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Method for fast identifying generation of EC (ethyl carbamate) precursor of citrulline from lactic acid bacteria

A lactic acid bacteria arginine deiminase and lactic acid bacteria technology, applied in the field of microbial detection, can solve the problems of long time consumption, poor universality, poor lactic acid bacteria detection effect, etc., and achieves good versatility, sensitive detection method, and good food safety detection. Foreground effect

Inactive Publication Date: 2017-05-10
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

This method takes a long time, not only needs to prepare the mobile phase, but also needs to make a standard curve, the detection cycle is about 1 week, it is not a fast detection method
Another method is to amplify partial sequences of arcA, arcB, and arcC genes with degenerate primers. This method is quick and easy, but has poor universality. Pediococcus pentosaceae and Leuconostoc enterococcus are derived from lactic acid bacteria in wine. The detection effect on lactic acid bacteria isolated from rice wine is very poor. The main performance is that many citrulline-producing lactic acid bacteria cannot expand Added arcA and arcB, these primers have been reported to have considerable limitations in practical application

Method used

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  • Method for fast identifying generation of EC (ethyl carbamate) precursor of citrulline from lactic acid bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] 1. Extraction of lactic acid bacteria genomic DNA: use the Ezup Column Bacterial Genomic DNA Extraction Kit (Shanghai Sangon Bioengineering Co., Ltd.) to extract the genomic DNA of the lactic acid bacteria strains isolated in the rice wine fermentation broth according to the instructions.

[0023] 2. Primer design and PCR amplification:

[0024] A degenerate primer pair A and a degenerate primer pair B were designed; the nucleotide sequences of the upstream and downstream primers of primer pair A were 5'-AACCAYGCHATGATGCAYYT-3' / 5'-TTKGABCCRTCRTTCCATTG-3', respectively, for the detection of arcA gene , the product length is 250bp; the nucleotide sequences of the upstream and downstream primers of the primer pair B are 5'-AYTCHGGKGTDGTTTGGAA-3' / 5'-TCVGTMACTTCCATTTCHGT-3', which are used to detect the arcB gene, and the product length is 500bp; among them, Y =C / T, K=G / T, R=A / G, B=G / T / C, H=A / C / T, D=A / G / T.

[0025] Lactic acid bacteria strain genomic DNA template <500ng, up...

Embodiment 2

[0027] Embodiment 2: Validity verification of the inventive method

[0028] The present invention will be further described in conjunction with the detection example of lactic acid bacteria in the rice wine fermentation process, but the protection scope of the present invention is not limited thereto, and can also be extended to the detection of lactic acid bacteria in other foods or beverages.

[0029] Such as figure 1 As shown, 33 strains of lactic acid bacteria (including Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus flexus, Lactobacillus brevis, Lactobacillus delbrueckii, Lactobacillus coryneform, Leuconostoc citricum, Lactococcus lactis, etc. genus of lactic acid bacteria), the method of Example 1 was used to process, 30 strains could amplify bands of corresponding size, and 3 strains could not detect corresponding bands.

[0030] Then, these lactic acid bacteria were fermented and the ability of lactic acid bacteria to degrade arginine and produce citr...

Embodiment 3

[0034] Embodiment 3: The impact of primer sequence on detection effectiveness and accuracy

[0035] The inventor has also adopted the primers from the literature of Detection ofarc Genes Related with the Ethyl Carbamate Precursors in Wine Lactic Acid Bacteria that have been reported to carry out PCR amplification to detect whether the lactic acid bacteria isolated from rice wine can produce citrulline, and the results show that many The high-performance liquid chromatography detection confirmed that the genome of the strain capable of producing citrulline could not amplify the corresponding PCR product, indicating that the primers reported in the literature were not suitable for the detection of lactic acid bacteria derived from rice wine. However, using the primers in the present invention to detect citrulline-producing lactic acid bacteria derived from rice wine is all applicable, and the present invention also detects lactic acid bacteria from other sources (such as yogurt),...

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Abstract

The invention discloses a method for fast identifying the generation of an EC (ethyl carbamate) precursor of citrulline from lactic acid bacteria, and belongs to the technical field of microbiological detection. By the method, whether a lactic acid bacterium strain has an arginine deiminase path or not and whether citrulline can be formed by using arginine or not can be judged, so that whether the lactic acid bacterium strain in the fermented food and beverage has the possibility to generate a cancerogen of EC or not can be determined. The method disclosed by the invention is fast, simple, convenient and accurate; the degenerate primer specificity is high; the universality is high; the lactic acid bacteria of different species from different food can be detected; good food safety detection prospects are realized.

Description

technical field [0001] The invention relates to a method for rapidly identifying citrulline, a precursor of urethane produced by lactic acid bacteria, and belongs to the technical field of microbial detection. Background technique [0002] Lactic acid bacteria are a kind of microorganism widely present in fermented food or beverage (such as soy sauce, fermented sausage, rice wine, etc.), and the lactic acid produced by its metabolism is a flavor substance, so it plays a positive role in fermentation. However, recent studies at home and abroad have found that some lactic acid bacteria can decompose arginine to produce citrulline, and citrulline is an important component of a carcinogen—ethyl carbamate (EC) widely present in fermented foods. Precursor substances, especially in soy sauce and rice wine, which consumers eat (drink) more on a daily basis, have a relatively high content of EC. Citrulline is formed by lactic acid bacteria degrading arginine through the arginine dei...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/689
Inventor 李晓敏禹伟陆健蔡国林
Owner JIANGNAN UNIV
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