KR101679121B1 - A novel bacterium Lactobacillus brevis THK-734, and a method for producing GABA using the same - Google Patents

Abstract

The present invention relates to novel Lactobacillus brevis THB-734 ( Lactobacillus < RTI ID = 0.0 > brevis THK-734) and a method for producing GABA using the same. By using the strain of the present invention, a culture containing a large amount of GABA can be efficiently obtained, and the culture can be effectively used as a food composition and a cosmetic composition because of its excellent anti-inflammatory effect and low cytotoxicity.

Description

A novel bacterium Lactobacillus brevis THK-734, and a method for producing GABA using the same method,

The present invention relates to novel Lactobacillus brevis THB-734 ( Lactobacillus < RTI ID = 0.0 > brevis THK-734) and a method for producing GABA using the same.

γ-aminobutyric acid (GABA) is a kind of non-protein amino acid. GABA is a major inhibitory neurotransmitter in the mammalian central nervous system. GABA has been reported to have antidepressant, sedative and diuretic effects and has been reported to be effective in neurological disorders such as epilepsy, Parkinson's disease, Alzheimer's disease, and Huntington's disease. Studies have also shown that systolic blood pressure is lowered in hypertensive animal model rats receiving 0.3 to 300 mg / kg of GABA over a long period of time. GABA itself is sold as a nutrient, and rice and cheese with increased GABA content are being developed and sold. In addition, cosmetics containing GABA are sold at high prices as functional cosmetics.

GABA can be synthesized by chemical or biochemical methods. Biochemical methods are more advantageous than chemical methods because of their simple reaction and high efficiency of enzymes. Therefore, various biochemical methods have been studied.

Korean Patent Publication No. 10-2012-0119195 Korean Patent Publication No. 10-2012-0008490

The present invention is to provide Lactobacillus brevis THK-734, a novel bacterium, and a method for producing GABA from glutamic acid using the same.

The present invention is intended to provide a pharmaceutical composition, a food composition, and a cosmetic composition comprising the culture of the strain.

In order to achieve the above object, the present invention provides Lactobacillus brevis THK-734 (Accession No. KCTC 12637BP), a novel bacterium.

Lactobacillus spp. Is a gram-positive bacterium that does not have motility and fermentes saccharides to obtain energy and produce a large amount of lactic acid.

The present invention provides a method for producing GABA from glutamic acid using the strain.

Biochemical synthesis of GABA consists of a one-step reaction in which L-glutamic acid is decarboxylated by a glutamate decarboxylase (GAD) as follows.

The Lactobacillus brevis THK-734 of the present invention can produce GABA by lowering glutamic acid with excellent efficiency.

The inventors of the present invention studied the optimal culture medium of Lactobacillus brevis THK-734, and found that both the cell growth and GABA production were excellent in the medium containing Dextrose and Yeast Extract. Preferably, Dextrose 1 to 3 It was confirmed that the cell growth and GABA production of Lactobacillus brevis THK-734 were maximized in a culture medium containing 0.1% to 3% yeast extract, more preferably 2% dextrose and 0.5% (w / v) yeast extract . However, the culture medium in which Lactobacillus brevis THK-734 of the present invention can be cultured is not limited thereto.

In another embodiment of the present invention, the method can be carried out by culturing the strain at 28-34 [deg.] C, preferably at 29-32 [deg.] C, most preferably at 30 [deg.] C. However, the incubation temperature is not limited thereto.

In another embodiment of the present invention, the method can be carried out by culturing the strain for 1 to 20 days, preferably for 5 to 10 days, more preferably for 5 days. However, the incubation period is not limited thereto.

The present invention also provides a food composition comprising a culture of Lactobacillus brevis THK-734.

The present invention also provides a cosmetic composition comprising a culture of Lactobacillus brevis THK-734.

The culture of Lactobacillus brevis THK-734 means a material cultured with Lactobacillus brevis THK-734.

In one embodiment of the present invention, the culture of Lactobacillus brevis THK-734 is obtained by culturing Lactobacillus brevis THK-734 in a culture medium containing cacao nip extract, which means that it contains cacao nip extract and GABA. The cacao nip extract may be prepared by extracting cacao powder with hot water and then filtering, but the extraction method is not limited thereto.

The culture is not limited thereto, and may include other materials other than cacao nip extract.

In one embodiment of the present invention, the culture may be a culture obtained by culturing the strain at 28 to 34 째 C, preferably at 29 to 32 째 C, most preferably at 30 째 C. However, the incubation temperature is not limited thereto.

In another embodiment of the present invention, the culture may be a culture obtained by culturing the strain for 1 to 20 days, preferably 5 to 10 days, more preferably 5 days. However, the incubation period is not limited thereto.

The culture of Lactobacillus brevis THK-734 contains a large amount of GABA. However, the content of GABA is not limited thereto.

The culture of Lactobacillus brevis THK-734 of the present invention can be applied to food compositions and cosmetic compositions. The matters mentioned in the uses, compositions and methods of the present invention are applied equally as far as they are not mutually contradictory. Hereinafter, the present invention will be described in detail.

Food composition

The food composition of the present invention comprises a culture of THK-734.

The content of the active ingredient can be suitably determined according to the intended use (preventive or therapeutic treatment). The culture of THK-734 in the composition of the present invention may be contained in an amount of 0.1 to 60% by weight, preferably 10% by weight, based on the total weight of the composition. However, the content is not limited thereto.

There is no particular limitation on the kind of the food. Examples of the food to which the food composition of the present invention can be added include tablets, capsules, powders, granules, liquids, pills, drinks, and the like.

When the food composition of the present invention is prepared by tablet, a mixture of the culture of THK-734 with excipients, binders, disintegrants, and other additives is granulated by a conventional method, followed by compression molding with a lubricant or the like , The mixture can be directly compression molded. The health functional food of the tablet form may contain a mating agent and the like if necessary, and may be sieved to a suitable skin care agent if necessary.

When the food composition of the present invention is prepared in the form of a drink, various flavors or natural carbohydrates may be added as an additional ingredient such as ordinary beverages. Examples of the natural carbohydrate include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose, and the like; And polysaccharides such as conventional sugars such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. Flavoring agents (saccharin, aspartame, etc.) other than those described above may also be used.

The food composition of the present invention can be used as a flavoring agent such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, colorants and enhancers (cheese, chocolate etc.), pectic acid and its salts, alginic acid and its salts, Organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like.

The food composition of the present invention can be used alone or in combination with methods for the treatment or amelioration of inflammation or using surgery, hormone therapy, chemotherapy and biological response modifiers.

Cosmetics  Composition

The present invention provides a cosmetic composition comprising a culture of THK-734.

In the present specification, the term 'cosmetic material' refers to a substance which can be a cosmetic or a quasi-drug.

Cosmetics refers to products that are used in the human body to maintain or improve the health of skin and hair by adding cleanliness and beauty to the body, changing appearance, brightening the appearance, and acting on the human body. Functional cosmetics are products that help to whiten the skin, help to improve the wrinkles of the skin, protect the skin from ultraviolet rays, and include those prescribed by the Ministry of Health, Welfare and Family Affairs. do.

Quasi-drugs refer to medicines classified by the Ministry of Health and Welfare for products that have a slight effect on the human body, rather than medicines used to treat or prevent diseases. According to the Pharmaceutical Affairs Law, textile or rubber products used for the treatment or prevention of diseases of humans or animals, excluding those used for medicinal purposes, , Sterilization and insecticides to prevent infectious diseases, and the like. Typical examples of quasi-drugs include toothpaste, functional soap, and functional shampoo.

The content of the active ingredient can be suitably determined according to the intended use (preventive or therapeutic treatment). The culture of THK-734 in the composition of the present invention may be contained in an amount of 0.1 to 60% by weight, preferably 10% by weight, based on the total weight of the composition. However, the content is not limited thereto.

The cosmetic composition of the present invention, in addition to the above-mentioned components, may be blended with other components, which are usually added to cosmetics as required. For example, stabilizers, solubilizers, vitamins, pigments and flavoring agents, and carriers.

In addition, the composition of the present invention can be manufactured into any formulations conventionally produced in the art, for example, emulsions, suspensions, creams, lotions, essences, packs, gels, powders, lipsticks, A formulation selected from a makeup base, foundation, lotion, ointment, patch, essence, cleansing foam, cleansing cream, cleansing water, body lotion, body cream, body oil, body essence, shampoo, rinse, body cleanser, soap, .

The preparation method of these formulations can be produced according to a conventional method of producing a cosmetic formulation.

How to improve or treat

The present invention also provides a method of preventing, ameliorating or treating inflammation by administering to a human or other animal a therapeutically effective amount of a culture of THK-734.

As used herein, "therapeutically effective amount" means the amount of active ingredient or pharmaceutical composition that elicits a biological or medical response in an animal or human considered by a researcher, veterinarian, physician or other clinician, And an amount that induces symptomatic relief of the disease or disorder being treated. It will be apparent to those skilled in the art that the therapeutically effective dose and the number of administrations of the active ingredient of the present invention will vary depending on the desired effect.

The novel Lactobacillus brevis THB-734 of the present invention can produce GABA by effectively lowering glutamic acid to low molecular weight. The culture of the strain has excellent anti-inflammatory effect and low cytotoxicity and thus has high safety, so that it can be usefully used as a food composition and a cosmetic composition.

Figure 1 is the 16s rRNA sequence of Lactobacillus brevis THK-734.
FIG. 2 is a view showing a carbon source suitable for the culture of THK-734 and determining the optimum concentration of dextrose.
FIG. 3 is a view showing an optimum concentration of a yeast extract among the nitrogen sources selected for the culture of THK-734.
FIG. 4 is a chart showing cell growth and GABA production of THK-734 with varying temperature.
FIG. 5 is a graph showing changes in pH and observation of cell growth and GABA production of THK-734 over time. FIG.
FIG. 6 is a graph showing the anti-inflammatory effect of the cacao nip extract cultured together with THK-734. FIG.
FIG. 7 shows cytotoxicity of cacao nip extract cultured with THK-734. FIG.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrating the present invention, and the scope of the present invention is not limited thereto.

Example  One. Shin Jong-kyun's  Isolation and identification

Kimchi was ground and diluted in saline, and then isolates were selected and strains with remarkably high GABA productivity were selected. The strain was named Lactobacillus brevis THK-734 (hereinafter referred to as THK-734). The above-mentioned THK-734 was deposited with the Korea Research Institute of Bioscience and Biotechnology (Accession No. KCTC 12637BP). The 16s rRNA sequence of THK-734 is shown in SEQ ID NO: 1 and is shown in Fig.

The general characteristics of THK-734 are shown in Table 1 below.

[Table 1]

Biochemical characteristics were also observed using an API kit (50CHL and Zym, Biomrieux Co., France). The observed characteristics are summarized in Table 2 below.

[Table 2]

Example  2. THK Identification of Optimal Culture Medium of -734

2-1. On carbon  Following GABA  Production verification

The following experiment was conducted to confirm which substance is a carbon source favorable to the growth of THK-734 of the present invention.

A medium containing 2% (w / v) of dextrose, maltose, starch and sucrose was prepared, and THK-734 was inoculated in each medium and cultured. After that, absorbance was measured at 600 nm to observe cell growth and measure GABA production.

The results are shown in Figure 2A. In FIG. 2A, when dextrose was used, both cell growth and GABA production were found to be high (black bars indicate cell growth and hatch bars indicate GABA production).

In order to confirm the best dextrose concentration of cell growth and GABA production, dextrose was added at 0, 0.5, 1, 1.5, 2, 3% (w / v) and beef extract 1 , Yeast extract 0.5%, manganese sulfate 0.005% and MSG 1% were prepared, and THK-734 was inoculated in each medium and cultured. After that, absorbance was measured at 600 nm to observe cell growth and measure GABA production.

The results are shown in Figure 2B. In FIG. 2B, cell growth and GABA production are highest when the dextrose concentration is 2% (black bars indicate cell growth and hatch bars indicate GABA production).

2-2. Nitrogen source GABA  Production verification

The following experiment was conducted to confirm which substance is a carbon source favorable to the growth of THK-734 of the present invention.

A medium containing 2.5% (w / v) of peptone, beef extract, tryptone, soytone and yeast extract was prepared, and each medium was inoculated with THK-734. After that, absorbance was measured at 600 nm to observe cell growth and measure GABA production.

The results are shown in Fig. 3A. In FIG. 3A, when yeast extract was used, both cell growth and GABA production were remarkably high (black bars indicate cell growth and hatch bars indicate GABA production).

In order to confirm the concentration of yeast extract which has the best effect on cell growth and GABA production, yeast extracts containing 0, 0.5, 1, 1.5, 2 and 3% (w / v) 1%, yeast extract 0.5%, manganese sulfate 0.005% and MSG 1% were prepared, and THK-734 was inoculated in each medium and cultured. After that, absorbance was measured at 600 nm to observe cell growth and measure GABA production.

The results are shown in Figure 3B. In Fig. 3B, cell growth and GABA production are both high when yeast extract concentration is 0.5% (black bars indicate cell growth and hatch bars indicate GABA production).

Example  3. THK Determination of Optimal Culture Temperature and Time of -734

3-1. Determine optimal culture temperature

To determine the optimum culture temperature of THK-734, cell growth and GABA production were monitored according to the incubation time while changing the temperature.

THK-734 was inoculated in a medium containing 2% of dextrose and 0.5% (w / v) of yeast extract, and THK-734 was grown at 20 ° C, 30 ° C and 37 ° C respectively and absorbance at 600 nm Cell growth was observed and GABA production was measured.

The results are shown in Fig. In FIG. 4, cell growth and GABA production were both high when cultured at 30 ° C.

3-2. Optimal incubation time and incubation time pH  Confirm change

To determine the optimal incubation time of THK-734, THK-734 was inoculated in a medium containing 2% of dextrose and 0.5% (w / v) of yeast extract, and the degree of cell growth and GABA production was observed , And the change in pH over time was also observed.

The results are shown in Fig. In Fig. 5, the square represents the growth of the strain, the triangle represents pH, and the circle represents GABA production.

In FIG. 5, GABA production was active 24 hours after incubation, and GABA production rapidly increased until 72 hours. After that, production of GABA hardly occurred.

The pH of the culture medium was initially reduced to pH 4.2 during the proliferation of THK-734 at about pH 6.0, and slightly to pH 4.5 after 72 hours, at which GABA production was terminated.

Cell growth occurred until the first 24 hours of culture and no further cell growth occurred after 24 hours of active GABA production.

Example  4. THK By -734 GABA  Increased content Cacao nip  Check the effect of the extract

4-1. Cacao nip THK -734 Culture production

Kana acid (purchased from roasted cacao noodles, roasted after removing the husks, ground cacao nip) was quenched with liquid nitrogen and crushed with a mortar to produce cacao powder. 10 times the weight of water was added to the cacao powder, and the mixture was subjected to primary hot water extraction at 121 ° C for 30 minutes, followed by secondary extraction at 80 ° C for 24 hours. After extraction, the mixture was centrifuged at 12,000 rpm at 4 ° C for 10 minutes and filtered using a filter paper to prepare a cacao nip extract.

10, 100 占 퐂 / ml of each of the cacao nip extracts was added to a medium containing 2% of dextrose and 0.5% (w / v) of yeast extract, and 1% (w / v) of THK- Lt; RTI ID = 0.0 > 30 C < / RTI > to produce cacao nip THK-734 cultures. On the other hand, cacao nip extract without the step of culturing with THK-734 was prepared as a comparative group.

4-2. Identification of anti-inflammatory activity

Raw 254.7 cells were treated with 10, 100 ㎍ / ml of cacao nip THK-734 culture and cacao nip extract, respectively, prepared in 4-1 above, together with LPS, an inflammation inducing substance. On the other hand, the positive control group (LPS) treated with LPS alone and the negative control group (Nor) treated with nothing were treated. And the production of NO ₂ , an inflammatory substance, was confirmed.

The results are shown in Fig. In FIG. 6, it can be seen that the culture of cacao nip THK-734 significantly reduced NO ₂ production in Raw 254.7 cells.

4-3. Cytotoxicity check

Some of the lactic acid bacteria produce cytotoxic substances, so the following experiment was conducted to confirm this.

Raw 254.7 cells were treated with 0.01, 0.1, 1, 10, and 100 쨉 g / ml of cacao nip THK-734 culture and cacao nip extract prepared in 4-1 above. On the other hand, the negative control group (Nor) was not treated. And cell viability was measured to confirm cytotoxicity.

The results are shown in Fig. 7, it can be seen that the culture of cacao nip THK-734 is almost similar to that of negative control and cacao nip extract. Therefore, it can be seen that the cacao nip THK-734 culture of the present invention is a safe substance having extremely low cytotoxicity.

Korea Biotechnology Research Institute KCTC12637BP 20140729

<110> University-Industry Cooperation Group of Kyung Hee University <120> A novel bacterium Lactobacillus brevis THK-734, and a method for          producing GABA using the same <130> P14-066-KHU <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 1365 <212> DNA <213> Artificial Sequence <220> <223> Lactobacillus brevis THK-734 16s rRNA <400> 1 tgacgtgctt gcactgattt caacaatgaa gcgagtggcg aactggtgag taacacgtgg 60 gaaatctgcc cagaagcagg ggataacact tggaaacagg tgctaatacc gtataacaac 120 aaaatctgca tggattttgt tgaaaggtgg cttcggctat cacttctgga tgatcccgcg 180 gcgtattagt tagttggtga ggtaaaggcc caccaagacg atgatacgta gccgacctga 240 gagggtaatc ggccacattg ggactgagac acggcccaaa ctcctacggg aggcagcagt 300 agggaatctt ccacaatgga cgaaagtctg atggagcaat gccgcgtgag tgaagaaggg 360 tttcggctcg taaaactctg ttgttaaaga agaacacctt tgagagtaac tgttcaaggg 420 ttgacggtat ttaaccagaa agccacggct aactacgtgc cagcagccgc ggtaatacgt 480 aggtggcaag cgttgtccgg atttattggg cgtaaagcga gcgcaggcgg ttttttaagt 540 ctgatgtgaa agccttcggc ttaaccggag aagtgcatcg gaaactggga gacttgagtg 600 cagaagagga cagtggaact ccatgtgtag cggtggaatg cgtagatata tggaagaaca 660 ccagtggcga aggcggctgt ctagtctgta actgacgctg aggctcgaaa gcatgggtag 720 cgaacaggat tagataccct ggtagtccat gccgtaaacg atgagtgcta agtgttggag 780 ggtttccgcc cttcagtgct gcagctaacg cattaagcac tccgcctggg gagtacgacc 840 gcaaggttga aactcaaagg aattgacggg ggcccgcaca agcggtggag catgtggttt 900 aattcgaagc tacgcgaaga accttaccag gtcttgacat cttctgccaa tcttagagat 960 aagacgttcc cttcggggac agaatgacag gtggtgcatg gttgtcgtca gctcgtgtcg 1020 tgagatgttg ggttaagtcc cgcaacgagc gcaaccctta ttatcagttg ccagcattca 1080 gttgggcact ctggtgagac tgccggtgac aaaccggagg aaggtgggga tgacgtcaaa 1140 tcatcatgcc ccttatgacc tgggctacac acgtgctaca atggacggta caacgagtcg 1200 cgaagtcgtg aggctaagct aatctcttaa agccgttctc agttcggatt gtaggctgca 1260 actcgcctac atgaagttgg aatcgctagt aatcgcggat cagcatgccg cggtgaatac 1320 gttcccgggc cttgtacaca ccgcccgtca caccatgaga gtttg 1365

Claims (9)

Lactobacillus brevis THK-734 ( Lactobacillus brevis THK-734) (Accession No. KCTC 12637BP). A method for producing GABA from glutamic acid using Lactobacillus brevis THK-734 (Accession No. KCTC 12637BP) strain. 3. The method of claim 2, wherein the strain is cultured in a medium comprising dextrose and yeast extract. 4. The method according to claim 3, wherein the medium contains 1 to 3% of the dextrose and 0.1 to 3% (w / v) of the yeast extract. 5. The method according to any one of claims 2 to 4, wherein the strain is cultured at 28 to 32 占 폚. A food composition comprising as an active ingredient a culture obtained by culturing a cacao nip extract and Lactobacillus brevis THK-734 (Accession No. KCTC 12637BP) together. A cosmetic composition comprising as an active ingredient a culture obtained by culturing a cacao nip extract and a lactobacillus brevis THK-734 (Accession No. KCTC 12637BP) together. 7. The composition of claim 6, wherein said composition is for preventing or ameliorating inflammation. 8. The composition of claim 7, wherein said composition is for preventing or ameliorating inflammation.

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