A method for extracting and purifying tylosin

Abstract

This invention relates to a method for the extraction and purification of tylosin. The method employs a polyamide-macroporous resin combination for adsorption and decolorization, combined with a gradient cooling secondary crystallization method. This establishes a novel process for separating and purifying tylosin from fermentation broth, optimizing the tylosin extraction and purification process and making it more suitable for industrial production. The extraction and purification method of this invention achieves a product yield of over 97.5% and a purity of over 95%.

Description

Technical Field

[0001] This invention belongs to the field of antibiotic purification technology, specifically relating to a method for extracting and purifying tylosin. Background Technology

[0002] Tylosin, also known as acetyl-4"-isovalery tylosin (AIV) and sometimes called super tylosin, is a macrolide antibiotic primarily produced by heat-resistant Streptomyces. Thermotolerans (Tylotolerans) are obtained through fermentation and were initially discovered by Eco Animal Health Products Ltd. in the UK. In 2002, the Ministry of Agriculture of my country approved the registration and sale of acetylisovaleryl tylosin in China. There are two dosage forms of acetylisovaleryl tylosin sold on the market: a premix, suitable for pigs, which effectively treats swine asthma and prevents and treats dysentery caused by *Treponema pallidum*, necrotic enteritis in piglets, and proliferative enteritis in pigs caused by *Laurinaria spp.*; and a water-soluble powder, administered via drinking water, suitable for chickens, and effective in treating and preventing respiratory infections in chickens. As a novel macrolide antibiotic, acetylisovaleryl tylosin overcomes the shortcomings of other macrolide drugs, possessing advantages such as high efficiency, low toxicity, low residue, and no drug resistance among macrolides. The structural formula of tylosin is as follows:

[0003]

[0004] Currently, research on tylvaporin mainly focuses on its biosynthetic pathway and fermentation process. The biosynthetic pathway shows that tylvaporin synthesis produces many intermediate products, such as 2'-O-acetyl-tylosin, 2',4”'-O-diacetyl-tylosin, and 2',4”'-O-triacetyl-tylosin. Isovalerate is then added to synthesize 2',4”'-O-diacetyl-4”-O-isovalerate tylosin and 3,2',4”'-O-triacetyl-4”-O-isovalerate tylosin, ultimately yielding 3-O-acetyl-4”-O-isovalerate tylosin (acetylisovalerate tylosin).

[0005] Tiamulin is weakly alkaline in aqueous solution and crystallizes out as a precipitate under alkaline conditions. Under acidic conditions, tiamulin forms salts with most acids and is readily soluble in water. Existing tiamulin purification processes primarily employ secondary crystallization and organic solvent extraction methods. However, in implementing the technical solutions described in this application, the present invention has discovered at least the following technical problems with these methods:

[0006] 1) The extraction process is prone to emulsification, has high toxicity, easily pollutes the environment, has low yield, and high production costs, which is not conducive to large-scale production.

[0007] 2) The secondary crystallization method generates too many impurities during the process, making it difficult to crystallize. This results in the finished product having excessive impurities and poor crystallization effect, with high purity but low yield. Summary of the Invention

[0008] The purpose of this invention is to provide a method for the extraction and purification of tivalmycin. By using polyamide-macroporous resin for adsorption and decolorization, combined with gradient cooling secondary crystallization, the technical problems of high impurity content and low purity of tivalmycin in extraction and purification methods are solved, effectively improving the purity of tivalmycin while increasing the yield.

[0009] To achieve the above-mentioned objectives, the technical solution adopted is as follows:

[0010] A method for extracting and purifying tylosin, comprising the following steps:

[0011] (1) Add acidic solution to the fermentation broth of tivamectin, acidify it, and then filter it through a plate and frame filter to obtain a primary acid solution of tivamectin.

[0012] (2) The tivacin acid solution obtained in step (1) is decolorized by adsorption with polyamide-macroporous resin to obtain tivacin decolorized solution.

[0013] (3) Add the tivacin decolorizing solution described in step (2) to an alkaline solution and cool it down gradually to crystallize, thereby obtaining tivacin primary crystals;

[0014] (4) Dissolve the primary crystallizer obtained in step (3) in an acidic solution to obtain a secondary acidic solution of tylosin;

[0015] (5) Add the tivamectin secondary acid solution obtained in step (4) to an alkaline solution for secondary crystallization to obtain the tivamectin finished product.

[0016] In step (1), the pH of the acidic solution is adjusted to 2.5-4.

[0017] The acidification time in step (1) is 1 to 2 hours.

[0018] In step (3), an alkaline solution is added to adjust the pH to 8-10.

[0019] In step (3), the gradient cooling rate is 5-15℃ / 10min.

[0020] In step (4), the pH of the acidic solution is adjusted to 2.5-4.

[0021] In step (5), an alkaline solution is added to adjust the pH to 8-10.

[0022] In step (5), the vacuum degree of secondary crystallization is 0.1 to 0.3 MPa, and the crystallization endpoint temperature is 0 to 8 °C.

[0023] The acidic solution mentioned above is selected from sulfuric acid, hydrochloric acid, tartaric acid, phosphoric acid, and citric acid solutions, with tartaric acid being preferred.

[0024] The alkaline solution mentioned above is selected from sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, and ammonia solution, preferably sodium hydroxide.

[0025] The technical solution of the present invention has at least the following beneficial technical effects:

[0026] 1) This invention uses polyamide-macroporous resin for adsorption and decolorization. First, polyamide effectively and accurately removes inorganic salts, most proteins, polysaccharides, lipids and pigments from the fermentation broth. Then, macroporous resin is used to further remove residual small molecule proteins, polysaccharides, lipids and pigments.

[0027] 2) In this invention, resin decolorization is used instead of activated carbon decolorization for the fermentation broth. While the decolorization effect of activated carbon decolorization and resin decolorization is similar, a significant problem with activated carbon decolorization is its adsorption of tiamulin in the fermentation broth, leading to a decrease in tiamulin concentration after adsorption. Therefore, using resin decolorization instead of activated carbon decolorization is beneficial for improving the final yield of tiamulin.

[0028] 3) The present invention employs a secondary crystallization method that uses gradient cooling crystallization and adjusts the pH of the fermentation broth to ensure that tivamectin exists in the form of free alkali in the fermentation broth rather than in the form of tivamectin salt after fermentation. At low temperatures, the solubility of tivamectin decreases sharply, which is more conducive to the precipitation of tivamectin crystals.

[0029] 4) The yield of the product obtained by the extraction and purification method of the present invention is over 97.5%, and the content is over 95%. Detailed Implementation

[0030] Example 1

[0031] Take 50 ml of tylosin fermentation broth with a potency of 28000 U / ml. 3The total amount was 1400U. It was acidified with 1mol / L sulfuric acid, with the final pH value being 2.5–3, and allowed to stand for 1.5 hours. After removing bacterial residue by plate and frame filtration, the filtrate was filtered through a polyamide column, washed with purified water, and the filtrate was collected. The resulting filtrate was then passed through a macroporous resin column, and finally washed with purified water, and the decolorized solution was collected. NaHCO3 was added to the decolorized solution to adjust the pH to 8–8.5, and crystallization was carried out at a cooling rate of 15℃ / 10min to obtain the crude product. The crude product was then dissolved in 6mol / L tartaric acid solution until the solution was clear. NaHCO3 was added to the resulting solution to adjust the pH to 8–8.5, and finally crystallization was carried out using a vacuum adiabatic crystallization method at a vacuum degree of 0.15 MPa, with the final crystallization temperature being 0–3℃. After crystallization, centrifugation yielded tylosin. The product yield was 98.1%, and the tylosin content was 95.2%.

[0032] Example 2

[0033] Take 50 m3 of tylosin fermentation broth with a potency of 27500 U / ml (total billion U 1375 U), add 2 mol / L hydrochloric acid for acidification, with the final pH value being 3-4, and let it stand for 2 hours. After removing bacterial residue by plate and frame filtration, filter the filtrate through a polyamide column, rinse with purified water and collect the filtrate. Then pass the filtrate through a macroporous resin column, finally rinse the macroporous resin column with purified water and collect the decolorized solution. Add NaOH to the decolorized solution to adjust the pH to 8.5-9, and crystallize at a cooling rate of 12℃ / 10min to obtain the crude product. Dissolve the crude product in 5 mol / L tartaric acid solution until the solution is clear, add NaOH to the resulting solution to adjust the pH to 8.5-9, and finally crystallize using a vacuum adiabatic crystallization method at a vacuum degree of 0.1 MPa, with the final crystallization temperature being 3-5℃. Centrifuge after crystallization to obtain tylosin. The product yield is 98.4%, and the tylosin content is 96.6%.

[0034] Example 3

[0035] Take 50 m3 of tylosin fermentation broth with a potency of 27500 U / ml (total billion U 1375 U), add 4 mol / L tartaric acid for acidification, with the final pH value being 3-4, and let it stand for 1.5 h. After removing the bacterial residue by plate and frame filtration, filter the filtrate through a polyamide column, rinse with purified water and collect the filtrate. Then pass the filtrate through a macroporous resin column, finally rinse the macroporous resin column with purified water and collect the decolorized solution. Add Na2CO3 to the decolorized solution to adjust the pH to 9-9.5, and crystallize at a cooling rate of 13℃ / 10 min to obtain the crude product. Dissolve the crude product in 6.5 mol / L tartaric acid solution until the solution is clear, add Na2CO3 to the solution to adjust the pH to 9-9.5, and finally crystallize using a vacuum adiabatic crystallization method at a vacuum degree of 0.2 MPa, with the final crystallization temperature being 4-6℃. Centrifuge after crystallization to obtain tylosin. The product yield was 97.6%, and the tylosin content was 95.7%.

[0036] Example 4

[0037] Take 50 m3 of tylosin fermentation broth with a potency of 28000 U / ml (total billion units: 1400 U), add 5 mol / L phosphoric acid for acidification, with the final pH value being 3.5–4, and let it stand for 1 hour. After removing bacterial residue by plate and frame filtration, filter the filtrate through a polyamide column, rinse with purified water and collect the filtrate. Then pass the filtrate through a macroporous resin column, finally rinse the macroporous resin column with purified water and collect the decolorized solution. Add KOH to the decolorized solution to adjust the pH to 9.5–10, and crystallize at a cooling rate of 10℃ / 10min to obtain the crude product. Dissolve the crude product in 8 mol / L tartaric acid solution until the solution is clear, add KOH to the resulting solution to adjust the pH to 9.5–10, and finally crystallize using a vacuum adiabatic crystallization method at a vacuum degree of 0.3 MPa and a final crystallization temperature of 0–4℃. Centrifuge after crystallization to obtain tylosin. The product yield is 98.3%, and the tylosin content is 95.3%.

[0038] Example 5

[0039] Take 50 ml of tylosin fermentation broth with a potency of 28500 U / ml. 3The total amount was 1425 U. It was acidified with 3 mol / L citric acid, with the final pH value being 2.5–3.5, and allowed to stand for 1.5 hours. After removing bacterial residue by plate and frame filtration, the filtrate was filtered through a polyamide column, washed with purified water, and the filtrate was collected. The resulting filtrate was then passed through a macroporous resin column, and finally washed with purified water, and the decolorized solution was collected. Ammonia was added to the decolorized solution to adjust the pH to 8–9, and crystallization was carried out at a cooling rate of 12℃ / 10min to obtain the crude product. The crude product was then dissolved in 5 mol / L tartaric acid solution until the solution was clear. Ammonia was added to the resulting solution to adjust the pH to 8–9, and finally crystallization was carried out using a vacuum adiabatic crystallization method at a vacuum degree of 0.1 MPa and a final crystallization temperature of 0–3℃. After crystallization, centrifugation yielded tylosin. The product yield was 97.9%, and the tylosin content was 96.2%.

[0040] Comparative Example 1

[0041] After the fermentation broth was acidified with hydrochloric acid and filtered, the filtrate was filtered through a polyamide column, washed with purified water and the filtrate was collected. The obtained filtrate was then passed through a macroporous resin column, and finally the macroporous resin column was washed with purified water and the decolorized solution was collected. The decolorized solution was added with NaOH to adjust the pH of the solution to 7-8, and crystallization was performed to obtain the crude product. The crude product was dissolved in tartaric acid until it became clear, and then NaOH was added to adjust the pH of the solution to 7-8. The final crystallization temperature was 0-3°C. The product was obtained by isothermal crystallization to obtain tylosin. The remaining conditions were the same as in Control Example 1. The yield was 96.0% and the content was 93.1%.

[0042] Comparative Example 2

[0043] After the fermentation broth was acidified with hydrochloric acid and filtered, it was decolorized with activated carbon. The resulting decolorized solution was adjusted to pH 7-8 with NaOH, and crystallized at a cooling rate of 12℃ / 10min. The crude product was dissolved in tartaric acid until clear, and then the pH of the solution was adjusted to 7-8 with NaOH again. Finally, it was crystallized using a vacuum adiabatic crystallization method with a vacuum degree of 0.1 MPa and a final crystallization temperature of 0-3℃. The remaining conditions were the same as in Control Example 1. The yield was 95.3% and the content was 93.9%.

[0044] Comparative Example 3

[0045] After the fermentation broth was acidified with hydrochloric acid and filtered, it was decolorized with activated carbon. The resulting decolorized solution was adjusted to pH 7-8 with NaOH, and the crude product was obtained by crystallization. The crude product was dissolved in tartaric acid until clear, and then the pH of the solution was adjusted to 7-8 with NaOH again. The final crystallization temperature was 0-3℃. The product was obtained by isothermal crystallization. The remaining conditions were the same as in Control Example 1. The yield was 94.1% and the content was 91.5%.

Claims

1. A method for extracting and purifying tylosin, characterized in that... The following steps are involved: (1) Add acidic solution to the fermentation broth of tivamectin, acidify it, and then filter it through a plate and frame filter to obtain a primary acid solution of tivamectin. (2) The tivacin acid solution obtained in step (1) is decolorized by adsorption with polyamide-macroporous resin to obtain tivacin decolorized solution. (3) Add the tivacin decolorizing solution from step (2) to an alkaline solution and crystallize by gradient cooling to obtain tivacin primary crystals, wherein the pH of the alkaline solution is adjusted to 8-10 and the gradient cooling rate is 5-15℃ / 10min. (4) Dissolve the primary crystallizer obtained in step (3) in an acidic solution to obtain a secondary acidic solution of tylosin; (5) Add the tivamectin secondary acid solution from step (4) to an alkaline solution for secondary crystallization to obtain the tivamectin product. The pH of the alkaline solution is adjusted to 8-10, the vacuum degree of the secondary crystallization is 0.1-0.3 MPa, and the crystallization endpoint temperature is 0-8°C.

2. The method for extracting and purifying tylosin according to claim 1, characterized in that... The pH of the acidic solution described in step (1) is adjusted to 2.5-4.

3. The method for extracting and purifying tylosin according to claim 1, characterized in that... The acidification time in step (1) is 1 to 2 hours.

4. The method for extracting and purifying tylosin according to claim 1, characterized in that... The pH of the acidic solution in step (4) is adjusted to 2.5-4.

5. The method for extracting and purifying tylosin according to claim 1, characterized in that... The acidic solution is selected from one of sulfuric acid, hydrochloric acid, tartaric acid, phosphoric acid, and citric acid solutions.

6. The method for extracting and purifying tylosin according to claim 1, characterized in that... The alkaline solution is selected from one of sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, and ammonia solution.

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