Novel lactic acid bacteria and use thereof

Abstract

The invention relates to a novel lactic acid bacterium, namely, bifidobacterium bifidum P61 KCCM13367P, lactobacillus paracasei P62 KCCM13368P or a mixture of the bifidobacterium bifidum and the lactobacillus paracasei. The novel lactic acid bacteria and mixtures 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 ameliorates cognitive disorders by inhibiting behaviors similar to cognitive disorders and modulating the expression of factors (BDNF, IL-10, etc.) associated with said disorders. In addition, the present invention has the effect of treating inflammation by reducing the expression of inflammatory markers (TNF-alpha, IL-6, IL-1beta, and MPO) and increasing the expression of an anti-inflammatory marker (IL-10).

Description

Technical Field

[0001] This disclosure relates to a novel lactic acid bacterium, Bifidobacterium bifidum (Bifidobacterium bifidum). Bifidobacterium bifidum P61KCCM13367P, Lactobacillus paracasei ( Lactobacillus paracasei P62 KCCM13368P or mixtures thereof, and their uses. Background Technology

[0002] As the world enters an aging society, people are exposed to various diseases, thus highlighting the increasing importance of age-related diseases. The most representative physiological change brought about by aging is the decline in muscle mass and strength. The human body is composed of approximately 600 muscles, which account for half of body weight. Muscles are made up of tens of thousands of muscle fibers (muscle cells), and while muscle fibers increase in size during development, their number decreases and their function gradually declines with age.

[0003] Sarcopenia is a disease characterized by a persistent decline in muscle mass due to an excess of muscle protein degradation over protein synthesis. This is primarily caused by reduced physical activity, malnutrition, environmental factors, disease, inflammation, mitochondrial abnormalities, and hormonal changes, with research also indicating genetic variations. Sarcopenia is a metabolic disorder that directly leads to decreased muscle strength, thus increasing the risk of death not only due to various functional declines and impairments but also due to a slowed metabolism and weakened immunity, leading to an increased prevalence of metabolic diseases such as hypertension, diabetes, arthritis, obesity, and cancer.

[0004] Another representative disease caused by population aging is cognitive impairment. Cognitive impairment is a condition characterized by cognitive impairment and behavioral changes, referring to diseases caused by a decline in functions such as memory, spatial perception, judgment, executive function, and language ability. Dementia is related to cognitive decline and is considered a disease of the elderly, but due to aging, it has become a disease that anyone can suffer from, and has become a social problem due to family care and economic burden.

[0005] Meanwhile, probiotics are live microbial agents that, as dominant bacteria, reside in the human gut, which contains a variety of microorganisms. They promote the growth of beneficial bacteria in the body and live in symbiosis with the human digestive system, playing a role in breaking down fiber and complex proteins into essential nutrients. Furthermore, they inhibit harmful bacteria (such as *Escherichia coli* and *Clostridium difficile*). Clostridium difficileLactic acid bacteria (LACBs) promote the reproduction of probiotics, improve diarrhea and constipation, and play a role in vitamin synthesis and lowering blood cholesterol. Among these probiotics, LACBs are bacteria that ferment sugars to obtain energy and produce large amounts of lactic acid. They are widely distributed in nature, including in agricultural products, food, and in the bodies of humans and animals. They are widely used in cheese fermentation, fermented milk, pickle making, bread making, etc. Generally, when LACBs are ingested, it is known that they not only inhibit harmful bacteria in the intestinal microbiota but also increase beneficial bacteria that help digestion, absorption, and breakdown of food. Therefore, various effects of consuming LACBs have been reported, such as lowering blood cholesterol levels, enhancing immunity, inhibiting endogenous infections, improving cirrhosis, and anti-cancer effects. In addition, recent studies have been conducted to enhance the efficacy of natural products fermented by LACBs.

[0006] Therefore, the inventors completed this disclosure by identifying novel lactic acid bacteria strains that exhibit ameliorative effects on sarcopenia, cognitive impairment, and inflammatory diseases. Summary of the Invention

[0007] The purpose of this disclosure is to provide Bifidobacterium bifidum P61 KCCM13367P.

[0008] Another objective of this disclosure is to provide Lactobacillus paracasei P62 KCCM13368P.

[0009] Another object of this disclosure is to provide compositions for the prevention, treatment or improvement of sarcopenia, cognitive impairment and inflammatory diseases, comprising Bifidobacterium bifidum P61 KCCM13367P, Lactobacillus paracasei P62 KCCM13368P or mixtures thereof.

[0010] In the following description, to avoid confusion caused by overlapping content, descriptions of overlapping content will be omitted. That is to say, the content of this invention is not limited to the following content, but should be interpreted according to the overall content of this invention.

[0011] This disclosure will now be described in detail.

[0012] This disclosure provides Bifidobacterium bifidum P61 (depository institution: Korean Culture Center of Microorganisms, deposit date: July 12, 2023, accession number: KCCM13367P).

[0013] The characteristic of the disclosed Bifidobacterium bifidum P61 is that it is a lactic acid bacterium isolated and identified from the intestinal microbiota of healthy individuals.

[0014] The 16S rDNA sequence used in this disclosure for the identification and classification of Bifidobacterium bifidum P61 is identical to SEQ ID NO: 1 appended to this specification. Therefore, Bifidobacterium bifidum P61 of this disclosure may include the 16S rDNA of SEQ ID NO: 1, as follows: 16S rDNA of Bifidobacterium bifidum P61 (SEQ ID NO: 1): TACGACTTAGTCCCAATCACGAGCCTCACCTTAGCGGCTCCATCCCACAAGGGGTTAGGCCACCGGCTTCGGGTGCTGCCCACTTTCATGACTTGACGGGCGGTGTGTACAAGGCCCGGGAACGCATTCACCGCGGCGTTGCTGATCCGCGATTACTAGCGACTCCGCCTTCACGGAGCCGGTTGCAGGCTCC GATCCGAACTGAGACCGGTTTTCAGGGATCCGCTCCATGTCGCCATGTCGCATCCCGCTGTACCGGCCATTGTAGCATGCGTGAAGCCCTGGACGTAAGGGGCATGATGATCTGACGTCATCCCCACCTTCCTCCGAGTTAACCCCGGCGGTCCCCCGTGAGTTCCCACCATAACGTGCTGGCAACACGGGGCGA GGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACGACCATGCACCACCTGTGAACCCGCCCCGAAGGGAAACGCCATCTCTGGCGTCGTCGGGAACATGTCAAGCCCAGGTAAGGTTCTTCGCGTTGCATCGAATTAATCCGCATGCTCCGCCGCTTGTGCGGGCCCCCGTCAAT TTCTTTGAGTTTTAGCCTTGCGGCCGTACTCCCCAGGCGGGACGCTTAACGCGTTAGCTCCGACACGGAACACGTGGAACGTGCCCCACATCCAGCGTCCACCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTGACGGCCCAGAGACCTGCCTTCG Analysis of the 16S rDNA nucleotide sequence of SEQ ID NO: 1 showed 99% homology with known Bifidobacterium strains (homological to Bifidobacterium bifidum strain KCTC3202), and whole-genome analysis also showed 98.8% homology (homological to Bifidobacterium JCM1255), thus demonstrating the highest molecular phylogenetic relationship with Bifidobacterium bifidum. Therefore, this lactic acid bacterium was identified as Bifidobacterium bifidum, named Bifidobacterium bifidum P61, and deposited at the Korean Center for Microbial Culture (KCCM13367P) on July 12, 2023.

[0015] The disclosed Bifidobacterium bifidum P61 is a Gram-positive bacterium with a rod-shaped cell morphology. More specifically, the physiological characteristics of Bifidobacterium bifidum P61 can be analyzed using methods commonly used in the art.

[0016] Specifically, the physiological characteristics of Bifidobacterium bifidum P61 were analyzed using a carbon source via an API kit, and the results are shown in Table 1 below.

[0017] [Table 1]

[0018] This disclosure provides Lactobacillus paracasei P62 (depository institution: Korea Center for Microbiology, deposit date: 2023.07.12, accession number: KCCM13368P).

[0019] The Lactobacillus paracasei P62 disclosed herein is characterized in that it is a lactic acid bacterium isolated and identified from the intestinal microbiota of healthy individuals.

[0020] The 16S rDNA sequence used in this disclosure for the identification and classification of *Lactobacillus paracasei* P62 is identical to SEQ ID NO: 2 appended to this specification. Therefore, *Lactobacillus paracasei* P62 of this disclosure may include the 16S rDNA of SEQ ID NO: 2, as follows: 16S rDNA of Lactobacillus paracasei P62 (SEQ ID NO: 2): ATGGCTCAGGATGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAACGAGTTCTCGTTGATGATCGGTGCTTGCACCGAGATTCAACATGGAACGAGTGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCCTTAAGTGGGGGATAACATTTGGAAACAGATGCTAATACCGCATAGATCCAAGAACCGCATGGTTCTTGGCTGAAAGATGGCGTAAGCTATCGCTTTTGGATGGACCCGCGGCGTATTAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCGATGATACGTAGCCGAACTGAGAGGTTGATGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGCAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTGGAGAAGAATGGTCGGCAGAGTAACTGTTGTCGGCGTGACGGTATCCAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCCTCGGCTTAACCGAGGAAGCGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCATG Analysis of the 16S rDNA sequence of SEQ ID NO: 2 showed 99% homology (homological to Lactobacillus paracasei strain R094) and 98.6% homology (homological to Lactobacillus paracasei subspecies JCM8130) with known Lactobacillus paracasei strains. Whole-genome analysis also showed 98.7% homology, demonstrating the highest molecular phylogenetic relationship with Lactobacillus paracasei. Therefore, this lactic acid bacterium was identified as Lactobacillus paracasei and named Lactobacillus paracasei P62, and deposited at the Korean Center for Microbial Culture (KCCM13368P) on July 12, 2023.

[0021] The *Lactobacillus paracasei* P62 disclosed herein is a Gram-positive bacterium with a rod-shaped cell morphology. More specifically, the physiological characteristics of *Lactobacillus paracasei* P62 can be analyzed using methods conventional in the art.

[0022] Specifically, the physiological characteristics of Lactobacillus paracasei P62 were analyzed using an API kit with carbon source, and the results are shown in Table 2 below.

[0023] [Table 2]

[0024] The present invention discloses Bifidobacterium bifidum P61 KCCM13367P, Lactobacillus paracasei P62 KCCM13368P, or a mixture thereof, which can treat sarcopenia by: (i) inhibiting the expression of myofibril degradation proteins MuRF-1 and MAFbx / Atrogin-1, as well as inflammatory cytokines TNF-α and IL-6; (ii) increasing the signaling activation of AKT, a myofibril synthesis protein; (iii) inhibiting the concentrations of blood corticosterone and endotoxins that induce sarcopenia; and (iv) promoting muscle strength and muscle formation.

[0025] It also exhibits therapeutic effects on cognitive impairment by: (i) inhibiting cognitive impairment-like behaviors; (ii) increasing the expression of BDNF and IL-10; and (iii) decreasing the expression of TNF-α, IL-6, and IL-1β and the concentration of endotoxins in the blood. Furthermore, it alleviates inflammation by reducing the expression of inflammatory markers (TNF-α, IL-6, IL-1β, and MPO) and increasing the expression of the anti-inflammatory marker (IL-10).

[0026] This disclosure provides pharmaceutical compositions comprising Bifidobacterium bifidum P61 KCCM13367P, Lactobacillus paracasei P62 KCCM13368P, or a mixture thereof.

[0027] This disclosure provides pharmaceutical compositions comprising Bifidobacterium bifidum P61 KCCM13367P, Lactobacillus paracasei P62 KCCM13368P, or mixtures thereof; and a pharmaceutically acceptable carrier.

[0028] This disclosure provides pharmaceutical compositions for the prevention or treatment of sarcopenia, cognitive impairment, and inflammatory diseases, comprising Bifidobacterium bifidum P61 KCCM13367P, Lactobacillus paracasei P62 KCCM13368P, or mixtures thereof.

[0029] As a specific embodiment for achieving the above objectives, this disclosure provides a pharmaceutical composition for the prevention or treatment of sarcopenia, comprising Bifidobacterium bifidum P61 KCCM13367P, Lactobacillus paracasei P62 KCCM13368P, or a mixture thereof.

[0030] In this disclosure, "sarcopenia" refers to a disease characterized by a decrease in muscle mass and a gradual decline in muscle volume and strength. Specifically, it is a general term for diseases in which the muscles of the limbs gradually atrophy in an almost bilaterally symmetrical manner, and may be associated with cancer, aging, kidney disease, genetic diseases, and various chronic diseases. Sarcopenia can be at least one selected from the group consisting of, but is not limited to, muscle atrophy, disuse atrophy, spinal muscular atrophy, malnutrition, muscle stiffness, hypotonia, muscle weakness, muscular dystrophy, amyotrophic lateral sclerosis, spinal-bulbar muscular atrophy, and myasthenia gravis.

[0031] According to one embodiment of this disclosure, it has been demonstrated that Bifidobacterium bifidum P61 KCCM13367P, Lactobacillus paracasei P62 KCCM13368P, or mixtures thereof promote muscle formation through, for example, the following actions, thereby exhibiting an alleviating effect on sarcopenia: (i) inhibiting the expression of myofibril degradation proteins MuRF-1 and MAFbx / Atrogin-1, as well as inflammatory cytokines TNF-α and IL-6; (ii) increasing muscle strength, muscle mass, and signaling activation of AKT as a myofibril synthesis protein; (iii) increasing the expression of MyHC, MyoG, PGC1-α, and SIRT-1; and (iv) reducing blood corticosterone and blood endotoxin.

[0032] As a specific embodiment for achieving the above objectives, this disclosure provides a pharmaceutical composition for the prevention or treatment of cognitive impairment, comprising Bifidobacterium bifidum P61 KCCM13367P, Lactobacillus paracasei P62 KCCM13368P, or a mixture thereof.

[0033] In this disclosure, "cognitive impairment" refers to a condition that displays cognitive impairment and behavioral changes, and specifically refers to a disease caused by a decline in functions such as memory, spatial perception, judgment, executive function, and language ability. Cognitive impairment can be any one or more of the following groups: for example, Alzheimer's disease, Huntington's disease, vascular dementia, Pick's disease, Parkinson's disease, Creutzfeldt-Jakob disease, and dementia, but is not limited thereto. Furthermore, cognitive impairment also encompasses memory impairment, dementia symptoms, etc., caused by the aforementioned symptoms.

[0034] According to one embodiment of this disclosure, it has been demonstrated that Bifidobacterium bifidum P61 KCCM13367P, Lactobacillus paracasei P62 KCCM13368P, or mixtures thereof have the effect of improving cognitive impairment by: (i) inhibiting cognitive impairment-like behaviors; (ii) increasing the expression of BDNF and IL-10; and (iii) decreasing the expression of TNF-α, IL-6 and IL-1β and the concentration of blood endotoxins.

[0035] As a specific embodiment for achieving the above objectives, this disclosure provides a pharmaceutical composition for the prevention or treatment of inflammatory diseases, comprising Bifidobacterium bifidum P61 KCCM13367P, Lactobacillus paracasei P62 KCCM13368P, or a mixture thereof.

[0036] In this disclosure, "inflammatory disease" is a general term for a disease in which inflammation is the primary lesion, wherein an inflammatory disease can be any one or more selected from the group consisting of: for example, inflammatory bowel disease (IBD), arthritis, gout, hepatitis, obesity, gastritis, nephritis, diabetes, tuberculosis, bronchitis, pleurisy, peritonitis, spondylitis, pancreatitis, urethritis, cystitis, vaginitis, atherosclerosis, sepsis, and periodontitis, but is not limited thereto.

[0037] Inflammatory bowel disease (IBD) refers to a group of inflammatory conditions affecting the colon and gastrointestinal tract. Representative examples of IBD include ulcerative colitis (UC) and Crohn's disease. The main difference between UC and Crohn's disease lies in the location and characteristics of the inflammatory changes. Crohn's disease can affect any part of the gastrointestinal tract, from the mouth to the anus, while the inflammation in UC is limited to the colon and rectum. Due to the specific presentation, it is often impossible to definitively diagnose Crohn's disease or ulcerative colitis. In such cases, an indeterminate colitis diagnosis 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.

[0038] In other words, in this disclosure, inflammatory bowel disease can be one or more of ulcerative colitis, Crohn's disease, collagenous colitis, lymphocytic colitis, ischemic colitis, diverted colitis, Behcet's disease, and undifferentiated colitis.

[0039] According to one embodiment of this disclosure, it has been demonstrated that Bifidobacterium bifidum P61 KCCM13367P, Lactobacillus paracasei P62 KCCM13368P, or a mixture thereof can reduce the expression of inflammatory markers (TNF-α, IL-6, IL-1β, and MPO) and increase the expression of anti-inflammatory marker (IL-10), thereby exhibiting an anti-inflammatory therapeutic effect.

[0040] In this disclosure, "muscle ring finger protein-1 (MuRF-1)" refers to a signaling molecule that degrades muscle proteins, and it is well known that muscle atrophy continues when MuRF-1 is activated.

[0041] In this disclosure, "Atrogin-1" is a muscle-specific F-box protein that is involved in the process of muscle protein degradation, and its increase precedes the decrease in muscle mass because it is induced in the initial process of muscle atrophy.

[0042] In this disclosure, "tumor necrosis factor-α (TNF-α)" is a cytokine produced by macrophages in response to various immune stimuli, including endotoxins, which are bacterial toxins, and plays an important role in the immune system. Aberrant regulation of TNF-α is known to occur in a variety of diseases, such as Alzheimer's disease, cancer, depression, and inflammatory bowel disease (IBD).

[0043] In this disclosure, "interleukin-6 (IL-6)" is an interleukin that can act as both a pro-inflammatory cytokine and an anti-inflammatory myocytokine, and plays the role of a differentiation factor in the activation of B lymphocytes and affects the function of various cells.

[0044] In this disclosure, "FOXO3a" refers to one of the forkhead box O family genes (FOXO1, FOXO3a, FOXO4, FOXO6), which are known as longevity genes, and regulates the expression of genes involved in cell death, cell cycle arrest, oxidative stress resistance, and autophagy.

[0045] In this disclosure, "nuclear factor-κB (NF-κB)" refers to a protein complex involved in DNA transcription, cytokine production and cell survival, and is involved in the regulation of inflammatory responses, the regulation of the immune system, cell death, cell proliferation, epithelial cell differentiation, etc.

[0046] In this disclosure, "AKT (protein kinase B, PKB)" is a general term for a group of three serine / threonine-specific protein kinases that play important roles in cell cycle progression, smooth muscle cell differentiation, promoting glucose uptake, angiogenesis, inhibiting apoptosis, and promoting cell growth.

[0047] In this disclosure, "mammalian target of rapamycin (mTOR)" is a mammalian serine / threonine kinase target of rapamycin, consisting of two independent protein complexes, mTOR complex 1 and mTOR complex 2, which regulate different cellular processes. mTOR can function as a serine / threonine protein kinase that regulates cell growth, cell proliferation, cell motility, cell survival, protein synthesis, autophagy, and transcription.

[0048] In this disclosure, "myosin heavy chain (MyHC)" is the main contractile protein of sarcomeres, which is an important component for maintaining muscle contraction.

[0049] In this disclosure, “MyoG” refers to the gene encoding myopoietin, which is involved in the regulation of skeletal muscle development or muscle formation and repair.

[0050] In this disclosure, “PGC1-α” is a key transcription factor activator that induces mitochondrial biosynthesis in cells and is known to regulate the composition and function of individual mitochondria.

[0051] In this disclosure, “SIRT-1” is the gene encoding Sirtuin1, which is a protein that plays a role in cellular responses to inflammatory, metabolic, and oxidative stress factors.

[0052] In this disclosure, "brain-derived neurotrophic factor (BDNF)" is a 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.

[0053] In this disclosure, "IL-10" refers to a potent immunosuppressive Th-2 cell cytokine produced by lymphocytes, which functions to inhibit the replication of macrophages / monocytes and T lymphocytes as well as the secretion of inflammatory cytokines.

[0054] In this disclosure, "IL-1β" is also referred to as leukocyte pyrogen, leukocyte endogenous mediator, monocyte cytokine, or lymphocyte activating factor, and acts as a key mediator of the inflammatory response.

[0055] In this disclosure, "myeloperoxidase (MPO)" is a dimerizing enzyme mainly found in azurophilic granules of neutrophils and lysosomes of monocytes, which promotes the formation of peroxides to primarily enhance the bactericidal activity of the innate immune response.

[0056] In this disclosure, "corticosterone" is a type of adrenocortical hormone and a representative glucocorticoid. Corticosterone is synthesized from pregnenolone or progesterone in the body and excreted through the kidneys. Corticosterone is known to protect the body from stress, promote protein breakdown, promote carbohydrate synthesis, suppress inflammation, and exhibit antagonistic effects on insulin.

[0057] In this disclosure, "endotoxin" is a component of the outer membrane of Gram-negative bacteria, known to react with the body's immune cells to induce an inflammatory response, and is also chemically known as lipopolysaccharide (LPS).

[0058] The results above show that Bifidobacterium bifidum P61 KCCM13367P, Lactobacillus paracasei P62 KCCM13368P, or mixtures thereof have outstanding efficacy in the prevention, treatment, or improvement of sarcopenia, cognitive impairment, and inflammatory diseases.

[0059] In addition, this drug composition can effectively prevent, treat or improve sarcopenia, cognitive impairment and inflammatory diseases by altering the composition of the gut microbiota.

[0060] The strains disclosed herein can be used in various forms, such as live cells, dead cells, cultures, lysates, or extracts. The strains disclosed herein, even under conditions including the strain, exhibit equivalent or higher levels of results in relation to the aforementioned effects (especially considering live cells).

[0061] Live cells refer to the living bacteria themselves, while dead cells refer to the effective components that are separated and extracted from viable bacteria under certain conditions through methods such as heat drying, pressurization, and drug treatment.

[0062] A culture is defined as a product obtained by culturing lactic acid bacteria in a known liquid or solid culture medium, and is a concept that includes the strains disclosed herein. The product may include lactic acid bacteria. The culture medium may be selected from known liquid or solid culture media, and may be, for example, MRS liquid medium, GAM liquid medium, MRS agar medium, GAM agar medium, BL agar medium, but is not limited thereto.

[0063] Lysate refers to the fragmented form obtained by separating and processing living cells, dead cells, or their cultures through mechanical or chemical methods. For example, fragmented forms can be prepared using bead mills, presses, ultrasonic disruptors, microfluidics, or enzyme treatment.

[0064] Extract refers to an extract obtained by using known extraction methods (known extraction solvents (e.g., water, C1 to C4 alcohols (methanol, ethanol, etc.))) from live cells, dead cells, and / or lysates.

[0065] In this disclosure, synergistic effects have been demonstrated in mixtures of strains, and this disclosure includes the use of any mixtures thereof.

[0066] In other words, excellent synergistic effects can be observed by mixing Bifidobacterium bifidum P61 KCCM13367P and Lactobacillus paracasei P62KCCM13368P as disclosed in this paper.

[0067] For example, a mixture of *Bifidobacterium bifidum* P61 KCCM13367P and *Lactobacillus paracasei* P62 KCCM13368P can be included based on the number of colony units (CFU) at a ratio of 10:1 to 1:10, preferably at a ratio of 1:6 to 1:1, more particularly at a ratio of 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, and more preferably at a ratio of 1:4.

[0068] According to one embodiment of this disclosure, the strain mixture exhibits excellent synergistic effects on sarcopenia, cognitive impairment, and inflammatory diseases, particularly inflammatory bowel disease.

[0069] In particular, in terms of gut microbiota normalization, strain mixtures may show a superior effect in maintaining gut health compared to single strains.

[0070] The pharmaceutical compositions according to this disclosure can be formulated into pharmaceutical preparations using methods well known in the art to provide immediate, sustained, or extended release of the active ingredient upon administration to mammals. During the preparation of the formulation, the pharmaceutical compositions according to this disclosure may additionally include a pharmaceutically acceptable carrier within the range that does not inhibit the activity of the compounds of this disclosure.

[0071] Pharmaceutically acceptable carriers include, but are not limited to, commonly used carriers such as lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylparaben, propylparaben, talc, magnesium stearate, and mineral oil. Furthermore, the pharmaceutical compositions disclosed herein may include diluents or excipients such as fillers, thickeners, binders, wetting agents, disintegrants, surfactants, and other pharmaceutically acceptable additives.

[0072] The pharmaceutical compositions disclosed in this invention can be administered at pharmaceutically effective amounts. Here, "pharmaceutically effective amount" means an amount sufficient to prevent or treat a disease, with a benefit / risk ratio suitable for medical treatment. The level of the effective amount can be selected by those skilled in the art based on factors such as formulation method, patient condition and weight, patient sex, age, disease severity, drug form, route and duration of administration, excretion rate, responsiveness, etc. The effective amount can vary depending on the route of treatment, the use of excipients, and the possibility of use with other drugs recognized by those skilled in the art. However, for optimal effect, for oral administration, the compositions of this disclosure can generally be administered to adults at a dose of 0.0001 mg / kg body weight to 100 mg / kg body weight daily, preferably 0.001 mg / kg body weight to 100 mg / kg body weight daily, but the dosage does not limit the scope of this disclosure in any way.

[0073] The pharmaceutical compositions according to this disclosure can be administered to mammals, such as mice, livestock, and humans, via various routes. In particular, the pharmaceutical compositions according to this disclosure can be administered orally or parenterally (e.g., topically, intravenously, subcutaneously, or intraperitoneally), but oral administration is preferred. Solid dosage forms for oral administration may include powders, granules, tablets, capsules, soft capsules, pills, etc. Liquid dosage forms for oral administration include suspensions, oral solutions, emulsions, syrups, aerosols, etc., which may include various excipients, such as wetting agents, sweeteners, flavoring agents, preservatives, etc., and also commonly used simple diluents, such as water and liquid paraffin. Preparations intended for parenteral administration may be formulated or used in the following forms: sterile aqueous solutions, solutions, non-aqueous solvents, suspensions, emulsions, eye drops, eye ointments, syrups, suppositories, aerosols, and other topical preparations, as well as sterile injections. Each preparation is sterilized according to conventional methods, and preferably, pharmaceutical compositions of creams, gels, patches, sprays, ointments, liniments, lotions, eye ointments, eye drops, pastes, or plasters are prepared and used, but are not limited thereto. Preparations for topical application may be anhydrous or aqueous formulations, depending on the clinical prescription. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils (such as olive oil), injectable esters (such as ethyl oleate), etc. Suppository bases may include white glue, polyethylene glycol, Tween 61, cocoa butter, glyceryl laurate, glycerin gelatin, etc.

[0074] The pharmaceutical compositions according to this disclosure can be administered alone as a single therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents. Furthermore, the pharmaceutical compositions of this disclosure can be administered as a single or multiple doses. Considering all of the above factors, it is important that the dosage is such that the maximum effect is achieved with the minimum possible dose, and that this can be readily determined by those skilled in the art.

[0075] As used herein, the term "subject" includes animals or humans whose symptoms can be relieved by administration of the pharmaceutical composition according to this disclosure. By administering the pharmaceutical composition according to this disclosure to a subject, sarcopenia, cognitive impairment, or inflammatory diseases can be effectively prevented and treated.

[0076] In this disclosure, the term "administration" means the introduction of a predetermined substance into a human or animal by any suitable method, and the therapeutic compositions according to this disclosure can be administered orally or parenterally via any common route, as long as it can reach the target tissue. Furthermore, the therapeutic compositions according to this disclosure can be administered via any device through which the active ingredient can move to target cells.

[0077] The preferred dosage of the pharmaceutical composition according to this disclosure varies depending on the patient's condition and weight, disease severity, form of the drug, route of administration, and duration of administration, and can be appropriately selected by those skilled in the art.

[0078] This disclosure provides Bifidobacterium bifidum P61 KCCM13367P, Lactobacillus paracasei P62 KCCM13368P, or mixtures thereof, for the prevention or treatment of one or more diseases selected from the group consisting of sarcopenia, cognitive impairment, and inflammatory diseases.

[0079] This disclosure provides Bifidobacterium bifidum P61 KCCM13367P, Lactobacillus paracasei P62 KCCM13368P, or a mixture thereof, for the prevention or treatment of sarcopenia.

[0080] This disclosure provides Bifidobacterium bifidum P61 KCCM13367P, Lactobacillus paracasei P62 KCCM13368P, or mixtures thereof for the prevention or treatment of cognitive impairment.

[0081] This disclosure provides Bifidobacterium bifidum P61 KCCM13367P, Lactobacillus paracasei P62 KCCM13368P, or mixtures thereof, for the prevention or treatment of inflammatory diseases.

[0082] This disclosure provides the use of Bifidobacterium bifidum P61 KCCM13367P, Lactobacillus paracasei P62 KCCM13368P, or mixtures thereof in the production of a medicament for treating one or more diseases selected from the group consisting of sarcopenia, cognitive impairment, and inflammatory diseases.

[0083] This disclosure provides the use of Bifidobacterium bifidum P61 KCCM13367P, Lactobacillus paracasei P62 KCCM13368P, or mixtures thereof in the production of a medicament for the treatment of sarcopenia.

[0084] This disclosure provides the use of Bifidobacterium bifidum P61 KCCM13367P, Lactobacillus paracasei P62 KCCM13368P, or mixtures thereof in the production of a medicament for the treatment of cognitive impairment.

[0085] This disclosure provides the use of Bifidobacterium bifidum P61 KCCM13367P, Lactobacillus paracasei P62 KCCM13368P, or mixtures thereof in the production of medicaments for treating inflammatory diseases.

[0086] This disclosure provides a method for the prevention or treatment of sarcopenia, cognitive impairment, and inflammatory diseases, comprising the step of administering Bifidobacterium bifidum P61 KCCM13367P, Lactobacillus paracasei P62 KCCM13368P, or a mixture thereof to a subject in need.

[0087] This disclosure provides compositions comprising Bifidobacterium bifidum P61 KCCM13367P, Lactobacillus paracasei P62 KCCM13368P, or mixtures thereof.

[0088] This disclosure provides a food composition for the prevention or improvement of sarcopenia, cognitive impairment and inflammatory diseases, comprising Bifidobacterium bifidum P61 KCCM13367P, Lactobacillus paracasei P62 KCCM13368P or a mixture thereof.

[0089] The terms “Bifidobacterium bifidum P61”, “Lactobacillus paracasei P62”, “sarcopenia”, “cognitive impairment” and “inflammatory disease” are the same as those described above.

[0090] In particular, the lactic acid bacteria included in the food composition according to this disclosure may be their live cells, their dead cells, their cultures, their lysates or extracts thereof, but any form of lactic acid bacteria that can achieve the effect of preventing or improving sarcopenia, cognitive impairment or inflammatory diseases may be used without limitation.

[0091] There are no specific restrictions on the types of food that can contain lactic acid bacteria. Foods for which lactic acid bacteria can be added include sausages, meats, bread, chocolate, snacks, candy, pastries, instant noodles, pizza, other noodles, chewing gum, dairy products (including ice cream), various soups, beverages, tea, alcoholic beverages, and vitamin complexes. When formulated into beverages, the added liquid components are not limited to these, in addition to novel lactic acid bacteria; they can also contain various flavorings or natural carbohydrates as additional components, just like regular beverages. These natural carbohydrates can be monosaccharides (e.g., glucose, fructose, etc.), disaccharides (e.g., maltose, sucrose, etc.), and polysaccharides (e.g., conventional sugars such as dextrin, cyclodextrin, etc.), as well as sugar alcohols (e.g., xylitol, sorbitol, erythritol, etc.).

[0092] The type of food may specifically be a functional food. Functional foods can contain various nutrients, vitamins, minerals (electrolytes), flavorings (such as synthetic and natural flavorings), colorings and flavor enhancers (cheese, chocolate, etc.), pectic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. These components can be used alone or in combination, and the proportions of these additives are typically chosen to be in the range of 0.001 parts by weight to 50 parts by weight per total component.

[0093] Health functional foods are foods that emphasize the bioregulatory functions of food, and are foods that are given added value through physical, biochemical, and biotechnological methods to perform specific functions and express specific purposes. The components of such health functional foods are designed and processed to fully exert bioregulatory functions related to biological defense and regulation of body rhythms, prevention and recovery from diseases, and may contain food additives, sweeteners, or food-acceptable functional ingredients.

[0094] When Bifidobacterium bifidum P61 KCCM13367P and / or Lactobacillus paracasei P62 KCCM13368P of this disclosure are used as health functional foods (or health functional beverage additives), Bifidobacterium bifidum P61 KCCM13367P and / or Lactobacillus paracasei P62 KCCM13368P can be added alone or used together with other foods or food components, and can be used appropriately according to conventional methods. The mixed amount of Bifidobacterium bifidum P61 KCCM13367P and / or Lactobacillus paracasei P62 KCCM13368P can be appropriately determined according to its intended use (prevention, health care or improvement, therapeutic treatment).

[0095] The novel lactic acid bacteria and mixtures disclosed herein regulate gut microbiota composition, thereby inhibiting the expression of sarcopenia markers (MuRF-1, MAFbx / Atrogin-1, etc.), increasing muscle strength and mass, and promoting muscle formation to alleviate sarcopenia. Furthermore, they inhibit cognitive impairment-like behaviors and regulate the expression of factors associated with these factors (BDNF, IL-10, etc.), thus exhibiting a therapeutic effect on cognitive impairment. In addition, they also alleviate inflammation by reducing the expression of inflammatory markers (TNF-α, IL-6, IL-1β, and MPO) and increasing the expression of the anti-inflammatory marker (IL-10). Attached Figure Description

[0096] Figure 1 This diagram illustrates 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). Figure 1 The data in the table are represented as mean ± SD (n = 4). # p <0.05 vs NC. p <0.05 vs. groups treated with dexamethasone or LPS alone. Figure 2This is a graph showing the changes in the number of NF-κB+CD11c+ cells in each mouse model (Yg, Vh, Lp, Bb, LB, and Cr) as observed by immunofluorescence staining.

[0097] Figure 3 This is a graph showing the changes in gastrocnemius muscle cell size (H&E staining), the number of MyHC positive cells (immunofluorescence staining), and muscle cross-sectional area (CSA) in each mouse model (Yg, Vh, Lp, Bb, LB, and Cr).

[0098] Figure 4 This is a graph showing the changes in the number of NF-κB+Iba1+ cells and BDNF+NeuN+ cells in each mouse model (Yg, Vh, Lp, Bb, LB, and Cr) as confirmed by immunofluorescence staining.

[0099] ( Figure 2 The data in the table are represented as mean ± SD (n = 7). # p <0.05 vs. Yg. p <0.05 vs. Vh / Ag. Detailed Implementation Preferred embodiments are presented below to aid in understanding this disclosure. However, these embodiments are provided for the purpose of facilitating understanding of this disclosure only, and the content of this disclosure is not limited thereto.

[0100] Experimental Example 1. Isolation and Culture of Lactic Acid Bacteria Bifidobacterium bifidum P61 and Lactobacillus paracasei P62, isolated from the human gut microbiota, were cultured in MRS medium (BD, Sparks, MD) at room temperature, then isolated and collected. The collected cells were freeze-dried, washed twice with physiological saline, and resuspended for in vitro experiments, and then resuspended in 1% maltose solution for in vivo experiments.

[0101] Experimental Example 2. C2C12 Cell Culture C2C12 cells (Korea Type Culture Collection) were cultured in DMEM medium containing 10% fetal bovine serum (FBS), 1% antibiotic-antifungal solution, and 3.7 g / L NaHCO3 in a 5% CO2 / 95% humidified air incubator at 37°C. The confluent myoblasts (80%) were then differentiated for 4 days in DMEM medium containing 2% horse serum.

[0102] Experimental Example 3. Preparation of a Mouse Model To establish mouse models, aged C57BL / 6 mice (male, 18 months old) and young C57BL / 6 mice (male, 3 months old) were purchased and used from Orientbio Co., Ltd. Furthermore, all animal experiments conducted in this disclosure were approved in advance by the Institutional Animal Care and Use Committee of Kyung Hee University (IACUC No. KHUASP(SE)-22-567) and conducted ethically in accordance with the university's guidelines on laboratory animal care and use.

[0103] The mice were divided into six groups, with eight mice in each group.

[0104] Yg(NC): Young C57BL / 6 mice treated with solvent.

[0105] Vh: Aged C57BL / 6 mice treated with solvent.

[0106] Bb: Use Bifidobacterium bifidum P61 (1×10⁻⁶) 9 Aged C57BL / 6 mice treated with CFU / mouse.

[0107] Lp: Lactobacillus paracasei P62 (1×10⁻⁶) 9 Aged C57BL / 6 mice treated with CFU / mouse.

[0108] LB: Aged C57BL / 6 mice treated with a 1:4 mixture of Bifidobacterium bifidum P61 and Lactobacillus paracasei P62.

[0109] Cr: Aged C57BL / 6 mice treated with creatine (75 mg / kg).

[0110] At this time, the preparations of all the above groups were administered orally once a day for 8 weeks (6 days a week).

[0111] Experimental Example 4. Methods for Measuring Motor Ability and Testing Cognitive Function In each group of Experimental Example 3, the mice's motor abilities were measured 20 hours after the last treatment with the formulation. Specifically, grip strength was tested by placing the mice's limbs on the grid of a grip strength meter and measuring their grip strength before the mice fell off the bar. Additionally, a treadmill test was conducted by acclimatizing the mice to a treadmill for one week and measuring their running time and distance by running at 23 m / min for 30 minutes.

[0112] Cognitive function was assessed using the Y-maze test, which employs a three-armed Y-maze measuring device. The three arms extend in a Y-shape, each measuring 25 cm in length, 14 cm in height, and 5 cm in width, and are positioned at the same angle.

[0113] Experimental Example 5. ELISA and Immunoblot Mouse gastrocnemius muscle, colon, brain tissue, and cells were homogenized on ice with RIPA lysis buffer containing a 1% phosphatase inhibitor mixture and a 1% protease inhibitor mixture (RPP), and centrifuged at 15,000 g for 15 minutes at 4°C. The supernatant was then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane for Western blotting. Proteins were detected using antibodies, HRP-conjugated secondary antibodies, and colorimetric analysis was performed using an ECL assay kit. Simultaneously, cytokines were analyzed using an ELISA kit (R&D Systems) by transferring the homogenate supernatant to 96-well plates.

[0114] The antibodies used at this time are as follows: phosphorylated 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), phosphorylated 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). The following are listed: signaling, p-FOXO3a (ser253, Cell signaling), FOXO3a (75D8, Cellsignaling), β-actin (sc-47778, Santa Cruz Biotechnology), and PGC1α (ab191838, Abcam).

[0115] Experimental Example 6: Real-time Polymerase Chain Reaction (qPCR) Analysis 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 purified RNA was reverse transcribed using the PrimeScript cDNA Synthesis Kit (Takara), and PCR was performed using the TB Green Premix Ex Taq II (Takara) and Rotor-GeneQ 5plex Platform (Qiagen).

[0116] Meanwhile, total DNA was purified from gastrocnemius muscle and C2C12 cells using the Genomic DNeasy kit (Qiagen), and mtDNA copy number was measured by qPCR.

[0117] The primers used in this study are shown in Table 3.

[0118] [Table 3]

[0119] Experimental Example 7. Hematoxylin and eosin (H&E) staining and immunofluorescence staining After paraformaldehyde was injected into the heart of mice, the gastrocnemius muscle of the hind limb was cut into 5 μm sections and stained with hematoxylin and eosin (H&E).

[0120] In addition, hypothalamic, gastrocnemius, and colonic tissues were collected from mice, sectioned, and incubated with primary antibodies (BDNF, NeuN, p65, NF-κB, Iba1, and / or CD11c) for 12 hours. Subsequently, the sections were treated with secondary antibodies conjugated to Alexa Fluor 594 or Alexa Fluor 488 and observed by confocal microscopy.

[0121] Implementation Plan 1. Bifidobacterium bifidum P61 and Lactobacillus paracasei P62 alleviate sarcopenia. (1) Confirm the changes in the expression of MuRF-1, MAFbx / Atrogin-1, TNF-α and IL-6 and the activation of NF-κB. C2C12 cells (1×10⁻⁶) from the mouse groups (Yg, Vh, Bb, Lp, LB, and Cr) prepared in Experimental Example 3 were treated with 0.3 mM dexamethasone or 100 ng / mL LPS. 5 The expression changes of myofibril degradation proteins (MuRF-1, MAFbx / Atrogin-1), inflammatory cytokines (TNF-α and IL-6), and NF-κB activation were observed.

[0122] The results confirmed that Bifidobacterium bifidum P61 and / or Lactobacillus paracasei P62 inhibited dexamethasone-induced expression of MuRF-1 and MAFbx / Atrogin-1. Figure 1 A and Figure 1 B). Furthermore, it was confirmed that lipopolysaccharide (LPS)-induced expression of TNF-α and IL-6, as well as NF-κB activation, were significantly inhibited. Figure 1 C to Figure 1 E).

[0123] In other words, it was confirmed that Bifidobacterium bifidum P61, Lactobacillus paracasei P62, or a mixture thereof improved the expression of sarcopenia-related factors and the activation of NF-κB, thereby alleviating sarcopenia.

[0124] (2) Confirm changes in muscle strength, muscle mass, and AKT signal activation. Experiments were conducted to verify the changes in muscle strength and muscle mass in the mouse groups (Yg, Vh, Bb, Lp, LB, and Cr) prepared in Experiment Example 3, and no significant differences in food intake and weight gain were observed among the aging mouse models during the experiment.

[0125] To verify changes in muscle strength, each mouse group underwent treadmill testing and grip strength testing.

[0126] Treadmill testing confirmed that treatment with Bifidobacterium P61 and / or Lactobacillus paracasei P62 increased running time and distance in aging mouse models (Tables 4A and 4B). The group treated with a 1:4 mixture of Bifidobacterium P61 and Lactobacillus paracasei P62 (LB) showed the most significant increase in running time and distance. Furthermore, grip strength testing confirmed that, compared to the Vh group, the groups treated with Bifidobacterium P61 and / or Lactobacillus paracasei P62 (Bb, Lp, and LB) showed approximately 1.15-fold, 1.14-fold, and 1.16-fold increases in grip strength, respectively (Table 4C).

[0127] 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) muscles in each group were observed. The results showed that treatment with Bifidobacterium bifidum P61 and / or Lactobacillus paracasei P62 significantly increased the weight of GA, SOL, QD, EDL, and TA in the aging mouse model (Tables 4D to 4H), and correspondingly, total muscle mass also showed an increasing pattern (Table 4I).

[0128] The specific results of the above experiments are shown in Table 4 below.

[0129] [Table 4]

[0130] Furthermore, it was also confirmed that in the gastrocnemius muscle of the groups treated with Bifidobacterium bifidum P61 and / or Lactobacillus paracasei P62 (Bb, Lp, and LB), the activation of AKT and mTOR, which are related to protein synthesis, was also increased, as shown in Table 5 below.

[0131] [Table 5]

[0132] In other words, it was confirmed that Bifidobacterium bifidum P61, Lactobacillus paracasei P62, or a mixture thereof increased muscle strength, muscle mass, and AKT signaling activation, and reduced FOXO3a and NF-κB activation, thereby showing a therapeutic effect on sarcopenia.

[0133] (3) Confirm changes in myogenin gene expression The mouse groups (Yg, Vh, Bb, Lp, LB, and Cr) prepared in Experimental Example 3 were subjected to qPCR, Western blotting, H&E staining, and immunofluorescence staining to confirm the changes in myogenic gene expression caused by the administration of Bifidobacterium bifidum P61 and / or Lactobacillus paracasei P62.

[0134] Specifically, qPCR results showed decreased expression of MuRF-1, MAFbx / Atrogin-1, TNF-α, and IL-6 in the groups treated with Bifidobacterium bifidum P61 and / or Lactobacillus paracasei P62 (Bb, Lp, and LB). Conversely, compared to the creatine-treated group (Cr), MyHC and MyoG expression was increased, particularly MyHC 2X, MyHC 2B, and MyHC. Furthermore, mtDNA levels in the gastrocnemius muscle recovered to levels similar to those in young mice, and significantly increased expression levels of PGC1-α and SIRT-1, which are involved in mitochondrial gene expression, were observed.

[0135] The specific results are shown in Table 6 below.

[0136] [Table 6]

[0137] According to ELISA, Bifidobacterium bifidum P61 and / or Lactobacillus paracasei P62 reduced age-related increases in blood corticosterone, as shown in Table 7 below.

[0138] [Table 7]

[0139] Furthermore, H&E staining or immunofluorescence staining of muscle cells revealed a decrease in the number of NF-κB+CD11c+ cells in the groups treated with Bifidobacterium bifidum P61 and / or Lactobacillus paracasei P62 (Bb, Lp, and LB). Figure 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 recovered to a level similar to that of the young mouse model (Yg). Figure 3 ).

[0140] In other words, it has been shown that muscle formation is promoted by Bifidobacterium bifidum P61, Lactobacillus paracasei P62, or a mixture thereof, thereby improving sarcopenia.

[0141] Implementation Plan 2. Bifidobacterium bifidum P61 and Lactobacillus paracasei P62 alleviate cognitive impairment. The mouse groups (Yg, Vh, Bb, Lp, LB, and Cr) prepared in Experimental Example 3 were subjected to the Y maze test to measure spontaneous alternation behavior and to confirm changes in the expression of cognitive function-related factors.

[0142] In the Y-maze test, aging mouse models (Bb, Lp, and LB) treated with *Bifidobacterium bifidum* P61 and / or *Lactobacillus paracasei* P62 showed a trend toward increased spontaneous alternation behaviors, confirming suppression of cognitive impairment-like behaviors (Table 8A). Furthermore, the hippocampus of the aging mouse models showed increased expression of BDNF and IL-10, and suppressed expression of TNF-α, IL-6, and IL-1β (Tables 8B to 8H), and F-κB... + Iba1 + Cells and BDNF + NeuN + Increase in cell number ( Figure 4 ).

[0143] The specific results are shown in Table 8 below. Figure 4 As shown.

[0144] [Table 8]

[0145] Furthermore, administration of Bifidobacterium bifidum P61 and / or Lactobacillus paracasei P62 reduced endotoxins that caused systemic inflammation and cognitive impairment. Specific results are shown in Table 9 below.

[0146] [Table 9]

[0147] In other words, it has been demonstrated that the application of Bifidobacterium bifidum P61, Lactobacillus paracasei P62, or a mixture thereof has a therapeutic effect on cognitive impairment.

[0148] Implementation Plan 3. Confirm that Bifidobacterium bifidum P61 and Lactobacillus paracasei P62 alleviate inflammation. To evaluate 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.

[0149] As shown in the qPCR results, the groups treated with Bifidobacterium bifidum P61 and / or Lactobacillus paracasei P62 (Bb, Lp and LB) showed decreased expression of inflammatory markers TNF-α, IL-6, IL-1β and myeloperoxidase (MPO), and increased expression of the anti-inflammatory marker IL-10.

[0150] The specific results are shown in Table 10 below.

[0151] [Table 10]

[0152] In other words, it was confirmed that Bifidobacterium bifidum P61, Lactobacillus paracasei P62, or a mixture thereof can alleviate inflammation by reducing the expression of inflammatory markers.

[0153] [Login ID] Preservation institution: Korean Center for Microbial Culture (KCCM) Login ID: KCCM13367P Deposit date: July 12, 2023 The Budapest Treaty on the International Recognition of the Preservation of Microorganisms for Patent Proceedings International Forms Original Preservation Acceptance Notice Signed by the international depositary authority identified at the bottom of this page in accordance with Article 7.1.

[0154] [Login ID] Preservation institution: Korean Center for Microbial Culture (KCCM) Login ID: KCCM13368P Deposit date: July 12, 2023 The Budapest Treaty on the International Recognition of the Preservation of Microorganisms for Patent Proceedings International Forms Original Preservation Acceptance Notice Signed by the international depositary authority identified at the bottom of this page in accordance with Article 7.1.

Claims

1. Bifidobacterium bifidum ( Bifidobacterium bifidum )P61 KCCM13367P.

2. The Bifidobacterium bifidum P61 KCCM13367P according to claim 1, wherein the Bifidobacterium bifidum P61 KCCM13367P comprises the 16S rDNA nucleotide sequence of SEQ ID NO:

1.

3. Lactobacillus paracasei ( Lactobacillus paracasei )P62 KCCM13368P.

4. The *Lactobacillus paracasei* P62KCCM13368P according to claim 3, wherein the *Lactobacillus paracasei* P62KCCM13368P comprises the 16S rDNA nucleotide sequence of SEQ ID NO:

2.

5. A pharmaceutical composition for the prevention or treatment of sarcopenia, cognitive impairment and inflammatory diseases, comprising Bifidobacterium bifidum P61 KCCM13367P, Lactobacillus paracasei P62 KCCM13368P or a mixture thereof.

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

7. The pharmaceutical composition of claim 5, wherein the cognitive impairment 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. The 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, atherosclerosis, sepsis, and periodontitis.

9. The pharmaceutical composition of claim 5, comprising Bifidobacterium bifidum P61 KCCM13367P and Lactobacillus paracasei P62 KCCM13368P in a colony forming unit (CFU) ratio of 10:1 to 1:

10.

10. The pharmaceutical composition of claim 9, comprising Bifidobacterium bifidum P61 KCCM13367P and Lactobacillus paracasei P62 KCCM13368P in a colony forming unit (CFU) ratio of 1:6 to 1:

1.

11. A food composition for the prevention or improvement of sarcopenia, cognitive impairment and inflammatory diseases, comprising Bifidobacterium bifidum P61 KCCM13367P, Lactobacillus paracasei P62 KCCM13368P or a mixture thereof.

12. The food composition of claim 11, wherein the sarcopenia is at least one selected from the group consisting of: muscle atrophy, disuse atrophy, spinal muscular atrophy, malnutrition, muscle stiffness, hypotonia, muscle weakness, muscular dystrophy, amyotrophic lateral sclerosis, spinal bulbar muscular atrophy, and myasthenia gravis.

13. The food composition of claim 11, wherein the cognitive impairment 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. The food composition of 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, atherosclerosis, sepsis, and periodontitis.

15. The food composition of claim 11, comprising Bifidobacterium bifidum P61 KCCM13367P and Lactobacillus paracasei P62 KCCM13368P in a colony forming unit (CFU) ratio of 10:1 to 1:

10.

16. The food composition of claim 15, comprising Bifidobacterium bifidum P61 KCCM13367P and Lactobacillus paracasei P62 KCCM13368P in a colony forming unit (CFU) ratio of 1:6 to 1:

1.

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 the prevention or treatment of sarcopenia, cognitive impairment, and inflammatory diseases, comprising administering to a subject in need Bifidobacterium bifidum P61 KCCM13367P, Lactobacillus paracasei P62 KCCM13368P, or a mixture thereof.

19. Use of Bifidobacterium bifidum P61 KCCM13367P, Lactobacillus paracasei P62 KCCM13368P, or mixtures thereof in the preparation of a medicament for the treatment of one or more diseases selected from the group consisting of sarcopenia, cognitive impairment, and inflammatory diseases.

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

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