Preparation method of hydroxycobalamin salt

Abstract

The invention relates to a preparation method of hydroxycobalamin salt, which comprises the following steps: carrying out reduction reaction on cobalamin adenosine fermentation liquor before photochemical reaction, carrying out macroporous resin separation and anion resin decoloration, and finally carrying out crystallization treatment to obtain the hydroxycobalamin salt. According to the method, the yield and the purity of the product are effectively improved, related substances of the product are reduced, the use of cyanide is avoided in the production process, the method is environment-friendly, the amount of wastewater is small, and green and environment-friendly industrial production of the hydroxycobalamin salt is easy to realize.

Description

Technical Field

[0001] The invention belongs to the technical field of biological fermentation and extraction, and particularly relates to a method for preparing a hydroxocobalamin salt. Background Art

[0002] Vitamin B12, also known as cobalamin, belongs to the cobalamin class of compounds, which is composed of a corrinoid ring and upper and lower ligands. Vitamin B12, or cobalamin, is a general term for a class of corrinoid compounds containing Co. According to the different Co-connected functional groups, it is divided into adenosylcobalamin, methylcobalamin, hydroxocobalamin, and cyanocobalamin. The structural formula is as follows:

[0003]

[0004] In the above structural formula, R=5'-deoxyadenosine is adenosylcobalamin; R=OH is hydroxocobalamin; R=CH3 is methylcobalamin; and R=CN is cyanocobalamin.

[0005] Hydroxocobalamin, also known as hydroxyvitamin B12, is a reddish-brown crystal or crystalline powder; odorless and tasteless; easily soluble in water and ethanol, insoluble in ether, chloroform, carbon tetrachloride and acetone; molecular formula C62H90ClCoN13O15P; molecular weight 1382.82. Hydroxocobalamin salts include hydroxocobalamin acetate, hydroxocobalamin hydrochloride and hydroxocobalamin sulfate, belonging to the vitamin B12 series of products. Hydroxocobalamin is called hydroxocobalamin because the central ion of the B12 molecule is connected to a hydroxyl group. Among the four B12 molecules, hydroxocobalamin has the best water solubility and is metabolized slowly in urine, also known as long-acting cobalamin; although its absorption rate is not as fast as cyanocobalamin and methylcobalamin, its concentration in the eye is very high, which can greatly relieve visual fatigue and nourish the optic nerve, so it has a certain effect on amblyopia caused by poisoning. Hydroxocobalamin has the efficacy of VB12 products and is used to treat pernicious anemia, megaloblastic anemia, neuralgia, liver parenchymal damage and contact dermatitis. In addition, hydroxocobalamin can quickly combine with free cyanide in aqueous solution to form non-toxic cyanocobalamin. Therefore, hydroxocobalamin is an efficient antidote for cyanide poisoning.

[0006] Hydroxocobalamin synthesis report has a method (CN1323746A) of preparing hydroxocobalamin with cyanocobalamin as precursor, in which cyanocobalamin is reduced to generate the reduced state of vitamin B12 through the reduction of a reducing agent (such as sodium metabisulfite, sodium nitrite) under an aqueous solution state, and the reduced state of B12 is oxidized (oxidant such as aminosulfonic acid) to generate hydroxocobalamin, and the reaction solution containing hydroxocobalamin is purified by a macroporous resin, and crystallized by acetone dissolution, and hydroxocobalamin crystals are obtained. There is also a report of using a methylcobalamin synthesis route, a method for preparing a hydroxocobalamin salt (CN102351933B), a methylcobalamin aqueous solution, under weak acidity, a photochemical reaction generates hydroxocobalamin, and purified by a macroporous resin, and crystallized by acetone dissolution, and hydroxocobalamin crystals are obtained. The problem of the above process is:

[0007] 1. The production of cyanocobalamin raw materials generally uses Pseudomonas denitrificans for aerobic fermentation to produce adenosylcobalamin fermentation broth, and then extracts, transforms, and purifies to obtain VB12 products. Among them, the process of removing the deoxyadenosine group from the cobalt atom in the adenosylcobalamin molecule and combining it with the -CN group to become cyanocobalamin is called conversion. In general production processes, conversion is achieved by adding sodium cyanide or potassium cyanide. Sodium cyanide and potassium cyanide are highly toxic drugs with great safety risks and high management costs. Special supervision is required in the procurement, transportation, use, storage, and packaging barrel recycling links, and the waste gas, wastewater, and waste solids generated during the conversion process need to be harmlessly treated to ensure the safety of the ecological environment.

[0008] 2. The production of methylcobalamin raw materials generally uses cyanocobalamin as a precursor, and generates the reduced state of vitamin B12 through reduction with a reducing agent (such as sodium borohydride). The reduced state of B12 is then subjected to a methylation reaction (methylation reagents such as methanol solution of methyl iodide or trimethyl sulfoxide iodide) to obtain a methylcobalamin reaction solution, which is then extracted and purified with a macroporous resin, and crystallized with acetone to obtain methylcobalamin. The fermentation broth of adenosylcobalamin is produced by aerobic fermentation of Pseudomonas denitrificans, and then methylcobalamin is obtained through cyanamide. The process is complex, with many production steps, high yield loss, increased three wastes, increased energy consumption, and high production costs.

[0009] 3. In the process of releasing the cobalt atom of vitamin B12 from the functional group (deoxyadenosine) through reduction reaction, the reducing agents used are sodium bisulfite and sodium nitrite, which are prone to produce harmful gases such as SO2 and NO. Sodium borohydride is used as a reducing agent, which is prone to produce dangerous gas H2, increasing the danger and harmfulness of the operating environment. Summary of the invention

[0010] The invention provides a method for preparing a hydroxocobalamin salt, which solves the problem that the existing photochemical reaction rate is uncontrollable, resulting in a significant increase in isomer impurities in the resulting hydroxocobalamin salt, by adding a reduction reaction before a photochemical reaction. Then, a macroporous resin separation step is combined with an anion resin decolorization step to achieve separation of the hydroxocobalamin salt and homologues, thereby effectively improving the purity of the product and reducing the related substances of the product.

[0011] The technical solutions adopted to achieve the above objectives are:

[0012] A method for preparing a hydroxocobalamin salt comprises the following steps:

[0013] 1) subjecting the adenosylcobalamin fermentation liquid to a reduction reaction to obtain a reduced adenosylcobalamin liquid;

[0014] 2) subjecting the reduced deoxyadenosylcobalamin solution obtained in step 1) to a photochemical reaction to obtain a hydroxocobalamin reaction solution;

[0015] 3) subjecting the hydroxocobalamin reaction solution obtained in step 2) to adsorption and analysis by a macroporous resin to obtain a hydroxocobalamin analysis solution;

[0016] 4) decolorizing the hydroxocobalamin solution obtained in step 3) by an anion resin to obtain a hydroxocobalamin salt refined solution;

[0017] 5) The hydroxocobalamin salt refined solution obtained in step 4) is crystallized to obtain the hydroxocobalamin salt.

[0018] Wherein, the adenosylcobalamin fermentation broth in step 1) is adenosylcobalamin fermentation broth produced by aerobic fermentation of Pseudomonas denitrificans.

[0019] The adenosylcobalamin fermentation broth is prepared by pre-treatment, and the pre-treatment includes: acidification and hydrolysis, flocculation and precipitation, and ultrafiltration treatment.

[0020] Wherein, the acidification uses oxalic acid, adjusts the pH to 3.5-4.5, and the acidification temperature is 30-40°C.

[0021] Wherein, the flocculant is polyaluminium chloride, which is added in an amount of 3.5-6.5 (g / L) according to the volume of the fermentation liquid.

[0022] Wherein, the pore size of the ultrafiltration membrane is 1 nm.

[0023] Wherein, the reducing agent in the reduction reaction of step 1) is vitamin C or isovitamin C.

[0024] The amount of the reducing agent added is adenosylcobalamin (w): reducing agent (w) = 1: 0.13-0.14, the reduction reaction temperature is 40-45° C., and the reaction time is 75-90 minutes.

[0025] Wherein, the photochemical reaction conditions in step 2) are: photochemical reaction wavelength 420-460nm, reaction temperature 30-40°C, and photochemical reaction time 50-80 minutes.

[0026] Wherein, the macroporous resin model in step 3) is DM1180,

[0027] Wherein, the analysis in step 3) adopts 0.5-0.8 mol / L HCL solution or 1.2-1.5 mol / L HAc solution.

[0028] Wherein, the anion resin model in step 4) is XAD761.

[0029] The method further comprises a pre-treatment step of the adenosylcobalamin fermentation broth, and the pre-treatment comprises: acidification and hydrolysis, flocculation and precipitation, and ultrafiltration treatment.

[0030] Wherein, the hydroxocobalamin salt is hydroxocobalamin acetate or hydroxocobalamin hydrochloride.

[0031] Wherein, in step 5), the crystallization treatment is performed by adding acetone in an amount of 1:3-5 of hydroxocobalamin hydrochloride purified liquid or hydroxocobalamin acetate purified liquid (V) and acetone (V), and the crystallization temperature is 5-8°C.

[0032] The present invention has at least the following beneficial effects:

[0033] 1. The present invention produces adenosylcobalamin fermentation broth by aerobic fermentation of Pseudomonas denitrificans, directly synthesizes hydroxocobalamin, does not undergo cyanocobalamin and methylcobalamin conversion, does not require cyanidation conversion, avoids the use of cyanide in the production process, has high conversion rate and yield, and produces less wastewater, and is a green and environmentally friendly production process;

[0034] 2. The present invention first performs a reduction reaction before the photochemical reaction, so that adenosylcobalamin is in a reduced state, and the cyanide position is in a free radical state, which is beneficial to improving the photochemical reaction rate of methylcobalamin, and greatly reduces the isomer impurities in the product hydroxycobalamin salt. In addition, the reducing agent uses vitamin C or isovitamin C, the reaction conditions are mild, the environment is friendly, and the process operation is safe;

[0035] 3. The present invention combines the macroporous resin separation step with the anion resin decolorization step to achieve the separation of hydroxocobalamin salt and homologues, the product purity is high, and the related substances of the product are effectively reduced. DETAILED DESCRIPTION

[0036] The present invention is described below with examples. It should be understood that the examples are used to illustrate the present invention rather than to limit the present invention. The scope and core content of the present invention are determined according to the claims.

[0037] 2000L of Pseudomonas denitrificans fermentation broth (titer 330 μg / ml) was taken and divided into three groups. The first group of 1000L was evenly divided into 5 parts for use as an example; the second group of 600L was divided into three parts, and adenosylcobalamin membrane filtration permeate was used as a raw material for a comparative case experiment; the third group of 400L was made into cyanocobalamin and divided into 2 parts, 1 part of which was made into methylcobalamin using cyanocobalamin as a raw material for a comparative case experiment, and 1 part of which was made into methylcobalamin using cyanocobalamin as a raw material, and then methylcobalamin was used as a raw material for a comparative case experiment.

[0038] Example 1

[0039] Add appropriate amount of oxalic acid to 200L of the above fermentation broth (titer 330μg / ml, equivalent to 66g of adenosylcobalamin mass), adjust the pH to 3.5, add 700g of polyaluminium chloride flocculant, acidify and hydrolyze at 30-32°C for 40 minutes, filter through plate and frame, collect the filtrate, filter through ultrafiltration membrane (filter element pore size 1nm), and collect the permeate.

[0040] The permeate was heated to 40° C., and vitamin C was added in an amount of adenosylcobalamin (w): vitamin C (w) = 1:0.13. The reaction time was 80 minutes to obtain a reduction reaction solution.

[0041] The above reduction reaction solution was controlled at a temperature of 30-40°C and irradiated with light at a wavelength of 420nm for 50 minutes to carry out a photochemical reaction. Afterwards, it was placed into a macroporous resin (model DM1180) and analyzed with a 0.5mol / L HCL solution.

[0042] The above macroporous resin solution was decolorized by anion resin (model: XAD761), top washed with 0.5 mol / L HCL solution, anion decolorized refined solution was collected, filtered, and acetone was added for crystallization. The added amount was according to the ratio of hydroxocobalamin hydrochloride refined solution (V): acetone (V) = 1:3, the crystallization temperature was 5-8°C, centrifuged and filtered, and dried to obtain 60.5 g of hydroxocobalamin hydrochloride, with a content of 98.22% and 1.54% of related substances. The total yield was 91.7%.

[0043] Example 2

[0044] Add appropriate amount of oxalic acid to 200L of the above fermentation broth (titer 330μg / ml, equivalent to 66g of adenosylcobalamin mass), adjust the pH to 3.5, add 800g of polyaluminium chloride flocculant, acidify and hydrolyze at 35-36°C for 50 minutes, filter through plate and frame, collect the filtrate, filter through ultrafiltration membrane (filter element pore size 1nm), and collect the permeate.

[0045] The permeate was heated to 43° C., and vitamin C was added in an amount of adenosylcobalamin (w): vitamin C (w) = 1:0.14. The reaction time was 75 minutes to obtain a reduction reaction solution.

[0046] The above reduction reaction solution was controlled at a temperature of 30-40°C and irradiated with light at a wavelength of 440nm for 60 minutes to carry out a photochemical reaction. Afterwards, it was placed into a macroporous resin (model DM1180) and analyzed with a 1 mol / L HCL solution.

[0047] The above macroporous resin solution was decolorized by anion resin (model: XAD761), top washed with 1.0 mol / L HCL solution, anion decolorized refined solution was collected, filtered, and acetone was added for crystallization. The added amount was according to the ratio of hydroxocobalamin hydrochloride refined solution (V): acetone (V) = 1:5, the crystallization temperature was 5-8°C, centrifuged and filtered, and dried to obtain 59.5 g of hydroxocobalamin hydrochloride, with a content of 98.73% and 1.49% of related substances. The total yield was 90.2%.

[0048] Example 3

[0049] Add appropriate amount of oxalic acid to 200L of the above fermentation broth (titer 330μg / ml, equivalent to 66g of adenosylcobalamin mass), adjust the pH to 3.5, add 1000g of polyaluminium chloride flocculant, acidify and hydrolyze at 38-40°C for 60 minutes, filter through plate and frame, collect the filtrate, filter through ultrafiltration membrane (filter element pore size 1nm), and collect the permeate.

[0050] The permeate was heated to 42° C., and isovitamin C was added in an amount of adenosylcobalamin (w): isovitamin C (w) = 1:0.13. The reaction time was 90 minutes to obtain a reduction reaction solution.

[0051] The above reduction reaction solution was controlled at a temperature of 30-40° C. and irradiated with light at a wavelength of 460 nm for 70 minutes to carry out a photochemical reaction. Thereafter, it was placed into a macroporous resin (model DM1180) and analyzed with a 1.2 mol / L HAc solution.

[0052] The above macroporous resin solution was decolorized by anion resin (model: XAD761), top washed with 1.2 mol / L HAc solution, and the anion decolorized refined solution was collected and crystallized by adding acetone in an amount of 1:3 of hydroxycobalamin acetate refined solution (V): acetone (V), and the crystallization temperature was 5-8°C. After centrifugation and drying, 60.9 g of hydroxycobalamin acetate was obtained, with a content of 98.19% and 1.56% of related substances. The total yield was 92.3%.

[0053] Example 4

[0054] Add appropriate amount of oxalic acid to 200L of the above fermentation broth (titer 330μg / ml, equivalent to 66g of adenosylcobalamin mass), adjust the pH to 3.5, add 1300g of polyaluminium chloride flocculant, acidify and hydrolyze at 33-35°C for 50 minutes, filter through plate and frame, collect the filtrate, filter through ultrafiltration membrane (filter element pore size 1nm), and collect the permeate.

[0055] The permeate was heated to 45° C., and isovitamin C was added in an amount of adenosylcobalamin (w): isovitamin C (w) = 1:0.14. The reaction time was 80 minutes to obtain a reduction reaction solution.

[0056] The above reduction reaction solution was controlled at a temperature of 30-40° C. and irradiated with light at a wavelength of 440 nm for 80 minutes to carry out a photochemical reaction. Afterwards, it was placed in a macroporous resin (model DM1180) and analyzed with a 1.5 mol / L HAc solution.

[0057] The above macroporous resin solution was decolorized by anion resin (model: XAD761), top washed with 1.5 mol / L HAc solution, anion decolorized refined solution was collected, acetone was added for crystallization, the added amount was according to the ratio of hydroxycobalamin acetate refined solution (V): acetone (V) = 1: 3-5, the crystallization temperature was 5-8°C, centrifuged and filtered, and dried to obtain 60.6 g of hydroxycobalamin acetate, with a content of 98.63% and 1.59% of related substances. The total yield was 91.9%.

[0058] Example 5

[0059] Add appropriate amount of oxalic acid to 200L of the above fermentation broth (titer 330μg / ml, equivalent to 66g of adenosylcobalamin mass), adjust the pH to 3.5, add 1000g of polyaluminium chloride flocculant, acidify and hydrolyze at 32-33°C for 50 minutes, filter through plate and frame, collect the filtrate, filter through ultrafiltration membrane (filter element pore size 1nm), and collect the permeate.

[0060] The permeate was heated to 45° C., and vitamin C was added in an amount of adenosylcobalamin (w): vitamin C (w) = 1:0.14. The reaction time was 90 minutes to obtain a reduction reaction solution.

[0061] The above reduction reaction solution was controlled at a temperature of 30-40°C and irradiated with light at a wavelength of 440nm for 70 minutes to carry out a photochemical reaction. Afterwards, it was placed into a macroporous resin (model DM1180) and analyzed with a 0.6mol / L HCL solution.

[0062] The above macroporous resin solution was decolorized by anion resin (model: XAD761), top washed with 0.6 mol / L HCL solution, anion decolorized refined solution was collected, acetone was added for crystallization, the added amount was according to the ratio of hydroxocobalamin hydrochloride refined solution (V): acetone (V) = 1: 3-5, the crystallization temperature was 5-8°C, centrifuged and filtered, and dried to obtain 60.3 g of hydroxocobalamin hydrochloride, with a content of 98.67% and related substances of 1.47%. The total yield was 91.4%.

[0063] Comparative Example 1

[0064] Add appropriate amount of oxalic acid to 200L of the above fermentation broth (titer 330μg / ml, equivalent to 66g of adenosylcobalamin mass), adjust the pH to 3.5, add 700g of polyaluminium chloride flocculant, acidify and hydrolyze at 30-32°C for 40 minutes, filter through plate and frame, collect the filtrate, filter through ultrafiltration membrane (filter element pore size 1nm), and collect the permeate.

[0065] The above permeate was controlled at a temperature of 30-40°C and irradiated with light at a wavelength of 420nm for 50 minutes to carry out a photochemical reaction. Afterwards, it was passed into a macroporous resin (model DM1180) and analyzed with a 0.5mol / L HCL solution.

[0066] The above macroporous resin solution was decolorized by anion resin (model: XAD761), top washed with 0.5 mol / L HCL solution, anion decolorized refined solution was collected, filtered, and acetone was added for crystallization. The added amount was according to the ratio of hydroxocobalamin hydrochloride refined solution (V): acetone (V) = 1:3, the crystallization temperature was 5-8°C, centrifuged and filtered, and dried to obtain 51.3 g of hydroxocobalamin hydrochloride, with a content of 98.26% and related substances of 1.36%. The total yield was 77.7%.

[0067] Comparative Example 2

[0068] Add appropriate amount of oxalic acid to 200L of the above fermentation broth (titer 330μg / ml, equivalent to 66g of adenosylcobalamin mass), adjust the pH to 3.5, add 700g of polyaluminium chloride flocculant, acidify and hydrolyze at 30-32°C for 40 minutes, filter through plate and frame, collect the filtrate, filter through ultrafiltration membrane (filter element pore size 1nm), and collect the permeate.

[0069] The permeate was heated to 40° C., and vitamin C was added in an amount of adenosylcobalamin (w): vitamin C (w) = 1:0.13. The reaction time was 80 minutes to obtain a reduction reaction solution.

[0070] The above reduction reaction solution was controlled at a temperature of 30-40°C and irradiated with light at a wavelength of 420nm for 50 minutes to carry out a photochemical reaction. Afterwards, it was placed into a macroporous resin (model DM1180) and analyzed with a 0.5mol / L HCL solution.

[0071] The above macroporous resin solution was filtered, and acetone was added for crystallization. The added amount was 1:3 of hydroxocobalamin hydrochloride purified solution (V): acetone (V), and the crystallization temperature was 5-8°C. Centrifugal filtration and drying were performed to obtain 59.6 g of hydroxocobalamin hydrochloride, with a content of 81.25% and 18.22% of related substances. The total yield was 90.3%.

[0072] Comparative Example 3

[0073] Add appropriate amount of oxalic acid to 200L of the above fermentation broth (titer 330μg / ml, equivalent to 66g of adenosylcobalamin mass), adjust the pH to 3.5, add 700g of polyaluminium chloride flocculant, acidify and hydrolyze at 30-32°C for 40 minutes, filter through plate and frame, collect the filtrate, filter through ultrafiltration membrane (filter element pore size 1nm), and collect the permeate.

[0074] The above permeate was controlled at a temperature of 30-40°C and irradiated with light at a wavelength of 420nm for 50 minutes to carry out a photochemical reaction. Afterwards, it was passed into a macroporous resin (model DM1180) and analyzed with a 0.5mol / L HCL solution.

[0075] The above macroporous resin solution was filtered, and acetone was added for crystallization. The added amount was 1:3 of hydroxocobalamin hydrochloride purified solution (V): acetone (V), and the crystallization temperature was 5-8°C. Centrifugal filtration and drying were performed to obtain 51.9 g of hydroxocobalamin hydrochloride, with a content of 80.92% and 19.27% ​​of related substances. The total yield was 78.6%.

[0076] Comparative Example Cyanocobalamin Sample Preparation

[0077] 20 g of anhydrous sodium sulfite was added to 400 L of the above fermentation broth (titer 330 μg / ml, equivalent to 132 g of adenosylcobalamin mass), pH was adjusted to 5.5 with 15% hydrochloric acid, heated to about 80°C, maintained for 30 minutes, acid hydrolyzed, 2000 g of polyaluminium chloride flocculant was added, stirred for 20 minutes, allowed to stand, filtered through a plate and frame filter, the filtrate was collected, fed to a cationic resin, analyzed with 10% ammonia water, the analyzed solution was collected, 112 g of sodium nitrite was added, 560 g of zinc chloride was added, pH was adjusted to 5.5 with 15% hydrochloric acid, the mixture was heated to about 80°C, maintained for 30 minutes, acid hydrolyzed, 2000 g of polyaluminium chloride flocculant was added, stirred for 20 minutes, allowed to stand, filtered through a plate and frame filter, the filtrate was collected, fed to a cationic resin, analyzed with 10% ammonia water, the analyzed solution was collected, 100 g of sodium nitrite was added, 500 g of zinc chloride was added, pH was adjusted with 15% hydrochloric acid, the mixture was heated to about 80°C, maintained for 30 minutes, acid hydrolyzed, 2000 g of polyaluminium chloride flocculant was added, the mixture was stirred for 20 minutes, the mixture was allowed to stand, the mixture was filtered through a plate and frame filter, the filtrate was collected, the mixture was filtered through a cationic resin, the mixture was analyzed with 10% ammonia water, the analyzed solution was collected, 100 g of sodium nitrite was added, 500 g of zinc chloride was added, the mixture was heated to about 80°C, pH was adjusted with 10% hydrochloric acid, the mixture was heated to about 10% H4.5, add 200g of sodium cyanide, heat to 90°C, keep warm for 30 minutes, cool to 50°C, adjust pH to 7.0 with 10% liquid caustic soda, add 1200g of polyaluminium chloride, stir for 20 minutes, filter to obtain cyanocobalamin conversion solution, purify with macroporous resin, analyze with 20% acetone aqueous solution, collect the analyzed solution, concentrate under reduced pressure to obtain cyanocobalamin solution, decolorize with activated carbon, filter, collect the refined solution, add acetone, precipitate crystals, filter and dry to obtain 122g of cyanocobalamin with a yield of 92.6%.

[0078] The above cyanocobalamin was divided into 2 portions, each with 61g, and the following comparative case experiments were carried out respectively.

[0079] Comparative Example 4

[0080] In a 100L reaction tank, add 61g of cyanocobalamin, dissolve it with 5L of distilled water, then add 122g of sodium metabisulfite, add 130ml of glacial acetic acid, let air react at 30℃ for 5 hours, adjust pH to 3.0-4.0 with 1M hydrochloric acid, add 30g of sodium nitrite, react at 35℃ for 2 hours, add aminosulfonic acid and react for 2 hours. Adjust pH to 3.5 with 2M sodium hydroxide, adsorb on macroporous resin, and wash with dry salt. Analyze with 50% acetone aqueous solution, collect the eluate. Adjust pH to 2.0-4.0 with 1M hydrochloric acid, add acetone, precipitate crystals, and filter. Wash and dry to obtain 52.6g of hydroxocobalamin hydrochloride, with a yield of 86.23%, a content of 97.88%, and 1.66% of related substances. The total yield is 79.8% (from adenosylcobalamin fermentation broth, synthesized by cyanocobalamin, the extraction yield is 92.6%, and then synthesized by hydroxocobalamin, the extraction yield is 86.23%).

[0081] Comparative Example 5

[0082] Take 61 grams of the above cyanocobalamin, add cyanocobalamin: cobalt chloride: trimethyl sulfoxide iodide: drinking water: ethanol in a 100L reaction tank according to the ratio of 1:0.05:0.35:15:0.6, dissolve into a mixed solution, pass nitrogen for deoxygenation, prepare 0.4% sodium borohydride solution according to cyanocobalamin (w): sodium borohydride (w) = 1:0.5, add 0.4% sodium borohydride solution dropwise, carry out methylation reaction at a temperature of 35±1°C, after addition, continue to react for 30 minutes to obtain methylcobalamin reaction solution, centrifuge and filter, concentrate the methylcobalamin reaction solution, adsorb on a macroporous resin, analyze with a 10% acetone aqueous solution, collect the analyzed solution, pass acetone until methylcobalamin crystals are precipitated, centrifuge and dry to obtain 56.8g of methylcobalamin with a yield of 93.2%.

[0083] 56.8g of methylcobalamin was prepared into a 2000μg / ml methylcobalamin solution, and the pH was adjusted to 3.3 with 7% hydrochloric acid. Air stirring was started, and the reaction solution was illuminated at 17000LX until the content of hydroxycobalamin hydrochloride in the reaction solution was 98.4%. The reaction solution was adsorbed on a styrene macroporous adsorption resin column at an adsorption rate of 100L / h, and after washing, it was analyzed with a 6% acetone aqueous solution at a layer development rate of 100L / h. When the outlet color changed from light red to colorless, 50% acetone aqueous solution was passed through the resin column at a rate of 100-250L / h, and a red crystallization stock solution was collected. Stirring was started, 95% acetone solution was added to the crystallization stock solution, stirred for 1h, allowed to stand for 4h, and filtered to obtain 54.1g of hydroxycobalamin hydrochloride. The product yield was 95.2%, the content was 98.62%, and the related substances were 1.73%. The total yield is 82.2% (from adenosylcobalamin fermentation broth, the yield of cyanocobalamin synthesis and extraction is 92.6%, the yield of methylcobalamin synthesis and extraction is 93.2%, and the yield of hydroxycobalamin synthesis and extraction is 95.2%).

[0084] Effect comparison:

[0085]

Claims

1. A method for preparing a hydroxocobalamin salt, comprising the following steps: 1) subjecting the adenosylcobalamin fermentation liquid to a reduction reaction to obtain a reduced adenosylcobalamin liquid; 2) subjecting the reduced deoxyadenosylcobalamin solution obtained in step 1) to a photochemical reaction to obtain a hydroxocobalamin reaction solution; 3) subjecting the hydroxocobalamin reaction solution obtained in step 2) to adsorption and analysis by a macroporous resin to obtain a hydroxocobalamin analysis solution; 4) decolorizing the hydroxocobalamin solution obtained in step 3) by an anion resin to obtain a hydroxocobalamin salt refined solution; 5) The hydroxocobalamin salt refined solution obtained in step 4) is crystallized to obtain the hydroxocobalamin salt.

2. The method for preparing the hydroxocobalamin salt according to claim 1, characterized in that Step 1) The adenosylcobalamin fermentation broth is adenosylcobalamin fermentation broth produced by aerobic fermentation of Pseudomonas denitrificans.

3. The method for preparing the hydroxocobalamin salt according to claim 2, characterized in that The adenosylcobalamin fermentation broth is prepared by pre-treatment, wherein the pre-treatment comprises acidification and hydrolysis, flocculation and precipitation, and ultrafiltration.

4. The method for preparing the hydroxocobalamin salt according to claim 1, characterized in that In step 1), the reducing agent in the reduction reaction is vitamin C or isovitamin C.

5. The method for preparing the hydroxocobalamin salt according to claim 4, characterized in that The amount of the reducing agent added is adenosylcobalamin (w): reducing agent (w) = 1: 0.13-0.14, the reduction reaction temperature is 40-45° C., and the reaction time is 75-90 minutes.

6. The method for preparing the hydroxocobalamin salt according to claim 1, characterized in that Step 2) The photochemical reaction conditions are: photochemical reaction wavelength 420-460nm, reaction temperature 30-40°C, photochemical reaction time 50-80 minutes.

7. The method for preparing the hydroxocobalamin salt according to claim 1, characterized in that Step 3) The macroporous resin model is DM1180.

8. The method for preparing the hydroxocobalamin salt according to claim 1, characterized in that Step 3) The analysis adopts 0.5-0.8 mol / L HCL solution or 1.2-1.5 mol / L HAc solution.

9. The method for preparing the hydroxocobalamin salt according to claim 1, characterized in that: Step 4) The anion resin model is XAD761.

10. The method for preparing a hydroxocobalamin salt according to claim 1, characterized in that The hydroxocobalamin salt is hydroxocobalamin acetate or hydroxocobalamin hydrochloride.

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