Primer for amplifying molecules of microbes in pit mud of Luzhou-flavor liquor and detection method

A technology for microbial flora and Luzhou-flavor liquor, which is applied in the field of winemaking, can solve the problems that the information of microbial population is small and cannot reflect the structure and richness of microbial flora in pit mud well, and achieves the effect of quick and timely monitoring.

Active Publication Date: 2011-02-16
LUZHOU PINCHUANG TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, because the amplified product is only 240bp, the amount of microbial population information reflected is less, that i...

Method used

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  • Primer for amplifying molecules of microbes in pit mud of Luzhou-flavor liquor and detection method
  • Primer for amplifying molecules of microbes in pit mud of Luzhou-flavor liquor and detection method
  • Primer for amplifying molecules of microbes in pit mud of Luzhou-flavor liquor and detection method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] The establishment of embodiment one inventive method

[0049] 1. Selection of primers

[0050] During the establishment of this method, primers for the amplification of the V6-V8 hypervariable region of 16S rDNA were used, and the product was about 470 bp, which was close to the optimal fragment length of DGGE. In addition, the primers used to amplify the V3 region which are widely used at present are also used, and the PCR products amplified by the two pairs of primers are about 240bp and 470bp respectively. Different primer sequences were amplified separately, the amplification of the V3 region referred to the existing literature, and the amplification of the V6-V8 region was optimized by gradient PCR.

[0051] 2. Optimization of amplification conditions

[0052] The amplification conditions of the V3 hypervariable region refer to the existing literature (Wang Qizan, Xu Qiufang, Jiang Peikun, etc. DGGE analysis of the 16S rDNA V3 region fragment PCR of the soil bact...

Embodiment 2

[0086] Embodiment 2 uses the method of the present invention to compare and detect the pit mud in different parts of the same cellar

[0087] (1) Extraction of microbial total DNA from pit mud, using E.Z.N.A.TM soil DNA kit to extract total microbial DNA.

[0088] (2) Primer sequences for amplifying 16S rDNA:

[0089] Primer I: 5'-CGC CCG GGG CGC GCC CCG GGC GGG GCG GGG GCA CGG GGG GAA CGC GAA GAACCT TAC-3',

[0090] Primer II: 5'-ACG GGC GGT GTG TAC-3'.

[0091] (3) Amplification of prokaryotic 16S rDNA V6-V8 region: PCR reaction system: 50 μL: 1.0 μL Taq enzyme (5U / μL), 5.0 μL 10× buffer, 3.0 μL MgCl2 (25 mmol / L), 4.0 μL dNTPs Mixture (2.5 mmol / L each), 1.0 μL each of primers (10 μmol / L), 1.0 μL template DNA (100 ng / L), 34.0 μL sterilized double distilled water.

[0092] PCR amplification program: pre-denaturation at 95°C for 4 minutes; denaturation at 95°C for 45s, application of falling PCR, annealing at 65°C, each round of 1°C drop, finally down to 49°C, annealing for ...

Embodiment 3

[0097] Embodiment 3 Using the method of the present invention to compare and detect different cellar muds with different cellar ages with large differences in wine production quality

[0098] (1) Extraction of total microbial DNA from pit mud, using E.Z.N.A.TM soil DNA ktt to extract total microbial DNA.

[0099] (2) Primer sequences for amplifying 16S rDNA:

[0100] Primer I: 5'-CGC CCG GGG CGC GCC CCG GGC GGG GCG GGG GCA CGG GGG GAA CGC GAA GAACCT TAC-3',

[0101] Primer II: 5'-ACG GGC GGT GTG TAC-3'.

[0102] (3) Amplification of prokaryotic 16S rDNA V6-V8 region: PCR reaction system: 50 μL: 1.0 μL Taq enzyme (5U / μL), 5.0 μL 10× buffer, 3.0 μL MgCl2 (25 mmol / L), 4.0 μL dNTPs Mixture ( 2.5 mmol / L each), 1.0 μL each primer (10 μmol / L), 1.0 μL template DNA (100 ng / L), 34.0 μL sterilized double distilled water.

[0103] PCR amplification program: pre-denaturation at 95°C for 4 minutes; denaturation at 95°C for 45s, application of falling PCR, annealing at 65°C, each round of...

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Abstract

The invention belongs to the technical field of wine making, which particularly relates to a primer for detecting molecules of microbes in pit mud of Luzhou-flavor liquor and a molecule detection method for microbes in pit mud of Luzhou-flavor liquor. The method is characterized in that protogenome of prokaryotic microbes in the microbes in pit mud is extracted by a culture-independent approach; the hypervariable regions V6-V8 of a microbe 16S rDNA are amplified through PCR (Polymerase Chain Reaction), and are processed by denaturing gradient gel electrophoresis (DGGE); and the diversity of the microbes and the variation of microbe structures can be analyzed through an electrophoretogram. By using the primer and the method, the 16S rDNA of the microbes in different kinds of pit mud in the process of making Luzhou-flavor liquor can be amplified quickly and accurately, the difference of microbes in different kinds of pit mud in the process of making Luzhou-flavor liquor can be further detected by DGGE, and thus realizing in-time monitoring on variations in the fermentation process of microbes in pit mud for making wine.

Description

Technical field: [0001] The invention belongs to the technical field of brewing, and in particular relates to a primer and a detection method for amplifying the microbial flora molecules of Luzhou-flavor liquor cellar mud. Background technique: [0002] Luzhou-flavor liquor is one of the three typical aroma liquors in China, and its unique technology has created the unique flavor characteristics of Luzhou-flavor liquor. One of the characteristics of Luzhou-flavor liquor is the strong aroma in the pit, and the microorganisms in the pit mud are the main source of the aroma substances in the brewing process. Cellars of different cellar ages, with the continuous fermentation process, the microbial flora and quantity tend to be balanced and stable, and finally reach their specific microbial flora structure, so that the quality of the wine tends to be stable, making the wine more Full-bodied and full-bodied. [0003] The brewing process of Luzhou-flavor liquor is through the col...

Claims

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Application Information

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IPC IPC(8): C12Q1/04C12Q1/68C12N15/11C12N15/10
Inventor 敖宗华陕小虎卢中明沈才洪易彬韩光敖灵
Owner LUZHOU PINCHUANG TECH CO LTD
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