Levilactobacillus brevis MG5552 strain having excellent GABA-producing ability and use thereof for alleviating seizures, convulsions, epilepsy, sleep disorders, and stress

Abstract

The present invention relates to a novel levilactobacillus brevis MG5552 strain and, more specifically, to the production of gamma-aminobutyric acid using the strain. Levilactobacillus brevis MG5552 according to the present invention was isolated from a fermented food sample, and was found to be far superior in the ability to produce GABA as compared to strains isolated together. Therefore, the strain according to the present invention can be variously used in the production of GABA and in the foods field.

Description

Levilactobacillus brevis MG5552 strain with excellent GABA production ability and its use in improving seizures, convulsions, epilepsy, sleep disorders, and stress

[0001] The present invention relates to a novel Lactobacillus brevis MG5552 strain, and more specifically, to the production of gamma-aminobutyric acid using the strain.

[0002] γ-Aminobutyric acid (GABA) is a non-protein amino acid found in many prokaryotic and eukaryotic cells. GABA regulates neural activity by inhibiting neurotransmission and has been linked to several neurological disorders, including epilepsy, depression, anxiety, Alzheimer's disease, Parkinson's disease, schizophrenia, and Huntington's disease.

[0003] Recent studies have shown that GABA improves plasma growth hormone levels, brain protein synthesis, memory, and cognitive abilities. Furthermore, GABA produced through microbial fermentation is environmentally friendly and safe, offering the potential to provide new health-promoting products.

[0004] GABA is primarily produced by microorganisms such as yeast, mold, and bacteria through food fermentation. The genus Levilactobacillus is a safe bacterium that produces higher levels of GABA than other microorganisms. Its unique properties, including resistance to bile and gastric acid and intestinal homeostasis, make it widely used as a probiotic. Furthermore, Levilactobacillus produces various organic acids, including lactic acid, and during fermentation, it produces antimicrobial molecules such as bacteriocins, antioxidants, and immunomodulators, as well as neurotransmitters such as GABA. Lactic acid bacteria (LAB) can also inhibit some pathogenic bacteria, thereby extending the shelf life of fermented foods.

[0005] Accordingly, the inventors of the present invention completed the present invention by isolating a strain from a fermented food sample and identifying Levi Lactobacillus brevis MG5552, which has excellent GABA production ability.

[0006] Accordingly, the purpose of the present invention is to provide a strain of Levilactobacillus brevis MG5552 deposited under the accession number KCTC15910BP.

[0007] Another object of the present invention is to provide a method for producing gamma-aminobutyric acid, comprising the step of culturing the strain Levilactobacillus brevis MG5552, deposited under the accession number KCTC15910BP, in a medium containing a GABA precursor.

[0008] Another object of the present invention is to provide a method for preventing or improving symptoms of deficiency of gamma-aminobutyric acid, comprising the step of administering to a subject in need thereof a composition comprising at least one selected from the group consisting of the strain Levilactobacillus brevis MG5552 deposited under the accession number KCTC15910BP, a culture medium of the strain, and a cell-free supernatant of the strain.

[0009] Another object of the present invention is to provide a composition comprising at least one selected from the group consisting of the strain Levilactobacillus brevis MG5552 deposited under the accession number KCTC15910BP, a culture medium of the strain, and a cell-free supernatant of the strain.

[0010] To achieve the above purpose, the present invention provides a strain of Levilactobacillus brevis MG5552 deposited under the accession number KCTC15910BP.

[0011] In addition, the present invention provides a method for producing gamma-aminobutyric acid, comprising the step of culturing the strain Levilactobacillus brevis MG5552, deposited under the accession number KCTC15910BP, in a medium containing a GABA precursor.

[0012] In addition, the present invention provides a method for preventing or improving symptoms of deficiency of gamma-aminobutyric acid, comprising the step of administering to a subject in need thereof a composition comprising at least one selected from the group consisting of the strain Levilactobacillus brevis MG5552 deposited under the accession number KCTC15910BP, a culture medium of the strain, and a cell-free supernatant of the strain.

[0013] In addition, the present invention provides a composition for preventing or improving symptoms of deficiency of gamma-aminobutyric acid, comprising at least one selected from the group consisting of the strain Levilactobacillus brevis MG5552 deposited under the accession number KCTC15910BP, a culture medium of the strain, and a cell-free supernatant of the strain.

[0014] In addition, the present invention provides a composition for producing gamma-aminobutyric acid, which comprises at least one selected from the group consisting of the strain Levilactobacillus brevis MG5552 deposited under the accession number KCTC15910BP, a culture medium of the strain, and a cell-free supernatant of the strain.

[0015] The Lactobacillus brevis MG5552 strain according to the present invention was isolated from a fermented food sample and was confirmed to have significantly superior GABA production ability compared to the strain isolated together. Therefore, the strain of the present invention can be utilized in various fields such as GABA production and food production.

[0016] Figure 1 is a diagram showing the results of confirming the GABA production ability of four strains (L. brevis MG5261, MG5405, MG5522, and MG5552) selected through screening using thin-layer chromatography (TLC).

[0017] Figure 2 is a diagram showing a phylogenetic tree of four strains (L. brevis MG5261, MG5405, MG5522, and MG5552) created based on 16S rRNA sequences.

[0018] Figure 3a is a diagram showing the results of confirming the effect of four strains (L. brevis MG5261, MG5405, MG5522, and MG5552) of the present invention on nitric oxide production.

[0019] Figure 3b is a diagram showing the results of confirming the cytotoxicity of four strains (L. brevis MG5261, MG5405, MG5522, and MG5552) of the present invention through MTT analysis.

[0020] Figure 4 is a diagram showing the results of investigating the effects of four strains (L. brevis MG5261, MG5405, MG5522, and MG5552) of the present invention on iNOS protein expression through Western blotting.

[0021] Figure 5 is a diagram showing the results of investigating the effects of four strains (L. brevis MG5261, MG5405, MG5522, and MG5552) of the present invention on NF-κB activation through Western blotting.

[0022] Hereinafter, the present invention will be described in detail.

[0023] According to an aspect of the present invention, the present invention provides a strain of Levilactobacillus brevis MG5552 deposited under the accession number KCTC15910BP.

[0024] In an embodiment of the present invention, a phylogenetic analysis was performed based on the base sequence of 16S rRNA of Reviractobacillus brevis MG5552, and as a result, it was confirmed that it is a new strain different from known strains of the same species and strains already in industrial use. Accordingly, Reviractobacillus brevis MG5552 was deposited with the Biological Resource Center of the Korea Research Institute of Bioscience and Biotechnology (Jeongeup-si, Jeollabuk-do, Korea) on May 17, 2024, and was assigned the accession number KCTC15910BP.

[0025] In a specific embodiment of the present invention, it is preferable that the strain comprises 16S rRNA represented by the base sequence of SEQ ID NO: 1. The 16S rRNA sequence is a sequence used to identify the Lactobacillus brevis MG5552 strain of the present invention.

[0026] In a specific example of the present invention, the strain may be used for producing gamma-aminobutyric acid (γ-aminobutyric acid, GABA).

[0027] In the present invention, as long as the purpose of the present invention can be achieved, the strain Levilactobacillus brevis MG5552 deposited under the accession number KCTC15910BP itself or a culture solution thereof can be used.

[0028] In addition, it is clear to those skilled in the art that any method known in the art can be used to obtain a culture solution of the above strain, and is not limited thereto.

[0029] The GABA produced by the above strain can be usefully utilized as an additive in the food and feed manufacturing industries, either alone or mixed with the strain itself. Furthermore, it can be provided as a pharmaceutical composition for the alleviation or treatment of seizures, convulsions, and epilepsy in individuals suffering from severe insufficient GABA levels in the body.

[0030] Therefore, the Levi Lactobacillus brevis MG5552 strain deposited under the accession number KCTC15910BP according to the present invention has a very excellent GABA production ability, and can be utilized in various ways in the production of GABA and in the food field.

[0031]

[0032] According to another aspect of the present invention, the present invention provides a composition comprising at least one selected from the group consisting of the strain Levilactobacillus brevis MG5552 deposited under accession number KCTC15910BP, a culture medium of the strain, and a cell-free supernatant of the strain. The composition may be a food, a health functional food, or a pharmaceutical composition.

[0033] In a specific embodiment of the present invention, the composition may be a composition for preventing or improving symptoms of gamma-aminobutyric acid deficiency. Specifically, the symptoms of gamma-aminobutyric acid deficiency may include seizures, convulsions, epilepsy, sleep disorders, increased stress, etc., and can be applied without limitation to any gamma-aminobutyric acid deficiency symptoms known in the art.

[0034] In a specific embodiment of the present invention, the composition may include a lyophilized product of the Lactobacillus brevis MG5552 strain, a culture medium of the strain, and a cell-free supernatant of the strain.

[0035] The above food composition may contain the strain, strain culture medium or cell-free supernatant alone as an active ingredient, and may additionally contain additional ingredients, i.e., pharmaceutically acceptable or nutritionally acceptable carriers, excipients, diluents or auxiliary ingredients, depending on the formulation, method of use and purpose of use.

[0036] In the present invention, food refers to food having a bioregulatory function, and is a concept that includes health functional foods.

[0037] In the present invention, the term "health functional food" refers to a food group or food composition that has been designed and processed to sufficiently exert its internal regulatory functions, such as regulating biological defense rhythms, disease prevention, and recovery, by using physical, biochemical, or bioengineering techniques to provide added value to the food so that the food's function can be performed and expressed for a specific purpose. For the purpose of the present invention, the above health functional food is intended to enhance activity.

[0038] In the present invention, the food composition can be manufactured using methods commonly used in the art, and during the manufacturing process, raw materials and ingredients commonly added in the art can be added. Furthermore, the formulation of the food composition can be manufactured without limitation as long as it is a formulation recognized as a food composition.

[0039] Foods according to the present invention include, for example, various foods, beverages, gums, tea, vitamin complexes, functional foods, etc. In addition, foods include, but are not limited to, special nutritional foods (e.g., formulated milk, infant and toddler food, etc.), processed meat products, fish products, tofu, jelly, noodles (e.g., ramen, noodles, etc.), bread, health supplements, seasoned foods (e.g., soy sauce, soybean paste, red pepper paste, mixed paste, etc.), sauces, confectionery (e.g., snacks), candies, chocolates, gums, ice cream, processed dairy products (e.g., fermented milk, cheese, etc.), other processed foods, kimchi, pickled foods (various kimchi, pickled vegetables, etc.), beverages (e.g., fruit drinks, vegetable drinks, soy milk, fermented drinks, etc.), natural seasonings (e.g., ramen soup, etc.), food additives, etc. The above foods, beverages, or food additives can be manufactured by a conventional manufacturing method.

[0040] When the composition of the present invention is used as a health functional food additive, the composition can be added as is or used together with other health functional food ingredients, and can be used appropriately according to a conventional method. The mixing amount of the active ingredient can be appropriately determined depending on the intended use. Generally, when manufacturing food or beverage, the composition of the present invention can be added in an amount of preferably 50 parts by weight or less, more preferably 25 parts by weight or less, relative to the raw material. However, in the case of long-term intake for the purpose of health control and hygiene, the amount can be below the above range, and since there is no problem in terms of stability, the active ingredient can also be used in an amount above the above range.

[0041] The food or health functional food composition of the present invention, in addition to containing the strain as an active ingredient, may contain various flavoring agents or natural carbohydrates as additional ingredients, like conventional food compositions. Examples of the aforementioned natural carbohydrates include monosaccharides such as glucose, fructose, etc.; disaccharides such as maltose, sucrose, etc.; and polysaccharides such as dextrin, cyclodextrin, etc., and conventional sugars, and sugar alcohols such as xylitol, sorbitol, erythritol, etc. As the flavoring agent, natural flavoring agents (thaumatin), stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used.

[0042] In addition, the food composition may contain, in addition to the strain, various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the food composition of the present invention may contain fruit pulp for producing natural fruit juice, fruit juice drinks, and vegetable drinks.

[0043] When the composition of the present invention is used as a pharmaceutical composition, the pharmaceutical composition of the present invention can be formulated and used in various forms according to conventional methods. For example, it can be formulated in oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, and syrups, and can be formulated and used in the form of topical preparations, suppositories, and sterile injectable solutions.

[0044] The composition of the present invention may contain at least one known effective ingredient having an effect of preventing or improving symptoms of gamma-aminobutyric acid deficiency together with Lactobacillus brevis MG5552.

[0045] The composition of the present invention may further include a pharmaceutically acceptable additive. At this time, the pharmaceutically acceptable additive may be starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose, mannitol, taffy, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, carnauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, white sugar, etc. The pharmaceutically acceptable additive according to the present invention is preferably included in the composition in an amount of 0.1 to 90 parts by weight, but is not limited thereto.

[0046] The composition of the present invention can be administered in various oral or parenteral dosage forms during actual clinical administration. When formulating, it can be prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants that are commonly used. It is preferable to use suitable formulations known in the relevant technical field disclosed in the literature.

[0047] The dosage of the pharmaceutical composition of the present invention may vary depending on the method of formulating the pharmaceutical composition, the method of administration, the time of administration, and / or the route of administration, and may vary depending on various factors including the type and degree of the response to be achieved by administration of the pharmaceutical composition, the type, age, weight, general health condition, symptoms or degree of the disease, sex, diet, excretion, drugs used simultaneously or simultaneously in the subject, other components of the composition, and similar factors well known in the medical field, and a person having ordinary knowledge in the relevant technical field can easily determine and prescribe an effective dosage for the desired treatment.

[0048] The dosage of the pharmaceutical composition of the present invention is preferably administered at a concentration of, for example, 0.05 to 5 mg / kg, more preferably 0.1 to 0.4 mg / kg, even more preferably 0.2 to 0.35 mg / kg, and even more preferably 0.25 mg / kg, but the dosage does not limit the scope of the present invention in any way.

[0049] The route and method of administration of the pharmaceutical composition of the present invention may be independent of each other, and are not particularly limited in their method, and any route and method of administration may be followed as long as the pharmaceutical composition can reach the target area.

[0050]

[0051] According to another aspect of the present invention, the present invention provides a composition for producing gamma-aminobutyric acid, comprising at least one selected from the group consisting of the strain Levilactobacillus brevis MG5552 deposited under the accession number KCTC15910BP, a culture medium of the strain, and a cell-free supernatant of the strain.

[0052] In a specific embodiment of the present invention, the composition may further comprise a medium comprising a GABA precursor.

[0053] In a preferred embodiment of the present invention, the medium containing the GABA precursor may be a MRS (de Man, Rogosa and Sharp) medium, and may be a medium containing 1 to 5% glucose, 1 to 5% yeast extract, and 0.5 to 3% peptone. In addition, the medium may further contain MSG (monosodium glutamate), and preferably contain 1% MSG.

[0054] In a preferred embodiment of the present invention, the GABA precursor may be glutamate or glutamic acid. The glutamate may be at least one selected from the group consisting of sodium glutamate, potassium glutamate, calcium glutamate, and ammonium glutamate, but is not limited thereto.

[0055]

[0056] The Levilactobacillus brevis MG5552 strain of the present invention has excellent gamma-aminobutyric acid production ability. Therefore, when a fermented food is manufactured using the Levilactobacillus brevis MG5552 strain, the content of gamma-aminobutyric acid in the manufactured fermented food can be significantly increased.

[0057]

[0058] According to another aspect of the present invention, the present invention provides a method for producing gamma-aminobutyric acid (GABA), comprising the step of culturing the strain Levilactobacillus brevis MG5552, deposited under accession number KCTC15910BP, in a medium containing a GABA precursor.

[0059] In a specific embodiment of the present invention, the GABA precursor may be glutamate or glutamic acid. The glutamate may be at least one selected from the group consisting of sodium glutamate, potassium glutamate, calcium glutamate, and ammonium glutamate, but is not limited thereto.

[0060] In a specific example of the present invention, when culturing the Lactobacillus brevis MG5552 strain, MRS medium can be used as a culture medium, and preferably, it can be a medium containing 1 to 5% glucose and 1 to 5% yeast extract.

[0061] In an embodiment of the present invention, MRS liquid medium containing 1% MSG (monosodium glutamate) was used.

[0062] In a specific embodiment of the present invention, it is preferable that the culturing is performed for 1 to 5 days.

[0063] In a specific example of the present invention, the culturing is preferably performed at a temperature of 20 to 40°C, and more preferably at a temperature of 37°C.

[0064] In a specific example of the present invention, the method for producing gamma-aminobutyric acid may further include a step of isolating and purifying GABA from a strain culture.

[0065]

[0066] According to another aspect of the present invention, a method for preventing or improving symptoms of deficiency of gamma-aminobutyric acid is provided, comprising the step of administering to a subject in need thereof a composition comprising at least one selected from the group consisting of the strain Levilactobacillus brevis MG5552 deposited under the accession number KCTC15910BP, a culture medium of the strain, and a cell-free supernatant of the strain.

[0067] In a specific embodiment of the present invention, the composition may include a lyophilized product of the Lactobacillus brevis MG5552 strain, a culture medium of the strain, and a cell-free supernatant of the strain.

[0068] In a specific embodiment of the present invention, the composition may be a food, a health functional food, or a pharmaceutical composition.

[0069] In a specific embodiment of the present invention, the subject may be, but is not limited to, a subject expected to develop symptoms of gamma-aminobutyric acid deficiency; a subject that has developed symptoms; or a subject that has improved after the onset of symptoms.

[0070]

[0071] Duplicate contents are omitted in consideration of the complexity of this specification, and terms not otherwise defined in this specification have meanings commonly used in the technical field to which the present invention belongs.

[0072] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0073]

[0074] Example 1. Isolation and identification of LAB (lactic acid bacteria)

[0075] Lactic acid bacteria (LAB) were isolated from various fermented foods sold in Korea. Ten grams of each food was suspended in 90 mL of sterile saline (0.85% NaCl) and homogenized. Each mixture was serially diluted 10-fold and streaked onto MRS (de Man, Rogosa, and Sharpe) agar plates. After 48 h of incubation, colonies were randomly selected from each plate and streaked onto bromocresol purple (BCP) agar plates and incubated at 37°C for 24 h. LAB were selected based on the yellow zone surrounding the colonies on the BCP agar plates. The isolated colonies were cultured in fresh MRS medium. Each culture was stored at -70°C with sterile 25% (v / v) glycerol until further use.

[0076] To identify the isolated strains, total DNA of each strain was extracted and purified using the PureLink Genomic DNA Mini Kit (Invitrogen, Carlsbad, CA, USA). 16S rRNA was amplified from the obtained total DNA using the universal primers 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'-TACGGYTACCTTGTTACGACTT-3'), and gene sequence analysis was performed (SolGent, Daejeon, Republic of Korea). Each strain was identified by comparing the 16S rRNA gene sequence of the isolated strains with those registered in the GenBank database using the Basic Local Alignment Search Tool of the National Center for Biotechnology Information (MD, USA).

[0077] As a result of strain identification, 33 strains were isolated, and they are as follows.

[0078] -Levilactobacillus brevis26 weeks

[0079] -Lactococcus lactis 2 weeks

[0080] -Lactiplantibacillus plantarum 1 week

[0081] -Lactobacillus acidophilus 1 strain

[0082] -Limosilactobacillus reuteri1 strain

[0083] -Enterococcus faecium strain 1

[0084] -Enterococcus faecalis 1 strain

[0085]

[0086] Example 2. GABA production LAB screening

[0087] - Preparation of supernatant

[0088] The 33 LAB strains identified in Example 1 were seeded on MRS containing MSG and cultured at 37°C for 48 h in a biochemical oxygen demand (BOD) incubator (Hanbaek Science, Bucheon, Korea). After culture, each culture was centrifuged at 4000 xg for 5 min, and the supernatant was collected and used for thin-layer chromatography (TLC) and GABA content analysis.

[0089]

[0090] - 1st screening

[0091] The primary screening of GABA-producing LAB was performed using TLC. Specifically, 1 mL of the supernatant was spotted onto a silica TLC plate (Sigma-Aldrich, St. Louis, MO, USA). A mixture of n-butanol (Daejung, Seoul, South Korea), acetic acid (Daejung), and distilled water in a volume ratio of 5:3:2 was used as the solvent. The standards used for TLC were L-glutamic acid (Daejung) and commercially available GABA (Sigma-Aldrich) with a purity of ≥99% dissolved in deionized water (final concentration 1%) and filtered. Each culture supernatant was spotted onto a TLC plate, and a 0.5% ninhydrin solution (Sigma-Aldrich) was dissolved in ethanol (99%) and dried at 90°C for 15 min. Red spots were observed on the TLC plate and compared with the standards for qualitative analysis.

[0092] To determine the GABA production capacity of the 33 selected LAB strains, the supernatants prepared by centrifugation of the cultures in MRS broth containing 1% MSG were analyzed using TLC. GABA-producing strains were selected by comparing the red spots produced on the TLC plate with those produced by a GABA standard. Eleven strains (L. brevisMG5342, L. brevisMG5522, L. brevisMG5250, L. brevisMG5306, L. brevisMG5524, L. brevisMG5286, L. brevisMG5354, L. brevisMG5261, L. brevisMG5263, L. brevisMG5405, and L. brevisMG5552) were selected through screening using TLC.

[0093]

[0094] - Second screening

[0095] A secondary screening of the 11 selected strains was performed using a synthetic medium containing 1% MSG. The synthetic medium contained 30 g / L glucose, 15 g / L peptone, 7 g / L yeast extract, 2.5 g / L Na-acetate, 1 g / L trisodium citrate, 1 g / L ammonium sulfate, 2 g / L K2HPO4, and 1 g / L KH2PO4. Each strain was seeded on the synthetic medium and cultured at 37°C for 48 hours. TLC was then performed to determine the amount of GABA produced by the 11 strains selected in the primary screening. The TLC results are shown in Figure 1.

[0096] As shown in Fig. 1, among the 11 strains screened in the first round, 4 strains (L. brevis MG5552, MG5405, MG5261, and MG5522) with excellent GABA production ability were selected.

[0097] The 16S rRNA gene sequences of the four selected strains were compared with those registered in the GenBank database using the Basic Local Alignment Search Tool of the National Center for Biotechnology Information (MD, USA), and a phylogenetic tree was created based on the comparison results. The created phylogenetic tree is shown in Figure 2.

[0098] As shown in Fig. 2, the four selected strains were confirmed to be new strains different from known strains of the same species and strains already in industrial use. In particular, the 16S rRNA sequence of Levilactobacillus brevisMG5552 is represented by the base sequence of SEQ ID NO: 1, and was deposited with the Biological Resource Center of the Korea Research Institute of Bioscience and Biotechnology (Jeongeup-si, Jeollabuk-do, Korea) on May 17, 2024, and was assigned the accession number KCTC15910BP.

[0099]

[0100] Example 3. Quantitative analysis of GABA using an amino acid analyzer

[0101] The amount of GABA produced by the four strains selected in Example 2 was quantitatively analyzed using an amino acid analyzer (HITACHI L-8900 amino acid analyzer) equipped with an ion exchange column (HITACHI HPLC packed column #2622PF). The elution buffer (KANTO HITACHI high-speed amino acid analyzer buffer [PF-1, 2, 3, 4, RG]) and coloring solution (Wako Ninhydrin Coloring Solution kit for HITACHI) were used in the experiment. The prepared samples were analyzed using a UV-VIS spectrophotometer. The analysis conditions were wavelengths of 570 nm (VIS1) and 440 nm (VIS2); and an injection volume of 20 μL. The GABA content in the samples was determined by calculating the peak area compared to the standard solution, and the results are shown in Table 1.

[0102]

[0103] As shown in Table 1, the GABA production amounts of strains MG5261, MG5405, MG5522, and MG5552 in the MRS medium were 0.304, 0.260, 0.280, and 0.322 mg / mL, respectively, and the GABA production amounts in the 1% MSG-containing medium were 0.624, 0.585, 0.591, and 0.979 mg / mL, respectively. In particular, GABA production was higher in the MRS medium containing 1% MSG than in the MRS medium, indicating that all selected strains had the ability to convert MSG into GABA. In particular, L. brevis MG5552 produced the most GABA under the same conditions.

[0104]

[0105] Example 4. Anti-inflammatory effect of selected strains on murine macrophages (RAW 246.7 cells)

[0106] 4-1. Preparation of cell-free supernatant (CFS)

[0107] To prepare cell-free supernatant CFS of the four selected strains, a single colony of each strain was inoculated onto de Man, Rogosa and Sharp (MRS, BD Bioscience, NJ, USA) medium and cultured in a BOD incubator at 37°C for 24 h. The cell concentration was 10 8 After adjusting to the cells / mL level, 2% of the culture solution was transferred to new MRS medium and cultured for 18 hours. The suspension was centrifuged at 4000xg for 10 minutes at 4°C, and the supernatant was prepared as CFS using a 0.22 μm syringe filter (Millipore Co., Bedford, MA, USA).

[0108]

[0109] 4-2. Culturing RAW 264.7 cells

[0110] RAW 264.7 cells were purchased from the American Type Culture Collection (Manassas, VA, USA). RAW 264.7 cells were cultured in DMEM (Gibco) medium containing 10% fetal bovine serum (FBS; Gibco, Waltham, MA, USA) and 1% penicillin-streptomycin (Gibco) at 37°C in an atmosphere of 5% CO2.

[0111]

[0112] 4-3. Nitric Oxide (NO) Production

[0113] NO production was measured using Griess reagent (1% sulfanilamide and 0.1% N-(1-naphthyl)-ethylenediamine dihydrochloride, Sigma-Aldrich, St. Louis, MO, USA). RAW 264.7 cells were seeded in 96-well plates (2 × 10 5 The cells / well were seeded and treated with CFS and lipopolysaccharide (LPS) of the four isolated strains for 1 hour and 24 hours, respectively. Afterwards, 100 μL of the supernatant was mixed with 100 μL of Griess reagent, reacted for 15 minutes, and the absorbance was measured at 540 nm to determine the amount of NO produced. The results are shown in Fig. 3a.

[0114] As shown in Fig. 3a, the LPS-treated group showed a significant increase in NO production compared to the control group. In contrast, the NO production levels of the four selected strains treated with CFS (MG5261, MG5405, MG5522, and MG5552) were 24.91, 9.58, 7.94, and 9.82 μM, respectively, indicating a significant decrease in NO production compared to the LPS-treated group. The above results suggest that the four selected strains may exhibit anti-inflammatory effects, which may be attributed to a decrease in NO production.

[0115]

[0116] 4-4. Cell viability analysis

[0117] Cell viability was determined using the MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) assay. Specifically, RAW 264.7 cells were suspended in DMEM containing 10% FBS, and the cell suspension was seeded into each well of a 96-well plate (Corning, NY, USA) at a density of 1X10 4Cells were seeded (100 μL / well). The plates were cultured in a CO2 incubator at 37°C for 24 h, pretreated with 5% CFS for 1 h, and then additionally treated with 0.5 μg / mL LPS for 24 h. The supernatant was removed from each well, and MTT solution (0.1 mg / mL, Sigma-Aldrich, St. Louis, MO, USA) was added to each well. The plates were incubated in a 37°C incubator for 4 h. After incubation, the supernatant was removed, 100 μL of dimethyl sulfoxy (DMSO) was added, and the absorbance was measured at 550 nm using an Epoch 2 microplate reader (Biotek Instruments Inc., Winooski, VT, USA). The MTT assay results are shown in Figure 3b.

[0118] As shown in Fig. 3b, the cell viability after CFS treatment of the four selected strains (MG5261, MG5405, MG5522, and MG5552) was confirmed to be 67.1, 85.1, 84.3, and 81.2%, respectively.

[0119]

[0120] 4-5. Protein extraction

[0121] Whole-cell proteins were extracted from the strains using radioimmunoprecipitation assay (RIPA) cell lysis buffer containing phosphatase and protease inhibitors (Gendepot, Katy, TX, USA). The extracted proteins were quantified using Bradford reagent (Gendepot). Protein samples were mixed with 5X sample buffer (iNtron Biotechnology, Seongnam, South Korea) and heated at 85°C for 10 min to prepare samples.

[0122]

[0123] 4-6. Western blotting

[0124] The effects of CFS of four selected strains on iNOS protein expression and NF-κB activation in RAW 264.7 cells were investigated using Western blotting. Specifically, the protein extracts (20 μg for each strain) isolated in Examples 4-5 were subjected to SDS-PAGE using 8% and 10% gels, and the proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Gendepot). The PVDF membrane was blocked for 5 minutes with Smart-Block™ 5 min-Fast Blocking buffer (Biomax, Seoul, Korea) and then incubated with iNOS, phospho-NF-κB p65, and NF-κB p65 antibodies (1:1000, Cell Signaling Technology, Danvers, MA, USA) at 4°C for more than 15 hours. After washing three times with TBST buffer for 10 min each, the membrane was treated with horseradish peroxydase-conjugated secondary antibody (1:5000, Gendepot) for 1 h. After secondary antibody treatment, the membrane was washed with TBST buffer for 10 min and treated with ECL (enhanced chemiluminescent, ATTO, Tokyo, Japan) solution for 1 min. Protein bands were identified using a LuminoGraph III Lite (ATTO) imaging system.

[0125] The results of Western blotting to determine the effect of CFS of the four selected strains on iNOS protein expression are shown in Figure 4. In addition, the results of determining the effect of CFS of the four selected strains on NF-κB activation are shown in Figure 5.

[0126] As shown in Figure 4, the LPS-treated group showed a significant increase in iNOS expression compared to the control group. In contrast, the CFS-treated groups of the four selected strains all showed a decrease in iNOS expression. In particular, a significant decrease in iNOS expression was confirmed in the CFS-treated groups of strains MG5405 and MG5552. The above results indicate that the four selected strains suppress the expression of iNOS, which is specifically expressed during the inflammatory response, i.e., suppress the inflammatory response.

[0127] As shown in Figure 5, the LPS-treated group showed a significant increase in the expression of NF-κB p65 compared to the control group. In contrast, the CFS-treated group of the four selected strains all showed a significant decrease in the expression of NF-κB p65. The above results indicate that the four selected strains positively regulate immune and inflammatory responses by suppressing the NF-κB signaling pathway.

[0128]

[0129] Example 5. Statistical Analysis

[0130] Statistical analysis was performed using SPSS version 21 (IBM Inc., Armonk, NY, USA) to analyze significant differences between samples. All data are expressed as the mean ± standard error. A p<0.05 was considered statistically significant.

[0131]

[0132] In summary, the present inventors isolated four novel strains of Reviractobacillus brevis (MG5261, MG5405, MG5522, and MG5552) from fermented food samples. The four strains were found to have significantly superior GABA production compared to strains isolated from the same sample. In particular, among the four strains, the Reviractobacillus brevis MG5552 strain exhibited the highest GABA production. Therefore, the strains of the present invention can be utilized in various fields, including GABA production and the food industry.

[0133]

[0134] While specific aspects of the present invention have been described in detail, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

[0135]

[0136] [Accession number]

[0137] Name of depositor: Korea Research Institute of Bioscience and Biotechnology, Biological Resource Center

[0138] Accession number: KCTC15910BP

[0139] Date of acceptance: 20240517

[0140]

[0141]

Claims

1. A method for producing gamma-aminobutyric acid, comprising the step of culturing the strain Levilactobacillus brevis MG5552, deposited under the deposit number KCTC15910BP, in a medium containing a GABA precursor.

2. A method for producing gamma-aminobutyric acid, wherein the strain comprises 16S rRNA represented by the base sequence of sequence number 1.

3. A method for producing gamma-aminobutyric acid in the first paragraph, wherein the GABA precursor is glutamate or glutamic acid.

4. A method for producing gamma-aminobutyric acid, wherein the culture is performed for 1 to 5 days in the first paragraph.

5. A method for preventing or improving symptoms of gamma-aminobutyric acid deficiency, comprising the step of administering to an individual in need thereof a composition comprising at least one selected from the group consisting of the strain Levilactobacillus brevis MG5552 deposited under the deposit number KCTC15910BP, a culture medium of the strain, and a cell-free supernatant of the strain.

6. A method for preventing or improving symptoms of gamma-aminobutyric acid deficiency in paragraph 5, wherein the symptoms of gamma-aminobutyric acid deficiency are seizures, convulsions, epilepsy, sleep disorders, or increased stress.

7. In the fifth paragraph, the composition is a method for preventing or improving symptoms of deficiency of gamma-aminobutyric acid, which is a food, health functional food or pharmaceutical composition.

8. A composition for preventing or improving symptoms of deficiency of gamma-aminobutyric acid, comprising at least one selected from the group consisting of Levilactobacillus brevis MG5552 strain deposited under the deposit number KCTC15910BP, a culture medium of the strain, and a cell-free supernatant of the strain.

9. In the 8th paragraph, the composition is a composition for preventing or improving symptoms of deficiency of gamma-aminobutyric acid, which is a food, health functional food or pharmaceutical composition.

10. A composition for producing gamma-aminobutyric acid, comprising at least one selected from the group consisting of the strain Levilactobacillus brevis MG5552 deposited under the deposit number KCTC15910BP, a culture medium of the strain, and a cell-free supernatant of the strain.

11. A composition for producing gamma-aminobutyric acid, wherein the composition further comprises a medium containing a GABA precursor.

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