Preparation method of vitamin D3

Abstract

The invention discloses a preparation method of vitamin D3. The preparation method comprises the following steps: S1, obtaining 7-dehydrocholesterol; s2, taking 7-dehydrocholesterol as a raw material, and carrying out primary photochemical reaction under the irradiation of an LED ultraviolet lamp, so that the 7-dehydrocholesterol is converted into pre-vitamin D3 and tachysterol to obtain a reaction solution; s3, adding a triplet-state photosensitizer into the reaction liquid, and carrying out secondary photochemical reaction under the irradiation of an LED (Light Emitting Diode) ultraviolet lamp, so that the tachysterol is converted into pre-vitamin D3, thereby obtaining a mixture containing the pre-vitamin D3; and S4, carrying out a thermal isomerization reaction on the mixture containing the pre-vitamin D3 to obtain the vitamin D3. Raw materials do not need to be recycled after the photochemical reaction, the process is simple, the process operation steps are reduced, the cost is reduced, and the process stability is good.

Description

Technical Field

[0001] The invention belongs to the technical field of vitamin preparation, and in particular relates to a method for preparing vitamin D3. Background Art

[0002] Vitamin D3 is a small variety of vitamins and is used as feed or food additives. Vitamin D3 has the following physiological functions: improve the body's absorption of calcium and phosphorus, so that the levels of plasma calcium and plasma phosphorus reach saturation; promote growth and bone calcification, and promote healthy teeth; increase phosphorus absorption through the intestinal wall and increase phosphorus reabsorption through the renal tubules; maintain normal levels of citrate in the blood; prevent amino acids from being lost through the kidneys. In recent years, research on feed vitamin D3 has gained further understanding, and it has gradually been found that in addition to the classic calcium and phosphorus regulation effects, feed vitamin D3 also participates in regulating the metabolic processes of many cells, which is of great significance to the body's immune function, reproduction and other aspects.

[0003] Vitamin D3 is mainly made from 7-dehydrocholesterol (hereinafter referred to as dehydrocholesterol) through two steps of photochemical reaction and thermal isomerization. The mechanism of preparing vitamin D3 is as follows Figure 1 As shown, dehydrocholesterol is irradiated with ultraviolet light and the raw materials are recovered through post-treatment to obtain a photochemical oil mainly containing previtamin D3 (P configuration) and tachysterol (T configuration), and then after thermal isomerization, a photochemical oil whose main components are vitamin D3 and tachysterol is obtained.

[0004] Under ultraviolet light, previtamin D3 (P configuration) is quickly converted into tachysterol (T configuration). When previtamin D3 is the main product, the reaction is stopped and then thermal isomerization is performed. At this time, most of the raw material (dehydrocholesterol) remains unreacted and the conversion rate is low (<20%), so the raw material needs to be recovered.

[0005] In addition, in the technology of preparing 7-dehydrocholesterol by fermentation of microorganisms (such as Saccharomyces cerevisiae), the extraction and purification of 7-dehydrocholesterol in the fermentation broth is a difficult point. Dehydrocholesterol is an intracellular fat-soluble compound. After fermentation, it exists in yeast cells. It is necessary to release the intracellular product, but the cell wall of Saccharomyces cerevisiae is extremely difficult to break. Therefore, the extraction of 7-dehydrocholesterol has the problem of difficult cell breaking and difficult selection of fat-soluble extraction solvents. Summary of the invention

[0006] The technical problem to be solved by the present invention is to overcome the deficiencies and defects mentioned in the above background technology and provide a method for preparing vitamin D3.

[0007] In order to solve the above technical problems, the technical solution proposed by the present invention is: A method for preparing vitamin D3, comprising the following steps: S1. Obtain 7-dehydrocholesterol; S2, using 7-dehydrocholesterol as a raw material, performing a photochemical reaction under the irradiation of an LED ultraviolet lamp to convert 7-dehydrocholesterol into previtamin D3 and tachysterol to obtain a reaction solution; S3, adding a triplet photosensitizer to the reaction solution, and performing a secondary photochemical reaction under the irradiation of an LED ultraviolet lamp to convert tachysterol into previtamin D3, thereby obtaining a mixture containing previtamin D3; The triplet photosensitizer is a triplet photosensitizer capable of absorbing light with a wavelength of 360-390 nm, and the wavelength of ultraviolet light for the secondary photochemical reaction is controlled at 360-390 nm; S4. Subjecting the mixture containing pre-vitamin D3 to thermal isomerization reaction to obtain vitamin D3.

[0008] As a further improvement, the 7-dehydrocholesterol described in S1 is obtained by the following steps: separating the 7-dehydrocholesterol fermentation broth to obtain bacterial residue; subjecting the bacterial residue to freeze-thaw treatment, and then adding an extraction solvent for reflux extraction.

[0009] As a further improvement, the extraction solvent includes one or more of tetrahydrofuran, ethyl acetate, methanol or ethanol.

[0010] As a further improvement, after adding the extraction solvent for reflux extraction, alkali is added to the extract and refluxed, and the crude 7-dehydrocholesterol is obtained through decolorization and post-treatment.

[0011] As a further improvement, the wavelength of ultraviolet light for the primary photochemical reaction in S2 is 280-300 nm.

[0012] As a further improvement, the photochemical reaction in S2 is stopped when the remaining 7-dehydrocholesterol is less than or equal to 10% of the initial added amount, so that the 7-dehydrocholesterol is basically converted into previtamin D3 and tachysterol.

[0013] As a further improvement, the triplet photosensitizer in S3 is one or more of 9-acetylanthracene, 1-acetylnaphthalene, or 2-acetylnaphthalene.

[0014] As a further improvement, the amount of triplet photosensitizer added in S3 is 0.5-1.5% of the mass of 7-dehydrocholesterol.

[0015] As a further improvement, the wavelength of ultraviolet light for the secondary photochemical reaction in S3 is 360-375 nm.

[0016] As a further improvement, the secondary photochemical reaction in S3 is carried out until the remaining tachysterol is less than or equal to 3% of the total amount of tachysterol after the primary photochemical reaction, and then the reaction is stopped to allow the tachysterol to be substantially converted into previtamin D3.

[0017] Compared with the prior art, the present invention has the following beneficial effects: The 7-dehydrocholesterol fermentation liquid obtained by the biological fermentation method is subjected to a primary photochemical reaction after dehydrocholesterol is extracted, so that dehydrocholesterol is basically converted into pre-vitamin D3 and tachysterol. Then a triplet photosensitizer is added, and a secondary photochemical reaction is carried out at a specific wavelength to convert all tachysterol into pre-vitamin D3, and then vitamin D3 is obtained by thermal isomerization. The present invention does not need to recover raw materials after the photochemical reaction, has a simple process, reduces process operation steps, reduces costs, and has good process stability.

[0018] In addition, for the extraction of dehydrocholesterol from 7-dehydrocholesterol fermentation broth, the present invention firstly uses the freeze-thaw method to break the yeast cell wall, and simultaneously uses a specific extraction solvent to extract 7-dehydrocholesterol, thereby significantly improving the extraction rate of 7-dehydrocholesterol. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] 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 some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 It is the reaction process in which 7-dehydrocholesterol undergoes photochemical reaction and thermal isomerization to obtain vitamin D3. DETAILED DESCRIPTION

[0021] In order to facilitate the understanding of the present invention, the present invention will be described more comprehensively and meticulously below in conjunction with the accompanying drawings and preferred embodiments of the present invention, but the protection scope of the present invention is not limited to the following specific embodiments.

[0022] Unless otherwise defined, all professional terms used below have the same meanings as those generally understood by those skilled in the art. The professional terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention.

[0023] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0024] In some specific embodiments, the method for preparing vitamin D3 of the present invention comprises the following steps: S1. Extracting 7-dehydrocholesterol fermentation broth to obtain crude 7-dehydrocholesterol.

[0025] The 7-dehydrocholesterol fermentation liquid of the present invention is a fermentation liquid containing 7-dehydrocholesterol and bacteria obtained by a microbial fermentation method. The present invention has no particular restrictions on the 7-dehydrocholesterol fermentation method, and can be applied to the dehydrocholesterol fermentation liquid obtained by various 7-dehydrocholesterol microbial fermentation methods known in the art, such as using Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) dehydrocholesterol fermentation broth obtained by fermentation.

[0026] In some embodiments, the extraction process includes: 1) Separate the solid and liquid of the 7-dehydrocholesterol fermentation broth to obtain bacterial residue.

[0027] 2) The mushroom residue is subjected to freeze-thaw treatment (freezing and then thawing). Preferably, the residue is frozen at a temperature of -30 to -15°C for 10 to 15 hours.

[0028] 3) adding an extraction solvent to perform reflux extraction, and then performing solid-liquid separation to obtain an extract, adding a base to the extract and performing reflux, and then decolorizing with activated carbon and post-treating to obtain a crude 7-dehydrocholesterol.

[0029] In some embodiments, the extraction solvent includes tetrahydrofuran, ethyl acetate, methanol, ethanol, etc., preferably tetrahydrofuran. In step 2), the yeast cell wall is broken by freeze-thaw method, but the cell wall breaking rate is only about 50%. Tetrahydrofuran is used for extraction. Compared with other solvents, tetrahydrofuran has strong penetration into the cell wall, which can greatly improve the extraction rate.

[0030] In some embodiments, the alkali can be sodium hydroxide, potassium hydroxide, etc., and the amount added is 0.5-1.5% of the mass of the mushroom residue. The alkali is added for decolorization.

[0031] In some embodiments, the reflux temperature is 55-65° C., tetrahydrofuran is added and refluxed for 2-3 hours, and a base is added and refluxed for 1-2 hours.

[0032] S2. Using crude 7-dehydrocholesterol as raw material, a photochemical reaction is carried out under the irradiation of LED ultraviolet lamp.

[0033] In some embodiments, the crude 7-dehydrocholesterol is first prepared into a 7-dehydrocholesterol solution with a concentration of 2-3 wt%, cooled to 10-20° C., and then reacted. The solvent of the 7-dehydrocholesterol solution can be a methanol / petroleum ether mixed solvent (preferably a volume ratio of methanol to petroleum ether of 1-5:1) or tetrahydrofuran, etc.

[0034] In some embodiments, the wavelength of ultraviolet light in a photochemical reaction is 280-300 nm, such as 285 nm, 295 nm, 300 nm, preferably 295 nm. The reaction is stopped when the remaining amount of the raw material (7-dehydrocholesterol) is ≤10%.

[0035] After one photochemical reaction, 7-dehydrocholesterol opens the ring, so that the raw material reacts completely and is basically converted into previtamin D3 and tachysterol, with tachysterol reaching about 60%.

[0036] S3. Add a triplet photosensitizer capable of absorbing light of 360-390 nm wavelength into the reaction solution, and carry out a secondary photochemical reaction under the irradiation of an LED ultraviolet lamp to obtain a mixture containing pre-vitamin D3.

[0037] In some embodiments, the triplet photosensitizer capable of absorbing light with a wavelength of 360 to 390 nm is one or more of 9-acetylanthracene, 1-acetylanthracene, and 2-acetylanthracene, and the amount added is 0.5 to 1.5% of the crude quality of 7-dehydrocholesterol. After the secondary photochemical reaction, tachysterol is completely converted into previtamin D3. If the above-mentioned triplet photosensitizer is not added, the conversion of tachysterol to previtamin D3 cannot be achieved. If other triplet photosensitizers that cannot absorb light with a wavelength of 360 to 390 nm are added, they cannot function, and therefore the conversion of tachysterol to previtamin D3 cannot be achieved.

[0038] In some embodiments, the wavelength of ultraviolet light for the secondary photochemical reaction is 360-390 nm, preferably 360-375 nm, such as 365 nm, 375 nm, 385 nm, preferably 365 nm. If it is not within the above wavelength range, the triplet photosensitizer cannot function and most of it cannot be converted into previtamin D3.

[0039] Therefore, only by using a triplet photosensitizer that can absorb light with a wavelength of 360-390 nm and matching it with an irradiation wavelength of 360-390 nm can the purpose of this step be achieved.

[0040] In some embodiments, the reaction is stopped when the amount of tachysterol remaining is ≤3% (ie, ≤3% of the total amount of tachysterol after one photochemical reaction), and the solvent is removed by concentration to obtain a mixture containing previtamin D3.

[0041] The LED ultraviolet lamp used in the present invention has a narrow spectrum, which can achieve precise control of the reaction. If a mercury lamp is used as a light source, due to its wide spectrum, light of different wavelengths will have a great influence on the reaction. Figure 1 The effects of the reactions shown are very complex, which may result in the products being difficult to control. For example, in patent CN114230500A, although the light is filtered, there are still wavelengths that are unfavorable to the reaction, so the conversion rate of the primary photochemical reaction needs to be controlled below 20%, otherwise it will be converted into tachysterol. In the secondary photochemical reaction, due to the presence of other wavelengths, the reaction proceeds in the direction of leucosterol.

[0042] S4. Subjecting the mixture containing pre-vitamin D3 to thermal isomerization reaction to obtain vitamin D3.

[0043] In some embodiments, the thermal isomerization temperature is 50-80° C. In some embodiments, the mixture containing previtamin D3 is thermally isomerized by keeping it warm at 50-80° C. under a vacuum degree of -0.08 to -0.095 MPa.

[0044] The preparation method of the dehydrocholesterol fermentation broth adopted in the following embodiment of the present invention is: 1. Bacteria culture: The medium (YPD) used for bacterial culture is composed of:

[0045] First-stage shake flask seeds: Use an inoculation loop to pick fresh Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) The colony was inoculated into a shake flask containing 40 mL YPD, and then glucose solution (final concentration 20 g / L) was added. Culture conditions: rotation speed 220 rpm, temperature 30°C, culture time 17-20 h.

[0046] Secondary shake flask seeds: 100 ml of YPD medium, sterilized and cooled, inoculated with 2 ml of primary seed solution into the seed bottle. Culture conditions: speed 200 rpm, temperature 30°C, culture time 17-24h.

[0047] Fermentation base The fermentation medium consists of:

[0048] Sterilize and cool the culture medium, and move the cultured first-level seeds into the fermentation tank with an inoculation volume of 100 ml. Culture conditions: dissolved oxygen is controlled at 30%, and pH is controlled at 5.4 with sodium hydroxide. When the sugar in the fermentation system is exhausted and the ethanol concentration is lower than 10g / L, start to supplement sugar, and control the residual sugar in the system to be less than 2g / L, and the residual ethanol to be less than 10g / L. When OD>50, start to add corn syrup solution at a uniform rate, with a supplementation rate of 8mL / h. Continue fermentation for 4 hours until glucose feeding is completed, and end the fermentation to obtain 7-dehydrocholesterol fermentation broth with a fermentation cell concentration greater than 200g / L.

[0049] Example 1 Extraction of 7-dehydrocholesterol The 7-dehydrocholesterol fermentation broth obtained above was centrifuged or filtered to obtain about 500g of fungus residue. After freezing at -20 degrees for 12 hours, thaw at room temperature, add 1L of tetrahydrofuran, reflux at 60 degrees for 2 hours, cool to room temperature and centrifuge, add 5g of sodium hydroxide to the filtrate and reflux for 1 hour, continue to add 20g of activated carbon and stir for 1 hour, and filter out the activated carbon. Concentrate tetrahydrofuran to a small volume, replace tetrahydrofuran with methanol, and the remaining volume is about 100ml. Filter to obtain 10g of crude 7-dehydrocholesterol, dry it, and the purity detected by HPLC is 96%. Compared with the 7-dehydrocholesterol standard, its content is detected to be 80%.

[0050] Example 2 Preparation of Vitamin D3 1. Prepare 7-dehydrocholesterol solution: dissolve 10 g of crude 7-dehydrocholesterol obtained in Example 1 in a mixed solvent (methanol and petroleum ether in a volume ratio of 3:1) at a concentration of 2.5 wt%, then cool to 15° C. and stir evenly; 2. Primary photochemical reaction: Add the prepared 7-dehydrocholesterol solution into the LED photochemical reactor; turn on the jacket to keep warm, start stirring, turn on the 295nm LED light source, perform HPLC monitoring during the reaction, and the conversion rate is about 95% (±1%); turn off the light.

[0051] 3. Secondary photochemical reaction: After the completion of the primary photochemical reaction, add 1wt% (0.1g) of 7-dehydrocholesterol and 9-acetylanthracene to the reaction solution, turn on the jacket for heat preservation, start stirring, switch to a 365nm light source, perform HPLC monitoring during the reaction, and turn off the light when the tachysterol ratio is less than 5%; 4. Thermal isomerization: After the secondary photochemical reaction is completed, the reaction solution is concentrated to an oily state without solvent, and kept at 60-80°C for 3 hours under a vacuum degree of -0.08~-0.095MPa to obtain about 10g of vitamin D3 photochemical oil. Samples are sent to HPLC for detection. The purity of vitamin D3 is 80%, pre-vitamin D3 is 10%, tachysterol is 3%, and raw material (7-dehydrocholesterol) is 7%. The external standard content measured by VD3 standard is 61%.

[0052] Comparative Example 1 Extraction of 7-dehydrocholesterol After centrifugation or pressure filtration of the 7-dehydrocholesterol fermentation broth, about 500g of bacterial residue was obtained. After freezing at -20 degrees for 12 hours, thaw at room temperature, add 1L of methanol, reflux at 60 degrees for 2 hours, cool to room temperature and centrifuge, add 5g of sodium hydroxide to the filtrate and reflux for 1 hour, continue to add 20g of activated carbon and stir for 1 hour, and filter out the activated carbon. Concentrate the methanol to a small volume, the remaining volume is about 100ml, filter, and obtain 8g of crude 7-dehydrocholesterol.

[0053] Comparative Example 2 Preparation of Vitamin D3 1. Prepare 7-dehydrocholesterol solution: dissolve 10 g of crude 7-dehydrocholesterol obtained in Example 1 in a mixed solvent (methanol and petroleum ether in a volume ratio of 3:1) at a concentration of 2.5 wt%, then cool to 15° C. and stir evenly; 2. Primary photochemical reaction: Add the prepared 7-dehydrocholesterol solution into the LED photochemical reactor; turn on the jacket to keep warm, start stirring, turn on the 295nm LED light source, perform HPLC monitoring during the reaction, and the conversion rate is about 95% (±1%); turn off the light.

[0054] 3. Secondary photochemical reaction: After the completion of the primary photochemical reaction, 9-acetylanthracene was not added, the jacket was opened for heat preservation, stirring was started, the light source was changed to 365 nm, and HPLC monitoring was performed during the reaction. Tachysterol did not react.

[0055] Comparative Example 3 Preparation of Vitamin D3 1. Prepare 7-dehydrocholesterol solution: dissolve 10 g of crude 7-dehydrocholesterol obtained in Example 1 in a mixed solvent (methanol and petroleum ether in a volume ratio of 3:1) at a concentration of 2.5 wt%, then cool to 15° C. and stir evenly; 2. Primary photochemical reaction: Add the prepared 7-dehydrocholesterol solution into the LED photochemical reactor; turn on the jacket to keep warm, start stirring, turn on the 295nm LED light source, perform HPLC monitoring during the reaction, and the conversion rate is about 95% (±1%); turn off the light.

[0056] 3. Secondary photochemical reaction: After the completion of the primary photochemical reaction, add 1wt% (0.1g) 9-acetylanthracene of 7-dehydrocholesterol to the reaction solution, turn on the jacket for insulation, start stirring, switch to a 340nm light source, and perform HPLC monitoring during the reaction. The reaction conversion is: dehydrocholesterol accounts for 31%, leucosterol is 63%, previtamin D3 is 6%, and tachysterol is 1%.

[0057] The above is only a preferred embodiment of the present invention and does not limit the present invention in any form. Therefore, any simple modification, equivalent change and modification made to the above embodiment according to the technical essence of the present invention without departing from the content of the technical solution of the present invention shall fall within the scope of protection of the technical solution of the present invention.

Claims

1. A method for preparing vitamin D3, characterized in that: The steps include: S1. Obtain 7-dehydrocholesterol; S2, using 7-dehydrocholesterol as a raw material, performing a photochemical reaction under the irradiation of an LED ultraviolet lamp to convert 7-dehydrocholesterol into previtamin D3 and tachysterol to obtain a reaction solution; S3, adding a triplet photosensitizer to the reaction solution, and performing a secondary photochemical reaction under the irradiation of an LED ultraviolet lamp to convert tachysterol into previtamin D3, thereby obtaining a mixture containing previtamin D3; The triplet photosensitizer is a triplet photosensitizer that can absorb light with a wavelength of 360-390 nm, and the wavelength of ultraviolet light for the secondary photochemical reaction is controlled at 360-390 nm; S4. Subjecting the mixture containing pre-vitamin D3 to thermal isomerization reaction to obtain vitamin D3.

2. The method for preparing vitamin D3 according to claim 1, characterized in that: The 7-dehydrocholesterol described in S1 is obtained by the following steps: separating the 7-dehydrocholesterol fermentation broth to obtain bacterial residue; subjecting the bacterial residue to freeze-thaw treatment, and then adding an extraction solvent for reflux extraction.

3. The method for preparing vitamin D3 according to claim 2, characterized in that: The extraction solvent includes one or more of tetrahydrofuran, ethyl acetate, methanol or ethanol.

4. The method for preparing vitamin D3 according to claim 2 or 3, characterized in that: After adding the extraction solvent and refluxing for extraction, adding alkali to the extract and refluxing, decolorizing and post-treatment to obtain crude 7-dehydrocholesterol.

5. The method for preparing vitamin D3 according to claim 1, characterized in that: The wavelength of ultraviolet light for the primary photochemical reaction in S2 is 280-300 nm.

6. The method for preparing vitamin D3 according to claim 1 or 5, characterized in that: The photochemical reaction in step S2 is performed until the remaining amount of 7-dehydrocholesterol is less than or equal to 10% of the initial amount added, and then the reaction is stopped to allow the 7-dehydrocholesterol to be substantially converted into previtamin D3 and tachysterol.

7. The method for preparing vitamin D3 according to claim 1, characterized in that: S3 The triplet photosensitizer is one or more of 9-acetylanthracene, 1-acetylnaphthalene, or 2-acetylnaphthalene.

8. The method for preparing vitamin D3 according to claim 1 or 7, characterized in that: The amount of triplet photosensitizer added in S3 is 0.5-1.5% of the mass of 7-dehydrocholesterol.

9. The method for preparing vitamin D3 according to claim 1, characterized in that: The wavelength of ultraviolet light for the secondary photochemical reaction in S3 is 360-375 nm.

10. The method for preparing vitamin D3 according to claim 1, characterized in that: The secondary photochemical reaction in S3 is continued until the remaining tachysterol is less than or equal to 3% of the total amount of tachysterol after the primary photochemical reaction, and then the reaction is stopped so that the tachysterol is basically converted into previtamin D3.

Images