A process for the production of vitamin b12

Abstract

The present application relates to the production process of vitamin B12, the process steps include pretreatment, ceramic membrane filtration, adsorption, adding hydrocyanic acid through pipeline reactor cyanidation, concentration, column chromatography and crystallization drying. The purpose of the present application is to optimize the extraction and purification process of vitamin B12, and provide a method with safe process steps, high extraction yield and product quality meeting the quality standards of foreign customers.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical extraction technology, and in particular relates to a method for extracting vitamin B12 using vitamin B12 fermentation broth. Background Technology

[0002] Vitamin B12 (C) 63 H 88 CoN 14 O 14 Vitamin B12 is a polycyclic compound containing trivalent cobalt. Four reduced pyrrole rings link together to form a corrin macrocycle (similar to porphyrin), which is the core of the vitamin B12 molecule. Vitamin B12 is a light red needle-like crystal, readily soluble in water and ethanol, decomposes in strong acid (pH < 2) or alkaline solutions, and is partially destroyed by heat. It is easily destroyed by strong light or ultraviolet light. Approximately 30% is lost during ordinary cooking. Vitamin B12 is one of the essential vitamins for the human body. It is mainly found in meat, and soybeans and some herbs also contain B12. Intestinal bacteria can synthesize it, so deficiency is generally not a problem. However, B12 is a vitamin that is easily deficient in people with digestive tract diseases, and it is also an essential element for red blood cell production.

[0003] The traditional extraction and purification process for vitamin B12 involves using vitamin B12 fermentation broth and combining methods such as organic solvent extraction, membrane filtration, cyanidation synthesis, ion exchange resin extraction, column chromatography, and crystallization to produce vitamin B12. The problem is:

[0004] 1. The cyanidation process uses sodium cyanide as raw material and a reaction vessel made of carbon steel or stainless steel as the synthesis equipment. The cyanidation reaction is highly corrosive, has a large thermal effect, and the synthesis yield is low, ranging from 85% to 90%.

[0005] 2. The cyanidation process uses sodium cyanide as a raw material, which generates a large amount of sodium chloride, increasing environmental costs.

[0006] 3. The effective content of the finished product is 98-99%, which cannot meet the requirements of foreign customers for its content (≥99.5%).

[0007] 4. During the cyanidation reaction, solid sodium cyanide is used, which is easy for workers to come into contact with, posing a certain safety risk. Summary of the Invention

[0008] The purpose of this invention is to overcome the defects in the prior art, optimize the vitamin B12 extraction and purification process, and provide a method with safe process steps, high extraction yield, low salt production, and finished product quality that meets the quality standards of foreign customers.

[0009] The technical solution adopted to achieve the above objectives is as follows:

[0010] A production process for vitamin B12 includes the following steps:

[0011] (1) Fermentation broth pretreatment

[0012] Vitamin B12 fermentation broth enters the pretreatment tank.

[0013] Start the stirring system, control the speed at 40-60 r / min, heat to 40-45℃, first add antioxidant, then add hydrochloric acid, adjust the pH to 3.2-3.6, and finally add zwitterionic polyacrylamide (ACPAM). Continue stirring for 40-60 min, cool to room temperature, let stand for 120-140 min, and then filter by plate and frame filter to obtain filtrate ①.

[0014] (2) Ceramic membrane filtration

[0015] The filtrate ① obtained in step (1) is heated to 35-40℃, and the processing volume is 6-7 ml. 3 / h, the running time is controlled within 20h, the temperature of the filtrate is below 35℃, the membrane filtration ends, and filtrate ② is obtained;

[0016] (3) Adjust pH

[0017] The pH of the filtrate ② obtained in step (2) was adjusted to 2-4 using an acid solution, and then allowed to stand for 60-100 minutes. After solid-liquid separation, the filtrate ③ was obtained.

[0018] (4) Adsorption ①

[0019] The filtrate ③ obtained in step (3) is adsorbed through an adsorption column. After the adsorption is completed, ammonia solution is added to the adsorption column for elution, and the eluent ① is collected.

[0020] (5) Cyanide

[0021] The eluent ① obtained in step (4) is introduced into a pipeline reactor, heated to 80-100℃, hydrogen cyanide is added, pH is adjusted to 1.5-2.3, the reaction time is controlled at 40-60 min, after the reaction is completed, alkali solution is added, pH is adjusted to 3.6-3.8, the reaction continues for 20-40 min, the reaction is completed, and the reaction solution is obtained.

[0022] (6) Adsorption ②

[0023] The reaction solution obtained in step (5) is introduced into the adsorption column through the pipeline for adsorption. After the adsorption is completed, acetone solvent is added to the adsorption column for elution, and the eluent ② is collected.

[0024] (7) Concentration

[0025] The eluent ② obtained in step (6) is subjected to vacuum distillation to remove acetone, and then concentrated to 40-50% of its original volume to obtain concentrated solution ①.

[0026] (8) Column Chromatography

[0027] Add adsorbent to the chromatography column, add ethanol solution to the column, and soak for 120-160 min. After soaking, rinse with ethanol 1-2 times, add the concentrated solution ① obtained in step (7) to the glass column, use acetic acid as the mobile phase, the flow rate is 3-5 L / min, and collect the chromatography solution;

[0028] (9) Concentration

[0029] The chromatographic solution obtained in step (8) was subjected to vacuum distillation to remove acetic acid, and then concentrated to 20-30% of the original volume to obtain concentrated solution ②.

[0030] (10) Crystallization and drying

[0031] Cool the concentrated solution ② obtained in step (9) to 0-5℃, add pure vitamin B12, stir for 120-150 min, let stand for 180-240 min, filter after completion, and separate the solid and liquid to obtain solid vitamin B12.

[0032] Solid vitamin B12 was dried under vacuum to obtain pure product.

[0033] In process step (1),

[0034] The concentration of hydrochloric acid should be controlled at 10-20%;

[0035] Antioxidants refer to butylated hydroxytoluene (BHT) or tea polyphenols, and their dosage is: W antioxidant (kg) = fermentation broth volume (m³) 3 *Fermentation broth potency (mg / L) / 1000;

[0036] The dosage of zwitterionic polyacrylamide (ACPAM) is: W (kg) = fermentation broth volume (m³). 3 )*4 / 100.

[0037] In process step (2), the ceramic membrane is made of titanium oxide with a pore size of 0.5 μm and an effective membrane area of ​​0.36 m². 2 .

[0038] In process step (3), the acid solution refers to hydrochloric acid, sulfuric acid, or phosphoric acid, with a concentration controlled at 10-20%.

[0039] In process step (4),

[0040] Adsorption column adsorption refers to the addition of strong acid cation exchange resin-001x7 (732) to a resin column, where the resin dosage is W (kg) = fermentation broth volume (m³). 3 *Fermentation broth potency (mg / L)*4.2 / 1000;

[0041] The ammonia concentration is between 22% and 24%.

[0042] In process step (5),

[0043] The concentration of hydrocyanic acid is 42-45%;

[0044] Alkaline solution refers to sodium hydroxide or sodium carbonate solution with a concentration of 10-20%.

[0045] In process step (6),

[0046] Adsorption column adsorption refers to the addition of macroporous cation exchange resin D113 to a resin column, where the resin dosage is W (kg) = fermentation broth volume (m³). 3 *Fermentation broth potency (mg / L)*3.1 / 1000;

[0047] In process step (8),

[0048] The adsorbent refers to modified urea-formaldehyde resin, and the dosage is W (kg) = fermentation broth volume (m³). 3 *Fermentation broth potency (mg / L)*2.4 / 1000;

[0049] The acetic acid content is 70-80%.

[0050] In process step (10), pure vitamin B12 refers to a content of 99.5% or higher, and the dosage is W (kg) = fermentation liquid volume (m³). 3 *Fermentation broth potency (mg / L)* / 100000.

[0051] The present invention has at least the following technical effects:

[0052] 1. Vitamin B12 content in the finished product: ≥99.5%, meeting the quality requirements of overseas customers.

[0053] 2. In the cyanidation reaction, a tubular reactor was used instead of a conventional synthesis reactor. Because the tubular reactor has a large heat exchange surface per unit reactor volume, it is particularly suitable for reactions with significant thermal effects. Furthermore, the reaction liquid reacts and flows rapidly in the tubular reactor, resulting in high productivity and a shorter synthesis time. The cyanidation synthesis yield reached over 95%.

[0054] 3. Hydrogen cyanide was used instead of sodium cyanide, avoiding direct contact with staff and reducing the risk of poisoning.

[0055] In the original cyanidation process, sodium cyanide was used in the reaction to produce 1 kg of vitamin B12, with an average sodium chloride yield of 134 kg. By using hydrogen cyanide instead of sodium cyanide, the average sodium chloride yield was reduced to 73 kg per kg of vitamin B12, a 45.5% reduction in sodium chloride production and thus lessening environmental impact. Detailed Implementation

[0056] The invention is illustrated below with examples. It should be understood that these examples are for illustrative purposes only and not for limiting the invention. The scope and core content of the invention are defined by the claims.

[0057] The resin used in adsorption process ① is a strong acid cation exchange resin-001x7(732), provided by Basler Chemical Technology Co., Ltd.

[0058] The resin used in adsorption process ② is macroporous cation exchange resin D113, provided by Basler Chemical Technology Co., Ltd.

[0059] The adsorbent used in the column chromatography process is modified urea-formaldehyde resin, which is provided by Mitsui Chemicals (Shandong) Co., Ltd.

[0060] The JY-DTR02 reactor was supplied by Jiuyuan Chemical.

[0061] Source of Vitamin B12 Fermentation Broth: The industrial production of Vitamin B12 primarily involves microbial fermentation. The strains used in industrial production mainly include anaerobic Propionibacterium fischeri and Propionibacterium shearer, as well as aerobic Pseudomonas monosodium spp. The Vitamin B12 fermentation broth described in the patent uses Pseudomonas monosodium spp. to produce Vitamin B12. The carbon source in the culture medium formulation is liquid sugar and beet molasses, while the nitrogen source is mainly ammonium sulfate and corn gluten meal.

[0062] Content detection method: Determined by "Ultraviolet-Vis Spectrophotometry (General Rule 0401)" in the 2020 edition of the Chinese Pharmacopoeia.

[0063] Example 1

[0064] Vitamin B12 fermentation broth 120ml 3 The potency was 283 mg / L.

[0065] (1) Fermentation broth pretreatment

[0066] Vitamin B12 fermentation broth entered 160m 3 Pretreatment tank.

[0067] Start the stirring system, control the speed at 40 r / min, heat to 40℃, first add 33.96 kg of BHT, then add 10% hydrochloric acid to adjust the pH to 3.2, and finally add 4.8 kg of zwitterionic polyacrylamide (ACPAM); continue stirring for 40 min, cool to room temperature, let stand for 120 min, and then filter by plate and frame filter to obtain filtrate ①.

[0068] (2) Ceramic membrane filtration

[0069] The filtrate is heated to 35°C, and the processing capacity is 6m³. 3 / h, with the running time controlled within 20h. When the temperature of the filtrate is below 35℃, membrane filtration ends, and filtrate ② is obtained.

[0070] (3) Adjust pH

[0071] Adjust the pH of filtrate ② to 2 using 10% hydrochloric acid solution, let it stand for 60 minutes, and then separate the solid and liquid to obtain filtrate ③;

[0072] (4) Adsorption ①

[0073] The filtrate ③ was adsorbed through an adsorption column (resin dosage 142.6 kg). After adsorption, 22% ammonia solution was added to the adsorption column for elution, and the eluent ① was collected.

[0074] (5) Cyanide

[0075] The eluent ① was introduced into a tubular reactor, heated to 80°C, and hydrogen cyanide was added to adjust the pH to 1.5. The reaction time was controlled at 40 min. After the reaction was completed, 10% sodium hydroxide was added to adjust the pH to 3.6, and the reaction was continued for another 20 min. The reaction was then completed, yielding the reaction solution. The cyanidation yield was 95.2%.

[0076] (6) Adsorption ②

[0077] The reaction solution enters the adsorption column through the pipeline for adsorption (resin dosage 105.3 kg). After adsorption is completed, acetone solvent is added to the adsorption column for elution, and the eluent ② is collected.

[0078] (7) Concentration

[0079] The eluent ② was distilled under reduced pressure to remove acetone, and then concentrated to 40% of its original volume.

[0080] (8) Column Chromatography

[0081] Add 81.5 kg of adsorbent to the chromatography column, then add ethanol solution and let it soak for 120 min. After soaking, rinse once more with ethanol. Add the concentrated solution to a glass column, using 70% acetic acid as the mobile phase at a flow rate of 3 L / min, and collect the chromatographic solution.

[0082] (9) Concentration

[0083] The chromatographic solution was distilled under reduced pressure to remove acetic acid, and then concentrated to 20% of its original volume.

[0084] (10) Crystallization and drying

[0085] The concentrate was cooled to 0°C, and 0.34 kg of pure vitamin B12 was added. The mixture was stirred for 120 min, allowed to stand for 180 min, filtered, and solid vitamin B12 was obtained by solid-liquid separation.

[0086] Solid vitamin B12 was vacuum dried to obtain 30.56 kg of pure product with an effective content of 99.5%, and the yield of sodium chloride as a byproduct was 2243.1 kg.

[0087] Example 2

[0088] Vitamin B12 fermentation broth 120ml 3 The potency was 297 mg / L.

[0089] (1) Fermentation broth pretreatment

[0090] Vitamin B12 fermentation broth entered 160m 3 Pretreatment tank.

[0091] Start the stirring system, control the speed at 45 r / min, and heat to 41℃. First, add 35.64 kg of tea polyphenols, then add 12% hydrochloric acid to adjust the pH to 3.3, and finally add 4.8 kg of zwitterionic polyacrylamide (ACPAM). Continue stirring for 45 min, cool to room temperature, let stand for 125 min, and then filter by plate and frame filter to obtain filtrate ①.

[0092] (2) Ceramic membrane filtration

[0093] The filtrate was heated to 36°C, and the processing capacity was 6.2 m³. 3 / h, with the running time controlled within 20h. When the temperature of the filtrate is below 35℃, membrane filtration ends, and filtrate ② is obtained.

[0094] (3) Adjust pH

[0095] The pH of filtrate ② was adjusted to 2.5 using 13% sulfuric acid solution, and allowed to stand for 70 minutes. Then, solid-liquid separation was performed to obtain filtrate ③.

[0096] (4) Adsorption ①

[0097] The filtrate ③ was adsorbed through an adsorption column (resin dosage 149.7 kg). After adsorption, 23% ammonia solution was added to the adsorption column for elution, and the eluent ① was collected.

[0098] (5) Cyanide

[0099] The eluent ① was introduced into a tubular reactor, heated to 85°C, and hydrogen cyanide was added to adjust the pH to 1.8. The reaction time was controlled at 45 min. After the reaction was completed, 12% sodium carbonate was added to adjust the pH to 3.7, and the reaction was continued for another 258 min. The reaction was then completed, yielding the reaction solution. The cyanidation yield was 95.4%.

[0100] (6) Adsorption ②

[0101] The reaction solution enters the adsorption column through the pipeline for adsorption (resin dosage 110.5 kg). After adsorption is completed, acetone solvent is added to the adsorption column for elution, and the eluent ② is collected.

[0102] (7) Concentration

[0103] The eluent ② was distilled under reduced pressure to remove acetone, and then concentrated to 42% of its original volume.

[0104] (8) Column Chromatography

[0105] Add 85.5 kg of adsorbent to the chromatography column, then add ethanol solution to the column and let it soak for 130 min. After soaking, rinse twice with ethanol. Add the concentrated solution to a glass column, using 72% acetic acid as the mobile phase at a flow rate of 3.5 L / min, and collect the chromatographic solution.

[0106] (9) Concentration

[0107] The chromatographic solution was distilled under reduced pressure to remove acetic acid, and then concentrated to 23% of its original volume.

[0108] (10) Crystallization and drying

[0109] The concentrate was cooled to 1°C, and 0.37 kg of pure vitamin B12 was added. The mixture was stirred for 130 min, allowed to stand for 190 min, filtered, and solid vitamin B12 was obtained by solid-liquid separation.

[0110] Solid vitamin B12 was vacuum dried to obtain 32.29 kg of pure product with an effective content of 99.6%, and the yield of sodium chloride by-product was 2360.4 kg.

[0111] Example 3

[0112] Vitamin B12 fermentation broth 120ml 3 The potency is 290 mg / L.

[0113] (1) Fermentation broth pretreatment

[0114] Vitamin B12 fermentation broth entered 160m 3 Pretreatment tank.

[0115] Start the stirring system, control the speed at 50 r / min, raise the temperature to 43℃, first add 34.8 kg of BHT, then add 15% hydrochloric acid to adjust the pH to 3.4, and finally add 4.8 kg of zwitterionic polyacrylamide (ACPAM); continue stirring for 50 min, cool to room temperature, let stand for 130 min, and then filter by plate and frame filter to obtain filtrate ①.

[0116] (2) Ceramic membrane filtration

[0117] The filtrate was heated to 37°C, and the processing capacity was 6.5 m³. 3 / h, with the running time controlled within 20h. When the temperature of the filtrate is below 35℃, membrane filtration ends, and filtrate ② is obtained.

[0118] (3) Adjust pH

[0119] The pH of filtrate ② was adjusted to 3 using 15% phosphoric acid solution, and the solution was allowed to stand for 80 minutes. Then, solid-liquid separation was performed to obtain filtrate ③.

[0120] (4) Adsorption ①

[0121] The filtrate ③ was adsorbed through an adsorption column (resin dosage 146.2 kg). After adsorption, 23% ammonia solution was added to the adsorption column for elution, and the eluent ① was collected.

[0122] (5) Cyanide

[0123] The eluent ① was introduced into a tubular reactor, heated to 90°C, and hydrogen cyanide was added to adjust the pH to 2.1. The reaction time was controlled at 50 min. After the reaction was completed, 15% sodium hydroxide solution was added to adjust the pH to 3.7, and the reaction was continued for another 30 min. The reaction was then completed, yielding the reaction solution. The cyanidation yield was 96.1%.

[0124] (6) Adsorption ②

[0125] The reaction solution enters the adsorption column through the pipeline for adsorption (resin dosage 107.9 kg). After adsorption is completed, acetone solvent is added to the adsorption column for elution, and the eluent ② is collected.

[0126] (7) Concentration

[0127] The eluent ② was distilled under reduced pressure to remove acetone, and then concentrated to 45% of its original volume.

[0128] (8) Column Chromatography

[0129] Add 83.5 kg of adsorbent to the chromatography column, then add ethanol solution and let it soak for 140 min. After soaking, rinse twice with ethanol. Add the concentrated solution to a glass column, using 75% acetic acid as the mobile phase at a flow rate of 4 L / min, and collect the chromatographic solution.

[0130] (9) Concentration

[0131] The chromatographic solution was distilled under reduced pressure to remove acetic acid, and then concentrated to 25% of its original volume.

[0132] (10) Crystallization and drying

[0133] The concentrate was cooled to 1°C, and 0.35 kg of pure vitamin B12 was added. The mixture was stirred for 140 min, allowed to stand for 210 min, filtered, and solid vitamin B12 was obtained by solid-liquid separation.

[0134] Solid vitamin B12 was vacuum dried to obtain 31.67 kg of pure product with an effective content of 99.8%, and the yield of sodium chloride as a byproduct was 2295.9 kg.

[0135] Example 4

[0136] Vitamin B12 fermentation broth 120ml 3 The potency is 300 mg / L.

[0137] (1) Fermentation broth pretreatment

[0138] Vitamin B12 fermentation broth entered 160m 3 Pretreatment tank.

[0139] Start the stirring system, control the speed at 55 r / min, raise the temperature to 44℃, first add 36 kg of tea polyphenols, then add 18% hydrochloric acid to adjust the pH to 3.5, and finally add 4.8 kg of zwitterionic polyacrylamide (ACPAM); continue stirring for 55 min, cool to room temperature, let stand for 135 min, and then filter by plate and frame filter to obtain filtrate ①.

[0140] (2) Ceramic membrane filtration

[0141] The filtrate was heated to 39°C, and the processing capacity was 6.8 m³. 3 / h, with the running time controlled within 20h. When the temperature of the filtrate is below 35℃, membrane filtration ends, and filtrate ② is obtained.

[0142] (3) Adjust pH

[0143] The pH of filtrate ② was adjusted to 3.5 using 18% hydrochloric acid solution, and allowed to stand for 90 minutes. Then, solid-liquid separation was performed to obtain filtrate ③.

[0144] (4) Adsorption ①

[0145] The filtrate ③ was adsorbed through an adsorption column (resin dosage 151.2 kg). After adsorption, 24% ammonia solution was added to the adsorption column for elution, and the eluent ① was collected.

[0146] (5) Cyanide

[0147] The eluent ① was introduced into a tubular reactor, heated to 95°C, and hydrogen cyanide was added to adjust the pH to 2.2. The reaction time was controlled at 55 min. After the reaction was completed, 18% sodium carbonate solution was added to adjust the pH to 3.8, and the reaction was continued for another 35 min. The reaction was then completed, yielding the reaction solution. The cyanidation yield was 95.8%.

[0148] (6) Adsorption ②

[0149] The reaction solution enters the adsorption column through the pipeline for adsorption (resin dosage 111.6 kg). After adsorption is completed, acetone solvent is added to the adsorption column for elution, and the eluent ② is collected.

[0150] (7) Concentration

[0151] The eluent ② was distilled under reduced pressure to remove acetone, and then concentrated to 47% of its original volume.

[0152] (8) Column Chromatography

[0153] Add 86.4 kg of adsorbent to the chromatography column, then add ethanol solution and let it soak for 150 min. After soaking, rinse twice with ethanol. Add the concentrated solution to a glass column, using 78% acetic acid as the mobile phase at a flow rate of 4.5 L / min, and collect the chromatographic solution.

[0154] (9) Concentration

[0155] The chromatographic solution was subjected to vacuum distillation to remove acetic acid, and then concentrated to 27% of its original volume.

[0156] (10) Crystallization and drying

[0157] The concentrate was cooled to 1°C, and 0.36 kg of pure vitamin B12 was added. The mixture was stirred for 145 min, allowed to stand for 220 min, filtered, and solid vitamin B12 was obtained by solid-liquid separation.

[0158] Solid vitamin B12 was vacuum dried to obtain 32.65 kg of pure product with an effective content of 99.7%, and the yield of sodium chloride as a byproduct was 2380.2 kg.

[0159] Example 5

[0160] Vitamin B12 fermentation broth 120ml 3 The potency was 273 mg / L.

[0161] (1) Fermentation broth pretreatment

[0162] Vitamin B12 fermentation broth entered 160m 3 Pretreatment tank.

[0163] Start the stirring system, control the speed at 60 r / min, raise the temperature to 45℃, first add 32.76 kg of BHT, then add 20% hydrochloric acid to adjust the pH to 3.6, and finally add 4.8 kg of zwitterionic polyacrylamide (ACPAM); continue stirring for 60 min, cool to room temperature, let stand for 140 min, and then filter by plate and frame filter to obtain filtrate ①.

[0164] (2) Ceramic membrane filtration

[0165] The filtrate is heated to 40°C, and the processing capacity is 7m³. 3 / h, with the running time controlled within 20h. When the temperature of the filtrate is below 35℃, membrane filtration ends, and filtrate ② is obtained.

[0166] (3) Adjust pH

[0167] The pH of filtrate ② was adjusted to 4.0 using 18% hydrochloric acid solution, and allowed to stand for 100 minutes. Then, solid-liquid separation was performed to obtain filtrate ③.

[0168] (4) Adsorption ①

[0169] The filtrate ③ was adsorbed through an adsorption column (resin dosage 137.6 kg). After adsorption, 24% ammonia solution was added to the adsorption column for elution, and the eluent ① was collected.

[0170] (5) Cyanide

[0171] The eluent ① was introduced into a tubular reactor, heated to 100℃, and hydrogen cyanide was added to adjust the pH to 2.3. The reaction time was controlled at 60 min. After the reaction was completed, 20% sodium hydroxide solution was added to adjust the pH to 3.8, and the reaction was continued for another 40 min. The reaction was then completed, yielding the reaction solution. The cyanidation yield was 95.5%.

[0172] (6) Adsorption ②

[0173] The reaction solution enters the adsorption column through the pipeline for adsorption (resin dosage 101.6 kg). After adsorption is completed, acetone solvent is added to the adsorption column for elution, and the eluent ② is collected.

[0174] (7) Concentration

[0175] The eluent ② was distilled under reduced pressure to remove acetone, and then concentrated to 50% of its original volume.

[0176] (8) Column Chromatography

[0177] 78.6 kg of adsorbent was added to the chromatography column, followed by ethanol solution, and the column was allowed to soak for 160 min. After soaking, the column was washed twice with ethanol. The concentrated solution was added to a glass column, using 80% acetic acid as the mobile phase at a flow rate of 5 L / min, and the chromatographic solution was collected.

[0178] (9) Concentration

[0179] The chromatographic solution was distilled under reduced pressure to remove acetic acid, and then concentrated to 30% of its original volume.

[0180] (10) Crystallization and drying

[0181] The concentrate was cooled to 1°C, and 0.33 kg of pure vitamin B12 was added. The mixture was stirred for 150 min, allowed to stand for 240 min, filtered, and solid vitamin B12 was obtained by solid-liquid separation.

[0182] Solid vitamin B12 was vacuum dried to obtain 29.68 kg of pure product with an effective content of 99.6%, and the yield of sodium chloride as a byproduct was 2172.6 kg.

[0183] Example 6

[0184] Vitamin B12 fermentation broth 120ml 3 The potency was 287 mg / L.

[0185] (1) Fermentation broth pretreatment

[0186] Vitamin B12 fermentation broth entered 160m 3 Pretreatment tank.

[0187] Start the stirring system, control the speed at 45 r / min, heat to 44℃, first add 34.44 kg of BHT, then add 10% hydrochloric acid to adjust the pH to 3.4, and finally add 4.8 kg of zwitterionic polyacrylamide (ACPAM); continue stirring for 40 min, cool to room temperature, let stand for 138 min, and then filter by plate and frame filter to obtain filtrate ①.

[0188] (2) Ceramic membrane filtration

[0189] The filtrate is heated to 39°C, and the processing capacity is 67m³. 3 / h, with the running time controlled within 20h. When the temperature of the filtrate is below 35℃, membrane filtration ends, and filtrate ② is obtained.

[0190] (3) Adjust pH

[0191] The pH of filtrate ② was adjusted to 3.7 using 10% hydrochloric acid solution, and allowed to stand for 72 minutes. Then, solid-liquid separation was performed to obtain filtrate ③.

[0192] (4) Adsorption ①

[0193] The filtrate ③ was adsorbed through an adsorption column (resin dosage 144.6 kg). After adsorption, 23% ammonia solution was added to the adsorption column for elution, and the eluent ① was collected.

[0194] (5) Cyanide

[0195] The eluent ① was introduced into a tubular reactor, heated to 83°C, and hydrogen cyanide was added to adjust the pH to 1.6. The reaction time was controlled at 42 min. After the reaction was completed, 20% sodium hydroxide solution was added to adjust the pH to 3.7, and the reaction was continued for another 24 min. The reaction was then completed, yielding the reaction solution. The cyanidation yield was 95.4%.

[0196] (6) Adsorption ②

[0197] The reaction solution enters the adsorption column through the pipeline for adsorption (resin dosage 106.7 kg). After adsorption is completed, acetone solvent is added to the adsorption column for elution, and the eluent ② is collected.

[0198] (7) Concentration

[0199] The eluent ② was distilled under reduced pressure to remove acetone, and then concentrated to 44% of its original volume.

[0200] (8) Column Chromatography

[0201] Add 82.6 kg of adsorbent to the chromatography column, then add ethanol solution and let it soak for 160 min. After soaking, rinse once more with ethanol. Add the concentrated solution to a glass column, using 77% acetic acid as the mobile phase at a flow rate of 3.4 L / min, and collect the chromatographic solution.

[0202] (9) Concentration

[0203] The chromatographic solution was subjected to vacuum distillation to remove acetic acid, and then concentrated to 22% of its original volume.

[0204] (10) Crystallization and drying

[0205] The concentrate was cooled to 1°C, and 0.34 kg of pure vitamin B12 was added. The mixture was stirred for 123 min, allowed to stand for 182 min, filtered, and solid vitamin B12 was obtained by solid-liquid separation.

[0206] Solid vitamin B12 was vacuum dried to obtain 31.13 kg of pure product with an effective content of 99.6%, and the yield of sodium chloride as a byproduct was 2281.8 kg.

[0207] Comparative Example 1

[0208] Vitamin B12 10mg 3 The fermentation broth (potency 292 mg / L) was hydrolyzed and filtered to obtain a filtrate, which was then adsorbed onto a 10m... 3The solution was washed and desorbed in macroporous resin to obtain a first solution, which was then concentrated to obtain a concentrated solution. 0.3 kg of zinc chloride was added to the concentrated solution, and the pH was adjusted to 7.5 with 30% sodium hydroxide. After filtration, the filter cake was suspended in water and filtered again. This operation was repeated twice, and all filtrates were combined to obtain a purified vitamin B12 solution. The purified solution was then introduced into a reaction vessel, and solid sodium cyanide was added to carry out the cyanidation reaction. After the reaction was completed, the reaction solution was adsorbed onto a 3m... 3 The solution was washed with water and then elute with 10% acetone in the chromatography resin. Development was performed to obtain developing solutions of other types of vitamin B12. After the bands of other types of vitamin B12 were completely removed, 85% acetone was used to obtain a secondary solution of vitamin B12. The secondary solution was adsorbed onto a 300L alumina column and desorbed with 85% acetone to obtain a crystalline solution of 10g of vitamin B12. Acetone was added to the crystalline solution under stirring to crystallize adenosylcobalamin. After filtration and drying, 2.37kg of vitamin B12 with a purity of 98.6% was obtained, and the total yield of vitamin B12 was 81.3%. The yield of sodium chloride as a byproduct was 317.6kg.

[0209] Comparative Example 2

[0210] A pure culture of *Pseudomonas denitrificans* was used to ferment a medium containing maltose, corn steep liquor, and cobalt chloride. The fermentation unit was 283 mg / L. 10 mL of the culture was used. 3The fermentation broth was subjected to solid-liquid separation by adding 60 kg of zinc sulfate flocculant and 48 kg of calcium chloride filter aid to obtain a filter cake. The filter cake was then suspended in water, and a settling agent (containing 2.7 kg each of polyaluminum chloride and magnesium chloride) was added to adjust the pH to 5.1. The mixture was heated to 87°C and held for 30 minutes. After filtration, a suspension filtrate was obtained. The filter cake was then suspended in 5000 kg of water and filtered again. The resulting filtrates were combined and added to the suspension filtrate. The suspension was then fed into a cyanide reaction vessel, sodium cyanide was added, and the synthesis reaction was completed. The mixture was then filtered to obtain the reaction solution. The reaction solution was exchanged at a flow rate of 500 L / h in a 600 L Amberlite™ Cobalamin cation exchange resin column. After elution with 250 L of 26% ammonia solution, a cation exchange column eluent was obtained. The pH was adjusted to 4.9, and the column was filtered to obtain a filtrate. This filtrate was then adsorbed at a flow rate of 20 L / h into a 50 L Amberlite™ 1180 macroporous resin column. After washing with water, the column was developed at a flow rate of 20 L / h with 150 L of developing agent to obtain a developing solution containing vitamin B12. This developing solution was then eluent at a flow rate of 20 L / h with 120 L of eluent to obtain a macroporous eluent. The macroporous eluent was vacuum evaporated until no acetone odor remained, yielding a concentrated solution. This concentrated solution was then passed through a 50 L Amberlite™ 1180 macroporous resin column at a flow rate of 50 L / h. Impurities and pigments were exchanged onto the Amberlite™ IRA900 anion exchange resin. Vitamin B12 was ejected from the resin column using 120L of water, yielding a decolorized solution. The pH of the solution was adjusted to 4.0 with 80% acetic acid. The decolorized solution was then adsorbed onto a 40L Amberlite™ 1600N macroporous resin column at a flow rate of 50L / h. The solution was then elute with 100L of acetone aqueous solution to obtain a crystallization stock solution. Acetone was added to the crystallization stock solution with stirring for 1 hour. After standing for 3 hours, the solution was filtered, washed, sieved, and dried to obtain 2.21kg of vitamin B12 product. The product was tested according to the 2020 edition of the Pharmacopoeia, and the content was 98.6%, with a total vitamin B12 yield of 78.1%. The yield of the byproduct sodium chloride was 298.4kg.

Claims

1. A production process for vitamin B12, comprising the following steps: (1) Fermentation broth pretreatment Vitamin B12 fermentation broth enters the pretreatment tank. Start the stirring system, control the speed at 40-60 r / min, heat to 40-45℃, first add antioxidant, then add hydrochloric acid, adjust the pH to 3.2-3.6, and finally add zwitterionic polyacrylamide (ACPAM). Continue stirring for 40-60 min, cool to room temperature, let stand for 120-140 min, and then filter by plate and frame filter to obtain filtrate ①. (2) Ceramic membrane filtration The filtrate ① obtained in step (1) is heated to 35-40℃, and the processing volume is 6-7 ml. 3 / h, the running time is controlled within 20h, the temperature of the filtrate is below 35℃, the membrane filtration ends, and filtrate ② is obtained; (3) Adjust pH The pH of the filtrate ② obtained in step (2) was adjusted to 2-4 using an acid solution, and the solution was allowed to stand for 60-100 minutes. Then, solid-liquid separation was performed to obtain filtrate ③. (4) Adsorption ① The filtrate ③ obtained in step (3) is adsorbed through an adsorption column. After the adsorption is completed, ammonia solution is added to the adsorption column for elution, and the eluent ① is collected. (5) Cyanide The eluent ① obtained in step (4) is introduced into a pipeline reactor, heated to 80-100℃, hydrogen cyanide is added, pH is adjusted to 1.5-2.3, the reaction time is controlled at 40-60 min, after the reaction is completed, alkali solution is added, pH is adjusted to 3.6-3.8, the reaction continues for 20-40 min, the reaction is completed, and the reaction solution is obtained. (6) Adsorption ② The reaction solution obtained in step (5) is introduced into the adsorption column through the pipeline for adsorption. After the adsorption is completed, acetone solvent is added to the adsorption column for elution, and the eluent ② is collected. (7) Concentration The eluent ② obtained in step (6) is subjected to vacuum distillation to remove acetone, and then concentrated to 40-50% of its original volume to obtain concentrated solution ①. (8) Column Chromatography Add adsorbent to the chromatography column, add ethanol solution to the column, soak for 120-160 min, after soaking, continue to rinse with ethanol 1-2 times, add the concentrated solution ① obtained in step (7) to the glass column, use acetic acid as the mobile phase, the flow rate is 3-5 L / min, and collect the chromatography solution. (9) Concentration The chromatographic solution obtained in step (8) was subjected to vacuum distillation to remove acetic acid, and then concentrated to 20-30% of the original volume to obtain concentrated solution ②. (10) Crystallization and drying Cool the concentrated solution ② obtained in step (9) to 0-5℃, add pure vitamin B12, stir for 120-150 min, let stand for 180-240 min, filter after completion, and separate the solid and liquid to obtain solid vitamin B12. Solid vitamin B12 was dried under vacuum to obtain the pure product.

2. The production process of vitamin B12 according to claim 1, characterized in that... In the process step (1), The concentration of hydrochloric acid should be controlled at 10-20%; Antioxidants refer to butylated hydroxytoluene (BHT) or tea polyphenols, and their dosage is: W antioxidant (kg) = fermentation broth volume (m³) 3 *Fermentation broth potency (mg / L) / 1000; The dosage of zwitterionic polyacrylamide (ACPAM) is: W (kg) = fermentation broth volume (m³). 3 )*4 / 100.

3. The production process of vitamin B12 according to claim 1, characterized in that... In process step (2), the ceramic membrane is made of titanium oxide, with a pore size of 0.5 μm and an effective membrane area of ​​0.36 m². 2 .

4. The production process of vitamin B12 according to claim 1, characterized in that... In process step (3), the acid solution refers to hydrochloric acid, sulfuric acid, or phosphoric acid, with a concentration controlled at 14-18%.

5. The production process of vitamin B12 according to claim 1, characterized in that... In the process step (4), Adsorption column adsorption refers to the addition of strong acid cation exchange resin-001x7 (732) to a resin column, where the resin dosage is W (kg) = fermentation broth volume (m³). 3 *Fermentation broth potency (mg / L)*4.2 / 1000; The ammonia concentration is between 22% and 24%.

6. The production process of vitamin B12 according to claim 1, characterized in that... In the process step (5), The concentration of hydrocyanic acid is 42-45%; Alkaline solution refers to sodium hydroxide or sodium carbonate solution with a concentration of 10-20%.

7. The production process of vitamin B12 according to claim 1, characterized in that... In the process step (6), Adsorption column adsorption refers to the addition of macroporous cation exchange resin D113 to a resin column, with the resin dosage W (kg) = fermentation broth volume (m³). 3 *Fermentation broth potency (mg / L)*3.1 / 1000.

8. The production process of vitamin B12 according to claim 1, characterized in that... In the process step (8), The adsorbent refers to modified urea-formaldehyde resin, and the dosage is W (kg) = fermentation broth volume (m³). 3 *Fermentation broth potency (mg / L)*2.4 / 1000; Acetic acid content 70-80%.

9. The production process of vitamin B12 according to claim 1, characterized in that... In the process step (10), pure vitamin B12 refers to a content of 99.5% or higher, and the dosage is W (kg) = fermentation liquid volume (m³). 3 *Fermentation broth potency (mg / L)* / 100000.