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Method for screening high-producing strains of Polyoxin B

A technology of polyoxin and high-yield strains, which is applied in the direction of microorganism-based methods, biochemical equipment and methods, and microbial measurement/inspection, which can solve the problems of low content of polyoxin B components and achieve low cost , a wide range of sources, the effect of important industrial and agricultural application potential

Pending Publication Date: 2020-05-05
陕西麦可罗生物科技有限公司
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  • Claims
  • Application Information

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Problems solved by technology

[0006] In order to solve the problem of low polyoxin B component content in the prior art, the present invention provides a method for screening high-yielding strains of polyoxin B component, comprising preparing the starting strain spore suspension, and diluting the diluted spore The suspension was subjected to ultraviolet mutagenesis first, then fluorescent lamp irradiation, and then after ARTP compound mutagenesis, it was spread on a medium plate and cultured, and half of the single colonies picked out were examined in shake flasks, and the polyoxin B component was selected Strains with high content

Method used

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  • Method for screening high-producing strains of Polyoxin B
  • Method for screening high-producing strains of Polyoxin B
  • Method for screening high-producing strains of Polyoxin B

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1: original bacterial strain spore preparation

[0038] Take Streptomyces aureus Chromogenes 98#, the starting strain of polyoxin, dilute and spread the slant medium, and culture at a constant temperature of 28°C for 7 days, select the fast-growing and plump single colony spores on the plate, cut off half of the single colony spores and add them to the physiological saline to make a spore suspension. The composition of 100mL slant medium is 0.5g of corn flour, 0.5g of maltose, 0.4g of yeast extract, 2.0g of agar, the rest is distilled water, and the pH before digestion is 7.0-7.2.

Embodiment 2

[0039] Embodiment 2: Mutagenesis of polyoxin starting strain

[0040] Add half of the single bacterial colony excised in Example 1 into physiological saline containing 20% ​​glycerol, vibrate and disperse to make a spore suspension. Pipette 15 μL of the spore suspension and drop it on the slide of the ARTP mutagenesis breeding instrument, and spread it evenly. Using the ARTP mutagenesis breeding instrument, first ultraviolet mutagenesis (15W ultraviolet lamp 30cm away from the suspension, irradiation for 30 seconds), then fluorescent lamp irradiation for 3 minutes, and then ARTP mutagenesis (irradiation power 120W, helium flow rate 8-12SLM, irradiation for 70 seconds) , to obtain the mutagen solution. Wash the mutagenesis solution with sterile saline under red light, and dilute the mutagenesis solution with normal saline gradient to 10 -4 、10 -5 、10-6 , smeared on the medium plate, and cultured in the dark at 26°C for 8 days. Select and cultivate the mutant single colony, ...

Embodiment 3

[0043] Embodiment 3: fermented liquid analysis

[0044] Use HPLC (high performance liquid chromatography) to detect the change of polyoxin components in the fermentation product, and screen the strains with differences in secondary metabolite components from the starting strain 98# through the increase or decrease of peak shape and the change of relative peak area.

[0045] Sample processing: take 1 mL of fermentation broth and centrifuge at 10,000 rpm for 5 minutes, take 200 μL of supernatant and add 200 μL of ultrapure water to dilute. Add 80 μL methanol aqueous solution containing 0.5% trifluoroacetic acid, mix thoroughly, and centrifuge at 14000 rpm for 10 minutes. Take 400 μL of the supernatant for HPLC analysis.

[0046] HPLC conditions: the chromatographic column adopts Thermo 250mm, 4.6μm C18 chromatographic column. The mobile phase of HPLC was 10% methanol aqueous solution (containing 0.1% trifluoroacetic acid), the detection wavelength was 260nm, the injection volu...

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Abstract

The invention relates to a method for screening high-producing strains of Polyoxin B. The method includes preparing spore suspension of starting strains; firstly performing ultraviolet mutagenesis onthe diluted spore suspension, and then performing daylight lamp irradiation; coating the spore suspension on a culture medium flat plate to perform cultivation after performing ARTP compound mutation;and performing shake flask assessment on half of each selected single colony, and selecting strains with high Polyoxin B content. The method can rapidly screen high content strains; and the fermentation broth of the screened strains with high Polyoxin B content has inhibition effects on apple alternaria leaf spot and alternaria alternata.

Description

technical field [0001] The invention belongs to the field of microbial pharmacy, and in particular relates to a method for screening high-yield strains of polyoxin B components. Background technique [0002] Polyoxin (polyoxin) is an agricultural antibiotic successfully developed by the Institute of Microbiology, Chinese Academy of Sciences in 1978, produced by Streptomyces aureus ( Streptomyces aureoch romogenes ) produced by fermentation, mainly used to control fungal diseases such as tobacco red spot, ginseng leaf black spot, sweet leaf brown spot, wheat powdery mildew, rice sheath blight, etc. It is an amphoteric water-soluble multi-component peptide pyrimidine Gan antibiotics. It is easily soluble in water, insoluble in organic solvents such as alcohols, acetone, chloroform, benzene and ether, stable in acid, inactivated in alkali, and stable to ultraviolet rays. It has good systemic conductivity, and mainly interferes with the biosynthesis of the cell wall composed o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N13/00C12Q1/04C12P17/16C12R1/49
CPCC12N13/00C12Q1/04C12P17/165
Inventor 杨玉旺邓钊潘忠成李蒲民
Owner 陕西麦可罗生物科技有限公司
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