Extraction reagent and extraction method for fermented bean curd microorganism flora DNA

A technology of microorganisms and fermented bean curd, which is applied to the extraction reagent and extraction of fermented bean curd microbial community DNA: 1) In the field of microbial separation and recovery in fermented bean curd, it can solve the problems of unsuitable fermented bean curd microbial community

Active Publication Date: 2019-12-20
UNIV OF ELECTRONICS SCI & TECH OF CHINA ZHONGSHAN INST
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  • Abstract
  • Description
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  • Application Information

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Problems solved by technology

The premise of the application of the above molecular biology methods is to obtain high-quality microbial DNA. However, since most of the fermented bean curd samples are soybean residues and contain a large amount of organic acids, amino acids and other substances, the existence of these substances makes the conventional microbial single-bacteria extraction difficult. Method not applicable to sufu microbiome

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  • Extraction reagent and extraction method for fermented bean curd microorganism flora DNA
  • Extraction reagent and extraction method for fermented bean curd microorganism flora DNA
  • Extraction reagent and extraction method for fermented bean curd microorganism flora DNA

Examples

Experimental program
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Embodiment 1

[0044] A set of reagents for extracting microbial DNA of fermented bean curd, including cell separation liquid, cell washing liquid, cell lysing liquid, PCR inhibitory substance precipitation liquid and cleaning liquid.

[0045] Preparation of cell separation solution: weigh 1.36g KH 2 PO 4 , 3.58 g Na 2 HPO 4 12H 2 O, 5.84g NaCl, 0.07g KCl, and 800ml of deionized water were added. After stirring evenly with a glass rod, the pH value was adjusted to 7.5, and the volume was adjusted to 1L by adding deionized water.

[0046] Preparation of cell washing solution: weigh 1.36g KH 2 PO 4 , 3.58 g Na 2 HPO 4 12H 2 O, add 1ml Tween 20 and add 800ml deionized water, stir evenly with a glass rod, adjust the pH value to 7.5, add deionized water to make up to 1L.

[0047] Preparation of cell lysate: Weigh 24.2g Tris (trishydroxymethylaminomethane), 74.5g KCl, 0.3g EDTA, add 800ml deionized water, stir evenly with a glass rod, add 5ml SDS, 5ml Triton x- 100, adjust the pH value t...

Embodiment 2

[0051] Extract commercialized fermented bean curd microbial DNA from different manufacturers, and use the extraction reagent provided in Example 1 to extract, a total of 12 fermented bean curd samples, the steps are as follows:

[0052] (1) 10g fresh fermented bean curd sample is mashed sample with Mixer Mill MM 400 stirring homogenizer of RETSCH company;

[0053] (2) Weigh 10 g of the crushed fermented bean curd sample, add 1 g of tungsten carbide beads with a diameter of 16 mm, and add 50 ml of cell separation medium SFS buffer, shake at room temperature for 15 minutes on a shaker at 120 rpm;

[0054] (3) Let the appeal sample stand at room temperature for 5 minutes, centrifuge at 13000×g for 1 minute, collect the precipitate, pour off all the supernatant, and weigh about 5 g of the precipitate to proceed to the next step;

[0055] (4) Add 25mL of cell washing solution SFW buffer, shake on a shaker at 120 rpm at room temperature for 10 minutes, let stand at room temperature ...

Embodiment 3

[0070] Six commercial red bean curd and six white bean curd were collected, their DNA was extracted, 16S rRNA gene was amplified by PCR, and the bacterial community composition of the bean curd samples was identified by high-throughput sequencing. Adopt the extraction reagent that embodiment 1 provides to extract, altogether 12 fermented bean curd samples, the steps are as follows:

[0071] (1) The DNA extraction method is the same as in Example 2, followed by PCR amplification of the 16S rRNA gene and high-throughput sequencing of the amplicon and bioinformatics analysis method;

[0072] (2) Take 1 μl of DNA as a template to amplify the V4 region of the 16S rRNA gene. 515F(5'-GTG CCA GCM GCC GCGGTA A-3')+806R(5'-GGA CTA CHV GGG TWT CTA AT-3') was used as primers for PCR amplification. The total volume of the PCR amplification system is 50 μl, and the composition of the reaction system is: 1 μl of upstream and downstream primers (concentration 10 μM), 2 μl of 10×Extag reactio...

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Abstract

The present invention discloses an extraction reagent and an extraction method for fermented bean curd microorganism flora DNA. The extraction reagent comprises a cell separation solution, a cell rinsing solution, a cell lysing solution, a PCR inhibitory substance precipitation solution and a washing solution. Based on characteristics of high content of soybean residues and richness of organic acids and amino acids in fermented bean curd samples, processes of fermented bean curd microorganism recovery, cell lysis, PCR reaction substance removal and highly-efficient DNA recovery are designed. For the PCR reaction substance removal, a combination method of ammonium sulfate, guanidine hydrochloride and CTAB is applied, and at the same time, polysaccharides, polyphenols and interfering proteins are removed. Therefore, high-quality DNA is acquired for subsequent molecular biology experiments. The method can simultaneously extract DNA of fermented bean curd bacteria and fungi, and the extracted DNA is successfully used for PCR reaction and high-throughput sequencing of 16S rRNA and ITS genes and is also successfully applied to sequencing and analysis of metagenomics.

Description

technical field [0001] The invention belongs to the technical field of genome extraction, and relates to a reagent and method for extracting fermented bean curd microbial community DNA, in particular to: 1) a method for separating and recovering microorganisms in fermented bean curd; Precipitation removal; 3) Microbial cell lysis and DNA recovery method; 4) Reagent formula used in the extraction process. Background technique [0002] Fermented bean curd is delicious and unique, and it is one of the soybean fermented foods with Chinese characteristics. Traditional sufu fermentation is divided into two stages, which is a complex biochemical process. After microbial fermentation, the protein contained in the bean curd embryo is gradually hydrolyzed under the action of microbial protease to produce small molecular substances such as peptides and amino acids; the starch is converted into monosaccharides and oligosaccharides, which are easily absorbed by the human body. degree m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/1006
Inventor 胡敏谭贵良
Owner UNIV OF ELECTRONICS SCI & TECH OF CHINA ZHONGSHAN INST
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