Novel lactic acid bacteria and use thereof

Abstract

The present invention relates to: Lactococcus lactis P135 and Bifidobacterium longum P142, which are novel strains of lactic acid bacteria, or a mixture thereof; and a use thereof. The strains and the mixture thereof according to the present invention can be effectively used for preventing, alleviating, or treating Helicobacter pylori infection, inflammatory diseases, depressive disorders, and / or cognitive dysfunction.

Description

Novel lactic acid bacteria and their uses

[0001] The present invention relates to novel lactic acid bacteria, specifically the Lactococcus lactis P135 strain, the Bifidobacterium longum P142 strain, or mixtures thereof, and their uses.

[0002] Helicobacter pylori infects the stomach and causes acute / chronic gastritis, gastric ulcers, duodenal ulcers, and gastric lymphoma. If gastritis caused by Helicobacter pylori persists, atrophic changes occur in the gastric mucosa, and subsequently, various factors can lead to gastric adenocarcinoma; therefore, in 1994, the World Health Organization (WHO) declared Helicobacter pylori a Class 1 carcinogen. In Korea, the infection rate of Helicobacter pylori is over 50% of the total population, and the infection rates among patients with duodenal ulcers and gastric ulcers are 90-95% and 60-80%, respectively.

[0003] The primary treatment for Helicobacter pylori infection is a combination of antibiotics and acid suppressants, and taking this medication for 7 to 14 days eradicates Helicobacter pylori with a probability of about 70 to 80 percent. However, recently, as antibiotic-resistant Helicobacter pylori has increased, there are many cases where treatment is difficult.

[0004] Furthermore, as drug use gradually increases, the incidence of gastrointestinal diseases such as gastritis is rising due to drug-induced damage to the gastric mucosa. Oral administration of analgesics and anti-inflammatory drugs, which are widely used in modern society, increases gastric acid secretion and inhibits the production of prostaglandins responsible for protecting the gastric mucosa; as this can lead to damage to the stomach wall and inflammation, the need for gastric protective agents is increasing.

[0005] Against this backdrop, there is a need to develop probiotic-based treatment methods and formulations that can suppress Helicobacter pylori infection, overcome the problem of resistance to existing drugs, and have minimal side effects even with long-term consumption.

[0006] The object of the present invention is to provide the Lactococcus lactis P135 KCCM13519P strain.

[0007] Another objective of the present invention is to provide the Bifidobacterium longum P142 KCCM13520P strain.

[0008] Another objective of the present invention is to provide a pharmaceutical composition for the prevention or treatment of inflammatory diseases, depressive disorders, or cognitive impairments comprising Lactococcus lactis P135 KCCM13519P, Bifidobacterium longum P142 KCCM13520P, or a mixture thereof.

[0009] Another objective of the present invention is to provide a food composition for the prevention or improvement of inflammatory diseases, depressive disorders, or cognitive impairments comprising Lactococcus lactis P135 KCCM13519P, Bifidobacterium longum P142 KCCM13520P, or a mixture thereof.

[0010] As one embodiment for carrying out the above purpose, the present invention provides Lactococcus lactis P135 (Depository: Korean Culture Collection of Microorganisms, Deposit date: September 19, 2024, Accession number: KCCM13519P).

[0011] Lactococcus lactis P135 of the present invention is characterized as a novel lactic acid bacterium isolated and identified from the feces of a healthy human.

[0012] First, whole-genome sequencing was performed to confirm the uniqueness of the strain at the strain level, and in particular, the results of the UPGMA (Unweighted Pair Group Method with Arithmetic Mean) phylogenetic analysis and heat map analysis are shown in Figures 1 and 2, respectively.

[0013] The aforementioned UPGMA phylogenetic analysis is a hierarchical clustering method that visually represents phylogenetic distances by forming clusters based on genomic similarity. Additionally, the heat map analysis calculates and displays homology at the genetic level by comparing target strains pairwise with the P135 strain; colors closer to blue indicate higher homology, while colors closer to red indicate lower homology.

[0014] Analysis results confirmed that strain P135 is a unique novel strain at the genomic level, as it belongs to the same clade as known Lactococcus lactis strains but exhibits an independent branching structure.

[0015] Therefore, the lactic acid bacteria were identified as Lactococcus lactis, named Lactococcus lactis P135, and deposited with the Korean Culture Collection of Microorganisms on September 19, 2024 (KCCM13519P).

[0016] The 16S rDNA sequence for the identification and classification of Lactococcus lactis P135 of the present invention is as shown in SEQ ID NO. 1 attached to this specification. Accordingly, Lactococcus lactis P135 of the present invention may include the 16S rDNA of SEQ ID NO. 1.

[0017] The physiological characteristics of Lactococcus lactis P135 of the present invention can be analyzed according to conventional methods in the art, and specifically, Lactococcus lactis P135 may use D-ribose, D-xylose, D-galactose, D-glucose, D-fructose, D-mannose, mannitol, N-acetyl-glucosamine, amygdalin, arbutin, esculin, salicin, cellobiose, maltose, lactose, sucrose, trehalose, starch, gentiobiose, and gluconate as carbon sources.

[0018] In another embodiment for carrying out the above purpose, the present invention provides Bifidobacterium longum P142 (Depository: Korean Culture Collection of Microorganisms, Deposit date: September 19, 2024, Accession number: KCCM13520P).

[0019] The Bifidobacterium longum P142 of the present invention is characterized as a novel lactic acid bacterium isolated and identified from the feces of a healthy human.

[0020] Likewise, whole-genome sequencing was performed to confirm the uniqueness of the strain at the strain level, and in particular, the results of the UPGMA (Unweighted Pair Group Method with Arithmetic Mean) phylogenetic analysis and heat map analysis are shown in Figures 3 and 4, respectively.

[0021] As a result of the analysis, strain P142 was confirmed to be a unique novel strain at the genomic level, as it belongs to the same clade as known Bifidobacterium longum strains but exhibits an independent branching structure. Therefore, the lactic acid bacteria were identified as Bifidobacterium longum, named Bifidobacterium longum P142, and deposited with the Korean Culture Collection Center on September 19, 2024 (KCCM13520P).

[0022] The 16S rDNA sequence for the identification and classification of Bifidobacterium longum P142 of the present invention is as shown in SEQ ID NO. 2 attached to this specification. Accordingly, Bifidobacterium longum P142 of the present invention may include the 16S rDNA of SEQ ID NO. 2.

[0023] The physiological characteristics of Bifidobacterium longum P142 of the present invention can be analyzed according to conventional methods in the art, and specifically, Bifidobacterium longum P142 may use urea, D-glucose, D-mannitol, D-lactose, D-sucrose, D-maltose, salicin, D-xylos, L-arabinose, esculin, D-mannose, D-meletchitose, D-raffinose, D-sorbitol, and D-trehalose as carbon sources.

[0024] Another aspect of the present invention relates to a pharmaceutical composition for the prevention or treatment of inflammatory diseases, depressive disorders, or cognitive impairments comprising Lactococcus lactis P135 KCCM13519P, Bifidobacterium longum P142 KCCM13520P, or a mixture thereof.

[0025] In the present invention, "inflammatory disease" refers to a general term for diseases in which inflammation is the primary lesion. The inflammatory diseases of the present invention may be one or more selected from the group comprising gastritis, arthritis, gout, hepatitis, obesity, keratitis, enteritis, nephritis, colitis, diabetes, tuberculosis, bronchitis, pleuritis, peritonitis, spondylitis, pancreatitis, inflammatory pain, urethritis, cystitis, vaginitis, arteriosclerosis, sepsis, and periodontitis. More specifically, the inflammatory disease may be gastritis, but is not limited thereto.

[0026] Preferably, it may be an inflammatory disease occurring in the digestive system. For example, it may be one or more selected from the group consisting of gastritis, hepatitis, enteritis, nephritis, colitis, and pancreatitis.

[0027] The above gastritis refers to a condition in which inflammation occurs in the inner lining of the stomach wall, and can be caused by Helicobacter pylori infection, alcohol consumption, smoking, taking drugs such as nonsteroidal anti-inflammatory drugs (NSAIDs), stress, etc., and includes acute gastritis and chronic gastritis.

[0028] The above non-steroidal anti-inflammatory drugs (NSAIDs) include, but are not limited to, salicylates (e.g., aspirin), propionic acid derivatives (e.g., ibuprofen, naproxen), acetic acid derivatives (e.g., indomethacin, diclofenac, etodolac), oxycam derivatives (e.g., meloxicam), phenamate derivatives (e.g., mefenamic acid), and selective or relatively selective COX-2 inhibitors (e.g., celecoxib, etroxicoxib, valdecoxib, nabumetone).

[0029] The aforementioned Helicobacter pylori primarily infects the gastric mucosa and is a direct cause of acute gastritis, chronic gastritis, gastric ulcers, duodenal ulcers, and gastric lymphoma. It is known that if chronic gastritis caused by Helicobacter pylori persists, atrophic changes occur in the gastric mucosa, and subsequently, various environmental and genetic factors act to increase the incidence of gastric adenocarcinoma. Furthermore, Helicobacter pylori infection is reported to affect not only gastrointestinal diseases but also symptoms of depression and anxiety, cognitive decline, or the development of dementia (Association of Helicobacter pylori Infection with Depression and Anxiety: A Systematic Review and Meta-Analysis, Li et al, International Journal of Clinical Practice, 2024; Helicobacter pylori infection and risk for developing dementia: an evidence-based meta-analysis of case-control and cohort studies, Liu et al., Aging 2021).

[0030] In the present invention, "depressive disorder" is a disease characterized by a decline in motivation and depression as major symptoms, causing various cognitive and psychosomatic symptoms and leading to a decline in daily functioning. It may be one or more selected from the group including, but not limited to, Major Depressive Disorder, Persistent Depressive Disorder, Dysthymia, Disruptive Mood Dysregulation Disorder, Premenstrual Dysphoric Disorder, Substance / Medication-Induced Depressive Disorder, Depressive Disorder due to Another Medical Condition, Other Specified Depressive Disorder, and Unspecified Depressive Disorder.

[0031] In the present invention, "cognitive impairment" refers to a state in which memory, attention, language ability, visuospatial ability, judgment ability, etc., are reduced, and includes cases ranging from mild to severe. Specifically, cognitive impairment may include memory loss, dementia, motor function decline, etc., and may be one or more selected from the group including anxiety, migraine, stress, Alzheimer's disease, Huntington's disease, vascular dementia, Pick's disease, Parkinson's disease, Creutzfeldt-Jakob disease, and dementia.

[0032] According to one embodiment of the present invention, the inflammatory disease, depressive disorder, or cognitive impairment of the present invention may be caused by Helicobacter pylori infection.

[0033] In one embodiment of the present invention, it was confirmed that Lactococcus lactis P135 and / or Bifidobacterium longum P142 reduce the expression of inflammatory cytokines, and thus can be utilized for therapeutic purposes for inflammatory diseases. In addition, it was confirmed through behavioral experiments that cognitive and motor functions are improved, and thus can be utilized for the treatment and improvement of impaired cognitive and motor functions.

[0034] Furthermore, it has been confirmed that Lactococcus lactis P135 and / or Bifidobacterium longum P142 of the present invention exhibit therapeutic and / or improvement effects not only on gastritis caused by Helicobacter pylori infection but also on gastritis caused by drugs, so the lactic acid bacteria of the present invention can be applied to treat gastritis with different causes.

[0035] The composition of the present invention comprises 1 x 10 of the Lactococcus lactis P135 strain or Bifidobacterium longum P142 strain. 3 CFU to 1X10 12 It may contain CFU. Preferably 1X10 6 CFU to 1X10 11 CFU, more preferably 1X10 9 CFU to 1X10 11 It may include CFU, but is not limited thereto.

[0036] Specifically, the mixture in the above pharmaceutical composition may be a mixture of Lactococcus lactis P135 strain and Bifidobacterium longum P142 strain in a Colony Forming Unit (CFU) ratio of 1:1 to 20:1 or 1:1 to 1:20, preferably 1:1 to 9:1 or 1:1 to 1:9, more preferably 1:1 to 4:1 or 1:1 to 1:4, and even more preferably 1:1 to 4:1, but is not limited thereto.

[0037] Additionally, specifically, the above Lactococcus lactis P135 or Bifidobacterium longum P142 may be their live cells, their dead cells, their cultures, their lysates, or their extracts, respectively, but may be applied without limitation as long as they are in a form that can achieve the desired effect.

[0038] In the present invention, "live cell" refers to the novel lactic acid bacteria of the present invention itself, "dead cell" refers to lactic acid bacteria that have been sterilized by heating, pressurization, or drug treatment, and "crushed material" refers to lactic acid bacteria that have been destroyed by enzyme treatment, homogenization, or ultrasonic treatment.

[0039] In the present invention, "extract" refers to a product obtained by extracting lactic acid bacteria with a known extraction solvent.

[0040] In the present invention, "culture" or "culture medium" refers to a product obtained by culturing lactic acid bacteria in a known medium, and said product may include novel lactic acid bacteria. The medium may be selected from known liquid or solid media, and may be, for example, MRS liquid medium, GAM liquid medium, MRS agar medium, GAM agar medium, BL agar medium, but is not limited thereto.

[0041] The pharmaceutical composition according to the present invention may be prepared into a pharmaceutical formulation using methods well known in the art to provide rapid, sustained, or delayed release of the active ingredient after administration to mammals. In preparing the formulation, the pharmaceutical composition according to the present invention may additionally include a pharmaceutically acceptable carrier to the extent that it does not inhibit the activity of the novel lactic acid bacteria.

[0042] The above pharmaceutically acceptable carriers include, but are not limited to, commonly used ones, such as lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. Additionally, the pharmaceutical composition of the present invention may include fillers, extenders, binders, wetting agents, disintegrants, diluents or excipients such as surfactants, and other pharmaceutically acceptable additives.

[0043] The dosage of the pharmaceutical composition according to the present invention must be a pharmaceutically effective amount. A "pharmaceutically effective amount" means an amount sufficient to prevent or treat the aforementioned disease or condition with a reasonable benefit / risk ratio applicable to medical treatment. The effective dose level may be selected by those skilled in the art according to various factors such as the method of formulation, the patient's condition and body weight, the patient's gender, age, severity of the disease, drug form, route and duration of administration, rate of excretion, response responsiveness, etc. As is recognized by those skilled in the art, the effective dose may vary depending on the route of treatment, the use of excipients, and the possibility of co-administration with other agents. However, for a desirable effect, in the case of oral formulations, the composition of the present invention may generally be administered to adults at a dose of 0.0001 to 100 mg / kg per day, preferably 0.001 to 100 mg / kg per day of body weight. Administration may be performed once a day or divided into several doses. The above dosage does not limit the scope of the present invention in any way.

[0044] The pharmaceutical composition of the present invention may be administered to mammals, such as mice, livestock, and humans, through various routes. Specifically, the pharmaceutical composition of the present invention may be administered orally or parenterally (e.g., topically or 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 may include suspensions, liquids, emulsions, syrups, aerosols, etc., and may include various excipients, such as humectants, sweeteners, flavorings, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. Preparations for parenteral administration may be formulated and used in the form of sterile aqueous solutions, liquids, non-aqueous solvents, suspensions, emulsions, eye drops, eye ointments, syrups, suppositories, aerosols, and other topical preparations, as well as sterile injectable preparations, according to conventional methods. Preferably, pharmaceutical compositions such as creams, gels, patches, sprays, ointments, warning agents, 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 depending on the clinical prescription. As non-aqueous solvents and suspensions, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleates may be used. Witepsol, Macrogol, Tween 61, Cacao, Laurin, Glycerozelatin, etc. can be used as bases for suppositories.

[0045] Another aspect of the present invention relates to a method for preventing or treating inflammatory diseases, depressive disorders, or cognitive impairments, comprising the step of administering Lactococcus lactis P135 KCCM13519P, Bifidobacterium longum P142 KCCM13520P, or a mixture thereof to a subject.

[0046] Another aspect of the present invention relates to the use of Lactococcus lactis P135 KCCM13519P, Bifidobacterium longum P142 KCCM13520P, or a mixture thereof in the manufacture of a drug for the treatment of inflammatory diseases, depressive disorders, or cognitive impairments.

[0047] Another aspect of the present invention relates to a pharmaceutical composition comprising Lactococcus lactis P135 KCCM13519P, Bifidobacterium longum P142 KCCM13520P or a mixture thereof; and a pharmaceutically acceptable carrier.

[0048] The above "inflammatory disease," "depressive disorder," and "cognitive impairment" are the same as previously described, and specifically, the above inflammatory disease may be gastritis.

[0049] The subject mentioned above refers to an animal and may typically be a mammal capable of exhibiting beneficial effects through treatment using the lactic acid bacteria of the present invention. Preferred examples of such a subject may include primates such as humans.

[0050] Another aspect of the present invention relates to a food composition for the prevention or improvement of inflammatory diseases, depressive disorders, or cognitive impairments comprising Lactococcus lactis P135 KCCM13519P, Bifidobacterium longum P142 KCCM13520P, or a mixture thereof.

[0051] The above "inflammatory disease," "depressive disorder," and "cognitive impairment" are the same as previously described, and specifically, the above inflammatory disease may be gastritis.

[0052] Specifically, the mixture of the strains may be a mixture of Lactococcus lactis P135 and Bifidobacterium longum P142 in a Colony Forming Unit (CFU) ratio of 1:1 to 20:1 or 1:1 to 1:20, preferably 1:1 to 9:1 or 1:1 to 1:9, more preferably 1:1 to 4:1 or 1:1 to 1:4, and even more preferably 1:1 to 4:1, but is not limited thereto.

[0053] In addition, specifically, the lactic acid bacteria included in the pharmaceutical composition or food composition of the present invention may be their live cells, their dead cells, their cultures, their lysates, or their extracts; however, any form of lactic acid bacteria capable of achieving a preventive or therapeutic effect for inflammatory diseases, depressive disorders, or cognitive impairments may be used without limitation. The "live cells," "dead cells," "cultures," "lysates," and "extracts" are the same as those described above.

[0054] Specifically, the above food may be a health functional food. The above health functional food is a food that emphasizes the biological regulatory function of the food and is a food that has been given added value to act and manifest for a specific purpose using physical, biochemical, or biotechnological methods. The ingredients of such health functional food are designed and processed to fully exert biological regulatory functions on the body related to biological defense, regulation of body rhythms, and prevention and recovery of diseases, and may contain food additives, sweeteners, or functional ingredients that are acceptable as food.

[0055] When the strains of the present invention are used as health functional foods (or additives for health functional beverages), the novel strains may be added as they are or used in combination with other foods or food ingredients, and may be used appropriately according to conventional methods. The mixing amount of the strains may be appropriately determined according to the purpose of use (prevention, health or improvement, therapeutic treatment).

[0056] Preferably, the strain according to the present invention may be in the form of a single strain, a mixture of two strains, or a mixture of three strains. Within the aforementioned range, the use of a mixed strain may be considered in light of synergistic effects.

[0057] The above food may contain various nutritional supplements, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, coloring agents and promoters (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. In addition, the health functional food of the present invention may contain fruit pulp for the production of fruit and vegetable beverages. These ingredients may be used alone or in combination, and the proportion of these additives is generally selected in the range of 0.001 to 50 parts by weight per total weight of the composition.

[0058] There are no specific restrictions on the types of food mentioned above. Foods to which the strains may be added include sausages, meat, bread, chocolates, snacks, candies, confectionery, ramen, pizza, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes. When formulated as a beverage, 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 ingredients, as in conventional beverages. The aforementioned natural carbohydrates may be monosaccharides (e.g., glucose, fructose, etc.), disaccharides (e.g., maltose, sucrose, etc.), polysaccharides (e.g., conventional sugars such as dextrin, cyclodextrin, etc.), and sugar alcohols such as xylitol, sorbitol, and erythritol.

[0059] Lactococcus lactis P135, Bifidobacterium longum P142, or a mixture thereof according to the present invention has excellent effects in preventing, treating, or improving Helicobacter pylori infection, inflammatory diseases, depressive disorders, and / or cognitive impairment.

[0060] The effects of the present invention are not limited to the effects described above, and should be understood to include all effects that can be inferred from the configuration of the invention described in the detailed description or claims of the present invention.

[0061] Figure 1 shows the results of whole-genome-based UPGMA (Unweighted Pair Group Method with Arithmetic Mean) phylogenetic analysis of Lactococcus lactis P135.

[0062] Figure 2 shows the results of a whole-genome similarity heat map analysis between Lactococcus lactis P135 and known Lactococcus lactis strains.

[0063] Figure 3 shows the results of whole-genome-based UPGMA (Unweighted Pair Group Method with Arithmetic Mean) phylogenetic analysis of Bifidobacterium longum P142.

[0064] Figure 4 shows the results of a whole-genome similarity heat map analysis between Bifidobacterium longum P142 and known Bifidobacterium longum strains.

[0065] The present invention will be explained in detail below by way of examples. However, the following examples are merely illustrative of the present invention, and the present invention is not limited by the following examples.

[0066] Example 1. Isolation and Identification of Lactic Acid Bacteria

[0067] 1-1. Isolation of Lactic Acid Bacteria from Human Feces

[0068] Human feces were placed in GAM liquid medium (GAM broth; Nissui Pharmaceutical, Japan) and suspended. Afterward, the supernatant was taken and transferred to GAM or BL agar medium (BL agar medium; Nissui Pharmaceutical, Japan), and after anaerobically incubated at 37°C for about 48 hours, the bacteria that formed colonies were isolated.

[0069] 1-2. Identification of Isolated Lactic Acid Bacteria

[0070] Physiological characteristics and 16S rDNA sequences of strains isolated from human feces were analyzed to determine the species of the strains and assign strain names. Table 1 below shows the control numbers and strain names of lactic acid bacteria isolated from the feces of healthy people.

[0071] Number Strain name Number Strain name 1Lactobacillus planantrumP13111Bifidobacterium longumP1412Lactobacillus planantrumP13212Bifidobacterium longumP1423Lactobacillus gasseriP13313Bifidobacterium longumP1434Lactococcus lactisP13414Bifidobacterium longumP1445Lactococcus lactisP13515Bifidobacterium adolescentisP1456Lactococcus lactisP13616Bifidobacterium adolescentisP1467Lactobacillus caseiP13717Bifidobacterium catenulatumP1478Lactobacillus reuteriP13818Bifidobacterium pseudocatenulatumP1489Lactobacillus acidophilusP13919Bifidobacterium animalisP14910Lactobacillus fermentumP14020Bifidobacterium animalisP150

[0072] 1-3. 16S rRNA Gene Analysis

[0073] Among the strains listed in Table 1 above, Lactococcus lactis P135 was 99.9% similar to Lactococcus lactisNCDO strain, and its 16S rDNA was found to have the nucleotide sequence of SEQ ID NO. 1.

[0074] As a result of performing whole-length gene analysis, P135 had a GC content of 35.0% and showed 98.8% similarity to Lactococcus lactisATCC19435 and 97.7% similarity to NBRC100931, confirming that P135 possesses a novel gene (Fig. 1).

[0075] The above-mentioned novel strain Lactococcus lactis P135 was deposited as a patent with the Korean Culture Collection of Microorganisms (Address: Yurim Building, 45 Hongjennae 2-ga-gil, Seodaemun-gu, Seoul, Republic of Korea), an accredited depositary institution, and was assigned the accession number KCCM13519P.

[0076] In addition, Bifidobacterium longum P142 was 99.5% similar to the Bifidobacterium longumsubsp. suillum Su851 strain, and its 16S rDNA was found to have the nucleotide sequence of SEQ ID NO. 2.

[0077] As a result of performing whole-length gene analysis, P142 had a GC content of 60.2% and showed 98.3% similarity to Bifidobacterium longumJCM1217 and 96.3% similarity to Bifidobacterium longumDSM20211, confirming that P142 possesses a novel gene (Fig. 2).

[0078] The above-mentioned novel strain Bifidobacterium longum P142 was deposited with the Korean Culture Collection of Microorganisms (address: Yurim Building, 45 Hongjenae 2-ga-gil, Seodaemun-gu, Seoul, Republic of Korea), an accredited depositary institution, and was assigned the accession number KCCM13520P.

[0079] 1-4. Analysis of Physiological Characteristics of Lactic Acid Bacteria

[0080] Carbon source availability among the physiological characteristics of Lactococcus lactis P135 and Bifidobacterium longum P142 was analyzed by sugar fermentation using the API 50 CH kit and API 20A kit (Manufacturer: BioMerieux's, USA). The results are shown in Table 2 below, where "+" indicates positive carbon source availability and "-" indicates negative carbon source availability.

[0081] Carbon Source P135API 50CH Kit Carbon Source P142API 20A Kit Glycerol-L-tryptophan-Erythritol-Urea+D-arabinose-D-glucose+L-arabinose-D-mannitol+D-ribose+D-lactose+D-xylose+D-sucrose+L- L-xylose-D-maltose+D-adonitol-Salicin+methyl-β-D-xylopyranoside-D-xylose+D-galactose+L-arabinose+D-glucose+Gelatin-D-Fructose (D-fructose) + Esculin + D-mannose + Glycerol - L-sorbose - D-cellobiose - L-rhamnose - D-mannose + Dulcitol - D-melezitose + Inositol - D-raffinose + Mannitol tol)+D-sorbitol+sorbitol-D-rhamnose-α-methyl-D-mannoside-D-trehalose+α-methyl-D-glucoside-CAT-N-acetyl-glucosamine+Spore-Amygdalin+Gram+Arbutin tin)+Cocci-esculin+salicin+cellobiose+maltose+lactose+melibiose-sucrose+trehalose+inulin-melezitose-raffinose-starch+glycogen-xylitol-genty Gentiobiose + D-turanose - D-lyxose - D-tagatose - D-fucose - L-fucose - D-arabitol - L-arabitol - GlucoNaTe + 2-keto-gluconate - 5-keto-gluconate

[0082] The biochemical characteristics of P135 and P142 were most similar to the previously known Lactobacillus lactis and Bifidobacterium longum, respectively, but not identical.

[0083] Experimental Example 1. Confirmation of the antioxidant activity of the lactic acid bacteria of the present invention

[0084] A DPPH solution was prepared by dissolving DPPH (2,2-Diphenyl-1-picrylhydrazyl) in ethanol to a concentration of 0.2 mM. A lactic acid bacteria suspension (1×10⁻⁶) was added to 0.1 ml of the above DPPH solution. 8 A solution of CFU / mL or vitamin C (1 g / mL) was added and incubated at 37°C for 20 minutes. The culture medium was centrifuged at 3000 rpm for 5 minutes to obtain the supernatant. Subsequently, the absorbance of the supernatant was measured at 517 nm, and the antioxidant activity of the lactic acid bacteria was calculated.

[0085] Inhibition rate (%) = 100 x (Absorbance of supernatant treated only with vehicle - Absorbance of supernatant treated with lactic acid bacteria) / (Absorbance of supernatant treated only with vehicle)

[0086] The results of the antioxidant effect measurement are shown in Table 3 below.

[0087] Experimental Example 2. Confirmation of the inhibitory activity of the lactic acid bacteria of the present invention against Helicobacter pylori (HP)

[0088] 2-1. Measurement of TNF-αIL-8 and IL-10 Expression in Macrophages

[0089] 2 mL of sterile 4% thioglycolate was administered into the peritoneal cavity of C57BL / 6 mice (male, 6 weeks old, 19-22 g), and the mice were anesthetized 4 days later. 8 mL of RPMI 1640 medium was administered into the peritoneal cavity of the mice, and after 5–10 minutes, the RPMI medium (macrophages) from the peritoneal cavity was extracted, centrifuged at 1000 xg for 10 minutes, and washed twice with RPMI 1640 medium. Macrophages were distributed at 0.5 x 10⁻⁶ per well. 6Inoculated into 24-well plates with a number of isolated lactic acid bacteria (final treatment concentration: 1×10⁻⁶ 5 cfu / mL) and heat-treated Helicobacter pylori (1X10⁻¹⁰), an inflammatory response inducing agent. 5 After treatment with cfu / mL at 90°C for 30 minutes, the samples were incubated in a CO2 / air incubator for 24 hours. Subsequently, the supernatant was obtained, and the expression levels of TNF-α, IL-8, and IL-10 were measured using an ELISA kit.

[0090] TNF-α Inhibition Rate (%) = 100 x (TNF-α expression level in cells treated only with Helicobacter pylori - TNF-α expression level in cells treated with both lactic acid bacteria and Helicobacter pylori) / (TNF-α expression level in cells treated only with Helicobacter pylori - TNF-α expression level in cells not treated with either lactic acid bacteria or Helicobacter pylori)

[0091] IL-8 Inhibition Rate (%) = 100 x (IL-8 expression level in cells treated only with Helicobacter pylori - IL-8 expression level in cells treated with both lactic acid bacteria and Helicobacter pylori) / (IL-8 expression level in cells treated only with Helicobacter pylori - IL-8 expression level in cells not treated with either lactic acid bacteria or Helicobacter pylori)

[0092] IL-10 Induction Rate (%) = 100 x (IL-10 expression in cells treated with both Lactobacillus and Helicobacter pylori - IL-10 expression in cells treated with only Helicobacter pylori) / (IL-10 expression in cells not treated with either Lactobacillus or Helicobacter pylori - IL-10 expression in cells treated with only Helicobacter pylori)

[0093] The measurement results are shown in Table 3 below.

[0094] 2-2. Inhibition of Helicobacter pylori growth

[0095] Lactic acid bacteria (1 x 10⁻¹⁰) pre-cultured in BHI medium in 10 mL of BHI medium 8Helicobacter pylori (1X10) together with CFU / mL) 8 CFU / mL) was inoculated and cultured for 24 hours under microaerotic conditions at 37°C (CampyPak Plus, BD). Afterward, 0.2 mL of the culture medium was inoculated into Columbia agar (5% sheep blood and antibiotics [Vancomycin 10.0 mg / L, Amphotericin B 5.0 mg / L] added) and cultured under microaerotic conditions for 3 days. Subsequently, the number of grown Helicobacter pylori colonies was measured and the inhibition rate of the lactic acid bacteria was measured.

[0096] Inhibition Rate (%) = 100 x (Number of agar colonies inoculated with medium containing only Helicobacter pylori - Number of agar colonies inoculated with medium containing both Lactobacillus and Helicobacter pylori) / (Number of agar colonies inoculated with medium containing only Helicobacter pylori)

[0097] The measurement results are shown in Table 3 below.

[0098] 2-3. Inhibition of Helicobacter pylori urease activity

[0099] Lactic acid bacteria (1 x 10⁻¹⁰) pre-cultured in BHI medium in 10 mL of BHI medium 8 Helicobacter pylori (1X10) together with CFU / mL) 8 CFU / mL) was inoculated, cultured for 24 hours under microaerophilic conditions at 37℃ (CampyPak Plus, BD), and collected (10,000g, 10 min, 4 o C). Only the precipitate was suspended in 1 mL of PBS, and urease enzyme activity was measured. Urease activity was measured using the MAK120 urease activity assay kit (Sigma Aldrich).

[0100] Inhibition rate (%) = 100 x (Urease activity of cells cultured with Helicobacter pylori only - Urease activity of cells cultured with lactic acid bacteria and Helicobacter pylori) / (Urease activity of cells cultured with Helicobacter pylori only)

[0101] The measurement results are shown in Table 3 below.

[0102] 2-4. Induction of Claudin-1 Expression

[0103] Caco2 colorectal cancer cells were obtained from the Korean Cell Line Bank and cultured in RPMI 1640 medium for 48 hours, after which the Caco2 cells were placed at a ratio of 1 x 10⁻¹⁰ per well. 6 Cells were dispensed into 24-well plates to achieve the required number. Each well was treated with either LPS 0.1 µg alone or LPS 0.1 µg and 1 x 10⁻¹⁰ lactic acid bacteria. 5 After co-treatment with CFU, the cells were cultured for 24 hours. Subsequently, cultured cells were collected from each well, and the expression level of the tight junction protein Claudin-1 was measured using an ELISA kit.

[0104] Claudin-1 Induction Rate (%) = 100 x (Claudin-1 expression in cells treated with both Lactobacillus and Helicobacter pylori - Claudin-1 expression in cells treated with only Helicobacter pylori) / (Claudin-1 expression in cells not treated with either Lactobacillus or Helicobacter pylori - Claudin-1 expression in cells treated with only Helicobacter pylori)

[0105] The measurement results are shown in Table 3 below.

[0106] Strain name Inhibitory activity Inducing activity DPPHHP Growth HP ureaseTNF-αIL-8IL-10Claudin-11Lactobacillus planantrumP131+--+++++2Lactobacillus planantrumP132+--+++++3Lactobacillus gasseriP133++--+++++4Lactococcus lactisP134+++++++++5Lactococcus lactisP135++++++++++++++++++++6Lactococcus lactisP136+++++++++7Lactobacillus caseiP137++--+++8Lactobacillus reuteriP138+-+++++9Lactobacillus acidophilusP139+--+++-+10Lactobacillus fermentumP140+--+++++11Bifidobacterium longumP141+--+++++12Bifidobacterium longumP142++++++++++++++++++++13Bifidobacterium longumP143+--++++14Bifidobacterium longumP144++-+++-+15Bifidobacterium adolescentisP145+++++++++16Bifidobacterium adolescentisP146+++++++++17Bifidobacterium catenulatumP147+--++++++18Bifidobacterium pseudocatenulatumP148++--++++++19Bifidobacterium animalisP149++-++++20Bifidobacterium animalisP150++-++++-+* Very high (+++; >50%); High (++; >30-50%); Low (+; >10-30%); Very low (-; <10%)

[0107] Experimental Example 3. Confirmation of Helicobacter pylori inhibitory activity of Lactococcus lactis P135, Bifidobacterium longum P142, or a mixture thereof

[0108] In 10 mL of BHI medium, mixtures (1×10⁻⁶) of Colony Forming Unit (CFU) ratios of Lactococcus lactis P135 and Bifidobacterium longum P142 in BHI medium were prepared beforehand with CFU ratios of 1:0, 0:1, 4:1, 1:1, and 1:4, respectively. 8 CFU / mL) was cultured and Helicobacter pylori (2x10) was cultured in each. 8 CFU / mL) was inoculated and cultured for 24 hours at 37°C under anaerobic conditions. 0.2 mL of the culture medium was inoculated into Columbia agar (5% sheep blood and antibiotics [Vancomycin 10.0 mg / L, Amphotericin B 5.0 mg / L] added) and cultured for 3 days under microaerotic conditions, after which the number of grown Helicobacter pylori colonies was measured and the inhibition rate of lactic acid bacteria was measured.

[0109] In addition, the precipitate was collected and suspended in 1 mL of PBS, and urease enzyme activity was measured. Urease activity was measured using the MAK120 urease activity assay kit (Sigma Aldrich).

[0110] The results of measuring the number of Helicobacter pylori colonies and Urease activity are shown in Table 4 below.

[0111] Number of HP colonies treated (1×10 8 CFU / mL) HP urease activity (unit / mL) Vehicle (physiological saline) 152149P135 (1×10 8 CFU / mL)0.258P142(1×10 8 CFU / mL)1265P135 / P142 [4:1] (1×10 8 CFU / mL)0.255P135 / P142 [1:1] (1×10 8 CFU / mL)0.557P135 / P142 [1:4] (1×10 8 CFU / mL)1.159

[0112] Experimental Example 4. Effect of improving depressive disorder, cognitive impairment, or inflammatory disease caused by Helicobacter pylori infection

[0113] 4-1. Production of Helicobacter pylori (HP) infected animals and administration of probiotics

[0114] Gastritis animals were administered orally to SPF C57BL / 6 mice (male, 19-12g, 6 weeks-old) at a rate of 1×10 per mouse. 9 It was produced by infecting the stomach with CFU (colony-forming unit) orally once a day for 7 days. For comparison, the control group (NC) used physiological saline instead of Helicobacter pylori.

[0115] For the Helicobacter pylori administration group, starting from the day after the final Helicobacter pylori administration, lactic acid bacteria P135, P142, or a mixture thereof (a 4:1 mixture of P135 and P142) were administered as the test substance at a rate of 1 x 10 9 Oral administration of omeprazole at 20 mg / kg for 7 days was performed at CFU / mouse / day, and each experimental group was designated as P135, P142, PLB, and Op below.

[0116] 4-2. Behavioral Experiment

[0117] Starting the day after the final administration of probiotics, one behavioral experiment was performed each day in the order of the Open field test, Y-maze test, and tail suspension test.

[0118] 4-2-1. Open field test (OFT)

[0119] To evaluate the basic motility and movement of mice, mice were placed in a white acrylic box measuring 50 x 50 x 50 cm, and their behavior was measured for 10 minutes using a video tracking system (smart v.2.5.21). The open field was divided into four sections, and the middle section was designated as the central zone. Subsequently, walking activity was measured using the voluntary movement of each group.

[0120] 4-2-2. Tail Suspension Test (TST)

[0121] A fixation device was attached to the end of a desk 50 cm high at about 1 cm from the tip of the mouse's tail, and the mouse was suspended. The immobility time (IT) of the experimental animals was measured for a total of 6 minutes.

[0122] 4-2-3. Y-maze test

[0123] The apparatus used for the Y-maze test consists of three branches, each measuring 42 cm in length, 3 cm in width, and 12 cm in height. The angle formed by each branch is 120°, and the structure is made of black polyvinyl resin. A mouse was carefully placed on one of the three branches of the Y-maze, designated as A, B, and C, and allowed to move freely for 8 minutes while the branch the mouse entered was recorded. The branch was recorded only when the mouse entered completely, including its tail, and it was also recorded if the mouse re-entered a branch it had previously visited. One point (actual alternation) was awarded for entering three different branches in succession. The maximum alternation was calculated by subtracting 2 from the total number of entries into the three branches.

[0124] Spontaneous alternation (SA)% = 100 x [actual alternation / maximum alternation]

[0125] The results of the above behavioral experiments are shown in Table 5 below. Mice infected with HP exhibited depression-like behaviors, with increased IT in the TST and decreased TC, DC, and TD in the OFT, and cognitive impairment-like behaviors, with decreased SA in the YMT. However, administration of P135, P142, or a mixture thereof (PLB: a 4:1 mixture of P135 and P142) generally improved depression-like and cognitive impairment-like behaviors more effectively than omeprazole (Op), which is used as a treatment for gastritis. Among these, the mixture of P135 and P142 showed the most superior effect.

[0126] NCHPP135P142PLBOpIT in TST (s)108174129115112117TC in OFT (s)823164637252DC in OFT (m)7.33.55.25.16.55.1TD in OFT (m)39.123.733.429.534.232.7SA in YMT (%)72.143.460.259.163.159IT, immobility time; TC, time spent in the central area; DC, distance spent in the central area; TD, total distance travelled; SA, spontaneous alternation

[0127] 4-3. Analysis of Biomarkers in Brain and Intestinal Tissues

[0128] Mice that had completed behavioral experiments were euthanized, and brain tissue (hippocampus) and intestinal tissue (stomach and gut / colon) were isolated. These tissues were homogenized with RIPA lysis buffer, and biomarkers in the supernatant after centrifugation were measured using an ELISA kit, immunoblotting, quantitative real-time polymerase chain reaction, and tissue staining.

[0129] 4-3-1. ELISA

[0130] Brain and intestines (gastrointestinal and large intestine) collected from mice were homogenized and lysed in RIPA buffer (150 mM sodium chloride, 1% sodium deoxycholate, 1% Triton X-100, 0.1% SDS, 50 mM Tris-HCl, 2 mM EDTA, pH 7.5) containing a phosphokinase inhibitor cocktail (Roche), and then centrifuged at 4°C and 10,000 g for 20 minutes.

[0131] After collecting the supernatant, the expression of TNF-α, IL-6, and IL-8 was measured using an ELISA kit (R&D system, Minneapolis, MN). The results are shown in Table 8.

[0132] 4-3-2. Immunoblotting

[0133] Brain tissues, hippocampal, gastric, and colon tissues isolated from mice were each homogenized in Radio Immunoprecipitation Assay Lysis (RIPA) lysis buffer (Pierce, Rockford, IL, USA) and centrifuged at 4°C and 10,000 g for 10 minutes. Proteins (p-p65, p65) in the supernatant (20 μg protein) were measured by immunoblotting. The results are shown in Table 8.

[0134] 4-3-3. Quantitative real-time-polymerase chain reaction (qPCR)

[0135] To analyze ZO-1, occluding, integrin α5, integrin β1, and β-actin, mRNA (2 μg) was first isolated from the stomach of mice using the RNeasy Mini kit (Qiagen), and cDNA was synthesized using the cDNA synthesis kit (TaKaRa). The qPCR analysis reaction conditions were as follows: initial denaturation at 95°C for 30 seconds, denaturation at 95°C for 15 seconds, binding at 60°C for 30 seconds, extension at 72°C for 30 seconds, and PCR was performed under 40 cycles; gene expression levels were calculated based on β-actin. The primer sequences for this are shown in Table 6 below.

[0136] Gene Direction Nucleotide Sequence (5' - 3') Sequence Number Integrin β1 Forward (F) 5'-GAG GTT CAT TTG AAA TTA GGC-3'3 Reverse (R) 5'-GGC TCT GCA CTG AAC ACA TTC-3'4 Integrin α5 Forward (F) 5'-GTG TGA GGA ACT GGT CGC CTA T-3'5 Reverse (R) 5'-CCG TTC TCT GGT CCA ACC GAT A-3'6 Occludin Forward (F) 5'-ACT CCT CCA ATG GAC AAG TG-3'7 Reverse (R) 5'-CCC CAC CTG TCG TGT AGT CT-3'8 ZO-1 Forward (F) 5'-CCA CCT CTG TCC AGC TCT TC-3'9 Reverse (R) 5'-CAC CGG AGT GAT GGT TTT CT-3'10β-actin Forward (F) 5'-TGTCCACCTTCCAGCAGATGT-3'11 Reverse (R) 5'- AGCTCAGTAACAGTCCGCCTAGA-3'12

[0137] In addition, to confirm Helicobacter pylori infection, DNA was isolated from gastric tissue using the QIAamp Fast DNA stool mini kit (Qiagen), and the VacA and CagA genes of Helicobacter pylori were analyzed by qPCR. The qPCR analysis reaction conditions were as follows: initial denaturation at 95°C for 30 seconds, denaturation at 95°C for 15 seconds, binding at 60°C for 30 seconds, extension at 72°C for 30 seconds, and 40 cycles; gene expression levels were calculated based on 16S rRNA. The primer sequences are shown in Table 7 below. The qPCR results are shown in Table 8.

[0138] Gene Direction Base Sequence (5' - 3') Sequence Number VacA Forward (F) 5'-ATG GAA ATA CAA CAA ACA CAC-3'13 Reverse (R) 5'- CTG CTT GAA TGC GCC AAA C-3'14 CagA Forward (F) 5'-CCA TGA ATT TTT GAT CCG TTC GG-3'15 Reverse (R) 5'-GAT AAC AGG CAA GCT TTT GAG AGG GA-3'16 16S rRNA Forward (F) 5'-CCA GCA GCC GCG GTA ATA CG-3'17 Reverse (R) 5'-TAC CAG GGT ATC TAC TCC-3'18

[0139] 4-3-4. Immunofluorescence staining

[0140] Mice were systemically perfused with 4% paraformaldehyde, brain tissue (hippocampus) and stomach were isolated and sedated, and incubated overnight with primary antibodies of the biomarkers to be measured (Helicobacter pylori [HP, 1:500, Invitrogen], NF-kB [1:500, Invitrogen], Iba1 [1:200, Cell Signaling], or / and glial fibrillary acidic protein (GFAP, Cell Signaling, 1:200)). Subsequently, color development was performed using secondary antibodies conjugated with Alexa Fluor 594 (1:200, Invitrogen) or Alexa Fluor 488, and the results were observed under a confocal microscope. The results are presented as fluorescence intensity values ​​in Table 8.

[0141] NCHPP135P142PLBOpInflammation score03.42.12.21.21.2HP Level (au)2.110.47.18.26.57.0Urease activity(unit / mL)-398153154143145VacA (fold change)12.61.21.31.11.2CagA17.32.23.11.92.3ZO-1 (fold change)10.150.820.810.840.83Occludin (fold change)10.340.840.820.990.91Integrin α5(fold change)18.74.14.33.73.9Integrin β1(fold change)16.11.61.91.21.6NF-kB+ cells (Immunofluorescence) 2.8 13.3 4.9 7.1 4.3 4.7 p-p6 5 / p6 5 0.7 2.7 0.8 1.0 0.7 0.9 IL-8 (pg / mg) 0.2 3 0.6 9 0.2 7 0.2 7 0.2 6 0.26 IL-6 (pg / mg) 0.1 8 0.4 7 0.2 6 0.2 7 0.2 3 0.24 TNF-α (pg / mg) 0.8 7 2.2 3 0.1 1 0.1 3 0.1 0 0.13 Inflammation score 0: No ulcer and no inflammation; 1: No ulceration and mild hyperemia; 2: Hyperemia without ulceration; 3: Severe hyperemia with ulceration; 4: Ulcer in only one site and inflammation (ulceration and inflammation at one site only); 5: ulceration and inflammation at two or more sites (two or more sites of ulceration and inflammation)

[0142] As shown in Table 8 above, in the gastrointestinal tract of mice infected with HP, compared to that of mice in the control group, macroscopic inflammation indices, HP+ and NF-kB+ cells observed by immunofluorescence staining, the expression of TNF-α, IL-6, and IL-8 (ELISA measurement), NF-kB activation (p-p65 / p65) (immunoblotting measurement), and HP-derived proteins VacA and CagA increased, while the expression of neutrophil-derived Integrin α5 and Integrin β1 increased (qPCR measurement). On the other hand, the expression levels of ZO-1 and occludin decreased (qPCR measurement).

[0143] When P135, P142, or a mixture thereof (PLB) was administered, inflammation indices, HP+ and NF-kB+ cells, the expression of TNF-α, IL-6, IL-8, VacA, CagA, Integrin α5, Integrin β1, and NF-kB activation (p-p65 / p65) generally decreased significantly compared to omeprazole (Op), which is used as a treatment for gastritis; however, the expression of ZO-1 and occludin increased. Among these, the mixture of P135 and P142 showed the most superior effect in improving gastritis.

[0144] 4-4. Analysis of Biomarkers in Blood

[0145] The experimental method is the same as described in 4-3 above. In the blood of mice infected with Helicobacter pylori, the concentrations of TNF-α and IL-6, measured by an ELISA kit, increased compared to those of mice in the control group. When P135, P142, or a mixture thereof were administered, the concentrations of TNF-α and IL-6 decreased generally more effectively than with omeprazole, which is used as a treatment for gastritis. Among these, the mixture of P135 and P142 showed the most superior effect in improving gastritis. The results are shown in Table 9.

[0146] NCHPP135P142PLBOpIL-6 (ng / mL)0.080.280.110.120.110.13TNF-α (ng / mL)0.881.690.921.020.890.92

[0147] 4-5. Biomarker Analysis at the Hippocampus

[0148] The experimental method is the same as in 4-3 above. In the hippocampus of mice infected with Helicobacter pylori, TNF-α expression increased compared to that of mice in the control group (ELISA measurement), and NF-κB activation (p-p65 / p65) also increased (immunoblotting measurement), but the amounts of BDNF and serotonin decreased (qPCR measurement).

[0149] On the other hand, when P135, P142, or a mixture thereof was administered, TNF-α expression and NF-κB activation (p-p65 / p65) decreased more significantly than with omeprazole, which is used as a treatment for gastritis, and the levels of BDNF and serotonin increased. Among these, the mixture of P135 and P142 showed the most superior effect in improving gastritis. The results are shown in Table 10.

[0150] NCHPP135P142PLBOpTNF-α (pg / mg)0.170.610.270.360.250.37BDNF (ng / mg)0.320.110.200.190.250.18Serotonin(pg / mg)8.96.58.48.38.97.8p-p65 / p650.41.40.60.70.50.9

[0151] 4-6. Analysis of Colon Biomarkers

[0152] The experimental method is the same as in 4-3 above. In the colon of mice infected with Helicobacter pylori, the intestinal length (cm) decreased compared to that of mice in the control group, and the expression of TNF-α and IL-6 (ELISA measurement) and NF-kB activation (p-p65 / p65) increased (immunoblotting measurement).

[0153] On the other hand, when P135, P142, or a mixture thereof was administered, intestinal length was generally improved superiorly compared to omeprazole, which is used as a treatment for gastritis, and the expression of TNF-α and IL-6 and NF-κB activation (p-p65 / p65) were reduced. Among these, the mixture of P135 and P142 showed the most superior effect in improving gastritis. The results are shown in Table 11.

[0154] NCHPP135P142PLBOp Length (cm) 6.15.65.85.85.95.7IL-60.210.770.320.350.290.45TNF-α0.320.530.330.360.320.36p-p65 / p650.52.20.70.90.71.0

[0155] Experimental Example 5. Effect of aspirin on improving depressive disorder, cognitive impairment, or inflammatory disease

[0156] 5-1. Preparation of aspirin-induced gastritis animals and administration of probiotics

[0157] Gastritis animals were prepared by orally administering aspirin (300 mg / kg, dissolved in physiological saline) once daily for 7 days to SPF C57BL / 6 mice (male, 19-12 g, 6 weeks-old) per mouse. For comparison, physiological saline was used instead of aspirin for the control group (NC).

[0158] For the aspirin administration group, starting from the day after the last aspirin administration, lactic acid bacteria P135, P142, or a mixture thereof (a 4:1 mixture of P135 and P142) were administered as the test substance at a rate of 1 x 10⁻¹⁰. 9 Oral administration of omeprazole at 20 mg / kg or saline at CFU / mouse / day or saline for 7 days was performed, and each experimental group was denoted as P135, P142, PLB, Op, Vh below.

[0159] 5-2. Behavioral Experiment

[0160] Starting from the day after the final administration of lactic acid bacteria, one behavioral test was performed daily in the order of Open field test, Y-maze test, and tail suspension test. The experimental method was the same as in 4-2 above, and the results are shown in Table 12.

[0161] Mice treated with aspirin showed depression-like behaviors compared to normal control mice, with increased IT in the TST and decreased TC, DC, and TD in the OFT, and decreased SA in the YMT, showing cognitive impairment-like behaviors.

[0162] However, the administration of P135, P142, or a mixture thereof generally improved depression-like behaviors and cognitive impairment-like behaviors superiorly compared to omeprazole, which is used as a treatment for gastritis. Among these, the mixture of P135 and P142 showed the most superior effect.

[0163] NCVhP135P142PLBOpIT in TST (s)98182115119105119TC in OFT (s)621848425139DC in OFT (m)7.12.24.84.56.14.2TD in OFT (m)37.22.43.4.13.4.03.4.83.4.0SA in YMT (%)73.64.395.925.816.255.7.0IT, immobility time; TC, time spent in the central area; DC, distance spent in the central area; TD, total distance travelled; SA, spontaneous alternation

[0164] 5-3. Analysis of Biomarkers in the Gastrointestinal Stomach

[0165] The experimental method is the same as in 4-3 above. In the gastrointestinal tract of mice treated with aspirin, compared to mice in the control group, macroscopic inflammation indices, NF-κB+ cells observed by immunofluorescence staining, the expression of TNF-α, IL-6, and IL-8 (ELISA measurement), and NF-κB activation (p-p65 / p65) (immunoblotting measurement) were increased. On the other hand, the expression levels of ZO-1 and occludin decreased (qPCR measurement).

[0166] When P135, P142, or a mixture thereof (PLB) was administered, inflammation indices, NF-kB+ cells, the expression of TNF-α, IL-6, IL-8, and NF-κB activation (p-p65 / p65) generally decreased significantly compared to omeprazole (Op), which is used as a treatment for gastritis; however, the expression of ZO-1 and occludin increased. Among these, the mixture of P135 and P142 showed the most superior effect in improving gastritis. The results are shown in Table 13.

[0167] NCVhP135P142PLBOpInflammation score03.71.72.61.33.1ZO-1(fold change)10.220.920.911.100.72Occludin (fold change)10.360.920.931.080.86NF-κB+ cells(Immunofluorescence)2.312.87.89.53.97.9p-p65 / p65(immunoblotting)0.72.70.81.00.70.9IL-8 (pg / mg)0.691.310.720.810.710.82IL-6 (pg / mg)0.190.480.260.300.250.29TNF-α (pg / mg)0.983.011.321.451.291.48

[0168] 5-4. Analysis of Biomarkers in Blood

[0169] The experimental method is the same as described in 4-3 above. In the blood of mice treated with aspirin, the concentrations of TNF-α and IL-6, measured by an ELISA kit, increased compared to those of mice in the control group. When P135, P142, or a mixture thereof was administered, the concentrations of TNF-α and IL-6 decreased more significantly than with omeprazole, which is used as a treatment for gastritis. Among these, the mixture of P135 and P142 showed the most superior effect in improving gastritis. The results are shown in Table 14.

[0170] NCVhP135P142PLBOpIL-6 (ng / mL)0.090.270.140.150.120.18TNF-α (ng / mL)0.911.590.981.020.911.32

[0171] 5-5. Biomarker Analysis at the Hippo Campus

[0172] The experimental method is the same as in 4-3 above. In the hippocampus of mice treated with aspirin, TNF-α expression increased compared to that of mice in the control group (ELISA measurement), and NF-κB activation (p-p65 / p65) also increased (immunoblotting measurement), but the amounts of BDNF and serotonin decreased (qPCR measurement).

[0173] On the other hand, when P135, P142, or a mixture thereof was administered, TNF-α expression and NF-κB activation (p-p65 / p65) were generally reduced more significantly compared to omeprazole, which is used as a treatment for gastritis, and the levels of BDNF and serotonin increased. Among these, the mixture of P135 and P142 showed the most superior effect in improving gastritis. The results are shown in Table 15.

[0174] NCVhP135P142PLBOpTNF-α (pg / mg)0.19 0.58 0.24 0.31 0.23 0.34BDNF (ng / mg)0.28 0.12 0.22 0.17 0.24 0.19Serotonin (pg / mg)4.6 3.6 4.3 4.2 4.3 3.9p-p65 / p65 0.5 1.3 0.7 0.8 0.6 0.9

[0175] 5-6. Analysis of Colon Biomarkers

[0176] The experimental method is the same as in 4-3 above. In the large intestines of mice treated with aspirin, the length of the intestine (cm) decreased compared to that of mice in the control group, and the expression of TNF-α and IL-6 (ELISA measurement) and NF-κB activation (p-p65 / p65) increased (immunoblotting measurement).

[0177] On the other hand, when P135, P142, or a mixture thereof was administered, intestinal length was generally improved superiorly compared to omeprazole, which is used as a treatment for gastritis, and the expression of TNF-α and IL-6 and NF-κB activation (p-p65 / p65) were reduced. Among these, the mixture of P135 and P142 showed the most superior effect in improving gastritis. The results are shown in Table 16.

[0178] NCVhP135P142PLBOp Length (cm) 6.25.75.95.96.05.8IL-60.090.620.190.240.180.27TNF-α0.180.610.340.390.310.52p-p65 / p650.42.30.70.70.61.0

[0179] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will understand that other specific forms can be easily modified without altering the technical spirit or essential features of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive. For example, each component described as a single unit may be implemented in a distributed manner, and components described as distributed may likewise be implemented in a combined form.

[0180] The scope of the present invention is defined by the claims set forth below, and all modifications or variations derived from the meaning and scope of the claims and equivalent concepts thereof should be interpreted as being included within the scope of the present invention.

[0181] [Consignment Number]

[0182] Depository Name: Korean Culture Collection Center (KCCM)

[0183] Trustee Number: KCCM13519P

[0184] Date of Trust: 20240919

[0185] Depository Name: Korean Culture Collection Center (KCCM)

[0186] Trustee Number: KCCM13520P

[0187] Date of Trust: 20240919

[0188]

[0189]

[0190]

Claims

1. Lactococcus lactis P135 (Lactococcus lactisP135) KCCM13519P.

2. Bifidobacterium longum P142 (Bifidobacterium longumP142) KCCM13520P.

3. A pharmaceutical composition for the prevention or treatment of inflammatory diseases, depressive disorders, or cognitive impairment comprising Lactococcus lactis P135 KCCM13519P, Bifidobacterium longum P142 KCCM13520P, or a mixture thereof.

4. In Paragraph 3, A pharmaceutical composition wherein the above-mentioned inflammatory disease is one or more selected from the group comprising gastritis, arthritis, gout, hepatitis, obesity, keratitis, enteritis, nephritis, colitis, diabetes, tuberculosis, bronchitis, pleuritis, peritonitis, spondylitis, pancreatitis, inflammatory pain, urethritis, cystitis, vaginitis, arteriosclerosis, sepsis, and periodontitis.

5. In Paragraph 4, A pharmaceutical composition wherein the above-mentioned inflammatory disease is one or more selected from the group consisting of gastritis, hepatitis, enteritis, nephritis, colitis, and pancreatitis.

6. In Paragraph 3, The above depressive disorder is one or more selected from the group including Major Depressive Disorder, Persistent Depressive Disorder, Dysthymia, Disruptive Mood Dysregulation Disorder, Premenstrual Dysphoric Disorder, Substance / Medication-Induced Depressive Disorder, Depressive Disorder due to Another Medical Condition, Other Specified Depressive Disorder, and Unspecified Depressive Disorder. A pharmaceutical composition wherein the above-mentioned cognitive impairment is one or more selected from the group comprising anxiety, migraine, stress, Alzheimer's disease, Huntington's disease, vascular dementia, Pick's disease, Parkinson's disease, Creutzfeldt-Jakob disease, and dementia.

7. In Paragraph 3, A pharmaceutical composition wherein the above mixture comprises Lactococcus lactis P135 KCCM13519P and Bifidobacterium longum P142 KCCM13520P mixed in a Colony Forming Unit (CFU) ratio of 1:1 to 20:1 or 1:1 to 1:

20.

8. In any one of paragraphs 3 through 7, A pharmaceutical composition in which the above-mentioned inflammatory disease, depressive disorder, or cognitive impairment is caused by Helicobacter pylori infection.

9. A food composition for the prevention or improvement of inflammatory diseases, depressive disorders, or cognitive impairment comprising Lactococcus lactis P135 KCCM13519P, Bifidobacterium longum P142 KCCM13520P, or a mixture thereof.

10. In Paragraph 9, A food composition wherein the above-mentioned inflammatory disease is one or more selected from the group comprising gastritis, arthritis, gout, hepatitis, obesity, keratitis, enteritis, nephritis, colitis, diabetes, tuberculosis, bronchitis, pleuritis, peritonitis, spondylitis, pancreatitis, inflammatory pain, urethritis, cystitis, vaginitis, arteriosclerosis, sepsis, and periodontitis.

11. In Paragraph 10, A food composition in which the above-mentioned inflammatory disease is one or more selected from the group consisting of gastritis, hepatitis, enteritis, nephritis, colitis, and pancreatitis.

12. In Paragraph 9, The above depressive disorder is one or more selected from the group including Major Depressive Disorder, Persistent Depressive Disorder, Dysthymia, Disruptive Mood Dysregulation Disorder, Premenstrual Dysphoric Disorder, Substance / Medication-Induced Depressive Disorder, Depressive Disorder due to Another Medical Condition, Other Specified Depressive Disorder, and Unspecified Depressive Disorder. A food composition wherein the above-mentioned cognitive impairment is one or more selected from the group comprising anxiety, migraine, stress, Alzheimer's disease, Huntington's disease, vascular dementia, Pick's disease, Parkinson's disease, Creutzfeldt-Jakob disease, and dementia.

13. In Paragraph 9, The above mixture is a food composition comprising Lactococcus lactis P135 KCCM13519P and Bifidobacterium longum P142 KCCM13520P mixed in a Colony Forming Unit (CFU) ratio of 1:1 to 20:1 or 1:1 to 1:

20.

14. In any one of paragraphs 9 through 13, A pharmaceutical composition in which the above-mentioned inflammatory disease, depressive disorder, or cognitive impairment is caused by Helicobacter pylori infection.

15. A method for the prevention or treatment of inflammatory diseases, depressive disorders, or cognitive impairments comprising the step of administering Lactococcus lactis P135 KCCM13519P, Bifidobacterium longum P142 KCCM13520P, or a mixture thereof to a subject.

16. In Paragraph 15, A method for prevention or treatment in which the above mixture is a mixture of Lactococcus lactis P135 KCCM13519P and Bifidobacterium longum P142 KCCM13520P in a Colony Forming Unit (CFU) ratio of 1:1 to 20:1 or 1:1 to 1:

20.

17. Use of Lactococcus lactis P135 KCCM13519P, Bifidobacterium longum P142 KCCM13520P, or a mixture thereof in the manufacture of a drug for the treatment of inflammatory diseases, depressive disorders, or cognitive impairments.

18. In Paragraph 17, The above mixture is a mixture of Lactococcus lactis P135 KCCM13519P and Bifidobacterium longum P142 KCCM13520P in a Colony Forming Unit (CFU) ratio of 1:1 to 20:1 or 1:1 to 1:

20.

19. Lactococcus lactis P135 KCCM13519P, Bifidobacterium longum P142 KCCM13520P or a mixture thereof; and a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

20. In Paragraph 19, A pharmaceutical composition wherein the above mixture comprises Lactococcus lactis P135 KCCM13519P and Bifidobacterium longum P142 KCCM13520P mixed in a Colony Forming Unit (CFU) ratio of 1:1 to 20:1 or 1:1 to 1:20.

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