KR20140087518A - New lactic acid bacteria having GABA-producing activity and composition comprising a cultured broth of the strain - Google Patents

Abstract

The present invention relates to a novel Lactobacillus strain having GABA-producing activity and a composition containing a fermented culture thereof and, more specifically, to a strain of Lactobacillus plantarum DSR GB150 (KCCM11343P) having a high GABA-producing activity, a fermented culture obtained by fermenting the strain, and a composition containing the fermented culture. The strain of Lactobacillus plantarum DSR GB150 (KCCM11343P) which is the novel Lactobacillus strain according to the present invention has excellent GABA-producing activity. Therefore, the fermented culture obtained by fermenting the strain can be practically used for a composition for food additives such as seasoning liquid for salted foods, a probiotic composition, a medicine for intestinal disorders, a feed composition, a cosmetic composition, etc.

Description

A novel lactic acid bacterial strain having a GABA production activity and a composition containing the fermentation culture,

The present invention relates to a novel lactic acid bacterial strain having a GABA production activity and a composition comprising the fermentation culture, and more particularly to a lactobacillus plantarum DSR GB150 (KCCM11343P) strain having high GABA production activity, And a culture containing the fermentation culture.

Gamma-aminobutyric acid (GABA) is a nonprotein constituent amino acid that is contained in the nervous system and blood in humans, and most of it is present in the bone marrow of the brain, thereby increasing neurotransmitters called acetylcholine, And physiological functions such as promoting the function.

GABA has not only a neurotransmitter, brain function, but also various other physiological activities. It has been known for a long time that it has blood pressure lowering action, diuretic effect and the like, and it is also registered as a medicine for stroke.

There is a report that lactic acid bacteria isolated from GABA-containing tea, rice and soybean fermented food, kimchi produce high concentration of GABA. In fact, in Japan, lactic acid bacteria isolated from kimchi are used to produce high concentration of GABA.

In addition, Japanese Soju from Lactobacillus brevis (Lactobacillus brevi ) to produce GABA directly. These effects of preventing hypertension and promoting brain function are expected to accelerate the industrial application of GABA at a time when the interest in health continues to increase

As a method for increasing the content of GABA in food, there is a method of increasing the content of GABA by treating tea leaves under anaerobic conditions, such as gabaron (tea), which is commercially available, or a method of immersing the embryo of rice in water , A significant increase in GABA and free amino acids has been developed.

Foods enriched with GABA using microbial fermentation methods have been developed. For example, a method of obtaining high concentration of GABA by inoculating two kinds of lactic acid bacteria, a method of producing glutamic acid as a starting material of GABA by treating dairy products with a protease, A method of fermenting lactic acid, a method of using lactic acid bacteria isolated from kimchi, a method of using lactic acid bacteria isolated from cruciferous sushi, a method of producing GABA-added soybean (soybean) using lactic acid bacteria having glutamic acid decarboxylase Is being reported.

There are several kinds of lactic acid bacteria in kimchi. Although there are reports of antimicrobial substances in these lactic acid bacteria, they are concentrated on the antibacterial effect, and there is no report on lactic acid bacteria having GABA production activity among kimchi - derived lactic acid bacteria.

Therefore, in order to solve the limitation of the GABA production ability so far, it is urgently required to search for a new microorganism having a high GABA production ability and to study the substance produced by the microorganism.

The inventors of the present invention have found that lactic acid bacteria isolated from kimchi containing a large amount of useful microorganisms have high GABA production activity while studying to overcome the problems of the prior art as described above, and completed the present invention.

Therefore, an object of the present invention is to provide a novel lactic acid bacteria having high GABA production activity.

Another object of the present invention is to provide a fermentation culture obtained by fermenting the above-mentioned lactic acid bacteria.

It is still another object of the present invention to provide a composition containing the culture obtained above.

In order to achieve the above object, the present invention provides Lactobacillus plantarum DSR GB150 (KCCM11343P) strain having GABA (gamma -aminobutyric acid) production activity.

The present invention also provides a fermentation culture obtained by fermenting the Lactobacillus plantarum DSR GB150 (KCCM11343P) strain.

The present invention also provides a composition containing said fermentation culture.

Since the lactobacillus plantarum DSR GB150 (KCCM11343P) strain of the novel lactic acid bacterium according to the present invention has excellent GABA production activity, the fermentation culture obtained by fermenting the fermented culture can be used as a food additive composition such as a seasoning for pickled foods, , A dressing agent, a feed composition, a cosmetic composition and the like.

Figures 1A-1D are photographs showing the Lactobacillus plantarum DSR GB150 (KCCM11343P) strain of the present invention and the known The results of analysis of the homology of 16S rRNA of Lactobacillus plantarum (GenBank accession No. HF562939) are shown.

One aspect of the present invention relates to a Lactobacillus plantarum DSR GB150 (KCCM11343P) strain having GABA (gamma -aminobutyric acid) production activity.

The strain according to the present invention has the 16S rRNA base sequence of SEQ ID NO: 1 and has 99% homology with 16S rRNA of Lactobacillus flutarium (GenBank Accession No. HF562939).

The strain according to the present invention has a GABA production activity of 300 mg / ml to 450 mg / ml, preferably 350 mg / ml to 400 mg / ml.

      The strain according to the present invention may be lactic acid bacteria isolated from kimchi, but the source of the isolate is not limited thereto.

Another aspect of the invention relates to a fermentation culture obtained by fermenting the Lactobacillus plantarum DSR GB150 (KCCM11343P) strain.

In the present invention, the term "fermented product" refers to a fermentation broth obtained by fermenting the strain, or a fermentation broth in which the broth component is removed by subjecting the fermentation broth to solid-liquid separation or a supernatant of the broth and the fermentation broth from which the strain has been removed do.

Another aspect of the present invention relates to a composition containing a fermentation culture obtained by fermenting the Lactobacillus plantarum DSR GB150 (KCCM11343P) strain.

The composition according to the present invention may be a probiotics composition.

In the present invention, the term " probiotic agent "means a live lactic acid bacterium for improving the disease resistance in humans through the enhancement of resistance to harmful microorganisms, or for feeding livestock to improve the productivity of livestock.

The composition according to the present invention may be a food additive composition, and the food additive composition may be a seasoning liquid for pickled foods such as bamboo shoots, radishes, pickles, and the like.

The composition according to the present invention may be a pharmaceutical composition for dressing.

In the present invention, the term "dressing" means to be used for cleansing the intestines and improving the intestinal function by, for example, killing intestinal bacteria and beneficial bacteria.

The pharmaceutical composition may be prepared in conventional formulations such as tablets, pills, capsules, and the like.

In the present invention, the composition may be a feed composition.

The feed composition is fed to the livestock to improve the productivity of the livestock through the enhancement of resistance to harmful microorganisms. The feed composition can be prepared by adding the fermentation culture of the present invention to a common feed.

The composition according to the present invention may be a cosmetic composition.

The cosmetic composition is preferably a formulation such as lotion, cream, gel and the like, but is not limited thereto.

Hereinafter, the present invention will be described in more detail by way of examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1 Isolation and Identification of Kimchi Lactic Acid Bacteria

Lactic acid bacteria were isolated from kimchi prepared by conventional kimchi manufacturing method.

Kimchi samples were diluted 10 times with 0.85% saline, and 0.1 ml each of MRS agar medium (MRS agar, Difco .; 10 g of bovine peptone, 10 g of beef extract, 5 g of yeast extract, 20 g of glucose, g, citric acid 2 g, potassium dibasic 2 g, sodium acetate 5 g, manganese sulfate 0.1 g, magnesium sulfate 0.05 g, agar 15 g / distilled water 1 l).

Plates were then incubated for 1 day in a 37 < 0 > C incubator. Each of the resulting colonies was inoculated on MRS agar plates and cultured at 37 ° C for 1 day to isolate lactic acid bacteria colonies.

Example 2: Isolation of GABA producing strains

Seventeen kinds of lactic acid bacteria isolated from kimchi according to Example 1 were cultured in MRS medium supplemented with 1% L-glutamic acid for 48 hours, followed by primary screening by TLC (thin layer cgromatography).

The MRS medium was MRS broth, Difco .; 10 g of bovine peptone, 10 g of beef extract, 5 g of yeast extract, 20 g of glucose, 80 g of tween, 2 g of citric acid, 2 g of dibasic potassium phosphate, 5 g of sodium acetate, 0.1 g of manganese sulfate, 15 g of agar / 1 l of distilled water.

Silica gel 60 (Merk) was used for the TLC, and the developing solvent was a mixture of butanol, acetic acid, and water at a ratio of 4: 1: 1. 2% ninhydrin reagent (Sigma) was used for color development. GABA-producing strains were isolated by selecting samples having the same Rf value as those of GABA standard reagents.

As a result, six candidate strains were selected. The cultures of the selected lactic acid bacteria were filtered with a 0.45um syringe filter, derivatized with AccQ-Tag method, and analyzed under linear gradient conditions. At this time, as the HPLC column, Nova-Pak 占 C18, 4 占 퐉 column was used. HPLC analysis showed that Lactobacillus plantarum DSR GB150 (KCCM11343P) strain produced the most GABA (see Table 1).

division DSR 121 DSR 122 GB150 (KCCM11343P) DSR 124 DSR 125 DSR 126 GABA content
(mg / ml) 141.3 176.5 389.1 157.8 98.2 239.4

Example 3 Morphological and Biochemical Characterization of Selected Lactic Acid Bacteria

a. Morphological characteristics

Using a microscope, the morphological characteristics of the strain identified above were observed. The isolated lactic acid bacteria were Gram - positive bacteria and had rod shape. The colonies were light yellow opaque and the surface of the colonies was smooth (Table 2).

Morphological and biochemical characteristics of lactic acid bacteria according to the present invention characteristic Lactobacillus Flora Room DSR GB150 Gram + Colony form Rod Colony color yellow Motility - Catalase (Catalase) - Fermentation form homo Spore - Growth at 16 ℃ + Growth in 4% NaCl +

b. Biochemical property

The isolated strain was identified by an API system (La Balme-les-Grottes, France). First, the sterilized platinum-fungus colonies were each taken and suspended in 2 ml of sterilized distilled water.

The bacterial solution was suspended in 5 ml of sterilized distilled water at a concentration of MccFaland Standard Solution No. 2 supplied by API 50CH kit (BioMerieux, France). The supernatant was added to the liquid medium of the API 50CH kit and homogenized, and 200 μl of each of the 50 tubes of the API 50CH kit was inoculated. The tube was overlaid with mineral oil and then cultured at 30 DEG C for 48 hours.

The culture solution was analyzed by an API system to identify 49 types of carbohydrate fermentation patterns, and the results were entered into an ATB identification computer system. As a result, DSR GB150 (KCCM11343P) was found to have a carbohydrate fermentation pattern of the following Table 3 (result of analysis of carbohydrate fermentation pattern of Lactobacillus plantarum GB150 (KCCM11343P) strain according to the present invention) It was identified.

The identified strain was named "Lactobacillus plantarum DSR GB150 ", and deposited on December 20, 2012 as KCCM 11343P at the Korean Society for Microbiological Care.

Example 4: Investigation of genetic characteristics of selected lactic acid bacteria

The base sequence of the 16S rRNA gene for the identification and classification of the lactic acid bacteria strain of the present invention was analyzed. Genomic DNA was isolated and the 16S ribosomal DNA fragment was amplified and the amplified DNA fragment was confirmed by electrophoresis.

DNA fragments were purified by Qiagen PCR purification kit (Giagen, Hilden, Germany) and mixed with a reaction solution containing d-Rhodamine dye-labeled dd-NTP and subjected to sequencing PCR. The obtained DNA was subjected to ethanol / sodium acetate precipitation Lt; / RTI >

The purified DNA was dissolved in Template Suppression Reagent (TSR) and analyzed with ABI prism 310 Genetic Analyzer (PE Applied Biosystems, USA). The analyzed base sequence was Genebank (http://www.ncbi.nlm.nih.gov/) Accession No. HF562939.

As shown in FIG. 1, the 16S rRNA nucleotide sequence (SEQ ID NO: 1) of the lactic acid bacteria of the present invention showed the highest homology (99%) with Lactobacillus plantarum.

<Preparation Example 1> Production of GABA-producing seasoning liquid using fermentation broth of lactic acid bacteria

5% of fructose, 0.5% of yeast extract, 1% of sodium citrate, 0.5% of sodium acetate, and 0.5% of MgSO ₄ were added to purified water 90.945% 0.005%, MnSO ₄ 0.05%, and then GABA-producing lactic acid bacteria GB150 (KCCM11343P) was inoculated at 2% and fermented for 48 hours.

Enzymatic treatment of stevia with 0.15%, vinegar with 5.3%, citric acid with 0.625%, salt with 2.75% and grapefruit seed extract with 0.075% was completed in 91.1% of the fermentation broth.

Table 2 shows the results of comparing the GABA content of the seasoning solution with commercially available samsam using HPLC in the same manner as in Example 2. [

division Company A Company B Invention GABA content (mg / ml) - - 52.1

As shown in Table 4, it was confirmed that GABA was produced at 52.1 mg / ml only in the fermented samsum supplemented with GABA producing strains.

Preparation Example 2 Preparation of a Probiotic Compound Using GABA-Producing Lactic Acid Fermentation Liquid

2% of the plant lactobacillus Lactobacillus plantarum DSR GB150 (KCCM11343P) of the present invention was inoculated into a medium containing 2.0% glucose, 1.0% peptone, 1.0% yeast extract, 0.2% ferric phosphate, 0.05% magnesium sulfate and 0.05% cysteine And cultured aerobically for 2 days at 37 ° C.

The culture broth of each of the above strains was inoculated at 10% by weight in a solid culture medium in which a defatted steel, soybean meal, corn and molasses were mixed at a weight ratio of 3: 1: 1: 1, Lt; / RTI &gt; The fermentation broth composition was prepared by anaerobically fermenting the fermentation broth with stirring at 40 ° C for 3 days. The broth composition prepared herein contained 1 × 10 ⁹ cfu / g or more of the lactic acid bacteria of the present invention.

<Preparation Example 3> Preparation of pharmaceutical composition using GABA-producing lactic acid fermentation broth

Each of the powders of the viable composition obtained in Preparation Example 2 was individually molded into tablets, pills and capsules according to a conventional method to prepare a pharmaceutical composition for correction. The composition prepared herein had a lactic acid bacterium of the present invention having a concentration of 1 × 10 ⁹ cfu / g or more .

<Preparation Example 4> Preparation of feed composition using GABA-producing lactic acid fermentation broth

Lactobacillus plantaium Lactobacillus plantarium of the present invention was inoculated into a medium containing 2.0% glucose, 1.0% peptone, 1.0% yeast extract, 0.2% ferric phosphate, 0.05% magnesium sulfate and 0.05% cysteine at 37 ° C for 2 days Were cultivated aerobically. The culture medium of each strain and the excipient degreasing solution were simply mixed at a weight ratio of 1: 1, followed by drying at 40 ° C or lower, and the dried mixture was pulverized to prepare a feed composition. At this time, the prepared feed composition contained the lactic acid bacteria of the present invention at a concentration of 1 × 10 ⁹ cfu / g or more.

PREPARATION EXAMPLE 5 Preparation of Cosmetic Composition Using GABA-Producing Lactic Acid Fermentation Liquid

The compositions of Table 5 below were weighed out in order and mixed uniformly. The remaining amount was filled with purified water to prepare a softened longevity (skin) containing the lactic acid fermentation supernatant prepared according to the examples.

Softening longevity weight(%) Purified water Balance glycerin 2 1,3-butylene glycol 2 Citric acid 0.01 ethanol 10 antiseptic 0.0001 Pigment 0.0001 Spices 0.0001 Lactic acid fermentation broth 10

Since the lactobacillus plantarum DSR GB150 (KCCM11343P) strain of the novel lactic acid bacterium according to the present invention has excellent GABA production activity, the fermentation culture obtained by fermenting the fermented culture can be used as a food additive composition such as a seasoning for pickled foods, , A dressing agent, a feed composition, a cosmetic composition and the like.

Korea Microorganism Conservation Center (overseas) KCCM11343P 20121220

&Lt; 110 > Daesang FNF Co., Ltd. <120> New lactic acid bacteria having GABA-producing activity and          composition of a cultured broth of the strain <160> 1 <170> Kopatentin 1.71 <210> 1 <211> 1358 <212> DNA <213> Lactobacillus plantarum <220> <221> gene &Lt; 222 > (1) .. 1358 <223> 16S rRNA <400> 1 gaactggtga gtaacacgtg ggaaacctgc ccagaagcgg gggataacac ctggaaacag 60 atgctaatac cgcataacaa cttggaccgc atggtccgag cttgaaagat ggcttcggct 120 atcacttttg gatggtcccg cggcgtatta gctagatggt ggggtaacgg ctcaccatgg 180 caatgatacg tagccgacct gagagggtaa tcggccacat tgggactgag acacggccca 240 aactcctacg ggaggcagca gtagggaatc ttccacaatg gacgaaagtc tgatggagca 300 acgccgcgtg agtgaagaag ggtttcggct cgtaaaactc tgttgttaaa gaagaacata 360 tctgagagta actgttcagg tattgacggt atttaaccag aaagccacgg ctaactacgt 420 gccagcagcc gcggtaatac gtaggtggca agcgttgtcc ggatttattg ggcgtaaagc 480 gagcgcaggc ggttttttaa gtctgatgtg aaagccttcg gctcaaccga agaagtgcat 540 cggaaactgg gaaacttgag tgcagaagag gacagtggaa ctccatgtgt agcggtgaaa 600 tgcgtagata tatggaagaa caccagtggc gaaggcggct gtctggtctg taactgacgc 660 tgaggctcga aagtatgggt agcaaacagg attagatacc ctggtagtcc ataccgtaaa 720 cgatgaatgc taagtgttgg agggtttccg cccttcagtg ctgcagctaa cgcattaagc 780 attccgcctg gggagtacgg ccgcaaggct gaaactcaaa ggaattgacg ggggcccgca 840 caagcggtgg agcatgtggt ttaattcgaa gctacgcgaa gaaccttacc aggtcttgac 900 atactatgca aatctaagag attagacgtt cccttcgggg acatggatac aggtggtgca 960 tggttgtcgt cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga gcgcaaccct 1020 tattatcagt tgccagcatt aagttgggca ctctggtgag actgccggtg acaaaccgga 1080 ggaaggtggg gatgacgtca aatcatcatg ccccttatga cctgggctac acacgtgcta 1140 caatggatgg tacaacgagt tgcgaactcg cgagagtaag ctaatctctt aaagccattc 1200 tcagttcgga ttgtaggctg caactcgcct acatgaagtc ggaatcgcta gtaatcgcgg 1260 atcagcatgc cgcggtgaat acgttcccgg gccttgtaca caccgcccgt cacaccatga 1320 gagtttgtaa cacccaaagt cggtggggta acctttta 1358

Claims (12)

Lactobacillus plantarum DSR GB150 (KCCM11343P) strain having GABA (γ-aminobutyric acid) production activity. 2. The strain of claim 1, wherein the strain has the 16S rRNA nucleotide sequence of SEQ ID NO: 1. The strain according to claim 1, wherein the strain has a GABA production activity of 300 mg / ml to 450 mg / ml. The strain according to claim 1, wherein the strain is isolated from kimchi. A fermentation culture obtained by fermenting a strain of Lactobacillus plantarum DSR GB150 (KCCM11343P) according to any one of claims 1 to 5. A composition containing the fermentation culture of claim 5. 7. The composition of claim 6, wherein the composition is a biocide composition. The composition according to claim 6, wherein the composition is a composition for food addition. The composition according to claim 8, wherein the composition for food addition is a seasoning for food. 7. The composition of claim 6, wherein the composition is a pharmaceutical composition for dressing. 7. The composition of claim 6, wherein the composition is a feed composition. 7. The composition of claim 6, wherein the composition is a cosmetic composition.

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