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Method for integrated detection of nine beer-spoilage bacteria

An integrated detection and contamination bacteria technology, applied in the field of beer, can solve the problems of cumbersome detection technology operation, time-consuming and labor-consuming detection types, etc., and achieve the effects of saving detection time, saving detection cost, high detection specificity and sensitivity

Active Publication Date: 2013-10-23
TSINGTAO BREWERY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The purpose of the present invention is to address the defects of the prior art, overcome the cumbersome operation of conventional beer contaminating bacteria detection technology, time-consuming and labor-consuming, and the lack of detection types, and provide a primer system composed of 9 pairs of primers, through the use of high-throughput Combining advanced multiplex PCR technology with high-resolution capillary electrophoresis separation technology, high-throughput, semi-quantitative scanning detection of various common pollutants in the beer production process can be realized

Method used

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  • Method for integrated detection of nine beer-spoilage bacteria
  • Method for integrated detection of nine beer-spoilage bacteria
  • Method for integrated detection of nine beer-spoilage bacteria

Examples

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Effect test

Embodiment 1

[0038] (1) Cultivation of beer-contaminated bacteria and DNA template extraction:

[0039]Take 1 bottle of finished wine from the production workshop of a brewery, and implant Lac.brevis, Lac.casei, Lac.coryniformis, Lac.plantarum, Lac.paracollinoides, Lac.lindneri, Ped.damnosus, Ped.inopinatus, Ped .claussenii strain, use MRS broth medium to enrich and culture beer and beer production process samples overnight at 26°C in an anaerobic environment; take 1ml of the culture solution and collect it in a 1.5ml centrifuge tube, centrifuge at 13,000×g for 2min, collect Cell pellet, remove supernatant; add 480ul of 50mM EDTA to resuspend cells thoroughly; add 120ul of 10mg / ml lysobacterium, incubate at 37°C for 60min, centrifuge at 13,000×g for 2min, remove supernatant. Afterwards, according to Promega's Instructions for use of the Genomic DNA Purification Kit kit Extract the DNA template of beer-contaminated bacteria, and use a UV spectrophotometer to measure the quality of the ext...

Embodiment 2

[0049] Take 1 bottle of finished wine from the production workshop of a brewery, and implant 10 bottles in the sterile room 5 Lactobacillus casei Lac.casei bacterial strain of CFU / mL concentration, and use same wine base to carry out gradient dilution to this bacterial suspension, dilute to 10 4 CFU / mL, 10 3 CFU / mL, 10 2 CFU / mL, 10 1 For the concentration of CFU / mL, take 1ml of the culture solution and collect it in a 1.5ml centrifuge tube, centrifuge at 13,000×g for 2min, collect the cell pellet, and remove the supernatant; add 480ul of 50mM EDTA to resuspend the cells thoroughly; add 120ul of 10mg / ml lysobacterium , incubate at 37°C for 60 minutes, centrifuge at 13,000×g for 2 minutes, and remove the supernatant. Promega Instructions for use of the Genomic DNA Purification Kit kit to extract the DNA template of beer-contaminated bacteria and use it in a multiplex PCR reaction system DNA Polymerase DNA kit, using 9-fold PCR reaction system, the primer system used is SE...

Embodiment 3

[0055] Pick a single colony of Lac.casei strain and add it to 10mL of finished beer liquid to make a bacterial suspension, and use the finished wine liquid to dilute the bacterial suspension 5 times 10 times, and take 1mL of the final dilution on the MRS-agar medium. After 7 days of anaerobic culture at 26°C, count the colonies after culture as the experimental control. The culture results are shown in Table 4; another 1 mL of the final dilution was added to a 1.5 ml centrifuge tube, centrifuged at 13,000×g for 2 min, and the cell pellet was collected. Remove the supernatant; add 480ul 50mM EDTA to resuspend the cells thoroughly; add 120ul 10mg / ml lysobacterium, incubate at 37°C for 60min, centrifuge at 13,000×g for 2min, and remove the supernatant. Promega Instructions for use of the Genomic DNA Purification Kit kit to extract the DNA template of beer-contaminated bacteria and use it in a multiplex PCR reaction system DNA Polymerase DNA kit, using 9-fold PCR reaction syste...

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Abstract

The invention relates to a method for integrated detection of nine beer-spoilage bacteria, belongs to the technical field of beers. According to the method, nine beer-spoilage bacteria of Lactobacillus brevis, Lactobacillus casei, Lactobacillus coryniformi, Lactobacillus plantarum, Lactobacillus paracollinoides, Lactobacillus lindneri, Pediococcus damnosus, Pediococcus inopinatus and Pediococcus clausenii are amplified simultaneously at one time by using a ninefold PCR; and then products amplified by the ninefold PCR is separated through capillary electrophoresis. A primer SEQIDNO. 1-18 is used in the multifold PCR reaction. The high throughput and semi-quantitative analysis method for detection of contamination microorganisms in the beer has important significance in aspects of the detection and traceability of the beer-spoilage bacteria.

Description

technical field [0001] The invention belongs to the technical field of beer and is used for the detection method of beer contaminating bacteria, in particular to a high-throughput and semi-quantitative analysis method for detecting common contaminating bacteria in beer based on multiple PCR technology and capillary electrophoresis separation technology. Background technique [0002] The brewing process of beer is based on the fermentation of pure beer yeast and the control of contaminating microorganisms. The main contaminating microorganisms in beer are lactic acid bacteria with hop-resistant genes. The detection methods for such microorganisms are mainly divided into two categories, The first type is the traditional cultivation method based on the culture medium. Most breweries at home and abroad adopt this method, but the detection time is long, and the species can only be quantified but cannot be qualitatively identified. Therefore, it is impossible to guide the productio...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/06
Inventor 董建华陈嵘陈璐尹花万秀娟赵玉祥贺扬孙力川杨梅刘佳
Owner TSINGTAO BREWERY
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