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A method for efficiently screening lactic acid bacteria that can fully utilize citrulline

A high-efficiency technology of citrulline, applied in the field of microorganisms, can solve the problems that there are no application examples of citrulline, and achieve the effect of reducing the content

Active Publication Date: 2018-08-28
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently, there are no examples of applications for effective screening of microorganisms that degrade citrulline

Method used

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  • A method for efficiently screening lactic acid bacteria that can fully utilize citrulline
  • A method for efficiently screening lactic acid bacteria that can fully utilize citrulline
  • A method for efficiently screening lactic acid bacteria that can fully utilize citrulline

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Cultivable microorganism isolation in embodiment 1 soy sauce

[0025] Sampling and dilution from the finished koji of brewed soy sauce were applied to MRS and nutrient broth medium plates respectively, and enriched and cultivated at 37°C for 5 days. Select a single colony with different colony forms and inoculate it on a medium plate containing MRS and nutrient broth, and separate it by streaking for pure culture. Four days later, the pure culture was inoculated into MRS and nutrient broth liquid medium and cultured for four days to enrich the cells and extract the genome. The 16S rDNA was obtained by PCR with common primers for 16S rDNA of bacteria, and the 16S rDNA sequencing results were compared with the database.

[0026] The deposit number of Pediococcus acidilacticiBBE 1120 is CCTCC NO:M 2013732.

[0027] Tetragenococcus halophilus BBE R23 was deposited in China Center for Type Culture Collection (CCTCC), Wuhan, China, Wuhan University on October 20, 2013, and ...

Embodiment 2AD

[0029] Embodiment 2ADI pathway gene analysis

[0030]According to the data published in NCBI GenBank about Weissella, Staphylococcus, and lactobacillus: Weissella confusa LBAE C39-2 GenBank Assembly ID: GCA_000239955.2, Weissella cibaria KACC 11862 )GenBank Assembly ID:GCA_000193635.2,葡萄球菌(Staphylococcus aureus subsp.aureusNCTC 8325)GenBank Assembly ID:GCA_000013425.1,乳酸片球菌(Pediococcus acidilactici DSM 20284)GenBank AssemblyID:GCA_000146325.1,嗜盐四联球菌(Tetragenococcus halophilus NBRC 12172) GenBank: AP012046.1, the analysis shows that the key genes of the ADI pathway of Weissella, Staphylococcus, and Lactobacillus only have a single copy, that is, only three genes, arcA, arcB, and arcC. The T.halophilusNBRC12172 pathway consists of arcA, arcB, arcC genes on arc operon and two additional copies of arcB, arcC genes other than arc operon, that is, arcA, arcB, arcC, arcB1, arcC1, arcB2, The seven genes of arcC2. The copy number of this ADI pathway gene may be related to the strain'...

Embodiment 3

[0031] Example 3 Analysis and Validation of ADI Pathway Gene Clusters of Tetradynococcus halophilus

[0032] Pick Tetragenococcus halophilus BBER23 (CCTCC NO:M 2013480) and Tetragenococcus halophilus BBE C3 (CCTCC NO:M 2013479) obtained from koji and preserved in glycerol tubes at -80°C, and streak on MRS solid medium with 10% NaCl . After culturing at 30°C for four days, pick a single colony and perform colony PCR. The primers are shown in Table 1 to verify whether there are arcA, arcB, arcC, arcB1, arcC1, arcB2, arcC2 genes. The PCR results showed that there was a complete arc operon (arcA, arcB, arcC) and two extra copies of arcB gene and arcC gene in the genome of T. halophilus R23; there was not a complete arc operon in the genome of T. halophilus C3 ( Deletion of arcA, arcB, arcC), but there are two copies of arcB1, arcC1 genes other than arc operon.

[0033] The primers used in the embodiment of table 1

[0034]

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Abstract

The invention discloses a method for efficiently screening lactic acid bacteria capable of sufficiently utilizing citrulline, belonging to the field of microorganisms. The method comprises the steps of starting with a genotype to screen bacteria with multicopy genes, inspecting the capability of metabolizing citrulline, and furthermore screening lactic acid bacteria capable of sufficiently utilizing citrulline. The content of citrulline is reduced through co-culture.

Description

technical field [0001] The invention relates to a method for efficiently screening lactic acid bacteria that can fully utilize citrulline, and belongs to the field of microorganisms. Background technique [0002] Ethyl carbamate (EC) is a carcinogen. Excessive and continuous intake by the human body may cause serious tumor diseases such as lung cancer and liver cancer, and cause damage to the human immune system. Urethane is widely found in various alcoholic beverages and fermented foods. Soy sauce is a fermented food in which ethyl carbamate was detected. [0003] The main formation pathway of ethyl carbamate in brewed soy sauce is the reaction of citrulline and ethanol, while the lactic acid bacteria in soy sauce consume arginine through the arginine deimidation pathway (ADI pathway) to produce citrulline. ADI pathway ( figure 1 ) consists of three enzyme-catalyzed reactions: ADI (encoded by arcA), OTC (encoded by arcB), and CK (encoded by arcC). Among them, ADI enzyme ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12Q1/686C12Q1/04
CPCC12Q1/04C12Q1/689
Inventor 方芳陈坚廖淡宜杨怡敏堵国成
Owner JIANGNAN UNIV
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