Method for separating and purifying fusidic acid through salting-out crystallization
The salting-out crystallization method for separating and purifying fusidic acid solves the problems of complex processes and high costs in existing technologies, and realizes a low-cost and efficient separation and purification process that is suitable for industrial production.
Patent Information
- Application Number
- CN202511209303.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-27
- Publication Date
- 2025-12-02
AI Technical Summary
Existing methods for separating and purifying fusidic acid are complex, costly, and difficult to meet market demands.
Fusidic acid was separated and purified by salting-out crystallization, which included steps such as fermentation broth filtration, salting-out crystallization, dissolution extraction, crystallization and drying. A single extraction solvent was used to reduce solvent loss and simplify the process.
This method achieves simple and low-cost separation and purification of fusidic acid, which is easy to industrialize and improves production efficiency and solvent recovery rate.
Smart Images
Figure CN121045306A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of chemical substance separation and purification technology, and in particular to a method for separating and purifying fusidic acid by salting out crystallization. Background Technology
[0002] Fusidic acid belongs to the clostridial antibiotic class. It was discovered and extracted in 1962 by Leo Pharmaceuticals of Denmark from the fermentation broth of *Clostridium clavatum*. Fusidic acid is a colorless crystalline solid insoluble in water, but soluble in alcohols, lipids, and other organic compounds. It is a weak acid with a pKa of 5.7. Clinically, its sodium salt form is generally used, as it is readily soluble in water.
[0003] In 2010, Jiangxi Jiuyang Biopharmaceutical Co., Ltd. published a patent entitled "A Method for Extracting and Separating Fusidic Acid." The fermentation broth was filtered through a membrane, adsorbed using macroporous resins D4020 and D312, and then elute with ethanol. The eluent was concentrated under reduced pressure after heating, and subsequently extracted and crystallized using lipid-based organic solvents. This method uses a large amount of organic solvents, requires a large volume of ethanol for eluent extraction, and necessitates a large amount of steam for ethanol recovery, resulting in high energy consumption and costs.
[0004] The 2012 master's thesis from Shanghai Normal University, titled "Preliminary Study on Fermentation and Separation and Purification Process of Fusidic Acid," describes a separation and purification method involving centrifugation and filtration of the fermentation broth under acidic conditions to obtain mycelia containing fusidic acid. After drying, the mycelia are extracted with ethanol or methanol. The extract is then adsorbed and desorbed using macroporous resin, and the resulting solution is concentrated under reduced pressure to obtain a white powder of fusidic acid with a purity greater than 90%. This process is cumbersome, involving tedious mycelial drying, high energy consumption (especially in southern my country where steam costs are even higher), large solvent consumption during extraction, low production safety, and high requirements for industrial-scale production, ultimately failing to meet market demands. Summary of the Invention
[0005] This invention proposes a method for separating and purifying fusidic acid through salting-out crystallization, which overcomes the shortcomings of existing methods for separating and purifying fusidic acid, which are complex and costly.
[0006] The technical solution of this invention is implemented as follows:
[0007] A method for separating and purifying fusidic acid by salting-out crystallization includes the following steps:
[0008] (1) Filtration of fermentation broth: The fermentation broth is filtered to obtain the filtrate;
[0009] (2) Salting out crystallization: Add salt for crystallization to the filtrate, then add acid to adjust the pH to 4.8-5.5, and carry out salting out. After the salt is fully separated, perform solid-liquid separation to obtain the salted-out product.
[0010] (3) Dissolution and extraction: Add extraction solvent to the salt-out and dissolve to a concentration of 100 g / L. After stirring and dissolving, add 0.5 BV volume of drinking water to extract. After standing and separating into layers, collect the extracted lipid phase.
[0011] (4) Crystallization: The extracted lipid phase is concentrated at a temperature of 50-55℃ to a concentration of 220-250 g / L. After cooling and stirring to crystallize, the crude fusidic acid is obtained by filtration.
[0012] (5) Recrystallization: Add the same extraction solvent as in step (3) to crude fusidic acid, dissolve to a concentration of 55-75 g / L, concentrate under reduced pressure to a concentration of 200 g / L after complete dissolution, cool to 10°C and stir to crystallize, then filter to obtain wet powder.
[0013] (6) Drying: The wet powder is dried under reduced pressure at 40-50℃ to obtain fusidic acid product.
[0014] Preferably, in step (1), the fermentation broth is filtered through a plate and frame filter or a membrane filter. The membrane filter is one of ultrafiltration, hollow fiber membrane filtration, or ceramic membrane filtration.
[0015] Preferably, the salt used for crystallization in step (2) is one of a sodium salt, a potassium salt, or an ammonium salt. Specifically, the salt used for crystallization in step (2) is one of sodium chloride, ammonium chloride, potassium chloride, sodium sulfate, or sodium acetate.
[0016] Preferably, oxalic acid, hydrochloric acid, acetic acid, formic acid or sulfuric acid are added in step (2) to adjust the pH.
[0017] Preferably, solid-liquid separation is achieved in step (2) by plate and frame filtration or centrifugation using a flat plate centrifuge.
[0018] Preferably, the extraction solvent is a lipid solvent. Specifically, the extraction solvent is ethyl acetate, butyl acetate, or isopropyl acetate.
[0019] Preferably, in step (4), the lipid phase is extracted for single-effect concentration.
[0020] The beneficial effects of this invention are: the production process is simple, the production cost is low, and it is easier to achieve industrial production. Furthermore, the salting-out crystallization and recrystallization steps use the same extraction solvent, reducing the use of multiple solvents, minimizing solvent loss, facilitating recovery, achieving a high recovery rate, and reducing production costs. Attached Figure Description
[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0022] Figure 1 This is a process diagram of the present invention. Detailed Implementation
[0023] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0024] like Figure 1 A method for separating and purifying fusidic acid by salting-out crystallization includes the following steps:
[0025] (1) Filtration of fermentation broth: The fermentation broth is filtered to obtain the filtrate;
[0026] (2) Salting out crystallization: Add salt for crystallization to the filtrate, then add acid to adjust the pH to 4.8-5.5, and carry out salting out. After the salt is fully separated, perform solid-liquid separation to obtain the salted-out product.
[0027] (3) Dissolution and extraction: Add extraction solvent to the salt-out and dissolve to a concentration of 100 g / L. After stirring and dissolving, add 0.5 BV volume of drinking water to extract. After standing and separating into layers, collect the extracted lipid phase.
[0028] (4) Crystallization: The extracted lipid phase is concentrated at a temperature of 50-55℃ to a concentration of 220-250 g / L. After cooling and stirring to crystallize, the crude fusidic acid is obtained by filtration.
[0029] (5) Recrystallization: Add the same extraction solvent as in step (3) to crude fusidic acid, dissolve to a concentration of 55-75 g / L, concentrate under reduced pressure to a concentration of 200 g / L after complete dissolution, cool to 10°C and stir to crystallize, then filter to obtain wet powder.
[0030] (6) Drying: The wet powder is dried under reduced pressure at 40-50℃ to obtain fusidic acid product.
[0031] Preferably, in step (1), the fermentation broth is filtered through a plate and frame filter or a membrane filter. The membrane filter is one of ultrafiltration, hollow fiber membrane filtration, or ceramic membrane filtration.
[0032] Preferably, the salt used for crystallization in step (2) is one of a sodium salt, a potassium salt, or an ammonium salt. Specifically, the salt used for crystallization in step (2) is one of sodium chloride, ammonium chloride, potassium chloride, sodium sulfate, or sodium acetate.
[0033] Preferably, oxalic acid, hydrochloric acid, acetic acid, formic acid or sulfuric acid are added in step (2) to adjust the pH.
[0034] Preferably, solid-liquid separation is achieved in step (2) by plate and frame filtration or centrifugation using a flat plate centrifuge.
[0035] Preferably, the extraction solvent is a lipid solvent. Specifically, the extraction solvent is ethyl acetate, butyl acetate, or isopropyl acetate.
[0036] Preferably, in step (4), the lipid phase is extracted for single-effect concentration.
[0037] The present invention features a simple production process, low production cost, and is easier to scale up for industrial production. Furthermore, the salting-out crystallization and recrystallization steps use the same extraction solvent, reducing the use of multiple solvents, minimizing solvent loss, facilitating easy recovery, achieving high recovery rates, and reducing production costs.
[0038] Example 1
[0039] A method for separating and purifying fusidic acid by salting-out crystallization includes the following steps:
[0040] (1) Filtration of fermentation broth: The fermentation broth was filtered through a ceramic membrane (pore size of 50 nm) to obtain filtrate. The content of the filtrate was detected by liquid chromatography, and the membrane filtration yield reached 92%.
[0041] (2) Salting out crystallization: Add 0.5 mol / L sodium chloride to the filtrate, then add oxalic acid to adjust the pH to 5.0, and carry out salting out. After stirring for 60 min to fully precipitate, filter with a plate and frame filter to obtain the salted-out product (wet salted-out powder). The yield of wet salted-out powder is 93%.
[0042] (3) Dissolution and extraction: Ethyl acetate was added to the salt-out and dissolved to a concentration of 100 g / L. After stirring and dissolving, 0.5 BV of drinking water was added to extract the ethyl acetate. After standing and separating the layers, the lipid phase was collected. The dissolution and extraction yield reached 94%.
[0043] (4) Crystallization: The extracted lipid phase was concentrated by single-effect concentration at a temperature of 50-55℃ to a concentration of 220-250 g / L. After cooling and stirring for 12 hours, the crude fusidic acid was obtained by filtration. The crude product crystallization yield was 81%, and the purity of the crude product was 97.8%.
[0044] (5) Recrystallization: Add ethyl acetate to crude fusidic acid and dissolve it to a concentration of 75 g / L. After complete dissolution, concentrate it under reduced pressure to a concentration of 200 g / L, cool it to 10°C and stir to crystallize for 3 hours. Filter to obtain wet powder.
[0045] (6) Drying: The wet powder is dried under reduced pressure at 40-50℃ for 12 hours to obtain fusidic acid product with a purity of 99.5%.
[0046] Example 2
[0047] A method for separating and purifying fusidic acid by salting-out crystallization includes the following steps:
[0048] (1) Fermentation broth filtration: The fermentation broth was filtered through a plate and frame filter to obtain the filtrate. The content of the filtrate was detected by liquid chromatography, and the yield of the plate and frame filtration reached 91%.
[0049] (2) Salting out crystallization: Add 1.0 mol / L sodium chloride to the filtrate, then add oxalic acid to adjust the pH to 5.5, and carry out salting out. Stir for 30 min until the precipitate is fully separated, then filter with a plate and frame filter to obtain the salting out product (wet salting out powder). The yield of wet salting out powder is 91%.
[0050] (3) Dissolution and extraction: Ethyl acetate was added to the salt-out and dissolved to a concentration of 100 g / L. After stirring and dissolving, 0.5 BV of drinking water was added to extract the ethyl acetate. After standing and separating the layers, the lipid phase was collected. The dissolution and extraction yield reached 96%.
[0051] (4) Crystallization: The extracted lipid phase was concentrated using a single-effect concentration method, with the concentration temperature controlled at 50-55℃, to a concentration of 220-250 g / L. After cooling and stirring for 12 hours, the mixture was filtered to obtain crude fusidic acid. The crude product crystallization yield was 79%. The purity of the obtained crude product was 98.1%.
[0052] (5) Recrystallization: Add ethyl acetate to crude fusidic acid and dissolve it to a concentration of 55 g / L. After complete dissolution, concentrate it under reduced pressure to a concentration of 200 g / L, cool it to 10°C and stir to crystallize for 3 hours. Filter to obtain wet powder.
[0053] (6) Drying: The wet powder is dried under reduced pressure at 40-50℃ for 12 hours to obtain fusidic acid product with a purity of 99.6%.
[0054] Example 3
[0055] A method for separating and purifying fusidic acid by salting-out crystallization includes the following steps:
[0056] (1) Filtration of fermentation broth: The fermentation broth was filtered through a ceramic membrane (pore size of 50 nm) to obtain filtrate. The content of the filtrate was detected by liquid chromatography, and the membrane filtration yield reached 90%.
[0057] (2) Salting out crystallization: Add 0.3 mol / L sodium sulfate to the filtrate, then add oxalic acid to adjust the pH to 4.8, and carry out salting out. Stir for 60 min until the precipitate is fully separated, then filter with a plate and frame filter to obtain the salted-out product (i.e., salted-out wet powder). The yield of salted-out wet powder is 94%.
[0058] (3) Dissolution and extraction: Ethyl acetate was added to the salt-out and dissolved to a concentration of 100 g / L. After stirring and dissolving, 0.5 BV of drinking water was added to extract the ethyl acetate. After standing and separating the layers, the lipid phase was collected. The yield of dissolution and extraction reached 93%.
[0059] (4) Crystallization: The extracted lipid phase was concentrated by single-effect concentration at 50-55℃ to a concentration of 220-250 g / L. After cooling to 10℃ and stirring for 6 hours, the crude fusidic acid was obtained by filtration. The crude product crystallization yield was 80%, and the purity of the crude product was 97.9%.
[0060] (5) Recrystallization: Add ethyl acetate to crude fusidic acid and dissolve it to a concentration of 60 g / L. After complete dissolution, concentrate it under reduced pressure to a concentration of 200 g / L, cool it to 10°C and stir to crystallize for 3 hours. Filter to obtain wet powder.
[0061] (6) Drying: The wet powder is dried under reduced pressure at 40-50℃ for 12 hours to obtain fusidic acid product with a purity of 99.4%.
[0062] Example 4
[0063] A method for separating and purifying fusidic acid by salting-out crystallization includes the following steps:
[0064] (1) Filtration of fermentation broth: The fermentation broth was filtered through a ceramic membrane (pore size of 50 nm) to obtain filtrate. The content of the filtrate was detected by liquid chromatography, and the membrane filtration yield reached 93%.
[0065] (2) Salting out crystallization: Add 1.0 mol / L sodium chloride to the filtrate, then add oxalic acid to adjust the pH to 5.2, and carry out salting out. Stir for 45 min until the precipitate is fully separated, then centrifuge to obtain the salted-out product (i.e., salted-out wet powder). The yield of salted-out wet powder is 93%.
[0066] (3) Dissolution and extraction: Ethyl acetate was added to the salt-out and dissolved to a concentration of 100 g / L. After stirring and dissolving, 0.5 BV of drinking water was added to extract the ethyl acetate. After standing and separating the layers, the lipid phase was collected. The yield of dissolution and extraction was 94%.
[0067] (4) Crystallization: The extracted lipid phase was concentrated by single-effect concentration at a temperature of 50-55℃ to a concentration of 220-250 g / L. After cooling and stirring for 12 hours, the crude fusidic acid was obtained by filtration. The crude product crystallization yield was 78%, and the purity of the crude product was 98.4%.
[0068] (5) Recrystallization: Add ethyl acetate to crude fusidic acid and dissolve it to a concentration of 75 g / L. After complete dissolution, concentrate it under reduced pressure to a concentration of 200 g / L, cool it to 10°C and stir to crystallize for 3 hours. Filter to obtain wet powder.
[0069] (6) Drying: The wet powder is dried under reduced pressure at 40-50℃ for 12 hours to obtain fusidic acid product with a purity of 99.6%.
[0070] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.
Claims
1. A method for separating and purifying fusidic acid by salting-out crystallization, characterized in that: Includes the following steps: (1) Filtration of fermentation broth: The fermentation broth is filtered to obtain the filtrate; (2) Salting out crystallization: Add salt for crystallization to the filtrate, then add acid to adjust the pH to 4.8-5.5, and carry out salting out. After the salt is fully separated, perform solid-liquid separation to obtain the salted-out product. (3) Dissolution and extraction: Add extraction solvent to the salt-out and dissolve to a concentration of 100 g / L. After stirring and dissolving, add 0.5 BV volume of drinking water to extract. After standing and separating into layers, collect the extracted lipid phase. (4) Crystallization: The extracted lipid phase is concentrated at a temperature of 50-55℃ to a concentration of 220-250 g / L. After cooling and stirring to crystallize, the crude fusidic acid is obtained by filtration. (5) Recrystallization: Add the same extraction solvent as in step (3) to crude fusidic acid, dissolve to a concentration of 55-75 g / L, concentrate under reduced pressure to a concentration of 200 g / L after complete dissolution, cool to 10°C and stir to crystallize, then filter to obtain wet powder. (6) Drying: The wet powder is dried under reduced pressure at 40-50℃ to obtain fusidic acid product.
2. The method for separating and purifying fusidic acid by salting-out crystallization as described in claim 1, characterized in that: Step (1) The fermentation broth is filtered through a plate and frame filter or a membrane filter.
3. The method for separating and purifying fusidic acid by salting-out crystallization as described in claim 2, characterized in that: The membrane filtration is one of ultrafiltration membrane filtration, hollow fiber membrane filtration, or ceramic membrane filtration.
4. The method for separating and purifying fusidic acid by salting-out crystallization as described in claim 1, characterized in that: The salt used for crystallization in step (2) is one of sodium, potassium, or ammonium salts.
5. The method for separating and purifying fusidic acid by salting-out crystallization as described in claim 4, characterized in that: The salt used for crystallization in step (2) is one of sodium chloride, ammonium chloride, potassium chloride, sodium sulfate, or sodium acetate.
6. The method for separating and purifying fusidic acid by salting-out crystallization as described in claim 1, characterized in that: In step (2), add oxalic acid, hydrochloric acid, acetic acid, formic acid or sulfuric acid to adjust the pH.
7. The method for separating and purifying fusidic acid by salting-out crystallization as described in claim 1, characterized in that: In step (2), solid-liquid separation is achieved by plate and frame filtration or centrifugation using a flat plate centrifuge.
8. The method for separating and purifying fusidic acid by salting-out crystallization as described in claim 1, characterized in that: The extraction solvent is a lipid solvent.
9. The method for separating and purifying fusidic acid by salting-out crystallization as described in claim 8, characterized in that: The extraction solvent is ethyl acetate, butyl acetate, or isopropyl acetate.
10. The method for separating and purifying fusidic acid by salting-out crystallization as described in claim 1, characterized in that: In step (4), the lipid phase is extracted for single-effect concentration.