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Screening method of colistin strains

A strain screening and colistin technology, applied in the biological field, can solve the problems of heavy workload, damage to strain activity, cumbersome detection and processing of fermentation samples, etc., and achieve the effect of improving screening speed and screening efficiency

Inactive Publication Date: 2013-04-24
HEBEI SHENGXUE DACHENG PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Since the screening of strains has gone through two stages of primary screening and retesting, there are more than 150-180 samples to be tested in each batch, and the detection and processing of fermentation samples is cumbersome, the workload is large, and the time required for liquid chromatography detection is long, which not only reduces the screening time Efficiency, and also affect the best period of preservation of high-yielding strains, causing certain damage to the activity of strains

Method used

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  • Screening method of colistin strains
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  • Screening method of colistin strains

Examples

Experimental program
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Effect test

Embodiment 1

[0021] a. Solid culture: Dilute Bacillus polymyxa to 10 -3 cells / mL, take 0.2 mL of evenly coated solid medium, culture at 28°C-30°C, relative humidity of 40% for 72 h, and obtain a single colony; the solid medium is L-asparagus Amino acid? g, glucose 1g, yeast extract 1g, peptone 0.1g, sodium chloride 0.1g, agar 2.2g, water? The component of g, prepared according to conventional methods, pH6.0-7.0;

[0022] b. Slant culture: Pick a single colony with regular shape on the solid medium containing L-aspartic acid, and apply it on the slant medium by streaking. The composition of the slant medium is consistent with the solid medium in step a; The culture conditions are also the same as step a.

[0023] c. Fermentation culture: the mature slant lawn on the slant medium is mixed with 20% glycerin solution for frozen preservation, and about 0.25 cm 2 The bacterial lawn was inoculated in 30 mL of fermentation medium, and cultured with shaking at 28°C-30°C and 200 rpm for 96 hours...

Embodiment 2

[0027] Example 2 Correspondence between the absorbance of the tested sample and the titer of the bacterial strain

[0028] Select three strains from the same batch for HPLC titer determination, and draw the histogram of average absorbance, HPLC titer and average biological potency (such as figure 1 ), the results show that the strains with high biological titer have low corresponding absorbance value and corresponding high actual titer in HPLC detection; the strains with low biological titer have high corresponding absorbance value and corresponding low actual titer in HPLC detection. Depend on figure 1 It can also be clearly seen that the absorbance of the indicator has a good correspondence with the potency of the fermentation broth, and the correlation coefficient R 2 up to 0.99 or more.

Embodiment 3

[0030] a. Solid culture: Dilute Bacillus polymyxa to 10 -6 cells / mL, take 0.2 mL of uniformly coated solid medium, and culture it for 96 h at 35°C-37°C, with a relative humidity of 60%, to obtain a single colony; the solid medium is L-asparagus Amino acid 0.2mg / mL, glucose 3g, yeast extract 3g, peptone 0.5g, sodium chloride 0.5g, agar 2.2g, the balance is water components, prepared according to conventional methods, pH6.0-7.0;

[0031] b. Slant culture: Pick a single colony with regular shape on the solid medium containing L-aspartic acid, and apply it on the slant medium by streaking. The composition of the slant medium is consistent with the solid medium in step a; Culture condition is also identical with a step;

[0032] c. Fermentation culture: take about 0.20 cm of the mature slant lawn on the slant medium 2 The bacterial lawn was inoculated in 20 mL of fermentation medium, and cultured with shaking at 32°C-35°C and 200 rpm for 72 hours to obtain a colistin fermentation...

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Abstract

The invention discloses a screening method of colistin strains. The screening method comprises the following steps of: adding Bacilluscolistinus into a solid culture medium containing L-asparaginic acid, transferring a single colony to an inclined plane for preservation after the single colony is mature, simultaneously inoculating into a fermentation culture medium, culturing till maturation, then centrifuging, taking supernatant liquid, adding into a bacterial solution of indicator bacteria for culture to prepare a detected sample, detecting the absorbance of the detected sample, and selecting the strains with low absorbance for preservation and later use. According to the method provided by the invention, the screening speed of polymyxin strains is greatly increased; and the screening period is shortened from original 3-4 weeks to one week, and the screening efficiency is improved by above 70%.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for screening colistin strains. Background technique [0002] Colistin ( Colistin ) is also known as colistin, anti-enemy, and Christine in China. It was discovered in 1947 by American and British scholars in Bacillus polymyxa ( Bacillus polymyxa ) culture medium with polymyxin B ( Polymyxin B ) were discovered together and were known at the time as polymyxin E ( Polymyxin E ). Later by colistin bacillus ( Bacillus colistinus ) Fermentation production, renamed colistin. It is a basic cyclic peptide antibiotic with an amino acid ring and a fatty acid side chain, which can combine with lipids on the bacterial cell membrane to disrupt the function of the cell membrane and destroy the integrity of the cell wall. It is effective against Gram-negative bacteria such as large intestine Bacillus, Salmonella, Shigella, Bacillus influenzae, Bacillus pertussis, Bac...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12Q1/04C12R1/07
Inventor 李琪刘聪洁王绘砖
Owner HEBEI SHENGXUE DACHENG PHARMA
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