Novel lactic acid bacteria and use thereof

Abstract

The present invention relates to novel lactic acid bacteria, Bifidobacterium bifidum P61 KCCM13367P, Lactobacillus paracasei P62 KCCM13368P, or a mixture thereof. The novel lactic acid bacteria and the mixture thereof of the present invention change the composition of intestinal microorganisms to inhibit the expression of sarcopenia markers (MuRF-1, MAFbx / Atrogin-1, etc.), increase muscle strength and muscle mass, and promote muscle formation, thereby alleviating sarcopenia. In addition, the present invention improves cognitive dysfunction by inhibiting behavior similar to cognitive dysfunction and regulating the expression of factors (BDNF, IL-10, etc.) associated with said dysfunction. In addition, the present invention has the effect of treating inflammation by reducing the expression of inflammatory markers (TNF-α, IL-6, IL-1ß, and MPO) and increasing the expression of an anti-inflammatory marker (IL-10).

Description

Novel lactic acid bacteria and their uses

[0001] The present invention relates to novel lactic acid bacteria, Bifidobacterium bifidum P61 KCCM13367P, Lactobacillus paracasei P62 KCCM13368P, or a mixture thereof, and uses thereof.

[0002] As the global population ages, people are increasingly exposed to various diseases, highlighting the importance of addressing age-related conditions. The most common physiological change associated with aging is the decline in muscle mass and strength. The human body consists of approximately 600 muscles, which account for half of body weight. Muscles are formed by tens of thousands of muscle fibers (muscle cells). Muscle fibers grow in size as they grow, but with age, their number decreases and their function declines.

[0003] Sarcopenia is a disease in which muscle protein breakdown exceeds protein synthesis, leading to a persistent decrease in muscle mass. This condition is primarily caused by reduced physical activity, malnutrition, environmental factors, disease, inflammation, mitochondrial abnormalities, and hormonal changes, although research suggests genetic differences may also play a role. Sarcopenia, a metabolic disease, directly causes muscle loss, which in turn increases the risk of death due to decreased and impaired bodily functions. Furthermore, it also increases the prevalence of metabolic diseases such as hypertension, diabetes, arthritis, obesity, and cancer due to its associated metabolic and immune system declines.

[0004] Another common condition resulting from an aging population is cognitive impairment. Cognitive impairment is a disorder characterized by cognitive impairment and behavioral changes, resulting from a decline in functions such as memory, spatial perception, judgment, executive function, and language abilities. Dementia, associated with cognitive decline, was once considered a disease of the elderly, but with the aging population, it has become a disease that can affect anyone. It is becoming a social problem due to the burden of caregiving and the financial burden on families.

[0005] Meanwhile, probiotics are live bacteria that are distributed as dominant bacteria in the human intestines, where various microorganisms exist, and promote the growth of beneficial bacteria in the body. They live symbiotically in the human digestive system, breaking down fiber and complex proteins to transform them into important nutrients. They also inhibit the growth of harmful bacteria such as Escherichia coli and Clostridium difficile, improve diarrhea and constipation, and play a role in vitamin synthesis and lowering blood cholesterol. Among these probiotics, lactic acid bacteria are bacteria that obtain energy by fermenting sugars and produce large amounts of lactic acid. They are widely distributed in nature, including agricultural products, foods, and the bodies of humans and animals. They are widely used in the fermentation of cheese, fermented milk, kimchi, and bread. In general, it is known that consuming lactic acid bacteria not only suppresses harmful bacteria among the intestinal microflora, but also increases beneficial bacteria that aid in the digestion, absorption, and breakdown of food. Therefore, it has been reported that consuming lactic acid bacteria has various benefits, including reducing blood cholesterol, boosting immunity, suppressing endogenous infections, improving liver cirrhosis, and having anticancer effects. Furthermore, recent research is also being conducted to enhance the efficacy of natural products fermented with lactic acid bacteria.

[0006] Accordingly, the inventors of the present invention completed the present invention by identifying novel lactic acid bacteria strains that exhibit improving effects on sarcopenia, cognitive dysfunction, and inflammatory diseases.

[0007] One object of the present invention is to provide Bifidobacterium bifidum P61 KCCM13367P.

[0008] Another object of the present invention is to provide Lactobacillus paracasei P62 KCCM13368P.

[0009] Another object of the present invention is to provide a composition for preventing, treating or improving sarcopenia, cognitive dysfunction and inflammatory diseases, comprising Bifidobacterium bifidum P61 KCCM13367P, Lactobacillus paracasei P62 KCCM13368P, or a mixture thereof.

[0010] To avoid confusion due to overlapping content, the description of redundant content will be omitted below. In other words, the content of the invention is not limited to the content described below, and the content of the invention should be interpreted based on the overall content of the invention.

[0011] Hereinafter, the present invention will be described in detail.

[0012] The present invention provides Bifidobacterium bifidum P61 (Deposit institution: Korean Culture Center of Microorganisms, Deposit date: 2023. 07.12, Accession number: KCCM13367P).

[0013] The Bifidobacterium bifidum P61 of the present invention is characterized as a lactic acid bacterium isolated and identified from the intestinal microflora of a healthy person.

[0014] The 16S rDNA base sequence for identification and classification of Bifidobacterium bifidum P61 of the present invention is as shown in SEQ ID NO: 1 attached to this specification. Therefore, Bifidobacterium bifidum P61 of the present invention may include the 16S rDNA of SEQ ID NO: 1 as follows.

[0015] 16S rDNA of Bifidobacterium bifidum P61 (SEQ ID NO: 1):

[0016] TACGACTTAGTCCCAATCACGAGCCTCACCTTAGACGGCTCCATCCCACAAGGGGTTAGGCCACCGGCTTCGGGTGCTGCCCACTTTCATGACTTGACGGGCGGTGTGTACAAGGCCCGGGAACGCATTCACCGCGGCGTTGCTGATCCGCGATTACTAGCGACTCCGCCTTCACGGAGCCGGTTGCAGGCTCCGATCCGAACTGAGACCGGTTTTCAGGGATCCGCTCCATGTCGCCATGTCGCATCCCGCTGTACCGGCCATTGTAGCATGCGTGAAGCCCTGGACGTAAGGGGCATGATGATCTGACGTCATCCCCACCTTCCTCCGAGTTAACCCCGGCGGTCCCCCGTGAGTTCCCACCATAACGTGCTGGCAACACGGGGCGAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACGACCATGCACCACCTGTGAACCCGCCCCGAAGGGAAACGCCATCTCTGGCGTCGTCGGGAACATGTCAAGCCCAGGTAAGGTTCTTCGCGTTGCATCGAATTAATCCGCATGCTCCGCCGCTTGTGCGGGCCCCCGTCAATTTCTTTGAGTTTTAGCCTTGCGGCCGTACTCCCCAGGCGGGACGCTTAACGCGTTAGCTCCGACACGGAACACGTGGAACGTGCCCCACATCCAGCGTCCACCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTGACGGCCCAGAGACCTGCCTTCG

[0017] As a result of analysis of the 16S rDNA base sequence of the above sequence number 1, it showed 99% homology (homology with Bifidobacterium bifidumKCTC3202 strain) with known Bifidobacterium bifidum strains, and as a result of whole-genome analysis, it also showed 98.8% homology (homology with Bifidobacterium bifidumJCM1255 strain), showing the highest molecular phylogenetic relationship with Bifidobacterium bifidum. Therefore, the above lactic acid bacteria was identified as Bifidobacterium bifidum, named Bifidobacterium bifidumP61, and deposited at the Korean Culture Center of Microorganisms on July 12, 2023 (KCCM13367P).

[0018] The Bifidobacterium bifidum P61 of the present invention is a gram-positive bacterium, and its cell morphology is a rod-shaped bacterium. More specific physiological characteristics of the Bifidobacterium bifidum P61 can be analyzed according to conventional methods in the art.

[0019] Specifically, the physiological characteristics of Bifidobacterium bifidum P61 were analyzed using an API kit test using a carbon source, and the results are shown in Table 1 below.

[0020] [Table 1]

[0021]

[0022] The present invention provides Lactobacillus paracasei P62 (Deposit institution: Korean Culture Center of Microorganisms, Deposit date: 2023.07.12, Accession number: KCCM13368P).

[0023] The Lactobacillus paracasei P62 of the present invention is characterized as a lactic acid bacterium isolated and identified from the intestinal microflora of a healthy person.

[0024] The 16S rDNA base sequence for identification and classification of Lactobacillus paracasei P62 of the present invention is as shown in SEQ ID NO: 2 attached to this specification. Therefore, Lactobacillus paracasei P62 of the present invention may include the 16S rDNA of SEQ ID NO: 2 as follows.

[0025] 16S rDNA of Lactobacillus paracasei P62 (SEQ ID NO: 2):

[0026] ATGGCTCAGGATGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAACGAGTTCTCGTTGATGATCGGTGCTTGCACCGAGATTCAACATGGAACGAGTGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCCTTAAGTGGGGGATAACATTTGGAAACAGATGCTAATACCGCATAGATCCAAGAACCGCATGGTTCTTGGCTGAAAGATGGCGTAAGCTATCGCTTTTGGATGGACCCGCGGCGTATTAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCGATGATACGTAGCCGAACTGAGAGGTTGATGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGCAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTGGAGAAGAATGGTCGGCAGAGTAACTGTTGTCGGCGTGACGGTATCCAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCCTCGGCTTAACCGAGGAAGCGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCATG

[0027] As a result of analysis of the 16S rDNA base sequence of the above sequence number 2, it showed 99% homology (homology with Lactobacillus paracaseiR094 strain) and 98.6% homology (homology with Lactobacillus paracaseisubsp.paracaseiJCM8130 strain) with known Lactobacillus paracasei strains, and the whole-genome analysis also showed 98.7% homology, showing the highest molecular phylogenetic relationship with Lactobacillus paracasei. Therefore, the above lactic acid bacteria was identified as Lactobacillus paracasei, named Lactobacillus paracasei P62, and deposited at the Korean Culture Center of Microorganisms on July 12, 2023 (KCCM13368P).

[0028] The Lactobacillus paracasei P62 of the present invention is a gram-positive bacterium, and its cell morphology is a rod-shaped bacterium. More specific physiological characteristics of the Lactobacillus paracasei P62 can be analyzed according to conventional methods in the art.

[0029] Specifically, the physiological characteristics of Lactobacillus paracasei P62 were analyzed using an API kit test using a carbon source, and the results are shown in Table 2 below.

[0030] [Table 2]

[0031]

[0032] The Bifidobacterium bifidum P61 KCCM13367P, Lactobacillus paracasei P62 KCCM13368P, or a mixture thereof of the present invention can treat sarcopenia by (i) inhibiting the expression of muscle fiber degradation proteins MuRF-1 and MAFbx / Atrogin-1 and inflammatory cytokines TNF-α and IL-6, (ii) increasing the activation of AKT signaling, which is a muscle fiber synthesis protein, (iii) inhibiting the concentration of blood corticosterone and endotoxin that induce sarcopenia, and (iv) promoting muscle strength and muscle formation.

[0033] In addition, it exhibits a therapeutic effect on cognitive dysfunction by (i) suppressing behaviors similar to cognitive dysfunction, (ii) increasing the expression of BDNF and IL-10, and (iii) reducing the expression of TNF-α, IL-6, and IL-1ß and the concentration of endotoxin in the blood. In addition, it has an effect of alleviating inflammation by reducing the expression of inflammatory markers (TNF-α, IL-6, IL-1ß, and MPO) and increasing the expression of anti-inflammatory markers (IL-10).

[0034] The present invention provides a pharmaceutical composition comprising Bifidobacterium bifidum P61 KCCM13367P, Lactobacillus paracasei P62 KCCM13368P, or a mixture thereof.

[0035] The present invention provides a pharmaceutical composition comprising Bifidobacterium bifidum P61 KCCM13367P, Lactobacillus paracasei P62 KCCM13368P, or a mixture thereof; and a pharmaceutically acceptable carrier.

[0036] The present invention provides a pharmaceutical composition for preventing or treating sarcopenia, cognitive dysfunction, and inflammatory diseases, comprising Bifidobacterium bifidum P61 KCCM13367P, Lactobacillus paracasei P62 KCCM13368P, or a mixture thereof.

[0037] As a specific embodiment for achieving the above purpose, the present invention provides a pharmaceutical composition for preventing or treating sarcopenia comprising Bifidobacterium bifidum P61 KCCM13367P, Lactobacillus paracasei P62 KCCM13368P, or a mixture thereof.

[0038] In the present invention, "sarcopenia" refers to a disease in which muscle mass decreases, resulting in a gradual decline in muscle volume and strength. Specifically, it refers to a general term for a disease in which the muscles of the limbs gradually atrophy, almost symmetrically, and can accompany cancer, aging, kidney disease, hereditary diseases, and various chronic diseases. The sarcopenia may be, but is not limited to, any one or more selected from the group including, for example, muscular atrophy, disuse atrophy, spinal muscular amyotrophy, muscular dystrophy, muscle rigidity, muscular hypotonia, muscle weakness, muscular dystrophy, amyotrophic lateral sclerosis, spinal bulbar muscular atrophy, and myasthenia gravis.

[0039] According to one embodiment of the present invention, it was confirmed that Bifidobacterium bifidum P61 KCCM13367P, Lactobacillus paracasei P62 KCCM13368P, or a mixture thereof (i) suppresses the expression of muscle fiber degradation proteins MuRF-1 and MAFbx / Atrogin-1 and inflammatory cytokines TNF-α and IL-6, (ii) increases muscle strength, muscle mass, and muscle fiber synthesis protein AKT signal activation, (iii) increases the expression of MyHC, MyoG, PGC1-α, and SIRT-1, and (iv) reduces blood corticosterone and blood endotoxin, thereby promoting muscle formation and thus alleviating sarcopenia.

[0040] As a specific embodiment for achieving the above purpose, the present invention provides a pharmaceutical composition for preventing or treating cognitive dysfunction comprising Bifidobacterium bifidum P61 KCCM13367P, Lactobacillus paracasei P62 KCCM13368P, or a mixture thereof.

[0041] In the present invention, "cognitive impairment" refers to a disorder that exhibits cognitive impairment and behavioral changes, and refers to a disease caused by a decline in functions such as memory, spatial perception, judgment, executive function, and language ability. The cognitive impairment may be, for example, at least one selected from the group including Alzheimer's disease, Huntington's disease, vascular dementia, Pick's disease, Parkinson's disease, Creutzfeldt-Jakob disease, and dementia, but is not limited thereto. In addition, cognitive impairment encompasses memory impairment, dementia symptoms, etc. caused by the above symptoms.

[0042] According to one embodiment of the present invention, it was confirmed that Bifidobacterium bifidum P61 KCCM13367P, Lactobacillus paracasei P62 KCCM13368P, or a mixture thereof (i) suppresses behaviors similar to cognitive dysfunction, (ii) increases the expression of BDNF and IL-10, and (iii) reduces the expression of TNF-α, IL-6, and IL-1ß and the concentration of endotoxin in the blood, thereby improving cognitive dysfunction.

[0043] As a specific embodiment for achieving the above purpose, the present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases, comprising Bifidobacterium bifidum P61 KCCM13367P, Lactobacillus paracasei P62 KCCM13368P, or a mixture thereof.

[0044] In the present invention, "inflammatory disease" is a general term for diseases in which inflammation is the main lesion, and the inflammatory disease may be at least one selected from the group including, but not limited to, inflammatory bowel disease (IBD), arthritis, gout, hepatitis, obesity, gastritis, nephritis, diabetes, tuberculosis, bronchitis, pleurisy, peritonitis, spondylitis, pancreatitis, urethritis, cystitis, vaginitis, arteriosclerosis, sepsis, and periodontitis.

[0045] The term "inflammatory bowel disease (IBD)" refers to a group of inflammatory conditions of the colon and gastrointestinal tract. Representative examples of inflammatory bowel disease include ulcerative colitis (UC) and Crohn's disease. The primary difference between ulcerative colitis and Crohn's disease is the location and nature of the inflammatory changes. Crohn's disease can affect any part of the gastrointestinal tract from the mouth to the anus, whereas ulcerative colitis' inflammation is limited to the colon and rectum. Due to the specific nature of the presentation, a definitive diagnosis of Crohn's disease or ulcerative colitis cannot be made. In such cases, a diagnosis of indeterminate colitis may be made. Other forms of inflammatory bowel disease include, but are not limited to, collagenous colitis, lymphocytic colitis, ischemic colitis, diversion colitis, Behçet's disease, and indeterminate colitis.

[0046] That is, in the present invention, the inflammatory bowel disease may be at least one selected from the group including ulcerative colitis, Crohn's disease, collagenous colitis, lymphocytic colitis, ischemic colitis, conversion colitis, Behcet's disease, and indeterminate colitis.

[0047] According to one embodiment of the present invention, it was confirmed that Bifidobacterium bifidum P61 KCCM13367P, Lactobacillus paracasei P62 KCCM13368P, or a mixture thereof exhibits a therapeutic effect on inflammation by reducing the expression of inflammatory markers (TNF-α, IL-6, IL-1ß, and MPO) and increasing the expression of anti-inflammatory markers (IL-10).

[0048] In the present invention, "MuRF-1 (Muscle RING-finger protein-1)" refers to a signaling substance that decomposes muscle proteins, and it is generally known that muscle atrophy progresses when MuRF-1 is activated.

[0049] In the present invention, "Atrogin-1" is a muscle-specific F-box protein that is involved in the process of decomposing muscle proteins, and is induced in the early stage of muscle atrophy, so it increases prior to the decrease in muscle mass.

[0050] In the present invention, "TNF-α (tumor necrosis factor-α)" is a cytokine produced by macrophages in response to various immune stimuli, including endotoxins, a toxic substance of bacteria, and plays a crucial role in the immune system. Abnormal regulation of TNF-α is known to occur in various diseases, such as Alzheimer's disease, cancer, depression, and inflammatory bowel disease (IBD).

[0051] In the present invention, "IL-6 (interleukin 6)" is an interleukin that can act as both a pro-inflammatory cytokine and an anti-inflammatory myokine, and acts as a differentiation factor involved in the activation of B lymphocytes, and affects the functions of various cells.

[0052] In the present invention, "FOXO3a" is one of the Forhead box O family genes (FOXO1, FOXO3a, FOXO4, FOXO6) known as longevity genes, and regulates the expression of genes involved in apoptosis, cell cycle arrest, oxidative stress resistance, and autophagy.

[0053] In the present invention, "NF-κB (nuclear factor-kappa B)" refers to a protein complex involved in the transcription process of DNA, production of cytokines, and cell survival, and is involved in inflammatory response regulation, immune system regulation, cell death, cell proliferation, and differentiation of epithelial cells.

[0054] In the present invention, "AKT (protein kinase B, PKB)" is a general term for a set of three serine / threonine-specific protein kinases, which play an important role in cell cycle progression, differentiation of smooth muscle cells, promotion of glucose uptake, angiogenesis, inhibition of apoptosis, and promotion of cell growth.

[0055] In the present invention, "mTOR (mammalian target of rapamycin)" refers to the serine-threonine kinase mammalian target of rapamycin, which is composed of two distinct protein complexes, mTOR complex 1 and mTOR complex 2, which regulate different cellular processes. The mTOR can function as a serine / threonine protein kinase that regulates cell growth, cell proliferation, cell motility, cell survival, protein synthesis, autophagy, and transcription.

[0056] In the present invention, “MyHC (myosin heavy chain)” is a major contractile protein of sarcomeric filaments and is an important component in maintaining muscle contraction.

[0057] In the present invention, “MyoG” refers to a gene encoding myogenin, which is involved in regulating skeletal muscle development or muscle production and repair.

[0058] In the present invention, "PGC1-α" is a key transcription factor activator that induces mitochondrial production in cells, and is known to regulate the composition and function of individual mitochondria.

[0059] In the present invention, "SIRT-1" is a gene encoding Sirtuin1, and Sirtuin1 is a protein that functions in cellular response to inflammatory, metabolic, and oxidative stress factors.

[0060] In the present invention, "BDNF (brain-derived neurotrophic factor)" is a brain-derived neurotrophic factor that acts on specific neurons in the central and peripheral nervous systems to help the survival of existing neurons and promote the growth and differentiation of new neurons and synapses.

[0061] In the present invention, "IL-10" refers to a potent immunosuppressive Th-2 cell cytokine produced by lymphocytes that exerts the function of inhibiting macrophage / monocyte and T-cell lymphocyte replication and secretion of inflammatory cytokines.

[0062] In the present invention, “IL-1ß” is also called leukocytic pyrogen, leukocytic endogenous mediator, mononuclear cell factor, or lymphocyte activating factor, and serves as a key mediator of the inflammatory response.

[0063] In the present invention, "MPO (myeloperoxidase)" is a dimeric enzyme mainly found in the azeotrophic granules of neutrophils and lysosomes of monocytes, which promotes the formation of superoxide and mainly increases the microbial bactericidal power of the innate immune response.

[0064] In the present invention, "corticosterone" is a type of adrenal cortex hormone and a representative glucocorticoid. Corticosterone is synthesized from pregnenolone or progesterone in the body and excreted by the kidneys. It is known to protect the body from stress, promote protein breakdown, promote carbohydrate synthesis, suppress inflammation, and exhibit an antagonistic effect on insulin.

[0065] In the present invention, "endotoxin" is a component of the outer membrane of gram-negative bacteria, known to react with immune cells in the body to cause an inflammatory response, and is also chemically called lipopolysaccharide (LPS).

[0066] The above results show that Bifidobacterium bifidum P61 KCCM13367P, Lactobacillus paracasei P62 KCCM13368P, or a mixture thereof is excellent in preventing, treating, or improving sarcopenia, cognitive dysfunction, and inflammatory diseases.

[0067] In addition, the pharmaceutical composition may exhibit excellent effects in preventing, treating, or improving sarcopenia, cognitive dysfunction, and inflammatory diseases by changing the composition of altered intestinal microorganisms.

[0068] The strain according to the present invention can be utilized in various forms, such as live cells, dead cells, cultures, lysates, or extracts. The strain according to the present invention, regardless of its form, exhibits results equivalent to or higher than the above-mentioned effects (especially considering live cells) under conditions involving the strain.

[0069] Probiotics refer to living bacteria, while dead bacteria refer to live bacteria cultured under certain conditions and then the effective ingredients are separated and extracted through methods such as heat drying, pressurization, and drug treatment.

[0070] A culture refers to a product obtained by culturing lactic acid bacteria in a known liquid medium or solid medium, and is a concept that includes a strain according to the present invention. The product may include lactic acid bacteria. The medium may be selected from known liquid or solid media, and may be, for example, MRS liquid medium, GAM liquid medium, MRS agar medium, GAM agar medium, or BL agar medium, but is not limited thereto.

[0071] A lysate refers to a form in which live cells, dead cells, or cultures thereof are separated and processed mechanically or chemically. For example, the lysate can be produced using bead mills, presses, sonicators, microfluidizers, enzyme treatment, etc.

[0072] An extract means a product obtained by extracting living cells, dead cells and / or fragments using a commonly known extraction method (a known extraction solvent (e.g., water, C1 to C4 alcohol (methanol, ethanol, etc.))).

[0073] In the present invention, it has been confirmed that a synergistic effect is exhibited in a mixture of strains, and any form of use of the mixture is included.

[0074] That is, by mixing Bifidobacterium bifidum P61 KCCM13367P and Lactobacillus paracasei P62 KCCM13368P according to the present invention, an excellent synergistic effect can be exhibited.

[0075] A mixture of Bifidobacterium bifidum P61 KCCM13367P and Lactobacillus paracasei P62 KCCM13368P can be included, for example, in a ratio of 10:1 to 1:10 based on the number of bacteria (CFU). Preferably, it can be included in a ratio of 1:6 to 1:1. More specifically, it can be 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10. More preferably, it can be 1:4.

[0076] According to one embodiment of the present invention, the mixture of the strains exhibits excellent synergistic effects on sarcopenia, cognitive dysfunction and inflammatory diseases (particularly, inflammatory bowel disease).

[0077] In particular, in terms of normalizing the intestinal microbiota, a mixture of the above strains may exhibit a superior effect in maintaining intestinal health compared to a single strain.

[0078] The pharmaceutical composition according to the present invention can be formulated into a pharmaceutical formulation using methods well known in the art to provide rapid, sustained, or delayed release of the active ingredient after administration to a mammal. In preparing the formulation, the pharmaceutical composition according to the present invention may additionally include a pharmaceutically acceptable carrier, provided that it does not inhibit the activity of the compound of the present invention.

[0079] The pharmaceutically acceptable carriers include, but are not limited to, those commonly used, such as lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition, the pharmaceutical composition of the present invention may include diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrating agents, and surfactants, and other pharmaceutically acceptable additives.

[0080] The pharmaceutical composition according to the present invention may be administered in a pharmaceutically effective amount. Here, "pharmaceutically effective amount" means an amount sufficient to prevent or treat a disease at a reasonable benefit / risk ratio applicable to medical treatment. The level of the effective amount may be variously selected by those skilled in the art according to factors such as the formulation method, the patient's condition and body weight, the patient's sex, age, the extent of the disease, the drug form, the route and duration of administration, the excretion rate, the sensitivity of the reaction, etc. As will be recognized by those skilled in the art, the effective amount may vary depending on the route of administration, the use of excipients, and the possibility of use together with other drugs. However, for a desirable effect, in the case of an oral administration agent, the composition of the present invention may generally be administered to an adult at 0.0001 to 100 mg / kg per kg of body weight per day, preferably 0.001 to 100 mg / kg, but the above dosage does not limit the scope of the present invention in any way.

[0081] The pharmaceutical composition according to the present invention can be administered to mammals such as mice, livestock, and humans through various routes. Specifically, the pharmaceutical composition according to the present invention can be administered orally or parenterally (e.g., by application or intravenous, subcutaneous, or intraperitoneal injection), but oral administration is preferred. Solid preparations for oral administration may include powders, granules, tablets, capsules, soft capsules, pills, etc. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, syrups, aerosols, etc. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. Preparations for parenteral administration may be formulated and used in the form of external preparations such as sterilized aqueous solutions, liquids, non-aqueous solvents, suspensions, emulsions, eye drops, eye ointments, syrups, suppositories, aerosols, etc., and sterilized injection preparations according to conventional methods, and preferably, pharmaceutical compositions such as creams, gels, patches, sprays, ointments, ointments, lotions, liniments, eye ointments, eye drops, pastes, or cataplasmas may be prepared and used, but are not limited thereto. Preparations for topical administration may be anhydrous or aqueous depending on the clinical prescription. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases that can be used include witepsol, macrogol, tween 61, cocoa butter, laurin butter, and glycerogelatin.

[0082] The pharmaceutical composition of the present invention can be administered alone as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents. Furthermore, the pharmaceutical composition of the present invention can be administered singly or in multiple doses. Taking all of the above factors into consideration, it is important to administer the amount that achieves maximum effect with the minimum amount possible without causing side effects, a determination readily made by those skilled in the art.

[0083] The term "subject" of the present invention includes an animal or human whose symptoms can be improved by administration of a pharmaceutical composition according to the present invention. By administering a pharmaceutical composition according to the present invention to a subject, sarcopenia, cognitive dysfunction, or inflammatory diseases can be effectively prevented and treated.

[0084] The term "administration" in the present invention refers to introducing a given substance into a human or animal by any suitable method. The therapeutic composition according to the present invention may be administered orally or parenterally via any common route, as long as it can reach the target tissue. Furthermore, the therapeutic composition according to the present invention may be administered by any device capable of transporting the active ingredient to target cells.

[0085] The preferred dosage of the pharmaceutical composition according to the present invention varies depending on the patient's condition and weight, the degree of disease, the drug form, the route and period of administration, and can be appropriately selected by a person skilled in the art.

[0086] The present invention provides Bifidobacterium bifidum P61 KCCM13367P, Lactobacillus paracasei P62 KCCM13368P, or a mixture thereof for use in the prevention or treatment of any one or more diseases selected from the group consisting of sarcopenia, cognitive dysfunction, and inflammatory diseases.

[0087] The present invention provides Bifidobacterium bifidum P61 KCCM13367P, Lactobacillus paracasei P62 KCCM13368P, or a mixture thereof for use in the prevention or treatment of sarcopenia.

[0088] The present invention provides Bifidobacterium bifidum P61 KCCM13367P, Lactobacillus paracasei P62 KCCM13368P, or a mixture thereof for use in the prevention or treatment of cognitive dysfunction.

[0089] The present invention provides Bifidobacterium bifidum P61 KCCM13367P, Lactobacillus paracasei P62 KCCM13368P, or a mixture thereof for use in the prevention or treatment of inflammatory diseases.

[0090] The present invention provides the use of Bifidobacterium bifidum P61 KCCM13367P, Lactobacillus paracasei P62 KCCM13368P, or a mixture thereof in the manufacture of a medicament for the treatment of any one or more diseases selected from the group consisting of sarcopenia, cognitive dysfunction, and inflammatory diseases.

[0091] The present invention provides the use of Bifidobacterium bifidum P61 KCCM13367P, Lactobacillus paracasei P62 KCCM13368P, or a mixture thereof in the manufacture of a medicament for treating sarcopenia.

[0092] The present invention provides the use of Bifidobacterium bifidum P61 KCCM13367P, Lactobacillus paracasei P62 KCCM13368P, or a mixture thereof in the manufacture of a medicament for treating cognitive dysfunction.

[0093] The present invention provides the use of Bifidobacterium bifidum P61 KCCM13367P, Lactobacillus paracasei P62 KCCM13368P, or a mixture thereof in the manufacture of a medicament for treating an inflammatory disease.

[0094] The present invention provides a method for preventing or treating sarcopenia, cognitive dysfunction and inflammatory diseases, comprising administering to a subject in need thereof Bifidobacterium bifidum P61 KCCM13367P, Lactobacillus paracasei P62 KCCM13368P, or a mixture thereof.

[0095] The present invention provides a composition comprising Bifidobacterium bifidum P61 KCCM13367P, Lactobacillus paracasei P62 KCCM13368P, or a mixture thereof.

[0096] The present invention provides a food composition for preventing or improving sarcopenia, cognitive dysfunction, and inflammatory diseases, comprising Bifidobacterium bifidum P61 KCCM13367P, Lactobacillus paracasei P62 KCCM13368P, or a mixture thereof.

[0097] The above "Bifidobacterium bifidum P61", "Lactobacillus paracasei P62", "sarcopenia", "cognitive dysfunction" and "inflammatory disease" are the same as described above.

[0098] Specifically, the lactic acid bacteria included in the food composition according to the present invention may be live cells thereof, dead cells thereof, culture thereof, lysate thereof, or extract thereof, but any form of lactic acid bacteria capable of achieving a preventive or improving effect on sarcopenia, cognitive dysfunction, or inflammatory disease disorders may be used without limitation.

[0099] There are no special restrictions on the types of the above foods. Foods to which the above lactic acid bacteria can be added include dairy products including sausages, meat, bread, chocolate, snacks, candies, confectionery, ramen, pizza, other noodles, gum, ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes. When formulated as a beverage, the liquid ingredients added in addition to the novel lactic acid bacteria are not limited thereto, but may contain various flavorings or natural carbohydrates as additional ingredients, as in conventional beverages. The above-mentioned natural carbohydrates may be monosaccharides (e.g., glucose, fructose, etc.), disaccharides (e.g., maltose, sucrose, etc.), and polysaccharides (e.g., conventional sugars such as dextrin, cyclodextrin, etc.), and sugar alcohols such as xylitol, sorbitol, and erythritol.

[0100] The type of the above food may be, specifically, a health functional food. The health functional food may contain various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and enhancers (cheese, chocolate, etc.), pectic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. These ingredients may be used alone or in combination, and the proportion of these additives is generally selected in the range of 0.001 to 50 parts by weight per total weight of the composition.

[0101] The above health functional foods emphasize the bioregulatory functions of food. They are foods with added value, utilizing physical, biochemical, and biotechnological methods to function and manifest for specific purposes. The ingredients of these health functional foods are designed and processed to fully exert their bodily regulatory functions related to biological defense, regulation of body rhythms, and disease prevention and recovery. They may also contain food additives, sweeteners, or functional ingredients permitted as food.

[0102] When the Bifidobacterium bifidum P61 KCCM13367P and / or the Lactobacillus paracasei P62 KCCM13368P of the present invention are used as a health functional food (or health functional beverage additive), the Bifidobacterium bifidum P61 KCCM13367P and / or the Lactobacillus paracasei P62 KCCM13368P may be added as is or used together with other foods or food ingredients, and may be used appropriately according to a conventional method. The mixing amount of the above Bifidobacterium bifidum P61 KCCM13367P and / or Lactobacillus paracasei P62 KCCM13368P can be appropriately determined depending on the purpose of use (prevention, health or improvement, therapeutic treatment).

[0103] The novel lactic acid bacteria and mixtures thereof of the present invention suppress the expression of sarcopenia markers (MuRF-1, MAFbx / Atrogin-1, etc.) by changing the composition of intestinal microorganisms, increase muscle strength and muscle mass, promote muscle formation, and alleviate sarcopenia. In addition, they exhibit a therapeutic effect on cognitive dysfunction by suppressing behaviors similar to cognitive dysfunction and regulating the expression of factors related to the disorder (BDNF, IL-10, etc.). In addition, they have the effect of alleviating inflammation by reducing the expression of inflammatory markers (TNF-α, IL-6, IL-1ß, and MPO) and increasing the expression of anti-inflammatory markers (IL-10).

[0104] Figure 1 shows the changes in the expression of sarcopenia-related factors (MuRF-1, MAFbx / Atrogin-1, TNF-α, and IL-6) and the activation of NF-κB (p65) in each mouse model (Yg(NC), Vh, Lp, Bb, LB, and Cr). (Data in Figure 1 are presented as mean ± SD (n = 4). #p<0.05 vs. NC. *p<0.05 vs. groups treated with dexamethasone or LPS alone.)

[0105] Figure 2 shows NF-κB for each mouse model (Yg, Vh, Lp, Bb, LB and Cr). + CD11c + This is a diagram showing changes in cell number observed through immunofluorescence staining.

[0106] Figure 3 shows the changes in the size of gastrocnemius muscle cells (H&E staining), the number of MyHC-positive cells (immunofluorescence staining), and the cross-sectional area (CSA) of the muscle for each mouse model (Yg, Vh, Lp, Bb, LB, and Cr).

[0107] Figure 4 shows NF-κB for each mouse model (Yg, Vh, Lp, Bb, LB, and Cr). + Iba1 + Cell count and BDNF + NeuN + This figure shows changes in cell number confirmed through immunofluorescence staining.

[0108] (Data in Figures 2 to 4 are presented as mean ± SD (n = 7). #p<0.05 vs Yg. *p<0.05 vs. Vh / Ag.)

[0109] Hereinafter, preferred examples are presented to aid in understanding the present invention. However, the following examples are provided solely to facilitate a better understanding of the present invention and are not intended to limit the scope of the present invention.

[0110] Experimental Example 1. Isolation and Cultivation of Lactic Acid Bacteria

[0111] Bifidobacterium bifidum P61 and Lactobacillus paracasei P62, lactic acid bacteria isolated from human intestinal microflora, were cultured at room temperature in MRS medium (BD, Sparks, MD), and then separated and collected. The collected cells were lyophilized, washed twice with saline solution and then suspended for in vitro experiments, and suspended in 1% maltose solution for in vivo experiments.

[0112] Experimental Example 2. C2C12 Cell Culture

[0113] C2C12 cells (4 Type Culture Collection) were cultured in DMEM medium containing 10% fetal bovine serum (FBS), 1% antibiotic-antimycotic solution, and 3.7 g / L NaHCO3 at 37°C in a humidified incubator with 5% CO2 / 95% air. Confluent myoblasts (80%) were differentiated for 4 days in DMEM medium containing 2% horse serum.

[0114] Experimental Example 3. Preparation of a Mouse Model

[0115] For the preparation of mouse models, aged C57BL / 6 mice (male, 18 months old) and young C57BL / 6 mice (male, 3 months old) were purchased from Orientbio Co. Ltd. In addition, all animal experiments conducted for the present invention were approved in advance by the Kyung Hee University Institutional Animal Care and Use Committee (IACUC No. KHUASP(SE)-22-567) and were conducted ethically in accordance with the university guidelines for the care and use of laboratory animals.

[0116] Mice were divided into the following six groups, each containing eight mice.

[0117] Yg(NC): Group treated with vehicle in young C57BL / 6 mice.

[0118] Vh: Group treated with vehicle in aged C57BL / 6 mice.

[0119] Bb: Aged C57BL / 6 mice were treated with Bifidobacterium bifidum P61 (1X10 9 Group treated with 100 CFU / mouse.

[0120] Lp: Aged C57BL / 6 mice were treated with Lactobacillus paracaseiP62 (1X10 9 Group treated with 100 CFU / mouse.

[0121] LB: Group treated with a 1:4 mixture of Bifidobacterium bifidum P61 and Lactobacillus paracasei P62 in aged C57BL / 6 mice.

[0122] Cr: Group treated with creatine (75 mg / kg) in aged C57BL / 6 mice.

[0123] At this time, the treatment of the preparation in each group was administered orally via gavage once a day for 8 weeks (6 days a week).

[0124] Experimental Example 4. Methods for Measuring Motor Ability and Testing Cognitive Function

[0125] Measurement of the exercise capacity of mice was conducted 20 hours after the last treatment with the preparation in each group of Experimental Example 3. Specifically, the grip strength test was conducted by placing the limbs of the mice on the grid of a grip dynamometer and measuring the grip strength just before they fell from the bar. In addition, the treadmill test was conducted by acclimating the mice to the treadmill for one week and having them run for 30 minutes at a speed of 23 m / min, and measuring the running time and distance.

[0126] Cognitive function was tested through a Y-maze test, which used a Y-maze measuring device with three passages (arms) extending in the shape of the letter Y, each passage having a length of 25 cm, a height of 14 cm, a width of 5 cm, and positioned at the same angle.

[0127] Experimental Example 5. ELISA and immunoblotting

[0128] Mouse gastrocnemius muscle, colon, brain tissues, and cells were homogenized on ice using RIPA lysis buffer containing 1% phosphatase inhibitor cocktail and 1% protease inhibitor cocktail (RPP), and centrifuged at 4°C and 15,000 g for 15 minutes. The supernatant of the homogenate was then subjected to SDS-PAGE (sulfate-polyacrylamide gel electrophoresis) and transferred to a PVDF membrane for immunoblotting. Proteins were detected using antibodies, detected with HRP-conjugated secondary antibodies, and visualized using an ECL detection kit. Meanwhile, cytokines were analyzed using an ELISA kit (R&D System) after transferring the supernatant of the homogenate to a 96-well plate.

[0129] The antibodies used at this time were as follows: phospho-AKT (Ser473) (193H12, Cell Signaling, Danvers, MA), AKT (11E7) (4685, Cell Signaling), MAFbx (F-9) (sc-166806, Santa Cruz Biotechnology, Santa Cruz, CA), MuRF-1 (C-11) (sc-398608, Santa Cruz) Biotechnology), phospho-mTOR (ser2448) (2971, Cell Signaling), mTOR(7C10) (2983, Cell Signaling), p-NF-κB-p65 (S536) (93H1, Cell Signaling), NF-κB-p65 (D14E12) (8242, Cell Signaling), MYH (B-5) (sc-376157, Santa Cruz Biotechnology), p16INK4A (E5F3Y, Cell signaling), p-FOXO3a (ser253, Cell signaling), FOXO3a (75D8, Cell signaling), β-actin (sc-47778, Santa Cruz Biotechnology), and PGC1α (ab191838, Abcam).

[0130] Experimental Example 6. Real-time polymerase chain reaction (qPCR) analysis

[0131] Total RNA was purified from gastrocnemius muscle and C2C12 cells using the RNeasy mini kit (Qiagen) and the RNeasy Fibrous Tissue mini kit (Qiagen), respectively. 2 μg of the purified RNA was reverse transcribed using the PrimeScript cDNA synthesis kit (Takara), and PCR was performed using TB Green Premix Ex Taq II (Takara) and the Rotor-Gene Q 5plex Platform (Qiagen).

[0132] Meanwhile, total DNA was purified from gastrocnemius muscle and C2C12 cells using the Genomic DNeasy kit (Qiagen), and the mtDNA copy number was measured by qPCR.

[0133] The primers used at this time are as shown in Table 3.

[0134] [Table 3]

[0135]

[0136] Experimental Example 7. Hematoxylin & Eosin (H&E) Staining and Immunofluorescence Staining

[0137] After paraformaldehyde was injected into the heart of the mouse, the gastrocnemius muscle of the hind limb was cut into 5 μm sections and stained with hematoxylin & eosin (H&E).

[0138] Additionally, hypothalamus, gastrocnemius muscle, and colon tissues were collected from the mice, sectioned, and incubated with primary antibodies (BDNF, NeuN, p65, NF-κB, Iba1, and / or CD11c) for 12 hours. Subsequently, they were treated with secondary antibodies conjugated with Alexa Fluor594 or Alexa Fluor 488 and observed using a confocal microscope.

[0139] Example 1. Alleviation of sarcopenia by Bifidobacterium bifidum P61 and Lactobacillus paracasei P62

[0140] (1) Confirmation of changes in the expression of MuRF-1, MAFbx / Atrogin-1, TNF-α, and IL-6, and activation of NF-κB

[0141] C2C12 cells (1X10) from mouse groups (Yg, Vh, Bb, Lp, LB and Cr) prepared in Experimental Example 3 5cells / mL) were treated with 0.3 mM dexamethasone or 100 ng / mL LPS, and the expression of myofibrillar degradation proteins (MuRF-1, MAFbx / Atrogin-1), inflammatory cytokines (TNF-α and IL-6), and changes in NF-κB activation were observed.

[0142] As a result, it was confirmed that Bifidobacterium bifidum P61 and / or Lactobacillus paracasei P62 inhibited the expression of MuRF-1 and MAFbx / Atrogin-1 induced by dexamethasone (Figs. 1a and 1b). In addition, it was confirmed that they significantly inhibited the expression of TNF-α and IL-6 and the activation of NF-κB induced by lipopolysaccharide (LPS) (Figs. 1c to 1e).

[0143] That is, it was confirmed that sarcopenia was alleviated by improving the expression of factors related to sarcopenia and the activation of NF-κB by Bifidobacterium bifidum P61, Lactobacillus paracasei P62, or a mixture thereof.

[0144] (2) Confirmation of changes in muscle strength, muscle mass, and AKT signal activation

[0145] An experiment was conducted to confirm changes in muscle strength and muscle mass for the mouse groups (Yg, Vh, Bb, Lp, LB, and Cr) prepared in Experimental Example 3. During the experiment, no significant differences were observed between the groups in food intake and body weight gain of the aging mouse model.

[0146] To confirm changes in muscle strength, treadmill tests and grip strength tests were conducted on each group of mice.

[0147] As a result of the treadmill test, it was confirmed that the running time and distance of the aging mouse model increased by treating with Bifidobacterium bifidum P61 and / or Lactobacillus paracasei P62 (Table 4(a) and (b)). Among them, the group (LB) treated with a 1:4 mixture of Bifidobacterium bifidum P61 and Lactobacillus paracasei P62 showed the most significant increase in running time and distance. In addition, in the grip strength test, it was confirmed that the grip strength of the groups (Bb, Lp, and LB) treated with Bifidobacterium bifidum P61 and / or Lactobacillus paracasei P62 increased by approximately 1.15-fold, 1.14-fold, and 1.16-fold, respectively, compared to the Vh group (Table 4(c)).

[0148] Next, to confirm changes in muscle mass, the weight changes of the gastrocnemius (GA), soleus (SOL), quadriceps femoris (QD), extensor digitorum longus (EDL), and tibialis anterior (TA) in each group were observed. As a result, the weights of the GA, SOL, QD, EDL, and TA in the aging mouse model were significantly increased by treatment with Bifidobacterium bifidum P61 and / or Lactobacillus paracasei P62 (Tables 4(d) to (h)), and the total muscle weight also increased accordingly (Table 4(i)).

[0149] The specific results of each of the above experiments are shown in Table 4 below.

[0150] [Table 4]

[0151]

[0152] In addition, it was confirmed that AKT and mTOR activation related to protein synthesis also increased in the gastrocnemius muscles of the groups (Bb, Lp, and LB) treated with Bifidobacterium bifidum P61 and / or Lactobacillus paracasei P62, and this is shown in Table 5 below.

[0153] [Table 5]

[0154]

[0155] That is, it was confirmed that Bifidobacterium bifidum P61, Lactobacillus paracasei P62, or a mixture thereof showed an effect in treating sarcopenia by increasing muscle strength, muscle mass, and AKT signaling activation, and decreasing FOXO3a and NF-κB activation.

[0156] (3) Confirmation of changes in the expression of myogenesis genes

[0157] The mouse groups (Yg, Vh, Bb, Lp, LB, and Cr) prepared in Experimental Example 3 were subjected to qPCR, immunoblotting, H&E staining, and immunofluorescence staining to confirm changes in the expression of myogenesis genes due to administration of Bifidobacterium bifidum P61 and / or Lactobacillus paracasei P62.

[0158] Specifically, qPCR results showed that the expression of MuRF-1, MAFbx / Atrogin-1, TNF-α, and IL-6 decreased in the groups (Bb, Lp, and LB) treated with Bifidobacterium bifidum P61 and / or Lactobacillus paracasei P62. On the other hand, the expression of MyHC and MyoG increased, and in particular, the expression of MyHC 2X, MyHC 2B, and MyHC was significantly increased compared to the group treated with creatine (Cr). In addition, the level of mtDNA in the gastrocnemius muscle was restored to a level similar to that of young mice, and the expression levels of PGC1-α and SIRT-1, which are involved in mitochondrial gene expression, were observed to significantly increase.

[0159] Specific results for this are shown in Table 6 below.

[0160] [Table 6]

[0161]

[0162] According to ELISA, Bifidobacterium bifidum P61 and / or Lactobacillus paracasei P62 reduced the blood corticosterone level increased with aging, as shown in Table 7 below.

[0163] [Table 7]

[0164]

[0165] In addition, when muscle cells were observed after H&E staining or immunofluorescence staining, NF-κB was observed in the groups (Bb, Lp, and LB) treated with Bifidobacterium bifidum P61 and / or Lactobacillus paracasei P62. + CD11c +While the number of cells decreased (Fig. 2, the size of gastrocnemius muscle cells (H&E staining) and the number of MyHC-positive cells (immunofluorescence staining) increased, and the cross-sectional area (CSA) of the muscle was confirmed to be restored to a level similar to that of the young mouse model (Yg) (Fig. 3).

[0166] That is, it was confirmed that sarcopenia could be improved by promoting muscle formation by Bifidobacterium bifidum P61, Lactobacillus paracasei P62, or a mixture thereof.

[0167] Example 2. Alleviation of cognitive dysfunction caused by Bifidobacterium bifidum P61 and Lactobacillus paracasei P62

[0168] The Y-maze test was performed on the mouse groups (Yg, Vh, Bb, Lp, LB, and Cr) prepared in Experimental Example 3 to measure spontaneous alternation and confirm changes in the expression of factors related to cognitive function.

[0169] Aging mouse models (Bb, Lp, and LB) treated with Bifidobacterium bifidum P61 and / or Lactobacillus paracasei P62 showed a tendency to increase spontaneous alternation in the Y-maze test, confirming that behaviors similar to cognitive dysfunction were suppressed (Table 8(a)). In addition, the expression of BDNF and IL-10 was increased in the hippocampus of the aging mouse models, and the expression of TNF-α, IL-6, and IL-1ß was suppressed (Tables 8(b) to (h)), and NF-κB + Iba1 + Cell count and BDNF + NeuN + The number of cells increased (Fig. 4).

[0170] Specific results for this are shown in Table 8 and Figure 4 below.

[0171] [Table 8]

[0172]

[0173] Additionally, administration of Bifidobacterium bifidum P61 and / or Lactobacillus paracasei P62 reduced endotoxins that induce systemic inflammation and cognitive dysfunction. The specific results are shown in Table 9 below.

[0174] [Table 9]

[0175]

[0176] That is, it was confirmed that administering Bifidobacterium bifidum P61, Lactobacillus paracasei P62, or a mixture thereof had a therapeutic effect on cognitive dysfunction.

[0177] Example 3. Confirmation of inflammation relief by Bifidobacterium bifidum P61 and Lactobacillus paracasei P62.

[0178] To confirm the anti-inflammatory effect, qPCR and immunofluorescence staining were performed on the colons of the mouse models (Yg, Vh, Bb, Lp, LB, and Cr) prepared in Experimental Example 3.

[0179] qPCR results showed that in the groups (Bb, Lp, and LB) treated with Bifidobacterium bifidum P61 and / or Lactobacillus paracasei P62, the expression of inflammatory markers TNF-α, IL-6, IL-1ß, and MPO (myeloperoxidase) decreased, and the expression of anti-inflammatory marker IL-10 increased.

[0180] Specific results for this are shown in Table 10 below.

[0181] [Table 10]

[0182]

[0183] That is, it was confirmed that Bifidobacterium bifidum P61, Lactobacillus paracasei P62, or a mixture thereof could alleviate inflammation by reducing the expression of inflammatory markers.

[0184]

[0185] [Accession number]

[0186] Name of depositor: Korea Center for Microbiological Conservation (KCCM)

[0187] Accession number: KCCM13367P

[0188] Date of acceptance: 20230712

[0189]

[0190]

[0191] [Accession number]

[0192] Name of depositor: Korea Center for Microbiological Conservation (KCCM)

[0193] Accession number: KCCM13368P

[0194] Date of acceptance: 20230712

[0195]

Claims

1. Bifidobacterium bifidum P61 KCCM13367P.

2. In the first paragraph, the Bifidobacterium bifidum P61 KCCM13367P comprises a 16S rDNA base sequence of sequence number 1.

3. Lactobacillus paracasei P62 KCCM13368P.

4. In the third paragraph, the Lactobacillus paracasei P62 (Lactobacillus paracaseiP62) KCCM13368P comprises a 16S rDNA base sequence of sequence number 2.

5. A pharmaceutical composition for preventing or treating sarcopenia, cognitive dysfunction and inflammatory diseases, comprising Bifidobacterium bifidum P61 KCCM13367P, Lactobacillus paracasei P62 KCCM13368P, or a mixture thereof.

6. A pharmaceutical composition according to claim 5, wherein the sarcopenia is at least one selected from the group consisting of muscular atrophy, disuse atrophy, spinal muscular amyotrophy, muscular dystrophy, muscle rigidity, muscular hypotonia, muscle weakness, muscular dystrophy, amyotrophic lateral sclerosis, spinal bulbar muscular atrophy, and myasthenia gravis.

7. A pharmaceutical composition according to claim 5, wherein the cognitive dysfunction is at least one selected from the group consisting of Alzheimer's disease, Huntington's disease, vascular dementia, Pick's disease, Parkinson's disease, Creutzfeldt-Jakob disease, and dementia.

8. A pharmaceutical composition according to claim 5, wherein the inflammatory disease is at least one selected from the group consisting of inflammatory bowel disease (IBD), arthritis, gout, hepatitis, obesity, gastritis, nephritis, diabetes, tuberculosis, bronchitis, pleurisy, peritonitis, spondylitis, pancreatitis, urethritis, cystitis, vaginitis, arteriosclerosis, sepsis, and periodontitis.

9. A pharmaceutical composition for sarcopenia, comprising Bifidobacterium bifidum P61 KCCM13367P and Lactobacillus paracasei P62 KCCM13368P in a ratio of 10:1 to 1:10 based on the number of bacteria (CFU) in paragraph 5.

10. A pharmaceutical composition for sarcopenia, comprising Bifidobacterium bifidum P61 KCCM13367P and Lactobacillus paracasei P62 KCCM13368P in a ratio of 1:6 to 1:1 based on the number of bacteria (CFU) in paragraph 9.

11. A food composition for preventing or improving sarcopenia, cognitive dysfunction, and inflammatory diseases, comprising Bifidobacterium bifidum P61 KCCM13367P, Lactobacillus paracasei P62 KCCM13368P, or a mixture thereof.

12. A food composition according to claim 11, wherein the sarcopenia is at least one selected from the group consisting of muscular atrophy, disuse atrophy, spinal muscular amyotrophy, muscular dystrophy, muscle rigidity, muscular hypotonia, muscle weakness, muscular dystrophy, amyotrophic lateral sclerosis, spinal bulbar muscular atrophy, and myasthenia gravis.

13. A food composition according to claim 11, wherein the cognitive dysfunction is at least one selected from the group consisting of Alzheimer's disease, Huntington's disease, vascular dementia, Pick's disease, Parkinson's disease, Creutzfeldt-Jakob disease, and dementia.

14. A food composition according to claim 11, wherein the inflammatory disease is at least one selected from the group consisting of inflammatory bowel disease, arthritis, gout, hepatitis, obesity, gastritis, nephritis, diabetes, tuberculosis, bronchitis, pleurisy, peritonitis, spondylitis, pancreatitis, urethritis, cystitis, vaginitis, arteriosclerosis, sepsis, and periodontitis.

15. A food composition for sarcopenia, comprising Bifidobacterium bifidum P61 KCCM13367P and Lactobacillus paracasei P62 KCCM13368P in a ratio of 10:1 to 1:10 based on the number of bacteria (CFU) in the 11th paragraph.

16. A sarcopenia food composition according to claim 15, comprising Bifidobacterium bifidum P61 KCCM13367P and Lactobacillus paracasei P62 KCCM13368P in a ratio of 1:6 to 1:1 based on the number of bacteria (CFU).

17. A pharmaceutical composition comprising Bifidobacterium bifidum P61 KCCM13367P, Lactobacillus paracasei P62 KCCM13368P, or a mixture thereof; and a pharmaceutically acceptable carrier.

18. A method for preventing or treating sarcopenia, cognitive dysfunction and inflammatory diseases, comprising administering to a subject in need thereof Bifidobacterium bifidum P61 KCCM13367P, Lactobacillus paracasei P62 KCCM13368P, or a mixture thereof.

19. Use of Bifidobacterium bifidum P61 KCCM13367P, Lactobacillus paracasei P62 KCCM13368P, or a mixture thereof in the manufacture of a medicament for treating one or more diseases selected from the group consisting of sarcopenia, cognitive dysfunction, and inflammatory diseases.

20. Bifidobacterium bifidum P61 KCCM13367P, Lactobacillus paracasei P62 KCCM13368P, or a mixture thereof, for use in the prevention or treatment of any one or more diseases selected from the group consisting of sarcopenia, cognitive dysfunction, and inflammatory diseases.

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