inoculum size was 7.5%, and the fermentation time was five days.

(2) Adding composite flocculant (polyaluminium chloride and 2 Na) into the fermentation liquor after the fermentation is finished according to the proportion that the concentration of the composite flocculant in the fermentation liquor is 45mg/L₂CO₃·3H₂O₂According to the mass ratio of 8: 1) stirring, standing, and filtering after the bacillus natto is settledThe supernatant is extracted by an energy tube to obtain the extract to be used.

(3) And (3) concentrating the extract to 50% of the original volume at 55 ℃ under reduced pressure to obtain a concentrated solution, then placing the concentrated solution in a freeze dryer, rapidly cooling to-30 ℃, vacuumizing to 0.1mbra, and sublimating water in the concentrated solution to obtain the freeze-dried powder containing the crude vitamin K2.

(4) According to the mass volume ratio of 1g of freeze-dried powder to acetone: dissolving the freeze-dried powder in acetone according to the proportion of 10ml, adding purified water serving as a precipitator (the adding volume of the purified water is 4 times of the volume of the acetone) after the freeze-dried powder is completely dissolved, centrifuging to separate out precipitates after no precipitate is separated out, and drying in vacuum to obtain the vitamin K2 product.

(5) Distilling the residual liquid separated from the precipitate in the step (4) at 60 ℃, and recovering the distilled acetone, wherein the residual liquid is mother liquid which is reserved to be combined with the concentrated solution prepared in the next step (3), so that the recovery and the utilization of the mother liquid are realized.

Example 3

A preparation method of vitamin K2 comprises the following steps:

(1) inoculating a bacillus natto culture medium into a fermentation tank as follows: glucose 2g/L, peptone 20g/L, ammonium sulfate 0.5%, K₂HPO₄ 2g/L、KH₂PO₄ 1g/、CaCl₂ 0.1g/L、MgSO₄1 g/L. Then inoculating bacillus natto into the culture medium, wherein the fermentation conditions are as follows: the fermentation temperature is 37 ℃, the initial pH value is 7.5, the inoculation amount is 5%, and the fermentation time is five days.

(2) Adding composite flocculant (polyaluminium chloride and 2 Na) into the fermentation liquor after the fermentation is finished according to the proportion that the concentration of the composite flocculant in the fermentation liquor is 60mg/L₂CO₃·3H₂O₂According to the mass ratio of 2.5: 1) and stirring, standing, and after the bacillus natto thalli are settled, extracting supernatant liquid by using a tube with a filtering function to obtain the extract for later use.

(3) And (3) concentrating the extract to 60% of the original volume at 75 ℃ under reduced pressure to obtain a concentrated solution, then placing the concentrated solution in a freeze dryer, rapidly cooling to-35 ℃, vacuumizing to 0.1mbra, and sublimating water in the concentrated solution to obtain the freeze-dried powder containing the crude product of vitamin K2.

(4) According to the mass volume ratio of 1g of freeze-dried powder to acetone: and (3) dissolving the freeze-dried powder in acetone according to the proportion of 6ml, adding purified water serving as a precipitator (the adding volume of the purified water is 2.5 times of the volume of the acetone) after the freeze-dried powder is completely dissolved, centrifuging to separate out a precipitate after no precipitate is separated out, and drying in vacuum to obtain the vitamin K2 product.

(5) Distilling the residual liquid separated from the precipitate in the step (4) at 65 ℃, and recovering the distilled acetone, wherein the residual liquid is mother liquid which is reserved to be combined with the concentrated solution prepared in the next step (3), so that the recovery and the utilization of the mother liquid are realized.

Example 4

A preparation method of vitamin K2 comprises the following steps:

(1) inoculating a bacillus natto culture medium into a fermentation tank as follows: glucose 2g/L, peptone 20g/L, ammonium sulfate 0.5%, K₂HPO₄ 2g/L、KH₂PO₄ 1g/、CaCl₂ 0.1g/L、MgSO₄1 g/L. Then inoculating bacillus natto into the culture medium, wherein the fermentation conditions are as follows: the fermentation temperature is 37 ℃, the initial pH value is 8.0, the inoculation amount is 6%, and the fermentation time is five days.

(2) After fermentation, the fermentation liquor is centrifuged, and the supernatant is taken as the liquid to be extracted for later use.

(3) And (3) concentrating the extract to 45% of the original volume at 60 ℃ under reduced pressure to obtain a concentrated solution, then placing the concentrated solution in a freeze dryer, rapidly cooling to-25 ℃, vacuumizing to 0.15mbra, and sublimating water in the concentrated solution to obtain the freeze-dried powder containing the crude vitamin K2.

(4) According to the mass volume ratio of 1g of freeze-dried powder to acetone: and (3) dissolving the freeze-dried powder in acetone according to the proportion of 8ml, adding purified water serving as a precipitator (the adding volume of the purified water is 3 times of the volume of the acetone) after the freeze-dried powder is completely dissolved, centrifuging to separate out precipitates after no precipitate is separated out, and drying in vacuum to obtain the vitamin K2 product.

(5) Distilling the residual liquid separated from the precipitate in the step (4) at 60 ℃, and recovering the distilled acetone, wherein the residual liquid is mother liquid which is reserved to be combined with the concentrated solution prepared in the next step (3), so that the recovery and the utilization of the mother liquid are realized.

Example 5

A preparation method of vitamin K2 comprises the following steps:

(1) inoculating a bacillus natto culture medium into a fermentation tank as follows: glucose 2g/L, peptone 20g/L, ammonium sulfate 0.5%, K₂HPO₄ 2g/L、KH₂PO₄ 1g/、CaCl₂ 0.1g/L、MgSO₄1 g/L. Then inoculating bacillus natto into the culture medium, wherein the fermentation conditions are as follows: the fermentation temperature is 37 ℃, the initial pH value is 8.0, the inoculation amount is 6%, and the fermentation time is five days.

(2) Adding composite flocculant (polyaluminium chloride and 2 Na) into the fermentation liquor after the fermentation is finished according to the proportion that the concentration of the composite flocculant in the fermentation liquor is 50mg/L₂CO₃·3H₂O₂According to the mass ratio of 5: 1) and stirring, standing, and after the bacillus natto thalli are settled, extracting supernatant liquid by using a tube with a filtering function to obtain the extract for later use.

(3) And carrying out gradient elution on the to-be-extracted liquid by using HPD100 type macroporous resin as a stationary phase and a citric acid aqueous solution with the mass concentration of 20% as an eluent through liquid chromatography to obtain a vitamin K2 product.

The vitamin K2 purity of the vitamin K2 product obtained in each example was measured, and the results are shown in table 1.

TABLE 1

From the above detection results, the purity of vitamin K2 in the production of vitamin K2 obtained in examples 4 and 5 is obviously lower than that in examples 1 to 3, wherein the bacillus natto in the fermentation broth separated by the high-speed centrifugation method in example 4 is easy to break, other metabolites in the bacillus natto are released, the complexity of components in the fermentation broth is increased, and the difficulty of separating vitamin K2 in the subsequent process is influenced. In example 5, a traditional liquid chromatography method is adopted, so that the separation effect is not ideal and the purity of the vitamin in the product is low compared with the method provided by the invention. The method proposed by the Penampy commercial product adopted in the examples 1 to 3 utilizes the solubility of the vitamin K2 in an organic solvent to realize the purification and separation of the vitamin K2, thereby effectively improving the purity of the vitamin K2 in the finished product.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.