METHOD FOR EXTRACTING MUPIROCIN

Abstract

A method for extracting mupirocin. The main steps thereof comprise: resin adsorption, desorption and concentration, multi-step extraction, and dehydration and decoloration, wherein the multi-step extraction comprises at least one ester solvent extraction and at least one alkaline water extraction. The method is suitable for industrialization, the extraction recovery rate of mupirocin is 80% or more, and the purity of the mupirocin is 80% or more.

Claims

1. A method for extracting mupirocin, comprising adsorbing a mupirocin solution with a resin, desorbing with a first organic solvent to obtain a desorption solution, concentrating the desorption solution, retaining an aqueous phase, and extracting the aqueous phase, wherein the extraction is a multi-step extraction comprising at least one ester solvent extraction and at least one alkaline water extraction.

2. The method according to claim 1, comprising: a. resin adsorption: adding the resin into a mupirocin fermentation broth and stirring for adsorption, filtering the fermentation broth, rinsing, and collecting the resin; b. desorption and concentration: loading the resin collected in step a on a desorption column; soaking the resin with ethyl acetate or acetone, desorbing, and collecting and concentrating the desorption solution; and c. extraction: extracting the concentrated desorption solution in step b, and separating a solvent phase; wherein the extraction is a multi-step extraction comprising at least one ester solvent extraction and at least one alkaline water extraction.

3. The method according to claim 2, wherein the solvent phase collected in step c is dehydrated and decolored in sequence.

4. The method according to claim 2, wherein the resin is a nonpolar macroporous adsorption resin and is selected from AB-8 resin, H103 resin, X-5 resin, H107 resin or D3520 resin; and/or the feeding amount of the resin is 0.01 to 0.05 g mupirocin/g resin; and/or the pH of the resin adsorption is 5.0 to 7.0.

5-10. (canceled)

11. The method according to claim 4, wherein the pH of the resin adsorption is 5.5 to 6.5.

12. The method according to claim 2, wherein the amount of ethyl acetate or acetone used for the desorption is 2 to 5 times the volume of the resin; and/or the flow rate of the desorption is ⅕ to 6/5 times the volume of the resin per hour.

13. The method according to claim 1, wherein the multi-step extraction comprises a first ester solvent extraction, an alkaline water extraction and a second ester solvent extraction.

14. The method according to claim 1, wherein the ester solvent is selected from diisooctyl phosphate, tributyl phosphate, ethyl acetate, isopropyl acetate and butyl acetate; and/or the alkaline water is selected from a sodium bicarbonate solution, an ammonium bicarbonate solution or a sodium hydroxide solution; and/or the pH of the alkaline water is 7.0 to 10.0.

15. The method according to claim 14, wherein the ester solvent is ethyl acetate.

16. The method according to claim 14, wherein the pH of the alkaline water is 7.5 to 9.0.

17. The method according to claim 13, wherein the alkaline water extraction is repeated once or twice or more times, and the alkaline water phases separated from the extractions are combined for later use in the second ester solvent extraction; and/or the pH of the second ester solvent extraction is 3.0 to 5.0.

18. The method according to claim 3, wherein the dehydrating agent is selected from anhydrous sodium sulfate, anhydrous magnesium sulfate or anhydrous calcium sulfate; and/or the amount of the dehydrating agent used is 0.02 to 0.08 kg/L solvent phase; and/or the decolorant is selected from activated carbon or activated clay; and/or the amount of the decolorant used is 1 to 6 g/g dehydrated solvent phase.

19. A method for inhibiting gram-positive bacteria, comprising administering mupirocin prepared by the method according to claim 1.

Description

DETAILED DESCRIPTION

[0047] The present invention will be further illustrated in detail with reference to the following specific examples. The following examples is only intended to help understand the method of the present invention and its core concepts. Any possible changes and substitutions can be made by those skilled in the art without departing from the spirit of the present invention, and these changes and substitutions are all within the scope of the present invention. Experimental procedures without specific conditions indicated in the following examples of the present invention are generally conducted according to conventional conditions, or according to conditions recommended by the manufacturers of the starting materials or commercial products. Reagents without specific sources indicated are generally commercially available conventional reagents.

Example 1. Extraction of Mupirocin

A. Resin Adsorption

[0048] A mupirocin fermentation broth was added into a pretreatment tank and the volume was measured. The broth was stirred, and the pH was adjusted to 6.0 with 2 N hydrochloric acid or 2 N sodium hydroxide.

[0049] Half an hour after pretreatment, H103 resin was added into the pretreatment tank at a feed amount of 0.04 g mupirocin/g resin. The mixture was stirred for 4.5 h at room temperature for adsorption. After the completion of the adsorption, the stir was stopped. The fermentation broth was filtered through a 40-mesh vibrating screen, and the surface of the resin was rinsed with drinking water to remove the adhered bacteria residues. The resin was collected (95.5% yield).

B. Desorption and Concentration

[0050] The resin was evenly loaded into a desorption column and washed reversely with drinking water more than 2 times the volume of the resin until the color of the water became light. The column was purged with air to remove the drinking water, and acetone was introduced reversely until the resin was completely soaked in acetone. The timing was started when the resin was completely soaked in acetone. After 2 hours, the desorption was performed at a flow rate of ⅗ times the volume of the resin per hour. The amount of acetone used for desorption was about 3 times the volume of the resin. The desorption solution was collected and the resin was washed with drinking water until the system was free of acetone smell. The mupirocin content in the desorption solution was 78.5%. The desorption solution was pumped into an acetone concentration tank and concentrated at an internal temperature below 50.0° C. until acetone was absent. The aqueous phase was retained.

C. Extraction

[0051] The first ester solvent extraction: The aqueous phase was pumped into an extraction tank. The pH was adjusted to 4.5 with 2 N hydrochloric acid. Ethyl acetate of ½ the aqueous phase volume was added. The mixture was stirred for 45 minutes at room temperature, and left to stand for 30 minutes. The ethyl acetate phase was separated. The above procedures were repeated once. The ethyl acetate phases separated from the two extractions were combined.

[0052] The first alkaline water extraction: The combined ethyl acetate phases were pumped into an extraction tank. A 2.5% ammonium bicarbonate solution of ⅓ the ethyl acetate phase volume was added. The mixture was stirred for 30 minutes at room temperature, and left to stand for 30 minutes. The alkaline water phase was separated. The above procedures were repeated thrice. The alkaline water phases separated from the four extractions were combined. The solvent of the ethyl acetate phase was recovered.

[0053] The second ester solvent extraction: The combined alkaline water phases were pumped into an extraction tank. The pH was adjusted to 4.0 by dropwise adding 2 N hydrochloric acid. Ethyl acetate of ½ the aqueous phase volume was added. The mixture was stirred for 45 minutes at room temperature, and left to stand for 30 minutes. The ethyl acetate phase was separated. The above procedures were repeated once. The ethyl acetate phases separated from the two extractions were combined. After extraction, the yield of mupirocin was 85.3%.

D. Dehydration and Decoloration

[0054] Anhydrous sodium sulfate was added at 2.3 kg/50 L combined ethyl acetate phases, and the mixture was stirred for dehydration for 30 minutes. The ethyl acetate phase was separated. After dehydration, the dehydrated anhydrous sodium sulfate was washed with fresh ethyl acetate in an amount of 1.5 times the weight of the anhydrous sodium sulfate added. The dehydrated ethyl acetate phase was decolorized according to a ratio of 3 g/g 8815 activated carbon. The activated carbon was washed with fresh ethyl acetate in an amount of 2 times the weight of the activated carbon added after decolorization. Finally, mupirocin was obtained with a yield of 83.3% and a content of 80.2%.

Example 2. Extraction of Mupirocin

A. Resin Adsorption

[0055] A fermentation broth was added into a pretreatment tank and the volume was measured. The broth was stirred, and the pH was adjusted to 6.0 with 2 N hydrochloric acid or 2 N sodium hydroxide. Half an hour after pretreatment, H103 resin was added into the pretreatment tank at a feed amount of 0.03 g mupirocin/g resin. The mixture was stirred for 4 h at room temperature for adsorption. After the completion of the adsorption, the stir was stopped. The fermentation broth was filtered through a 40-mesh vibrating screen, and the surface of the resin was rinsed with drinking water to remove the adhered bacteria residues. The resin was collected (94.7% yield).

B. Desorption and Concentration

[0056] The resin was evenly loaded into a desorption column and washed reversely with drinking water more than 2 times the volume of the resin until the color of the water became light. The column was purged with air to remove the drinking water, and acetone was introduced reversely until the resin was completely soaked in acetone. The timing was started when the resin was completely soaked in acetone. After 2 hours, the desorption was performed at a flow rate of 7/10 times the volume of the resin per hour. The amount of acetone used for desorption was about 3.5 times the volume of the resin. The desorption solution was collected and the resin was washed with drinking water until the system was free of acetone smell. The desorption solution was pumped into an acetone concentration tank and concentrated at a vacuum pressure below -0.08 MPa, a hot water tank temperature at 70.0° C. and an internal temperature below 50.0° C. until acetone was absent. The aqueous phase was retained.

C. Extraction

[0057] The first ester solvent extraction: The aqueous phase was pumped into an extraction tank. The pH was adjusted to 3.5 with 2 N hydrochloric acid. Ethyl acetate of ½ the aqueous phase volume was added. The mixture was stirred for 45 minutes at room temperature, and left to stand for 30 minutes. The ethyl acetate phase was separated. The above procedures were repeated once. The ethyl acetate phases separated from the two extractions were combined.

[0058] The first alkaline water extraction: The combined ethyl acetate phases were pumped into an extraction tank. A 2% ammonium bicarbonate solution of ⅓ the ethyl acetate phase volume was added. The mixture was stirred for 30 minutes at room temperature, and left to stand for 30 minutes. The alkaline water phase was separated. The above procedures were repeated thrice. The alkaline water phases separated from the four extractions were combined. The solvent of the ethyl acetate phase was recovered.

[0059] The second ester solvent extraction: The combined alkaline water phases were pumped into an extraction tank. The pH was adjusted to 4.0 by dropwise adding 2 N hydrochloric acid. Ethyl acetate of ½ the aqueous phase volume was added. The mixture was stirred for 45 minutes at room temperature, and left to stand for 30 minutes. The ethyl acetate phase was separated. The above procedures were repeated once. The ethyl acetate phases separated from the two extractions were combined. After the extraction, the yield of mupirocin was 87.3%, and the content of mupirocin was 82.5%.

Example 3. Investigation on Resin Adsorption of Mupirocin

[0060] Resin adsorption: A fermentation broth was added into a pretreatment tank and the volume was measured. The broth was stirred, and the pH was adjusted to 6.0 with hydrochloric acid or sodium hydroxide. Half an hour after pretreatment, H103 resin was added into the pretreatment tank at a feed amount of 0.02 g mupirocin/g resin. The mixture was stirred for 4 h at room temperature for adsorption. After the completion of the adsorption, the stir was stopped. The fermentation broth was filtered through a 40-mesh vibrating screen, and the surface of the resin was rinsed with drinking water to remove the adhered bacteria residues. The resin was collected (94.5% yield).

[0061] The other procedures were the same as those in Example 2, with an overall mupirocin yield of 82.3% and a purity of 82.0%.

Example 4. Investigation on Desorption and Concentration of Mupirocin

[0062] In the desorption step of the process for extracting mupirocin, mupirocin in the desorption column is eluted from the resin into acetone or ethyl acetate. To make the adsorption method applicable to industrial mass production, in addition to the adsorption and desorption described above, other conditions such as space velocity (volume flow rate/resin volume), the geometry of resin column bed (height/diameter ratio), the structure of the column bed, operation procedures and the like are also required to cooperate, wherein space velocity tends to be a main influencing factor.

[0063] Desorption and concentration: The resin was evenly loaded into a desorption column and washed reversely with drinking water more than 2 times the volume of the resin until the color of the water became light. The column was purged with air to remove the drinking water, and ethyl acetate was introduced reversely until the resin was completely soaked in ethyl acetate. The timing was started when the resin was completely soaked in ethyl acetate. After 2 hours, the desorption was performed at a flow rate of 7/10 times the volume of the resin per hour for 5 h. The amount of ethyl acetate used for desorption was about 4 times the volume of the resin. The desorption solution was collected and the resin was washed with drinking water. The mupirocin content in the desorption solution was 86.9%. The other procedures were the same as those in Example 1, with an overall mupirocin yield of 86.0% and a purity of 80.5%.

Example 5. Investigation on Desorption and Concentration of Mupirocin

[0064] Desorption and concentration: The resin was evenly loaded into a desorption column and washed reversely with drinking water more than 2 times the volume of the resin until the color of the water became light. The column was purged with air to remove the drinking water, and acetone was introduced reversely until the resin was completely soaked in acetone. The timing was started when the resin was completely soaked in acetone. After 2 hours, the desorption was performed at a flow rate of ⅕ times the volume of the resin per hour for 10 h. The amount of acetone used for desorption was about 4 times the volume of the resin. The desorption solution was collected and the resin was washed with drinking water until the system was free of acetone smell. The mupirocin content in the desorption solution was 88.5%.

Example 6. Investigation on Extraction of Mupirocin

[0065] The first ester solvent extraction: The aqueous phase was pumped into an extraction tank. The pH was adjusted to 5.0 with hydrochloric acid. Ethyl acetate of ½ the aqueous phase volume was added. The mixture was stirred for 45 minutes at room temperature, and left to stand for 30 minutes. The ethyl acetate phase was separated. The above procedures were repeated once. The ethyl acetate phases separated from the two extractions were combined.

[0066] The first alkaline water extraction: The combined ethyl acetate phases were pumped into an extraction tank. A 2% ammonium bicarbonate solution of ⅓ the ethyl acetate phase volume was added. The mixture was stirred for 30 minutes at room temperature, and left to stand for 30 minutes. The alkaline water phase was separated. The above procedures were repeated four times. The alkaline water phases separated from the four extractions were combined. The solvent of the ethyl acetate phase was recovered.

[0067] The second ester solvent extraction: The combined alkaline water phases were pumped into an extraction tank. The pH was adjusted to 4.0 by dropwise adding hydrochloric acid. Ethyl acetate of ½ the aqueous phase volume was added. The mixture was stirred for 45 minutes at room temperature, and left to stand for 30 minutes. The ethyl acetate phase was separated. The above procedures were repeated once. The ethyl acetate phases separated from the two extractions were combined. After extraction, the relative yield of mupirocin was 95.5%.

[0068] The other procedures were the same as those in Example 1, with an overall mupirocin yield of 82.0% and a purity of 82.5%.

Example 7. Necessity of Multi-step Extraction in Mupirocin Extraction

[0069] 1. Only one ester solvent extraction in the extraction step: The aqueous phase after desorption and concentration was pumped into an extraction tank. The pH was adjusted to 3.5 with hydrochloric acid. A proper amount of ethyl acetate was added. The mixture was stirred and left to stand. The ethyl acetate phase was separated. The above procedures were repeated once, and the ethyl acetate phases separated from the two extractions were combined. After one ester solvent extraction, the yield of mupirocin was 80.5%, and the purity was 78.1%.

[0070] 2. Only one alkaline water extraction in the extraction step: The aqueous phase after desorption and concentration was pumped into an extraction tank. The alkaline water and the aqueous phase are of the same polarity and thus cannot be separated, and the extraction cannot be performed.

[0071] 3. More than one ester solvent extraction in the extraction step: The aqueous phase after desorption and concentration was pumped into an extraction tank. The pH was adjusted to 3.5 with hydrochloric acid. A proper amount of ethyl acetate was added. The mixture was stirred and left to stand. The ethyl acetate phase was separated. The above procedures were repeated twice, and the ethyl acetate phases separated from the three extractions were combined. After two ester solvent extractions, the yield of mupirocin was 82.0%, and the purity was 78.5%.

Example 8. Investigation on Amount of Resin Used in Resin Adsorption of Mupirocin

[0072] A mupirocin fermentation broth was taken and the mupirocin content was calculated in the fermentation broth. The resin was added at an amount of 0.02, 0.03 or 0.04 g mupirocin/g resin. The mixture was stirred for adsorption for 4, 6, 8 or 24 hours and then filtered. The content of mupirocin in the filtrate was detected. The fermentation titers after adsorption were 278, 217, 137, 85, 385, 293, 238, 194, 446, 386, 340 and 322, respectively.

Example 9. Reproduction of Method for Extracting Pseudomonic Acid A Described in CN101591333B

[0073] (Example 1, i.e., the method described in paragraphs [0041] to [0044], which reports that the content of pseudomonic acid A in the desorption solution reaches 70% and the yield of pseudomonic acid A reaches 90%) 25 L of mupirocin fermentation broth (the concentration of pseudomonic acid A was 6017 .Math.g/mL) was taken and adjusted to a neutral pH with 1 M NaOH. The fermentation broth was centrifuged at 4500 rpm and 25° C. for 20 minutes with a high-speed low-temperature centrifuge to obtain a supernatant. The residues were washed with water and then discarded. The supernatant was adjusted to pH 4.0 with 1 M HCl and loaded on an H103 resin column (pre-treated, 2.2 L) for absorption. The H103 pretreatment was performed according to the manual provided by the manufacturer. After absorption, the column was washed with deionized water until the effluent was nearly colorless.

[0074] The desorption solution containing pseudomonic acid A was obtained from the resin with 8.3 L of a solvent containing ethanol, salt and water (3320 mL of ethanol, 410 g of ammonium chloride, and the remaining of water), and the ratio of the solvent containing ethanol, salt and water to pseudomonic acid A was 3.0 mL: 22 mg.

[0075] After desorption, the content of pseudomonic acid A in the desorption solution was 72%, and the yield of pseudomonic acid A was 52%.

[0076] The desorption indexes described in Example 1 of Patent No. CN101591333B are: the content of pseudomonic acid A in the desorption solution collected reaches 70%, and the yield of pseudomonic acid A reaches 90%. The yield reported in the document is significantly different from that in the reproduction experiment.