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Method for breeding low-sugar-consumption strain capable of producing polymyxin E

A technology of polymyxin and bacterial strains, applied in the field of biopharmaceuticals, can solve the problems of hazardous waste safety management and increased production costs, and achieve the effect of alleviating the pressure of three wastes treatment, low utilization, and long-term stable use in large-scale production

Active Publication Date: 2021-08-17
HEBEI SHENGXUE DACHENG PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, most of the relevant research is aimed at improving potency, but with the upgrading of environmental management, the safety management of hazardous waste, and the increase in production costs have become the lifeblood of enterprise survival, and cost reduction has become the solution for most enterprises. Top priority

Method used

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  • Method for breeding low-sugar-consumption strain capable of producing polymyxin E
  • Method for breeding low-sugar-consumption strain capable of producing polymyxin E
  • Method for breeding low-sugar-consumption strain capable of producing polymyxin E

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] ARTP mutagenesis

[0061] 1), the polymyxin E bacterial strain SC1605 used in production is inoculated into the activation bottle and cultivated

[0062] The components of the activated medium used are (g / L): yeast powder 30.0g, malt extract 30.0g, sodium chloride 5g, medium pH7.0-7.2;

[0063] Culture conditions: shaker with an amplitude of 5 cm, a rotation speed of 200-220 rpm, a culture temperature of 30.0°C-32.0°C, and a culture period of 16 hours.

[0064] 2), absorb 10mL in 1) and centrifuge for 5 minutes / 3000rpm, pour out the supernatant, add 10% glycerin solution to mix and centrifuge again, after pouring out the supernatant, add 5mL10% glycerin solution, shake well to form spore suspension solution, so that the spore concentration reaches 10 7 -10 8 . Under sterile conditions, drop it on the sterile iron plate used for mutagenesis, and carry out ARTP mutagenesis, using high-purity helium as the gas source, processing power 80W, gas flow 10SLM (standard lite...

Embodiment 2

[0073] 1), inoculate the activated freezing solution preserved in 6) in Example 1 into the Z medium,

[0074] The composition and ratio of the basic medium (Z) of the fermentation bottle used are (g / L): starch 90.0, soybean cake powder 20.0, calcium carbonate 14.0, ammonium sulfate 20.0, potassium dihydrogen phosphate 1.2, amylase 0.05, soybean oil 10.0, pH7.2-7.4,

[0075] Culture conditions: shaker with an amplitude of 5 cm, a rotation speed of 200-220 rpm, a temperature of 30.0-32.0° C., and a period of 96-120 hours of culture.

[0076] 2) The fermented broth matured in 1) is tested for potency by liquid chromatography, and the residual sugar is detected by the formaldehyde method.

[0077] Table 1: Titer and residual sugar data of strains obtained by ARTP mutagenesis on Z medium.

[0078]

[0079] As can be seen in Table 1, the sugar consumption of strains after ARTP mutagenesis was equal to that of the starting strain, and the titer was generally higher than that of ...

Embodiment 3

[0081] 1), inoculate the activated freezing solution preserved in 6) in Example 1 into the D2 medium,

[0082] The composition and ratio of the medium used in the D2 fermentation bottle are (g / L): starch 54, soybean meal powder 20.0, calcium carbonate 14.0, ammonium sulfate 20.0, potassium dihydrogen phosphate 1.2, amylase 0.05, soybean oil 10.0, pH 7.2 -7.4,

[0083] D2 culture conditions: shaker with an amplitude of 5 cm, a rotation speed of 200-220 rpm, a temperature of 30.0-32.0° C., and periodical culture for 18-22 hours.

[0084] 2), inoculating the D2 fermentation broth grown to the period in 1) into the Z medium,

[0085] The composition and ratio of the basic medium (Z) of the fermentation bottle used are (g / L): starch 90.0, soybean cake powder 20.0, calcium carbonate 14.0, ammonium sulfate 20.0, potassium dihydrogen phosphate 1.2, amylase 0.05, soybean oil 10.0, pH7.2-7.4,

[0086] Culture conditions: shaker with an amplitude of 5 cm, a rotation speed of 200-220 r...

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Abstract

The invention belongs to the technical field of biological pharmacy, and particularly relates to a method for breeding a low-sugar-consumption strain capable of producing polymyxin E. The method comprises the following steps: A, preservation of an ARTP mutant strain; B, verification and determination of the low-sugar-consumption strain; C, breeding verification; and D, hereditary detection. The novel strain obtained by using such a scheme has lower utilization amount of a carbon source, production cost can be directly reduced, and direct economic benefits are brought; the residual sugar content of the obtained strain in fermentation is lower, and the pressure of three-waste treatment can be relieved to a certain extent; and the strain obtained by the method has genetic stability and can be used for large-scale production.

Description

technical field [0001] The invention belongs to the technical field of biopharmaceuticals, and in particular relates to a method for breeding polymyxin E strains with low sugar consumption. Background technique [0002] Colistin Sulfate, also known as colistin, antienemy, polymyxin E, is a basic polypeptide extracted from the culture medium of Bacillus polymyxa var.colistimus antibiotic. In the past ten years, due to the increasingly prominent potential harm to humans caused by drug residues and drug resistance, developed countries have successively banned several feed antibiotics. Colistin sulfate is not easily absorbed by the gastrointestinal tract when taken orally, and it is not easy to produce drug resistance. It has a strong antibacterial effect on Gram-negative bacteria, such as Escherichia coli, Salmonella, Pasteurella, and Pseudomonas aeruginosa. focus on. It is approved as a veterinary drug by the United States and the European Union, and is also used as a feed ...

Claims

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Application Information

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IPC IPC(8): C12N15/01C12N13/00C12N1/20C12R1/12
CPCC12N15/01C12N13/00C12N1/20
Inventor 郑万静徐珍刘聪洁韩冰
Owner HEBEI SHENGXUE DACHENG PHARMA
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