KR20130027604A - Primer kit for detection of lactic acid bacteria in kimchi and detection method to lactic acid bacteria in kimchi including the same - Google Patents

Primer kit for detection of lactic acid bacteria in kimchi and detection method to lactic acid bacteria in kimchi including the same Download PDF

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KR20130027604A
KR20130027604A KR1020110042063A KR20110042063A KR20130027604A KR 20130027604 A KR20130027604 A KR 20130027604A KR 1020110042063 A KR1020110042063 A KR 1020110042063A KR 20110042063 A KR20110042063 A KR 20110042063A KR 20130027604 A KR20130027604 A KR 20130027604A
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kimchi
lactic acid
acid bacteria
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strain
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박기범
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위니아만도 주식회사
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    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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Abstract

As described above, according to the method for detecting kimchi lactic acid bacteria using the primer set for detecting kimchi lactic acid bacteria represented by the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2 according to the present invention and a polymerization chain reaction comprising the same, a polymerization chain reaction (PCR ) To accurately and quickly determine the existence of Weissella koreensis during the fermentation of Kimchi by time and time to reduce the time and cost of microbial identification as well as determine the taste and flavor of Kimchi. By suggesting the standardization of Weissella koreensis strain to be used as a tool of kimchi quality control.

Description

Primer kit for detection of Lactic Acid Bacteria in Kimchi and detection method to Lactic Acid Bacteria in Kimchi including the same}

The present invention relates to a kimchi lactic acid bacteria detection primer set and a kimchi lactic acid bacteria detection method comprising the same.

Lactic acid bacteria are bacteria that break down sugars such as glucose to produce lactic acid. Also called lactic acid bacteria, the lactic acid produced by lactic acid fermentation prevents the growth of pathogens and harmful bacteria in food production such as dairy products, kimchi and brewed foods. Doing. In addition, lactic acid bacteria are used as intestinal agents by preventing abnormal fermentation caused by various bacteria by living in the intestine of mammals. The lactic acid bacteria are Gram-positive bacteria, having anaerobic or anaerobic properties, and are known in the genus Lactobacillus and Streptococcus.

Kimchi is a Korean traditional food fermented by lactic acid bacteria (lactic acid bacteria). Lactic acid bacteria (lactic acid bacteria) classified in kimchi include Leuconostoc mesenteroides , Leuconostoc dextranicum and Lactobacillus. Lactobacillus brevis , Lactobacillus plantarum , Pediococcus pentosacues , and the like.

In general, as kimchi is fermented, leuconosstocks including Leuconostoc mesenteroides proliferate predominantly, leading to early acid production, and are involved in kimchi fermentation. The number of active leukonostock strains will decrease. On the other hand, in the late fermentation, lactobacillus bacteria, such as Lactobacillus plantarum and Lactobacillus brevis , which are resistant to acid, are actively active, so that kimchi is soured as the number of Lactobacillus bacteria gradually increases.

Since the quality of kimchi is affected by the distribution of various lactic acid bacteria involved in the fermentation process, in order to control the fermentation process or quality of kimchi, the distribution patterns of lactic acid bacteria involved in the kimchi fermentation and the strains acting according to the fermentation time can be analyzed. There is a need.

In order to analyze lactic acid bacteria involved in conventional kimchi fermentation, the form and number of colonies formed by smearing lactic acid bacteria present in fermented products such as kimchi under culture conditions specific to lactic acid bacteria have been identified. However, smear culture can only measure the number of microorganisms that can survive under artificial culture conditions, and microorganisms present in kimchi that play an important role in fermentation can be killed in preparation or incubation of samples. Even when cultured, the strain takes some time to form colonies, and has a disadvantage of low specificity and sensitivity.

The present invention as having a major effect on the quality of kimchi flow Pocono stock (Leuconostoc) in and Lactobacillus bacteria (Lactobacillus) in addition to the present from the initial fermentation stage of kimchi that being aged and stored at low temperature to affect the taste of kimchi Weissella koreensis was detected as a strain, and it is an object of the present invention to provide a primer set and a method for detecting the strain.

In order to accurately detect the Weissella koreensis strain, the present invention is a primer for detecting kimchi lactic acid bacteria that can overcome the excessive time required and the inaccuracy of detection according to the confirmation of the form or number of colonies by smear culture. It is intended to provide a set and a method for detecting kimchi lactic acid bacteria comprising the same.

The present invention provides a primer set for detecting kimchi lactic acid bacteria represented by the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2.

The present invention provides a method for detecting kimchi lactic acid bacteria using a polymerase chain reaction comprising a primer set represented by a nucleotide sequence of SEQ ID NO: 1 and SEQ ID NO: 2.

In the present invention, the kimchi lactic acid bacteria are characterized in that the Weissella koreensis strain (405).

Kimchi lactic acid bacteria detection primer set according to the present invention and the kimchi lactic acid bacteria detection method using a polymerized chain reaction including the same is a time-consuming and inaccurate disadvantage of the conventional methods of detecting lactic acid bacteria found in kimchi and pickles, etc. Only the strain can be detected quickly and accurately.

1 relates to the sequence of 16S rRNA of Weissella koreensis strain 405.

The present invention provides a primer set for detecting kimchi lactic acid bacteria represented by the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2.

Lactic acid bacteria are involved in the kimchi fermentation, but a number of, the Kimchi lactic acid bacteria is related to a typical lactic acid bacteria is by Cellar Corey N-Sys (Weissella koreensis) strain (strain) 405 that exists in the Effective initial continuously acts on the fermentation and ripening of kimchi .

In the present invention, the Bisella koreensis strain affecting the flavor of kimchi may be used by directly separating the sample from the fermented kimchi at regular intervals, Korea Gene Bank (KCTC), Korea spawn association (KCCM) and the Korea Agricultural Microbial Resources Center (KACC) may be used to obtain the strain.

The primer set is a primer set capable of specifically binding only to the 16S rRNA sequence of the Weissella koreensis strain 405. The known bisellar corensis sequences, Lactobacillus strain and leukonostock strain The 16S rRNA sequences were compared to select a high conserved region to design a primer set consisting of the nucleotide sequence represented by SEQ ID NO: 1 and SEQ ID NO: 2.

Forward primer (SEQ ID NO: 1): AGAGTTTGATCMTGGCTCAG

Reverse primer (SEQ ID NO: 2): AAGGAGGTGWTCCARCC

The present invention also provides a method for detecting kimchi lactic acid bacteria using a polymerase chain reaction comprising a primer set represented by the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2.

In the present invention, the kimchi lactic acid bacteria are continuously present from the beginning of fermentation, and are related to the strain 405, which is a representative lactic acid bacterium, Weissella koreensis , which acts on fermentation and ripening of kimchi.

In the present invention, in order to perform the polymerase chain reaction, genomic DNA is extracted from Weissella koreensis strain 405 obtained by directly separating from kimchi fermentation, or used by Korea Gene Bank (KCTC), Genomic DNA can be extracted and used from strains distributed from KCCM) and Korea Agricultural Microbial Resources Center (KACC).

The polymerization chain reaction is carried out including a primer set consisting of a nucleotide sequence represented by SEQ ID NO: 1 and SEQ ID NO: 2, wherein the primer set is specific only to the 16S rRNA sequence of the Weissella koreensis strain 405 A set of primers that can bind.

In addition, the polymerase chain reaction can be carried out using a kimchi lactic acid bacteria detection kit comprising a primer set represented by the nucleotide sequence of SEQ ID NO: 1 and SEQ ID NO: 2, the kit is a common component of the lactic acid bacteria detection kit It may include, and typical components may include reaction buffer, DNA polymerase and dNTP.

The detection method of the kimchi lactic acid bacteria in the present invention, as well as the polymerase chain reaction (Polymerase Chain Reaction), multiplex polymerase chain reaction (Multiplex polymerase chain reaction), reverse transcriptase chain reaction (Reverse transcriptase PCR), DNA sequencing (DNA) sequencing, hybridization by microarray, restriction fragment length polymerphism (PCR-RFLP), randomly amplified polymorphic DNA (RAPD), DNA amplification fingerprinting (DAF), arbitrarily primed PCR (AP-PCR), sequence tagged site, expressed sequence tag (EST), sequence characterized amplified regions (SCAR), inter-simple sequence repeat amplication (ISSR), amplified fragment length polymorphism (AFLP), cleaved amplified polymorphic sequence (CAPS), single- PCR One or more of the methods selected from the group consisting of strand conformation polymorphism, Northern blot and Southern blot.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are intended to illustrate the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention.

Example 1 Securing Weissella koreensis Strains

In the present invention, the fermented kimchi was collected at regular intervals to isolate the strain. For isolation and counting of strains, kimchi was diluted to 10 -1 to 10 -8 concentration using 1% peptone water, 1 ml of the diluted solution was incubated for 24 hours at 37 ° C in plate culture with agar for plate counting (PCA). The colonies shown were the total bacterial count. After culturing these colonies at 37 ° C. for 24 hours, genomic DNA was extracted from the cultured strains using Genomic DNA Extraction kit (iNtRON Biotechnology, Suwon, Korea), which was used for polymerase chain reaction (PCR). Used as template DNA.

Example 2 Preparation of Specific Primer

There are a number of lactic acid bacteria involved in kimchi fermentation, but the present invention is a 16S rRNA sequence of the strain 405 Weissella koreensis strain 405 (see Fig. 1), which is a lactic acid bacterium that exists continuously from the beginning of fermentation to determine the flavor of kimchi. The primer set that can specifically bind only to the 16S rRNA of the strain was prepared as shown in Table 1.

As a result, the obtained primer sets are shown in Table 1 below as nucleotide sequences represented by SEQ ID NO: 1 and SEQ ID NO: 2.

Accession number of 16S rRNA sequence SEQ ID NO: 1 SEQ ID NO: 2 Weissella koreensis strain 405 16S ribosomal RNA gene (DQ883229.1) AGAGTTTGATCMTGGCTCAG AAGGAGGTGWTCCARCC

Example 3 Polymerization Chain Reaction Using a Specific Primer

A polymerase chain reaction (PCR) was performed using a primer set consisting of SEQ ID NO: 1 and SEQ ID NO: 2 designed in Example 2. As the DNA used as a template, genomic DNA extracted from the Weissella koreensis strain 405 of Example 1 was used.

Polymerase chain reaction (PCR) was carried out using 20 μl of the total reaction solution, and 10 μl of PCR premix (1 μM of Taq DNA polymerase, 200 μM of dNTP, 1.5 mM of MgCl 2 ), template DNA, and 1 μM of SEQ ID NO: 1 and The primers of SEQ ID NO: 2 were mixed to make 10 μl. The polymerase chain reaction (PCR) conditions were preheated at 94 ° C. for 1 minute, then denatured at 94 ° C. for 2 minutes, annealed at 55 ° C. for 1 minute, and at 72 ° C. for 1 minute. Extension was made.

The obtained PCR amplification product (electrophoresis product) was electrophoresed on 1.5% agarose gel, and analyzed by detecting the amount of color of Etbr (Ethidium bromide) on UV, and the molecular weight was compared with the molecular weight ladder marker.

As a result, as shown in Table 2, it was confirmed that the amplified gene product of Weissella koreensis strain 405 was 1200bp by the primer set consisting of SEQ ID NO: 1 and SEQ ID NO: 2.

Lactobacillus Species of 16S rRNA Sequences SEQ ID NO: 1 SEQ ID NO: 2 DNA size Weissella koreensis strain 405 16S ribosomal RNA gene (DQ883229.1) AGAGTTTGATCMTGGCTCAG AAGGAGGTGWTCCARCC
1200 bp

The present invention can be easily and accurately detected by PCR method of Bisella koreensis strain which exists from the beginning of fermentation of kimchi and affects the flavor of kimchi, and according to the confirmation of the form or number of colonies by smear culture. It could overcome the excessive disturbance and the inaccuracy of detection.

Example 4 Monitoring of Weissella koreensis Strains According to Fermentation Time in Kimchi

Genomic DNA was extracted from Kimchi with different fermentation time as in Example 2 for monitoring the strain of Weissella koreensis according to fermentation time, and used as template DNA for polymerase chain reaction (PCR). Used.

As a result of PCR using the primers of SEQ ID NOs: 1 and 2 of Example 3, it can be confirmed that the strain of Bisella koreensis was continuously present from the beginning of fermentation of kimchi, and continuously existed during the fermentation of kimchi until 60 days of fermentation. (Not shown).

Thus, the present example confirmed that the strain of Weissella koreensis was a strain capable of monitoring fermentation of kimchi, and the monitoring by PCR method was performed by conventional smear culture. It can easily and accurately monitor the presence of the strain in a way to overcome the excessive time required and the inaccuracy of the detection according to the confirmation.

Example 5 Monitoring of Weissella koreensis Strains According to Storage Temperature in Kimchi

After storing kimchi at different storage temperatures of kimchi, genomic DNA was extracted from these kimchi in the same manner as in Example 2. As a result of PCR using genomic DNA of kimchi obtained by varying the storage temperature, it was confirmed that the detection amount of the strain of Weissella koreensis varies depending on the storage temperature (not shown). The method of overcoming the excessive time required and the inaccuracy of detection according to the confirmation of the form or number of colonies by smear culture has the advantage of easy and accurate monitoring of the presence of the strain.

Claims (4)

Primer for detecting kimchi lactic acid bacteria represented by the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2
The method of claim 1, wherein the kimchi lactic acid bacteria Weissella koreensis strain (strain) Wetella lactic acid bacteria detection primer set characterized in that the strain (405)
Method for detecting kimchi lactic acid bacteria using polymerase chain reaction comprising primer sets represented by nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2
The method of claim 3, wherein the kimchi lactic acid bacteria is Weissella koreensis strain 405.
KR1020110042063A 2011-05-03 2011-05-03 Primer kit for detection of lactic acid bacteria in kimchi and detection method to lactic acid bacteria in kimchi including the same KR20130027604A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20170098075A (en) 2016-02-19 2017-08-29 엘지전자 주식회사 Optical Sensor for Measuring Degree of Fermentation of Kimchi
KR20180056053A (en) * 2016-11-18 2018-05-28 한국식품연구원 Use of 2-hydroxyisocaproic acid for indicator of fermentation

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20170098075A (en) 2016-02-19 2017-08-29 엘지전자 주식회사 Optical Sensor for Measuring Degree of Fermentation of Kimchi
KR20180056053A (en) * 2016-11-18 2018-05-28 한국식품연구원 Use of 2-hydroxyisocaproic acid for indicator of fermentation

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