Method for extracting polymyxin B from fermentation broth

A technology of polymyxin and fermentation liquid, which is applied in the direction of preparation methods of polymyxin and peptides, chemical instruments and methods, etc., and can solve the problems of low purity of polymyxin B, large one-time investment, and easy pollution of resins, etc. problems, to achieve the effects of less drug resistance, shorter time consumption, and improved purity

Inactive Publication Date: 2013-06-05
SHANGHAI INST OF PHARMA IND +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The one-time investment of these two processes is relatively large, and the resin is easy to pollute and needs to be regenerated.
Therefore, the resi

Method used

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  • Method for extracting polymyxin B from fermentation broth

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] a. Fermentation broth pretreatment:

[0039] After the fermentation, a total of 1000 mL of fermented liquid was collected, the titer of polymyxin B was 7.7 IU / mL, the pH was adjusted to 2.3 with sulfuric acid, the fermented liquid was heated to 96° C., and maintained for 45 minutes to obtain a fermentation pretreatment liquid.

[0040] The determination of the potency of the fermentation broth is determined by the tube-and-dish method (with reference to the 2010 edition of "Chinese Pharmacopoeia (Part II)"). The specific method is as follows:

[0041] Indicator bacteria: Bordetella Bronchiseptica, CMCC NO: 58403.

[0042] Incline medium: beef extract 3g; peptone 8g; sodium chloride 5g; glucose 5g; pH 7.0; deionized water 1000mL; agar 22g.

[0043] Test medium: 6g of peptone; 4g of trypticase; 3g of yeast extract; 1.5g of beef extract; 1.0g of glucose; pH6.5~6.7;

[0044] Verification method:

[0045] Pick the well-grown thalli from the slant culture medium, insert i...

Embodiment 2

[0062] The fermentation broth preparation method is the same as in Example 1.

[0063] a. Fermentation broth pretreatment:

[0064] After the fermentation, a total of 1000 mL of fermentation liquid was collected, the titer of polymyxin B was 8.6 IU / mL, the pH was adjusted to 2.0 with sulfuric acid, and the fermentation liquid was heated to 90° C. for 30 minutes to obtain a fermentation pretreatment liquid.

[0065] The fermentation pretreatment liquid is separated into a clear filtrate by solid-liquid separation, and the filtrate is used for extraction.

[0066] b. Extraction:

[0067] Add 480ml of n-butanol to the 1000ml filtrate, mix thoroughly, adjust the pH to 12.0 with sodium hydroxide solution, and transfer the polymyxin B from the filtrate to the extractant. Take the n-butanol extract phase, wash it fully with water, then add an equal volume of water, adjust the pH to 1.6 with sulfuric acid, take the water phase, and obtain 480 ml of an aqueous solution containing pol...

Embodiment 3

[0074] The method and fermentation broth used are the same as in Example 1, except that after adding n-butanol to the filtrate, the pH is adjusted to 12.5 with sodium hydroxide solution to obtain about 648 mg of polymyxin B. Afterwards, about 635 mg of polymyxin B sulfate powder with a purity of 86.3% was obtained, and the titer determined by the tube-and-dish method was 8222 IU / g. The yield was 67.8%.

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Abstract

The invention discloses a method for extracting polymyxin B from a fermentation broth of the polymyxin B. The method for extracting the polymyxin B from the fermentation broth of the polymyxin B comprises the following steps: a, conducting solid-liquid separation on the fermentation broth of the polymyxin B and obtaining a filtrate; b, extracting the filtrate with a polar organic solvent, and extracting an extract liquor reversely with water when the extract liquor is at the potential hydrogen (pH) value of 1.0-2.0; c, adding the reversal extract liquor which is obtained from the step b into inorganic salt, wherein the volume of the reversed extract liquor of the inorganic salt is 3-8% (w/v), and adjusting the pH value to be 9.0-10.0 so that the polymyxin B is obtained after precipitation and sediment. Preferably, the method for extracting the polymyxin B from the fermentation broth further comprises a step of d: reacting the polymyxin B and sulfuric acid to form salt, and dissolving and precipitating, thereby obtaining the polymyxin B sulfate. The polymyxin B or the sulfate prepared by means of the method is higher in yield and purity, the purity of a product is above 85%, the tilter is above 8100IU/g, and the yield is above 60%. The method for extracting polymyxin B from the fermentation broth has the advantages of being easy and convenient to operate, short in time consuming, low in requirement of equipment, and capable of recycling the organic solvent and being used for large-scale industrial production.

Description

technical field [0001] The invention belongs to the field of methods for extracting substances from organisms, in particular to a method for extracting polymyxin B from polymyxin B fermentation broth. Background technique [0002] Polymyxin B is a basic cyclic polypeptide with good antibacterial activity synthesized and secreted by Bacillus polymyxa during the metabolic process, with a molecular weight of about 1200 Da; Salt, the molecular weight is about 1300Da. Among the known antibiotics, polymyxin has the strongest effect on Gram-negative bacteria, especially it can inhibit various infections caused by Pseudomonas aeruginosa and Escherichia coli, such as meningitis, dysentery, respiratory system infection , peritonitis, bile duct infection, urinary tract infection, burn infection, corneal infection and sepsis, etc., and it is not easy to produce drug resistance. [0003] Polymyxin B can be obtained by separating, extracting, purifying and drying the fermented liquid pr...

Claims

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Application Information

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IPC IPC(8): C07K7/62C07K1/36C07K1/30A61K38/12A61P31/04
Inventor 张元钦那可赵文杰赵波程晴华
Owner SHANGHAI INST OF PHARMA IND
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