Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Two pairs of specific primers for detecting acid-resistant lactobacillus content during Baijiu fermentation and application thereof

A fermentation process, Lactobacillus technology, applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc. Conducive to quality control, advanced design methods

Inactive Publication Date: 2018-02-16
HARBIN INST OF TECH AT WEIHAI +1
View PDF3 Cites 4 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But too much lactic acid will make the fermented grains rancid, not only the yield of wine will be low, but the quality of wine will also deteriorate
[0004] Due to the differences in the construction time and geographical location of the liquor brewing plant area, the composition and content of acid substances in different cellars are quite different, and the composition of some microorganisms is also significantly different in the content of high-quality cellars and ordinary cellars. These differences are closely related to the brewing quality, and are important factors for quantitative determination in the brewing quality control process. Among them, there is a large difference in the content of Lactobacillus acetotolerans between ordinary cellars and high-quality cellars, and a highly specific method is needed for the bacteria The bacterium was found to be an absolute dominant strain in the fermented grains during the fermentation process of various liquors. However, the genus Lactobacillus is currently known to have hundreds of different species, and it is generally involved in the fermentation process of liquor There are nearly 50 different species of Lactobacillus. Most Lactobacillus have not been carefully studied, and their genome information is not comprehensive, so there are few reports on specific primer design for Lactobacillus acetotolerans

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Two pairs of specific primers for detecting acid-resistant lactobacillus content during Baijiu fermentation and application thereof
  • Two pairs of specific primers for detecting acid-resistant lactobacillus content during Baijiu fermentation and application thereof
  • Two pairs of specific primers for detecting acid-resistant lactobacillus content during Baijiu fermentation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Extraction of metagenomics from pit mud or fermented grains

[0026] Use the Shanghai Sangon B518233-0100 kit to extract the metagenome, weigh 100-300 mg of cellar mud (if it is wine grains, you need to crush it in advance, and weigh 100-300 mg after crushing), add 400ul Buffer SCL preheated at 65°C, Shake well and place in a 65°C water bath for 5 minutes. Centrifuge at room temperature at 12000rpm for 3min, and pipette the supernatant into a clean 1.5ml centrifuge tube. Add an equal volume of Buffer SP, invert to mix, and ice-bath for 10 minutes. After adding Buffer SP and mixing, the solution becomes white or light yellow turbid. Centrifuge at room temperature at 12000rpm for 3min, and pipette the supernatant into a clean 1.5ml centrifuge tube. Add 200ul of chloroform, mix well, centrifuge at 12000rpm for 5min, draw the upper layer of the aqueous phase into a clean 1.5ml centrifuge tube. Add 1.5 times the volume of Buffer SB, mix well, add all of it to the adsorpti...

Embodiment 2

[0028]Primer design and amplification of target DNA

[0029] Use Primer-Blast to design species-specific primers for the whole genome of Lactobacillus acetotolerans. The purpose of designing specific primers for a certain species is generally based on two reasons. One is to identify the species and detect whether the species is contained in a certain biological community. Another aim was to determine the abundance of the species in the overall microbial community.

[0030] Table 1 Species-specific primers for Lactobacillus acetotolerans

[0031]

[0032] Table 2 Amplified sequences of two pairs of primers

[0033]

[0034] PCR reaction system: NPK02buffer (2×) 6ul, species-specific primer LA2F LA2R (or LA3F LA3R) 1.5ul, template 0.15ul (use fermented grains metagenomic mixed sample as template), Taq enzyme 0.25ul, ddH2O 5.1ul. 94°C-4min, (94°C-30s, 57°C-40s, 72°C-40s)×35 cycles, 72°C-2min 37 cycles. The purified PCR products were used for TA cloning and blue-white scr...

Embodiment 3

[0036] Validation of primer specificity by TA cloning

[0037] PMD19-T vector 0.3ul, LA2 (or LA3) PCR purified product 0.5ul, sterilized ddH2O 4.2ulSolution I 5ul, mix well, add 30ul Escherichia coli competent cells DH5α ice bath for 40 minutes, then shake at 37°C Incubate and activate for 1 h. Mix X-gal 40ul, IPTG 40ul, 160ul dH2O, spread on LB ampicillin (100ug / ml) solid medium (LBA), wait for it to dry, continue to spread 200ul of activated bacteria solution on X-gal+IPTG Dried LBA solid medium. Incubate overnight at a constant temperature of 37°C. Randomly pick 15 single colonies and send them to the sequencing company for full-length sequencing of the inserted sequence. The sequencing results were subjected to BLAST analysis and RDP analysis (http: / / rdp.cme.msu.edu / ) to conduct qualitative and quantitative analysis on whether the amplified product belonged to the target Lactobacillus. All 15 cloned sequences are target sequences to pass the specificity test.

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses two pairs of specific primers for detecting acid-resistant lactobacillus content during Baijiu fermentation and application thereof. The specific primers include a forward primer of LA2 shown as a sequence 1, a reverse primer of LA2 shown as a sequence 2, a forward primer of LA3 shown as a sequence 3 and a reverse primer of LA3 shown as a sequence 4. The application includes application of the specific primers in quantitatively detecting wine spoilage bacteria content and application of the same in differentiating high-quality pit mud, common pit mud and low-quality pitmud. The invention discloses two pairs of specific primers for detecting wine spoilage bacteria content. Measuring of microorganism wine spoilage bacteria content in pit mud in Baijiu high-quality pits and common pits and in fermented grains finds that content of the wine spoilage bacteria can be used for differentiating pits different in grade.

Description

technical field [0001] The invention belongs to the field of biological technology, in particular the invention relates to two pairs of species-specific primers for detecting Lactobacillus acetotolerans and their application in liquor quality control. Background technique [0002] Liquor is a traditional fermented food in my country, has a long history, and has been liked by people since ancient times. Traditional Chinese liquor is an alcoholic beverage made from grains and grains rich in starch, using Chinese koji as a saccharification agent, fermented in solid, semi-solid or liquid state, and distilled, stored and blended. [0003] Lactobacillus refers to bacteria that can ferment sugars to produce lactic acid. It is the largest genus of lactic acid bacteria. Lactobacillus belongs to the Lactobacillus family and exists in Daqu, Xiaoqu and their fermented grains. In the production of liquor, lactic acid bacteria play an important role in the taste of liquor. And fragrance ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6851C12Q1/06C12N15/11
CPCC12Q1/6851C12Q1/689C12Q2531/113
Inventor 李俊薇曹润洁何宏魁张会敏李安军张治洲
Owner HARBIN INST OF TECH AT WEIHAI
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com