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Absolute quantification method for bacterial flora and application of absolute quantification method in Chinese liquor fermentation process

An absolute quantitative, liquor technology, applied in the fields of fermentation engineering and biology, it can solve the problems of interference, difficulty in finding, and inconvenient operation of bacterial flora structure sequencing results.

Active Publication Date: 2019-07-05
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it is difficult to find bacterial species that absolutely do not exist in the original system, and it is difficult to achieve; the method of adding internal standards is not only inconvenient to operate, but also interferes with the original bacterial group structure sequencing results

Method used

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  • Absolute quantification method for bacterial flora and application of absolute quantification method in Chinese liquor fermentation process
  • Absolute quantification method for bacterial flora and application of absolute quantification method in Chinese liquor fermentation process
  • Absolute quantification method for bacterial flora and application of absolute quantification method in Chinese liquor fermentation process

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1: Design of Lactobacillus acetotolerans-specific primers

[0069] (1) Use the Blast function of the National Center for Biotechnology Information (NCBI) website (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) to manually screen from the whole genome of Lactobacillus acetotolerans (AP014808.1) The specific gene WP_082137158.1 (NCBI accession number) is functionally annotated as a transcriptional regulator. According to the comparison results, only the genome sequence of Lactobacillus acetotolerans (AP014808.1) has a 100% similarity, indicating that the nucleotide sequence of the transcriptional regulator is a highly specific sequence of Lactobacillus acetotolerans.

[0070] (2) Use Batchprimer online software to design specific primers. The parameters are set as follows: primer length 18-27bp, optimal 20bp; annealing temperature 57-62°C, optimal 60°C; GC content 40-60%, optimal 50% ; The temperature difference between the primer pair is set to be less than 5°C; the ...

Embodiment 2

[0074] Example 2: Design of Lactobacillus sp.-specific primers

[0075] (1) Since Lactobacillus sp. is an uncultivated microorganism, the only 16S rRNA differential region sequence (GenBank: KU674948.1) was selected for specific primer design. The differential sequence was obtained by comparing the following sequences: 1) According to the Blast comparison To the result (similarity is shown in parentheses after the bacterial species name), select the 16S rRNA sequence of the strain with high similarity: Lactobacillus caviae (90%), Lactobacillus fructivorans (90%), Lactobacillushomohiochii (90%), Lactobacillus ixorae (89%) ), Lactobacillus reuteri (89%), Lactobacillus vespulae (89%), Lactobacillus ozensis (88%); 2) Lactobacillus brevis (88%) and Lactobacillus acetotolerans (87%) that appear frequently in the liquor brewing system. The screening of the 16S rRNA nucleic acid sequence difference region was completed by using the DNAMAN bioinformatics software to compare with the 16...

Embodiment 3

[0079] Example 3: Validation of Lactobacillus acetotolerans-specific primers

[0080](1) Select a target Lactobacillus acetotolerans for qPCR, and the negative control is 36 microorganisms from mouse intestine, liquor Xiaoqu, Daqu, commercial starter, yogurt, and silage: Lactobacillus buchneri, Lactobacillus dioilvorans, Lactobacillus brevis, Lactobacilluscrustorum, Lactobacillusplantarum ,Lactobacillus harbinensis,Lactobacillusacidiliscis,Pediococcus ethanolidurans,Pediococcus acidilactici,Pediococcuspentosaceus,Lactobacillus murinus,Lactobacillus curvatus,Lactobacillus casei,Lactobacillus reuteri,Lactobacilluspanis,Lactobacillusfermentum,Lactobacillusjohnsonii,Lactobacillus delbrueckii,Lactococcus lactis,Weissella confusa,Weissella paramesenteroides,Weissellaviridescens,Leuconostoc citreum,Leuconostoc lactis, Leuconostoc mesenteroides, Leuconostocpseudomesenteroides, Enterococcus italicus, Enterococcus lactis, Enterococcusfaecalis, Bacillus coagulans, Bacillus licheniformis, ...

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Abstract

The invention discloses an absolute quantification method for bacterial flora and application of the absolute quantification method in a Chinese liquor fermentation process, and belongs to the field of biology and fermentation engineering. The invention firstly constructs a method for calculating the absolute content of microbial flora based on a natural internal standard. The specific primers ofthe natural internal standards Lactobacillus acetotolerans and Lactobacillus sp. provided by the invention can be used for detecting and quantifying internal standards in a Chines liquor fermentationsystem. Furthermore, according to the invention, statistics is carried out on the distribution range of the internal standards, and the result shows that the internal standards are widely distributedin fragrant type, soy sauce flavor type, strong flavor type, soybean flavor type and sesame flavor type Chinese liquor fermentation systems. By absolute quantification of the internal standards, the flora content of different flavor types can be calculated, and the method has the characteristics of convenience in operation and wide application range.

Description

technical field [0001] The invention relates to a method for absolute quantification of bacterial flora and its application in the fermentation process of Chinese liquor, belonging to the fields of biology and fermentation engineering. Background technique [0002] Chinese liquor has a history of thousands of years of development and is a valuable cultural wealth in China. Chinese liquor belongs to the solid-state multi-species mixed fermentation system, in which the bacterial community plays an important role in the fermentation process, such as Bacillus, Lactobacillus, Lactococcus, etc. Absolute quantification of bacterial flora is the basis for studying the function of flora. In past studies, high-throughput amplicon sequencing technology was used to effectively reveal the composition and structure of bacterial flora during liquor fermentation. However, only relative abundance can be used to quantify The bacterial community cannot truly reflect the dynamic change charact...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/06C12Q1/689C12Q1/6851C12N15/11C12R1/225
CPCC12Q1/06C12Q1/6851C12Q1/689C12Q2600/158C12Q2600/166C12Q2600/178C12Q2531/113C12Q2545/101C12Q2563/107
Inventor 徐岩吴群杜如冰
Owner JIANGNAN UNIV
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