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Method for detecting microbial community structure of pu'er tea

A microbial community and Pu'er tea technology, applied in the direction of microbial measurement/inspection, biochemical equipment and methods, etc., can solve the problems of insufficient comprehensiveness and system, lack of simultaneous quantitative analysis of microbial quantity, etc.

Inactive Publication Date: 2013-10-23
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is very little research on the bacterial community structure of Puer tea. Puer tea fermentation process and finished tea contain a large number of bacteria. The composition and changes of the bacterial flora are also closely related to the formation of Puer tea quality. It is obviously not enough to study only the structure of fungi comprehensive and systematic
Secondly, most studies at home and abroad only analyze the microbial community structure through DGGE maps, lacking simultaneous quantitative analysis of the number of microorganisms

Method used

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  • Method for detecting microbial community structure of pu'er tea
  • Method for detecting microbial community structure of pu'er tea
  • Method for detecting microbial community structure of pu'er tea

Examples

Experimental program
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Embodiment 1

[0033] A method for detecting Pu'er tea microbial community structure and relative quantity, comprising the steps of:

[0034] 1. Take a Pu-erh tea product for daily drinking as a sample;

[0035] 2. Collect the microbial cells of tea samples by glass bead shaking method: Weigh 5g of tea samples and place them in 45mL sterile phosphate buffer solution filled with acid-washed glass beads, shake at 200rpm for 2 hours at room temperature; Filter with a layer of gauze, transfer the filtrate to a 2ml EP tube, centrifuge at 12000rpm for 3min, remove the supernatant, wash the precipitate with 1mL of sterile deionized water, centrifuge at 12000rpm for 3min, the obtained precipitate is the microbial cell on the tea;

[0036] 3. Extraction of total microbial DNA: use a combination of liquid nitrogen freeze-thaw + enzymatic method and CTAB method, 600 μL cell lysate to fully suspend bacterial sediment, freeze-thaw repeatedly in liquid nitrogen and 65°C water bath for 6 times, and 37°C wa...

Embodiment 2

[0043] A method for detecting microbial community structure and relative quantity in the Pu'er tea fermentation process, comprising the steps of:

[0044] 1. Use the 5-point sampling method to sample the Pu’er tea during the fermentation process in a Pu’er tea processing factory in Yunnan. Before the Pu’er tea is fermented and turned over, take 10g of the surface and inner tea leaves, and fully mix them in a sterile sealed bag. Turn the pile every 6 days, and sample 4 times in total.

[0045] 2. Collect microbial cells of tea samples at various stages of fermentation by glass bead shaking method: Weigh 10 g of tea samples and place them in 40 mL of sterile phosphate buffer solution with acid-washed glass beads, shake at 200 rpm for 1.5 h at room temperature; Filter with sterile double gauze, transfer the filtrate to a 2ml EP tube, centrifuge at 12000rpm for 3min, remove the supernatant, wash the precipitate with 1mL sterile deionized water, centrifuge at 12000rpm for 3min, the...

Embodiment 3

[0054] A method for detecting the microbial community structure and relative quantity of Pu'er tea from different origins, comprising the steps of:

[0055] 1. Select three Pu-erh tea products from different origins of A, B, and C from the market as samples;

[0056] 2. Collect the microbial cells of tea samples by glass bead shaking method: Weigh 8g of tea samples and place them in 42mL sterile phosphate buffer solution filled with acid-washed glass beads, shake at 200rpm for 1h at room temperature; Filter with a layer of gauze, transfer the filtrate to a 2ml EP tube, centrifuge at 12000rpm for 3min, remove the supernatant, wash the precipitate with 1mL of sterile deionized water, centrifuge at 12000rpm for 3min, the obtained precipitate is the microbial cell on the tea;

[0057] 3. Extraction of total microbial DNA: use liquid nitrogen freeze-thaw + enzymatic method and CTAB method, 600 μL cell lysate to fully suspend bacterial sediment, freeze-thaw repeatedly in liquid nitr...

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Abstract

The invention discloses a method for detecting the microbial community structure of pu'er tea. The method is implemented through collecting microbial bacteria of tea leaves of the pu'er tea by using a glass bead oscillating method; then, extracting a total microbial DNA by using the combination of a liquid nitrogen freezing-thawing and enzymatic method and a CTAB (cetyl trimethyl ammonium bromide) method; carrying out PCR (polymerase chain reaction) amplification on the extracted genomic DNA (deoxyribonucleic acid) by respectively using universal primers of bacterial 16S rRNA and fungal 18S rRNA variable regions; separating a PCR product by using DGGE (denaturing gradient gel electrophoresis) and dyeing the obtained product so as to obtain a band map, and carrying out relative quantitative analysis on bands by using Quantity One software; carrying out cut gel extraction on the DNA of the bands, carrying out the PCR amplification on extracted nucleic acids, connecting a product with a T carrier, and selecting positive clones to carry out community PCR with a GC-clamp; carrying out comparison again by using DGGE electrophoresis, and selecting a PCR product corresponding to a target band to carry out sequencing identification. The method disclosed by the invention can be used for detecting the changes of the microbial community structure and relative quantity of the pu'er tea in the process of fermentation.

Description

technical field [0001] The invention relates to the analysis and detection of the microbial community structure and relative quantity of Pu'er tea, in particular to a method for detecting the microbial community structure and relative quantity of Pu'er tea by combining PCR-DGGE technology and Quantity One software analysis. Background technique [0002] Pu-erh tea is produced in Yunnan, my country. It is a type of tea produced by a special post-fermentation process using the large-leaf sun-dried green tea from Yunnan as raw material. The formation of the special flavor of Pu-erh tea is related to the enzymatic action of microorganisms involved in the post-fermentation process and the secondary metabolism of the microorganisms themselves. Therefore, the research on the structure and diversity of microbial communities in Pu-erh tea plays a very important role in the realization and strength of the high-sustained and stable characteristics of the unique quality of Pu-erh tea. ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/06C12Q1/04
Inventor 林炜铁罗剑飞杨晓苹
Owner SOUTH CHINA UNIV OF TECH
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