Levilactobacillus parabrevis LBMB320041 strain and composition comprising same for preventing, alleviating or treating degenerative brain diseases

Abstract

The present invention relates to Levilactobacillus parabrevis LBMB320041 strain and a composition comprising same for preventing, alleviating or treating degenerative brain diseases, and exhibits excellent preventive and therapeutic effects on various degenerative brain diseases, and thus can be effectively utilized as a composition for uses of the treatment, prevention, alleviation, or the like for degenerative brain diseases in humans or animals.

Description

Revillactobacillus parabrevis strain LBMB320041 and composition for preventing, improving or treating degenerative brain diseases comprising the same

[0001] The present invention relates to a strain of Revillactobacillus parabrevis LBMB320041 and a composition comprising the strain for preventing, improving or treating degenerative brain diseases.

[0002] Memory is increasingly important in modern society, driven by the rapidly changing demands of daily life. It is a major concern for individuals ranging from adolescence, who often experience significant learning, to the elderly. Degenerative brain diseases are known to cause memory impairment, and cerebral inflammatory responses have been identified as a key factor in degenerative brain diseases of the central nervous system, such as Alzheimer's disease, Parkinson's disease, and Huntington's disease (Jin DQ et al., Biochemical and Biophysical Research Communications, 331: 1264-1269, 2005; Lim CS et al., Biological and Pharmaceutical Bulletin, 29: 1212-1216, 2006).

[0003] Parkinson's disease (PD) is the second most common neurodegenerative disease after Alzheimer's disease. It is a progressive neurodegenerative disorder characterized by the progressive degeneration of dopaminergic neurons distributed from the substantia nigra to the striatum. Currently, dopamine replacement therapy, such as L-DOPA, and dopamine receptor agonists are used to treat PD. However, long-term administration of these drugs is known to cause serious side effects, such as dyskinesia.

[0004] Recently, due to the increase in the elderly population and the rapid increase in the number of patients with degenerative brain diseases such as dementia, various treatment strategies are being established and efforts are being made to develop effective drugs to improve and enhance cognitive and learning functions that have declined due to dementia and other diseases. Memory-enhancing drugs developed to date include acetylcholine precursors, receptor agonists, and acetylcholine esterase inhibitors. However, there is still no treatment that can treat the fundamental cause of degenerative brain diseases. Available general treatments include acetylcholine esterase inhibitors such as Pfizer's Aricept, Novartis' Exelon, and Janssen's Reminyl, as well as Lundbeck's Ebixa (Memantine), an NMDA receptor antagonist that recently received approval from the US FDA. However, acetylcholinesterase inhibitors only improve cognitive decline and have limitations in treating the underlying cause of Alzheimer's disease. Furthermore, they only provide temporary symptom relief in some patients, and their effects are short-lived, making it difficult to expect a fundamental therapeutic effect. Furthermore, the nature of degenerative brain diseases necessitates long-term use of these medications, which can cause various side effects, including liver toxicity, vomiting, and loss of appetite. Therefore, the development of new treatments that can halt the progression of degenerative brain diseases is becoming an urgent task.

[0005] Meanwhile, lactic acid bacteria are widely distributed in the oral cavity, intestines, vagina, feces, and fermented foods like kimchi of humans and animals, and are closely related to human and animal health. Lactic acid bacteria exhibit various health-promoting effects, including intestinal intestinal health, suppression of harmful bacteria, immune regulation, lowering blood cholesterol, and anticancer effects. Furthermore, recent research has revealed that gut microbes actively influence not only human intestinal health and intestinal diseases, but also brain function regulation and the development of brain diseases.

[0006] Meanwhile, Korean Patent No. 10-2404016 discloses 'Lactococcus lactis subsp. Hordniae strain and its use for preventing, improving or treating degenerative brain disease', and Korean Patent No. 10-1799830 discloses 'Agathobaculum strain having a preventive or therapeutic effect on degenerative brain disease and its use', but the preventive or therapeutic effect on degenerative brain disease using a novel Revillactobacillus parabrevis strain or a culture thereof as an active ingredient has not been revealed.

[0007] One object of the present invention is to provide a strain of Levilactobacillus parabrevis LBMB320041 (Accession No.: KCTC15607BP) having excellent alpha-synuclein aggregation inhibitory activity.

[0008] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating degenerative brain diseases, which comprises the strain or a culture thereof as an active ingredient.

[0009] Another object of the present invention is to provide a food composition for preventing or improving degenerative brain diseases, which comprises the strain or a culture thereof as an effective ingredient.

[0010] Another object of the present invention is to provide a food additive composition for preventing or improving degenerative brain diseases, which comprises the strain or a culture thereof as an effective ingredient.

[0011] Another object of the present invention is to provide a feed composition for preventing or improving degenerative brain disease in animals, which comprises the strain or a culture thereof as an effective ingredient.

[0012] Another object of the present invention is to provide a feed additive composition for preventing or improving degenerative brain diseases in animals, comprising the strain or a culture thereof as an effective ingredient.

[0013] One aspect of the present invention provides a strain of Levilactobacillus parabrevis LBMB320041 (Accession No.: KCTC15607BP) having excellent alpha-synuclein aggregation inhibitory activity.

[0014] Another aspect of the present invention provides a pharmaceutical composition for preventing or treating a degenerative brain disease, comprising the strain or a culture thereof as an active ingredient.

[0015] According to one specific example of the present invention, the degenerative brain disease may be any one disease selected from the group consisting of Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, Creutzgeldt-Jacob disease, stroke, and multiple sclerosis.

[0016] According to one specific example of the present invention, the composition can be administered orally or parenterally.

[0017] According to one specific example of the present invention, the parenteral administration may be intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, intradermal administration, topical administration, intranasal administration, intrapulmonary administration, or rectal administration.

[0018] Another aspect of the present invention provides a food composition for preventing or improving degenerative brain diseases, comprising the strain or a culture thereof as an active ingredient.

[0019] According to one specific example of the present invention, the food may be a health functional food.

[0020] Another aspect of the present invention provides a food additive composition for preventing or improving degenerative brain diseases, comprising the strain or a culture thereof as an active ingredient.

[0021] Another aspect of the present invention provides a feed composition for preventing or improving degenerative brain disease in animals, comprising the strain or a culture thereof as an effective ingredient.

[0022] Another aspect of the present invention provides a feed additive composition for preventing or improving degenerative brain disease in animals, comprising the strain or a culture thereof as an active ingredient.

[0023] According to the Revillactobacillus parabrevis strain LBMB320041 and the composition for preventing, improving or treating degenerative brain diseases comprising the same, it exhibits excellent preventive and therapeutic effects on various degenerative brain diseases, and therefore can be usefully utilized as a composition for the treatment, prevention or improvement of degenerative brain diseases in humans or animals.

[0024] Figure 1 is a graph showing the results of taking pictures using a fluorescence microscope to confirm the alpha-synuclein aggregation inhibition effect by treating a composition according to the present invention after inducing alpha-synuclein aggregation, and calculating the GFP fluorescence value area ratio compared to RFP.

[0025] Figure 2 is a graph showing the results of measuring fluorescence values ​​to confirm the degree of inhibition of Mao-B activity by treating the composition according to the present invention.

[0026] Figure 3 is a diagram showing the results of confirming cell viability to confirm the degree of protection of nerve cells by treating the composition according to the present invention.

[0027] Figure 4 is a graph showing the results of measuring the amount of dopamine (DA) in brain proteins to confirm the degree of increase in dopamine levels in a Parkinson's mouse model following treatment with a composition according to the present invention.

[0028] FIG. 5 is a diagram and graph showing the relative expression level of alpha-synuclein protein compared to tubulin to confirm the degree of alpha-synuclein inhibition in a Parkinson's mouse model according to treatment with a composition according to the present invention.

[0029] FIG. 6 is a graph showing the results of staining brain tissue with tyrosine hydroxylase (TH) to confirm the degree of inhibition of dopaminergic neuron death in a Parkinson's mouse model according to treatment with a composition according to the present invention, and calculating the relative optical density of the striatum or the number of dopaminergic neurons in the substantia nigra pars compacta of the midbrain.

[0030] One aspect of the present invention provides a strain of Levilactobacillus parabrevis LBMB320041 (Accession No.: KCTC15607BP) having excellent alpha-synuclein aggregation inhibitory activity.

[0031] The Revillactobacillus parabrevis LBMB320041 strain according to the present invention was isolated from kimchi and deposited at the Biological Resource Center of the Korea Research Institute of Bioscience and Biotechnology on September 14, 2023 (Accession number: KCTC15607BP).

[0032] The above-mentioned Lactobacillus parabrevis strain LBMB320041 has a 16S rRNA gene nucleic acid sequence of SEQ ID NO: 1 to SEQ ID NO: 6 (Table 1).

[0033]

[0034] Sequence number: DNA sequence of the 16S rRNA gene of Lactobacillus parabrevis LBMB320041 (5'→3') Sequence number: Sequence number2ATGCAAATCCTAGAGATAGGACGTTCCCTTCGGGGACATGGATACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTATCAGTTGCCAGCATTAAGTTGGGCACTCTGGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGGTACAACGAGTCGCGAAGTCGTGAGGCTAAGCTAATCTCTTAAAGCCGTTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCAAAGCCGGTGAGATAACCTTAGGGAGTCAGCCGTCTAAGGTGGGACAGATGATTAGGGTGAAGTCGTAACAAGGTAGCCGTAGGAGAACCTGCGGCTGGATCACCTCCTT서열번호3ATGCAAATCCTAGAGATAGGACGTTCCCTTCGGGGACATGGATACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTATCAGTTGCCAGCATTAAGTTGGGCACTCTGGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGGTACAACGAGTCGCGAAGTCGTGAGGCTAAGCTAATCTCTTAAAGCCGTTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCAAAGCCGGTGAGATAACCTTCGGGAGTCAGCCGTCTAAGGTGGGACAGATGATTAGGGTGAAGTCGTAACAAGGTAGCCGTAGGAGAACCTGCGGCTGGATCACCTCCTT서열번호4ATGCAAATCCTAGAGATAGGACGTTCCCTTCGGGGACATGGATACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTATCAGTTGCCAGCATTAAGTTGGGCACTCTGGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGGTACAACGAGTCGCGAAGTCGTGAGGCTAAGCTAATCTCTTAAAGCCGTTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCAAAGCCGGTGAGATAACCTTCGGGAGTCAGCCGTCTAAGGTGGGACAGATGATTAGGGTGAAGTCGTAACAAGGTAGCCGTAGGAGAACCTGCGGCTGGATCACCTCCTT서열번호5ATGCAAATCCTAGAGATAGGACGTTCCCTTCGGGGACATGGATACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTATCAGTTGCCAGCATTAAGTTGGGCACTCTGGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGGTACAACGAGTCGCGAAGTCGTGAGGCTAAGCTAATCTCTTAAAGCCGTTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCAAAGCCGGTGAGATAACCTTCGGGAGTCAGCCGTCTAAGGTGGGACAGATGATTAGGGTGAAGTCGTAACAAGGTAGCCGTAGGAGAACCTGCGGCTGGATCACCTCCTT서열번호

[0035]

[0036] The above Revillactobacillus parabrevis LBMB320041 strain can be used by inoculating 0.1 to 10% of the MRS liquid medium and culturing it at 25 to 37°C for 4 to 48 hours.

[0037] The above cultivation method is preferably a static cultivation method, but is not limited thereto.

[0038] Another aspect of the present invention provides a pharmaceutical composition for preventing or treating a degenerative brain disease, comprising the strain or a culture thereof as an active ingredient.

[0039] A composition comprising the Revillactobacillus parabrevis LBMB320041 strain of the present invention or a culture thereof as an active ingredient has a preventive or therapeutic effect on degenerative brain diseases and can be used as a pharmaceutical composition.​

[0040] The Revillactobacillus parabrevis LBMB320041 strain of the present invention is a lactic acid bacteria strain. The Revillactobacillus parabrevis LBMB320041 strain is a probiotic and has the general intestinal and immune-enhancing effects of lactic acid bacteria. It is a well-known fact that lactic acid bacteria of the genus Revillactobacillus have intestinal and immune-enhancing effects.

[0041] In the present invention, "probiotics" are understood to mean live microorganisms that improve the intestinal microbial environment of the host within the gastrointestinal tract of animals, including humans, and thus have a beneficial effect on the health of the host. Probiotics are live microorganisms with probiotic activity. When administered to humans or animals in the form of dried cells or fermented products, either as single or complex strains, they can have a beneficial effect on the host's intestinal flora.

[0042] According to one specific example of the present invention, the degenerative brain disease may be any one disease selected from the group consisting of Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, Creutzgeldt-Jacob disease, stroke, and multiple sclerosis.

[0043] The above degenerative brain disease may be, but is not limited to, Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, Creutzgeldt-Jacob disease, stroke, or multiple sclerosis.

[0044] The Revillactobacillus parabrevis LBMB320041 strain included in the composition according to the present invention may exist as a live or dead cell body, and may also exist in a dried or lyophilized form. In addition, a culture of the Revillactobacillus parabrevis LBMB320041 strain may be the active ingredient, and the culture may include a live cell culture solution or a dead cell supernatant. The form and formulation method of lactic acid bacteria suitable for inclusion in various compositions are well known to those skilled in the art.

[0045] According to one specific example of the present invention, the composition can be administered orally or parenterally.

[0046] According to one specific example of the present invention, the parenteral administration may be intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, intradermal administration, topical administration, intranasal administration, intrapulmonary administration, or rectal administration.

[0047] The composition may be administered orally or parenterally. Parenteral administration may include intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, intradermal administration, topical administration, intranasal administration, intrapulmonary administration, and rectal administration. Preferably, intravenous injection is used, but is not limited thereto.

[0048] The appropriate dosage of the above composition can be prescribed in various ways depending on factors such as the formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and reaction sensitivity.

[0049] When the composition of the present invention is used as a pharmaceutical composition, the pharmaceutical composition of the present invention can be prepared using pharmaceutically suitable and physiologically acceptable auxiliary agents in addition to the above-mentioned effective ingredient, and the auxiliary agents can include excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, glidants, or flavoring agents.

[0050] The above pharmaceutical composition may be preferably formulated as a pharmaceutical composition by including one or more pharmaceutically acceptable carriers in addition to the above-described effective ingredients for administration.

[0051] For example, for formulation in the form of tablets or capsules, the active ingredient may be combined with an orally acceptable, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. In addition, if desired or necessary, suitable binders, lubricants, disintegrants, and coloring agents may also be included in the mixture. Suitable binders include, but are not limited to, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, trackacanth, or sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. Disintegrants include, but are not limited to, starch, methylcellulose, agar, bentonite, xanthan gum, and the like. In the composition to be formulated as a liquid solution, acceptable pharmaceutical carriers include those that are sterile and biocompatible, such as saline solution, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and mixtures of one or more of these components. If necessary, other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate the composition into injectable formulations such as aqueous solutions, suspensions, and emulsions, pills, capsules, granules, or tablets.

[0052] Furthermore, it can be preferably formulated according to each disease or ingredient using the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA, as an appropriate method in the relevant field.

[0053] Another aspect of the present invention provides a food composition for preventing or improving degenerative brain diseases, comprising the strain or a culture thereof as an active ingredient.

[0054] Another aspect of the present invention provides a food additive composition for preventing or improving degenerative brain diseases, comprising the strain or a culture thereof as an active ingredient.

[0055] According to one specific example of the present invention, the food may be a health functional food.

[0056] The above food composition may be in the form of a health functional food.

[0057] The above “health functional food” refers to a food manufactured and processed using raw materials or ingredients with functionality useful to the human body in accordance with the Health Functional Food Act (Article 3, Paragraph 1), and functionality means obtaining a useful effect for health purposes, such as regulating nutrients or physiological actions for the structure and function of the human body (Article 2, Paragraph 2).

[0058] The above food composition may additionally contain food additives, and its suitability as a food additive is determined by the specifications and standards for the relevant item in accordance with the general provisions and general test methods of the Food Additives Codex approved by the Ministry of Food and Drug Safety, unless otherwise specified.

[0059] Examples of items listed in the above food additive code include chemically synthesized products such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamic acid; natural additives such as persimmon pigment, licorice extract, crystalline cellulose, and guar gum; and mixed preparations such as sodium L-glutamate preparations, alkaline agents for noodles, preservative preparations, and tar color preparations.

[0060] Foods containing the active ingredient of the present invention include confectionery such as bread, rice cakes, dried fruits, candies, chocolates, chewing gums, and jams; ice cream products such as ice cream, frozen desserts, and ice cream powders; dairy products such as milk, low-fat milk, lactose-hydrolyzed milk, processed milk, goat milk, fermented milk, buttermilk, condensed milk, milk cream, buttermilk, natural cheese, processed cheese, powdered milk, and whey; meat products such as processed meat products, processed egg products, and hamburgers; fish products such as processed fish products such as fish cakes, ham, sausage, and bacon; noodles such as ramen, dried noodles, fresh noodles, fried noodles, luxurious dried noodles, improved boiled noodles, frozen noodles, and pasta; beverages such as fruit drinks, vegetable drinks, carbonated drinks, soy milk, and yogurt, and mixed drinks; soy sauce, soybean paste, red pepper paste, soybean paste, cheonggukjang, mixed sauce, vinegar, Condiments such as sauces, tomato ketchup, curry, dressings, margarine, shortening, and pizza are included, but are not limited to these.

[0061] In addition to the above, the composition of the present invention may include various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the composition of the present invention may include fruit pulp for the production of natural fruit juice, fruit juice drinks, and vegetable drinks. These ingredients may be used independently or in combination.

[0062] The beverage composition including the active ingredient of the present invention has no particular limitations on other ingredients, and may contain various flavoring agents or natural carbohydrates as additional ingredients, like conventional beverages. Examples of the above-mentioned natural carbohydrates include conventional sugars such as monosaccharides (e.g., glucose, fructose, etc.); disaccharides (e.g., maltose, sucrose, etc.); and polysaccharides (e.g., dextrin, cyclodextrin, etc.), and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (thaumatin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used.

[0063] Another aspect of the present invention provides a feed composition for preventing or improving degenerative brain disease in animals, comprising the strain or a culture thereof as an effective ingredient.

[0064] Another aspect of the present invention provides a feed additive composition for preventing or improving degenerative brain disease in animals, comprising the strain or a culture thereof as an active ingredient.

[0065] When the composition is manufactured as a feed additive, the composition may be a highly concentrated solution of 20 to 90% or may be manufactured in the form of a powder or granules. The feed additive may further include one or more of organic acids such as citric acid, fumaric acid, adipic acid, lactic acid, and malic acid; phosphates such as sodium phosphate, potassium phosphate, acid pyrophosphate, and polyphosphate (polyphosphate); and natural antioxidants such as polyphenol, catechin, alpha-tocopherol, rosemary extract, vitamin C, green tea extract, licorice extract, chitosan, tannic acid, and phytic acid. When manufactured as a feed, the composition may be formulated in the form of a conventional feed and may include conventional feed ingredients together.

[0066] The feed and feed additives may further include grains, such as ground or shredded wheat, oats, barley, corn, and rice; plant-based protein feeds, such as feeds mainly composed of rapeseed, soybeans, and sunflower; animal-based protein feeds, such as blood meal, meat meal, bone meal, and fish meal; dry ingredients composed of sugars and dairy products, such as various types of milk powder and whey powder, and may further include nutritional supplements, digestion and absorption enhancers, growth promoters, and the like.

[0067] The feed additive may be administered to animals alone or in combination with other feed additives in an edible carrier. Furthermore, the feed additive may be readily administered to animals as a top dressing, by mixing it directly into animal feed, or in an oral formulation separate from the feed. When the feed additive is administered separately from animal feed, it may be prepared as an immediate-release or sustained-release formulation by combining it with a pharmaceutically acceptable edible carrier, as is well known in the art. Such edible carriers may be solid or liquid, such as cornstarch, lactose, sucrose, soybean flakes, peanut oil, olive oil, sesame oil, and propylene glycol. When a solid carrier is used, the feed additive may be in the form of a tablet, capsule, powder, troche, or saccharide tablet, or a top dressing in a microdispersible form. When a liquid carrier is used, the feed additive may be in the form of a gelatin soft capsule, or a syrup, suspension, emulsion, or solution.

[0068] Additionally, the feed and feed additives may contain auxiliary agents, such as preservatives, stabilizers, wetting or emulsifying agents, and solution accelerators. The feed additives may be added to animal feed by injecting, spraying, or mixing.

[0069] The feed or feed additive of the present invention can be applied to the diets of many animals, including mammals, poultry and fish.

[0070] As the mammals, it can be used not only for pigs, cows, horses, sheep, rabbits, goats, rodents and laboratory rodents such as rats, hamsters, and guinea pigs, but also for pets (e.g., dogs, cats), as well as for poultry such as chickens, turkeys, ducks, geese, pheasants, and quail, as well as for fish such as carp, crucian carp, and trout, but is not limited thereto.

[0071] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples, in accordance with the gist of the present invention.

[0072]

[0073] Example 1. Cultivation of strains

[0074] The strain Revillactobacillus parabrevis LBMB320041 was deposited at the Biological Resource Center of the Korea Research Institute of Bioscience and Biotechnology on September 14, 2023 (Accession number: KCTC15607BP).

[0075] Levilactobacillus parabrevis LBMB320041 strain was inoculated with 0.1% (v / v) of the strain stock in 30 ml of MRS liquid medium and cultured at 30°C for 48 hours.

[0076]

[0077] Example 2. Preparation of strain culture solution

[0078] The Revillactobacillus parabrevis LBMB320041 strain of Example 1 was washed three times with PBS (phosphate buffered saline) solution to remove remaining medium components, divided into three parts, and cultured in 10 ml of cell culture media (RPMI1640, DMEM, MEM) at 30°C for 24 hours. After culture, the culture solution was separated by centrifugation at 3500 rpm for 10 minutes, adjusted to pH 7.2, and passed through a 0.2 μm filter to prepare a strain culture solution. The prepared strain culture solution was used in the experiments of Examples 3 to 5 thereafter.

[0079]

[0080] Example 3. Confirmation of alpha-synuclein aggregation inhibition efficacy.

[0081] To determine the inhibitory effect of Revillactobacillus parabrevis strain LBMB320041 on alpha-synuclein aggregation, 1 x 10 SH-SY5Y cell line stably expressing red fluorescent alpha-synuclein was incubated with 5Cells / well were cultured in a 4-well chamber, and PFF (pre-formed fibrils) were diluted in the medium and used at 4 μg / ml to induce alpha-synuclein aggregation. The Revillactobacillus parabrevis LBMB320041 strain cultured in Example 2 was diluted to 5% of the total volume of the medium and used, and UCB0599, an alpha-synuclein aggregation inhibitor, was used at 2 μM as a control group. After incubation in a CO2 incubator for 48 hours and fixation with 4% PFA (paraformaldehyde), aggregated alpha-synuclein was stained with ThT (Thioflavin T) and nuclei were stained with DAPI. GFP, RFP, and DAPI were measured at 400x magnification using a fluorescence microscope. The GFP and RFP fluorescence values ​​were calculated by calculating the area above a threshold of 30 using ImageJ, and the results of calculating the GFP area ratio to RFP are shown in Figure 1.

[0082] As shown in Fig. 1, compared to the control group, alpha-synuclein aggregation increased in the group treated with PFF, and the group treated with the Revylactobacillus parabrevis LBMB320041 strain culture showed a 60% aggregation inhibition rate. In light of the above results, it was confirmed that a composition containing the Revylactobacillus parabrevis LBMB320041 strain as an active ingredient or a culture thereof as an active ingredient can alleviate Parkinson's disease by inhibiting the aggregation of alpha-synuclein in nerve cells.

[0083]

[0084] Example 4. Confirmation of Mao-B activity inhibition efficacy

[0085] To determine the inhibitory effect of Revillactobacillus parabrevis strain LBMB320041 on monoamine oxidase B activity, 5 x 10 C8-D1A cell line was cultured. 4Cells / well were cultured in 96-well plates, and the Revillactobacillus parabrevis LBMB320041 strain culture of Example 2 was diluted to 5% of the total volume of the medium and used. After culturing and reacting in a CO2 incubator for 24 hours, the cells were washed with PBS and the fluorescence value was measured at 530-560 nm (excitation) and 590 nm (emission) after 3 hours according to the experimental method using the Amplex Red Monoamine Oxidase Assay Kit. The graph that quantifies the results of the fluorescence value measurement is shown in Fig. 2.

[0086] As shown in Fig. 2, it was confirmed that the Bao-B activity was reduced by 22% in the group treated with the Revylactobacillus parabrevis LBMB320041 strain culture. Based on the above results, it was confirmed that a composition containing the Revylactobacillus parabrevis LBMB320041 strain as an active ingredient or a culture thereof as an active ingredient can inhibit dopamine degradation in Parkinson's disease by reducing the Mao-B activity of nerve cells.

[0087]

[0088] Example 5. Confirmation of neuroprotective efficacy

[0089] To confirm the neuroprotective effect of Revillactobacillus parabrevis LBMB320041, SH-SY5Y cell line was cultured at 4 x 10 4Cells / well were cultured in 96-well plates, and the Revillactobacillus parabrevis LBMB320041 strain culture solution of Example 2 was diluted to 5% of the total volume of the medium and used. 6-OHDA (6-hydroxydopamine hydrobromide) was diluted in the medium and used at 50 μM. 10 nM of NaHS was used as a control group. After culturing and reacting in a CO2 incubator for 24 hours, cell viability was confirmed using CellTiter 96 Non-Radiocative Cell Proliferation Assay kt, and the results are shown in Fig. 3.

[0090] As shown in Fig. 3, the cell viability of the 6-OHDA-treated group was 15% of the control, whereas the group treated with the culture medium of the Revillactobacillus parabrevis LBMB320041 strain showed a viability of 72%. Based on the above results, it was confirmed that the Revillactobacillus parabrevis LBMB320041 strain can have a neuroprotective effect.

[0091]

[0092] Example 6. Induction of Parkinson's disease using MPTP and probenecid in a mouse model and administration of Revillactobacillus parabrevis strain.

[0093] To determine the preventive, ameliorating, or therapeutic effects of Revillactobacillus parabrevis LBMB320041 strain in a Parkinson's disease mouse model, C57BL / 6 mice were intraperitoneally injected with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and probenecid at doses of 25 mg / kg and 250 mg / kg, respectively. Probenecid was injected 30 minutes in advance, and the injections were performed twice a week for a total of 11 times. Revillactobacillus parabrevis LBMB320041 strain cultured in Example 1 was injected at a density of 1 x 10 9The solution was diluted to 100 μl of colony forming unit (CFU) per 100 μl and administered orally five times a week for a total of 26 times. As a control group, UCB0599, an alpha-synuclein aggregation inhibitor, was injected intraperitoneally at a dose of 1 mg / kg.

[0094]

[0095] Example 7. Confirmation of the efficacy of restoring dopamine levels in a mouse model of Parkinson's disease.

[0096] In order to confirm the efficacy of the Revillactobacillus parabrevis LBMB320041 strain in improving dopamine levels in a Parkinson's disease mouse model, the brains of the Parkinson's disease mice administered the Revillactobacillus parabrevis LBMB320041 strain in Example 6 were extracted, and striatum tissue was isolated from the left brain. The tissue was pulverized in a solution containing 0.01 N hydrogen chloride (HCl) and 1 mM ethylene-diamine-tetraacetic acid (EDTA), and then centrifuged to separate the protein, which was quantified as bovine serum albumin (BSA). The same amount of protein was assayed according to the experimental method of the DA (Dopamine) ELISA kit, and the optical density was measured at 450 nm. The results of calculating the dopamine level per 1 mg of albumin are shown in Figure 4.

[0097] As shown in Fig. 4, in the group in which Parkinson's disease was induced by treatment with MPTP and probenecid, dopamine levels decreased by 28% compared to the control group, whereas in the group administered the Revylactobacillus parabrevis LBMB320041 strain, dopamine levels increased by 46% compared to the Parkinson's disease-induced group. In light of the above results, it was confirmed that administration of the Revylactobacillus parabrevis LBMB320041 strain can treat or improve Parkinson's disease by restoring dopamine levels.

[0098]

[0099] Example 8. Confirmation of alpha-synuclein inhibition efficacy in a Parkinson's disease mouse model.

[0100] To confirm the inhibitory effect of Revillactobacillus parabrevis LBMB320041 strain on alpha-synuclein in a Parkinson's disease mouse model, the brains of Parkinson's disease mice administered Revillactobacillus parabrevis LBMB320041 strain in Example 6 were extracted, and then substantia nigra (SN) tissue was isolated from the left brain. The tissue was pulverized in a solution containing 1% Triton X-100, 2 mM EDTA, 1% protease inhibitor, and 1% phosphatase inhibitor, and then sonicated and centrifuged to separate proteins, which were quantified using BSA. For the same amount of protein, a sample buffer containing sodium dodecyl sulfate (SDS) and β-mercaptoethanol was added and boiled at 98°C for 10 minutes, followed by electrophoresis and transfer. The membrane was blocked with 5% skim milk, and the primary antibody was attached at 4°C overnight or at room temperature for 2 hours. After washing three times with Tris-buffered saline with Tween 20 (TBST), the secondary antibody was attached at room temperature for 1 hour. The protein bands were treated with enhanced chemiluminescence (ECL) solution and analyzed using Imagequant software. The expression values ​​of alpha-synuclein relative to gamma-tubulin were calculated, and the results, normalized to the Parkinson's disease-induced group, are shown in Figure 5.

[0101] As shown in Fig. 5, in the group in which Parkinson's disease was induced by treatment with MPTP and probenecid, the expression of alpha-synuclein polymers increased by 111% compared to the control group, whereas in the group administered with the Revylactobacillus parabrevis LBMB320041 strain, the expression decreased by 12% compared to the Parkinson's disease-induced group. In light of the above results, it was confirmed that administration of the Revylactobacillus parabrevis LBMB320041 strain can treat or improve Parkinson's disease by inhibiting the aggregation of alpha-synuclein.

[0102]

[0103] Example 9. Confirmation of dopaminergic neuron improvement efficacy in a Parkinson's disease mouse model.

[0104] In order to confirm the dopaminergic neuron-improving efficacy of the Revillactobacillus parabrevis LBMB320041 strain in a Parkinson's disease mouse model, the brains of the Parkinson's disease mice administered the Revillactobacillus parabrevis LBMB320041 strain in Example 6 were extracted, and the right brain was fixed in 4% paraformaldehyde at 4°C. The fixed tissues were embedded in paraffin blocks, and the striatum and substantia nigra were each attached to slides at 5 μm thickness and stained with tyrosine hydroxylase, a marker of dopaminergic neurons. The staining results and optical density of the striatum region were measured and normalized to the Parkinson's disease-induced group, and the staining results of the substantia nigra pars compacta (SNpc) region and the number of dopaminergic neurons are shown in Fig. 6b.

[0105] As shown in Fig. 6a, in the group in which Parkinson's disease was induced by treatment with MPTP and probenecid, the expression of dopaminergic neurons in the striatum was reduced by 68% compared to the control group, whereas in the group administered with the Revillactobacillus parabrevis LBMB320041 strain, it was increased by 51% compared to the Parkinson's disease-induced group.

[0106] As shown in Fig. 6b, in the group in which Parkinson's disease was induced by treatment with MPTP and probenecid, the number of dopaminergic neurons in the substantia nigra pars compacta decreased by 67% compared to the control group, whereas in the group administered the Revylactobacillus parabrevis LBMB320041 strain, the number increased by 89% compared to the Parkinson's disease-induced group. In light of the above results, it was confirmed that administration of the Revylactobacillus parabrevis LBMB320041 strain can treat or improve Parkinson's disease by inhibiting the death of dopaminergic neurons.

[0107]

[0108] In summary of the results of the above examples, it was confirmed that a composition containing the Revillactobacillus parabrevis LBMB320041 strain or a culture thereof as an active ingredient can be used as a composition capable of preventing or treating degenerative brain diseases including Parkinson's disease.

[0109]

[0110] The present invention has been described above, focusing on specific embodiments thereof. Those skilled in the art will appreciate that the present invention can be implemented in modified forms without departing from its essential characteristics. Therefore, the disclosed embodiments should be considered illustrative rather than limiting. The scope of the present invention is set forth in the claims, not the foregoing description, and all differences within the scope equivalent thereto should be construed as being encompassed by the present invention.

[0111]

[0112] [Accession number]

[0113] Name of depositor: Korea Research Institute of Bioscience and Biotechnology, Biological Resource Center (KCTC)

[0114] Accession number: KCTC15607BP

[0115] Date of acceptance: 20230914

[0116]

[0117]

Claims

1. Levilactobacillus parabrevis LBMB320041 (Accession Number: KCTC15607BP) strain with excellent alpha-synuclein aggregation inhibition activity.

2. A pharmaceutical composition for the prevention or treatment of degenerative brain disease, containing the strain of Article 1 or a culture thereof as an active ingredient.

3. A pharmaceutical composition for preventing or treating a degenerative brain disease, wherein the degenerative brain disease in the second paragraph is any one disease selected from the group consisting of Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, Creutzgeldt-Jacob disease, stroke, and multiple sclerosis.

4. In the second paragraph, the composition is a pharmaceutical composition for preventing or treating a degenerative brain disease, which is administered orally or parenterally.

5. A pharmaceutical composition for preventing or treating degenerative brain disease, wherein the parenteral administration in paragraph 4 is intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, intradermal administration, local administration, intranasal administration, intrapulmonary administration, or rectal administration.

6. A food composition for preventing or improving degenerative brain disease, containing the strain of Article 1 or a culture thereof as an effective ingredient.

7. A food composition for preventing or improving degenerative brain disease, wherein the food in paragraph 6 is a health functional food.

8. A food additive composition for preventing or improving degenerative brain diseases, comprising the strain of Article 1 or a culture thereof as an effective ingredient.

9. A feed composition for preventing or improving degenerative brain disease in animals, containing the strain of Article 1 or a culture thereof as an effective ingredient.

10. A feed additive composition for preventing or improving degenerative brain disease in animals, comprising the strain of Article 1 or a culture thereof as an effective ingredient.

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