Method for extracting microorganism total DNA (Deoxyribonucleic Acid) in pu'er tea piling fermentation process

A fermentation process and microbial technology, applied in the field of microorganisms, can solve the problems of difficulty in extracting total microbial DNA, insufficient cell lysis rate, high DNA impurity content, etc., and achieve the effect of low cost, simple and efficient method, and high purity

Inactive Publication Date: 2012-10-03
KUNMING UNIV OF SCI & TECH
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Problems solved by technology

[0003] At present, the main problem in the extraction of the total microbial DNA of Pu’er tea is that the microorganisms use Pu’er tea as a substrate to perform complex secondary metabolism, and a large amount of tea polyphenols, tea polysaccharides, quinones, etc. remain on the surface and even in the body of the microorganisms. Makes it very difficult to extract high-quality microbial total DNA
According to the current method, the microbial total DNA of Puer tea is extracted. It is difficult to avoid the combination of tea polyphenols and quinones with DNA during the extraction process. The extracted DNA has a high content of impurities and cannot be directly used for subsequent molecular biology research such as PCR amplification. The current method still has insufficient cell lysis rate in the extraction process, and the combination of DNA and tea polyphenols, quinones, etc. leads to excessive loss in the extraction process, etc., resulting in a low extraction rate

Method used

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  • Method for extracting microorganism total DNA (Deoxyribonucleic Acid) in pu'er tea piling fermentation process
  • Method for extracting microorganism total DNA (Deoxyribonucleic Acid) in pu'er tea piling fermentation process
  • Method for extracting microorganism total DNA (Deoxyribonucleic Acid) in pu'er tea piling fermentation process

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Effect test

Embodiment 1

[0026] Embodiment 1: the method for extracting the total DNA of microorganisms in the Pu'er tea stack fermentation process, the specific operations are as follows:

[0027] The samples of Pu-erh tea pile fermented were collected to the Pu-erh tea production workshop in the process of production. The sampling point was 3cm below the surface of the Pu-erh tea pile. Ferment for 10 days (3 days after turning over), the core temperature is 64°C, and the water content is 23.88%.

[0028](1) Washing of samples: Weigh 3g of Pu’er tea samples for each sample, cut them into pieces to a size between 0.5-1cm, transfer them to a 50mL centrifuge tube, add 30mL of washing buffer to the samples, let them stand for 20min, and shake them in an ultrasonic cleaner After 10 min, vortex shaking for 60 s, take the supernatant; place the supernatant in a 60°C water bath for 9 min, centrifuge at 100×g for 2 min, collect the supernatant, centrifuge the supernatant at 6000×g for 8 min, discard the super...

Embodiment 2

[0042] Embodiment 2: The method for extracting the total DNA of microorganisms in the Pu'er tea stack fermentation process, the specific operations are as follows:

[0043] The samples of Pu-erh tea stack fermentation were collected in the Pu-erh tea production workshop in the process of production. The sampling point was 3cm below the surface of the Pu-erh tea stack. Multi-point sampling and mixing 1 sample (number 2), and the sampling time was the stack fermentation In 20 days (4 days after the second turning over), the core temperature was 63°C and the water content was 23.24%.

[0044] (1) Washing of samples: Weigh 1g of Pu’er tea samples for each sample, cut them into pieces to a size between 0.5-1cm, transfer them into a 50mL centrifuge tube, add 20mL of washing buffer to the samples, let them stand for 20min, and shake them in an ultrasonic cleaner After vortex shaking for 45 s, take the supernatant; place the supernatant in a 70°C water bath for 3 min, centrifuge at 20...

Embodiment 3

[0053] Embodiment 3: the method for extracting the total DNA of microorganisms in the Pu'er tea stack fermentation process, the specific operations are as follows:

[0054] The samples of Pu-erh tea pile fermented were collected to the Pu-erh tea production workshop in the process of production. The sampling point was 3cm below the surface of the Pu-erh tea pile. Multi-point sampling was mixed to form one sample (number 3). Fermentation for 30 days (5 days after triple turning), the core temperature is 62°C, and the water content is 23.11%.

[0055] (1) Washing of samples: Weigh 2g of Pu’er tea samples for each sample, cut them into pieces to a size between 0.5-1cm, transfer them into a 50mL centrifuge tube, add 25mL of washing buffer to the samples, let them stand for 20min, and shake them in an ultrasonic cleaner After 12 minutes, vortex and shake for 50 seconds, take the supernatant; place the supernatant in a 65°C water bath for 6 minutes, centrifuge at 150×g for 1.5 minut...

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Abstract

The invention discloses a method for extracting microorganism total DNA (Deoxyribonucleic Acid) in a pu'er tea piling fermentation process at high quality. The method comprises the following steps of: separating microorganisms on the surface of pu'er tea by using ultrasonic wave and vortex oscillation; washing the microorganisms by using washing buffer solution; performing rough extraction on the microorganism total DNA by using DNA extract; and purifying the roughly extracted DNA. The method provided by the invention is low in cost and high in DNA extraction rate; the extracted DNA has high completeness and high purity and can meet the requirements of PCR (Polymerase Chain Reaction) without purification or dilution; the purified DNA can meet the requirements of molecular biology research; and meanwhile, the extracted DNA covers bacteria and fungi and the requirements of subsequent researches in microorganism diversity, functional genes, metagenomics and the like can be met.

Description

technical field [0001] The invention relates to a method for extracting the total DNA of microorganisms in the fermentation process of Pu'er tea, which belongs to the field of microorganisms. Background technique [0002] Pu-erh tea is a large leaf tea from Yunnan ( C. sinensis var. assamica ) and other large-leaf teas (Pu-erh green tea) as raw materials, loose tea and pressed tea produced by post-fermentation process. Stacking fermentation in the modern Pu'er tea production process is a key process for producing Pu'er tea. The types and quantities of microorganisms in the production process directly affect the formation of Pu'er tea flavor. Therefore, microorganisms play an important role in the stacking fermentation of Pu'er ripe tea. effect. However, the current detection and classification methods based on morphological and physiological and biochemical indicators are limited by culture conditions and simulated culture techniques, so there is still a big difference bet...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
Inventor 陈朝银吕昌勇刘迪秋葛锋韩本勇熊向峰
Owner KUNMING UNIV OF SCI & TECH
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