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Method for improving forward mutation probability of strains for producing polymyxin

A technology of forward mutation and polymyxin, applied in the direction of microorganism-based methods, biochemical equipment and methods, bacteria, etc., can solve the problems of cumbersome and complicated screening methods, reduce production costs, increase probability, and be easy to operate Effect

Active Publication Date: 2017-01-04
HEBEI SHENGXUE DACHENG PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Zhou Xigui, Dai Penggao and others disclosed a method of "breeding colistin high-yielding strains" (China Knowledge Network), using nitrosoguanidine to mutagenize Paenibacillus polymyxa AS1 5 4 1, and using its The positive mutation rate of its own secondary metabolite colistin is 32.3%, but the screening method is still cumbersome and complicated, although the probability of obtaining high-yield strains can be further improved

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] (1) Prepare aseptic separation plate medium, make a plate on an ultra-clean table, and then cultivate it empty for 3 days under the condition of a temperature of 30-35°C; the separation plate medium is composed of the following components and weight percentages: glucose 1.0 %, yeast powder 1.0%, beef extract 0.5%, sodium chloride 0.2%, agar 2.2%, water balance, pH6.0-7.2;

[0022] (2) Use sterile water to dilute the cryopreservation tube of the production strain, and select a dilution of 10 -3 -10 -8 The dilutions are sequentially applied to the separation plate culture medium in step (1), and cultivated for 70h-100h at a temperature of 28-35°C and a humidity of 30%-60%, to obtain colonies;

[0023] (3) Pick 135 single bacterial colonies in step (2), apply them on 135 slant mediums by streaking, and cultivate them at a temperature of 28-35°C and a humidity of 30%-60%, to obtain Bacterial lawn; slant medium is composed of the following components and weight percentages...

Embodiment 2

[0028] (1) Prepare aseptic separation plate medium, make a plate on an ultra-clean table, and then cultivate it empty for 3 days under the condition of a temperature of 30-35°C; the separation plate medium is composed of the following components and weight percentages: glucose 1.0 %, yeast powder 1.0%, beef extract 0.5%, sodium chloride 0.2%, light calcium carbonate 0.1%, agar 2.2%, water balance, pH6.0-7.2;

[0029] (2) Put the separation plate culture medium after 3 days of hollow culture in step (1) into the refrigerator, and refrigerate at 2-10°C for 6 days;

[0030] (3) Use sterile water to dilute the cryopreservation tube of the production strain, and select a dilution of 10 -2 -10 -8 The diluted solution is coated successively on the separation plate culture medium after refrigeration in step (2), at a temperature of 28-35°C and a humidity of 30%-60%, culture for 70h-100h to obtain N (viscosity Thick) and G (dry) type colonies;

[0031] (4) Pick 128 colonies of G (st...

Embodiment 3

[0036] (1) Prepare aseptic separation plate medium, make a plate on an ultra-clean table, and then cultivate it empty for 3 days under the condition of a temperature of 30-35°C; the separation plate medium is composed of the following components and weight percentages: glucose 1.0 %, yeast powder 1.0%, beef extract 0.5%, sodium chloride 0.2%, light calcium carbonate 0.15%, agar 2.2%, water balance, pH6.0-7.2;

[0037] (2) Put the separation plate culture medium after 3 days of hollow culture in step (1) into the refrigerator, and refrigerate at 2-10°C for 8 days;

[0038] (3) Use sterile water to dilute the cryopreservation tube of the production strain, and select a dilution of 10 -2 -10 -8 The diluted solution is coated successively on the separation plate culture medium after refrigeration in step (2), at a temperature of 28-35°C and a humidity of 30%-60%, culture for 70h-100h to obtain N (viscosity Thick) and G (dry) type colonies;

[0039] (4) Pick 144 colonies of G (s...

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Abstract

The invention provides a method for improving probability of selecting forward mutation strains of polymyxin and relates to the field of screening of differential bacteria of bacillus polymyxa. The method comprises the following steps: adding light calcium carbonate into a separation plate culture medium; after preparing a plate, carrying out low-temperature refrigeration; carrying out strain culture on the separation plate; transferring to a slant culture medium added with the light calcium carbonate for culturing according to an appearance of a single colony; inoculating a bacterial lawn cultured on the slant culture medium into a seed culture medium for culturing, so as to obtain the strains with the forward mutation probability being more than 50 percent. The invention adopts a screening method combining metabolic regulation on colistin producing strains and regulation of initial humidity of a solid culture medium, so that a direct and distinguishable screening method of high-yield bacterial colonies is realized; the probability of obtaining the forward mutation strains by screening colistin is improved and the screening efficiency is improved. The method is simple in process and easy to operate. Therefore, large-scale screening can be obtained and more high-yield strains are obtained, and furthermore, the production cost is reduced.

Description

technical field [0001] The invention relates to the field of Bacillus polymyxa screening for differential bacteria, in particular to a method for increasing the probability of selecting polymyxin forward mutant strains. Background technique [0002] Colistin Sulfate, also known as colistin, antienemy, polymyxin E, is a basic polypeptide extracted from the culture solution 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 Un...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/02C12N1/20C12R1/12
Inventor 郑万静闫彩杰
Owner HEBEI SHENGXUE DACHENG PHARMA
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