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Micro-culture method of pleuromutilin producing bacteria and high-throughput screening method of high-yield bacteria of pleuromutilin

A technology of pleuromutilin and its culture method, which is applied in the field of micro-cultivation of pleuromutilin-producing bacteria and high-throughput screening of its high-yielding bacteria, can solve problems such as inapplicability, improve screening efficiency, ensure consistency, The effect of reducing errors

Active Publication Date: 2013-04-10
LANZHOU INST OF ANIMAL SCI & VETERINARY PHARMA OF CAAS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Only the combination of micro-culture and microplate reader detection can solve this problem well, and the current micro-culture and high-throughput detection technologies are not suitable for higher basidiomycete fungi such as pleuromutilin-producing bacteria, and Its special diterpene structure

Method used

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  • Micro-culture method of pleuromutilin producing bacteria and high-throughput screening method of high-yield bacteria of pleuromutilin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] The microculture steps of pleuromutilin-producing bacteria are as follows:

[0047] (1) Dilute the pleuromutilin-producing bacteria 10 times with sterile water to make a bacterial suspension, spread it on a plate, incubate at 25°C for 3-5 days, and isolate a single colony;

[0048](2) Prepare a 96-well microtiter plate containing 1 / 2 of the solid fermentation medium for later use: Autoclave the solid fermentation medium at 121°C for 15 minutes, and add it to the 96-well microtiter plate in an ultra-clean bench aseptically. The amount added to the well is 150 μl;

[0049] (3) Pick up the single-colony mycelia of the pleuromutilin-producing bacteria formed in step (1) with a bamboo stick, inoculate it in a 96-well microtiter plate containing solid fermentation medium, repeat the plate once, and leave Used for identification of shake flasks;

[0050] (4) Incubate the inoculated 96-well ELISA plate at 25°C for 7 days to complete the microculture of pleuromutilin-producing...

Embodiment 2

[0065] The microculture steps of pleuromutilin-producing bacteria are as follows:

[0066] (1) Dilute the pleuromutilin-producing bacteria to 100 times with sterile water to make a bacterial suspension, spread it on a plate, incubate at 25°C for 3-5 days, and isolate a single colony;

[0067] (2) Prepare a 96-well microtiter plate containing 1 / 3 of the solid fermentation medium for later use: Autoclave the solid fermentation medium at 121°C for 15 minutes, and add it to the 96-well microtiter plate in an ultra-clean bench aseptically. The amount added to the well is 100 μl;

[0068] (3) Use an inoculation loop to pick up the single colony mycelia of the pleuromutilin-producing bacteria formed in step (1), and inoculate it in a 96-well microtiter plate containing solid fermentation medium;

[0069] (4) Incubate the inoculated 96-well ELISA plate at 25°C for 8 days to complete the microculture of pleuromutilin-producing bacteria.

[0070] The high-throughput screening method o...

Embodiment 3

[0075] The microculture steps of pleuromutilin-producing bacteria are as follows:

[0076] (1) Dilute the pleuromutilin-producing bacteria to 1000 times with sterile water to make a bacterial suspension, spread it on a plate, culture at 25°C for 3-5 days, and isolate a single colony;

[0077] (2) Prepare a 96-well microtiter plate containing 2 / 3 of the solid fermentation medium for later use: Autoclave the solid fermentation medium at 121°C for 15 minutes, and add it to the 96-well microtiter plate in an ultra-clean bench aseptically. The amount added to the well is 200 μl;

[0078] (3) Pick up the single colony mycelium of the pleuromutilin-producing bacteria formed in step (1) with an inoculation needle, and inoculate it in a 96-well microtiter plate containing solid fermentation medium;

[0079] (4) Incubate the inoculated 96-well ELISA plate at 25°C for 9 days to complete the microculture of pleuromutilin-producing bacteria.

[0080] The high-throughput screening method ...

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Abstract

The invention provides a micro-culture method of pleuromutilin producing bacteria. The method comprises the following steps of: culturing the pleuromutilin producing bacteria and then separating a single bacterial colony; preparing a solid fermentation culture medium and sterilizing at a high pressure, and then adding the sterilized solid fermentation culture medium into each pore of an enzyme label plate; inoculating mycelia of the single bacterial colony generated by the pleuromutilin producing bacteria into the enzyme label plate and culturing for 7-9 days. The invention also provides a high-throughput screening method of high-yield bacteria of the pleuromutilin. The high-throughput screening method comprises the following steps of: adding extraction liquid into the pores of the enzymelabel plate of the micro-cultured pleuromutilin producing bacteria to extract; sucking the extraction liquid for each pore and transferring to another enzyme label plate; adding a color-developing agent to develop; measuring by using an enzyme label meter and analyzing the result to obtain the screened high-yield bacteria of the pleuromutilin. As the solid fermentation is adopted, the step of culturing seeds is simplified and the fermentation time is greatly shortened. Compared with the common screening method, the high-throughput screening method has a good related coefficient, thereby beingapplied to screening of the high-yield bacteria of the pleuromutilin.

Description

technical field [0001] The invention relates to a micro-cultivation method for pleuromutilin-producing bacteria and a high-throughput screening method for high-yielding bacteria. Background technique [0002] Pleuromutilin derivatives include warnimulin, tiamulin, etc., which are mainly added to animal feed as a premix, which not only prevents swine malaria and pneumonia, but also increases the output of pigs. The role of pleuromutilin antibiotics in production practice has become increasingly prominent, and its fermentation research has gradually become a research hotspot. In order to increase fermentation yield, breeding high-yielding strains is a breakthrough point for the application of pleuromutilin antibiotics. [0003] At present, high-throughput microbial micro-cultivation and detection methods are gradually becoming the main method for strain screening, but the micro-culture methods of various microorganisms are different, and there is no micro-culture method for a ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/00
Inventor 梁剑平赵晓彬陶蕾郭文柱王学红郭志廷尚若峰郝宝成
Owner LANZHOU INST OF ANIMAL SCI & VETERINARY PHARMA OF CAAS
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