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Method for identifying structure of yeast colony of Daqu starter or fermented grain of distilled spirit by using denaturing gradient electrophoresis

A technique of denaturing gradient and electrophoresis, which is applied in the direction of microorganism-based methods, biochemical equipment and methods, and microorganism determination/inspection, etc.

Inactive Publication Date: 2009-11-04
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The purpose of the present invention is to provide a method for identifying the yeast community structure in liquor Daqu and fermented grains by using denaturing gradient electrophoresis analysis to solve the problems existing in existing research methods

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  • Method for identifying structure of yeast colony of Daqu starter or fermented grain of distilled spirit by using denaturing gradient electrophoresis
  • Method for identifying structure of yeast colony of Daqu starter or fermented grain of distilled spirit by using denaturing gradient electrophoresis
  • Method for identifying structure of yeast colony of Daqu starter or fermented grain of distilled spirit by using denaturing gradient electrophoresis

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Experimental program
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Embodiment 1

[0027] Daqu, a strong-flavor liquor from northern Jiangsu, was used to sample the center and skin of the koji. The genomes of all microorganisms in the samples were extracted by the Beadbeater method. Use a Bio-rad PCR instrument for PCR amplification. The yeast PCR conditions are as follows: use universal primers to amplify the DNA fragments in the yeast 26S rDNAD1 region. The primers are:

[0028] NL1-GC: 5'-CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCATATCAATAAGCGGAGGAAAAG-3',

[0029] LS2: 5'-ATTCCCAAACAACTCGACTC-3';

[0030]The 25 μL reaction system includes 2.5 μL 10×Buffer, 2 μL 25 mmol / L dNTP mixture, 1U TaqDNA polymerase, 25 pmol of each primer, about 10 ng of DNA template, and 25 μL of double distilled water. PCR reaction program: 94°C pre-denaturation for 4 min; 94°C for 1 min, 65°C for 1 min, 72°C for 1 min, 30 cycles; finally 72°C for 10 min.

[0031] The DGGE conditions are: the acrylamide gel concentration is 8%, the denaturing gradient range is 10%-50% (aqueous solution...

Embodiment 2

[0034] Fen-flavor liquor fermented grains were used, and samples were taken at three points, the upper, middle, and lower points in the fermentation vat, and mixed after the sampling was completed. The genomes of all microorganisms in the samples were extracted by the Beadbeater method. PCR amplification was performed using a Bio-rad PCR instrument. Yeast PCR conditions are as follows: use universal primers to amplify the DNA fragment of yeast 26S rDNAD1 region. Primers are:

[0035] NL1-GC: 5'-CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCATATCAATAAGCGGAGGAAAAG-3',

[0036] LS2: 5'-ATTCCCAAACAACTCGACTC-3';

[0037] The 25 μL reaction system includes 2.5 μL 10×Buffer, 2 μL 25 mmol / L dNTP mixture, 1U TaqDNA polymerase, 25 pmol of each primer, about 10 ng of DNA template, and 25 μL of double distilled water. PCR reaction program: 94°C pre-denaturation for 4 min; 94°C for 1 min, 65°C for 1 min, 72°C for 1 min, 30 cycles; finally 72°C for 10 min.

[0038] The DGGE conditions are: the acryl...

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Abstract

The invention relates a method for identifying a structure of a yeast colony of a Daqu starter or a fermented grain of distilled spirit by using denaturing gradient electrophoresis, which belongs to the field of microbial ecological techniques. The method comprises the following steps: extracting a genome DNA of the Daqu starter or fermented grain sample of the distilled spirit by using a bead milling method; by designing a general DGGE primer of yeasts in a common wine-making microbe, performing PCR amplification on a specific specificity DNA segment in a yeast ribosome; separating corresponding DNA segments of different varieties by DGGE electrophoresis; gel-cutting and recovering straps corresponding to preponderant microbes; performing the PRC application again, connecting a T carrierand selecting a positive clone; and after re-comparison by the DGGE electrophoresis, checking orders and identifying microbe information corresponding to gel-cutting straps. The method adopts a molecular technique without depending on cultivation, and has the characteristics of simplicity, convenience, quick speed, sensitivity and the like; besides, the method provides a detection method for identifying the structure of the yeast colony of the Daqu stater or the fermented grain of the distilled spirit, and further provides a reference frame for developing wine-brewing microbes oriented to distilled spirit flavors with different odor types.

Description

technical field [0001] Using denaturing gradient electrophoresis to identify the yeast community structure of liquor Daqu or fermented grains, involving the use of PCR-DGGE modern molecular ecological technology to overcome the defects of traditional microbial isolation methods to analyze the community structure of liquor Daqu and fermented grains yeast and guide the orientation of superiority and high-efficiency flavor The technology of brewing microorganism screening belongs to the technical field of microbial ecology. Background technique [0002] Denaturing gradient electrophoresis (DGGE) technique is an electrophoresis technique first proposed by Fischer and Lerman in 1979 for detecting DNA mutations. In 1985, Myers et al. used "GC splint" and heteroduplex technology in DGGE for the first time, making the technology perfect day by day. DNA fragments of different lengths can be distinguished, but DNA fragments of the same length have the same migration behavior in the g...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N27/447C12R1/645
Inventor 高亦豹徐岩王海燕
Owner JIANGNAN UNIV
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