Novel probiotics and their uses

Abstract

The present invention relates to novel lactic acid bacteria, Lactobacillus rhamnosus, Bifidobacterium longum, and Lactococcus lactis. The strain according to the present invention has high value as a medicine or food due to its excellent antioxidant effect, inflammation improving effect, neuroimproving effect, cognition improving effect, intestinal microbiota imbalance improving effect, etc., and may be used in particular for the prevention, treatment or improvement of inflammatory diseases and nervous system diseases. In addition, the strain according to the present invention may be used for immune regulation and enhancement by increasing the activity of the immune system due to its immune function improving and immune enhancement effects.

Description

[Technical field]

[0001] The present invention relates to novel probiotics Lactobacillus rhamnosus, Bifidobacterium longum and Lactococcus lactis. [Background technology]

[0002] Probiotics are live bacteria activators that are distributed as dominant bacteria in the human intestine where various microorganisms exist and promote the growth of beneficial bacteria in the body. They play a role in symbiosis with the human digestive system and breaking down fiber and complex proteins to produce important nutritional components. They also suppress the growth of harmful bacteria such as Escherichia coli and Clostridium difficile, improve diarrhea and constipation, synthesize vitamins, and lower blood cholesterol. Among such probiotics, lactic acid bacteria are bacteria that ferment sugars to obtain energy and produce large amounts of lactic acid. They are widely distributed in nature, such as agricultural products, foods, and the bodies of humans and animals. They are often used in the fermentation of cheese, fermented milk, kimchi, bread, etc. It is generally known that consuming lactic acid bacteria not only suppresses harmful bacteria in the intestinal microorganisms, but also increases beneficial bacteria that help digest, absorb, and decompose food. Therefore, it has been reported that ingesting lactic acid bacteria has various effects such as reducing blood cholesterol, boosting immunity, suppressing endogenous infections, improving liver cirrhosis, and having anticancer effects. In addition, research has recently been conducted into the enhancement of the efficacy of natural products produced by lactic acid bacteria. The word "microbiome" is a combination of "microbes" and "biome." Scholars sometimes refer to the microbiome as the "second genome." This is because each person has a different microbiome, like a fingerprint. These differences affect the incidence of various diseases, from allergies, atopy, and obesity to enteritis and heart disease. Incidentally, more than 95% of the microbiome lives in the intestines, drawing even more attention to intestinal bacteria. When the healthy ratio of beneficial and harmful bacteria in the intestinal flora is not met, immunity is greatly weakened. This is because beneficial bacteria in the intestine induce the activation of immune cells. Furthermore, when the ratio of harmful bacteria increases, the function of the intestinal defense wall weakens and the intestinal mucosa is damaged. This can cause pathogens and toxins present in the intestinal tract to flow into the bloodstream, resulting in infectious diseases and autoimmune diseases. There is also a claim that intestinal bacteria increase the risk of inducing dementia. Therefore, it is very important to change the intestinal microflora and increase the level of the desired beneficial bacteria in order to improve multiple health functions. To do this, it is necessary to find the appropriate strain from the countless number of strains, but it is very difficult to find such a specific strain. Under these circumstances, the present inventors have identified a novel strain that is capable of exhibiting preventive, ameliorative and therapeutic effects on inflammatory diseases and nervous system diseases, and have completed the present invention. Summary of the Invention [Problem to be solved by the invention]

[0003] The object of the present invention is to provide Lactobacillus rhamnosus, Bifidobacterium longum, and Lactococcus lactis as novel probiotics. Another object of the present invention is to provide uses of said novel probiotics. [Means for solving the problem]

[0004] As one aspect of achieving the above object, the present invention provides Lactobacillus rhamnosus NK210 (Depositary: Korea Microorganism Collection, Date of Deposition: September 15, 2021, Accession No.: KCCM13049P). The Lactobacillus rhamnosus NK210 of the present invention is characterized as a novel probiotic of Lactobacillus rhamnosus isolated and identified from human feces. The base sequence of 16S rDNA for identification and classification of Lactobacillus rhamnosus NK210 of the present invention is as shown in SEQ ID NO: 1 attached hereto. Therefore, Lactobacillus rhamnosus NK210 of the present invention may contain the 16S rDNA of SEQ ID NO: 1. Analysis of the 16S rDNA base sequence of SEQ ID NO: 1 showed 99% homology with known Lactobacillus rhamnosus strains and the highest molecular phylogenetic relationship with Lactobacillus rhamnosus. Therefore, the lactic acid bacterium was identified as Lactobacillus rhamnosus, named Lactobacillus rhamnosus NK210, and deposited at the Korea Microorganism Collection on September 15, 2021 (accession number KCCM13049P). The Lactobacillus rhamnosus NK210 of the present invention is a gram-positive bacterium, and its cell morphology is rod-shaped. More specifically, the physiological properties of Lactobacillus rhamnosus NK210 may be analyzed according to the conventional method in the art, and the results are shown in Table 3 below. Specifically, Lactobacillus rhamnosus NK210 may use any one or more selected from the group consisting of D-galactose, D-glucose, D-fructose, D-mannose, dulcitol, mannitol, sorbitol, N-acetyl-glucosamine, amygdalin, arbutin, esculin, salicin, cellobiose, maltose, lactose, trehalose, melezitose, gentiobiose, D-tagatose, gluconate and L-fucose as a carbon source. In another aspect of the present invention to achieve the above object, the present invention provides Bifidobacterium longum NK219 (Depository: Korea Microorganism Collection, Date of Deposit: September 15, 2021, Accession No.: KCCM13050P). The Bifidobacterium longum NK219 of the present invention is characterized as a novel lactic acid bacterium of Bifidobacterium longum isolated and identified from human feces. The base sequence of 16S rDNA for identification and classification of Bifidobacterium longum NK219 of the present invention is as shown in SEQ ID NO: 2 attached hereto. Therefore, Bifidobacterium longum NK219 of the present invention may include the 16S rDNA of SEQ ID NO: 2. Analysis of the 16S rDNA base sequence of SEQ ID NO:2 showed 99% homology with known Bifidobacterium longum strains and the highest molecular phylogenetic relationship with Bifidobacterium longum. Therefore, the lactic acid bacterium was identified as Bifidobacterium longum, named Bifidobacterium longum NK219, and deposited at the Korea Microorganism Collection on September 15, 2021 (accession number KCCM13050P). The Bifidobacterium longum NK219 of the present invention is a Gram-positive bacterium, and its cell morphology is rod-shaped. More specifically, the physiological properties of Bifidobacterium longum NK219 may be analyzed according to a conventional method in the art, and the results are shown in Table 4 below. Specifically, Bifidobacterium longum NK219 may use any one or more selected from the group consisting of D-glucose, D-lactose, D-saccharose, D-maltose, salicin, D-xylose, L-arabinose, gelatin, D-mannose, D-raffinose, and D-trehalose as a carbon source. In another aspect of the present invention to achieve the above object, the present invention provides Lactococcus lactis NK209 (Depository: Korea Microorganism Collection, Date of Deposit: September 15, 2021, Accession No.: KCCM13048P). The Lactococcus lactis NK209 of the present invention is characterized as a novel lactic acid bacterium of Lactococcus lactis isolated and identified from human feces. The base sequence of 16S rDNA for identification and classification of Lactococcus lactis NK209 of the present invention is as shown in SEQ ID NO: 3 attached hereto. Therefore, Lactococcus lactis NK209 of the present invention may include the 16S rDNA of SEQ ID NO: 3. Analysis of the 16S rDNA base sequence of SEQ ID NO: 3 showed 98% homology with known Lactococcus lactis strains, indicating the highest molecular phylogenetic relationship with Lactococcus lactis. Therefore, the lactic acid bacterium was identified as Lactococcus lactis, named Lactococcus lactis NK209, and deposited at the Korea Microorganism Collection on September 15, 2021 (accession number KCCM13048P). The Lactococcus lactis NK209 of the present invention is a Gram-positive bacterium, and its cell morphology is rod-shaped. More specifically, the physiological properties of Lactococcus lactis NK209 may be analyzed according to a conventional method in the art, and the results are shown in Table 5 below. Specifically, Lactococcus lactis NK209 may use any one or more selected from the group consisting of D-ribose, D-galactose, D-glucose, D-fructose, D-mannose, mannitol, esculin, salicin, cellobiose, maltose, melibiose, sucrose, trehalose, raffinose, starch, gentiobiose, N-acetyl-glucosamine, amygdalin, and arbutin as a carbon source. The present invention provides a composition comprising Lactobacillus rhamnosus NK210 KCCM13049P, Bifidobacterium longum NK219 KCCM13050P, Lactococcus lactis NK209 KCCM13048P or any mixture thereof. The present invention provides an immunomodulatory composition comprising Lactobacillus rhamnosus NK210 KCCM13049P, Bifidobacterium longum NK219 KCCM13050P, Lactococcus lactis NK209 KCCM13048P or any mixture thereof. The present invention provides an immune enhancing composition comprising Lactobacillus rhamnosus NK210 KCCM13049P, Bifidobacterium longum NK219 KCCM13050P, Lactococcus lactis NK209 KCCM13048P or any mixture thereof. The present invention provides a composition for improving immune function, comprising Lactobacillus rhamnosus NK210 KCCM13049P, Bifidobacterium longum NK219 KCCM13050P, Lactococcus lactis NK209 KCCM13048P or any mixture thereof. In another aspect of the present invention, there is provided a pharmaceutical composition comprising Lactobacillus rhamnosus NK210 KCCM13049P, Bifidobacterium longum NK219 KCCM13050P, Lactococcus lactis NK209 KCCM13048P, or any mixture thereof. The pharmaceutical composition of the present invention may be a pharmaceutical composition containing any one, two or three of the probiotics mentioned above. In another aspect of the present invention for achieving the above object, there is provided a pharmaceutical composition comprising Lactobacillus rhamnosus NK210 KCCM13049P, Bifidobacterium longum NK219 KCCM13050P, Lactococcus lactis NK209 KCCM13048P or any mixture thereof, and a pharma- ceutically acceptable carrier. In another aspect of the present invention to achieve the above object, there is provided a pharmaceutical composition for preventing or treating an inflammatory disease, comprising Lactobacillus rhamnosus NK210 KCCM13049P, Bifidobacterium longum NK219 KCCM13050P, Lactococcus lactis NK209 KCCM13048P, or any mixture thereof. In another aspect of the present invention for achieving the above object, there is provided a pharmaceutical composition for preventing or treating cognitive impairment, comprising Lactobacillus rhamnosus NK210 KCCM13049P, Bifidobacterium longum NK219 KCCM13050P, Lactococcus lactis NK209 KCCM13048P, or any mixture thereof. The present invention provides an immunomodulatory pharmaceutical composition comprising Lactobacillus rhamnosus NK210 KCCM13049P, Bifidobacterium longum NK219 KCCM13050P, Lactococcus lactis NK209 KCCM13048P or any mixture thereof. The present invention provides a pharmaceutical composition for enhancing immunity, comprising Lactobacillus rhamnosus NK210 KCCM13049P, Bifidobacterium longum NK219 KCCM13050P, Lactococcus lactis NK209 KCCM13048P or any mixture thereof. The present invention provides a pharmaceutical composition for improving immune function, comprising Lactobacillus rhamnosus NK210 KCCM13049P, Bifidobacterium longum NK219 KCCM13050P, Lactococcus lactis NK209 KCCM13048P or any mixture thereof. Specifically, the immune enhancing and / or functional improving composition is intended to enhance the health of humans or animals, and may be administered to healthy humans or animals. In addition, the composition for enhancing immunity and / or improving function is intended to enhance and / or improve the immunity of a human body or animal whose immune system has been weakened, for example, by exposure to radiation or administration of an antibiotic or anticancer drug, and may be administered to any of the immune-weakened human bodies or animals mentioned above. In the present invention, the inflammatory disease is a collective term for diseases whose main lesion is inflammation, and may be any one or more selected from the group including inflammatory bowel disease, arthritis, gout, hepatitis, asthma, obesity, keratitis, gastritis, nephritis, colitis, diabetes, tuberculosis, bronchitis, pleurisy, peritonitis, spondylitis, pancreatitis, urethritis, cystitis, vaginitis, arteriosclerosis, sepsis, burns, dermatitis, periodontitis, and gingivitis. The term inflammatory bowel disease (IBD) refers to a class of inflammatory conditions of the colon and digestive tract. The main types of IBD are ulcerative colitis (UC) and Crohn's disease. The main difference between UC and Crohn's disease is the location and nature of the inflammatory changes. Crohn's disease can affect any part of the digestive tract from the mouth to the anus, whereas UC is restricted to the colon and rectum. Due to the specificity of presentation, a definitive diagnosis of Crohn's disease or UC cannot be made. In such cases, a diagnosis of atypical colitis can be made. Other forms of IBD include, but are not limited to, collagenous colitis, lymphocytic colitis, ischemic colitis, diversion colitis, Behçet's disease, atypical colitis, antibiotic-induced colitis, and chemotherapy-induced colitis. That is, the inflammatory bowel disease according to the present invention may be any one or more selected from the group consisting of ulcerative colitis, Crohn's disease, collagenous colitis, lymphocytic colitis, ischemic colitis, diversion colitis, Behcet's disease, atypical colitis, antibiotic-induced colitis, and anticancer drug-induced colitis. Antibiotic-induced colitis occurs after the administration of antibiotics, and is caused by the destruction of normal bacteria present in the intestine after exposure to antibiotics. Normal bacteria ferment non-absorbed carbohydrates in the intestine to produce short chain fatty acids. If the normal bacteria are reduced by antibiotics and carbohydrate fermentation becomes abnormal, the osmotic pressure and acidity in the intestine change, inducing diarrhea and causing inflammation in the intestine. Approximately 20% of antibiotic-induced colitis is caused by abnormal proliferation of C. difficile, and in a small number of cases, C. perfringens, S. aureus, E. coli, Candida albicans, etc. can also be causative bacteria. Examples of such antibiotics include clindamycin, ampicillin, amoxicillin, cephalosporin, chloramphenicol, erythromycin, etc. Anticancer drug-induced colitis is an inflammatory condition caused by side effects in the large intestine caused by the administration of anticancer drugs, such as 5-fluorouracil, cyclophosphamide, capecitabine, oxaliplatin, irinodecan, cetuximab, and bevacizumab. The cognitive dysfunction in the present invention refers to a disorder exhibiting cognitive damage and behavioral changes, which is a disease caused by a decline in functions such as memory, spatial perception, judgment, executive function, and language ability. The cognitive dysfunction may be selected from the group consisting of, for example, anxiety, depression, migraine, stress, Alzheimer's disease, Huntington's disease, vascular dementia, Pick's disease, Parkinson's disease, Creutzfeldt-Jakob disease, dementia, and combinations thereof, but is not limited thereto. In the present invention, "neuroinflammation" refers to inflammation occurring in the brain, which is an important factor in causing cognitive impairment-related diseases. It is known that excessive activation of inflammatory cells in the brain leads to increased secretion of proinflammatory cytokines, and the overactivation of such inflammatory responses in the brain leads to damage to brain cells, thereby inducing cognitive impairment. In the present invention, improvement of immune function means activation of immunoregulatory activity. Here, immune regulation means resolving immune imbalance in the human body and maintaining immune homeostasis. Maintenance of immune homeostasis refers to a balanced state between immune tolerance, which suppresses immunity, and immune response, which enhances immunity. Such immune regulation may improve immune function, and may preferably correspond to immune enhancement. For example, it may be characterized by acting as a supplement for the host's gut microflora, improving the intestinal barrier function, and regulating the host's immune system. More specifically, the term "improvement of immune function" in this specification may also mean "immune enhancement," which may be interpreted as including immune regulating effects in addition to the dictionary meaning. That is, the terms "improvement of immune function" and "immune enhancement" can be used equivalently in the present invention. Improving immune function and / or enhancing immunity may involve increasing or modulating the activity of an immunoregulator to increase the activity of the immune system. Specifically, the compound may exhibit preventive and ameliorative effects against immunosuppression, and preventive, ameliorative and therapeutic effects against immune-related dysfunction or associated diseases, by suppressing the generation of new blood vessels, increasing the ability to inhibit the growth of pathogenic bacteria, and the like. Specifically, the compound may enhance the body's defense against antigens, thereby increasing cellular and humoral immunity against antigens, and may exhibit preventive and ameliorative effects against immunosuppression, and preventive, ameliorative and therapeutic effects against immune-related dysfunction or related diseases. The immune function improving and / or immune enhancing mechanisms may include, but are not limited to, for example, promoting the activity of antigen presenting cells such as macrophages, promoting specific activity against lymphocytes, removing dead cells, regulating the secretory capacity of immune mediators, and the like. The immunity enhancement according to the present invention is not limited thereto, but may be for enhancing immunity in a subject having normal immunity in order to enhance health, or for enhancing immunity weakened by radiation therapy or exposure, antibiotic therapy, anticancer drug therapy, etc. Specifically, the purpose may be to improve and / or recover from immune deficiency, immune depression, or immune system damage caused by the administration of an antibiotic or anticancer drug. The anti-cancer agent may be a drug, such as a compound or protein, that promotes the regression of cancer or even prevents the growth of tumors, and may also include other anti-cancer therapies, radiation therapy, other anti-cancer therapies other than drug therapy, etc. The other therapeutic agent may be, but is not limited to, an immune checkpoint inhibitor, a chemo-anti-cancer agent, or a targeted anti-cancer agent. Antibiotics include any member of the antibiotic class, including, but not limited to, clindamycin, ampicillin, amoxicillin, cephalosporins, chloramphenicol, erythromycin, and the like. According to one embodiment of the present invention, Lactobacillus rhamnosus NK210 KCCM13049P, Bifidobacterium longum NK219 KCCM13050P, Lactococcus lactis NK209 KCCM13048P or any mixture thereof exhibits excellent antioxidant activity and is effective in improving inflammatory diseases, cognitive dysfunction and immune function. According to one embodiment of the present invention, Lactobacillus rhamnosus NK210 KCCM13049P, Bifidobacterium longum NK219 KCCM13050P, Lactococcus lactis NK209 KCCM13048P or any mixture thereof exhibits excellent effects in improving inflammation by regulating the expression of cytokines, such as TNF-α and IL-10. According to one embodiment of the present invention, Lactobacillus rhamnosus NK210 KCCM13049P, Bifidobacterium longum NK219 KCCM13050P, Lactococcus lactis NK209 KCCM13048P or any mixture thereof exhibits excellent effects in improving inflammation by regulating the expression of cytokines, such as regulating the expression of inflammatory cytokines, e.g., TNF-α, and increasing the expression of anti-inflammatory cytokines, e.g., IL-10. According to one embodiment of the present invention, Lactobacillus rhamnosus NK210 KCCM13049P, Bifidobacterium longum NK219 KCCM13050P, Lactococcus lactis NK209 KCCM13048P, or any mixture thereof, exhibits excellent effects in preventing and treating cognitive dysfunction by increasing the expression of brain-derived neurotrophic factor (BDNF) and increasing the expression of Neuropeptide Y, which is closely related to stress, anxiety, depression, and cognitive impairment. According to one embodiment of the present invention, Lactobacillus rhamnosus NK210 KCCM13049P, Bifidobacterium longum NK219 KCCM13050P, Lactococcus lactis NK209 KCCM13048P, or any mixture thereof, suppresses inflammatory diseases, regulates immune function, and regulates nervous system and cognitive function by regulating the intestinal microbiota. Specifically, it regulates the intestinal microbiota, including suppressing intestinal Clostridium difficile, which is highly related to enteritis, and reduces LPS levels in the intestine. It also promotes beneficial bacteria in the intestine and reduces harmful bacteria to improve intestinal health, thereby showing an effect on improving nerve, especially cognitive function, via the gut-brain axis. According to one embodiment of the present invention, the strain according to the present invention was administered to animals with LPS-induced cognitive impairment to improve intestinal function, improve systemic inflammatory response, improve cognitive impairment, and reduce inflammation levels in the nerves. Specifically, the strain restored the decrease in intestinal length in inflammatory bowel disease induced by E. coli-derived LPS, reduced MPO activity, reduced the level of inflammatory cytokines, and increased the level of anti-inflammatory cytokines. In addition, in the systemic inflammatory response confirmed by the spleen, the strain regulated the phagocytic ability of macrophages and the cell killing ability of NK cells, reduced the level of inflammatory cytokines, and increased the level of anti-inflammatory cytokines. In addition, the strain showed behavioral characteristics related to memory improvement in the nervous system, especially in cognitive impairment, and also showed excellent improving effects on neuroinflammation. According to one embodiment of the present invention, the strain according to the present invention was administered to an animal model of immune damage (suppression) induced by an anticancer drug, and improved intestinal function, improved systemic inflammatory response, improved immune function, improved cognitive function damage, and reduced inflammation levels in the nerves. Specifically, in an animal model in which immune function was damaged by the administration of an anticancer drug, the strain restored the decrease in intestinal length, reduced MPO activity, and normalized and regulated the expression ratio of inflammatory cytokines and anti-inflammatory cytokines. In addition, in the systemic inflammatory response confirmed by the spleen, the strain increased the phagocytic ability of macrophages and the killing ability of NK cells, and increased the level of anti-inflammatory cytokines compared to inflammatory cytokines. In addition, the strain showed behavioral characteristics related to memory improvement in the nervous system, especially in cognitive dysfunction, and also showed excellent improving effects on neuroinflammation. According to one embodiment of the present invention, the strain according to the present invention was administered to an animal model with an imbalance of gut microbiota induced by antibiotics to improve gut function, improve systemic inflammatory response, improve immune function, improve cognitive function damage, and reduce inflammation levels in the nerves. Specifically, in an animal model with an imbalance of gut microbiota caused by the administration of antibiotics such as ampicillin, the strain restores the decrease in gut length, reduces MPO activity, and normalizes and regulates the expression ratio of inflammatory cytokines and anti-inflammatory cytokines. In addition, in the systemic inflammatory response confirmed by the spleen, the strain regulated the phagocytic ability of macrophages and the killing ability of NK cells to reduce the level of inflammatory cytokines and increase the level of anti-inflammatory cytokines. In addition, the strain exhibits behavioral characteristics related to memory improvement in the nervous system, especially in cognitive dysfunction, and also exhibits excellent improving effects on neuroinflammation. According to one embodiment of the present invention, the strain according to the present invention was administered to a healthy animal model, and preventive or ameliorative effects were observed, including improving intestinal function, improving systemic inflammatory response, improving immune function, and improving cognitive impairment. According to one embodiment of the present invention, the strain according to the present invention was administered to an animal model with intestinal disease and cognitive dysfunction induced by TNBS to improve intestinal function, improve systemic inflammatory response, improve immune function, improve cognitive impairment, and reduce inflammation levels in the nerves. Specifically, in an animal model with intestinal disease and cognitive dysfunction caused by administration of TNBS, the strain restores the decrease in intestinal length, reduces MPO activity, and normalizes and regulates the expression ratios of inflammatory cytokines and anti-inflammatory cytokines. In addition, the strain exhibits behavioral characteristics related to memory improvement in the nervous system, especially in cognitive dysfunction, and also exhibits excellent improving effects on neuroinflammation. Specifically, the strain contained in the pharmaceutical composition of the present invention may be used in various forms such as live cells, killed cells, cultures, homogenates or extracts thereof. The term "viable cells" refers to cells that are still alive. Killed bacteria are formed by harvesting live bacteria that have been cultured under certain conditions, and then preventing further bacterial growth by methods such as heat drying, pressure, and chemical treatment. The culture means a product obtained by culturing lactic acid bacteria in a known liquid or solid medium, and is a concept including the strain according to the present invention. The product may contain lactic acid bacteria. The medium may be selected from known liquid or solid media, such as, but not limited to, MRS liquid medium, GAM liquid medium, MRS agar medium, GAM agar medium, and BL agar medium. The term "disintegrated material" refers to a form in which live or dead cells or a culture thereof have been separated and processed by mechanical or chemical methods and disrupted, for example, by bead mills, presses, sonicators or microfluidizers, enzyme treatment, etc. The term "extract" refers to a form of "water (liquid)" obtained by extracting live or dead bacteria using a commonly known extraction method, for example, by extracting live or dead bacteria and / or crushed bacteria using a commonly known extraction method (using a commonly known extraction solvent (e.g., water, C1 to C4 alcohol (methanol, ethanol, etc.))). According to the present invention, the above mentioned strains may be used alone. In addition, since a synergistic effect can be exhibited by using strains in combination, one or more strains may be used in combination. Such combination forms include, for example, Lactobacillus rhamnosus NK210 KCCM13049P and Bifidobacterium longum NK219 KCCM13050P; Lactobacillus rhamnosus NK210 KCCM13049P and Lactococcus lactis NK209 KCCM13048P; Bifidobacterium longum NK219 KCCM13050P and Lactococcus lactis NK209 KCCM13048P; or Lactobacillus rhamnosus NK210 KCCM13049P, Bifidobacterium longum NK219 KCCM13050P and Lactococcus lactis NK209 KCCM13048P. Preferably, it may be a combination of Lactobacillus rhamnosus NK210 KCCM13049P and Bifidobacterium longum NK219 KCCM13050P, or Bifidobacterium longum NK219 KCCM13050P and Lactococcus lactis NK209 KCCM13048P, or a combination of Lactobacillus rhamnosus NK210 KCCM13049P, Bifidobacterium longum NK219 KCCM13050P and Lactococcus lactis NK209 KCCM13048P. As an example, the combination of Lactobacillus rhamnosus NK210 KCCM13049P and Bifidobacterium longum NK219 KCCM13050P strains may be, for example, 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 based on the number of bacteria (CFU). Preferably, 9:1 to 1:1, more preferably 4:1 to 1:1, more particularly 4:1. As an example, the combination of Lactococcus lactis NK209 KCCM13048P and Bifidobacterium longum NK219 KCCM13050P strains may be, for example, 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 based on the number of bacteria (CFU). Preferably, 9:1 to 1:1, more preferably 4:1 to 1:1, more particularly 4:1. As an example, the combination of Lactococcus lactis NK209 KCCM13048P and Lactobacillus rhamnosus NK210 KCCM13049P strains may be, for example, 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 based on the number of bacteria (CFU). Preferably, 9:1 to 1:1, more preferably 4:1 to 1:1 may be used. As an example, when three kinds are used in combination, the combination may be, for example, 4-8:4-8:1-2, (NK209:NK210:NK219) based on the number of bacteria (CFU). Preferably, it may be used in a ratio of 2:2:1. The pharmaceutical composition according to the present invention may be prepared into a pharmaceutical dosage form using a method well known in the art so as to provide a quick, sustained or delayed release of the active ingredient after administration to a mammal. In preparing the dosage form, the pharmaceutical composition according to the present invention may further contain a pharma- ceutical acceptable carrier within a range that does not inhibit the activity of the novel lactic acid bacteria. The pharma- ceutically acceptable carriers include, but are not limited to, commonly used ones, such as lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil, etc. The pharmaceutical composition of the present invention may also include a diluent or excipient such as a filler, extender, binder, wetting agent, disintegrant, or surfactant, or other pharma- ceutically acceptable additive. The dosage of the pharmaceutical composition according to the present invention must be a pharmaceutical effective amount. A "pharmaceutical effective amount" means an amount sufficient to prevent or treat the disease or condition mentioned above at a reasonable benefit / risk ratio applicable to medical treatment. The effective dosage level may be selected by those skilled in the art in various ways depending on factors such as the formulation method, the condition and weight of the patient, the sex and age of the patient, the degree of the disease, the drug form, the route and duration of administration, the excretion rate, the reaction sensitivity, and the like. The effective amount may vary depending on the route of treatment, the use of excipients, and the possibility of using with other drugs, as recognized by those skilled in the art. However, for a favorable effect, in the case of oral administration, the composition of the present invention may generally be administered to an adult at 0.0001 to 100 mg / kg, preferably 0.001 to 100 mg / kg, of body weight per day. The administration may be administered once a day or in several divided doses. The dosage amount does not limit the scope of the present invention in any aspect. The pharmaceutical composition of the present invention may be administered to mammals such as mice, livestock, and humans via various routes. Specifically, the pharmaceutical composition of the present invention may be administered orally or parenterally (e.g., by application or intravenous, subcutaneous, or intraperitoneal injection), with oral administration being preferred. Solid preparations for oral administration may include powders, granules, tablets, capsules, soft capsules, pills, and the like. Liquid preparations for oral administration include suspensions, liquids for internal use, emulsions, syrups, aerosols, and the like, and may include various excipients such as wetting agents, sweeteners, flavorings, preservatives, and the like in addition to water and liquid paraffin, which are commonly used simple diluents. Preparations for parenteral administration may be prepared and used in the form of external preparations such as sterilized aqueous solutions, solutions, non-aqueous solvents, suspensions, emulsions, eye drops, eye ointments, syrups, suppositories, aerosols, and sterile injections, each of which is sterilized according to a conventional method. Preferably, pharmaceutical compositions such as creams, gels, patches, sprays, ointments, plasters, lotions, liniments, eye ointments, eye drops, pastes, or cataplasms may be prepared and used, but are not limited thereto. Preparations for topical administration may be anhydrous or aqueous according to clinical prescriptions. As non-aqueous solvents and suspensions, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like may be used. As suppository bases, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin, and the like may be used. In another aspect for achieving the above object, the present invention provides a method for preventing or treating cognitive impairment, comprising the step of administering to a subject Lactobacillus rhamnosus NK210 KCCM13049P, Bifidobacterium longum NK219 KCCM13050P, Lactococcus lactis NK209 KCCM13048P, or any mixture thereof. In another aspect for achieving the above object, the present invention provides a method for preventing or treating an inflammatory disease, comprising the step of administering to a subject Lactobacillus rhamnosus NK210 KCCM13049P, Bifidobacterium longum NK219 KCCM13050P, Lactococcus lactis NK209 KCCM13048P, or any mixture thereof. In another aspect for achieving the above object, the present invention provides a method for immunomodulation, comprising the step of administering to a subject Lactobacillus rhamnosus NK210 KCCM13049P, Bifidobacterium longum NK219 KCCM13050P, Lactococcus lactis NK209 KCCM13048P, or any mixture thereof. In another aspect for achieving the above object, the present invention provides a method for improving immune function, comprising the step of administering to a subject Lactobacillus rhamnosus NK210 KCCM13049P, Bifidobacterium longum NK219 KCCM13050P, Lactococcus lactis NK209 KCCM13048P, or any mixture thereof. In another aspect for achieving the above object, the present invention provides a method for enhancing immunity, comprising the step of administering to a subject Lactobacillus rhamnosus NK210 KCCM13049P, Bifidobacterium longum NK219 KCCM13050P, Lactococcus lactis NK209 KCCM13048P, or any mixture thereof. In the present invention, the explanations of the strains, diseases, administration, etc. mentioned above may appropriately reflect the contents mentioned above. The subject may be an animal, typically a mammalian animal, that may benefit from treatment with the novel probiotics of the present invention. Preferred examples of such subjects may include primates, such as humans. The present invention provides Lactobacillus rhamnosus NK210 KCCM13049P, Bifidobacterium longum NK219 KCCM13050P, Lactococcus lactis NK209 KCCM13048P or any mixture thereof for use in the prevention or treatment of an inflammatory disease. The present invention provides Lactobacillus rhamnosus NK210 KCCM13049P, Bifidobacterium longum NK219 KCCM13050P, Lactococcus lactis NK209 KCCM13048P or any mixture thereof for use in the prevention or treatment of cognitive impairment. The present invention provides Lactobacillus rhamnosus NK210 KCCM13049P, Bifidobacterium longum NK219 KCCM13050P, Lactococcus lactis NK209 KCCM13048P or any mixture thereof for use in immunomodulation. The present invention provides Lactobacillus rhamnosus NK210 KCCM13049P, Bifidobacterium longum NK219 KCCM13050P, Lactococcus lactis NK209 KCCM13048P or any mixture thereof for use in improving immune function. The present invention provides Lactobacillus rhamnosus NK210 KCCM13049P, Bifidobacterium longum NK219 KCCM13050P, Lactococcus lactis NK209 KCCM13048P or any mixture thereof for use in immune enhancement. The present invention provides the use of Lactobacillus rhamnosus NK210 KCCM13049P, Bifidobacterium longum NK219 KCCM13050P, Lactococcus lactis NK209 KCCM13048P or any mixture thereof in the manufacture of a medicament for the treatment of an inflammatory disease. The present invention provides the use of Lactobacillus rhamnosus NK210 KCCM13049P, Bifidobacterium longum NK219 KCCM13050P, Lactococcus lactis NK209 KCCM13048P or any mixture thereof in the manufacture of a medicament for the treatment of cognitive impairment. The present invention provides the use of Lactobacillus rhamnosus NK210 KCCM13049P, Bifidobacterium longum NK219 KCCM13050P, Lactococcus lactis NK209 KCCM13048P or any mixture thereof in the manufacture of a medicament for use in immunomodulation. The present invention provides the use of Lactobacillus rhamnosus NK210 KCCM13049P, Bifidobacterium longum NK219 KCCM13050P, Lactococcus lactis NK209 KCCM13048P or any mixture thereof in the manufacture of a medicament for use in improving immune function. The present invention provides the use of Lactobacillus rhamnosus NK210 KCCM13049P, Bifidobacterium longum NK219 KCCM13050P, Lactococcus lactis NK209 KCCM13048P or any mixture thereof in the manufacture of a medicament for use in immune enhancement. In another aspect, the present invention provides a food composition for preventing or ameliorating an inflammatory disease, comprising Lactobacillus rhamnosus NK210 KCCM13049P, Bifidobacterium longum NK219 KCCM13050P, Lactococcus lactis NK209 KCCM13048P, or any mixture thereof. In another aspect, the present invention provides a food composition for preventing or improving cognitive impairment, comprising Lactobacillus rhamnosus NK210 KCCM13049P, Bifidobacterium longum NK219 KCCM13050P, Lactococcus lactis NK209 KCCM13048P, or any mixture thereof. In another aspect, the present invention provides an immunomodulatory food composition comprising Lactobacillus rhamnosus NK210 KCCM13049P, Bifidobacterium longum NK219 KCCM13050P, Lactococcus lactis NK209 KCCM13048P or any mixture thereof. In another aspect, the present invention provides a food composition for improving immune function, comprising Lactobacillus rhamnosus NK210 KCCM13049P, Bifidobacterium longum NK219 KCCM13050P, Lactococcus lactis NK209 KCCM13048P, or any mixture thereof. The present invention provides an immunity enhancing food composition comprising Lactobacillus rhamnosus NK210 KCCM13049P, Bifidobacterium longum NK219 KCCM13050P, Lactococcus lactis NK209 KCCM13048P or any mixture thereof. A health functional food is a food that emphasizes the bioregulatory function of the food, and is a food that is added with added value to act and express a specific purpose using physical, biochemical, and biotechnological methods. The food composition of the present invention may be used as a health functional food. The ingredients of such a health functional food are designed and processed to fully exert the body regulation function related to biodefense and regulation of body rhythms, and prevention and recovery of diseases in the living body, and may include food supplement additives, sweeteners, or functional ingredients that are acceptable as foods. When the strain of the present invention is used as a functional health food (or functional health drink additive), the novel strain may be added as it is or may be used together with other foods or food ingredients, and may be used appropriately according to conventional methods. The amount of the strain to be mixed may be appropriately determined depending on the purpose of use (prevention, health or improvement, therapeutic treatment). Preferably, the strain according to the present invention may be in the form of a single strain, a mixture of two or three strains, and the use of a mixture of strains may be considered in consideration of the synergistic effect within the ranges mentioned above. The food may contain various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and enhancers (cheese, chocolate, etc.), pectinic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonation agents used in carbonated drinks, etc. The health functional food of the present invention may also contain fruit pulp for the production of fruit and vegetable drinks. Such ingredients may be used alone or in combination, and the proportion of such additives is generally selected in the range of 0.001 to 50 parts by weight per total weight of the composition. There is no particular limit to the type of food. Foods to which the strain can be added include sausages, meat, bread, chocolates, snacks, candies, confectionery, ramen, pizza, other noodles, gums, dairy products including ice cream, various soups, drinking water, tea, drink preparations, alcoholic beverages, and vitamin complexes. When formulated into drinking water, liquid components added in addition to the novel lactic acid bacteria may include, but are not limited to, various flavoring agents or natural carbohydrates as additional components, as in ordinary beverages. The above-mentioned natural carbohydrates may be monosaccharides (e.g., glucose, fructose, etc.), disaccharides (e.g., maltose, sucrose, etc.), and polysaccharides (e.g., ordinary sugars such as dextrin, cyclodextrin, etc.), and sugar alcohols such as xylitol, sorbitol, and erythritol. Thus, unless otherwise specified, the numerical values ​​set forth in this specification should be interpreted as including an equivalent range. In the following, preferred examples are presented to aid in understanding the present invention. However, the following examples are provided only to facilitate understanding of the present invention, and are not intended to limit the scope of the present invention. Effect of the Invention

[0005] The novel probiotics according to the present invention have excellent antioxidant effects, inflammation-improving effects, neurological effects, cognition-improving effects, intestinal microbiota imbalance improving effects, immunomodulatory effects, and the like, making them highly valuable as pharmaceuticals and foods, and may be used in particular for the prevention, treatment, or improvement of inflammatory diseases, immune function improvement, and cognitive dysfunction. [Brief description of the drawings]

[0006] [Figure 1] The results show that the imbalance of the intestinal microbiota caused by ampicillin was improved by treatment with the strain of the present invention (AP: ampicillin, LR: Lactobacillus rhamnosus NK210, BL: Bifidobacterium longum NK219, Mx: NK210+NK219). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0007] In the following, preferred examples are presented to aid in understanding the present invention. However, the following examples are provided only to facilitate understanding of the present invention, and are not intended to limit the scope of the present invention. Example 1: Isolation and identification of lactic acid bacteria (1) Isolation of lactic acid bacteria from human feces Human feces was suspended in GAM broth (Nissui Pharmaceutical, Japan) and left at 4°C for 10 minutes. The supernatant was then transferred to MRS agar medium (BD, USA) and cultured anaerobically at 37°C for approximately 48 hours, and the grown colonies were isolated. (2) Identification of selected lactic acid bacteria The physiological characteristics and 16S rDNA sequences of the isolated strains were analyzed. Among the physiological characteristics, carbon source utilization was analyzed using an API Kit (model name: API 50 CHL; manufacturer: BioMerieuxs, USA). The species of the strains was confirmed and they were given names. The strains identified through the above procedures are listed in Table 1 below. [Table 1] JPEG2024535901000003.jpg182148 Example 2: Analysis of strain characteristics In Example 1, a total of 50 strains were isolated, and the strains with excellent characteristics were selected by checking the antioxidant activity, changes in cytokine expression, BDNF expression, and NPY expression in macrophages, and the effect on the proliferation and LPS production of Clostridium difficile in Gut microbiota. The specific experimental method and measurement results are as follows. (1) Effects on antioxidant activity (in vitro) DPPH (2,2-Diphenyl-1-picrylhydrazyl) was dissolved in ethanol to a concentration of 0.2 mM to prepare a DPPH solution. 8 CFU / ml) or vitamin C solution (1 g / ml) was added and incubated at 37°C for 20 minutes. The culture solution was centrifuged at 3000 rpm for 5 minutes to obtain the supernatant. The absorbance of the supernatant was measured at 517 nm, and the antioxidant activity of the lactic acid bacteria was calculated. (2) Isolation of macrophages and the effect on the expression of inflammatory markers TNF-α and IL-10 2 ml of sterilized 4% thioglycolate was administered into the abdominal cavity of a C57BL / 6 mouse (male, 6 weeks old, 20-23 g). After 96 hours, the mouse was anesthetized and 8 ml of RPMI 1640 medium was administered into the abdominal cavity of the mouse. 5-10 minutes later, the RPMI medium (macrophages) in the abdominal cavity of the mouse was removed, centrifuged at 1000 g for 10 minutes, and washed twice with RPMI 1640 medium again. Macrophages were added to each well at 0.5 × 10 6The cells were placed on a 24-well plate in groups of 100 and treated with lactobacillus bacteria and LPS, an inflammatory response inducer, for 24 hours. The expression levels of TNF-α and IL-10 cytokines in the culture supernatant were then measured using an ELISA kit. (3) Effect on BDNF expression in SH-SY5Y cells SH-SY5Y cells provided by the Korea Cell Line Bank were cultured in DMEM medium supplemented with 10% FBS and 1% antibiotics, and plated in a 12-well plate at 2 × 10 cells per well. 6 Then, each well was filled with lactic acid bacteria (1 × 10 4 CFU / ml) and LPS (200 ng / ml) were added and cultured for 24 hours. The cells and supernatant were collected, sonicated, and centrifuged, and the amount of BDNF in the supernatant was measured using an ELISA kit. (4) Effect on neuropeptide Y (NPY) expression in PC12 phaeochromocytoma cells PC12 cells provided by the Korea Cell Line Bank were cultured in DMEM medium supplemented with 10% FBS and 1% antibiotics, and plated in a 12-well plate at 1 × 10 cells per well. 6 Then, each well was filled with lactic acid bacteria (1 × 10 4 CFU / ml) and LPS (200 ng / ml) were added and cultured for 24 hours, and the cells and supernatant were collected, sonicated, centrifuged, and the NPY amount in the supernatant was measured using an ELISA kit. (5)Effect of gut microbiota on the growth and LPS production of Clostridium difficile Fresh human feces was suspended in anaerobic medium (GAM, Nissui Pharmaceutical Co., Ltd., Japan) and allowed to stand for 10 minutes. The supernatant was then diluted 10,000 times with anaerobic medium, and 0.05 mL of the solution was transferred to 5 mL of freshly prepared anaerobic medium. Lactic acid bacteria (1 × 10 6CFU / mL) was inoculated and cultured anaerobically for 24 hours. The culture was divided into two aliquots. One was centrifuged (5000 rpm, 20 min) and the precipitate was used to isolate DNA using a QIAamp DNA stool mini kit (Qiagen, Germany). Clostridium difficile (primer: forward, 5′-GGG AGC TTC CCA TAC GGG TTG-3′ (sequence number 4); reverse, 5′-TTG ACT GCC TCA ATG CTT GGG C-3′ (sequence number 5) was quantified by qPCR. The reaction was first treated at 95°C for 30 s, followed by 42 cycles of reaction at 95°C for 5 s and 72°C for 30 s. One of the aliquots was sonicated, centrifuged, and sterilized by filtration, and the amount of LPS in the supernatant was measured using an ELISA kit. The results are shown in Table 2. [Table 2] JPEG2024535901000005.jpg207152*very strongly(+++;>90%);strongly(++;>60~90%);weakly(+;>20~60%);not or less than 20%(-;<20%) As can be seen from Table 2, Lactobacillus rhamnosus NK210 and Bifidobacterium longum NK219 were found to have excellent antioxidant activity, anti-inflammatory activity, neurological improvement, and improvement effects on the proliferation of intestinal Clostridium difficile and LPS production. In addition to the above strains, it was also confirmed that Lactococcus lactis NK209 strain showed a certain level of activity, especially a significant effect in inhibiting the proliferation of intestinal Clostridium difficile. Based on the above results, further experiments were carried out focusing on Lactobacillus rhamnosus NK210, Bifidobacterium longum NK219, and Lactococcus lactis NK209 strains. The 16S rDNA sequence of Lactobacillus rhamnosus NK210 is specifically shown in SEQ ID NO: 1, the 16S rDNA sequence of Bifidobacterium longum NK219 is specifically shown in SEQ ID NO: 2, and the 16S rDNA sequence of Lactococcus lactis NK209 is specifically shown in SEQ ID NO: 3. In addition, a phylogenetic tree was created to confirm that all of these strains are novel strains. Therefore, the present inventors deposited Lactobacillus rhamnosus NK210, Bifidobacterium longum NK219, and Lactococcus lactis NK209 with the Korea Microorganism Collection (Address: Yurim Building, 45, Hongjenae 2-ga-gil, Seodaemun-gu, Seoul, Republic of Korea), an accredited depository institution, and were assigned the accession numbers KCCM13049P, KCCM13050P, and KCCM13048P, respectively, on September 15, 2021. Example 3: Verification of the physiological properties of the strain The physiological properties of the deposited strain were confirmed. Specifically, the carbon source utilization among the physiological characteristics was analyzed by a sugar fermentation test using an API 50 CHL kit (API 50 CHL; manufacturer: BioMerieux, USA) or an API 20A kit (API 20A, BioMerieux's, USA). The results are shown in Tables 3 (Lactobacillus rhamnosus NK210), 4 (Bifidobacterium longum NK219), and 5 (Lactococcus lactis NK209) below. In Tables 3 to 5 below, "+" indicates a positive carbon source utilization, and "-" indicates a negative carbon source utilization. [Table 3] [Table 4] [Table 5] Example 4: Production of killed and disrupted bacteria Example 4-1. Production of killed bacteria Lactobacillus rhamnosus NK210 KCCM13049P, Bifidobacterium longum NK219 KCCM13050P, or Lactococcus lactis NK209 KCCM13048P were heat-treated three times at 90°C for 10 min to produce killed cells. Example 4-2. Production of crushed material (soluble and insoluble materials) Viable cells obtained by centrifugation of Lactobacillus rhamnosus NK210 KCCM13049P, Bifidobacterium longum NK219 KCCM13050P, or Lactococcus lactis NK209 KCCM13048P were collected at 1 × 10 6 The bacteria were suspended in sterile distilled water to obtain a CFU / mL concentration, sonicated at 4°C (1 min treatment, 1 min break, repeated 5 times), and centrifuged (10,000 g, 4°C, 15 min) to obtain a supernatant (soluble matter) and a precipitate (insoluble matter). These were freeze-dried and used. Example 5. Confirmation of activity of killed cells and disrupted cells The killed cells and lysates prepared in Example 4 were used to confirm the anti-inflammatory activity in macrophages in the same manner as in Example 2. As a result, it was confirmed that killed bacteria produced by heat treatment (90℃, 10 minutes, 3 times) and homogenized bacteria produced by ultrasonic treatment (1 minute treatment, 1 minute break, repeated 5 times) all showed IL-10 expression induction activity in comparison to TNF-α expression, similar to that observed when live bacteria were treated. Test Example The measurement methods in the tests described below were performed according to the following test method standards. (1) Measurement of myeloperoxidase (MPO) activity 100 mg of colon tissue was homogenized in 200 μl of 10 mM potassium phosphate buffer (pH 7.0) containing 0.5% hexadecyl trimethyl ammonium bromide. The tissue was then centrifuged at 4°C and 10,000 g for 10 minutes to obtain the supernatant. 50 μl of the supernatant was added to 0.95 ml of reaction solution (containing 1.6 mM tetramethyl benzidine and 0.1 mM H2O2) and reacted at 37°C while measuring the absorbance at 650 nm over time. (2) Measurement of IFN(interferon)-γ, TNF-α, and IL-10 indicators The hippocampus, colonic tissue, and splenic tissue were homogenized in 1 ml of RIPA buffer (Gibco) containing a protease inhibitor cocktail, and centrifuged at 13,000 rpm at 4°C for 15 minutes to obtain the supernatant. The indicators of this supernatant were measured using an ELISA kit (Ebioscience). (3) Memory experiment 1) Y-maze task experiment method: The Y-maze measurement device had three arms, each of which was 25 cm long, 14 cm high, and 5 cm wide, and was positioned at the same angle. The animal was placed with its head facing the end of one arm of the Y-maze, and allowed to move freely around the arm for 8 minutes. The animal's movements were recorded, and an arm entry was considered to have occurred when the animal's hind paws entered the arm. The animal's movements were expressed as the number of alternations, which was defined as one crossing when the animal passed through three arms consecutively. The amount of spontaneous crossing behavior was expressed as the percentage of the actual number of alternations and the maximum possible number of alternations (i.e., the total number of alternations minus 2). 2) Object recognition experiment: The experiment was carried out in a box (40 x 40 x 40 cm) that was designed so that the outside could not be seen from the inside. Two objects of the same shape and size (A, A') were fixed in the box, and the mouse was started from the center of the box. The number of times the mouse touched the two objects was recorded for 10 minutes. After 24 hours, one of the two objects was replaced with a new object (A, B), and the original object and the new opposing object were prepared, and the time the animal spent exploring was measured. (4)qPCR (Quantitative real time polymerase chain reaction) mRNA was isolated from spleen tissue using the RNA Isolation Kit: RNeasy Mini Kit (QIAGEN), converted to cDNA, and qPCR was performed to measure the expression rates of Tbet, Foxp3, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The reaction was first treated at 95°C for 30 s, followed by 38 repeated reactions at 95°C for 5 s and 72°C for 30 s. Primers for qPCR [Table 6] (5) Analysis of gut microbiota First, fecal DNA was isolated using the QIAamp DNA stool mini kit (Qiagen, Germany), and pyrosequencing was performed by amplifying barcoded primers (V4 region of the bacterial 16S rRNA gene) to produce amplicons, and 16S rDNA was analyzed using an Illumina iSeq 100 (San Diego, CA) to analyze occupancy rates. Example 6: Analysis of the efficacy of strain administration in animals with LPS-induced cognitive impairment Six C57BL / 6 male mice (5 weeks old, 19-21 g) were grouped in each group and allowed to adapt to the laboratory for one week. To induce intestinal and brain diseases, E. coli-derived LPS (10 μg / kg) was intraperitoneally administered once a day for five days. From the next day, lactobacillus bacteria were administered at a dose of 1 × 10 9 CFU / mouse concentration (NK210 alone: ​​1 × 10 9 CFU / mouse, NK219 alone: ​​1 × 10 9 CFU / mouse, combination (210+219): 210, 8 × 10 8 CFU / mouse and 219, 2 × 10 8 The animals were administered 50 mg / kg (50 CFU / mouse) and 50 mg / kg (50 mg / mouse) of sulfasalazine once a day for five days. The normal experimental animals were orally administered the same saline solution used to suspend the lactobacillus. The experimental animals were sacrificed the day after the sample administration was completed, and the hippocampus, colon, and spleen were obtained. In the hippocampus, the inflammation indicators IFN-γ, TNF-α, and IL-10 were measured by ELISA, the intestinal length and the inflammation indicators IFN-γ, TNF-α, and IL-10 were measured in the colon, and Th1 cells and Treg cells were measured in the spleen by Pax. (1) Confirmation of effectiveness in improving colitis [Table 7] As can be seen from Table 7, the administration of single strains and combined strains showed an effect of improving colitis, as confirmed by intestinal length, changes in expression of inflammatory factors, and changes in MPO activity. Specifically, the decrease in intestinal length was improved, MPO activity was reduced, the expression levels of interferon-gamma and TNF-α were reduced, and the expression level of IL-10, an anti-inflammatory cytokine, was increased. That is, it inhibited IFN-γ and TNF-α increased by endotoxin, increased IL-10 expression decreased by endotoxin, and in particular, increased IL-10 expression relative to IFN-γ and IL-10 expression relative to TNF-α, thereby greatly improving the symptoms of colitis. It was confirmed that this action acts as a synergy with the combined strains, and in particular, the most excellent effect was observed when NK210 and NK219 were administered at a mixture ratio of 4:1. (2) Confirmation of improvement effect on the spleen [Table 8] JPEG2024535901000012.jpg25146As can be seen from Table 8, it was confirmed that the phagocytic ability of macrophages, which are natural immune cells, and the cell killing effect of NK cells were improved. In addition, it suppressed IFN-γ and TNF-α, which were increased by endotoxin, and increased IL-10 expression, which was decreased by endotoxin, and in particular, it increased IL-10 expression compared to IFN-γ and IL-10 expression compared to TNF-α (i.e., it means a decrease in the IFN-γ / IL-10 and TNF-α / IL-10 ratios, hereinafter the same as above). Such an action on the spleen, which is involved in the inflammatory response systemically, indicates the excellent inflammation-improving effect of the strain according to the present invention. Furthermore, such an action was shown to be highly excellent, showing a synergistic effect not only when used alone but also when used in combination, and in particular, when NK210 and NK219 were administered at a mixture ratio of 4:1, the most excellent effect was shown. (3) Confirmation of the effect of improving cognitive function [Table 9] As can be seen from Table 9, administration of the strain according to the present invention showed excellent improving effects in both the Y-maze experiment and the NOR task. In particular, this improving effect showed a significant synergistic effect when the strain was used in combination. In addition, it was confirmed that the strain improved inflammation indicators in the hippocampus, in particular, lowered IFN-γ and TNF-α increased by endotoxin, increased IL-10 expression, and increased IL-10 expression compared to IFN-γ and IL-10 expression compared to TNF-α, thereby improving neuritis. Example 7: Analysis of the efficacy of the administration of the strain in immunosuppressed animals induced by anticancer drug (cyclophosphamide) Six C57BL / 6 male mice (5 weeks old, 19-21g) were divided into groups and allowed to adapt to the laboratory for one week. To induce intestinal and brain disease, cyclophosphamide (CP, 150mg / kg, dissolved in saline) was administered for one day, followed by a day of rest, and then intraperitoneally administered the following day. In contrast, 0.1ml of saline was administered intraperitoneally to the normal group. After the final administration of the anticancer drug, the test sample, lactic acid bacteria, was suspended in saline and administered once a day for five days, at 1×10 per mouse. 9 CFU (NK210 alone: ​​1 x 10 9 CFU / mouse, NK219 alone: ​​1 × 10 9 CFU / mouse, combination (210+219): 210, 8 × 10 8 CFU / mouse and 219, 2 × 10 8 CFU / mouse) and sulfasalazine 50mg / kg (mouse) were orally administered to the animals. The day after the administration of the samples was completed, the experimental animals were sacrificed and the hippocampus, colon, and spleen of the brain were obtained. In the hippocampus, the inflammation indicators IFN-γ, TNF-α, and IL-10 were measured by ELISA, the intestinal length and the inflammation indicators IFN-γ, TNF-α, and IL-10 were measured in the colon, and Th1 cells and Treg cells were measured in the spleen by Pax. (1) Confirmation of effectiveness in improving colitis [Table 10] As can be seen from Table 10, the administration of single strains and combined strains showed an effect of improving colitis, as confirmed by intestinal length, changes in expression of inflammatory factors, changes in MPO activity, etc. Specifically, the decrease in intestinal length was improved, MPO activity was reduced, and the expression of IFN-γ, TNF-α, and IL-10, all of which were suppressed by anticancer drugs, increased. In particular, the expression of IL-10 compared to IFN-γ and IL-10 compared to TNF-α was increased, improving colitis. It was confirmed that this action acts as a synergy with the combined strains, and in particular, the most excellent effect was observed when NK210 and NK219 were administered at a mixed ratio of 4:1. (2) Confirmation of improvement effect in the spleen [Table 11] As can be seen from Table 11, the phagocytic ability of macrophages, which are natural immune cells, and the cell killing effect of NK cells were improved. The expression of IFN-γ, TNF-α, and IL-10, which were all suppressed by anticancer drugs, increased. In particular, the expression of IL-10 was increased compared to IFN-γ and IL-10 compared to TNF-α, demonstrating excellent therapeutic effects. Furthermore, this action was shown to be highly excellent, showing synergistic effects not only when used alone but also when used in combination, and in particular, the most excellent effect was observed when NK210 and NK219 were administered in a mixture ratio of 4:1. (3) Confirmation of the effect of improving cognitive function [Table 12] As can be seen from Table 12, administration of the strain according to the present invention showed excellent improving effects in both the Y-maze experiment and the NOR task. In particular, this improving effect showed a significant synergistic effect when the strain was used in combination. In addition, the expression of IFN-γ, TNF-α, and IL-10, which were all suppressed by anticancer drugs, was increased, and in particular, the expression of IL-10 was increased in comparison to IFN-γ, and IL-10 in comparison to TNF-α, showing a therapeutic effect (immunomodulatory effect). Example 8: Analysis of the efficacy of strain treatments in animals with antibiotic-induced gut microbiota imbalance Six C57BL / 6 male mice (5 weeks old, 19-21 g) were divided into groups and allowed to adapt to the laboratory for one week. To induce intestinal and brain diseases, ampicillin (AP, 100 mg / kg, dissolved in saline) was orally administered for three days. Meanwhile, 0.1 ml of saline was orally administered to the normal group. Starting from the day after the final antibiotic administration, the test sample, lactic acid bacteria, was suspended in saline and administered at 1 × 10 per mouse once a day for five days. 9 CFU (NK210 alone: ​​1 x 10 9 CFU / mouse, NK219 alone: ​​1 × 10 9 CFU / mouse, combination (210+219): 210, 8 × 10 8 CFU / mouse and 219, 2 × 10 8 CFU / mouse) and sulfasalazine 50mg / kg (mouse) were orally administered to the animals. The experimental animals were sacrificed the day after the end of sample administration, and the hippocampus, colon, and spleen of the brain were obtained. In the hippocampus, inflammation indicators IFN-γ, TNF-α, and IL-10 were measured by ELISA, intestinal length and inflammation indicators IFN-γ, TNF-α, and IL-10 were measured in the colon, Th1 cells and Treg cells were measured in the spleen by Pax, and endotoxin (LPS) and intestinal microbial community were measured in the feces. (1) Confirmation of effectiveness in improving colitis [Table 13] As can be seen from Table 13, the administration of single strains and combined strains showed an effect of improving colitis, as confirmed by intestinal length, changes in expression of inflammatory factors, changes in MPO activity, etc. Specifically, the decrease in intestinal length was improved, MPO activity was reduced, the expression levels of interferon-gamma and TNF-α were reduced, and the expression level of IL-10, an anti-inflammatory cytokine, was increased. That is, it inhibited IFN-γ and TNF-α increased by endotoxin, and increased IL-10 expression decreased by endotoxin, and in particular, it increased IL-10 expression relative to IFN-γ and IL-10 expression relative to TNF-α, thereby greatly improving the symptoms of colitis. It was confirmed that this action acts as a synergy with the combined strains, and in particular, the most excellent effect was observed when NK210 and NK219 were administered at a mixture ratio of 4:1. (2) Confirmation of improvement effect in the spleen [Table 14] As can be seen from Table 14, it was confirmed that the phagocytic ability of macrophages, which are natural immune cells, and the cell killing effect of NK cells were improved. In addition, IFN-γ and TNF-α, which were increased by endotoxin, were suppressed, and IL-10 expression, which was decreased by endotoxin, was increased, and in particular, IL-10 expression was increased in comparison with IFN-γ and IL-10 expression in comparison with TNF-α. Such an action on the spleen, which is involved in inflammatory reactions systemically, indicates the excellent inflammation-improving effect of the strain according to the present invention. Furthermore, such an action was shown to be highly excellent, showing a synergistic effect not only when used alone but also when used in combination, and in particular, the most excellent effect was shown when NK210 and NK219 were administered at a mixing ratio of 4:1. (3) Confirmation of the effect of improving cognitive function [Table 15] As can be seen from Table 15, administration of the strain according to the present invention showed excellent improving effects in both the Y-maze experiment and the NOR task. In particular, this improving effect showed a significant synergistic effect when the strain was used in combination. In addition, it was confirmed that the strain improved inflammation indicators in the hippocampus, in particular, lowered IFN-γ and TNF-α that were increased by endotoxin, and increased IL-10 expression, and thus had an improving effect on neuritis by increasing IL-10 expression compared to IFN-γ and IL-10 expression compared to TNF-α. (4) Improving gut microbiota imbalance The results of improving the imbalance of the intestinal microbiota are shown in Figure 1. As can be seen from Figure 1, the imbalance in the intestinal microbiota, which had a decrease in beta diversity, Proteobacteria, and Bacteroidetes due to the administration of the antibiotic ampicillin, was improved by administering LR (NK210), BL (NK219), and a mixture of these (Mx), resulting in a balanced intestinal microbiota. Example 9: Analysis of the efficacy of strain administration in healthy animals Six C57BL / 6 male mice (5 weeks old, 19-21 g) were divided into groups and allowed to adapt to the laboratory for one week. The test sample, lactic acid bacteria, was suspended in saline and administered to each mouse once a day for five days. 9 CFU (NK210 alone (LR): 1 x 10 9 CFU / mouse, NK219 alone (BL): 1 × 10 9 CFU / mouse, combination (MX, 210+219): 210, 8 × 10 8 CFU / mouse and 219, 2 × 10 8The animals were orally administered 100 mg / kg of lactic acid bacteria (CFU / mouse). The positive control group was orally administered sulfasalazine, a colitis treatment drug, instead of lactic acid bacteria, at 50 mg / kg, and the normal group was orally administered physiological saline used to suspend the lactic acid bacteria. The animals were sacrificed the day after the sample administration was completed, and the hippocampus, large intestine, and spleen were obtained. In the hippocampus, inflammation indicators IFN-γ, TNF-α, and IL-10 were measured by ELISA, intestinal length and inflammation indicators IFN-γ, TNF-α, and IL-10 were measured in the large intestine, and Th1 cells and Treg cells were measured in the spleen by Pax. (1) Confirmation of effectiveness in improving colitis [Table 16] As can be seen from Table 16, administration of a single strain in healthy animals increased the expression of interferon gamma, and when used in combination, it was confirmed that the expression was increased significantly. In particular, the most excellent effect was observed when NK210 and NK219 were administered in a mixture ratio of 4:1. (2) Confirmation of improvement effect in the spleen [Table 17] As can be seen from Table 17, it was confirmed that the phagocytic ability of macrophages, which are natural immune cells, and the cell killing effect of NK cells were improved. In addition, the expression of interferon gamma was increased. Furthermore, when used in combination, this effect was significantly increased with respect to the expression of IFN-γ. In particular, the expression of IL-10 compared to IFN-γ and IL-10 compared to TNF-α were improved. From the above results, it was confirmed that the strain according to the present invention has excellent immune-enhancing effects. Example 10: Creation of animals with intestinal and brain diseases induced by TNBS and confirmation of efficacy of administering the strain to them C57BL / 6 male mice (5 weeks old, 19-21g) were divided into groups of 6 mice and allowed to adapt to the laboratory for 1 week. To induce intestinal and brain diseases, 5% 2,4,6-trinitrobenzenesulfonic acid (TNBS, Sigma, USA) solution was diluted 1:1 with 50% ethanol, and 0.1 ml of the solution was administered into the large intestine via the anus using a 1 ml round-tipped syringe, which was held vertically for 30 seconds. In contrast, the normal group was orally administered 0.1 ml of saline. From the next day, the test sample, lactic acid bacteria, was suspended in saline and administered at 1 × 10 per mouse once a day for 5 days. 9 CFU (NK210 alone (LR): 1 x 10 9 CFU / mouse, NK219 alone (BL): 1 × 10 9 CFU / mouse, NK209 alone (LL): 1 × 10 9 CFU / mouse, combination (MX, 210+219): NK210, 8×10 8 CFU / mouse and NK219, 2 × 10 8 CFU / mouse, combination (MX, 209+219): NK209, 8 × 10 8 CFU / mouse and NK219, 2 × 10 8 CFU / mouse, LR+BL+LL:NK210, 4×10 8 CFU / mouse, and NK219, 2 × 10 8 CFU / mouse and NK209, 4 × 10 8 CFU / mouse, heat treatment (H-): 1 × 10 9 The animals were orally administered 50 mg / kg of sulfasalazine, a colitis treatment drug, instead of lactobacilli, at a dose of 100 mg / kg per mouse. The animals in the positive control group were orally administered 50 mg / kg of sulfasalazine, a colitis treatment drug, instead of lactobacilli, and the animals in the normal group were orally administered the same saline solution used to suspend lactobacilli. The animals were sacrificed the day after the administration of the samples, and the hippocampus, large intestine, and spleen were obtained. In the hippocampus, inflammation indicators TNF-α and IL-10 were measured by ELISA, intestinal length and inflammation indicators TNF-α and IL-10 were measured in the large intestine, and Th1 cells and Treg cells were measured in the spleen by Pax. (1) Confirmation of the effect of improving cognitive function [Table 18] As can be seen from Table 18, both NK210 and NK219 improved cognitive function damage caused by TNBS. In particular, they suppressed hippocampal inflammation indicators IFN-gamma and TNF-alpha, which were increased by TNBS, and increased IL-10. This was a remarkable effect not only in live bacteria but also in heat-killed bacteria. It was also confirmed that the synergistic effect was the best when two types of bacteria were used together (NK210 and NK219), and especially when three types were used together (NK210, NK219, and NK209), rather than when used alone. All of the above effects were confirmed to be superior to those of sulfasalazine currently used as a pharmaceutical. (2) Confirmation of improvement effect on the large intestine [Table 19] As can be seen from Table 19, in the case of colitis damaged by TNBS, the treatment with the strain of the present invention showed an improved effect. In particular, it suppressed hippocampal inflammation indicators myeloperoxidase (MPO) activity, IFN-gamma, and TNF-alpha, which were increased by TNBS, and increased IL-10, and this effect was confirmed not only with live bacteria but also with dead bacteria. In particular, the effect of the combination of NK210, NK219, and NK209 was the most excellent, and not only each but all of the combinations showed better results than sulfasalazine, which is currently used as a medicine. <Information on lactic acid bacteria consignment> The inventors of the present invention deposited Lactobacillus rhamnosus NK210 with the Korea Microorganism Collection, an accredited depository institution (Address: Yurimville, 45, Hongjenae 2gagil, Seodaemun-gu, Seoul, Republic of Korea) on September 15, 2021, and it was assigned the accession number KCCM13049P. The inventors of the present invention deposited Bifidobacterium longum NK219 with the Korea Microorganism Collection, an accredited depository institution (Address: Yurimville, 45, Hongjenae 2gagil, Seodaemun-gu, Seoul, Republic of Korea) on September 15, 2021, and it was assigned the accession number KCCM13050P. The inventors of the present invention deposited Lactococcus lactis NK209 with the Korea Microorganism Collection (Address: Yurimville, 45, Hongjenae 2gagil, Seodaemun-gu, Seoul, Republic of Korea), an accredited depository institution, on September 15, 2021, and it was assigned the accession number KCCM13048P. [Accession number] Name of depository institution: Korea Microorganism Collection Center (overseas) Accession number: KCCM13048P Date of acceptance: 20210915 Name of depository institution: Korea Microorganism Collection Center (overseas) Accession number: KCCM13049P Date of acceptance: 20210915 Name of depository institution: Korea Microorganism Collection Center (overseas) Accession number: KCCM13050P Date of acceptance: 20210915

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[0009] JPEG2024535901000026.jpg208144JPEG2024535901000027.jpg9144

[0010] JPEG2024535901000028.jpg207144JPEG2024535901000029.jpg17144 [Industrial Applicability]

[0011] The novel probiotic according to the present invention has excellent antioxidant effects, inflammation improving effects, neurological improving effects, cognitive improving effects, intestinal microbiota imbalance improving effects, immunomodulatory effects, and the like, making it highly valuable as a pharmaceutical or food product, and may be particularly useful for preventing, treating, or improving inflammatory diseases, improving immune function, and cognitive dysfunction.

Claims

1. Bifidobacterium longum NK219 KCCM13050P.

2. The Bifidobacterium longum NK219 according to claim 1, wherein the Bifidobacterium longum NK219 comprises the 16S rDNA base sequence of SEQ ID NO:

2. Bifidobacterium longum NK219 KCCM13050P.

3. Lactobacillus rhamnosus NK210 KCCM13049P.

4. The Lactobacillus rhamnosus NK210 (Lactobacillus rhamnosus NK210) according to claim 3, which comprises the 16S rDNA base sequence of SEQ ID NO: 1, Lactobacillus rhamnosus NK210 (Lactobacillus rhamnosus NK210) KCCM13049P.

5. A pharmaceutical composition for preventing or treating inflammatory diseases or cognitive impairment, comprising Bifidobacterium longum NK219 KCCM13050P, Lactobacillus rhamnosus NK210 KCCM13049P, or a mixture thereof.

6. The pharmaceutical composition for preventing or treating an inflammatory disease or cognitive impairment according to claim 5, further comprising Lactococcus lactis NK209 KCCM13048P.

7. 7. The pharmaceutical composition for preventing or treating an inflammatory disease or cognitive impairment according to claim 5 or 6, wherein the inflammatory disease is any one or more selected from the group consisting of inflammatory bowel disease, arthritis, gout, hepatitis, asthma, obesity, keratitis, gastritis, nephritis, colitis, diabetes, tuberculosis, bronchitis, pleuritis, peritonitis, spondylitis, pancreatitis, urethritis, cystitis, vaginitis, arteriosclerosis, sepsis, burns, dermatitis, periodontitis, and gingivitis.

8. 8. The pharmaceutical composition for preventing or treating an inflammatory disease or cognitive impairment according to claim 7, wherein the inflammatory bowel disease is any one or more selected from the group consisting of ulcerative colitis, Crohn's disease, collagenous colitis, lymphocytic colitis, ischemic colitis, diversion colitis, Behcet's disease, atypical colitis, antibiotic-induced colitis, and anticancer drug-induced colitis.

9. 7. The pharmaceutical composition for preventing or treating an inflammatory disease or cognitive dysfunction according to claim 5 or 6, wherein the cognitive dysfunction is any one or more selected from the group consisting of anxiety, depression, migraine, stress, Alzheimer's disease, Huntington's disease, vascular dementia, Pick's disease, Parkinson's disease, Creutzfeldt-Jakob disease, dementia, and combinations thereof.

10. A food composition for preventing or improving inflammatory diseases or cognitive impairment, comprising Bifidobacterium longum NK219 KCCM13050P, Lactobacillus rhamnosus NK210 KCCM13049P, or a mixture thereof.

11. The food composition for preventing or ameliorating inflammatory diseases or cognitive impairment according to claim 10, further comprising Lactococcus lactis NK209 KCCM13048P.

12. 12. The food composition for preventing or ameliorating an inflammatory disease or cognitive impairment according to claim 10 or 11, wherein the inflammatory disease is any one or more selected from the group consisting of inflammatory bowel disease, arthritis, gout, hepatitis, asthma, obesity, keratitis, gastritis, nephritis, colitis, diabetes, tuberculosis, bronchitis, pleuritis, peritonitis, spondylitis, pancreatitis, urethritis, cystitis, vaginitis, arteriosclerosis, sepsis, burns, dermatitis, periodontitis and gingivitis.

13. 13. The food composition for preventing or ameliorating inflammatory diseases or cognitive impairment according to claim 12, wherein the inflammatory bowel disease is any one or more selected from the group consisting of ulcerative colitis, Crohn's disease, collagenous colitis, lymphocytic colitis, ischemic colitis, diversion colitis, Behcet's disease, atypical colitis, antibiotic-induced colitis, and anticancer drug-induced colitis.

14. 12. The food composition for preventing or ameliorating inflammatory diseases or cognitive dysfunction according to claim 10 or 11, wherein the cognitive dysfunction is any one or more selected from the group consisting of anxiety, depression, migraine, stress, Alzheimer's disease, Huntington's disease, vascular dementia, Pick's disease, Parkinson's disease, Creutzfeldt-Jakob disease, dementia, and combinations thereof.

15. A composition for improving immune function, comprising Bifidobacterium longum NK219 KCCM13050P, Lactobacillus rhamnosus NK210 KCCM13049P, or a mixture thereof.

16. The composition for improving immune function according to claim 15, further comprising Lactococcus lactis NK209 KCCM13048P.

17. The composition for improving immune function according to claim 15 or 16, wherein the improvement of immune function is recovery from immune deficiency, immune depression or immune system damage caused by radiation therapy or exposure, or administration of antibiotics or anticancer drugs.

18. 17. The composition for improving immune function according to claim 15 or 16, wherein the improvement of immune function is achieved by enhancing the activity of the immune system.

19. The composition for improving immune function according to claim 15 or 16, wherein the improvement of immune function is intended to enhance the immune strength of a subject having normal immune strength.

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