CN108977482B - Preparation method of polymyxin B sulfate - Google Patents

Preparation method of polymyxin B sulfate Download PDF

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CN108977482B
CN108977482B CN201810952403.4A CN201810952403A CN108977482B CN 108977482 B CN108977482 B CN 108977482B CN 201810952403 A CN201810952403 A CN 201810952403A CN 108977482 B CN108977482 B CN 108977482B
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polymyxin
culture
sulfate
culture medium
tank
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CN108977482A (en
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单含文
秦涛
吕稼锋
斯晓忠
张涛铸
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Zhejiang Funuo Pharmaceutical Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/50—Cyclic peptides containing at least one abnormal peptide link
    • C07K7/54—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring
    • C07K7/60—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring the cyclisation occurring through the 4-amino group of 2,4-diamino-butanoic acid
    • C07K7/62—Polymyxins; Related peptides

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Abstract

The invention provides a preparation method of polymyxin B sulfate, which at least comprises the following steps: (1) preparing polymyxin B seeds; (2) fermenting; (3) refining; the method controls the single impurities in the fermentation liquor which may affect the quality of the finished product to a level lower than 1.5%, and simultaneously combines an effective refining method to ensure that the quality of the finished product obtained by extracting the fermentation liquor meets the standards of EP8.0 and CP 2015.

Description

Preparation method of polymyxin B sulfate
Technical Field
The invention relates to the technical field of microbial fermentation, in particular to a preparation method of polymyxin B sulfate.
Background
Polymyxin B sulfate (polymyxin B sulfate), also known as colistin B sulfate, is a polypeptide antibiotic that has a strong inhibitory or bactericidal effect on almost all gram-negative bacteria, such as escherichia coli, pseudomonas aeruginosa, paraescherichia coli, klebsiella pneumoniae, acidophilus, bordetella pertussis, shigella dysenteriae, and the like, and particularly has a significant effect on pseudomonas aeruginosa. Many countries around the world have approved this drug for use as a feed additive. Polymyxin B sulfate is also used as a medicine and is mainly used for infection caused by sensitive bacteria, urinary system infection, meningitis, septicemia, burn infection, skin mucosa infection and the like caused by pseudomonas aeruginosa clinically.
The production enterprises of polymyxin B sulfate in China are still few, and no enterprise for obtaining the production batch of the raw material medicine in China mainly depends on the import from foreign countries to meet the requirements, so that the accelerated development and production of the product with excellent market prospect in China becomes a critical affair, and the development of the polymyxin raw material medicines is listed in a priority support plan. The control of related substances of polymyxin B fermentation products is a difficult point, any single impurity cannot exceed 3.0 percent and the total impurity cannot exceed 17.0 percent in the regulations of the related substances of polymyxin B in Chinese pharmacopoeia, and the problem to be solved is urgently needed to be solved how to effectively control the fermentation impurities and simultaneously consider the fermentation yield.
Therefore, in view of the above problems, the present invention provides a method for preparing polymyxin B sulfate, which controls the maximum single impurities in the fermentation broth that may affect the quality of the finished product to a level below 1.5%, and simultaneously combines an effective refining method to make the quality of the finished product obtained by extraction of the fermentation broth meet the standards of EP8.0 and CP 2015.
Disclosure of Invention
In order to solve the above problems, the present invention provides a preparation method of polymyxin B sulfate, which at least comprises the following steps:
(1) polymyxin B seed preparation: putting the polymyxin B producing strain into the culture medium A for recovery culture, and then putting the polymyxin B producing strain into the culture medium B for shaking table culture to obtain an activated polymyxin B producing strain;
(2) fermentation: adding the activated polymyxin B producing strain obtained in the step (1) into a culture tank C, and culturing in a seeding tank; then placing the polymyxin B strain in a culture tank D for fermentation tank culture to obtain fermentation liquor of polymyxin B strain;
(3) refining: and (3) filtering the fermentation liquor of the polymyxin B strain obtained in the step (2), treating by using ion exchange resin, treating by using macroporous resin, desorbing alkali crystallization of liquid, transferring salt, decoloring, concentrating by nanofiltration, and carrying out vacuum freeze drying to obtain the polymyxin B sulfate.
In one embodiment, said medium A in said step (1) contains the following components per 100ml of medium: 1-3g of sucrose, 1-5g of peptone, 0.1-0.5g of dipotassium phosphate, 0.2-1g of magnesium sulfate heptahydrate, 1-3g of agar and the balance of water.
In one embodiment, said medium B in said step (1) contains the following components per 100ml of medium: 7-10g of wheat flour, 0.002-0.01g of amylase, 0.2-0.6g of yeast powder, 0.1-0.5g of calcium carbonate, 0.01-0.03g of disodium hydrogen phosphate, 0.2-0.7g of ammonium sulfate and the balance of water.
In one embodiment, said step (2) comprises the following composition per 100ml of the culture medium in said culture tank C: 7-10g of wheat flour, 0.002-0.01g of amylase, 0.2-0.6g of yeast powder, 0.1-0.5g of calcium carbonate, 0.01-0.03g of disodium hydrogen phosphate, 0.2-0.7g of ammonium sulfate, 0.01-0.05g of defoaming agent and the balance of water.
In one embodiment, said step (2) comprises the following composition for every 100ml of the culture medium in said culture tank D: 10-15g of wheat flour, 0.002-0.01g of amylase, 0.5-1g of yeast powder, 0.1-0.5g of calcium carbonate, 0.01-0.03g of disodium hydrogen phosphate, 0.2-0.7g of ammonium sulfate, 0.01-0.05g of defoaming agent, 0.01-0.1g of L-threonine and the balance of water.
In one embodiment, the fermenter culturing process in step (2) further comprises a feeding process, wherein the feeding formulation comprises the following components per 100ml of the culture medium: 0.01-0.1g of L-threonine, 0.1-0.5g of ammonium sulfate and the balance of water.
In one embodiment, the ion exchange resin in step (3) is a strong acid cation resin.
In one embodiment, the strong acid cation resin is a 4-sulfonamide benzoylimine and a 4-sulfopyridine-3-sulfonamide modified 732 cation exchange resin.
In one embodiment, the macroporous resin in step (3) comprises one of D001, D113, D201, D301, D370, and D152.
In one embodiment, the polymyxin B-producing strain of step (1) is Paenibacillus polymyxa.
The above-described and other features, aspects, and advantages of the present application will become more apparent with reference to the following detailed description.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention provides a preparation method of polymyxin B sulfate, which at least comprises the following steps:
(1) polymyxin B seed preparation: putting the polymyxin B producing strain into the culture medium A for recovery culture, and then putting the polymyxin B producing strain into the culture medium B for shaking table culture to obtain an activated polymyxin B producing strain;
(2) fermentation: adding the activated polymyxin B producing strain obtained in the step (1) into a culture tank C, and culturing in a seeding tank; then placing the polymyxin B strain in a culture tank D for fermentation tank culture to obtain fermentation liquor of polymyxin B strain;
(3) refining: and (3) filtering the fermentation liquor of the polymyxin B strain obtained in the step (2), treating by using ion exchange resin, treating by using macroporous resin, desorbing alkali crystallization of liquid, transferring salt, decoloring, concentrating by nanofiltration, and carrying out vacuum freeze drying to obtain the polymyxin B sulfate.
In one embodiment, said medium A in said step (1) contains the following components per 100ml of medium: 1-3g of sucrose, 1-5g of peptone, 0.1-0.5g of dipotassium phosphate, 0.2-1g of magnesium sulfate heptahydrate, 1-3g of agar and the balance of water; preferably, the culture medium A in the step (1) contains the following components per 100ml of culture medium: 1-3g of sucrose, 1-3g of peptone, 0.2-0.3g of dipotassium phosphate, 0.3-0.8g of magnesium sulfate heptahydrate, 1-3g of agar and the balance of water; more preferably, said medium A in said step (1) contains the following components per 100ml of medium: 2g of sucrose, 2g of peptone, 0.25g of dipotassium phosphate, 0.6g of magnesium sulfate heptahydrate, 2g of agar and the balance of water.
In one embodiment, said medium B in said step (1) contains the following components per 100ml of medium: 7-10g of wheat flour, 0.002-0.01g of amylase, 0.2-0.6g of yeast powder, 0.1-0.5g of calcium carbonate, 0.01-0.03g of disodium hydrogen phosphate, 0.2-0.7g of ammonium sulfate and the balance of water; preferably, the medium B in the step (1) contains the following components per 100ml of the medium: 8-10g of wheat flour, 0.003-0.008g of amylase, 0.3-0.5g of yeast powder, 0.2-0.4g of calcium carbonate, 0.01-0.03g of disodium hydrogen phosphate, 0.3-0.5g of ammonium sulfate and the balance of water; more preferably, said medium B in said step (1) contains the following components per 100ml of medium: 9g of wheat flour, 0.005g of amylase, 0.45g of yeast powder, 0.3g of calcium carbonate, 0.015g of disodium hydrogen phosphate, 0.45g of ammonium sulfate and the balance of water.
The recovery culture in the step (1) of the invention is concretely as follows:
sterilizing the prepared culture medium A with steam at 121 deg.C for 25min, placing the slant, inoculating polymyxin B strain, and culturing at 29 + -0.5 deg.C for 48 h.
The shaking culture in the step (1) of the invention is specifically as follows:
steam sterilizing the prepared culture medium B at 121 ℃ for 25min, scraping the lawn from the culture medium A by using a glass rod, placing the lawn in the culture medium B, performing shake cultivation for 15-17h at 29 +/-0.5 ℃ in a shake manner, and performing smear microscopy on the lawn without other strains to obtain qualified strains serving as production strains.
In one embodiment, said step (2) comprises the following composition per 100ml of the culture medium in said culture tank C: 7-10g of wheat flour, 0.002-0.01g of amylase, 0.2-0.6g of yeast powder, 0.1-0.5g of calcium carbonate, 0.01-0.03g of disodium hydrogen phosphate, 0.2-0.7g of ammonium sulfate, 0.01-0.05g of defoaming agent and the balance of water; preferably, the culture tank C in the step (2) contains the following components per 100ml of the culture medium: 8-10g of wheat flour, 0.003-0.008g of amylase, 0.3-0.5g of yeast powder, 0.2-0.4g of calcium carbonate, 0.01-0.03g of disodium hydrogen phosphate, 0.3-0.5g of ammonium sulfate, 0.02-0.04g of defoaming agent and the balance of water; more preferably, said culture tank C in said step (2) contains the following components per 100ml of the culture medium: 9g of wheat flour, 0.005g of amylase, 0.45g of yeast powder, 0.3g of calcium carbonate, 0.015g of disodium hydrogen phosphate, 0.45g of ammonium sulfate, 0.03g of defoaming agent and the balance of water.
In one embodiment, said step (2) comprises the following composition for every 100ml of the culture medium in said culture tank D: 10-15g of wheat flour, 0.001-0.01g of amylase, 0.5-1g of yeast powder, 0.1-0.5g of calcium carbonate, 0.01-0.03g of disodium hydrogen phosphate, 0.2-0.7g of ammonium sulfate, 0.01-0.05g of defoaming agent, 0.01-0.1g of L-threonine and the balance of water; preferably, the culture tank D in the step (2) contains the following components per 100ml of the culture medium: 11-13g of wheat flour, 0.005-0.008g of amylase, 0.6-0.9g of yeast powder, 0.2-0.4g of calcium carbonate, 0.01-0.02g of disodium hydrogen phosphate, 0.2-0.6g of ammonium sulfate, 0.02-0.04g of defoaming agent, 0.05-0.09g of L-threonine and the balance of water; more preferably, said culture tank D in said step (2) contains the following components per 100ml of the culture medium: 12g of wheat flour, 0.006g of amylase, 0.8g of yeast powder, 0.3g of calcium carbonate, 0.015g of disodium hydrogen phosphate, 0.45g of ammonium sulfate, 0.03g of defoaming agent, 0.08g of L-threonine and the balance of water.
The defoaming agent is GPE type polyoxyethylene polyoxypropylene glycerol ether.
The seeding tank culture in the step (2) of the invention is concretely as follows:
sterilizing the culture tank C prepared on site with steam at 120 deg.C for 20min, cooling to 29 + -0.5 deg.C, inoculating the strain producing polymyxin B obtained in step (1) into the culture tank C by flame inoculation method at 29 + -0.5 deg.C and under 0.045-0.055 MPa; air flow rate is 0.3-0.5 VVM; stirring time is 15-17h, stirring speed: the speed is 300rpm for 0-5h and 500rpm after the reaction is finished for 6 h; and (5) reducing the pH value to 6.0 +/-0.2 to obtain pot seeds, and transplanting the seeds.
The fermentation tank culture in the step (2) of the invention is specifically as follows:
sterilizing the culture tank D with steam at 120 deg.C for 20min, cooling to 29 + -0.5 deg.C, transferring the cultured seeds to the culture tank D by differential pressure method, culturing at 29 + -0.5 deg.C under 0.045-0.055 MPa; air flow rate is 0.3-0.5 VVM; the culture time is 18-20h, and the stirring speed is as follows: 400 rpm.
In one embodiment, the fermenter culturing process in step (2) further comprises a feeding process, wherein the feeding formulation comprises the following components per 100ml of the culture medium: 0.01-0.1g of L-threonine, 0.1-0.5g of ammonium sulfate and the balance of water; preferably, the fermenter culture process in step (2) further comprises a feeding process, and the feeding formula comprises the following components per 100ml of culture medium: 0.05-0.1g of L-threonine, 0.3-0.4g of ammonium sulfate and the balance of water; more preferably, the fermenter culturing process in step (2) further comprises a feeding process, wherein the feeding formulation comprises the following components per 100ml of the culture medium: 0.08g of L-threonine, 0.3g of ammonium sulfate and the balance of water.
In the invention, the feed supplement is slowly added into a fermentation tank for continuous fermentation at the temperature of 29 +/-0.5 ℃ and the tank pressure of 0.045-0.055 MPa; air flow 0.5 VVM; continuing culturing for 18-22h, stirring: 400 rpm.
Feeding is generally a selective supplementation of nutrients during the fermentation phase up to the point where the product is produced in large quantities, as is required for the synthesis of the product and for the maintenance of cell activity. Suitable feeding processes are effective in controlling the intermediary metabolism of the microorganism towards favoring product accumulation. The nutrient substances supplemented by the feed-batch fermentation are roughly five types: supplementing energy and carbon sources required by the thalli, such as glucose, liquefied starch and the like; ② supplementing nitrogen sources required by the thalli, such as organic nitrogen sources of peptone, bean cake powder, corn steep liquor, yeast powder, urea and the like. Some fermentations also adopt the introduction of ammonia gas or the addition of ammonia water and the like. And adding some trace elements and inorganic salts, such as phosphate, sulfate, etc. necessary for thallus to grow and metabolize. And fourthly, for microorganisms producing the induced enzyme, a substrate for the enzyme is added properly in the feed to improve the yield of the enzyme. For some antibiotic fermentation, the precursor formed by antibiotic is often needed to be supplemented.
The precursor supplement is to add chemical precursors in the antibiotic fermentation process, which is used for controlling the biosynthesis direction of antibiotics and increasing the antibiotic yield, and can be taken by antibiotic producing bacteria and incorporated into an antibiotic synthesis structure.
Polymyxin B has a chemical composition of a cyclic structure consisting of 7 amino acids and a side chain of 3 amino acids, and the end of the chain is linked with methyloctanoic acid. The whole structure contains 6 structures of 2, 4-diaminobutyric acid (Dab), and research finds that L-2, 4-diaminobutyric acid C is used14As a tracer label, a marker compound may be incorporated into polymyxin B, indicating that the former is a precursor of polymyxin B. And the L-Dab activating enzyme is separated from the polymyxin, participates in the synthesis of polymyxin B, and researches show that all variants losing the polymyxin synthesis capacity cannot activate the L-Dab; when the polymyxa is grown in an environment unfavorable for the growth of polymyxin, the activity of the L-Dab activating enzyme is reduced.
The L-threonine structure is 2-amino-3-hydroxybutyric acid, and is similar to L-Dab structure and uses L-threonine-C14And the trace label is added, and then chloramphenicol and actinomycin D inhibition labels are added to be doped into protein synthesis, and more actinomycin D inhibition labels are utilized by production bacteria to stimulate the formation of polymyxin. Therefore, L-threonine is a potential substrate for polymyxin synthesis and can be used in a fermentation feeding mode to increase yield and impurity control.
Additive material Is free of Chloromycetin Actinomycin D
Polymyxin C14 6950 9650 7900
Intra-protein C14 334000 7250 21500
In one embodiment, the ion exchange resin in step (3) is a strong acid cation resin.
In one embodiment, the strong acid cation resin is a 732 cation exchange resin modified with 4-sulfonamide benzoylimine (CAS number: 54951-45-0) and 4-sulfopyridine-3-sulfonamide (CAS number: 72810-60-7).
The preparation method of the strong acid cation resin comprises the following steps:
(1) adding 732 hydrogen ion type polystyrene-divinylbenzene sulfonic acid cationic resin and chloroform into a reactor, swelling for 30h at room temperature, dropwise adding a mixed solution of thionyl chloride/chloroform (the mass volume ratio of the thionyl chloride to the chloroform is 1: 3), heating to 75 ℃, carrying out heat preservation reaction for 10h, cooling to room temperature, filtering, and washing for 3 times by using dichloromethane to obtain 732 hydrogen ion type polystyrene-divinylbenzene sulfonic acid cationic resin; the weight ratio of the 732 hydrogen ion type polystyrene-divinylbenzene sulfonic acid cationic resin to the trichloromethane is 1: 6; the mole ratio of the 732 hydrogen ion type polystyrene-divinylbenzene sulfonic acid cation resin to the thionyl chloride is 1: 1.1;
(2) adding 732 hydrogen ion type polystyrene-divinylbenzene sulfonic acid cationic resin obtained in the step (1), a mixed solution of 4-sulfopyridine-3-sulfonamide and trichloromethane (the weight volume ratio of the 4-sulfopyridine-3-sulfonamide to the trichloromethane is 1: 3) and the trichloromethane into a reactor, heating to 70 ℃, carrying out heat preservation reaction for 6 hours, adding a mixed solution of 4-sulfonamide benzoylimine and the trichloromethane (the weight volume ratio of the 4-sulfonamide benzoylimine to the trichloromethane is 1: 2), carrying out heat preservation reaction for 8 hours, cooling to room temperature, filtering, washing for 2 times with dichloromethane, washing for 3 times with acetone, and drying for 24 hours under reduced pressure at 60 ℃ to obtain strong acid cationic resin; the mole ratio of the 732 hydrogen chloride ionic polystyrene-divinylbenzene sulfonic acid cationic resin to the 4-sulfonic pyridine-3-sulfonamide and the 4-sulfonamide benzoylimine is 1: 0.3: 0.7; the weight ratio of the 732 hydrogen chloride ion type polystyrene-divinylbenzene sulfonic acid cation resin to the trichloromethane is 1: 6.
in the invention, the strong acid cation resin obtained by modifying 732 cation exchange resin by 4-sulfamide benzoylimine and 4-sulfonic pyridine-3-sulfamide has excellent separation performance on metal ions, and 4-sulfonic pyridine-3-sulfamide introduces the complexation of coordination atoms in groups of sulfonic acid and pyridyl on the metal ions; meanwhile, the electrostatic interaction between the sulfonic acid group with negative charge and the metal ion with positive charge in the 4-sulfamide benzoyl imine and the 4-sulfonic pyridine-3-sulfamide and the synergistic interaction of the two promote the strong acid cation resin to separate the metal ion; in addition, the 4-sulfamide benzoyl imine and the 4-sulfonic pyridine-3-sulfamide modified 732 cation exchange resin can improve the regeneration performance of the strong acid cation resin and the separation effect on impurities, and improve the quality of the product.
The metal ions in the present invention include at least calcium ions.
The water in the present invention is sterile purified water.
In one embodiment, the macroporous resin in step (3) comprises one of D001, D113, D201, D301, D370, and D152; preferably, the macroporous resin in the step (3) is D152.
The filtration in the step (3) of the invention is specifically as follows:
centrifuging the fermentation liquid with a refrigerated centrifuge for 10-30min, collecting the primary supernatant, adjusting the pH of the supernatant to 3.0 with oxalic acid, stirring for 0.5-1h, centrifuging with a refrigerated centrifuge for 10-30min, collecting the secondary supernatant, and cooling to 5-10 deg.C.
The ion exchange resin treatment in step (3) of the present invention is specifically as follows:
loading ion exchange resin into a column, treating with 1mol/L hydrochloric acid until the pH of the outlet feed liquid is less than or equal to 2.0, soaking for 2 hours, washing with purified water until the pH is more than 5.0, treating with 1mol/L sodium hydroxide aqueous solution until the pH of the eluate is more than or equal to 10.0, soaking for 2 hours, washing with purified water until the pH is less than 9.0, and performing column treatment on the secondary supernatant cooled to 5-10 ℃ to obtain a decalcification solution; the proportion of the secondary supernatant to the ion exchange resin is (5-10): 1; the mass of the secondary supernatant is Kg, and the volume of the ion exchange resin is L; the proportion of the hydrochloric acid solution to the ion exchange resin is (1-3): 1; the mass of the hydrochloric acid solution is Kg, and the volume of the ion exchange resin is L; the ratio of the sodium hydroxide aqueous solution to the ion exchange resin is (1-3): 1; the mass of the sodium hydroxide aqueous solution is measured by Kg, and the volume of the ion exchange resin is measured by L; the proportion of the purified water to the ion exchange resin is (3-6): 1; the mass of the purified water is measured by Kg, and the volume of the ion exchange resin is measured by L;
the macroporous resin treatment in the step (3) of the invention is as follows:
loading macroporous resin into a column, treating the macroporous resin with 1mol/L hydrochloric acid until the pH value of outlet feed liquid is less than or equal to 2.0, soaking for 2 hours, washing the outlet feed liquid with purified water to more than 5.0, treating the outlet feed liquid with 1mol/L sodium hydroxide aqueous solution until the pH value of the outlet feed liquid is more than or equal to 10.0, soaking for 2 hours, washing purified water with alkali until the pH value is less than 9.0, adjusting the pH value of the decalcified liquid to 6.0-6.2 with 1mol/L sodium hydroxide aqueous solution, cooling to 5-10 ℃, carrying out column passing adsorption on the decalcified liquid, washing the column with purified water until the light transmittance of effluent liquid under 470nm is more than 95%, desorbing with 0.2mol/L sulfuric acid solution until the effluent liquid unit is less than 2000 units, and collecting to obtain desorption liquid, wherein the ratio of the decalcified liquid to the macroporous resin is (: 1; the weight of the decalcifying liquid is Kg, and the volume of the macroporous resin is L; the ratio of the sodium hydroxide aqueous solution to the macroporous resin is (1-3): 1; the mass of the sodium hydroxide aqueous solution is Kg, and the volume of the macroporous resin is L; the ratio of the purified water to the macroporous resin is (3-6): 1; the mass of the purified water is Kg, and the volume of the macroporous resin is L; the proportion of the hydrochloric acid solution to the macroporous resin is (1-3): 1; the mass of the hydrochloric acid solution is measured by Kg, and the volume of the macroporous resin is measured by L.
The desorption liquid alkali crystallization in the step (3) of the invention is concretely as follows:
cooling the desorption solution to 0-5 ℃, adjusting the pH value to 12.0 by using 1mol/L aqueous solution of sodium hydroxide, heating to 20 ℃ at the speed of 0.05-0.15 ℃/min, keeping stirring at 20-30rpm in the heating process for 2-6h, filtering, and washing for 3 times by using purified water at 70 ℃ which is 1/3 times of the volume of the desorption solution to obtain a crude polymyxin B product.
The salt conversion in the step (3) of the invention is specifically as follows:
regulating the value to 4.8-5.2 by using 9.5-10.5 mass percent of sulfuric acid in the polymyxin B crude product, and stirring until the polymyxin B crude product is completely dissolved to obtain a polymyxin B sulfate solution.
The decoloring in the step (3) of the invention is specifically as follows:
heating the polymyxin B sulfate solution to 50-60 ℃, adding activated carbon, keeping the temperature and stirring for 0.5-1h, and filtering to obtain polymyxin B sulfate decolorized solution, wherein the absorbance value measured at 470nm is lower than 0.015/10000 mug/ml. The weight ratio of the polymyxin B sulfate to the activated carbon is 1: 0.02.
the nanofiltration concentration in the step (3) of the invention is specifically as follows:
and (3) carrying out nanofiltration concentration on the polymyxin B sulfate decolorized solution, pressurizing by a pressure pump at 1.0-1.5MPa, returning the concentrated discharge solution to a mother tank, circulating through a column, carrying out top washing on purified water until the conductivity of the effluent waste liquid is reduced to below 30, and collecting the concentrated solution to obtain the polymyxin B sulfate solution after nanofiltration concentration.
The vacuum freeze drying in step (3) of the invention is as follows:
and (2) carrying out vacuum freeze drying on the polymyxin B sulfate solution after nanofiltration concentration, wherein the temperature of the vacuum freeze drying is-30-35 ℃ (in the first stage, the temperature is kept at-30 ℃ for 8h, and the temperature change rate is 0.7 ℃/min, in the second stage, the temperature is kept at-10 ℃ for 6h, and the temperature change rate is 0.1 ℃/min, in the third stage, the temperature is kept at 0 ℃ for 7h, and the temperature change rate is 0.1 ℃/min, in the fourth stage, the temperature is kept at 15 ℃ for 4h, and the temperature change rate is 0.1 ℃/min, in the fifth stage, the temperature is kept at 35 ℃ for 3h, and the temperature change rate is 0.1 ℃/min), and the vacuum.
In one embodiment, the polymyxin B-producing strain of step (1) is Paenibacillus polymyxa Paenibacillus.
The strain preservation number of the Paenibacillus myxomicus is as follows: CPCC 140855.
In the present invention, all the raw materials are commercially available and purchased from national chemical reagents unless otherwise specified.
To further illustrate the present invention, the following examples are provided for illustration.
Example 1
A preparation method of polymyxin B sulfate at least comprises the following steps:
(1) polymyxin B seed preparation: putting the polymyxin B producing strain into the culture medium A for recovery culture, and then putting the polymyxin B producing strain into the culture medium B for shaking table culture to obtain an activated polymyxin B producing strain; the culture medium A in the step (1) contains the following components in every 100ml of culture medium: 2g of sucrose, 2g of peptone, 0.25g of dipotassium phosphate, 0.5g of magnesium sulfate heptahydrate, 2g of agar and the balance of water; the culture medium B in the step (1) contains the following components in every 100ml of culture medium: 9g of wheat flour, 0.005g of amylase, 0.45g of yeast powder, 0.3g of calcium carbonate, 0.015g of disodium hydrogen phosphate, 0.45g of ammonium sulfate and the balance of water;
(2) fermentation: adding the activated polymyxin B producing strain obtained in the step (1) into a culture tank C, and culturing in a seeding tank; then placing the polymyxin B strain in a culture tank D, performing fermentation tank culture, supplementing materials, and continuing fermentation tank culture to obtain fermentation liquor of polymyxin B strain; the culture tank C in the step (2) contains the following components in every 100ml of culture medium: 9g of wheat flour, 0.005g of amylase, 0.45g of yeast powder, 0.3g of calcium carbonate, 0.015g of disodium hydrogen phosphate, 0.45g of ammonium sulfate, 0.03g of defoaming agent and the balance of water; the culture tank D in the step (2) contains the following components per 100ml of culture medium: 12g of wheat flour, 0.006g of amylase, 0.8g of yeast powder, 0.3g of calcium carbonate, 0.015g of disodium hydrogen phosphate, 0.45g of ammonium sulfate, 0.03g of defoaming agent, 0.08g of L-threonine and the balance of water; the feed supplement formula contains the following components in each 100ml of culture medium: 0.08g of L-threonine, 0.3g of ammonium sulfate and the balance of water; the defoaming agent is GPE type polyoxyethylene polyoxypropylene glycerol ether;
(3) refining: filtering the fermentation liquor of the polymyxin B strain obtained in the step (2), treating by ion exchange resin, treating by macroporous resin, desorbing alkali crystallization of liquid, transferring salt, decoloring, concentrating by nanofiltration, and freeze-drying in vacuum to obtain polymyxin B sulfate; the ion exchange resin in the step (3) is strong acid cation resin; the strong acid cation resin is a 732 cation exchange resin modified by 4-sulfamide benzoyl imine and 4-sulfonic pyridine-3-sulfamide; the macroporous resin in the step (3) is D152;
the recovery culture in the step (1) of the invention is concretely as follows:
sterilizing the prepared culture medium A with steam at 121 deg.C for 25min, placing the slant, inoculating polymyxin B strain, and culturing at 29 + -0.5 deg.C for 48 h.
The shaking culture in the step (1) of the invention is specifically as follows:
steam sterilizing the prepared culture medium B at 121 ℃ for 25min, scraping the lawn from the culture medium A by using a glass rod, placing the lawn in the culture medium B, performing shake cultivation for 15-17h at 29 +/-0.5 ℃ in a shake manner, and performing smear microscopy on the lawn without other strains to obtain qualified strains serving as production strains.
The seeding tank culture in the step (2) of the invention is concretely as follows:
sterilizing the culture tank C prepared on site with steam at 120 deg.C for 20min, cooling to 29 + -0.5 deg.C, inoculating the strain producing polymyxin B obtained in step (1) into the culture tank C by flame inoculation method at 29 + -0.5 deg.C and under 0.045-0.055 MPa; air flow 0.5 VVM; incubation time 17h, stirring speed: the speed is 300rpm for 0-5h and 500rpm after the reaction is finished for 6 h; and (5) reducing the pH value to 6.0 +/-0.2 to obtain pot seeds, and transplanting the seeds.
The fermentation tank culture in the step (2) of the invention is specifically as follows:
sterilizing the culture tank D with steam at 120 deg.C for 20min, cooling to 29 + -0.5 deg.C, transferring the cultured seeds to the culture tank D by differential pressure method, culturing at 29 + -0.5 deg.C under 0.045-0.055 MPa; air flow 0.5 VVM; culture time 20h, stirring speed: 400 rpm.
The filtration in the step (3) of the invention is specifically as follows:
centrifuging the fermentation liquid with a refrigerated centrifuge for 30min, collecting primary supernatant, adjusting pH of the supernatant to 3.0 with oxalic acid, stirring for 0.5h, centrifuging with a refrigerated centrifuge for 30min, collecting secondary supernatant, and cooling to 6 deg.C.
The ion exchange resin treatment in step (3) of the present invention is specifically as follows:
loading ion exchange resin into a column, treating with 1mol/L hydrochloric acid until the pH of outlet feed liquid is 2.0, soaking for 2h, washing with purified water until the pH is 5.0, treating with 1mol/L sodium hydroxide aqueous solution until the pH of leacheate is 10.0, soaking for 2h, washing with purified water until the pH is 9.0, and performing column treatment on secondary supernatant cooled to 5-10 ℃ to obtain decalcification solution; the ratio of the secondary supernatant to the ion exchange resin is 8: 1; the mass of the secondary supernatant is Kg, and the volume of the ion exchange resin is L; the ratio of the hydrochloric acid solution to the ion exchange resin is 2.4: 1; the mass of the hydrochloric acid solution is Kg, and the volume of the ion exchange resin is L; the ratio of the sodium hydroxide aqueous solution to the ion exchange resin is 2: 1; the mass of the sodium hydroxide aqueous solution is measured by Kg, and the volume of the ion exchange resin is measured by L; the ratio of the purified water to the ion exchange resin is 5: 1; the mass of the purified water is measured by Kg, and the volume of the ion exchange resin is measured by L;
the macroporous resin treatment in the step (3) of the invention is as follows:
loading macroporous resin into a column, treating the macroporous resin with 1mol/L hydrochloric acid until the pH value of outlet feed liquid is 2.0, soaking for 2 hours, washing with purified water until the pH value is 5.0, treating the outlet feed liquid with 1mol/L sodium hydroxide aqueous solution until the pH value of the outlet feed liquid is 10.0, soaking for 2 hours, washing with purified water until the pH value is 9.0, adjusting the pH value of the decalcified liquid to 6.0 with 1mol/L sodium hydroxide aqueous solution, cooling to 5-10 ℃, performing column adsorption on the decalcified liquid, washing the column with purified water until the light transmittance of effluent liquid under 470nm is more than 95%, desorbing the decalcified liquid with 0.2mol/L sulfuric acid solution until the effluent liquid unit is less than 2000 units, and stopping collecting to obtain desorption liquid, wherein the ratio of the decalcified liquid to the macroporous resin is 8: 1; the weight of the decalcifying liquid is Kg, and the volume of the macroporous resin is L; the ratio of the sodium hydroxide aqueous solution to the macroporous resin is 1.2: 1; the mass of the sodium hydroxide aqueous solution is Kg, and the volume of the macroporous resin is L; the ratio of the purified water to the macroporous resin is 4.5: 1; the mass of the purified water is Kg, and the volume of the macroporous resin is L; the ratio of the hydrochloric acid solution to the macroporous resin is 1.3: 1; the mass of the hydrochloric acid solution is measured by Kg, and the volume of the macroporous resin is measured by L.
The desorption liquid alkali crystallization in the step (3) of the invention is concretely as follows:
cooling the desorption solution to 2 ℃, adjusting the pH value to 12.0 by using 1mol/L aqueous solution of sodium hydroxide, heating to 20 ℃ at the speed of 0.1 ℃/min, keeping stirring at 20-30rpm in the heating process for 4h, filtering, and washing for 3 times by using purified water at 70 ℃ with the volume being 1/3 times that of the desorption solution to obtain a crude polymyxin B product.
The dissolution in step (3) of the present invention is specifically as follows:
regulating the value to 5.0 by using 9.5 mass percent sulfuric acid in the crude polymyxin B product, stirring for 1h, and completely dissolving the crude polymyxin B product to obtain a polymyxin B sulfate solution.
The decoloring in the step (3) of the invention is specifically as follows:
heating the polymyxin B sulfate solution to 55 ℃, adding activated carbon, and stirring for 1h under heat preservation to obtain polymyxin B sulfate decoloration solution; the weight ratio of the polymyxin B sulfate to the activated carbon is 1: 0.02.
the nanofiltration concentration in the step (3) of the invention is specifically as follows:
and (3) carrying out nanofiltration concentration on the polymyxin B sulfate decolorized solution, pressurizing by a pressure pump of 1.0-1.5Mpa, returning the concentrated discharge solution to a mother tank to circulate through a column, washing purified water by jacking until the conductivity of the effluent waste liquid is reduced to below 30, and collecting the concentrated solution to obtain the polymyxin B sulfate solution after nanofiltration concentration.
The vacuum freeze drying in step (3) of the invention is as follows:
and (2) carrying out vacuum freeze drying on the polymyxin B sulfate solution after nanofiltration concentration, wherein the temperature of the vacuum freeze drying is-30-35 ℃ (in the first stage, the temperature is kept at-30 ℃ for 8h, and the temperature change rate is 0.7 ℃/min, in the second stage, the temperature is kept at-10 ℃ for 6h, and the temperature change rate is 0.1 ℃/min, in the third stage, the temperature is kept at 0 ℃ for 7h, and the temperature change rate is 0.1 ℃/min, in the fourth stage, the temperature is kept at 15 ℃ for 4h, and the temperature change rate is 0.1 ℃/min, in the fifth stage, the temperature is kept at 35 ℃ for 3h, and the temperature change rate is 0.1 ℃/min), and the vacuum.
The preparation method of the strong acid cation resin comprises the following steps:
(1) adding 732 hydrogen ion type polystyrene-divinylbenzene sulfonic acid cationic resin and chloroform into a reactor, swelling for 30h at room temperature, dropwise adding a mixed solution of thionyl chloride/chloroform (the mass volume ratio of the thionyl chloride to the chloroform is 1: 3), heating to 75 ℃, carrying out heat preservation reaction for 10h, cooling to room temperature, filtering, and washing for 3 times by using dichloromethane to obtain 732 hydrogen ion type polystyrene-divinylbenzene sulfonic acid cationic resin; the weight ratio of the 732 hydrogen ion type polystyrene-divinylbenzene sulfonic acid cationic resin to the trichloromethane is 1: 6; the mole ratio of the 732 hydrogen ion type polystyrene-divinylbenzene sulfonic acid cation resin to the thionyl chloride is 1: 1.1;
(2) adding 732 hydrogen ion type polystyrene-divinylbenzene sulfonic acid cationic resin obtained in the step (1), a mixed solution of 4-sulfopyridine-3-sulfonamide and trichloromethane (the weight volume ratio of the 4-sulfopyridine-3-sulfonamide to the trichloromethane is 1: 3) and the trichloromethane into a reactor, heating to 70 ℃, carrying out heat preservation reaction for 6 hours, adding a mixed solution of 4-sulfonamide benzoylimine and the trichloromethane (the weight volume ratio of the 4-sulfonamide benzoylimine to the trichloromethane is 1: 2), carrying out heat preservation reaction for 8 hours, cooling to room temperature, filtering, washing for 2 times with dichloromethane, washing for 3 times with acetone, and drying for 24 hours under reduced pressure at 60 ℃ to obtain strong acid cationic resin; the mole ratio of the 732 hydrogen chloride ionic polystyrene-divinylbenzene sulfonic acid cationic resin to the 4-sulfonic pyridine-3-sulfonamide and the 4-sulfonamide benzoylimine is 1: 0.3: 0.7; the weight ratio of the 732 hydrogen chloride ion type polystyrene-divinylbenzene sulfonic acid cation resin to the trichloromethane is 1: 6.
example 2
A preparation method of polymyxin B sulfate at least comprises the following steps:
(1) polymyxin B seed preparation: putting the polymyxin B producing strain into the culture medium A for recovery culture, and then putting the polymyxin B producing strain into the culture medium B for shaking table culture to obtain an activated polymyxin B producing strain; the culture medium A in the step (1) contains the following components in every 100ml of culture medium: 2g of sucrose, 2g of peptone, 0.25g of dipotassium phosphate, 0.5g of magnesium sulfate heptahydrate, 2g of agar and the balance of water; the culture medium B in the step (1) contains the following components in every 100ml of culture medium: 9g of wheat flour, 0.005g of amylase, 0.45g of yeast powder, 0.3g of calcium carbonate, 0.015g of disodium hydrogen phosphate, 0.45g of ammonium sulfate and the balance of water;
(2) fermentation: adding the activated polymyxin B producing strain obtained in the step (1) into a culture tank C, and culturing in a seeding tank; then placing the polymyxin B strain in a culture tank D, performing fermentation tank culture, supplementing materials, and continuing fermentation tank culture to obtain fermentation liquor of polymyxin B strain; the culture tank C in the step (2) contains the following components in every 100ml of culture medium: 9g of wheat flour, 0.005g of amylase, 0.45g of yeast powder, 0.3g of calcium carbonate, 0.015g of disodium hydrogen phosphate, 0.45g of ammonium sulfate, 0.03g of defoaming agent and the balance of water; the culture tank D in the step (2) contains the following components per 100ml of culture medium: 12g of wheat flour, 0.006g of amylase, 0.8g of yeast powder, 0.3g of calcium carbonate, 0.015g of disodium hydrogen phosphate, 0.45g of ammonium sulfate, 0.03g of defoaming agent, 0.08g of L-threonine and the balance of water; the feed supplement formula contains the following components in each 100ml of culture medium: 0.01g of L-threonine, 0.37g of ammonium sulfate and the balance of water; the defoaming agent is GPE type polyoxyethylene polyoxypropylene glycerol ether;
(3) refining: filtering the fermentation liquor of the polymyxin B strain obtained in the step (2), treating by ion exchange resin, treating by macroporous resin, desorbing alkali crystallization of liquid, transferring salt, decoloring, concentrating by nanofiltration, and freeze-drying in vacuum to obtain polymyxin B sulfate; the ion exchange resin in the step (3) is strong acid cation resin; the strong acid cation resin is a 732 cation exchange resin modified by 4-sulfamide benzoyl imine and 4-sulfonic pyridine-3-sulfamide; the macroporous resin in the step (3) is D152;
resuscitating culture in step (1) of the present invention, shaking culture in step (1) of the present invention, seeding tank culture in step (2) of the present invention, fermenter culture in step (2) of the present invention, filtering in step (3) of the present invention, ion exchange resin treatment in step (3) of the present invention, macroporous resin treatment in step (3) of the present invention, alkali crystallization of desorbent solution in step (3) of the present invention, dissolution in step (3) of the present invention, decoloring in step (3) of the present invention, nanofiltration concentration in step (3) of the present invention, vacuum freeze-drying in step (3) of the present invention, and a method for preparing the strong acid cation resin is the same as in example 1.
Example 3
A preparation method of polymyxin B sulfate at least comprises the following steps:
(1) polymyxin B seed preparation: putting the polymyxin B producing strain into the culture medium A for recovery culture, and then putting the polymyxin B producing strain into the culture medium B for shaking table culture to obtain an activated polymyxin B producing strain; the culture medium A in the step (1) contains the following components in every 100ml of culture medium: 2g of sucrose, 2g of peptone, 0.25g of dipotassium phosphate, 0.5g of magnesium sulfate heptahydrate, 2g of agar and the balance of water; the culture medium B in the step (1) contains the following components in every 100ml of culture medium: 9g of wheat flour, 0.005g of amylase, 0.45g of yeast powder, 0.3g of calcium carbonate, 0.015g of disodium hydrogen phosphate, 0.45g of ammonium sulfate and the balance of water;
(2) fermentation: adding the activated polymyxin B producing strain obtained in the step (1) into a culture tank C, and culturing in a seeding tank; then placing the polymyxin B strain in a culture tank D, performing fermentation tank culture, supplementing materials, and continuing fermentation tank culture to obtain fermentation liquor of polymyxin B strain; the culture tank C in the step (2) contains the following components in every 100ml of culture medium: 9g of wheat flour, 0.005g of amylase, 0.45g of yeast powder, 0.3g of calcium carbonate, 0.015g of disodium hydrogen phosphate, 0.45g of ammonium sulfate, 0.03g of defoaming agent and the balance of water; the culture tank D in the step (2) contains the following components per 100ml of culture medium: 12g of wheat flour, 0.006g of amylase, 0.8g of yeast powder, 0.3g of calcium carbonate, 0.015g of disodium hydrogen phosphate, 0.45g of ammonium sulfate, 0.03g of defoaming agent, 0.08g of L-threonine and the balance of water; the feed supplement formula contains the following components in each 100ml of culture medium: 0.1g of L-threonine, 0.28g of ammonium sulfate and the balance of water; the defoaming agent is GPE type polyoxyethylene polyoxypropylene glycerol ether;
(3) refining: filtering the fermentation liquor of the polymyxin B strain obtained in the step (2), treating by ion exchange resin, treating by macroporous resin, desorbing alkali crystallization of liquid, transferring salt, decoloring, concentrating by nanofiltration, and freeze-drying in vacuum to obtain polymyxin B sulfate; the ion exchange resin in the step (3) is strong acid cation resin; the strong acid cation resin is a 732 cation exchange resin modified by 4-sulfamide benzoyl imine and 4-sulfonic pyridine-3-sulfamide; the macroporous resin in the step (3) is D152;
resuscitating culture in step (1) of the present invention, shaking culture in step (1) of the present invention, seeding tank culture in step (2) of the present invention, fermenter culture in step (2) of the present invention, filtering in step (3) of the present invention, ion exchange resin treatment in step (3) of the present invention, macroporous resin treatment in step (3) of the present invention, alkali crystallization of desorbent solution in step (3) of the present invention, dissolution in step (3) of the present invention, decoloring in step (3) of the present invention, nanofiltration concentration in step (3) of the present invention, vacuum freeze-drying in step (3) of the present invention, and a method for preparing the strong acid cation resin is the same as in example 1.
Comparative example 1
A preparation method of polymyxin B sulfate at least comprises the following steps:
(1) polymyxin B seed preparation: putting the polymyxin B producing strain into the culture medium A for recovery culture, and then putting the polymyxin B producing strain into the culture medium B for shaking table culture to obtain an activated polymyxin B producing strain; the culture medium A in the step (1) contains the following components in every 100ml of culture medium: 2g of sucrose, 2g of peptone, 0.25g of dipotassium phosphate, 0.5g of magnesium sulfate heptahydrate, 2g of agar and the balance of water; the culture medium B in the step (1) contains the following components in every 100ml of culture medium: 9g of wheat flour, 0.005g of amylase, 0.45g of yeast powder, 0.3g of calcium carbonate, 0.015g of disodium hydrogen phosphate, 0.45g of ammonium sulfate and the balance of water;
(2) fermentation: adding the activated polymyxin B producing strain obtained in the step (1) into a culture tank C, and culturing in a seeding tank; then placing the polymyxin B strain in a culture tank D, performing fermentation tank culture, supplementing materials, and continuing fermentation tank culture to obtain fermentation liquor of polymyxin B strain; the culture tank C in the step (2) contains the following components in every 100ml of culture medium: 9g of wheat flour, 0.005g of amylase, 0.45g of yeast powder, 0.3g of calcium carbonate, 0.015g of disodium hydrogen phosphate, 0.45g of ammonium sulfate, 0.03g of defoaming agent and the balance of water; the culture tank D in the step (2) contains the following components per 100ml of culture medium: 12g of wheat flour, 0.006g of amylase, 0.8g of yeast powder, 0.3g of calcium carbonate, 0.015g of disodium hydrogen phosphate, 0.45g of ammonium sulfate, 0.03g of defoaming agent, 0.08g of L-threonine and the balance of water; the feed supplement formula contains the following components in each 100ml of culture medium: 0.08g of L-threonine, 0.3g of glucose and the balance of water; the defoaming agent is GPE type polyoxyethylene polyoxypropylene glycerol ether;
(3) refining: filtering the fermentation liquor of the polymyxin B strain obtained in the step (2), treating by ion exchange resin, treating by macroporous resin, desorbing alkali crystallization of liquid, transferring salt, decoloring, concentrating by nanofiltration, and freeze-drying in vacuum to obtain polymyxin B sulfate; the ion exchange resin in the step (3) is strong acid cation resin; the strong acid cation resin is a 732 cation exchange resin modified by 4-sulfamide benzoyl imine and 4-sulfonic pyridine-3-sulfamide; the macroporous resin in the step (3) is D152;
resuscitating culture in step (1) of the present invention, shaking culture in step (1) of the present invention, seeding tank culture in step (2) of the present invention, fermenter culture in step (2) of the present invention, filtering in step (3) of the present invention, ion exchange resin treatment in step (3) of the present invention, macroporous resin treatment in step (3) of the present invention, alkali crystallization of desorbent solution in step (3) of the present invention, dissolution in step (3) of the present invention, decoloring in step (3) of the present invention, nanofiltration concentration in step (3) of the present invention, vacuum freeze-drying in step (3) of the present invention, and a method for preparing the strong acid cation resin is the same as in example 1.
Comparative example 2
A preparation method of polymyxin B sulfate at least comprises the following steps:
(1) polymyxin B seed preparation: putting the polymyxin B producing strain into the culture medium A for recovery culture, and then putting the polymyxin B producing strain into the culture medium B for shaking table culture to obtain an activated polymyxin B producing strain; the culture medium A in the step (1) contains the following components in every 100ml of culture medium: 2g of sucrose, 2g of peptone, 0.25g of dipotassium phosphate, 0.5g of magnesium sulfate heptahydrate, 2g of agar and the balance of water; the culture medium B in the step (1) contains the following components in every 100ml of culture medium: 9g of wheat flour, 0.005g of amylase, 0.45g of yeast powder, 0.3g of calcium carbonate, 0.015g of disodium hydrogen phosphate, 0.45g of ammonium sulfate and the balance of water;
(2) fermentation: adding the activated polymyxin B producing strain obtained in the step (1) into a culture tank C, and culturing in a seeding tank; then placing the polymyxin B strain in a culture tank D, performing fermentation tank culture, supplementing materials, and continuing fermentation tank culture to obtain fermentation liquor of polymyxin B strain; the culture tank C in the step (2) contains the following components in every 100ml of culture medium: 9g of wheat flour, 0.005g of amylase, 0.45g of yeast powder, 0.3g of calcium carbonate, 0.015g of disodium hydrogen phosphate, 0.45g of ammonium sulfate, 0.03g of defoaming agent and the balance of water; the culture tank D in the step (2) contains the following components per 100ml of culture medium: 12g of wheat flour, 0.006g of amylase, 0.8g of yeast powder, 0.3g of calcium carbonate, 0.015g of disodium hydrogen phosphate, 0.45g of ammonium sulfate, 0.03g of defoaming agent, 0.08g of L-threonine and the balance of water; the feed supplement formula contains the following components in each 100ml of culture medium: 0.08g of L-threonine, 0.2g of ammonium sulfate, 0.1g of glucose and the balance of water; the defoaming agent is GPE type polyoxyethylene polyoxypropylene glycerol ether;
(3) refining: filtering the fermentation liquor of the polymyxin B strain obtained in the step (2), treating by ion exchange resin, treating by macroporous resin, desorbing alkali crystallization of liquid, transferring salt, decoloring, concentrating by nanofiltration, and freeze-drying in vacuum to obtain polymyxin B sulfate; the ion exchange resin in the step (3) is strong acid cation resin; the strong acid cation resin is a 732 cation exchange resin modified by 4-sulfamide benzoyl imine and 4-sulfonic pyridine-3-sulfamide; the macroporous resin in the step (3) is D152;
resuscitating culture in step (1) of the present invention, shaking culture in step (1) of the present invention, seeding tank culture in step (2) of the present invention, fermenter culture in step (2) of the present invention, filtering in step (3) of the present invention, ion exchange resin treatment in step (3) of the present invention, macroporous resin treatment in step (3) of the present invention, alkali crystallization of desorbent solution in step (3) of the present invention, dissolution in step (3) of the present invention, decoloring in step (3) of the present invention, nanofiltration concentration in step (3) of the present invention, vacuum freeze-drying in step (3) of the present invention, and a method for preparing the strong acid cation resin is the same as in example 1.
Comparative example 3
A preparation method of polymyxin B sulfate at least comprises the following steps:
(1) polymyxin B seed preparation: putting the polymyxin B producing strain into the culture medium A for recovery culture, and then putting the polymyxin B producing strain into the culture medium B for shaking table culture to obtain an activated polymyxin B producing strain; the culture medium A in the step (1) contains the following components in every 100ml of culture medium: 2g of sucrose, 2g of peptone, 0.25g of dipotassium phosphate, 0.5g of magnesium sulfate heptahydrate, 2g of agar and the balance of water; the culture medium B in the step (1) contains the following components in every 100ml of culture medium: 9g of wheat flour, 0.05g of amylase, 0.45g of yeast powder, 0.3g of calcium carbonate, 0.015g of disodium hydrogen phosphate, 0.45g of ammonium sulfate and the balance of water;
(2) fermentation: adding the activated polymyxin B producing strain obtained in the step (1) into a culture tank C, and culturing in a seeding tank; then placing the polymyxin B strain in a culture tank D for fermentation tank culture to obtain fermentation liquor of polymyxin B strain; the culture tank C in the step (2) contains the following components in every 100ml of culture medium: 9g of wheat flour, 0.05g of amylase, 0.45g of yeast powder, 0.3g of calcium carbonate, 0.015g of disodium hydrogen phosphate, 0.45g of ammonium sulfate, 0.03g of defoaming agent and the balance of water; the culture tank D in the step (2) contains the following components per 100ml of culture medium: 12g of wheat flour, 0.6g of amylase, 0.8g of yeast powder, 0.3g of calcium carbonate, 0.015g of disodium hydrogen phosphate, 0.45g of ammonium sulfate, 0.03g of defoaming agent, 0.08g of L-threonine and the balance of water; the defoaming agent is GPE type polyoxyethylene polyoxypropylene glycerol ether;
(3) refining: filtering the fermentation liquor of the polymyxin B strain obtained in the step (2), treating by ion exchange resin, treating by macroporous resin, desorbing alkali crystallization of liquid, transferring salt, decoloring, concentrating by nanofiltration, and freeze-drying in vacuum to obtain polymyxin B sulfate; the ion exchange resin in the step (3) is strong acid cation resin; the strong acid cation resin is a 732 cation exchange resin modified by 4-sulfamide benzoyl imine and 4-sulfonic pyridine-3-sulfamide; the macroporous resin in the step (3) is D152;
performing resuscitation culture in the step (1) of the present invention, performing shake culture in the step (1) of the present invention, performing seeding tank culture in the step (2) of the present invention, performing fermentation tank culture in the step (2) of the present invention, performing filtration in the step (3) of the present invention, performing ion exchange resin treatment in the step (3) of the present invention, performing macroporous resin treatment in the step (3) of the present invention, performing desorbent alkali crystallization in the step (3) of the present invention, performing dissolution in the step (3) of the present invention, performing decolorization in the step (3) of the present invention, performing nanofiltration concentration in the step (3) of the present invention, performing vacuum freeze drying in the step (3) of the present invention, and preparing the strong acid cation resin in the same manner as in the step (1); the difference from example 1 is that no feed is used.
Comparative example 4
A preparation method of polymyxin B sulfate at least comprises the following steps:
(1) polymyxin B seed preparation: putting the polymyxin B producing strain into the culture medium A for recovery culture, and then putting the polymyxin B producing strain into the culture medium B for shaking table culture to obtain an activated polymyxin B producing strain; the culture medium A in the step (1) contains the following components in every 100ml of culture medium: 2g of sucrose, 2g of peptone, 0.25g of dipotassium phosphate, 0.5g of magnesium sulfate heptahydrate, 2g of agar and the balance of water; the culture medium B in the step (1) contains the following components in every 100ml of culture medium: 9g of wheat flour, 0.005g of amylase, 0.45g of yeast powder, 0.3g of calcium carbonate, 0.015g of disodium hydrogen phosphate, 0.45g of ammonium sulfate and the balance of water;
(2) fermentation: adding the activated polymyxin B producing strain obtained in the step (1) into a culture tank C, and culturing in a seeding tank; then placing the polymyxin B strain in a culture tank D, performing fermentation tank culture, supplementing materials, and continuing fermentation tank culture to obtain fermentation liquor of polymyxin B strain; the culture tank C in the step (2) contains the following components in every 100ml of culture medium: 9g of wheat flour, 0.005g of amylase, 0.45g of yeast powder, 0.3g of calcium carbonate, 0.015g of disodium hydrogen phosphate, 0.45g of ammonium sulfate, 0.03g of defoaming agent and the balance of water; the culture tank D in the step (2) contains the following components per 100ml of culture medium: 12g of wheat flour, 0.006g of amylase, 0.8g of yeast powder, 0.3g of calcium carbonate, 0.015g of disodium hydrogen phosphate, 0.45g of ammonium sulfate, 0.03g of defoaming agent, 0.08g of L-threonine and the balance of water; the feed supplement formula contains the following components in each 100ml of culture medium: 0.08g of L-threonine, 0.3g of ammonium sulfate and the balance of water; the defoaming agent is GPE type polyoxyethylene polyoxypropylene glycerol ether;
(3) refining: filtering the fermentation liquor of the polymyxin B strain obtained in the step (2), treating by ion exchange resin, treating by macroporous resin, desorbing alkali crystallization of liquid, transferring salt, decoloring, concentrating by nanofiltration, and freeze-drying in vacuum to obtain polymyxin B sulfate; the ion exchange resin in the step (3) is strong acid cation resin; the strong acid cation resin is a 732 cation exchange resin modified by 4-sulfonic pyridine-3-sulfonamide; the macroporous resin in the step (3) is D152;
resuscitating culture in step (1) of the present invention, shaking culture in step (1) of the present invention, seeding tank culture in step (2) of the present invention, fermenter culture in step (2) of the present invention, filtering in step (3) of the present invention, ion exchange resin treatment in step (3) of the present invention, macroporous resin treatment in step (3) of the present invention, alkali crystallization of desorbent solution in step (3) of the present invention, dissolution in step (3) of the present invention, decoloring in step (3) of the present invention, nanofiltration concentration in step (3) of the present invention, vacuum freeze-drying in step (3) of the present invention, and a method for preparing the strong acid cation resin is the same as in example 1.
Comparative example 5
A preparation method of polymyxin B sulfate at least comprises the following steps:
(1) polymyxin B seed preparation: putting the polymyxin B producing strain into the culture medium A for recovery culture, and then putting the polymyxin B producing strain into the culture medium B for shaking table culture to obtain an activated polymyxin B producing strain; the culture medium A in the step (1) contains the following components in every 100ml of culture medium: 2g of sucrose, 2g of peptone, 0.25g of dipotassium phosphate, 0.5g of magnesium sulfate heptahydrate, 2g of agar and the balance of water; the culture medium B in the step (1) contains the following components in every 100ml of culture medium: 9g of wheat flour, 0.005g of amylase, 0.45g of yeast powder, 0.3g of calcium carbonate, 0.015g of disodium hydrogen phosphate, 0.45g of ammonium sulfate and the balance of water;
(2) fermentation: adding the activated polymyxin B producing strain obtained in the step (1) into a culture tank C, and culturing in a seeding tank; then placing the polymyxin B strain in a culture tank D, performing fermentation tank culture, supplementing materials, and continuing fermentation tank culture to obtain fermentation liquor of polymyxin B strain; the culture tank C in the step (2) contains the following components in every 100ml of culture medium: 9g of wheat flour, 0.005g of amylase, 0.45g of yeast powder, 0.3g of calcium carbonate, 0.015g of disodium hydrogen phosphate, 0.45g of ammonium sulfate, 0.03g of defoaming agent and the balance of water; the culture tank D in the step (2) contains the following components per 100ml of culture medium: 12g of wheat flour, 0.006g of amylase, 0.8g of yeast powder, 0.3g of calcium carbonate, 0.015g of disodium hydrogen phosphate, 0.45g of ammonium sulfate, 0.03g of defoaming agent, 0.08g of L-threonine and the balance of water; the feed supplement formula contains the following components in each 100ml of culture medium: 0.08g of L-threonine, 0.3g of ammonium sulfate and the balance of water; the defoaming agent is GPE type polyoxyethylene polyoxypropylene glycerol ether;
(3) refining: filtering the fermentation liquor of the polymyxin B strain obtained in the step (2), treating by ion exchange resin, treating by macroporous resin, desorbing alkali crystallization of liquid, transferring salt, decoloring, concentrating by nanofiltration, and freeze-drying in vacuum to obtain polymyxin B sulfate; the ion exchange resin in the step (3) is strong acid cation resin; the strong acid cation resin is a 4-sulfonamide benzoylimine modified 732 cation exchange resin; the macroporous resin in the step (3) is D152;
resuscitating culture in step (1) of the present invention, shaking culture in step (1) of the present invention, seeding tank culture in step (2) of the present invention, fermenter culture in step (2) of the present invention, filtering in step (3) of the present invention, ion exchange resin treatment in step (3) of the present invention, macroporous resin treatment in step (3) of the present invention, alkali crystallization of desorbent solution in step (3) of the present invention, dissolution in step (3) of the present invention, decoloring in step (3) of the present invention, nanofiltration concentration in step (3) of the present invention, vacuum freeze-drying in step (3) of the present invention, and a method for preparing the strong acid cation resin is the same as in example 1.
Comparative example 6
A preparation method of polymyxin B sulfate at least comprises the following steps:
(1) polymyxin B seed preparation: putting the polymyxin B producing strain into the culture medium A for recovery culture, and then putting the polymyxin B producing strain into the culture medium B for shaking table culture to obtain an activated polymyxin B producing strain; the culture medium A in the step (1) contains the following components in every 100ml of culture medium: 2g of sucrose, 2g of peptone, 0.25g of dipotassium phosphate, 0.5g of magnesium sulfate heptahydrate, 2g of agar and the balance of water; the culture medium B in the step (1) contains the following components in every 100ml of culture medium: 9g of wheat flour, 0.005g of amylase, 0.45g of yeast powder, 0.3g of calcium carbonate, 0.015g of disodium hydrogen phosphate, 0.45g of ammonium sulfate and the balance of water;
(2) fermentation: adding the activated polymyxin B producing strain obtained in the step (1) into a culture tank C, and culturing in a seeding tank; then placing the polymyxin B strain in a culture tank D, performing fermentation tank culture, supplementing materials, and continuing fermentation tank culture to obtain fermentation liquor of polymyxin B strain; the culture tank C in the step (2) contains the following components in every 100ml of culture medium: 9g of wheat flour, 0.005g of amylase, 0.45g of yeast powder, 0.3g of calcium carbonate, 0.015g of disodium hydrogen phosphate, 0.45g of ammonium sulfate, 0.03g of defoaming agent and the balance of water; the culture tank D in the step (2) contains the following components per 100ml of culture medium: 12g of wheat flour, 0.006g of amylase, 0.8g of yeast powder, 0.3g of calcium carbonate, 0.015g of disodium hydrogen phosphate, 0.45g of ammonium sulfate, 0.03g of defoaming agent, 0.08g of L-threonine and the balance of water; the feed supplement formula contains the following components in each 100ml of culture medium: 0.08g of L-threonine, 0.3g of ammonium sulfate and the balance of water; the defoaming agent is GPE type polyoxyethylene polyoxypropylene glycerol ether;
(3) refining: filtering the fermentation liquor of the polymyxin B strain obtained in the step (2), treating by ion exchange resin, treating by macroporous resin, desorbing alkali crystallization of liquid, transferring salt, decoloring, concentrating by nanofiltration, and freeze-drying in vacuum to obtain polymyxin B sulfate; the ion exchange resin in the step (3) is strong acid cation resin; the strong acid cation resin is 732 cation exchange resin; the macroporous resin in the step (3) is D152;
resuscitating culture in step (1) of the present invention, shaking culture in step (1) of the present invention, seeding tank culture in step (2) of the present invention, fermenter culture in step (2) of the present invention, filtration in step (3) of the present invention, ion exchange resin treatment in step (3) of the present invention, macroporous resin treatment in step (3) of the present invention, alkali crystallization of desorbent solution in step (3) of the present invention, dissolution in step (3) of the present invention, decolorization in step (3) of the present invention, nanofiltration concentration in step (3) of the present invention, and vacuum freeze-drying in step (3) of the present invention are the same as in example 1.
Comparative example 7
A preparation method of polymyxin B sulfate at least comprises the following steps:
(1) polymyxin B seed preparation: putting the polymyxin B producing strain into the culture medium A for recovery culture, and then putting the polymyxin B producing strain into the culture medium B for shaking table culture to obtain an activated polymyxin B producing strain; the culture medium A in the step (1) contains the following components in every 100ml of culture medium: 2g of sucrose, 2g of peptone, 0.25g of dipotassium phosphate, 0.5g of magnesium sulfate heptahydrate, 2g of agar and the balance of water; the culture medium B in the step (1) contains the following components in every 100ml of culture medium: 9g of wheat flour, 0.005g of amylase, 0.45g of yeast powder, 0.3g of calcium carbonate, 0.015g of disodium hydrogen phosphate, 0.45g of ammonium sulfate and the balance of water;
(2) fermentation: adding the activated polymyxin B producing strain obtained in the step (1) into a culture tank C, and culturing in a seeding tank; then placing the polymyxin B strain in a culture tank D for fermentation tank culture to obtain fermentation liquor of polymyxin B strain; the culture tank C in the step (2) contains the following components in every 100ml of culture medium: 9g of wheat flour, 0.005g of amylase, 0.45g of yeast powder, 0.3g of calcium carbonate, 0.015g of disodium hydrogen phosphate, 0.45g of ammonium sulfate, 0.03g of defoaming agent and the balance of water; the culture tank D in the step (2) contains the following components per 100ml of culture medium: 12g of wheat flour, 0.006g of amylase, 0.8g of yeast powder, 0.3g of calcium carbonate, 0.015g of disodium hydrogen phosphate, 0.45g of ammonium sulfate, 0.03g of defoaming agent, 0.08g of L-threonine and the balance of water;
(3) refining: filtering the fermentation liquor of the polymyxin B strain obtained in the step (2), treating by ion exchange resin, treating by macroporous resin, desorbing alkali crystallization of liquid, transferring salt, decoloring, concentrating by nanofiltration, and freeze-drying in vacuum to obtain polymyxin B sulfate; the ion exchange resin in the step (3) is strong acid cation resin; the strong acid cation resin is 732 cation exchange resin; the macroporous resin in the step (3) is D152;
performing resuscitation culture in the step (1) of the present invention, performing shake culture in the step (1) of the present invention, performing seeding tank culture in the step (2) of the present invention, performing fermenter culture in the step (2) of the present invention, performing filtration in the step (3) of the present invention, performing ion exchange resin treatment in the step (3) of the present invention, performing macroporous resin treatment in the step (3) of the present invention, performing base crystallization of a desorption solution in the step (3) of the present invention, performing dissolution in the step (3) of the present invention, performing decolorization in the step (3) of the present invention, performing nanofiltration concentration in the step (3) of the present invention, and performing vacuum freeze-drying in the step (3) of the present invention are the same as in example 1; the difference from example 1 is that there is no feed and the strong acid cation resin is 732 cation exchange resin.
And (3) performance testing:
table 1 results of performance testing
Figure BDA0001771843020000211
Figure BDA0001771843020000221
As can be seen from the above data, glucose and (NH) were fed in a fed-batch fashion at the end of logarithmic growth (20h)4)2SO4The effect on polymyxin B fermentation titers was examined. The data in the experimental examples show that the titer of the fermentation liquor can be improved to different degrees by adding glucose and ammonium sulfate separately, but the effect of the ammonium sulfate is obviously better than that of the glucose. When glucose and ammonium sulfate are supplemented simultaneously, the effect is not the superposition of the two, and is slightly lower than that of the single supplement of ammonium sulfate. Therefore, ammonium sulfate is added when polymyxin B is fermented for 20h, so that the titer of the fermentation liquid is remarkably improved. The preparation method of polymyxin B sulfate provided by the invention controls single impurities possibly influencing the quality of finished products in fermentation liquor to a level lower than 1.5%, and simultaneously combines an effective refining method to ensure that the quality of the finished products extracted from the fermentation liquor meets the standards of EP8.0 and CP 2015.
The foregoing examples are merely illustrative and serve to explain some of the features of the method of the present invention. The appended claims are intended to claim as broad a scope as is contemplated, and the examples presented herein are merely illustrative of selected implementations in accordance with all possible combinations of examples. Accordingly, it is applicants' intention that the appended claims are not to be limited by the choice of examples illustrating features of the invention. Also, where numerical ranges are used in the claims, subranges therein are included, and variations in these ranges are also to be construed as possible being covered by the appended claims.

Claims (6)

1. A preparation method of polymyxin B sulfate is characterized by at least comprising the following steps:
(1) polymyxin B seed preparation: putting the polymyxin B producing strain into the culture medium A for recovery culture, and then putting the polymyxin B producing strain into the culture medium B for shaking table culture to obtain an activated polymyxin B producing strain;
(2) fermentation: adding the activated polymyxin B producing strain obtained in the step (1) into a culture tank C, and culturing in a seeding tank; then placing the polymyxin B strain in a culture tank D for fermentation tank culture to obtain fermentation liquor of polymyxin B strain;
(3) refining: filtering the fermentation liquor of the polymyxin B strain obtained in the step (2), treating by ion exchange resin, treating by macroporous resin, desorbing alkali crystallization of liquid, transferring salt, decoloring, concentrating by nanofiltration, and freeze-drying in vacuum to obtain polymyxin B sulfate; the ion exchange resin is strong acid cation resin; the strong acid cation resin is a 732 cation exchange resin modified by 4-sulfamide benzoyl imine and 4-sulfonic pyridine-3-sulfamide; the fermentation tank culture process in the step (2) also comprises a feeding process, and each 100ml of culture medium in the feeding formula contains the following components: 0.01-0.1g of L-threonine, 0.1-0.5g of ammonium sulfate and the balance of water; the polymyxin B producing strain in the step (1) is Paenibacillus polymyxa (Paenibacillus polymyxa).
2. The process for producing polymyxin B sulfate according to claim 1, wherein the medium A in step (1) contains the following components per 100ml of the medium: 1-3g of sucrose, 1-5g of peptone, 0.1-0.5g of dipotassium phosphate, 0.2-1g of magnesium sulfate heptahydrate, 1-3g of agar and the balance of water.
3. The process for producing polymyxin B sulfate according to claim 1, wherein the medium B in step (1) contains the following components per 100ml of the medium: 7-10g of wheat flour, 0.002-0.01g of amylase, 0.2-0.6g of yeast powder, 0.1-0.5g of calcium carbonate, 0.01-0.03g of disodium hydrogen phosphate, 0.2-0.7g of ammonium sulfate and the balance of water.
4. The process for producing polymyxin B sulfate according to claim 1, wherein the culture tank C in step (2) contains the following components per 100ml of the culture medium: 7-10g of wheat flour, 0.002-0.01g of amylase, 0.2-0.6g of yeast powder, 0.1-0.5g of calcium carbonate, 0.01-0.03g of disodium hydrogen phosphate, 0.2-0.7g of ammonium sulfate, 0.01-0.05g of defoaming agent and the balance of water.
5. The process for producing polymyxin B sulfate according to claim 1, wherein the culture tank D in step (2) contains the following components per 100ml of the culture medium: 10-15g of wheat flour, 0.002-0.01g of amylase, 0.5-1g of yeast powder, 0.1-0.5g of calcium carbonate, 0.01-0.03g of disodium hydrogen phosphate, 0.2-0.7g of ammonium sulfate, 0.01-0.05g of defoaming agent, 0.01-0.1g of L-threonine and the balance of water.
6. The method for preparing polymyxin B sulfate according to claim 1, wherein the macroporous resin in step (3) comprises one of D001, D113, D201, D301, D370 and D152.
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