Method for obtaining high-yield curdlan bacterial strain through high-flux screening

A high-throughput, high-yield technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problems of time-consuming, labor-intensive, limited number of screening, high cost, etc.

Inactive Publication Date: 2017-11-24
EAST CHINA NORMAL UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The traditional screening of high-yield curdlan gum strains is to directly measure their dry weight through shake flask fermentation. This method not only takes a long time, is time-consuming, laborious, and costly, but also greatly limits the number of screens.

Method used

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  • Method for obtaining high-yield curdlan bacterial strain through high-flux screening
  • Method for obtaining high-yield curdlan bacterial strain through high-flux screening
  • Method for obtaining high-yield curdlan bacterial strain through high-flux screening

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Effect test

Embodiment 1

[0066] Correlation between different concentrations of curdlan gum standard substance and aniline blue color development in embodiment 1

[0067] Prepare 2mg / ml curdlan standard substance, dilute to different concentrations, successively 0, 0.1, 0.2, 0.4, 0.6, 0.8, 1, 1.2, 1.4, 1.6, 1.8, 2mg / ml, dilute the different concentrations Standard samples of curdlan gum and 2 mg / ml aniline blue solution with pH 7.0, shake and mix well, react at room temperature for 1-2 hours in the dark, and measure with a microplate reader at 590 nm. The results are shown in Table 1 and figure 1 shown by figure 1 It can be seen that there is a good correlation between curdlan content and absorbance value, R 2 is 0.9891.

[0068] Table 1 Different concentrations of standard substances and aniline blue color reaction measurement results

[0069]

Embodiment 2

[0070] The measurement of embodiment 2 aniline blue assay method coefficient of variation in 96 orifice plates

[0071] Select a strain and spread it on the plate, after 3 days of culture, pick a single colony into a 96-well plate, and use the above method for fermentation and measurement. The results are as follows: image 3 shown by image 3 It can be seen that the coefficient of variation of the 96-well plate fermentation is 13.8%, which is within a reasonable range (less than 20%), and there is no edge well effect. Therefore, positive mutants whose absorbance value measured by a microplate reader is higher than the coefficient of variation can be selected for re-screening.

Embodiment 3

[0072] Application of embodiment 3 aniline blue color development method in NTG mutagenesis screening

[0073] Carry out NTG mutagenesis using the method in step (1) above, pick 40 strains and carry out the 96-well plate primary screening described in step (2) for fermentation, and simultaneously carry out the shake flask verification fermentation described in step (4) After the experiment, the correlation between the two was determined after the fermentation. Experimental results such as figure 2 shown by figure 2 It can be seen that using the aniline blue chromogenic method established above to screen the mutagenized strains on a 96-well plate, and at the same time as a shake flask control, the results show that there is a good correlation between the chromogenic method and the shake flask assay. Therefore, the method established by the present invention can be well used for the screening of high-yield curdlan gum strains.

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Abstract

The invention discloses a method for obtaining high-yield curdlan bacterial strain through high-flux screening. The method comprises the following steps: 1) selecting NTC-mutagenic soil agrobacterium single colony; 2) employing a 96 orifice plate for primary screening; 3) performing secondary screening by a test tube; and 4) swing a bottle for verification. According to the invention, aniline blue is taken as a curdlan color developing substance while mutagenesis primary screening. The method has the advantages of rapidity and accuracy, can produce the curdlan bacterial strain with high yield through high-flux screening, can greatly reduce the cost, and increases the screening efficiency.

Description

technical field [0001] The invention relates to the technical field of microbial breeding, in particular to a novel high-throughput screening method for high-yield curdlan gum strains. Background technique [0002] Curdlan gum is a new type of biopolymer composed of glucose structural units linked by β-1,3-D-glucosidic bonds. It has the unique property of forming an irreversible gel under heating conditions. It has broad application prospects in fields such as medicine and food processing. [0003] In 1996, the U.S. Food and Drug Administration approved and allowed curdlan gum to be used in the food industry as a food additive. The Ministry of Health of my country also officially approved curdlan gum as a new type of food additive in 2006. As a stabilizer, coagulant, and thickener, curdlan gum can significantly improve the quality of foods including soy products, flour products, and meat products, and is deeply loved by the public. At present, the output of available rubbe...

Claims

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Application Information

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IPC IPC(8): C12N1/02C12N15/01C12P19/04C12R1/05
CPCC12N1/02C12N15/01C12P19/04
Inventor 金明飞常忠义高红亮陈璐田苏刘晓霞陈中山步国建
Owner EAST CHINA NORMAL UNIVERSITY
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