KR100454591B1 - A novel strain of Weisella, Weisella koreensis, and method for preparation of kimchi using this strain - Google Patents

Abstract

The present invention relates to a Weissella koreensis strain, a new strain of the genus Weissella, and a kimchi production method using the strain. Using the strain and the kimchi production method of the present invention shortened the maturation period of the kimchi and gave reproducibility to the production of kimchi having a good flavor, and also because the delay of rancidity of kimchi can maintain the flavor for a long time, of excellent quality It can be usefully used to make kimchi.

Description

A novel strain of Weisella, Weisella koreensis, and method for preparation of kimchi using this strain

The present invention relates to a kimchi fermentation new strain and a kimchi production method using the strain, and more particularly to a kimchi fermentation bisella genus new strain and a low-temperature kimchi production method using the strain.

Kimchi, a traditional Korean fermented food, is not only an indispensable food in Koreans' diets, but has recently become one of Korea's representative foods in the world market. As the interest in the health of the developed nations has been amplified, the dietary changes from meat eating to vegetarian diets have been active, and the nutritional values such as the kimchi's unique flavor and the inhibitory effect of various cancers caused by lactic acid bacteria and the immune enhancing effect have been recognized. It is expected to expand its market further in the future (Ahn Soon-cheol et al., Korean Journal of Microbiology, 37 (3): 234, 2001). However, in order for Korean-made kimchi to be more competitive than the present in the world market, it is produced and supplied with standardized kimchi with a certain flavor to emphasize the nutritional benefits of kimchi, and also to the taste of those who consume it. The flavor should be used to get used to. To this end, not only the quality of the product should be improved, but also the production method of kimchi should be systematically manufactured to produce standardized kimchi with a certain flavor, and the preservation during distribution.

The researches on kimchi have been conducted mainly on food studies to enhance the taste of kimchi, research on packaging methods to increase the value of kimchi, and on microorganisms related to fermenting kimchi. Factors that can influence the fermentation of kimchi are known to be pretreatment, salt concentration, fermentation temperature, etc. These are all factors affecting the growth of kimchi microorganisms (Kim Myung-hwan and Jang Moon-jeong, Korean Journal of Agricultural Chemistry, 43 (1): 7, 2000). Among them, fermentation temperature, especially low temperature fermentation, is known to be important, and the empirical fact that kimchi kimchi prepared in early winter and matured at low temperature during winter tastes better than kimchi fermented rapidly in summer is well supported. According to the results of research showing that the different types of lactic acid bacteria involved in kimchi fermented at low temperature and kimchi fermented at medium or normal temperature, the good flavor of low-temperature fermented kimchi is reflected in the taste of the product The results are as follows (So Myung Hwan and Young Bae Kim, Korean Society of Food Science and Technology, 27 (4): 495, 1995). The results of the observation of the behavior of lactic acid bacteria during the fermentation of Kimchi showed that during the early stage of fermentation, leukostock , including Leuconostoc mesentroides , proliferated predominantly, leading to early acid production, Lactobacillus bacteria such as Lactobacillus plantarum are known to proliferate vigorously (Min Tae-ik and Kwon Tae-wan, Korean Journal of Food Science and Technology, 16 (4): 443, 1984). Lactobacillus plantarum ( Lactobacillus plantarum ) and Lactoacilus brevis ( Lactoacilus brevis ) acts as a rancidum bacterium in kimchi fermentation, it is known that the bacteria of the genus Lukonostok are the main fermentation bacteria of kimchi that influences the flavor of kimchi. Therefore, many studies have been conducted to find a method for maintaining the strains of the genus Lukonostok in kimchi for a long time, but no good results have been reported so far (Kang Sang-mo, Korean Patent Application No. 1996-033361).

Until now, lactic acid bacteria of the genus Leukonostok and Lactobacillus have been known as dominant species of kimchi. Therefore, there were cases where these strains were intended to be used as a starter strain for kimchi manufacturing, but did not obtain good results. The reason is that in the conventional R & D, the microorganisms that can be separated from the kimchi samples in the limited artificial medium and the environment are limited, so the changes of microbial diversity affecting the fermentation of kimchi in the case of low temperature fermentation are not properly identified. .

In order to overcome the limitations of the low temperature fermentation kimchi microbial separation method according to the limitation of microbial analysis due to the culture method, the present inventors directly isolate DNA of microorganisms present in kimchi samples and analyze the diversity of microorganisms from these microbial DNAs. (Denaturing Gradient Gel Electrophoresis) method was used, and as a result, unlike the results of the previous studies, kimchi fermented at low temperature has a lactic acid bacterium which is continuously dominated from the beginning of fermentation. The present inventors confirmed that the present invention is a strain of bisella genus Weissella koreensis , and by inoculating the strain with a starter of kimchi fermentation, showing that it can quickly produce excellent flavor kimchi at low temperature The invention has been completed.

An object of the present invention to provide a kimchi fermentation strains and kimchi production method using the strain.

1 is a graph showing the change of pH according to the fermentation period in the case of low-temperature (4 ℃) kimchi fermentation,

Figure 2 is an electrophoresis picture of the change pattern of 16S rRNA gene fragments derived from various microorganisms in kimchi according to the fermentation in the case of low temperature (4 ℃) kimchi fermentation, DGGE method,

Figure 3 is a graph showing the change in pH according to the low temperature (4 ℃) fermentation period of kimchi when the bisella corriensis strain of the present invention is added as a starter (starter) for kimchi fermentation.

■: control kimchi,

●: Kimchi with bisella corriensis as starter

In order to achieve the above object, the present invention provides a bisella corriensis strain and kimchi production method using the strain.

Hereinafter, the present invention will be described in detail.

The present invention provides a bisella corriensis strain. The bisella corriensis strain of the present invention, a new strain of bisella genus that affects the flavor of kimchi, is isolated from kimchi fermented at low temperature. Specifically, the cabbage is cut to a normal size and pickled in brine, and then mixed kimchi ingredients including leek, garlic, onion, ginger, salted fish and red pepper powder are added, and samples are regularly taken while fermenting at low temperature. 10 to 1000 microorganism colonies (colony) are formed, and then diluted with sterile saline, plated on MRS plate medium and incubated. Strains with different colony shapes are selected for the strains that form the colonies, and each of these strains is identified and identified. By the above method, the bisella corriensis strain of the present invention is isolated. The inventors of the present invention deposited the strain at the Korea Biotechnology Research Institute Gene Bank on Dec. 10, 2002 (Accession No .: KCTC 1040BP).

Various taxonomic properties are investigated to identify bisella corriensis strains of the present invention. Specifically, whether acid is produced from various kinds of sugars, whether the sugar is available as a carbon source, whether ammonia is produced from arginine, the types of amino acids constituting murine, whether dextran is produced, and lactic acid produced The taxonomic position of the strain is investigated from the form of the optical isomer of the isomer, the G + C content of the nucleic acid and the form of the bacterium (see Table 1 ). In addition, the strains of the present invention were purified and cultured to extract DNA, and 16S rRNA gene sequence was determined to confirm homology to various strains and 16S rRNA sequences. As a result, the strain of the present invention had a similarity of 97.2% with Weissella kandleri , and also 25% with Bisella kandleri and 16% or less with DNA of Weissella viridescens . Reassociation value of DNA-DNA hybridization is shown (see Table 2 and Sequence Listing 3 ). Thus, we have named the strain identified above as Bisella coriensis as the Bisella genus strain.

In addition, the present inventors noticed that the flavor of kimchi fermented at low temperature is better than the flavor of kimchi fermented at room temperature, and identified a dominant strain that influences the flavor of kimchi when fermenting kimchi at low temperature. Specifically, the microbial diversity according to the fermentation period in the low-temperature fermented kimchi of the present invention extracted DNA of the microorganisms directly from the kimchi sample and then confirmed the region corresponding to their 16S rRNA gene region through DGGE analysis. As a result, it was confirmed that the bicellar corriensis of the present invention is a dominant species microorganism of low temperature fermented kimchi.

In addition, the present invention provides a kimchi production method using the above-mentioned bisella corriensis strain.

At this time, the bisella corriensis strain is characterized in that inoculated with a starter strain of kimchi fermentation. More specifically, the method of preparing kimchi of the present invention is characterized by inoculating bisella corriensis as a starter strain after mixing seasonings including red pepper powder, garlic, salted fish, and the like with pickled cabbage.

In this case, it is preferable to inoculate the cellar by Corey N-Sys at a concentration of kimchi 110 ^{4-10 7} cells per g, preferably of kimchi fermentation at 0 to 10 ℃ after inoculation of the strain.

In the kimchi manufacturing method of the present invention, typically in the kimchi immersion method addition ratio of the starter strains, regardless of differences in the addition of the spices and the ratio of Chinese cabbage or radish kimchi method is adjusted to 1 to 10 ⁷ cells per ^4-10 g Can be added.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Isolation of Kimchi Mycelia

Cut the Chinese cabbage into 3 cm of each side, immerse in salted water to make a final salt concentration of 2%, and then add kimchi ingredients mixed with green onion, garlic, onion, ginger, salted fish and red pepper powder, and ferment at 4 ℃. Samples were taken at 5 day intervals. 10 to 1000 microorganism colonies (colony) were formed, and then diluted with 10 fold dilution method with sterile saline solution (M.F., Difco, Becton Dickinson Co. Sparke, MC. USA) (1 L of distilled water, protease peptone (Bacto protease peptone) No.3 10 g, Bacto beef extract 10.0 g, Bacto yeast extract 5.0 g, Bacto dextrose 20.0 g, Polysorbate 80 1.0 g, ammonium citrate 2.0 g, sodium acetate 5.0 g, magnesium sulfate 0.1 g, manganese sulfate 0.005 g, dipotassium phosphate 2.0 g and 20.0 g of bacto agar) was incubated at 25 ° C. for 2-3 days. Strains with different colonies were selected for the colony-forming strains, and each of these strains was cultured by pure separation.

<Example 2> Bisella corriensis ( Weissella koreensis Identification of Strains

As a result of separating the strains using the MRS medium among the strains isolated in Example 1, strains having the taxonomic characteristics as shown in Table 1 were separated.

Taxonomic Characteristics of Strains Statue Property Statue Property L-Arabinose Cellobiogalactose Maltose Mellibiose + ---- Acid-produced yumurafinose ribose sucrose stress haloxylose -+-+ Escurin Resolution - Dextran generation + Ammonia-producing ability from arginine + Photoisomer of Producing Lactic Acid ^* D Murine formation Lys-ala-ser Morphology Irregular, short or spherical bacilli Nucleic Acid G + C Content (mol%) 37

^* Form D: when at least 90% of the lactic acid produced is D (-),

In addition, the present inventors extracted the DNA after the pure separation of the strain to determine the 16S rRNA gene sequence ( Table 2) . Specifically, DNA was extracted from the culture medium obtained by inoculating the strain into a single colony of MRS liquid medium and then standing at 30 ° C. overnight. To use the extracted DNA as template DNA and to amplify the 16S rRNA gene region, 27f (5'-AGAGTTTGATCCTGGCTCAG) as set out in SEQ ID NO: 1 and 1492r (5'-TGACTGACTGAGGYTACCTTGTTACGACTT) as set out in SEQ ID NO: 2 (Lane, et al ., Nucleic acid techniques inbacterial systematics (p115, 1991) were used as primers to amplify the region corresponding to the 16S rRNA gene region by PCR, and then sequence was determined for the product. As a result, the strain had a 16S rRNA sequence as set forth in SEQ ID NO: 3, and confirmed homology to several strains and the 16S rRNA sequence, and showed a similarity of 97.2% to Weissella kandleri . The DNA-DNA hybridization was less than 25% with Sella candelli and 16% with Weissella viridescens ( Table 2 ).

DNA-DNA Hybridization with Isolates and 16S rRNA Gene Sequences Strain % Recombination with Marked DNA Strains of the Invention Weisella Kandleri Bisella Viridesense Strains of the Invention 100 25 16 Bisella KandleriKCTC 3610T 20 100 14 Vicella ViridesenseKCTC 3504T 16 9 100

Therefore, the present inventors identified the strain as a new strain belonging to the genus Bisella and named it Bisella corriensis, and deposited it on the Korea Biotechnology Research Institute Gene Bank on December 10, 2002 (Accession No .: KCTC 10400BP).

Example 3 Changes of Kimchi Microbial Community During Fermentation

DGGE analysis was performed to investigate how the microbial diversity in kimchi changes with fermentation time at low temperature. Specifically, after preparing kimchi by the method described in Example 1, the kimchi sample obtained by filtering the solids of the collected kimchi first with gauze and again filtered with Whatman International Ltd. Maidstone, UK DNA and RNA were extracted from them to investigate changes in microbial diversity.

<3-1> Extraction of Genomic DNA from Kimchi Samples

3 ml of Kimchi samples were centrifuged to collect microbial cells in the sample and suspended in 200 µl of STES buffer (0.2 M Tris-Cl, 0.5 M NaCl, 0.01 M EDTA, 1% SDS, pH 7.0). To the suspension was added 200 mg of 0.1 mm glass beads, followed by shaking to break up the cell walls, and to the suspension with the same amount (v / v) of phenol: chloroform: isoamyl alcohol (25: 24: 1) solution. After mixing, the supernatant was transferred to a new tube by centrifugation at 10,000 g for 5 minutes. 1 μl of RNaseA (Promega Co. Madison, Wis. USA) was added to the solution and reacted at 37 ° C. for 30 minutes to remove RNA, and 1/10 times (v / v) of 3 M sodium acetate (v / v). Two times (v / v) ethanol was added to precipitate DNA and then suspended in 50 μl of sterile water.

<3-2> touch down PCR

To amplify the 16S rRNA gene region of various microorganisms present in the kimchi sample, gc338f primer (5'-CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACCGGCGGGCGGGGCGGGGGCACGGGGGGACTCCTAC GGGAGGCAGCAG) and SEQ ID NO: 5 (518r primer) described in SEQ ID NO: 4 corresponding to the universal site of 16S rDNA) 5'-ATTACCGCGGCTGCTGG) (Ampe, et al ., Appl. Environ. Microbiol ., 65 (12): 5464, 1999). 100 ng of genomic DNA extracted from the Kimchi sample of Example <3-1>, 5 pmole of gc338f and 518r primers, 40 mM KCl, 1.5 mM MgCl ₂ , 250 μM dNTP and 1 U of Taq polymerase, respectively, were added. After a total volume of 20 μl, heat treatment at 94 ° C. for 5 minutes, followed by 1 minute at 94 ° C. for DNA denaturation, and the temperature for annealing of the primers first starts at 65 ° C. It was set to decrease by 1 ℃ every 2 cycles to react for 1 minute and touchdown PCR was performed by performing 15 more cycles when reaching 55 ℃. In order to elongate the PCR fragment, the reaction was carried out at 72 ° C. for 1 minute, and finally at 72 ° C. for 10 minutes, and then the reaction was stopped.

<3-3> PCR-DGGE Analysis

PCR fragments prepared as above were analyzed via DGGE. The gel was prepared to continuously form a concentration gradient from 20% to 50% of urea and formamide as a modifier on 7.5% polyacrylamide gel. Electrophoresis was performed for 16 hours at a voltage of 60 volts for the touchdown PCR product in the gel prepared as above. In order to analyze the sequencing of the bands of the PCR product shown by staining the gel with SYBR Green I (Promega Co. Madison, USA), each band was separated from the gel and immersed in 10 μl of sterile water overnight. Extracted. The extracted PCR products were subjected to touchdown PCR once again to amplify the fragments, and the fragments were then analyzed for nucleic acid sequences using an ABI automated sequencer (Perkin Elmer Applied Biosystems. Foster city, California, USA). . Homology using the databank of NCBI (http://www.ncbi.nlm.nih.gov) and RDP (http://rdp.cme.msu.edu/html/) for the analyzed sequences The search confirmed that each band is a 16S rRNA gene derived from which microorganism.

As a result, 16S rRNA gene region bands of various lactic acid bacteria appear depending on the fermentation period, and when the kimchi was prepared at 4 ° C, it was confirmed that the lactic acid bacteria were variously changed. In particular, bisella corriensis appeared continuously during the fermentation of kimchi from the beginning of fermentation to 60 days of fermentation, and during that period, it was confirmed that it had a significant influence on the flavor of kimchi ( Fig. 2 ).

Example 4 Preparation of Low Temperature Fermented Kimchi

A low-temperature fermented kimchi was prepared using a starter strain of Bisella corriensis strain prepared in Example 2 above. Specifically, on the basis of the weight of cabbage 100g onions by weight ratio 3.0%, scallion 1.5%, garlic 1.5%, red pepper powder 1.5%, ginger 1.0% and refined salt (Korean industry, flower salt) so that the final salt concentration of 2% Used. At this time, the cabbage was cut into 3 cm in length to produce the cabbage kimchi, as a starter strain for fermentation Kimchi 1 kg immersed in a bisella Corriensis strain (10 ⁹ ~ ^{10 10} cells / ㎖) sufficiently cultured in MRS medium 10 ml of sugar was added to make 10 ^{6-10 7} cells / g kimchi, and the fermentation temperature of kimchi was maintained at 4 ° C.

<4-1> Sensory effect of low temperature fermented kimchi

The degree of kimchi ripening was determined by measuring the pH of the kimchi sample at the same time taste. pH was measured using a pH meter (Fisher Science, Model 25, USA) after taking 20 ml of kimchi samples.

Sensory testing was performed by a five-point scale (5: very good, 4: good, 3: normal, 2: bad, 1: very bad). 8 people).

As a result, in the case of kimchi using the Bisella Corriensis strain as a starter, the sensory test result was much better than the control group without the starter ( Table 3 ). In addition, the kimchi fermentation period can be efficiently shortened during fermentation of kimchi at low temperature, and after fermentation, an appropriate pH was maintained like kimchi of the control, and rancidity was also delayed ( FIG. 3 ).

Sensory Test of Kimchi Added with Starter Fermentation period (days) Bisella Coriensis Non-Added Kimchi Bicella Coriensis Addition Kimchi 0 3.40 3.40 5 3.70 3.90 10 3.95 4.15 15 4.05 4.20 20 4.10 4.25 25 4.15 4.20 30 3.90 4.10 35 3.65 4.00 40 3.20 3.90 45 2.60 3.70 50 2.40 3.00 55 2.15 2.50 60 2.05 2.10

As described above, the bisella corriensis strain of the present invention is a predominant strain in low-temperature kimchi fermentation when the strain is used as a starter strain of kimchi fermentation to reduce the maturation period of kimchi and to prepare a kimchi having a good flavor The reproducibility is imparted to the kimchi, and since the rancidity of kimchi can be delayed and the flavor can be maintained for a long time, it can be usefully used for producing high quality kimchi.

<110> Korea Research Institute of Bioscience and Biotechnology <120> A novel strain of Weisella, Weisella koreensis, and method for preparation of kimchi using this strain <130> 2p-12-25 <160> 5 <170> KopatentIn 1.71 <210 > 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 27f primer <400> 1 agagtttgat cctggctcag 20 <210> 2 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> 1492r primer <400> 2 tgactgactg aggytacctt gttacgactt 30 <210> 3 <211> 1491 <212> DNA <213> Weissella koreensis <400> 3 tggcggcgtg cctaatacat gcaagtcgaa cgcattgtgg ttgaaatgat atgaagaact 60 tgttcgtgtgtggtgtgt taacatttgg aaacaaatgc taataccgta taataattaa 180 aaccgcatgg ttttagttta aaagatggtc ttgctatcac taagagatgg tcccgcggtg 240 tattagttag t tggtgaggt aatggctcac caagacaatg atacatagcc gagttgagag 300 actgaccggc cacaatggga ctgagacacg gcccatactc ctacgggagg cagcagtagg 360 gaatcttcca caatggacga aagtctgatg gagcaacgcc gcgtgtgtga tgaagggttt 420 cggctcgtaa aacactgttg taagagaaga atgacattga gagtaactgt tcaatgtgtg 480 acggtatctt accagaaagg aacggctaaa tacgtgccag cagccgcggt aatacgtatg 540 ttccaagcgt tatccggatt tattgggcgt aaagcgagcg cagacggtta tttaagtctg 600 aagtgaaagc cctcggctca accgaggaat tgctttggaa actggataac ttgagtgcag 660 tagaggaaag tggaactcca tgtgtagcgg tgaaatgcgt agatatatgg aagaacacca 720 gtggcgaagg cggctttctg gactgtaact gacgttgagg ctcgaaagtg tgggtagcaa 780 acaggattag ataccctggt agtccacact gtaaacgatg agtgctagtt gttcgagggt 840 ttccgccctt gagtgacgaa gctaacgcat taagcactcc gcctggggag tacgaccgca 900 aggttgaaac tcaaaggaat tgacggggac ccgcacaagc ggtggagcat gtggtttaat 960 tcgaagcaac gcgaagaacc ttaccaggtc ttgacatcct ttgaccactc cagagatgga 1020 gctttccctt cggggacaaa gtgacaggtg gtgcatggtt gtcatcagct cgtgtcgtga 1080 gatgttgggt taagtcccgc aacgagcgca acccttattg ttagttgcca gcatttagtt 1140 gggcactcta gcaagactgc cggtgacaaa ccggaggaag gcggggatga cgtcaaatca 1200 tcatgcccct tatgacctgg gctacacacg tgctacaatg gcaagtacaa cgagtcgcca 1260 acccgcgagg gtgcgcaaat ctcttaaagc ttgtctcagt tcggactgta ggctgcaact 1320 cgcctacacg aagtcggaat cgctagtaat cgcggatcag cacgccgcgg tgaatacgtt 1380 cccgggtctt gtacacaccg cccgtcacac catgagagtt tgtaacaccc aaagtcggtg 1440 aggtaacctt ttattaggag ccagccgcnt aaggtggaca gatgattagg g 1491 <210> 4 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> gc338f primer <400> 4 cgcccgccgc gcgcggcggg cggggcgggg gcacgggggg actcctacgg gaggcagcag 60 60 <210> 5 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> 518r primer <400> 5 attaccgcgg ctgctgg 17

Claims (4)

Weissella koreensis (Accession No .: KCTC 10400BP) strain. Kimchi production method characterized by inoculating the strain of claim 1 with the starter strain of kimchi fermentation. 3. The method of claim 2, characterized in that the strain so that the inoculated kimchi 110 ^{4-10 7} cells per g. According to claim 2 or 3, characterized in that the kimchi is aged at 0 to 10 ℃ after inoculation of the strain.