Quantitative analysis method of lactic acid bacteria and bacilli in yeast microbial community

A microbial community, Bacillus technology, applied in the field of bioengineering, can solve the problems of lack of stability and reliability of detection results, time-consuming and labor-intensive, etc., to strengthen monitoring, improve detection efficiency, and simplify detection procedures.

Active Publication Date: 2014-06-04
LUZHOU PINCHUANG TECH CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The traditional quantitative detection of microorganisms in Daqu still adopts the method of screening of selective medium and pure culture isolation and counting. However, this method is affected by external environmental factors, strain characteristics, etc., and the detection results lack stability and reliability, and are time-consuming. laborious

Method used

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  • Quantitative analysis method of lactic acid bacteria and bacilli in yeast microbial community
  • Quantitative analysis method of lactic acid bacteria and bacilli in yeast microbial community
  • Quantitative analysis method of lactic acid bacteria and bacilli in yeast microbial community

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] The preparation of embodiment 1 standard curve

[0028] 1. Synthesize specific primers for lactic acid bacteria and bacillus, and specifically amplify the lactic acid bacteria and bacillus screened in Daqu

[0029] Lactic acid bacteria specific primer sequence (5'→3'):

[0030] Lacto-F: AGAACACCAGTGGCGAAGG

[0031] Lacto-R: CAGGCGGAGTGCTTAATGC

[0032] Bacillus-specific primer sequence (5′→3′)

[0033] Bacil-F: ATGGCTGTCGTCAGCT

[0034] Bacil-R: ACGGGCGGTGTGTAC

[0035] 2. Screening of lactic acid bacteria and bacillus in Daqu and determination of standard strains:

[0036] a. Screening of lactic acid bacteria:

[0037] Screening medium for lactic acid bacteria: glucose 2%w / w, peptone 1%w / w, beef extract 1%w / w, yeast extract 0.5%w / w, K 2 HPO 4 0.2%w / w, Triammonium Citrate 0.2%w / w, Sodium Acetate 0.5%w / w, Tween 800.1%v / v, MgSO 4 ·7H 2 O0.05%w / w, MnSO 4 4H 2 O0.025%w / w, pH6.2~6.4, agar 1.5%w / w, 121℃, after 20min sterilization, add sterilized CaCO 3 Solution 2...

Embodiment 2

[0065] The extraction of microbial total DNA in the Daqu microbial flora of embodiment 2

[0066] 1. Daqu sample collection

[0067] Collect Daqu samples. If the DNA cannot be extracted in time after the sample is collected, it should be stored in a -80°C refrigerator immediately.

[0068] 2. Extraction of microbial total DNA from Daqu

[0069] Weigh 1 g of Daqu sample, add liquid nitrogen to the mortar and grind thoroughly, and transfer to a 50 mL centrifuge tube. Transfer to a centrifuge tube, add 1mL CTAB extract (2%CTAB, 5mol / L NaCl, 1mol / LTris-HCl(pH8.0), 0.5mol / L EDTA) and 20μL mercaptoethanol to the centrifuge tube, and mix at 65°C. Shake for 30min with a homogenizer, add 5μL of proteinase k (20mg / mL), centrifuge at 6000×g for 10min at 37°C at 220r / min, and collect the supernatant (1mL).

[0070] Add an equal volume of Tris-saturated phenol (500 μL) and chloroform-isoamyl alcohol (volume ratio 24:1) (500 μL), extract once, centrifuge at 12,000 rpm for 10 min, take th...

Embodiment 3

[0071] Quantitative Analysis of Lactic Acid Bacteria and Bacillus in Embodiment 3 Daqu Microflora

[0072] Quantification of lactic acid bacteria in Daqu samples: use the total DNA of Daqu obtained in Example 2, and use specific primers for lactic acid bacteria and Bacillus in Example 1. Daqu fluorescent quantitative PCR analysis was performed according to the fluorescent quantitative PCR reaction system and PCR program in Example 1.

[0073] According to the fluorescent quantitative PCR of lactic acid bacteria standard sample, the linear equation y=-0.3411x+14.07(R 2 =0.9978) to calculate the copy number of lactic acid bacteria in Daqu samples at different time points, so as to obtain the change trend of lactic acid bacteria biomass in Daqu (such as figure 1 Note as shown: figure 1 Days on the x-axis shown represent Daqu production and storage days).

[0074] Quantification of Bacillus in Daqu samples: according to the linear equation y=-0.3387x+13.152(R 2 =0.9974) to cal...

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Abstract

The invention belongs to the technical field of biological engineering, in particular relates to a quantitative analysis method of lactic acid bacteria and bacilli in a yeast microbial community and aims to provide a new choice for analysis of the lactic acid bacteria and the bacilli in a yeast. According to the technical scheme, the quantitative analysis method of the lactic acid bacteria and the bacilli in the yeast microbial community comprises the following steps: a, preparation of a standard curve; b, extraction of a genome DNA (Deoxyribonucleic Acid); c, amplification; d, quantitative analysis. The method can be used for analyzing the variation trend of the lactic acid bacteria and the bacilli in pit mud.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a quantitative analysis method for lactic acid bacteria and bacillus in Daqu microbial community. Background technique [0002] Daqu is a large block made of wheat as the main raw material and contains multi-bacteria enzymes, and is used as a saccharifying agent and a leavening agent in the brewing process of liquor. As the saying goes: "There must be a good song for good wine", "the song is the bone of the wine", from this we can know the importance of Daqu to the brewing of liquor. The production of Daqu to the storage of finished Qu is an open process. The microorganisms in the environment participate in the whole process of Daqu production. Under the growth and metabolism of different microorganisms, Daqu gradually becomes Micro-ecological products of compound aroma substances. Therefore, the type and quantity of microorganisms in Daqu are important facto...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/06C12R1/07
CPCC12Q1/6851C12Q2531/113C12Q2545/114
Inventor 许正宏史劲松陆震鸣肖辰王松涛
Owner LUZHOU PINCHUANG TECH CO LTD
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