KR20240015823A - Novel probiotic Lactobacillus brevis strains and uses thereof - Google Patents

Abstract

The present invention relates to a novel probiotic Lactobacillus brevis strain (KU15152) isolated from Chonggak kimchi and its use. The strain has only probiotic properties such as stability to the stomach, enzyme production ability, and intestinal fixation ability. In addition, it showed an anti-inflammatory effect on intestinal cell models and a protective effect against oxidative stress in nerve cells, confirming that it had excellent nerve cell protection. Therefore, the strain can be used in health functional foods and treatments for neurodegenerative diseases/depression, etc.

Description

Novel probiotic Lactobacillus brevis strains and uses thereof}

The present invention relates to a novel probiotic Lactobacillus brevis strain and its use, and more specifically to the prevention of neurodegenerative diseases including the Lactobacillus brevis KU15152 strain, the strain, its dead cells, and/or its metabolites. Or it relates to pharmaceutical compositions for treatment, pharmaceutical compositions for preventing or treating depression, health functional foods, and probiotic compositions.

The human gut microbiota consists of trillions of diverse microorganisms, which are essential for nutrient absorption, intestinal barrier function, and immune regulation. Intestinal microorganisms act as a barrier to external stimuli such as lipopolysaccharide (LPS) or inflammatory cytokines, which stimulate intestinal epithelial cells and cause intestinal inflammation. Abnormalities in the composition of intestinal microorganisms facilitate the invasion of pathogenic bacteria, causing harmful immune responses and various intestinal diseases.

Probiotics are live microorganisms that have beneficial effects on the host's health when consumed. They can colonize as part of the intestinal microflora and protect the intestines from external invasion. Probiotics have been reported to inhibit the expression of inflammatory cytokines and inhibit the development of intestinal inflammatory diseases. Research on the immune-boosting and anti-inflammatory effects of probiotics is also actively underway around the world.

Meanwhile, reactive oxygen species (ROS) can cause oxidative stress and cause several neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, and Huntington's disease. Hydrogen peroxide (H ₂ O ₂ ) is a representative ROS that activates apoptosis-related factors and causes apoptosis of nerve cells.

Additionally, morphological changes in hippocampal neurons or loss of neurons caused by oxidative stress can cause depression. It is known that antidepressants promote the expression of brain-derived neurotrophic factor (BDNF) in the hippocampus. BDNF is mainly involved in the differentiation and survival of nerve cells during brain development, and long-term treatment with antidepressants has been shown to restore atrophy of nerve cell projections caused by stress.

Accordingly, the present inventors demonstrated that the novel probiotic Lactobacillus brevis KU15152 strain, isolated and identified from Chonggak kimchi, not only has probiotic properties such as acid resistance, bile resistance, and intestinal adhesion, but also exhibits anti-inflammatory effects in cell models, as well as neuronal viability and nerve function. The present invention was completed by confirming that it has the effect of protecting nerve cells from death caused by oxidative stress by improving cell-derived substances and growth factors.

The purpose of the present invention is to provide a novel probiotic Lactobacillus brevis strain and its use.

To achieve the above object, the present invention provides Lactobacillus brevis KU1512 strain (KCCM 13177P), which has a protective effect against nerve cell death.

In the present invention, the neuronal cell death may be characterized as being induced by oxidative stress.

In the present invention, the protective effect against nerve cell death may be characterized as being due to the action of metabolites produced through reactions between the strain and enterocytes.

In the present invention, the strain may include the 16S rRNA base sequence of SEQ ID NO: 1.

The present invention also provides a pharmaceutical composition for preventing or treating neurodegenerative diseases comprising Lactobacillus brevis KU1512 strain (KCCM 13177P), dead cells thereof, and/or metabolites thereof as active ingredients.

In the present invention, the neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, Lou Gehrig's disease, Huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis, immune system abnormality and brain dysfunction, progressive neurodegenerative disease, metabolic brain disease, Niemann-Pick disease, brain It may be characterized as one or more selected from the group consisting of dementia caused by ischemia and cerebral hemorrhage.

The present invention also provides a pharmaceutical composition for preventing or treating depression containing Lactobacillus brevis KU1512 strain (KCCM 13177P), dead cells thereof, and/or metabolites thereof as active ingredients.

The present invention also provides a health functional food containing Lactobacillus brevis KU1512 strain (KCCM 13177P), its dead cells, and/or its metabolites as active ingredients.

The present invention also provides a probiotic composition containing Lactobacillus brevis KU1512 strain (KCCM 13177P), its dead cells, and/or its metabolites as active ingredients.

The novel probiotic Lactobacillus brevis KU15152 strain of the present invention, its dead cells, and its metabolites have excellent protective properties against intestinal inflammatory reactions and neuronal protective effects, and can be used in health functional foods and treatments for neurodegenerative diseases/depression.

Figure 1 shows the 16S rRNA partial sequencing results of the novel Lactobacillus brevis KU1512 strain isolated and identified from Chonggak kimchi.
Figure 2 is a schematic diagram of Lactobacillus brevis KU1512 strain.
Figure 3 relates to the effect of Lactobacillus brevis KU1512 strain on (A) cell viability and (B) expression of IL-8 in HT-29 cells.
Figure 4 relates to the effect of Lactobacillus brevis KU1512 strain on the activity of NF-κB in HT-29 cells.
Figure 5 relates to the effect of Lactobacillus brevis KU1512 strain on MEK/ERK signaling mechanism in HT-29 cells.
Figure 6 relates to the effect of Lactobacillus brevis KU1512 strain on Akt/GSK 3β signaling mechanism in HT-29 cells.
Figure 7 relates to the evaluation of neuronal toxicity using MTT assay of strain-derived metabolites (conditioned medium; CM).
Figure 8 relates to the neuronal protective effect of strain-derived metabolites (conditioned medium; CM) using the MTT assay.
Figure 9 relates to morphological changes in SH-SY5Y cells treated with H ₂ O ₂ and CM.
Figure 10 relates to the level of BDNF expression when human-derived colon cancer cells are treated with dead Lactobacillus cells.
Figure 11 shows the results of treating nerve cells with CM, inducing toxicity _{with H2O2} , and measuring the gene expression levels of BDNF and TH in CM nerve cells.
Figure 12 shows the results of treating nerve cells with CM, inducing toxicity with _{H2O2 ,} and measuring the gene expression ratios of Bax and Bcl-2 in CM nerve cells.
Figure 13 shows the results of treating nerve cells with CM, inducing toxicity with H ₂ O ₂ and measuring the activity of Caspase-3 on CM nerve cells.
Figure 14 shows the results of comparing IL-8 production when human-derived colon cancer cells were treated with aLTA.

Hereinafter, the present invention will be described in detail.

Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by a person skilled in the art to which the present invention pertains. In general, the nomenclature used herein is well known and commonly used in the art.

The present invention relates to Lactobacillus brevis KU1512 strain (KCCM 13177P), which consistently has a protective effect against neuronal cell death.

In the present invention, the nerve cell death may be characterized as being induced by oxidative stress, but is not limited thereto.

In the present invention, the protective effect against nerve cell death may be characterized as being due to the action of metabolites produced through a reaction between the strain and enterocytes, but is not limited thereto.

In the present invention, the strain may include the 16S rRNA base sequence of SEQ ID NO: 1.

From another point of view, the present invention relates to a pharmaceutical composition for preventing or treating neurodegenerative diseases comprising Lactobacillus brevis KU1512 strain (KCCM 13177P), dead cells thereof, and/or metabolites thereof as active ingredients.

In the present invention, the neurodegenerative disease includes Alzheimer's disease, Parkinson's disease, Lou Gehrig's disease, Huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis, immune system abnormality and brain dysfunction, progressive neurodegenerative disease, metabolic brain disease, Niemann-Pick disease, brain It may be characterized as one or more selected from the group consisting of dementia caused by ischemia and cerebral hemorrhage, but is not limited thereto.

From another aspect, the present invention relates to a pharmaceutical composition for preventing or treating depression comprising Lactobacillus brevis KU1512 strain (KCCM 13177P), dead cells thereof, and/or metabolites thereof as active ingredients.

From another perspective, the present invention relates to a health functional food containing Lactobacillus brevis KU1512 strain (KCCM 13177P), its dead cells, and/or its metabolites as active ingredients.

From another perspective, the present invention relates to a probiotic composition comprising Lactobacillus brevis KU1512 strain (KCCM 13177P), its dead cells, and/or its metabolites as active ingredients.

In the present invention, the term “pharmaceutical composition” refers to the Lactobacillus brevis KU1512 strain (KCCM 13177P) of the present invention, its dead cells and/or its metabolites, and pharmaceutically acceptable excipients such as diluents or carriers. means a mixture containing Pharmaceutical compositions include cosmetic compositions as well as compositions for therapeutic use. According to some embodiments, a method of administering a pharmaceutical composition comprising the composition of the present invention to a subject as needed is provided. In some embodiments, compositions of the present invention can be administered to humans.

In the present invention, the description of pharmaceutical compositions principally relates to pharmaceutical compositions for administration to humans, but those skilled in the art will understand that such compositions are generally suitable for administration to all types of animals. A skilled veterinary pharmacologist with a good understanding of the modifications of pharmaceutical compositions for administration to various animals can design and/or perform such modifications, if necessary, simply by routine experimentation.

In the present invention, the pharmaceutical composition may be prepared by any method known in the field of pharmacology or discussed later in the text. Generally, these methods for tableting involve the steps of associating the active ingredient with excipients and/or one or more other auxiliary ingredients, followed by shaping and/or packaging the product into the desired single- or multi-dose units, if necessary or desired. Includes steps.

In the present invention, the pharmaceutical composition may be manufactured, packaged, and/or sold unpackaged as a single unit dose and/or multiple single unit doses. “Unit dose” is a discrete amount of pharmaceutical composition containing a predetermined amount of active ingredient. The amount of active ingredient is generally equal to the dosage of active ingredient administered to the subject and/or a convenient fraction of such dosage such as, for example, one-half or one-third of the dosage.

In the present invention, the relative amounts of the active ingredient, pharmaceutically acceptable excipients, and/or any additional ingredients in the pharmaceutical composition may vary depending on the identity, size, and/or disorder of the subject being treated and the route by which the composition is administered. It will change. By way of example, the composition may include 0.1% to 100% (w/w) active ingredient.

For the purposes of the present invention, pharmaceutically acceptable excipients include any solvent, dispersion medium, diluent, or other liquid vehicle, dispersion or suspension aid, surface active agent, isotonic agent, thickener or emulsifier, preservative, or solid that is suitable for the purpose of the particular dosage form. Includes binders, lubricants, etc. Remington's (The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro, (Lippincott, Williams & Wilkins, Baltimore, MD, 2006) describes various excipients used in the preparation of pharmaceutical compositions and known methods for their preparation. The technology is disclosed, except that any conventional carrier medium is incompatible with the substance or its derivatives, for example by imparting any undesirable biological effect or otherwise interacting in a deleterious manner with any other component of the pharmaceutical composition. and its uses are considered to be within the scope of the present invention. Pharmaceutically acceptable excipients are at least 95%, 96%, 97%, 98%, 99%, or 100% pure.

The excipients are approved for human and veterinary use. In some embodiments, the excipient is approved by the Food and Drug Administration. In some embodiments, the excipient is pharmaceutical grade. In some embodiments, the excipient meets the standards of the United States Pharmacopeia (USP), European Pharmacopoeia (EP), British Pharmacopoeia, and/or International Pharmacopoeia (EP).

In some embodiments, excipients are approved for human and veterinary use. In some embodiments, the excipient is approved by the Food and Drug Administration. In some embodiments, the excipient is pharmaceutical grade. In some embodiments, the excipient meets the standards of the United States Pharmacopeia (USP), European Pharmacopoeia (EP), British Pharmacopoeia, and/or International Pharmacopoeia (EP).

Pharmaceutically acceptable excipients used in the preparation of pharmaceutical compositions include, but are not limited to, inert diluents, dispersants and/or granulating agents, surface active agents and/or emulsifiers, disintegrants, binders, preservatives, buffers, lubricants, and/or oils. Not limited.

Such excipients may optionally be included in the formulations of the present invention. Excipients such as cocoa butter and suppository waxes, colorants, coating agents, sweeteners, flavors, and perfumers may be present in the composition at the discretion of the formulator.

Exemplary diluents include calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dried starch, Including, but not limited to, corn starch, powdered sugar, and combinations thereof.

Exemplary granulating and/or dispersing agents include potato starch, corn starch, tapioca starch, sodium starch glycolate, clay, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponges, cation-exchange resins. , Calcium Carbonate, Silicate, Sodium Carbonate, Cross-Linked Poly(vinyl-pyrrolidone) (Crospovidone), Sodium Carboxymethyl Starch (Sodium Starch Glycolate), Carboxymethyl Cellulose, Cross-Linked Sodium Carboxymethyl Cellulose ( croscarmellose), methylcellulose, pregelatinized starch (Starch 1500), microcrystalline starch, water-insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (vegeum), sodium lauryl sulfate, quaternary ammonium compounds, and these. Including, but not limited to, combinations of.

Exemplary surface active agents and/or emulsifiers include natural emulsifiers (e.g. acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, waxes, and lecithin), colloidal clays (e.g. bentonite [aluminum silicate] and beegum [magnesium aluminum silicate]), long chain amino acid derivatives, high molecular weight alcohols (e.g. stearyl alcohol, cetyl alcohol, oleyl alcohol) , triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g. carboxy polymethylene, polyacrylic acid, acrylic acid polymer, and carboxyvinyl polymers), carrageenans, cellulose derivatives (e.g. carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty acid esters (e.g. polyoxymethylcellulose) Ethylene Sorbitan Monolaurate [Tween 20], Polyoxyethylene Sorbitan [Tween 60], Polyoxyethylene Sorbitan Monooleate [Tween 80], Sorbitan Monopalmitate [Span 40], Sorbitan Monostearate [ span 60], sorbitan tristearate [span 65], glyceryl monooleate, sorbitan monooleate [span 80]), polyoxyethylene ester (e.g. polyoxyethylene monostearate [mirz 45]) , polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and solutol), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g. cremophor), polyoxyethylene ethers, ( For example, polyoxyethylene lauryl ether [Brise 30]), poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl. Including, but not limited to, lauray, sodium lauryl sulfate, pluronic F 68, poloxamer 188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, and/or combinations thereof. No.

Exemplary binders include starches (e.g. corn starch and starch paste); gelatin; sugars (e.g. sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol); Natural and synthetic gums (e.g. acacia, sodium alginate, extract of Irish moss, Farnwer gum, Shatty gum, Isapol Husk's slime, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropyl) cellulose, hydroxypropyl methylcellulose, microcrystalline cellulose, cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate (bigum), and lachi arabogalactan); alginate; polyethylene oxide; polyethylene glycol; inorganic calcium salt; silicic acid; polymethacrylate; wax; water; Alcohol; and combinations thereof.

Exemplary preservatives may include antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and other preservatives. Exemplary antioxidants include alpha tocopherol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, bisulfite. Including, but not limited to, sodium sulfate, sodium metabisulfite, and sodium sulfite. Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium edetate, disodium edetate, edetic acid, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and trisodium edetate. . Exemplary antimicrobial preservatives include benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, Includes, but is not limited to, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and thimerosal. Exemplary antifungal preservatives include, but are not limited to, butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and sorbic acid. Exemplary alcohol preservatives include, but are not limited to, ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and phenylethyl alcohol. Exemplary acidic preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and phytic acid. Other preservatives include tocopherol, tocopherol acetate, deteroxyme mesylate, cetrimide, butylated hydroxyanisole (BHA), butylated hydroxytoluende (BHT), ethylenediamine, sodium lauryl sulfate (SLS), Sodium Lauryl Ether Sulfate (SLES), Sodium Bisulfite, Sodium Metabisulfite, Potassium Sulfite, Potassium Metabisulfite, Glydant Plus, Fenonib, Methylparaben, Low Mol 115, Germaben II, Neolon, Katon, and E Including, but not limited to, Uxil. In certain embodiments, the preservative is an antioxidant. In another embodiment, the preservative is a chelating agent.

Exemplary buffering agents include citrate buffer solution, acetate buffer solution, phosphate buffer solution, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium gluvionate, calcium gluceptate, calcium gluconate, D-gluconic acid, glyceroside. Calcium phosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixture, dibasic potassium phosphate, monobasic Basic potassium phosphate, potassium phosphate mixture, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixture, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen -Includes, but is not limited to, free water, isotonic saline, Ringer's solution, ethyl alcohol, and combinations thereof.

Exemplary lubricants include magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behanate, hydrogenated vegetable oil, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium. Including, but not limited to, lauryl sulfate, and combinations thereof.

Exemplary oils include almond, apricot kernel, avocado, babassu palm, bergamot, black currant seed, borage, cayenne, chamomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee. , corn, cotton seed, emu, eucalyptus, evening primrose, fish, flax seed, geraniol, pumpkin, grape seed, hazelnut, hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon, litthea cucumber. beba, macadamia nut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange-orange rappi, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower. , sandalwood, sasquana, savory, sea buckthorn, sesame, shea butter, silicone, soybean, sunflower, tea tree, thistle, tsubaki, vetiver, walnut, and wheat germ oil. Exemplary oils include butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil. , and combinations thereof.

Liquid dosage forms for oral and parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active ingredient, liquid dosage forms may contain inert diluents such as, for example, water or other solvents, solubilizers and emulsifiers commonly used in the art such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl. Alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (especially cottonseed, peanut, corn, sprout, olive, castor, and sesame oil), glycerol, tetrahydrofurfuryl alcohol, It may also include polyethylene glycol and fatty acid esters of sorbitan, and mixtures thereof. In addition to inert diluents, oral compositions may include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents. In certain embodiments for parenteral administration, the Lactobacillus brevis KU1512 strain (KCCM 13177P) of the present invention, its dead cells, and/or its metabolites are dissolved in a solubilizing agent such as cremophor, alcohol, oil, denatured oil, glycol, poly Mixed with sorbates, cyclodextrins, polymers, and combinations thereof.

Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In these solid dosage forms, the active ingredient may be combined with one or more inert pharmaceutically acceptable excipients or carriers such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starch, lactose, sucrose, glucose, mannitol, and silicic acid, b ) binders such as, for example, carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrants such as agar, calcium carbonate, potato or tapioca starch. , alginic acid, certain silicates, and sodium carbonate, e) dissolution retarders such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) humectants such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof.

In the case of capsules, tablets and pills, dosage forms may contain buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using lactose or milk sugar as well as high molecular weight polyethylene glycols and the like as such excipients. Solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulation art. They may optionally contain opacifying agents and may be compositions that release the active ingredient only, or preferentially, in a specific part of the intestinal tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using lactose or milk sugar as well as high molecular weight polyethylene glycols and the like as such excipients.

The active ingredient may be in micro-encapsulated form with one or more of the excipients described above. Solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, controlled release coatings, and other coatings well known in the pharmaceutical formulation art. In these solid dosage forms the active ingredient may be mixed with one or more inert diluents such as sucrose, lactose or starch. These dosage forms may contain additional substances other than inert diluents as in common practice, such as tablet lubricants and other tablet auxiliaries such as magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, dosage forms may also contain buffering agents. They may optionally contain opacifying agents and may be compositions that release the active ingredient only, or preferentially, in a specific part of the intestinal tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes.

In the present invention, dosage forms for topical and/or transdermal administration of the Lactobacillus brevis KU1512 strain (KCCM 13177P), its dead cells, and/or its metabolites include ointment, paste, cream, lotion, gel, powder, and solution. , sprays, inhalants, and/or patches. Generally, the active ingredient is admixed under conditions of sterility with a pharmaceutically acceptable carrier and/or any necessary preservatives and/or buffering agents that may be required. Additionally, the present invention contemplates the use of transdermal patches, which often have the additional advantage of providing controlled delivery of the active ingredient to the body. Such dosage forms can be prepared, for example, by dissolving and/or dispersing the active ingredient in a suitable medium. Alternatively or additionally, the rate may be controlled by providing a rate controlling membrane and/or dispersing the active ingredient in a polymer matrix and/or gel.

Formulations for topical administration include, but are not limited to, liquid and/or semi-liquid preparations such as liniments, lotions, oil-in-water and/or water-in-oil emulsions such as creams, ointments/or pastes, and/or solutions and/or suspensions. Although the concentration of the active ingredient may be as high as the solubility limit of the active ingredient in the solvent, topically-administrable formulations may also comprise, for example, from about 1% to about 10% (w/w) of the active ingredient. Formulations for topical administration may further comprise one or more additional ingredients described herein.

In the present invention, Lactobacillus brevis KU1512 strain (KCCM 13177P), its dead cells and/or its metabolites are typically prepared in dosage unit form for easy administration and uniform administration. However, it will be understood that the total daily dosage of the composition of the present invention will be determined by the attending physician within the scope of sound medical judgment. The particular therapeutically effective dose level for any particular subject will depend on a variety of factors, including the disease, disorder, or disorder being treated and the severity of the disorder; the activity of the specific active ingredient employed; the specific composition employed; The subject's age, weight, general health, gender, and diet; the time of administration, route of administration, and rate of excretion of the particular active ingredient employed; duration of treatment; Drugs used in combination or simultaneously with the specific active ingredients employed; and factors well known in the medical field.

The pharmaceutical composition containing the Lactobacillus brevis KU1512 strain (KCCM 13177P) of the present invention, its dead cells, and/or its metabolites may be administered by any route. In some embodiments, pharmaceutical compositions comprising Lactobacillus brevis KU1512 strain (KCCM 13177P), dead cells thereof, and/or metabolites thereof are administered orally, intravenously, intramuscularly, intraarterially, intramedullarily, intrathecally, subcutaneously, intracerebroventricular, transdermal, intradermal, rectal, intravaginal, intraperitoneal, topical (by powder, ointment, cream, and/or drop), mucosal, nasal, oral, enteral, sublingual; endotracheal instillation, bronchial instillation, and/or inhalation; and/or administered by various routes, including oral spray, nasal spray, and/or aerosol. Particularly contemplated routes are permeable intravenous injection, local administration via the blood and/or lymphatic supply, and/or direct administration to the affected area. In general, the most appropriate route of administration will depend on a variety of factors, including the properties of the agent (e.g., stability in the environment of the gastrointestinal tract), and the disorder of the subject (e.g., whether the subject can tolerate oral administration). will be. Currently, oral and/or nasal spray and/or aerosol routes are most commonly used to deliver therapeutic agents directly to the lungs and/or respiratory system. However, the present invention encompasses the delivery of the pharmaceutical composition according to the invention by any suitable route taking into account advances in the field of drug delivery.

In certain embodiments, the pharmaceutical composition comprising the Lactobacillus brevis KU1512 strain (KCCM 13177P) of the present invention, its dead cells, and/or its metabolites is administered at an amount of about 0.001 mg/kg to about 100 mg/kg of the subject's body weight daily, About 0.01 mg/kg to about 50 mg/kg, about 0.1 mg/kg to about 40 mg/kg, about 0.5 mg/kg to about 30 mg/kg, about 0.01 mg/kg to about 10 mg/kg, about 0.1 The desired therapeutic effect may be achieved by administering from about 10 mg/kg to about 10 mg/kg, or from about 1 mg/kg to about 25 mg/kg, at a dosage level sufficient to deliver at least once a day. The intended dosage may be delivered three times a day, twice a day, daily, every two days, every three days, weekly, every two weeks, every three weeks, or every four weeks. In certain embodiments, the desired dosage may be delivered via multiple administrations (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more administrations). .

In the present invention, it will be understood that the dosage ranges provide guidance for administration of the provided pharmaceutical composition to adults. For example, the amount administered to a child or adolescent may be determined by a physician or person skilled in the art, and may be less or the same as that administered to an adult. The exact amount of a peptide according to the invention required to achieve an effective amount will vary from subject to subject, depending, for example, on the subject's species, age, and overall disorder, severity of side effects or disorders, identity of the specific compound, mode of administration, etc.

It will be understood that in the present invention, a pharmaceutical composition containing Lactobacillus brevis KU1512 strain (KCCM 13177P), its dead cells, and/or its metabolites can be used in combination therapy. The particular combination of treatments (treatments or procedures) for use in combination therapy will take into account the desired therapeutic effect to be achieved and the suitability of the desired treatments and/or procedures.

The pharmaceutical compositions of the invention may be administered alone or in combination with one or more therapeutically active agents. As for "combination", it is not intended to imply that the agents must be administered at the same time and/or be formulated for delivery together, although the following delivery methods are within the scope of the present invention. The composition may be administered concurrently with, prior to, or subsequent to one or more other therapeutic agents or medical procedures. Generally, each agent will be administered at a dose and/or time schedule established for that agent. Additionally, the present invention provides a pharmaceutical form of the present invention in combination with agents capable of improving its bioavailability, reducing and/or modifying its metabolism, inhibiting its secretion, and/or modifying its distribution within the body. It encompasses delivering the composition. It will be further understood that the Lactobacillus brevis KU1512 strain (KCCM 13177P) of the invention, its dead cells, its metabolites and the therapeutically active agent used in this combination may be administered together in a single composition or administered separately in different compositions.

The specific combination used in combination therapy determines the intended therapeutic effect to be achieved and/or the suitability of the procedure and/or therapeutically active agent comprising Lactobacillus brevis KU1512 strain (KCCM 13177P), its dead cells and/or its metabolites of the invention. will consider. The combination used can achieve the desired effect for the same disorder (e.g., the Lactobacillus brevis KU1512 strain (KCCM 13177P) of the present invention, its dead cells and/or its metabolites are used to treat the same disorder may be administered in combination with another therapeutically active agent), and/or they may achieve different effects (e.g., control of any side effects).

As used herein, "therapeutically active agent" refers to any substance used as a medicine to treat, prevent, delay, reduce or ameliorate a disorder, and refers to substances used in treatment, including prophylactic and curative treatments. .

In some embodiments, the pharmaceutical compositions of the present invention treat, alleviate, ameliorate, alleviate, delay the onset, inhibit the progression, or reduce the severity of one or more symptoms or features of neurodegenerative disease/depression; and/or in combination with any therapeutically active agent or procedure (e.g., surgery, radiation therapy) useful for reducing its incidence.

In the present invention, health functional foods can be manufactured in various forms according to conventional methods known in the art. Health functional foods are not limited to this, but for example, the Lactobacillus brevis KU1512 strain (KCCM 13177P) of the present invention, its dead cells and/or its metabolites are prepared in the form of tea, juice and drinks for drinking (health drinks) ) can be consumed by liquefying, granulating, encapsulating, and powdering. In addition, the nutritional supplement is not limited to this, but can be prepared by adding the Lactobacillus brevis KU1512 strain (KCCM 13177P) of the present invention, its dead cells, and/or its metabolites to capsules, tablets, pills, etc. In addition, in order to use the Lactobacillus brevis KU1512 strain (KCCM 13177P), its dead cells, and/or its metabolites of the present invention in the form of a food additive, it can be prepared and used in the form of a powder or concentrate. In addition, the Lactobacillus brevis KU1512 strain (KCCM 13177P) of the present invention, its dead cells and/or its metabolites, and a known active ingredient known to prevent or improve neurodegenerative diseases/depression are mixed to form a composition. can do.

In the present invention, when Lactobacillus brevis KU1512 strain (KCCM 13177P), its dead cells, and/or its metabolites are used as a health drink, the health drink composition contains various flavors or natural carbohydrates like a normal drink. May contain additional ingredients. The above-mentioned natural carbohydrates include monosaccharides such as glucose and fructose; Disaccharides such as maltose and sucrose; polysaccharides such as dextrins and cyclodextrins; It may be a sugar alcohol such as xylitol, sorbitol, or erythritol. Sweeteners include natural sweeteners such as thaumatin and stevia extract; Synthetic sweeteners such as saccharin and aspartame can be used. The proportion of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g, per 100 mL of the composition of the present invention.

In the present invention, Lactobacillus brevis KU1512 strain (KCCM 13177P), its dead cells, and/or its metabolites may be contained as an active ingredient in a food composition for preventing or improving neurodegenerative diseases/depression, and the amount thereof is suitable for neurodegenerative diseases. /The amount effective to prevent or improve depression is not particularly limited, but is preferably 0.01 to 100% by weight based on the total weight of the entire composition.

In the present invention, the composition for health functional food is prepared by mixing Lactobacillus brevis KU1512 strain (KCCM 13177P), its dead cells, and/or its metabolites with other active ingredients known to be effective in neurodegenerative diseases/depression. can be manufactured.

In the present invention, in addition to the above, the food of the present invention contains various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid, salts of pectic acid, alginic acid, salts of alginic acid, organic acids, protective colloidal thickeners, pH adjusters, and stabilizers. It may contain topicals, preservatives, glycerin, alcohol, or carbonating agents. In addition, the food of the present invention may contain pulp for the production of natural fruit juice, fruit juice beverage, or vegetable beverage. These ingredients can be used independently or in combination.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it is obvious to those skilled in the art that the scope of the present invention should not be construed as limited by these examples.

[Example]

Experimental materials and methods

(Example 1-1) Strains used

Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ) GG strain was used as a standard strain. The neuronal protective effect of Lactobacillus brevis KU1512 strain was confirmed. Lactobacillus brevis KU1512 strain was isolated from Chonggak Kimchi using MRS medium. Lactobacillus brevis KU15153 and Tobacillus brevis KU15176 strains isolated from Chonggak kimchi were used as comparative strains, respectively, to verify the anti-inflammatory activity and brain cell protection effect of Lactobacillus brevis KU1512 strain.

(Example 1-2) Measurement of acid resistance and bile resistance

The acid resistance of the strain was adjusted to pH 2.5, the strain was inoculated into MRS liquid medium containing 0.3% pepsin, and the number of bacteria after culturing for 3 hours at 37°C was compared with the initial number of inoculated bacteria. For bile resistance, the strain was inoculated into MRS liquid medium containing 0.3% oxgall and the number of bacteria after culturing at 37°C for 24 hours was compared with the initial number of inoculated bacteria.

(Example 1-3) Measurement of tenon ability

The HT-29 cell line, a human-derived intestinal epithelial cell, was inoculated into a 24-well plate (2×10 ⁵ cells/well) and cultured until a single layer was formed, followed by Lactobacillus brevis KU1512 strain (7 log CFU/mL). Inoculated and incubated at 37°C for 2 hours. After washing three times with PBS to remove bacteria not attached to the cells, the attached cells were lysed with 1% Triton X-100 solution. The solution diluted to the appropriate ratio was plated on MRS agar medium and cultured at 37°C for 24 hours to measure the number of attached bacteria.

(Example 1-4) Measurement of enzyme production ability

Enzyme production was evaluated using the API ZYM kit. Lactobacillus brevis KU1512 strain and Lactobacillus rhamnosus GG, cultured for 18 hours, were diluted in PBS to 9 log CFU/mL before use. Enzyme activity was evaluated based on color change (+, Production; -, Non-production).

(Example 1-5) Anti-inflammatory effect of the strain in intestinal cell model

Lactobacillus brevis KU1512 strain was cultured in MRS medium at 37°C for 18 hours. The supernatant was removed using a centrifuge and samples were prepared by dissolving in RPMI medium at a concentration of 7 log CFU/mL.

HT-29 cells were inoculated into a 24-well plate (2 × 10 cells/well) and cultured in a 5% CO ₂ environment at 37°C until a monolayer was formed. The above sample was pretreated for 2 hours, then treated with 50 μg/mL of Staphylococcus aureus lipoteichoic acid (aLTA) and cultured for 22 hours. The supernatant was collected and the expression level of IL-8 was quantified using an ELISA kit.

MTT [4,5-dimethyldiazol-2-yl-2,5-dephenylterazolium promid] reagent was dispensed onto the plate to a final concentration of 0.5 mg/mL and incubated for 30 minutes. After completely removing the supernatant, the produced formagen component was dissolved in DMSO and the absorbance was calculated at 570 nm. Cell viability was expressed as a percentage relative to the absorbance of the negative control group.

(Example 1-6) Western blotting

HT-29 enterocytes were inoculated into a 6-well plate (5 × 10 cells/well) and cultured in a 5% CO ₂ environment at 37°C until a monolayer was formed. The above sample was pretreated for 2 hours, then treated with 50 μg/mL of Staphylococcus aureus lipoteichoic acid (aLTA) and cultured for a certain period of time. Cultured cells were lysed with Pro-Prep lysis buffer, centrifuged, and only the supernatant was collected. 20-30 μg of protein quantified using the DC Protein Assay Kit was separated by electrophoresis on a 10% sodium dodecyl sulfate-polyacrylamide gel and then transferred to a polyvinyl difluoride membrane. After reacting with 5% skim milk for 1 hour, an appropriate antibody was added. After washing three times with TBS-T solution, the protein band was analyzed by reacting with an enhanced chemiluminescence detection kit and exposing it to an X-ray film in the dark. Quantification of protein bands was performed using ImageJ software.

(Example 1-7) Preparation of dead cells and strain-derived metabolites (conditioned medium; CM)

Lactobacillus strain was inoculated into MRS medium and cultured for 18 hours. After washing twice with PBS, dead cells were prepared by heat treatment at 80°C for 30 minutes.

Human-derived intestinal epithelial cells HT-29 were inoculated into a 6-well plate (5×10 ⁶ cells/mL), cultured until a single layer was formed, and then inoculated with dead Lactobacillus cells (1×10 ⁹ CFU/mL). and culture for 24 hours. The supernatant was collected, centrifuged at 14,000 × g, 4°C for 10 minutes, and then filtered using a 0.45 μM filter to prepare strain-derived metabolites (conditioned medium; CM).

(Example 1-8) Measurement of antioxidant activity of dead cells

1) DPPH scavenging ability

500 μL of dead lactic acid bacteria cells (1× ¹⁰⁹ CFU/mL) were added to 500 μL of 0.1 M DPPH solution dissolved in ethanol, and reacted in the dark for 30 minutes at room temperature. The solution was centrifuged at 14,240 × g for 1 minute, the supernatant was taken, and the absorbance was measured at 517 nm. The equation for DPPH scavenging ability is as follows.

DPPH radical scavenging activity (%)=(1-(absorbance for sample)/(absorbance for control))×100

2) ABTS erasing ability

After preparing the ABTS solution, it was reacted in the dark at room temperature and stored for 18 hours. The ABTS solution was diluted using distilled water so that the absorbance was 0.7 at 734 nm. 200 μL of dead cells were mixed with 800 μL of ABTS solution and incubated in the dark for 15 minutes at room temperature. After the reaction, centrifugation was performed at 14,240 × g for 1 minute, the supernatant was taken, and the absorbance was measured at 734 nm. The equation for ABTS scavenging ability is as follows.

ABTS radical scavenging activity (%)=(1-(absorbance for sample)/(absorbance for control))×100

(Example 1-9) Measurement of neuronal cell death protective effect

MTT assay was performed to measure the cytotoxicity of CM on neuronal SH-SY5Y cells and the cytoprotective effect against oxidative stress, H ₂ O ₂ . SH-SY5Y cells (1×10 ⁶ cells/mL) are cultured in a 96-well plate for 24 hours. To evaluate the toxicity of CM, CM is treated and cultured for 24 hours. To confirm the protective effect of H ₂ O ₂ on nerve cells, CM was treated with 150 μM H ₂ O ₂ for 4 hours and cultured for 20 hours. Afterwards, the medium is removed and treated with MTT (5 mg/mL) for 4 hours. Remove the MTT solution, add 100 μL of DMSO solution, and react for 20 minutes to dissolve the crystals. Afterwards, the absorbance was measured at 570 nm and the cell viability was calculated according to the following equation.

Cell viability (%)=(1-(cell viability for treatment group)/(cell viability for control group))×100

(Example 1-10) Morphological changes in nerve cells

SH-SY5Y cells (1×10 ⁶ cells/mL) were cultured for 24 hours, then treated with CM for 4 hours and H ₂ O ₂ for 20 hours, and the morphology of the cells was observed under a microscope.

(Example 1-11) Gene expression investigation of human-derived colon cancer cells and nerve cells

The increase or decrease in BDNF when colon cancer cells were treated with lactic acid bacteria and the increase or decrease in BDNF, TH, Bax, and Bcl-2 when CM was treated in nerve cells were measured through RT-PCR. HT-29 cells were cultured at 5×10 ⁶ cells/mL in a 6-well plate and treated with dead lactic acid bacteria for 24 hours. SH-SY5Y cells were cultured at 1×10 ⁶ cells/well in a 6-well plate and treated with CM for 4 hours and H ₂ O ₂ for 6 hours. Afterwards, RNA was extracted from each cell, converted to cDNA, and RT-PCR was performed to measure changes in gene expression.

(Example 1-12) Measurement of Caspase-3 activity

Caspase-3 activity was measured using the Caspase-3/cpp32 colorimetric assay kit. SH-SY5Y cells were cultured in a 6-well plate at 1×10 ⁶ cells/well, first treated with CM for 4 hours, and then treated with H ₂ O ₂ for 3 hours. Cells were lysed with cell lysis buffer and centrifuged at 10,000 × g for 1 minute to obtain lysates. Each sample protein is quantified, and the lysate containing 50 μg of protein is diluted in 50 μL of cell lysis buffer and added to 50 μL of 2×reaction buffer containing 10 mM DTT. Add 5 μL of 4 mM DEVD-pNA substrate and react at 37°C for 2 hours. Caspase-3 activity was determined by measuring absorbance at 405 nm compared to a control group that did not induce oxidative stress.

Identification and phylogenetic tree confirmation of Lactobacillus brevis KU1512 strain

The 16S rRNA of a new strain (KU15152) isolated from Chonggak kimchi was obtained and its base sequence was analyzed. The 16S rRNA base sequence of the Lactobacillus brevis KU1512 strain represented by SEQ ID NO: 1 is shown in Figure 1. Through blast, it was confirmed that it showed 98.04% similarity to the Lactobacillus brevis strain. Therefore, the strain was named Lactobacillus brevis KU1512 strain (accession number: KCCM 13177P).

Confirmation of acid resistance, bile resistance and intestinal adhesion of Lactobacillus brevis KU1512 strain

To evaluate the probiotic properties of Lactobacillus brevis KU1512 strain, acid resistance and bile resistance were measured.

As a result, when treated for 3 hours at pH 2.5 and 0.3% pepsin, the Lactobacillus brevis KU1512 strain was 99.36% and the comparative strain Lactobacillus rhamnosus GG was 98.35% (Table 1). In addition, to evaluate resistance to bile, when treated with 0.3% Oxgall for 24 hours, the Lactobacillus brevis KU15152 strain showed 108.24% and the comparative strain Lactobacillus rhamnosus GG showed 100.48% (Table 1). When the intestinal adhesion of Lactobacillus brevis KU15152 strain was measured, it showed an adhesion ability of 12.92% (Table 1). This was found to be similar to the attachment ability of Lactobacillus rhamnosus GG, which was a comparative strain of 12.04%.

Probiotic properties of Lactobacillus rhamnosus GG and Lactobacillus brevis KU1512 strains L. rhamnosus GG L. brevis KU15152 Tolerance to artificial gastric acid 98.35±2.65% 99.36±0.81% Tolerance to artificial bile salt 100.48±1.11% 108.24±3.74% Adhesion ability 12.04±1.71% 12.92±2.61%

Confirmation of enzyme production ability of Lactobacillus brevis KU1512 strain

An enzyme production ability experiment was conducted to determine whether Lactobacillus brevis KU15152 strain produces useful or harmful enzymes.

As a result, Lactobacillus brevis KU15152 strain and Lactobacillus rhamnosus GG did not produce β-glucuronidase, which is known to be a carcinogenic enzyme (Table 2).

Enzyme production capacity of Lactobacillus rhamnosus GG and Lactobacillus brevis KU1512 strains Enzymes L. rhamnosu sGG L. brevis KU15152 Control - - Esterase (C4) + + Esterase lipase (C8) + + Leucine arylamidase + + Valine arylamidase + + Cystine arylamidase + + Acid phosphatase + + Naphthol-AS-BI-phosphohydrolase + + α-Galactosidase + - β-Galactosidase + + β-Glucuronidase - - α-Glucosidase - + β-Glucosidase + + N-Acetyl-β-glucosaminidase + -

+, Production; -, Non-production

Confirmation of anti-inflammatory activity of Lactobacillus brevis KU1512 strain in intestinal cell model

It was confirmed whether Lactobacillus brevis KU1512 strain has anti-inflammatory activity in an intestinal cell model. To confirm the cytotoxicity potential, the cell survival rate of HT-29 enterocytes was measured. Additionally, the expression level of IL-8 was measured in HT-29 cells in which an inflammatory response was induced by aLTA.

As a result, strains of 7 log CFU/mL did not negatively affect the survival rate of enterocytes (Figure 3A), and the effect of cytotoxicity was excluded in subsequent experiments. Although the expression level of IL-8 was significantly increased by aLTA, it was confirmed that Lactobacillus brevis KU15152 strain suppressed it most significantly (Figure 3B).

Confirmation of mechanism related to anti-inflammatory activity of Lactobacillus brevis KU15152 strain

The mechanism through which the anti-inflammatory activity of Lactobacillus brevis KU15152 strain is achieved was verified in an intestinal cell model. When the immune system receives external stimulation, signal transduction begins, and activated NF-κB moves into the nucleus and mediates the transcription of inflammatory factors. In addition, MEK and ERK1/2 are key factors in the MEK/ERK signaling mechanism and react with various substrates in the cytoplasm and nucleus. Their activation catalyzes the activation of transcription factors, which results in the expression of several genes related to cell differentiation, proliferation, and apoptosis. Akt can activate lymphoma-related X protein in B cells and inhibit mitochondrial membrane potential and apoptosis. Phosphorylated Akt mediates the phosphorylation of GSK 3β, which plays a major role in apoptosis.

Figure 4 shows how Lactobacillus brevis KU15152 strain affects the activation of NF-κB in HT-29 cells. Treatment with aLTA activated p65, a component of NF-κB, and Lactobacillus brevis KU15152 strain inhibited it. In addition, both MEK and ERK 1/2 were phosphorylated by aLTA, but it was confirmed that Lactobacillus brevis KU15152 strain significantly inhibited this (Figure 5). aLTA phosphorylated Akt and GSK 3β, but Lactobacillus brevis KU15152 strain effectively inhibited this activity (Figure 5). Inhibition was confirmed (Figure 6). This indicates that the anti-inflammatory activity shown in the intestinal cell model of Lactobacillus brevis KU15152 strain was achieved by inhibiting the activation of NF-κB by regulating MEK/ERK and Akt/GSK 3β.

Confirmation of antioxidant activity of dead cells of Lactobacillus brevis KU15152 strain

The antioxidant activity of dead cells of the strain was evaluated by DPPH radical scavenging activity and ABTS radical scavenging activity.

As a result, in the DPPH radical scavenging ability test, Lactobacillus rhamnosus GG and Lactobacillus brevis KU15152 showed similar levels, and Lactobacillus brevis KU15159 strain and Lactobacillus brevis KU15176 strain had lower activity than Lactobacillus rhamnosus GG and Lactobacillus brevis KU15152 strain. It showed antioxidant activity. In the case of ABTS radical scavenging ability, Lactobacillus brevis KU15152 and Lactobacillus brevis KU15159 showed similar scavenging ability. Overall, Lactobacillus brevis KU15152 showed excellent antioxidant activity.

Antioxidant activity evaluation results of dead cells
Antioxidant activity LAB strains L. rhamnosus GG L. brevis KU15152 L. brevis KU15159 L. brevis KU15176 DPPH radical scavenging activity (%) 12.60± ^1.61a 14.25± ^1.86a 9.29±1.24 ^b 7.66±2.49 ^b ABTS radical scavenging activity (%) 50.42±1.54 ^b 52.85± ^3.15a 52.68± ^1.27a 46.20± ^1.42c

Confirmation of neuronal cell death protective function of metabolite (CM) derived from Lactobacillus brevis KU15152 strain

Before confirming the protective effect of CM on neuronal cell death by inducing oxidative stress, MTT assay was performed to evaluate the cytotoxicity of CM. In addition, MTT assay was performed to measure the neuronal protective effect of lactic acid bacteria CM by treating nerve cells with H ₂ O ₂ , which acts as oxidative stress.

As a result, all four strains, including the comparison strain, showed no toxicity to nerve cells (Figure 7). In addition, when the cell survival rate of the control group not treated with H ₂ O ₂ was set to 100%, the survival rate of cells treated with only H ₂ O ₂ was measured to be 50.26 ± 4.26% (FIG. 8). When Lactobacillus rhamnosus GG, Lactobacillus brevis KU15152, Lactobacillus brevis KU15159, and Lactobacillus brevis KU15176 were treated with CM and H2O2, they were 63.64±3.03%, 64.45±5.73%, 53.90±1.83%, and 53.93±2.03%, respectively. survival rate of was shown (Figure 8).

Confirmation of morphological changes in nerve cells

In control cells that were not treated with H ₂ O ₂ , cell bodies and neurites were clearly observed (Figure 9). In cells treated with H ₂ O ₂ , shrinkage and aggregation of the cell body were found. In cells treated with CM of Lactobacillus rhamnosus GG and Lactobacillus brevis KU15152 strains, cell contraction and aggregation were weakened, showing a similar appearance to the control group (Figure 9).

Confirmation of gene expression in human-derived colon cancer cells and nerve cells

(Example 10-1) Confirmation of BDNF expression level in human-derived colon cancer cells HT-29

RT-PCR was performed to measure the amount of change in the BDNF gene when HT-29 cells were treated with dead cells of Lactobacillus brevis KU15152 strain. In the control group, the experiment was conducted by treating the cells with PBS instead of dead cells.

As a result, when HT-29 cells were treated with Lactobacillus rhamnosus GG and Lactobacillus brevis KU15152, the increase was 1.35-fold and 1.45-fold, respectively, compared to the control group, whereas Lactobacillus brevis KU15159 and Lactobacillus brevis KU15176 increased 1.12-fold and 1.07-fold, respectively. The expression level of the embryo was shown (Figure 10). Through this, it was confirmed that Lactobacillus brevis KU15152 showed higher BDNF performance than Lactobacillus rhamnosus GG.

(Example 10-2) H ₂ O ₂ Confirmation of BDNF and TH expression levels in human neuroblastoma SH-SY5Y induced cytotoxicity

To measure the neuroprotective effect of CM, SH-SY5Y was treated with CM of Lactobacillus brevis KU15152 strain, cytotoxicity was added with H ₂ O ₂ , and changes in BDNF and TH were evaluated by RT-PCR.

As a result, when neurons were treated with H ₂ O ₂ alone, the expression level of BDNF decreased to 0.75-fold compared to the control group, but when treated with CM of Lactobacillus rhamnosus GG and Lactobacillus brevis KU15152 strains, it increased to 1.36-fold and 3.96-fold, respectively ( Figure 11). The expression level of TH decreased by 0.74 times when nerve cells were treated with H ₂ O ₂ alone, but when treated with CM of Lactobacillus rhamnosus GG and Lactobacillus brevis KU15152 strains, the expression level was 1.22 times and 4.62 times higher, respectively. (Figure 11). Through this, it was confirmed that CM of Lactobacillus brevis KU15152 strain alleviates the reduction of BDNF and TH caused by H ₂ O ₂ .

(Example 10-3) H ₂ O ₂ Confirmation of expression level of apoptosis-related genes (Bax, Bcl-2) in human neuroblastoma SH-SY5Y cells with induced cytotoxicity

To measure the neuronal protective effect of CM, after treatment with CM and H ₂ O ₂ , changes in Bax, an apoptosis-inducing factor, and Bcl-2 genes, an apoptosis-inhibiting factor, were evaluated by RT-PCR to determine Bax/Bcl-2. Expressed as a ratio.

As a result, when nerve cells were treated with H ₂ O ₂ alone, the ratio increased 2.40 times compared to the control group, but when treated with CM of Lactobacillus rhamnosus GG and Lactobacillus brevis KU15152 strains, expression was confirmed to be 1.43-fold and 0.76-fold, respectively. (Figure 12). Through this, it was confirmed that CM prepared from Lactobacillus brevis KU15152 strain alleviates the apoptosis-inducing action caused by H ₂ O ₂ .

Caspase-3 activity measurement results

When nerve cells were treated with H ₂ O ₂ alone, the activity of caspase-3 increased to 305.53% compared to the control group (FIG. 13). Lactobacillus rhamnosus GG and Lactobacillus brevis KU15152 strains showed activities of 273.11% and 233.33%, respectively (Figure 13). Through this, it was confirmed that treatment with CM of the Lactobacillus brevis KU15152 strain exhibited a neuronal protective effect.

Lactobacillus brevis strain comparison results

The neuroprotective effect of the gut-brain axis is related to the antioxidant, anti-inflammatory, and BDNF production capacity of enterocytes. After confirming the anti-inflammatory effect in enterocytes, we compared the ability of enterocytes to produce IL-8 when inflammation was induced using aLTA. Lactobacillus brevis 15151 strain and Lactobacillus brevis 15153 strain were used as comparison strains.

As a result, the anti-inflammatory effect appeared in the following order: Lactobacillus brevis KU15152> Lactobacillus rhamnosus GG> Lactobacillus brevis KU15151> Lactobacillus brevis KU15153.

In addition, in relation to its ability to protect nerve cells, the neurotoxicity of Lactobacillus brevis strain CM was confirmed using the MTT assay on nerve cells (SH-SY5Y cells) damaged by oxidative stress (H ₂ O ₂ ).

As a result, cell viability was reduced to 48.36% due to oxidative stress, while Lactobacillus brevis KU15152 strain showed the highest cytoprotective efficacy at 64.45% (Table 4).

Comparison of CM's neuronal protection ability Treatments Cell viability(%) Control 100.00±1.50 Control + H ₂ O ₂ (150 μM) 48.36±4.95 Lactobacillus rhamnosus GG 43.71±9.38
Lactobacillus brevis G1 45.19±3.69 Lactobacillus brevis KU15152 64.45±5.73 Lactobacillus brevis KU15176 53.93±2.03 Lactobacillus brevis KU15006 52.51±0.87 Lactobacillus brevis B7 39.29±5.16 Lactobacillus brevis KU15159 53.90±1.83

Korea Center for Microbial Conservation (KCCM) KCCM13177P 20220511

Claims (9)

Lactobacillus brevis KU1512 strain (KCCM 13177P), which has a protective effect against neuronal cell death.
According to clause 1,
A strain characterized in that the neuronal cell death is induced by oxidative stress.
According to clause 1,
A strain, characterized in that the protective effect against nerve cell death is due to the action of metabolites produced through a reaction between the strain and enterocytes.
According to clause 1,
The strain includes the 16S rRNA base sequence of SEQ ID NO: 1.
A pharmaceutical composition for preventing or treating neurodegenerative diseases comprising Lactobacillus brevis KU1512 strain (KCCM 13177P), dead cells thereof, and/or metabolites thereof as active ingredients.
According to clause 5,
The above-described neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, Lou Gehrig's disease, Huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis, immune system abnormalities, brain dysfunction, progressive neurodegenerative diseases, metabolic brain diseases, Niemann-Pick disease, cerebral ischemia, and cerebral hemorrhage. A composition comprising at least one selected from the group consisting of dementia.
A pharmaceutical composition for preventing or treating depression comprising Lactobacillus brevis KU1512 strain (KCCM 13177P), dead cells thereof, and/or metabolites thereof as active ingredients.
A health functional food comprising Lactobacillus brevis KU1512 strain (KCCM 13177P), its dead cells, and/or its metabolites as active ingredients.
A probiotic composition comprising Lactobacillus brevis KU1512 strain (KCCM 13177P), its dead cells, and/or its metabolites as active ingredients.