KR20150138746A - Lactobacillus brevis llb5238 strain producing γ-aminobutyric acid and method for manufacturing γ-aminobutyric caid by the same - Google Patents

Abstract

The present invention relates to a novel Lactobacillus brevis LLB5238 (accession no. KCCM11538P) strain separated from kimchi having excellent ability to produce gamma-aminobutyric acid (GABA). The Lactobacillus brevis LLB5238 strain has excellent ability to produce GABA from a material such as glutamic acid, monosodium glutamate (MSG), a kelp extract, etc, thereby efficiently providing GABA useful for stressed modern people, examinees, people who drink excessive alcohol, etc.

Description

Lactobacillus brevis LLB 5238 strain having gamma aminobutyric acid production ability and production method of gamma aminobutyric acid using the same Lactobacillus brevis LLB 5238 strain producing γ-aminobutyric acid and method for manufacturing γ-aminobutyric acid by the same method,

The present invention relates to a strain of Lactobacillus brevis LLB5238 (accession number KCCM11538P) having gamma-aminobutyric acid (GABA) production ability and use thereof. Specifically, the strain according to the present invention is a strain producing glutamic acid decarboxylase (GAD), which is obtained by decarboxylating glutamic acid to produce gamma-aminobutyric acid Wherein the conversion rate of gamma aminobutyric acid is 95% or more, and a method for producing gamma aminobutyric acid using the same.

Gamma aminobutyric acid (GABA) is an inhibitory neurotransmitter, which is known to suppress blood pressure rise, improve visual acuity, and calm anxiety and nervousness. In animals, it is distributed in the brain, heart, lung, kidney, etc. In the case of plants, it is mainly found in germinated brown rice and green tea. It is reported that modern people who are stressed, examinees, and overeating alcohol have low brain and blood GABA levels, and the extreme shortage of GABA concentration in the body is known to cause seizures, convulsions, and epilepsy. Efforts to develop foods or medicines for increasing GABA concentration in the body have been demanded.

Studies have been actively conducted to increase the content of GABA naturally in various materials in accordance with such interest. Korean Laid-Open Patent Application No. 2009-0123070 discloses a method of inoculating and cultivating a medium containing glutamic acid as a main raw material Followed by culturing to increase the content of the Gabba component and the organic acid.

Also, green tea, GABA-containing rice embryo and germinated brown rice, which have increased GABA content, are produced by allowing fresh green leaves of green tea to stand in N 2 gas for 6 to 8 hours or immersing rice embryos and brown rice at constant temperature have.

However, the GABA content in the product manufactured by this method is not soluble in the form of the insoluble form and the problem that the product can not exceed 0.5% (w / w) based on the weight of the product is a limit for the development of the product using GABA .

Recently, in Japan, MSG (Monosodium glutamate, MSG), which is added to the substrate using lactic acid bacteria, has been converted to GABA by using decarboxylase (GAD), and then GABA accumulated in the culture solution A product was recovered and concentrated. Thus, GABA produced by using lactic acid bacteria can produce high concentration products according to the post-fermentation process, and it is possible to produce products that are completely dissolved.

On the other hand, the GABA production ability of the strain is determined by the conversion of glutamic acid to GABA by adding glutamic acid, which is a substrate of glutamic acid decarboxylase (GAD), to the medium and culturing.

In addition, it is predicted that the GABA production ability of the strain may greatly vary depending on the degree of activation of GAD possessed by the strain. Therefore, in order to produce GABA at a high concentration, it is necessary to obtain a strain having a high activity of GAD. Unfortunately, in Korea, there is very little case of developing a lactic acid bacteria producing GABA at a high concentration so far.

Therefore, the present inventors have studied a method for producing GABA, and have succeeded in isolating new strains which have been confirmed to produce GABA, thereby completing the present invention.

An object of the present invention is to provide a strain of Lactobacillus brevis LLB5238 (accession number KCCM11538P) having gamma-aminobutyric acid (GABA) production ability.

Another object of the present invention is to provide a method for producing gamma aminobutyric acid using the novel Lactobacillus brevis LLB5238 (Accession No. KCCM11538P) strain.

It is yet another object of the present invention to provide a food composition comprising a strain of Lactobacillus brevis LLB5238 (accession number KCCM11538P).

In order to achieve the above object, the present invention provides Lactobacillus brevis LLB5238 (Accession No. KCCM11538P) having gamma-aminobutyric acid (GABA) production ability.

The novel Lactobacillus brevis LLB5238 (Accession No. KCCM11538P) strain according to the present invention can be isolated from kimchi. From the kimchi, the strain is identified and belongs to Lactobacillus brevis, which is a kind of lactic acid bacteria.

(A) culturing a strain of Lactobacillus brevis LLB5238 (accession number KCCM11538P); And (b) adding the culture medium to a medium containing glutamic acid and fermenting the same to produce gamma-aminobutyric acid (GABA).

Glutamic acid decarboxylase (GAD) produced by the novel strain is converted into gamma aminobutyric acid by using glutamic acid contained in the medium as a substrate to provide gamma aminobutyric acid.

More specifically, the medium of step (a) may be a medium in which Lactobacillus brevis LLB 5238 strain can grow well. Typically, commercially available MRS medium may be used, but the present invention is not limited thereto.

At this time, the culturing conditions are preferably carried out at 25 to 42 ° C for 18 to 36 hours. When the incubation temperature is lower than 25 ° C and higher than 42 ° C, there is a problem that the growth of the LLB 5238 strain of the present invention is slower than the growth in the range of 25 to 42 ° C.

When the incubation time is less than 18 hours, the LLB 5238 strain of the present invention fails to grow sufficiently, and since the activity (microbial strain activity) of the LLB 5238 strain gradually decreases at the highest level for more than 36 hours, It is better to do it within.

The basic composition of the culture medium of step (a) or (b) was 10 g / L of proteose peptone, 10 g / L of beef extract, 5 g / L of yeast extract, 20 g / L of Dextrose, 1 g / L of polysorbate- / L, sodium acetate 5 g / L, magnesium sulfate 0.1 g / L, manganese sulfate 0.05 g / L, and dipotassium phosphate 2 g / L.

The medium may be typically a commercially available MRS medium. The medium may include monosodium glutamate (MSG) or sea tangle extract, which is a material of glutamic acid, and is not limited as long as it contains glutamic acid.

At this time, when glutamic acid is contained, it is preferable that it contains 1 to 10 wt% with respect to the total weight of the culture medium. When monosodium glutamate (MSG) or sea tangle extract or a mixture thereof is contained, it is preferable that it contains 2 to 5% by weight based on the total weight of the medium.

The culture medium of step (a) is preferably added in an amount of 0.5 to 2% by volume based on the total volume of the culture medium. When the amount is less than 0.5% by volume, there is a problem that the growth of the LLB 5238 strain may be delayed somewhat, and when the amount is more than 2% by volume, the seeds may be overdosed.

In addition, the culturing conditions are preferably 24 to 72 hours at 24 to 42 ° C. When the incubation temperature is lower than 25 ° C and higher than 42 ° C, there is a problem that the growth of the LLB 5238 strain of the present invention is slower than the growth in the range of 25 to 42 ° C. Further, when the incubation time is less than 24 hours, the LAB 5238 strain of the present invention does not grow sufficiently and thus the GABA production ability is not sufficiently exhibited. In the case of over 72 hours, the GABA production amount approaches the maximum value, It is better to do it within.

The present invention also relates to a method for producing Lactobacillus brevis Lactobacillus brevis Lactobacillus brevis LLB 5238 (accession number KCCM11538P) having a gamma-aminobutyric acid (GABA) production ability, a culture of the strain, a concentrate of the culture solution, As an active ingredient, at least one selected from the group consisting of < RTI ID = 0.0 >

The fermented food may be prepared by using the strain as a fermentation seed. The food can be variously applied in the form of fermented food or fermented beverage. It can also be used as various food additives or medical physiologically active substances.

In the present invention, the term "medium" means a solid or a liquid containing nutrients necessary for growing animal cells, plant cells or microorganisms. The term "culture liquid" means a culture obtained by inoculating a strain into a liquid medium, and includes a culture filtrate which refers to a supernatant obtained by removing a strain from a liquid medium. The term " concentrated liquid of the culture liquid " means a liquid concentrate of the culture liquid, and the term " dried material of the culture liquid " means that the liquid is removed from the culture liquid.

The present invention relates to a method for producing Lactobacillus brevis LLB5238 (accession number KCCM11538P) having gamma-aminobutyric acid (GABA) production ability and gamma-aminobutyric acid (GABA) using the strain And a food composition comprising the strain. Therefore, it is very useful for people with low GABA concentration, such as modern people who are stressed, examinees, alcohol-overdose.

Fig. 1 shows a strain having GABA production ability by performing thin layer chromatography of Example 1-2. Fig.
Fig. 2 shows the result of measuring the sugar availability of Lactobacillus brevis LLB5238 strain.
Fig. 3 shows the result of sequence homology analysis of Lactobacillus brevis LLB5238.
4 is a graph showing measurement of GABA production and consequently glutamic acid conversion in MRS medium containing 1% by weight of glutamic acid using Lactobacillus brevis LLB5238 strain and Comparative strain strain.
5 shows the GABA production and consequently the conversion of glutamic acid in the MRS medium containing 2% by weight of monosodium L-glutamate (MSG) or sea tangle extract, using Lactobacillus brevis LLB5238 strain and Comparative strain strain As shown in FIG.

Hereinafter, the structure of the present invention will be described in more detail with reference to examples, but the scope of the present invention is not limited to the following examples.

Example

Materials and methods

1. Isolation of lactic acid bacteria from kimchi

1-1. Establishment of Kimchi Sample

A total of 150 kimchi samples were collected from traditional traditional markets all over the country. The Kimchi samples collected are shown in Table 1. All kimchi were collected in February and stored under the same storage conditions (4 ℃).

Separation area Seoul Gyeonggi-do Chungcheongnam-do Chung-cheong bukdo Jeollanam-do all Sampling sample 29 46 18 26 31 150

1-2. How to separate

Kimchi samples were diluted with sterilized distilled water and homogenized in a homogenizer using a homogenizer (Stomacher, Pro-Media SH-001, ELMEX). The diluted solution was diluted stepwise with sterile saline solution to obtain 0.1 ml of the sample. The diluted solution was applied to a MRS solid medium supplemented with 0.002% by weight of BCP (Bromocresol purple) and 1.5% by weight of agar And incubated for 48 hours in an incubator maintained at 37 ° C.

After cultivation, colonies showing yellow circles were selected and purified by subculturing 2-3 times in the same medium. The selected strains were inoculated into MRS liquid medium for each individual, and cultured at 37 ° C for 48 hours.

1-3. Securing seed culture

The selected strains were cultured in DifcoTMLactobacilli MRS solid medium (10 g / L of proteose peptone, 10 g / L of beef extract, 5 g / L of yeast extract, 20 g / L of Dextrose, 1 g / L of polysorbate 80, 2 g / L of ammonium citrate, L of magnesium sulfate, 0.1 g / L of magnesium sulfate, 0.05 g / L of manganese sulfate, 2 g / L of dipotassium phosphate, 15 g / L of Agar) and incubated at 37 ° C for 24 hours. Colonies were collected in an appropriate amount (2 to 3 colonies) and inoculated into DifcoTMLactobacilli MRS liquid medium (the agar was excluded from the MRS solid medium constituents) and cultured at 37 ° C for 24 hours to obtain a culture medium.

2. GABA Exploring the producing strains

2-1. Enzyme reaction

Each of the cultures of Examples 1-3 was added to 1 volume% of MRS liquid medium containing 1% by weight of glutamic acid (G1251, Sigma-Aldrich, USA) and fermented at 37 ° C for 48 hours. Followed by centrifugation (10,000 g, 10 min) to obtain a supernatant.

2-2. Thin layer  Chromatography ( thin layer chromatography , TLC ) Perform

Thin layer chromatography (TLC) was performed to search for strains producing GABA. The supernatant obtained in the above step was used as a sample for TLC development. As a solvent for TLC, mixed solvent of Butanol: Acetic acid: Distiled water = 4: 1: 1 was used and confirmed by Ninhydrin staining method. After heating, it was confirmed that a spot having the same Rf value was produced as compared with the standard material. The standard reagent used was Sigma's GABA standard, and the TLC development time was about 40 minutes. Some of these TLC progress results are shown in FIG. 1, and eight strains among the whole strains isolated from Kimchi through Example 1-2 were confirmed to have GABA converting ability and were selected as primary strain.

3. Ultra-fast liquid chromatography ( UPLC ) Perform

Glucamic acid (L-Glutamic acid, G1251, Sigma-Aldrich, USA) was added to the culture solution prepared in Example 1-3 using 8 strains selected first through Example 2-2 (MRS) medium supplemented with 10% by weight of the culture medium and incubated for 48 hours in a 37 ° C incubator. Then, the content of GABA was measured using UPLC (Acquity ™ Ultra Performance Liquid Chromatography, Waters, USA).

To measure the GABA content in the culture sample, the aqueous solution layer containing GABA was centrifuged (4 ° C, 12,000 g, 15 minutes) and recovered.

(AccQ-Tag ™ Ultra Derivatization Kit, Cat No. 186003836, Waters, USA) which is composed of ACQ (6-Aminoquinolyl-N-hydroxysuccimidyl carbamate) and acetonitrile and borate buffer for fluorescence derivatization of GABA ), And an Accq-Tag column (AccQ-Tag ™ Ultra C18, 2.1 mm x 100 mm, 1.7 μm, Waters, USA) was used for separation of these derivatives. The column temperature was 35 ° C, and was measured at 260 nm using a PDA detector (Photodiode detector, Waters, USA) for the detection of derivatized GABA.

The standard reagent was compared and calculated in comparison with Sigma's standard (γ-Aminobutyric acid, A2129, Sigma-Aldrich, USA). As a result, the strain producing the most GABA was finally selected as the strain according to the present invention. As shown in Table 2 below, it was confirmed that strain G-238 had the highest GABA conversion.

Strain G-17 G-55 G-61 G-109 G-238 G-272 G-276 G-294 Conversion Rate (%) 6.3 26.5 15.3 35.7 98.2 55.1 1.6 5.5

4. Final selection strain

The final selected strain (G-238) with the highest GABA conversion rate was inoculated on MRS liquid medium and cultured at 37 ° C for 48 hours. After completion of the culture, centrifugation (10,000 g, 10 min) Respectively. The obtained cells were added to 1 ml of a freezing solution (MRS liquid medium: Glycerol) in a ratio of 4: 1, and the mixture was placed in a cryo-tube and frozen at -70 ° C. Respectively. The frozen strain was used as a starter for subsequent testing.

5. Detached GABA  Identification of the producing strains

5-1. Identification of strains 1

The morphological and biochemical characteristics of the LLB 5238 strain were confirmed and shown in Table 3. As shown in Table 3, it can be confirmed that the LLB 5238 strain is negative for spore formation and catalase by Gram-positive, bacillus.

Morphology and biochemical characteristics reaction Colony Shape Small round shape Colony color Light Ivory Shape Rod Gram staining Gram positive Expiratory condition Flowable anaerobic Endospore voice Spore shape voice Catalase test voice

In addition, the sugar availability of LLB 5238 strain was analyzed using an API 50 CHL kit (Biomerieux, France). The results are shown in Tables 4 and 5 and FIG. 2 below.

NO. Strip 0-19
Tube / substrate +/- NO. Strips 20-39
Tube / substrate +/- NO. Strip 40-49
Tube / substrate +/- 0 Control group - 20 alpha -methyl-D-mannoside
(Mannoside) - 40 D-Turanos
(TURANose) + One  Glycerol - 21 alpha -methyl-D-glucoside - 41 D-lyros
(LYXose) - 2 Erythritol
(ERYthritol) - 22 N-acetyl-glucosamine + 42 D-tagatose
(TAGatose) - 3 D-arabinose
(Arabinose) - 23 Amigalline
(AMYgdalin) - 43 D-Fucos
(FUCose) - 4 L-arabinose + 24 ARBUTIN - 44 L-Fucos - 5 Ribose + 25 Esculin - 45 D-albitol
(ArabitoL) - 6 D-xylose
(XYLose) + 26 Salicin - 46 L-Alvitol - 7 L-xylose - 27 CELlobiose - 47 Gluconate
(GlucoNaTe) - 8 Adonitol
(ADOnitol) - 28 MALtose + 48 2-keto-gluconate - 9 beta -methyl-D-xylose - 29 Lactose (LACtose) - 49 5-keto-gluconate - 10 Galactose
(GALactose) - 30 MELibiose -




11 Glucose
(GLUcose) + 31 Sucrose -


12 Fructose
(FRUctose) + 32 TREHALOSE -




13 Manose - 33 Inulin (INUlin) -




14 Surobos
(SroBose) - 34 Meligitos
(MeLeZitose) -




15 Rams North
(RHAmnose)  - 32 RAFfinose -




16 DULcitol - 36 Starch -




17 Inositol
(INOsitol) - 37 Glycogen (GLYcoGen) -




18 MANITOL - 38 Xylitol (XyLiTol) -




19 Sorbitol - 39 Zentio Bios
(GENtiobiose) -

(GAL) 89%, Melibiose (MEL) 76%, D-Turanose (TUR) 1% and Gluconic acid (GNT) 87% were found to be identical with the existing Lactobacillus brevis Of the patients were found to be slightly different.

Identification result % T Index The opposite biochemical characteristics (Test against) Lactobacillus brevis 99.9 0.58 GAL 89% MEL 76% TUR 1% GNT 87%

5-2. Identification of strain 2

The 16S rRNA gene of the LLB 5238 strain was identified and sequenced. Genomic DNA was extracted from the isolated strain using a genomic DNA preparation kit (Promega Co., USA), and then a universal primer 27F (5'-AGA GTT TGA TCC TGG CTC AG-3 ) And 1492R (5'-TAC GGY TAC CTT GTT ACG ACT T-3 ') were amplified by Polymerase Chain Reaction (PCR).

The amplified product was purified using acetate and 70% ethanol, and then used as a template for sequencing primer 518F (5'-CCA GCA GCC GCG GTA ATA CG-3 '), 800R (5'-TAC CAG GGT ATC TAA The amplification product was purified with ethanol using an ABI PRISM 3730XL Analyzer (96 capillary type) with a base (TCC-3 ') and a sequencing kit (ABI PRISM BigDye ™ TerminatorCycleSequencingKits) The sequence was determined.

The 16S rRNA gene sequence of the novel strain is shown in SEQ ID NO: 1. The 16S rRNA gene sequences of these isolates were aligned with the sequences of the strains obtained from the EMBL / DDBJ / PDB / GenBank sequence databases and compared with their homologous sequences.

As a result, as shown in Table 6 and FIG. 3, it showed 97% homology with the strain of Lactobacillus brevis. Therefore, the present inventor named the LLB 5238 strain as Lactobacillus brevis LLB5238, and deposited it on May 21, 2014 with the Korean Society for Microbiological Care (KCCM) under accession number KCCM11538P.

Description Max score Total score Query cover E
value Ident Lactobacillus brevis strain p-18 16S ribosomal RNA gene, partial sequence 2049 2049 93% 0.0 97% Lactobacillus brevis strain NJ14 16S ribosomal RNA gene, partial sequence 2049 2049 94% 0.0 97% Lactobacillus brevis strain NJ-13 16S ribosomal RNA gene, partial sequence 2049 2049 93% 0.0 97% Lactobacillus brevis gene for 16S partial sequence, strain ZU31 2049 2049 93% 0.0 97% Lactobacillus brevis strain bh3 16S ribosomal RNA gene, partial sequence 2049 2049 94% 0.0 97% Lactobacillus brevis strain NSe1 16S ribosomal RNA gene, partial sequence 2047 2047 94% 0.0 97% Lactobacillus brevis strain CGMCC 1-2028 16S ribosomal RNA gene, partial sequence 2047 2047 94% 0.0 97% Lactobacillus brevis KB290 DNA, complete genome 2045 10193 93% 0.0 97% Lactobacillus brevis ATCC 367 strain ATCC 367, 16s ribosomal RNA complete sequence 2045 2045 93% 0.0 97% Lactobacillus brevis strain 877G 16S ribosomal RNA gene, partial sequence 2045 2045 93% 0.0 97% (Omitted below)

Experimental Example

One. Lactobacillus Brevis ( Lactobacillus brevis ) Of strain LLB5238 Acid resistance  And of mine Bile acid measurement

The acidity and bile acid tolerance of Lactobacillus brevis LLB5238 strain were investigated. This is one of the general characteristics of lactic acid bacteria, and it is possible to use GABA as a starting material In the case of the acid resistance test, Lactobacillus brevis LLB5238 strain was cultured as in Example 1-3, followed by inoculation in an MRS liquid medium at 1 volume% of the prepared culture broth and incubation at 37 ° C for 24 hours. The resulting culture was adjusted to pH 3.0 with 10% H2SO4 and left for 2 hours. After dilution of each sample, the diluted solution was spread on a MRS-BCP solid medium and cultured at 37 ° C for 24 hours. The number of viable cells after the initial viable cell count and pH 3.0 was measured. The acid resistance was expressed as a ratio of (number of viable cells / number of viable cells after pH treatment).

For the bile test, the strain was inoculated on a MRS-BCP solid medium supplemented with 0.5% bile (bile) and cultured at 37 ° C. for 24 hours.

As shown in Table 7 below, Comparative Examples 1 to 3 used strains obtained from Korean Biotechnology Research Institute (KCTC) for application for sale. The acid resistance and bile acid test were carried out in the same manner as in the examples.

division sample My biliary * Acid resistance (%) Example L. brevis LLB5238 + 55.6 Comparative Example 1 L. brevis KCTC3103 - 0.02 Comparative Example 2 L. brevis KCTC41028 - 71.5 Comparative Example 3 L. brevis KCTC41029 + 42.3 * +: Can grow / -: Can not grow

As shown in Table 7, it was confirmed that the Lactobacillus brevis LLB5238 strain and the KCTC41029 strain of the present invention were able to grow even when 0.5% bile was added in case of bile resistance.

In the case of acid resistance, it was confirmed that even when 1.0% bile was added to all the strains, growth was possible. In particular, LLB 5238 showed a survival rate of about 56% when treated at pH 3.0 for about 2 hours, which was confirmed to be similar to other standard strains.

Therefore, when GABA production is proceeded using Lactobacillus brevis LLB5238 strain of the present invention, in addition to the method of separately producing and purifying only GABA in the culture medium, it was confirmed that it could be provided as probiotics when the cells and GABA were also supplied.

2. Depending on culture conditions GABA Production capacity  Measure

Lactobacillus brevis LLB5238 strain was used to measure actual GABA production ability according to culture conditions.

Firstly, the culture solution prepared in the step of seed culture of the example was inoculated into the MRS liquid medium containing 1% by weight of L-glutamic acid, and cultured at 37 ° C for 72 hours. Based on the inoculation time, some of the samples were taken after 24 hours and analyzed for GABA production. In Comparative Examples 1 to 3, strains of Table 7 were used and the experiment was conducted under the same conditions. The GABA content in the culture medium was determined by UPLC, and the sample pretreatment and UPLC analysis were carried out in the same manner as in the above example.

The GABA conversion rates of the Lactobacillus brevis LLB5238 strain and the Comparative strain strains with respect to the concentration of 1 wt% of L-Glutamic acid according to fermentation culture time are shown in Table 8 and FIG.

Culture time (hours) Strain GABA conversion (%) 24 L. brevis LLB5238 97.3 L. brevis KCTC3103 0.16 L. brevis KCTC41028 0.00 L. brevis KCTC41029 0.00 48 L. brevis LLB5238 98.1 L. brevis KCTC3103 0.23 L. brevis KCTC41028 0.00 L. brevis KCTC41029 0.01 72 L. brevis LLB5238 99.4 L. brevis KCTC3103 0.25 L. brevis KCTC41028 0.01 L. brevis KCTC41029 0.01

As shown in Table 8 and FIG. 1, it was confirmed that 97% or more of L-Glutamic acid was converted into GABA by using Lactobacillus brevis LLB5238 strain according to the present invention after 24 hours of culture. On the other hand, as a comparative example, it was confirmed that the standard strains of Lactobacillus brevis were much lower than those of strain LLB5238.

3. From various materials GABA  production

Actual GABA production ability was measured from various glutamic acid materials such as MSG or kelp extract. When Lactobacillus brevis LLB5238 was applied to industrial scale, it was confirmed whether glutamic acid could be replaced by economical material instead of reagent grade L-Glutamic acid. Based on this, it was confirmed that high-concentration GABA production.

Lactobacillus brevis LLB5238 strain was cultured in the same manner as in the above example, and the MSG or kelp extract was inoculated into the MRS liquid medium containing 5 to 10 wt% of the extract, followed by culturing at 37 DEG C for 72 hours.

Based on the inoculation time, some of the samples were taken after 24 hours and analyzed for GABA production. The inoculation of the seed culture was 1 vol% and the culture was carried out at 37 ° C for 24 hours. In Comparative Examples 1 to 3, the lactobacillus standard strains of Table 6 were used and the experiment was conducted under the same conditions. The GABA content in the culture medium was measured by UPLC, and the sample pretreatment and UPLC analysis were carried out in the same manner as in the above example.

division Strain GABA production (nmol / ml) MSG
2 wt% Example L. brevis LLB5238 2864.81 Comparative Example 1 L. brevis KCTC3103 18.76 Comparative Example 2 L. brevis KCTC41028 1.13 Comparative Example 3 L. brevis KCTC41029 5.79 Seaweed extract
2 wt% Example L. brevis LLB5238 1654.31 Comparative Example 1 L. brevis KCTC3103 4.90 Comparative Example 2 L. brevis KCTC41028 1.22 Comparative Example 3 L. brevis KCTC41029 2.45

division Strain GABA production (nmol / ml) MSG
5 wt% Example L. brevis LLB5238 4939.33 Comparative Example 1 L. brevis KCTC3103 36.07 Comparative Example 2 L. brevis KCTC41028 2.05 Comparative Example 3 L. brevis KCTC41029 7.83 Seaweed extract
5 wt% Example L. brevis LLB5238 2954.13 Comparative Example 1 L. brevis KCTC3103 10.21 Comparative Example 2 L. brevis KCTC41028 1.85 Comparative Example 3 L. brevis KCTC41029 4.38

As shown in Tables 9, 10 and 4, when MSG and sea tangle extract were added as glutamic acid, the LLB 5238 strain was significantly higher than those of other comparative strains similarly to the case of adding reagent grade L-Glutamic acid Concentration of GABA was produced.

In particular, the results of the present invention of the present invention in which 5% by weight of MSG was added to MRS medium showed that 2,500 nmol / ml of GABA in a previous study (Patent Publication No. 10-2006-0006342) It was confirmed that a significantly higher content of GABA was produced than that produced, which is presumably due to the activity of glutamic acid decarboxylase (GAD) of the Lactobacillus brevis LLB5238 strain of the present invention.

In addition, since it exists in a water-soluble form in the culture solution, it can be formulated into various forms such as powder drying and tableting, and it is considered to be very useful when it is provided for food and medical use.

It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

Korea Microorganism Conservation Center (overseas) KCCM11538P 20140521

<110> LOTTE FOODS CO., LTD. <120> Lactobacillus brevis LLB5238 strain producing gamma-aminobutyric          acid and method for manufacturing gamma-aminobutyric acid by the          same <130> DP-2014-0214 <160> 1 <170> Kopatentin 1.71 <210> 1 <211> 1257 <212> DNA &Lt; 213 > Lactobacillus brevis LLB5238 <400> 1 ggcttccgt atttcaacaa tgaagcgagt ggcgaactgg tgagtaacac gtggggaatc tgcccagaag 120 caggggataa cacttggaaa caggtgctaa taccgtataa caacaaaatc cgcatggatt 180 ttgtttgaaa ggtggcttcg gctatcactt ctggatgatc ccgcggcgta ttagttagtt 240 ggtgaggtaa aggcccacca agacgatgat acgtagccga cctgagaggg taatcggcca 300 cattgggact gagacacggc ccaaactcct acgggaggca gcagtaggga atcttccaca 360 atggacgaaa gtctgatgga gcaatgccgc gtgagtgaag aagggtttcg gctcgtaaaa 420 ctctgttgtt aaagaagaac acctttgaga gtaactgttc aagggttgac ggtatttaac 480 cagaaagcca cggctaacta cgtgccagca gccgcggtaa tacgtaggtg gcaagcgttg 540 tccggattta ttgggcgtaa agcgagcgca ggcggttttt taagtctgat gtgaaagcct 600 tcggcttaac cggagaagtg catcggaaac tgggagactt gagtgcagaa gaggacagtg 660 gaactccatg tgtagcggtg gaatgcgtag atatatggaa gaacaccagt ggcgaaggcg 720 gctgtctagt ctgtaactga cgctgaggct cgaaagcatg ggtagcgaac aggattagat 780 accctggtag tccatgccgt aaacgatgag tgctaagtgt tggagggttt ccgcccttca 840 gtgctgcagc taacgcatta agcactccgc ctggggagta cgaccgcaag gttgaaactc 900 aaaggaattg acgggggccc gcacaagcgg tggagcatgt ggtttaattc gaagctacgc 960 gaagaacctt accaggtctt gacatcttct gccaatctta gagataagac gttcccttcg 1020 gggacagaat gacaggtggn gcatggnnnt cgtcagctcg tgtcgtgaga tgttgggtta 1080 agtcccgcan gagcgcaacc nnnattatca gttgccagca ttcanttggg nnncnctggn 1140 gnnactgcgn gacnaacngn nannngggga tgacgtcaat catcatgccc ttatgaccnn 1200 nnannnnnnt acannnnnnn nacangannn ncnnnnnnnc gnnnanggct taaannn 1257

Claims (4)

Lactobacillus brevis LLB5238 (Accession No. KCCM11538P) strain having ability to produce gamma-aminobutyric acid (GABA).
The Lactobacillus brevis LLB5238 (Accession No. KCCM11538P) strain according to claim 1, wherein the strain is isolated from kimchi.
A process for the preparation of gamma aminobutyric acid comprising the steps of:
(a) culturing a strain of Lactobacillus brevis LLB5238 (Accession No. KCCM11538P); And
(b) adding the culture medium to a medium containing glutamic acid and fermenting to produce gamma-aminobutyric acid (GABA).
A strain of Lactobacillus brevis LLB5238 (accession number KCCM11538P) having ability to produce gamma-aminobutyric acid (GABA), a culture of the strain, a concentrate of the culture, and a dried product of the culture At least one of which is contained as an active ingredient.

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