A method for improving the filtration efficiency of vitamin B12 fermentation broth
By treating the vitamin B12 filtrate after ammonia desorption with a molecular sieve filter, the problem of pH increase in the filtrate caused by ammonia desorption was solved, the recovery rate and purity of vitamin B12 were improved, the impurity content was reduced, and a stable, safe and economical production process was achieved.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- NINGXIA JINMEIYINO ANIMAL BEVERAGE CO LTD
- Filing Date
- 2024-11-25
- Publication Date
- 2026-05-26
AI Technical Summary
Existing technologies that use ammonia to decompose vitamin B12 result in an increase in the pH of the filtrate, which destroys the vitamin B12 and increases the content of impurities, leading to unstable production and potential safety hazards.
A molecular sieve filter is used to remove ammonia molecules and other small molecule impurities by adjusting the reaction parameters of the eluent, avoiding the use of hydrochloric acid and maintaining an acidic environment in the filtrate. After eluent is used, the filtrate is filtered through a molecular sieve.
It effectively protects vitamin B12 from degradation, reduces impurity content, improves product quality, reduces subsequent processing volume, lowers production costs, and enables continuous operation.
Smart Images
Figure BDA0005149865920000031 
Figure BDA0005149865920000041
Abstract
Description
Technical Field
[0001] This invention relates to a method for improving the filtration efficiency of vitamin B12 in fermentation broth, belonging to the field of fermentation product extraction technology. Background Technology
[0002] Vitamin B12, also known as cobalamin, is an essential vitamin for the life activities of eukaryotes, playing a crucial role in cellular metabolism, particularly in DNA synthesis, methylation modification, and mitochondrial metabolism. Cobalamin mainly includes cyanocobalamin, adenosylcobalamin, hydroxycobalamin, and methylcobalamin. Adenosylcobalamin is one of the naturally synthesized active forms in prokaryotes and is also known as coenzyme B12. The chemical structure of cobalamin includes a central cobalt porphyrin ring, upper ligand groups (cyano, adenosine, hydroxyl, methyl, etc.), and a lower ligand 5,6-dimethylbenzimidazole group. Vitamin B12 is soluble in water, sparingly soluble in ethanol, and insoluble in acetone and ether.
[0003] Due to the complexity and cumbersome operation of chemical synthesis, vitamin B12 production currently relies on microbial fermentation. Industrially, vitamin B12 is mainly produced through bacterial culture fermentation. The basic extraction process includes: fermentation broth, flocculation, plate and frame filtration, filter cake conditioning, heating hydrolysis, plate and frame filtration, cation exchange resin adsorption, ammonia desorption, sodium cyanide conversion, macroporous resin adsorption, acetone desorption, alumina chromatography, crystallization, and drying. After removing a large amount of macromolecular impurities such as proteins from the fermentation broth, the filtrate is adsorbed onto a 50-type cation exchange resin column for impurity removal. This process effectively removes cationic impurities from the filtrate. However, the use of ammonia during desorption introduces ammonia molecules, raising the pH of the filtrate. Vitamin B12 is destroyed in an alkaline environment, thus reducing yield and increasing impurity content. Currently, to address this issue, hydrochloric acid is used to adjust the pH of the desorption solution during the process. This process requires operators to constantly monitor the filtrate pH and involves the use of the hazardous reagent hydrochloric acid, both of which introduce uncertainties into production.
[0004] Molecular sieve filters are filtration devices based on the principle of molecular sieves, primarily used to separate substances with small molecular sizes. Their principle involves selectively separating molecules of different sizes through the pore size and channel structure of the molecular sieve material. Currently, there are no literature reports on using molecular sieves to obtain vitamin B12 after ammonia desorption. Summary of the Invention
[0005] In view of this, the technical problem to be solved by the present invention is to provide a reaction system that, by adjusting the relevant reaction parameters of the eluent in the system, utilizes molecular sieves to efficiently remove ammonia molecules and other small molecule impurities from vitamin B12 filtrate.
[0006] This invention provides a method for efficiently removing ammonia molecules and other small molecule impurities from vitamin B12 filtrate using molecular sieves, comprising the following steps:
[0007] a) The filtrate after plate and frame filtration is adsorbed into the resin column of the cation exchange resin;
[0008] b) Use ammonia water for analysis;
[0009] c) The eluent obtained from the analysis is filtered through a molecular sieve.
[0010] d) Wash the remaining filtrate with purified water and determine the vitamin B12 content in the filtrate using high performance liquid chromatography.
[0011] Preferably, in step a), the adsorption capacity is 0.5-10 g / L.
[0012] Preferably, in step b), the concentration of ammonia water is 5%-25%, more preferably 8%-12%.
[0013] Preferably, in step b), the volume of the ammonia water desorption solution is 0.2-2L, and more preferably 1.2-2L.
[0014] Preferably, in step c), the molecular sieve pore size is 4.1A-20A, and more preferably 8.6-12A.
[0015] Compared with existing technologies, this invention effectively removes small molecule impurities such as ammonia from the 50-column eluent, alters the alkaline environment of the filtrate to prevent vitamin B12 from being damaged, reduces impurity content, and improves the quality of vitamin B12. Furthermore, the molecular sieve filtration process concentrates the filtrate, significantly reducing the volume of feed solution required for subsequent processes, and allows for continuous operation, greatly saving production costs. Detailed Implementation
[0016] Example 1
[0017] At room temperature, the filtrate from plate and frame filtration was adsorbed onto a resin column containing 100 ml of 50-column cation exchange resin at an adsorption capacity of 3 g / L. The filtrate was then eluented with 500 ml of 5% ammonia solution at -3 to -7 °C. The eluent was filtered through a molecular sieve with a pore size of 4.1 Å. The remaining filtrate was washed with purified water to a volume of 100 ml. The pH of the filtrate was determined to be 6.5. High-performance liquid chromatography (HPLC) determined the vitamin B12 content in the filtrate to be 2870.6 mg / L, with a recovery rate of 95.69% and a total impurity content of 16.8%.
[0018] Comparative Example 1
[0019] At room temperature, the filtrate from plate and frame filtration was adsorbed onto a resin column containing 100 ml of 50-column cation exchange resin at an adsorption capacity of 3 g / L. The filtrate was then eluented with 500 ml of 5% ammonia solution at -3 to -7°C. The pH of the eluent was adjusted to 6.5 with hydrochloric acid, and the vitamin B12 content in the filtrate was determined by high performance liquid chromatography to be 550.6 mg / L, with a recovery rate of 91.77% and a total impurity content of 28.6%.
[0020] Example 2
[0021] At room temperature, the filtrate from plate and frame filtration was adsorbed onto a resin column containing 100 ml of 50-column cation exchange resin at an adsorption capacity of 10 g / L. The filtrate was then eluented with 2000 ml of 8% ammonia solution at -3 to -7 °C. The eluent was filtered through a molecular sieve with a pore size of 8.6 Å. The remaining filtrate was washed with purified water to a volume of 100 ml. The pH of the filtrate was determined to be 7.2. High-performance liquid chromatography (HPLC) determined the vitamin B12 content in the filtrate to be 9865.6 mg / L, with a recovery rate of 98.66% and a total impurity content of 13.6%.
[0022] Comparative Example 2
[0023] At room temperature, the filtrate from plate and frame filtration was adsorbed onto a resin column containing 100 ml of 50-column cation exchange resin at an adsorption capacity of 10 g / L. The filtrate was then eluented with 2000 ml of 8% ammonia solution at -3 to -7°C. The pH of the eluent was adjusted to 7.2 with hydrochloric acid, and the vitamin B12 content in the filtrate was determined by high performance liquid chromatography to be 450.6 mg / L, with a recovery rate of 90.12% and a total impurity content of 33.6%.
[0024] Example 3
[0025] At room temperature, the filtrate from plate and frame filtration was adsorbed onto a resin column containing 100 ml of 50-column cation exchange resin at an adsorption capacity of 8 g / L. The filtrate was then eluented with 1200 ml of 12% ammonia solution at -3 to -7°C. The eluent was filtered through a molecular sieve with a pore size of 12 Å. The remaining filtrate was washed with purified water to a volume of 100 ml. The pH of the filtrate was determined to be 6.8. High-performance liquid chromatography (HPLC) determined the vitamin B12 content in the filtrate to be 7866.4 mg / L, with a recovery rate of 98.33% and a total impurity content of 15.3%.
[0026] Example 4
[0027] At room temperature, the filtrate from plate and frame filtration was adsorbed onto a resin column containing 100 ml of 50-column cation exchange resin at an adsorption capacity of 0.5 g / L. The filtrate was then eluented with 200 ml of 25% ammonia solution at -3 to -7 °C. The eluent was filtered through a molecular sieve with a pore size of 20 Å. The remaining filtrate was washed with purified water to a volume of 20 ml. The pH of the filtrate was determined to be 6.3. High-performance liquid chromatography (HPLC) determined the vitamin B12 content in the filtrate to be 2427.5 mg / L, with a recovery rate of 97.1% and a total impurity content of 16.9%.
[0028] Comparative Example 3
[0029] At room temperature, the filtrate from plate and frame filtration was adsorbed onto a resin column containing 100 ml of 50-column cation exchange resin at an adsorption capacity of 0.5 g / L. The filtrate was then eluented with 200 ml of 25% ammonia at -3 to -7 °C. The pH of the eluent was adjusted to 6.3 with hydrochloric acid, and the vitamin B12 content in the filtrate was determined by high performance liquid chromatography to be 231.6 mg / L, with a recovery rate of 92.64% and a total impurity content of 26.9%.
[0030] Table 1. Comparison of Vitamin B12 content and product quality obtained from Examples 1-4 and Comparative Examples 1-3.
[0031]
[0032]
[0033] As can be seen from the table above, this invention can effectively solve the problem of damage to vitamin B12 caused by ammonia water desorption, change the alkaline environment of the filtrate, and avoid damage to vitamin B12; significantly reduce the impurity content and improve the quality of vitamin B12; the production process is more stable and safe, and has no pollution to the environment; and after molecular sieve filtration, the filtrate is concentrated, which greatly reduces the volume of the feed liquid in subsequent processes, saves production costs, and is easy to promote for large-scale industrial production.
Claims
1. A method for improving the filtration efficiency of vitamin B12 fermentation broth, characterized in that, Includes the following steps: a) The filtrate after plate and frame filtration is adsorbed into the resin column of the cation exchange resin; b) Use ammonia water for analysis; c) The eluent obtained from the analysis is filtered through a molecular sieve. d) Wash the remaining filtrate with purified water and determine the vitamin B12 content in the filtrate using high performance liquid chromatography.
2. The method for improving the filtration efficiency of vitamin B12 fermentation broth according to claim 1, characterized in that: The ammonia concentration in step b) is 5%-25%.
3. The method for improving the filtration efficiency of vitamin B12 fermentation broth according to claim 2, characterized in that: The ammonia concentration in step b) is 8%-12%.
4. The method for improving the filtration efficiency of vitamin B12 fermentation broth according to claim 1, characterized in that: In step b), the volume of the ammonia water eluent is 0.2-2L.
5. The method for improving the filtration efficiency of vitamin B12 fermentation broth according to claim 4, characterized in that: In step b), the volume of the ammonia water desorption solution is 1.2-2L.
6. The method for improving the filtration efficiency of vitamin B12 fermentation broth according to claim 1, characterized in that: In step c), the molecular sieve pore size is 4.1A-20A.
7. The method for improving the filtration efficiency of vitamin B12 fermentation broth according to claim 6, characterized in that: In step c), the molecular sieve pore size is 8.6A-12A.