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Method for analyzing Bacillus community structure in white spirit fermentation system

A technology of community structure and liquor, applied in the direction of biochemical equipment and methods, microbial determination/inspection, etc.

Inactive Publication Date: 2012-10-10
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The present invention aims at the defects of the existing traditional microorganism separation method, and discloses a method using nested PCR combined with denaturing gradient gel electrophoresis to semi-quantitatively analyze the microorganisms in the fermentation system of liquor. Bacillus The composition of the structure and its proportion

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  • Method for analyzing Bacillus community structure in white spirit fermentation system
  • Method for analyzing Bacillus community structure in white spirit fermentation system
  • Method for analyzing Bacillus community structure in white spirit fermentation system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] p1-F: 5'-CGATGCGTAGCCGACCTGAG-3',

[0039] p1-R: 5'-AAGGAGGTGATCCAGCCGCA-3'.

[0040] The first-round PCR reaction system is 25 μL, including 12.5 μL 2×Taq PCR MasterMix enzyme, 0.5 μL (25 pmol) of each primer, 10.5 μL sterilized ultrapure water, and the amount of DNA template is about 10 ng. The first round of PCR reaction program was: 95°C pre-denaturation for 5 minutes, followed by 25 cycles, 95°C for 1 minute; 68°C for 90 seconds; 72°C for 2 minutes, and finally 72°C for 10 minutes. PCR products were detected by 1% agarose gel electrophoresis. The first-round PCR product was diluted 100-fold, and 1 μL was used as the template for the second-round PCR.

[0041] The primers used in the second round of PCR were:

[0042] p2-F: 5'-ATGGC TGTCGTCAGCT-3',

[0043] p2-R: 5'-CGCCCGCCGC GCGGCGGGCG GGGCGGGGGC ACGGGCGGTG TGTAC-3'.

[0044] The reaction system of the second round of PCR was the same as that of the first round. The second round of PCR reaction conditions wa...

Embodiment 2

[0048] p1-F: 5'-CGATGCGTAGCCGACCTGAG-3',

[0049] p1-R: 5'-AAGG AGGTGATCCAGCCGCA-3'.

[0050] The first-round PCR reaction system is 25 μL, including 12.5 μL 2×Taq PCR MasterMix enzyme, 0.5 μL (25 pmol) of each primer, 10.5 μL sterilized ultrapure water, and the amount of DNA template is about 10 ng. The first round of PCR reaction program was: 95°C pre-denaturation for 5 minutes, followed by 25 cycles, 95°C for 1 minute; 68°C for 90 seconds; 72°C for 2 minutes, and finally 72°C for 10 minutes. PCR products were detected by 1% agarose gel electrophoresis. The first-round PCR product was diluted 100-fold, and 1 μL was used as the template for the second-round PCR.

[0051] The primers used in the second round of PCR were:

[0052] p2-F:5'-ATGGCTGTCGTCAGCT-3',

[0053] p2-R: 5'-CGCCCGC CGCGCGGCGGGCGGGGCGGGGGCACGGGCGGTGTGTAC-3'.

[0054] The reaction system of the second round of PCR was the same as that of the first round.

[0055] The second round of PCR reaction conditio...

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Abstract

The invention discloses a method for analyzing the Bacillus community structure in a white spirit fermentation system, belonging to the technical field of microbial ecology. According to the invention, the diversity of a Bacillus community structure in the white spirit fermentation system is semi-quantitatively analyzed by a nested PCR-DGGE (polymerase chain reaction-denaturing gel gradient electrophoresis) method. The method comprises the following steps of: extracting the total genome DNA (deoxyribonucleic acid) in a yeast or fermented grains sample by a bead milling method; performing two steps of amplification of the Bacillus specific segment through the nested PCR; performing DGGE electrophoretic analysis; determining the microorganism species corresponding to the DNA stripe by use of a standard strain; performing glue cutting of the unknown stripe and recycling and then performing PCR again; connecting the T vector and cloning; performing DGGE electrophoresis again; performing positive cloning sequencing and performing blast comparison in the GenBank databae to obtain the microorganism information; digitally processing DGGE map by use of quantityone software; and calculating the proportion of different Bacillus strains in the total Bacillus flora according to the brightness of the stripe. The method disclosed by the invention has the advantages of simplicity, strong specificity and good repeatability, and lays foundation for further studying the importance of Bacillus in white spirit production.

Description

technical field [0001] A method for analyzing liquor fermentation system Bacillus The method of community structure, the present invention involves the use of modern molecular biology technology-polymerase chain reaction-deformation gradient gel electrophoresis (PCR-DGGE) to overcome the defects of traditional microbial isolation methods, qualitative and semi-quantitative analysis of microbial fermentation of Chinese liquor in the system Bacillus The structure and composition of the population and its dynamic evolution rules belong to the technical field of microbial ecology. Background technique [0002] The production of Chinese liquor is an open solid-state fermentation process. It can be said that the production of liquor actually includes a complex metabolic process of a huge microbial flora, which is greatly affected by the environment. As the environmental conditions change, the microflora will also change and affect the flavor and taste of liquor. Daqu and ferment...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/06
Inventor 徐岩王海燕吴莉莉
Owner JIANGNAN UNIV
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