A method for extracting vitamin k2

Abstract

The application provides an extraction method of vitamin K2, through the action of a composite enzyme on a vitamin K2 polysaccharide protein complex in a fermentation liquor and the lysis of bacterial cells, intracellular vitamin K2 is released to the extracellular, the vitamin K2 is in a free state and is no longer soluble in water. Then according to the melting point of the vitamin K2 being 52-54 DEG C, the temperature of the feed liquid is reduced to below 52 DEG C, the vitamin K2 is solidified and then floats on the surface of the concentrated liquid. A small amount of anhydrous ethanol is further used for purification, and a high-purity vitamin K2 product is prepared; the application overcomes the defects in the prior art, such as many steps, complex operation, use of toxic and harmful organic solvents, extraction of only intracellular vitamin K2 products and the like.

Description

Technical Field

[0001] This invention relates to the field of vitamin extraction technology, specifically to a method for extracting vitamin K2. Background Technology

[0002] Vitamin K2, also known as menadione, is an important fat-soluble vitamin and a necessary cofactor for osteocalcin carboxylation, enabling the transfer of calcium ions to bones and playing a crucial role in preventing osteoporosis. It can promote the synthesis of prothrombin in the liver, regulate the synthesis of various clotting factors, and, as an electron carrier in respiratory metabolism, participate in many important metabolic reactions in the body. Recent studies have found that vitamin K2 has many other functions in clinical applications. For example, it can not only be used to prevent and treat cardiovascular and cerebrovascular diseases, Parkinson's disease, and type II diabetes, but also promote liver function recovery, showing broad application prospects.

[0003] Currently, microbial fermentation is the preferred method for producing vitamin K2 due to its advantages such as low pollution, low cost, high product activity, and ease of scaling up production. However, current research on vitamin K2 production processes mainly focuses on cell selection and genetic modification, while research on the extraction and purification of fermentation broth is relatively limited.

[0004] Existing technologies for extracting vitamin K2 from fermentation broth mostly employ organic solvent extraction, transferring vitamin K2 from the bacterial cells to an organic solvent to facilitate subsequent purification processes. Patent application CN109824496A discloses a vitamin K2 extraction method involving filtering and concentrating the fermentation broth through a ceramic membrane, bombarding the bacterial cells with a nitrogen ion beam to disrupt the cell walls, extracting the disrupted cells with ethanol, and then centrifuging to collect the supernatant to obtain the vitamin K2 extract. Patent application CN106631748A discloses a vitamin K2 preparation method where vitamin K2 extraction involves first obtaining wet bacterial cells through centrifugation for solid-liquid separation, repeatedly freezing and thawing the wet bacterial cells to disrupt the cell walls, adding n-hexane for extraction, and purifying the resulting extract using resin and silica gel columns, ultimately crystallizing to obtain pure K2. Because the fermentation unit of vitamin K2 produced by fermentation is very low, the content of vitamin K2 in the bacterial cells is extremely low. Traditional organic solvent extraction processes require the addition of large amounts of organic solvents to improve the extraction rate. This results in excessively large extract volumes, very low vitamin K2 content, and severe accumulation of other intracellular metabolic impurities, increasing the difficulty of subsequent purification processes.

[0005] Vitamin K2 is a coenzyme in the respiratory chain of Bacillus subtilis natto, acting as a carrier in the electron transport chain. It binds to proteins and polysaccharides to form a water-soluble complex, which is then secreted extracellularly. Therefore, vitamin K2 is present in both the fermentation broth and the bacterial cells. The patents mentioned above address the extraction and purification of vitamin K2 from within the bacterial cells. Although patent application CN112321408A discloses an apparatus and method for extracting vitamin K2 from microbial fermentation broth, the equipment requires significant investment and involves complex maintenance.

[0006] Therefore, providing a vitamin K2 extraction method that involves fewer steps, is simpler to operate, and is environmentally friendly has become an urgent problem to be solved. Summary of the Invention

[0007] To address the aforementioned issues, this invention provides a simple method for extracting vitamin K2 from the inside of bacterial cells and fermentation broth. The extraction process does not use toxic or harmful organic solvents, is simple, suitable for large-scale production, and achieves green production.

[0008] To achieve the above objectives, this invention utilizes a complex enzyme to act on the vitamin K2 polysaccharide-protein complex in the fermentation broth and lyse bacterial cells, releasing intracellular vitamin K2 into the extracellular space, thus rendering it free and insoluble in water. Then, based on the melting point of vitamin K2 (52-54℃), the temperature of the fermentation solution is lowered below 52℃, causing the vitamin K2 to solidify and float on the surface of the concentrated solution. Further purification with a small amount of anhydrous ethanol yields a high-purity vitamin K2 product. The specific steps include:

[0009] Step 1: Heat the bacterial fermentation broth rich in vitamin K2 and add a compound enzyme to enzymatically hydrolyze the bacterial fermentation broth to obtain the enzymatically hydrolyzed fermentation broth.

[0010] Preferably, the vitamin K2-rich bacterial fermentation broth is Bacillus subtilis fermentation broth for natto;

[0011] Preferably, the mass ratio of the added compound enzyme is 0.01%-0.05%;

[0012] Preferably, the complex enzyme comprises lysozyme, neutral protease, papain, lipase, and β-glucanase in a mass ratio of 5:3:2:2:1.

[0013] Preferably, the temperature is heated to 50-55℃; enzymatic hydrolysis is performed for 2-6 hours.

[0014] Step 2: After the enzymatic fermentation broth is filtered twice and concentrated once, the concentrated broth is collected. At this time, the volume of the concentrated broth is reduced to 1 / 10-1 / 20 of the original volume, and the resulting liquid is obtained.

[0015] Preferably, the first step is to use a membrane with a pore size of 50-100nm for filtration, the second step is to use an organic membrane with a pore size of 10000Da-3000Da for filtration, and the third step is to use an organic membrane with a pore size of 1000Da-500Da for concentration.

[0016] Step 3: After cooling the liquid, stir it. After stirring, let it stand for a period of time until a dark yellow solid appears in the liquid.

[0017] Preferably, the temperature is lowered to 10℃-15℃; stirring is performed for 10-15 minutes; and the mixture is allowed to stand for 8-16 hours.

[0018] Step 4: Filter out the dark yellow solid, add anhydrous ethanol, and then heat until the dark yellow solid is completely dissolved. Then cool down, and a light yellow powder will precipitate out.

[0019] Preferably, the amount of anhydrous ethanol added is 5-10 times the mass of the deep yellow solid; heating to 65℃-70℃; cooling to 5℃-10℃;

[0020] Step 5: Filter out the light yellow powder, dry it at 40℃-45℃ to obtain vitamin K2 powder.

[0021] The present invention has the following advantages:

[0022] (1) This invention overcomes the shortcomings of existing vitamin K2 fermentation broth extraction and purification processes, such as multiple steps, complex operation, use of toxic and harmful organic solvents, and extraction of only intracellular vitamin K2 products;

[0023] (2) This invention provides a vitamin K2 extraction technology, which uses fermentation broth containing vitamin K2 as raw material, and performs solid-liquid separation and membrane concentration; taking advantage of the low freezing point of vitamin K2 itself, the temperature is lowered to precipitate vitamin K2. It has the advantages of fewer extraction steps, lower process temperature, no use of toxic solvents, and green and environmentally friendly process. Detailed Implementation

[0024] The technical solutions in the embodiments of the invention are described clearly and completely below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] The following examples and test cases all used the high performance liquid chromatography method in the National Health and Family Planning Commission Announcement No. 8 of 2016 to detect the vitamin K2 content.

[0026] Example 1

[0027] Step 1: Take 50L of Bacillus subtilis fermentation broth for natto. The vitamin K2 content was tested to be 421ug / ml. The fermentation broth was heated to 50℃, and a compound enzyme with a mass ratio of 0.02% was added. Enzymatic hydrolysis was carried out for 2 hours to obtain the enzymatic fermentation broth. The compound enzyme included lysozyme, neutral protease, papain, lipase, and β-glucanase in a mass ratio of 5:3:2:2:1.

[0028] Step 2: The enzymatic fermentation broth is filtered through a membrane with a pore size of 100 nm to collect the clear liquid. The clear liquid is then filtered through a 10000 Da organic membrane to collect the clear liquid again. After the clear liquid is concentrated through a 1000 Da organic membrane, the volume is reduced to 1 / 10 of the original volume. The concentrated liquid is collected again to obtain the feed solution.

[0029] Step 3: Cool the liquid to 10°C, stir for 15 minutes, and let it stand for 8 hours until a dark yellow solid appears on the surface of the liquid.

[0030] Step 4: Filter out the dark yellow solid, add 6 times the mass of the dark yellow solid in anhydrous ethanol, heat to 70°C until the solid is completely dissolved, and then slowly cool to 10°C. A large amount of pale yellow powder will precipitate out.

[0031] Step 5: Filter and collect the precipitate, and dry it to obtain 19.67g of powder;

[0032] Step six: The purity of vitamin K2 was determined to be 97.51%, with a yield of 89.26%.

[0033] Example 2

[0034] Step 1: Take 50L of Bacillus subtilis fermentation broth for natto. The vitamin K2 content was tested to be 413ug / ml. The fermentation broth was heated to 55℃, and a compound enzyme with a mass ratio of 0.03% was added. Enzymatic hydrolysis was carried out for 3 hours. The compound enzyme included lysozyme, neutral protease, papain, lipase, and β-glucanase in a mass ratio of 5:3:2:2:1.

[0035] Step 2: The enzymatic fermentation broth is filtered through a membrane with a pore size of 100 nm to collect the clear liquid. The clear liquid is then filtered through a 3000 Da organic membrane to collect the clear liquid again. After the clear liquid is concentrated through a 1000 Da organic membrane, the volume is reduced to 1 / 10 of the original volume. The concentrated liquid is collected again to obtain the feed solution.

[0036] Step 3: Cool the liquid to 10°C, stir for 15 minutes, and let it stand for 8 hours until a dark yellow solid appears on the surface of the liquid.

[0037] Step 4: Filter out the dark yellow solid, add 8 times the mass of the dark yellow solid in anhydrous ethanol, heat to 70°C until the solid is completely dissolved, and then slowly cool to 10°C. A large amount of pale yellow powder will precipitate out.

[0038] Step 5: Filter and collect the precipitate, and dry it to obtain 18.96g of powder;

[0039] Step six: The purity of vitamin K2 was determined to be 98.22%, with a yield of 90.2%.

[0040] Example 3

[0041] Step 1: Take 50L of Bacillus subtilis fermentation broth for natto. The vitamin K2 content was tested to be 396ug / ml. The fermentation broth was heated to 55℃, and a compound enzyme with a mass ratio of 0.04% was added. Enzymatic hydrolysis was carried out for 4 hours. The compound enzyme included lysozyme, neutral protease, papain, lipase, and β-glucanase in a mass ratio of 5:3:2:2:1.

[0042] Step 2: The enzymatic fermentation broth is filtered through a membrane with a pore size of 50 nm to collect the clear liquid. The clear liquid is then filtered through a 10000 Da organic membrane to collect the clear liquid again. The clear liquid is then concentrated through a 1000 Da organic membrane, at which point the volume is reduced to 1 / 10 of the original volume. The concentrated liquid is collected again to obtain the feed solution.

[0043] Step 3: Cool the liquid to 10°C, stir for 15 minutes, and let it stand for 10 hours until a dark yellow solid appears on the surface of the liquid.

[0044] Step 4: Filter out the dark yellow solid, add 10 times the mass of the dark yellow solid in anhydrous ethanol, heat to 70°C until the solid is completely dissolved, and then slowly cool to 10°C. A large amount of pale yellow powder will precipitate out.

[0045] Step 5: Filter and collect the precipitate, then dry it to obtain 19.11g of powder;

[0046] Step six: The purity of vitamin K2 was tested and found to be 98.45%, with a yield of 95.0%.

[0047] Test case

[0048] A three-factor, four-level orthogonal experiment was designed using three factors: enzymatic hydrolysis temperature, the mass ratio of the compound enzyme, and the enzymatic hydrolysis time, and the experiment was optimized. The selection of factors and levels for the orthogonal experiment is shown in Table 1, and the experimental design and results are shown in Table 2.

[0049] Table 1. Factor Levels in Orthogonal Experiments

[0050] factor Enzymatic hydrolysis temperature (A) Complex enzyme mass ratio (B) Enzymatic hydrolysis time (C) Level 1 45℃ 0.01％ 1h Level 2 50℃ 0.02％ 2h Level 3 55℃ 0.03％ 3h Level 4 60℃ 0.04％ 4h

[0051] Table 2. Orthogonal experiment table and experimental results

[0052]

[0053]

[0054] Table 2 shows that the R-value indicates the order of factors affecting enzymatic hydrolysis efficiency as follows: hydrolysis temperature > compound enzyme mass ratio > hydrolysis time, with hydrolysis temperature having the most significant impact. After adding the compound enzyme to the solution, the bacterial cells break down and lyse, and some intracellular substances dissolve in the solution, which lowers the OD600 detection value. The lower the detection value, the better the enzymatic hydrolysis effect. Therefore, based on orthogonal and single-factor experiments, the optimal conditions for enzymatic hydrolysis are: hydrolysis temperature A3: 55℃, compound enzyme mass ratio B3: 0.04%, and hydrolysis time C4: 4h.

[0055] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those 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 invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for extracting vitamin K2, characterized in that, Includes the following steps: Step 1: Heat the bacterial fermentation broth rich in vitamin K2 and add a compound enzyme to enzymatically hydrolyze the bacterial fermentation broth to obtain an enzymatically hydrolyzed fermentation broth. Step 2: After the enzymatic fermentation broth is filtered twice and concentrated once, the concentrated broth is collected. At this time, the volume of the concentrated broth is reduced to 1 / 10-1 / 20 of the original volume, and the resulting liquid is obtained. Step 3: After cooling the liquid, stir it. After stirring, let it stand for a period of time until a dark yellow solid appears in the liquid. Step 4: Filter out the dark yellow solid, add anhydrous ethanol, and then heat until the dark yellow solid is completely dissolved. Then cool down, and a light yellow powder will precipitate out. Step 5: Filter out the light yellow powder, dry it at 40℃-45℃ to obtain vitamin K2 powder; In step one, the amount of compound enzyme added is 0.01%-0.05% of the fermentation broth mass; the compound enzyme includes lysozyme, neutral protease, papain, lipase, and β-glucanase, with a mass ratio of 5:3:2:2:

1. In step one, the heating temperature is 50-55℃ and the enzymatic hydrolysis time is 2-6 hours.

2. The method for extracting vitamin K2 according to claim 1, characterized in that, In step one, the fermentation broth rich in vitamin K2 is Bacillus subtilis fermentation broth for natto.

3. The method for extracting vitamin K2 according to claim 1, characterized in that, In step two, the first filtration is done using a membrane with a pore size of 50-100nm, the second filtration is done using an organic membrane with a pore size of 10000Da-3000Da, and the third filtration is done using an organic membrane with a pore size of 1000Da-500Da for concentration.

4. The method for extracting vitamin K2 according to claim 1, characterized in that, In step three, cool down to 10℃-15℃; stir for 10min-15min; let stand for 8-16h.

5. The method for extracting vitamin K2 according to claim 1, characterized in that, In step four, the amount of anhydrous ethanol added is 5-10 times the mass of the dark yellow solid; heat to 65℃-70℃; cool to 5℃-10℃.

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