Bifidobacterium longum subsp. suillum strain and immunity enhancement uses thereof

Abstract

The present application relates to a Bifidobacterium longum subsp. suillum strain and immunity enhancement uses thereof. The Bifidobacterium longum subsp. suillum KBL2967 strain of the present application increases the expression of IL-6, TNF-α, and IL-1β, which are active marker cytokines of macrophages, thereby enhancing immunity, does not produce bioamines, and does not retain antibiotic resistance, and thus can be widely used as safe probiotics for enhancing immunity in the development of food additives, health functional foods, and therapeutic agents for immune diseases.

Description

Bifidobacterium longum subsp. suilum strain and its use for enhancing immunity

[0001] Cross-citation with related application(s)

[0002] This application claims the benefit of priority from Republic of Korea Patent Application No. 10-2024-0012704, dated January 26, 2024, the entire contents of which are incorporated herein by reference.

[0003] Numerous papers and patents are referenced and cited throughout this application. The disclosures of these cited papers and patents are incorporated into this application in their entirety by reference, providing a clearer understanding of the state of the art and the content of this application.

[0004] The present application relates to a Bifidobacterium longum subsp. suilum strain and its use for enhancing immunity.

[0005]

[0006] As the correlation between the microbiome and health has been widely revealed through numerous studies, the probiotic product market has also grown, and a wide range of lactic acid bacteria with diverse and effective functions are being developed and distributed. Bifidobacterium strains, first isolated from the feces of breastfed infants, have long been recognized for their beneficial effects on the human body and are among the most sought-after strains as probiotic components. Bifidobacterium plays a key role in the large intestine, and among them, Bifidobacterium longum is known to exhibit antimicrobial activity, including inhibition of Escherichia coli growth, as well as effects such as increased intestinal motility and enhanced bowel movements. While the functions of various species and subspecies of Bifidobacterium have been elucidated, research on the function of Bifidobacterium longum subsp. suillum remains insufficient.

[0007]

[0008] [Prior Art Literature]

[0009] [Patent Document]

[0010] (Patent Document 1) Republic of Korea Patent No. 10-0142615 (registered on April 2, 1998)

[0011]

[0012] Against this backdrop, the present inventors isolated and identified the Bifidobacterium longumsubsp.suillum KBL2967 strain and determined that the strain has immune-enhancing activity, thereby completing the present invention.

[0013] Accordingly, the purpose of the present application is to provide a Bifidobacterium longumsubsp.suillum KBL2967 strain and its use for enhancing immunity.

[0014]

[0015] According to one aspect of the present application, the present application provides a Bifidobacterium longumsubsp.suillum KBL2967 strain having the accession number KCTC 15739BP.

[0016] According to another aspect of the present application, the present application provides an immune-enhancing composition comprising at least one selected from the group consisting of Bifidobacterium longumsubsp.suillum KBL2967 strain having accession number KCTC 15739BP, a culture of the strain, a lysate of the strain, an extract of the strain, and a combination thereof.

[0017] According to another aspect of the present application, the present application provides a composition for preventing, treating or improving an immune disease, comprising at least one selected from the group consisting of Bifidobacterium longumsubsp.suillum KBL2967 strain having accession number KCTC 15739BP, a culture of the strain, a lysate of the strain, an extract of the strain, and a combination thereof.

[0018] According to another aspect of the present application, the present application provides a food comprising at least one selected from the group consisting of Bifidobacterium longumsubsp.suillum KBL2967 strain having accession number KCTC 15739BP, a culture of the strain, a lysate of the strain, an extract of the strain, and a combination thereof.

[0019] According to another aspect of the present application, the present application provides at least one immune-enhancing use selected from the group consisting of Bifidobacterium longumsubsp.suillum KBL2967 strain having accession number KCTC 15739BP, a culture of the strain, a lysate of the strain, an extract of the strain, and a combination thereof.

[0020] According to another aspect of the present application, the present application provides a use for producing an immune-enhancing composition and / or an immune-enhancing food, wherein the composition is selected from the group consisting of Bifidobacterium longumsubsp.suillum KBL2967 strain having the accession number KCTC 15739BP, a culture of the strain, a lysate of the strain, an extract of the strain, and a combination thereof.

[0021] According to another aspect of the present application, the present application provides a use for preventing, treating or improving one or more immune diseases selected from the group consisting of Bifidobacterium longumsubsp.suillum KBL2967 strain having accession number KCTC 15739BP, a culture of the strain, a lysate of the strain, an extract of the strain, and a combination thereof.

[0022] According to another aspect of the present application, the present application provides a use for manufacturing a pharmaceutical composition for preventing or treating an immune disease and / or a food for preventing or improving an immune disease, wherein the pharmaceutical composition is selected from the group consisting of Bifidobacterium longumsubsp.suillum KBL2967 strain having accession number KCTC 15739BP, a culture of the strain, a lysate of the strain, an extract of the strain, and a combination thereof.

[0023]

[0024] Hereinafter, the present application will be described in more detail.

[0025] According to one aspect of the present application, the present application provides a Bifidobacterium longumsubsp.suillum KBL2967 strain having the accession number KCTC 15739BP.

[0026] Considering that approximately 80% of all immune-related cells in the human body are located around the intestines, the present inventors have made extensive research efforts to utilize Bifidobacterium, which has a high intestinal colonization ability, to enhance immunity. Accordingly, we newly isolated Bifidobacterium longumsubsp.suillum KBL2967 strain from a healthy adult, and analyzed the 16S rDNA base sequence and phylogenetic tree to investigate the phylogenetic characteristics and identify the strain. In addition, we analyzed the biochemical characteristics (saccharolytic properties and antibiotic resistance) of the identified strain. In addition, the present inventors demonstrated through experiments that when the strain was treated to macrophages, the production of cytokines such as IL-6, IL-1β, and TNF-α was promoted by the activation of macrophages, thereby enhancing immunity. Accordingly, the Bifidobacterium longumsubsp.suillum KBL2967 strain according to an example of the present application can be widely used as a safe probiotic for enhancing immunity, in the development of food additives, health functional foods, and immune disease treatments.

[0027] In this specification, the term “probiotics” is recommended by the Food and Agriculture Organization of the United Nations (FAO) and the World Health Organization (WHO), and refers to beneficial bacteria that have a beneficial effect on the health of the host when consumed in adequate amounts.

[0028] The strain Bifidobacterium longumsubsp.suillum KBL2967 having the accession number KCTC 15739BP of this application was deposited with the Korean Collection for Type Culture of the Korea Research Institute of Bioscience and Biotechnology under the accession number KCTC 15739BP on December 13, 2023.

[0029] In one embodiment, the strain described above may have a 16S rRNA sequence of sequence number 1.

[0030] In one embodiment, the strain described above can use at least one selected from the group consisting of D-glucose, D-lactose, D-saccharose, D-maltose, D-xylose, L-arabinose, D-mannose, D-raffinose, and D-sorbitol as a carbon source, but is not limited thereto.

[0031] In one embodiment, the strain described above may have one or more of the following (1) to (11) sugar-decomposing properties, but is not limited thereto:

[0032] (1) D-Glucose degradation capacity;

[0033] (2) D-Lactose decomposition capacity;

[0034] (3) D-Saccharose decomposition capacity;

[0035] (4) D-Maltose decomposition capacity;

[0036] (5) D-Xylose resolution;

[0037] (6) L-Arabinose degradation capacity;

[0038] (7) Non-degradability of glycerol;

[0039] (8) Non-degradability of D-Cellobiose;

[0040] (9) D-Melezitose non-degradable;

[0041] (10) D-Rhamnose non-degradability; and

[0042] (11) Non-degradability of D-Trehalose.

[0043] In one embodiment, the strain described above may be susceptible to antibiotics. Accordingly, the strain described above may not exhibit resistance to antibiotics. The resistance may be extrinsic resistance, and extrinsic resistance refers to resistance that can be induced when a resistance gene for antibiotics is introduced into another external bacterium through a mobile means such as a plasmid or transposon. Therefore, the strain of the present application that does not exhibit extrinsic resistance to antibiotics does not horizontally transfer the resistance gene to harmful bacteria existing in the intestine, so there is no concern that harmful bacteria in the intestine will acquire extrinsic resistance and cause resistance problems to antibiotics. The antibiotics to which the strain of the present application described above does not exhibit resistance are not particularly limited, as long as they are known in the technical field to which the present invention pertains, and may be, for example, penicillin antibiotics, tetracycline antibiotics, macrolide antibiotics, sulfonamide antibiotics, amphenicol antibiotics, aminoglycoside antibiotics, etc. In this case, the antibiotic may be one or more antibiotics selected from the group consisting of ampicillin, vancomycin, gentamycin, streptomycin, erythromycin, clindamycin, tetracycline, and chloramphenicol, but is not limited thereto.

[0044] In the present invention, which will be described later, it was confirmed that the Bifidobacterium longumsubsp.suillum KBL2967 strain having the accession number KCTC 15739BP according to an example of the present application did not show resistance to the antibiotic, and thus could be used as a safe probiotic.

[0045]

[0046] In one embodiment, the strain described above may not produce biogenic amines.

[0047] As used herein, the term “biogenic amine” refers to a substance containing one or more amine groups produced by a living organism. Biogenic amines are low-molecular-weight organic bases that are synthesized through metabolism in microorganisms, plants, and animals. Biogenic amines are mainly produced by chemical reactions such as decarboxylation and transamination of free amino acids by microorganisms when protein-containing foods spoil or during the fermentation and maturation process. Examples of biogenic amines include histamine, tyramine, tryptamine, serotonin, and catecholamine neurotransmitters (norepinephrine, epinephrine, and dopamine). Lactic acid bacteria also decarboxylate histidine and tyrosine, among the various amino acids absorbed in their growth environment, to produce biogenic amines such as histamine and tyramine, respectively. These biogenic amines cause various allergic reactions such as itching, nausea, shortness of breath, fever, flushing, sweating, headache, burning mouth, diarrhea, cramps, erythema, increased blood pressure, and hives. Since existing probiotic products do not consider the inherent biogenic amine-producing ability of the strains that make up the products, there is a potential risk of inducing allergic reactions due to biogenic amine hypersensitivity when taken. Antihistamines are representative drugs that suppress allergic reactions, but since antihistamines inhibit the functional group of histamine, continuous use is required as long as residual histamine exists in the body. Therefore, the production of biogenic amines has a significant impact on the safety of probiotic products.

[0048] In the present invention, which will be described later, through genome analysis of the Bifidobacterium longumsubsp.suillum KBL2967 strain having the accession number KCTC 15739BP according to an example of the present application, it was confirmed that genes encoding histidine decarboxylase and tyrosine decarboxylase are not present in the genome of the strain described above, and therefore the strain described above does not produce histamine and tyrosine. Accordingly, the strain according to an example of the present application can be used for the development of safe probiotic products that do not cause allergic reactions.

[0049] In one embodiment, the strain described above may have immune enhancing activity.

[0050] In this specification, the term “immunity enhancement” means enhancing the activity of a series of in vivo immune responses to protect the body from antigens, which are foreign substances such as bacteria, viruses, cancer cells, blood and tissues of other humans or animals, etc.

[0051] In one embodiment, the above-described immune enhancement may be due to activation of macrophages.

[0052] In one embodiment, the aforementioned immune enhancement may be due to increased cytokine production by macrophages.

[0053] Activated macrophages produce cytokines such as tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β), and IL-6, which can regulate secondary immune responses such as proliferation of T and B cells, activation of macrophages for phagocytosis, and defense against microbial infections. TNF-α (tumor necrosis factor-alpha) is a cell signaling protein that mainly plays a role in inflammatory and immune responses. In the body's immune response, it acts alone or in combination with cytokines such as IL-1β to damage tumor blood vessels, resulting in tumor necrosis or enhancing resistance to microbial infection. IL-6 (interleukin 6) is a major response mediator produced in the initial immune response to infection and tissue damage, promoting acute inflammatory responses and activating B cells. In addition, IL-1β (interleukin 1β) promotes inflammation, activates vascular endothelial cells, and plays an important role in various cellular activities including cell proliferation, differentiation, and apoptosis.

[0054] In the present invention, which will be described later, it was confirmed that the expression levels of IL-6, TNF-α, and IL-1β in macrophages significantly increased when the KBL2967 strain according to an example of the present application was treated, thereby confirming that the strain described above has immune-enhancing activity.

[0055]

[0056] According to another aspect of the present application, the present application provides an immune-enhancing composition comprising at least one selected from the group consisting of Bifidobacterium longumsubsp.suillum KBL2967 strain having accession number KCTC 15739BP, a culture of the strain, a lysate of the strain, and an extract of the strain.

[0057] In one embodiment, the above-described immune enhancement may be due to activation of macrophages.

[0058] In one embodiment, the immune enhancement may be due to increased cytokine production by macrophages.

[0059] In one embodiment, the strain may be a live or heat-inactivated strain according to an example of the present application.

[0060] In this specification, the term “culture of a strain” means a product obtained by culturing a strain according to an example of the present application, and the culture may be a whole culture of the strain according to an example of the present application, a dilution thereof, a concentrate, a dried product, a lyophilized product, a lyophilized product, a fragment, and / or a fraction thereof, and the concentrate may be obtained by centrifuging or evaporating the culture, the dried product may be obtained by drying the culture using a dryer or the like, the lyophilized product may be obtained by lyophilizing the culture using a freeze dryer or the like, the fragment may be obtained by physically or ultrasonically treating the strain or culture, and the fraction may be obtained by subjecting the culture, the fragment, or the like to a method such as centrifugation, chromatography, or the like. The culture may be in a solid phase (solid, for example, a dried product), a liquid phase (liquid), or a fluid phase, but is not limited thereto. In one embodiment, the culture may refer to the entire medium including the cultured strain, its metabolites, and / or extra nutrients obtained by culturing the strain according to an example of the present application for a certain period of time. In one embodiment, the culture may be one in which the strain according to an example of the present application is removed or not removed. In one embodiment, the culture may refer to the remaining components excluding the strain (bacterial cells) in a culture obtained by culturing the strain according to an example of the present application in a medium. In one embodiment, the culture may be a culture (or culture) obtained by removing the strain (bacterial cells) from a culture solution obtained by culturing the strain according to an example of the present application in a medium. The culture solution (or culture) obtained by removing the strain may be a cell-free culture solution (or culture) or a culture solution containing dead cells, and may be, for example, a filtrate (centrifuged supernatant) obtained by removing the strain by filtration or centrifugation, and / or a culture solution (or dried product of the culture solution) containing dead cells.Specifically, the culture may exhibit an immune-enhancing activity equivalent to the activity exhibited by the strain according to an example of the present application.

[0061] In this specification, the term “strain lysate” may mean a product obtained by disrupting a strain according to an example of the present application by chemical or physical force. The lysate may be a lysate, a dilution thereof, a concentrate, a dried product, a lyophilized product, and / or a fraction thereof of the strain according to an example of the present application. The concentrate may be obtained by centrifuging or evaporating the culture, the dried product may be obtained by drying the culture using a dryer, etc., the lyophilized product may be obtained by freeze-drying the culture using a freeze dryer, etc., and the fraction may be obtained by subjecting the culture, lysate, etc. to a method such as centrifugation or chromatography. Specifically, the lysate may exhibit an immune-enhancing activity at a level equivalent to the activity exhibited by the strain according to an example of the present application.

[0062] In this specification, the term “extract” may refer to a product obtained by extracting a strain according to an example of the present application, a culture of the strain, a lysate of the strain, or a mixture thereof, regardless of the extraction method, extraction solvent, extracted component, or form of the extract, and is a broad concept that includes all materials that can be obtained by processing or handling by another method after extraction. For example, the extract may be an extract of the strain according to an example of the present application, an extract of a culture of the strain, or an extract of a lysate of the strain. The above extract may be an extract, a dilution thereof, a concentrate thereof, a dried product, a lyophilized product, a lyophilized product, a fragment thereof, and / or a fraction thereof of a strain according to an example of the present application. The concentrate may be obtained by centrifuging or evaporating the culture. The dried product may be obtained by drying the culture using a dryer or the like. The lyophilized product may be obtained by lyophilizing the culture using a freeze dryer or the like. The lyophilized product may be obtained by physically or ultrasonically treating the strain or culture. The fraction may be obtained by subjecting the culture, fragment thereof, or the like to a method such as centrifugation or chromatography. Specifically, the extract may exhibit an immune-enhancing activity at a level equivalent to the activity exhibited by the strain according to an example of the present application, the culture of the strain, or the lyophilized product of the strain.

[0063] In one embodiment, the immune-enhancing composition described above may be provided as a pharmaceutical composition. In this case, the pharmaceutical composition may include, as an active ingredient, the Bifidobacterium longumsubsp.suillum KBL2967 strain having the accession number KCTC 15739BP according to an example of the present application.

[0064] In the examples of the present invention described below, it was confirmed that the expression levels of IL-6, TNF-α, and IL-1β in macrophages significantly increased when treated with a strain according to an example of the present application, thereby confirming that the strain described above has immune-enhancing activity. Accordingly, the strain according to an example of the present application can be used as a pharmaceutical composition for preventing, improving, or treating immune diseases.

[0065] As used herein, the term “immune disease” refers to a disease caused by a decrease in immune function, and may include, but is not limited to, autoimmune diseases such as rheumatoid arthritis, type 1 diabetes, multiple sclerosis, and systemic lupus erythematosus, asthma, inflammatory bowel disease, chronic obstructive pulmonary disease, septic shock, pulmonary fibrosis, undifferentiated spondyloarthropathies, undifferentiated arthropathy, arthritis, inflammatory osteolysis, and inflammatory diseases such as chronic inflammation caused by chronic viral or bacterial infections, various cancers, and viral diseases such as the common cold.

[0066] In this specification, the term “prevention” means any act of suppressing, inhibiting or delaying the onset of an immune disease by administering a composition containing, as an active ingredient, the Bifidobacterium longumsubsp.suillum KBL2967 strain having the accession number KCTC 15739BP according to an example of the present application.

[0067] In this specification, the term “treatment” means partially or completely alleviating, improving, alleviating, inhibiting or delaying symptoms of an immune disease or disease, reducing the severity or reducing the occurrence of one or more symptoms or characteristics by administering a composition comprising, as an active ingredient, the Bifidobacterium longumsubsp.suillum KBL2967 strain having the accession number KCTC 15739BP according to an example of the present application.

[0068] A composition according to an example of the present application, for example, a pharmaceutical composition, may additionally contain one or more active ingredients exhibiting the same or similar function in addition to the strain according to an example of the present application.

[0069] A composition according to an example of the present application, for example, a pharmaceutical composition, may additionally contain a pharmaceutically acceptable carrier in addition to the strain, which is an active ingredient of the present invention. The pharmaceutically acceptable carrier contained in the composition of the present invention is one commonly used in formulations, and includes, but is not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above components, the composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like.

[0070] A composition according to an example of the present application, for example, a pharmaceutical composition, may be administered orally or parenterally, and is preferably administered orally. The pharmaceutical composition of the present invention may be formulated into various oral or parenteral dosage forms, but is not limited thereto.

[0071] Oral dosage forms include, for example, tablets, pills, hard / soft capsules, liquids, suspensions, emulsifiers, syrups, granules, elixirs, etc., and these dosage forms may use, in addition to the above-mentioned active ingredients, one or more diluents or excipients such as fillers, bulking agents, wetting agents, disintegrants, lubricants, binders, and surfactants that are commonly used. Disintegrants that can be used include agar, starch, alginic acid or its sodium salt, calcium hydrogen phosphate anhydride, etc., and lubricants that can be used include silica, talc, stearic acid or its magnesium or calcium salt, polyethylene glycol, etc., and binders that can be used include magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidine, low-substituted hydroxypropyl cellulose, etc. In addition, lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, glycine, etc. can be used as diluents, and in some cases, commonly known boiling mixtures, absorbents, colorants, flavoring agents, sweeteners, etc. can be used together.

[0072] A composition according to an example of the present application, for example a pharmaceutical composition, may be sterilized or contain auxiliary agents such as preservatives, stabilizers, wetting agents or emulsifying agents, salts for regulating osmotic pressure, buffers, and other therapeutically useful substances, and may be formulated according to conventional mixing, granulating or coating methods.

[0073] The appropriate dosage of a composition according to an example of the present application, such as a pharmaceutical composition, may vary depending on factors such as the formulation method, administration method, patient age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity. A typical dosage of the pharmaceutical composition of the present application is within the range of 0.001-1000 mg / kg for adults. For example, the composition according to the present application may be administered in an amount of 0.001 to 10,000 mg, 0.001 to 5,000 mg, 0.001 to 1,000 mg, 0.001 to 500 mg, 0.001 to 300 mg, 0.001 to 100 mg, 0.001 to 50 mg, 0.001 to 30 mg, 0.001 to 10 mg, 0.001 to 5 mg, 0.001 to 1 mg, 0.001 to 0.5 mg, 0.001 to 0.1 mg, 0.001 to 0.05 mg, 0.001 to 0.01 mg, 0.01 to 10,000 mg, 0.01 to 5,000 mg, per kg of body weight. 0.01 to 1,000 mg, 0.01 to 500 mg, 0.01 to 300 mg, 0.01 to 100 mg, 0.01 to 50 mg, 0.01 to 30 mg, 0.01 to 10 mg, 0.01 to 5 mg, 0.01 to 1 mg, 0.01 to 0.5 mg, 0.01 to 0.1 mg, 0.01 to 0.05 mg, 0.1 to 10,000 mg, 0.1 to 5,000 mg, 0.1 to 1,000 mg, 0.1 to 500 mg, 0.1 to 300 mg, 0.1 to 200 mg, 0.1 to 100 mg, 0.1 to 50 mg, 0.1 to 30 mg, 0.1 to 10 mg, 0.1 to 5 mg, 0.1 to 1 mg, 0.1 to 0.5 mg, 1 to 10,000 mg, 1 to 5,000 mg, 1 to 1,000 mg, 1 to 500 mg, 1 to 300 mg, 1 to 200 mg, 1 to 100 mg, 1 to 50 mg, 1 to 10 mg, 1 to 5 mg, 10 to 10,000 mg, 10 to 5,000 mg, 10 to 1,000 mg, 10 to 500 mg, 10 to 300 mg, 10 to 200 mg, 10 to 100 mg, 10 to 50 mg, 10 to 40 mg, 10 to 30 mg, 10 to 20 mg, 100 to 10,000 mg, 100 to 5,000 mg, 100 It can be administered in a daily dosage of, but is not limited to, 1,000 mg, 100 to 500 mg, 100 to 300 mg, or 100 to 200 mg. In addition, the human dosage can be converted based on animal testing.

[0074] A composition according to an example of the present application, for example, a pharmaceutical composition, may be prepared in a unit dosage form or may be prepared by inserting it into a multi-dose container by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method that can be easily performed by a person having ordinary skill in the art to which the present invention pertains. In this case, the formulation may be in the form of a solution, suspension, syrup or emulsion in an oil or aqueous medium, or in the form of an extract, powder, granule, tablet or capsule, and may additionally include a dispersant or stabilizer.

[0075] The composition for enhancing immunity and the pharmaceutical composition for preventing or treating immune diseases of the present application comprise, as an active ingredient, the Bifidobacterium longumsubsp.suillum KBL2967 strain having the accession number KCTC 15739BP, which is one embodiment of the present application described above. Therefore, overlapping contents are commonly applied, and their description is omitted to avoid excessive complexity of the present specification.

[0076]

[0077] According to another aspect of the present application, the present application provides a food comprising at least one selected from the group consisting of Bifidobacterium longumsubsp.suillum KBL2967 strain having accession number KCTC 15739BP, a culture of the strain, a lysate of the strain, an extract of the strain, and a combination thereof.

[0078] In one embodiment, the food described above may be for enhancing immunity.

[0079] In one embodiment, the food described above may be a food for preventing or improving an immune disease.

[0080] In this specification, the term “improvement” means any act of reducing the degree of an immune disease or symptoms due to a disease by administering a composition and / or food containing, as an active ingredient, the Bifidobacterium longumsubsp.suillum KBL2967 strain having the accession number KCTC 15739BP according to an example of the present application.

[0081] In one embodiment, the food may be a health functional food, and the health functional food may be a health functional food for enhancing immunity or preventing or improving an immune disease.

[0082] In one embodiment, the food may be in the form of, but is not limited to, meat, bread, chocolate, candy, jelly, snacks, confectionery, kimchi, soy sauce, cheese, dairy products, powders, beverages, vitamin complexes, water-soluble films, or food additives.

[0083] The food of the present application may additionally contain one or more effective ingredients exhibiting the same or similar function in addition to the strain according to an example of the present application.

[0084] The food of the present application may include not only the KBL2967 strain according to an example of the present application as an effective ingredient, but also ingredients commonly added during food manufacturing. The added ingredients include, for example, proteins, carbohydrates, fats, nutrients, seasonings, and flavoring agents. The carbohydrates include common sugars such as monosaccharides (e.g., glucose, fructose, etc.), disaccharides (e.g., maltose, sucrose, oligosaccharides, etc.), and polysaccharides (e.g., dextrin, cyclodextrin, etc.), and sugar alcohols such as xylitol, sorbitol, and erythritol). Natural flavoring agents (thaumatin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used.

[0085] For example, when the food of the present application is manufactured as a drink, in addition to the strain according to an example of the present application, citric acid, high fructose corn syrup, sugar, glucose, acetic acid, malic acid, juice, jujube extract, or licorice extract, etc. may be additionally included.

[0086] The food of this application includes processed forms of all natural ingredients, such as foods, functional foods, nutritional supplements, health foods, and food additives. These types of foods can be manufactured in various forms using conventional methods known in the art. For example, as health foods, the strains described above can be manufactured into tea, juice, and drinks for consumption, or granulated, encapsulated, or powdered for consumption. In addition, foods such as beverages (including alcoholic beverages), fruits and processed foods thereof (e.g., canned fruits, bottled fruits, jams, marmalades, etc.), fish, meats and processed foods thereof (e.g., ham, sausages, corned beef, etc.), breads and noodles (e.g., udon, buckwheat noodles, ramen, spaghetti, macaroni, etc.), fruit juices, various drinks, cookies, taffy, dairy products (e.g., yogurt, fermented milk, butter, cheese, etc.), edible vegetable oils, margarine, vegetable proteins, retort foods, frozen foods, various seasonings (e.g., soybean paste, soy sauce, sauces, etc.) can be produced by adding the strain according to an example of the present application. In addition, in order to use the strain according to an example of the present application in the form of a food additive, it can be produced in the form of a powder or a concentrate.

[0087] The preferred dosage of the food of the present application may vary depending on the patient's condition and weight, age, sex, health status, dietary constitution, nature of the preparation, degree of disease, administration time of the composition, administration method, administration period or interval, excretion rate, and drug form, and may be appropriately selected by a person skilled in the art.

[0088]

[0089] Another aspect of the present application relates to a method for enhancing immunity, comprising administering to a subject a composition comprising at least one selected from the group consisting of Bifidobacterium longumsubsp.suillum KBL2967 strain having accession number KCTC 15739BP, a culture of the strain, a lysate of the strain, an extract of the strain, and a combination thereof.

[0090] Another aspect of the present application relates to a method for preventing, improving or treating an immune disease, comprising administering to a subject a composition comprising at least one selected from the group consisting of Bifidobacterium longumsubsp.suillum KBL2967 strain having accession number KCTC 15739BP, a culture of the strain, a lysate of the strain, an extract of the strain, and a combination thereof.

[0091] In the case of the method for enhancing immunity and the method for preventing, improving or treating immune diseases of the present application, since it is a method using the Bifidobacterium longumsubsp.suillum KBL2967 strain having the accession number KCTC 15739BP according to an example of the present application described above, a culture of the strain, a lysate of the strain, an extract of the strain, and a combination thereof, overlapping contents are commonly applied, and their description is omitted to avoid excessive complexity of the present specification.

[0092]

[0093] Another aspect of the present application provides at least one immune-enhancing use selected from the group consisting of Bifidobacterium longumsubsp.suillum KBL2967 strain having accession number KCTC 15739BP, a culture of the strain, a lysate of the strain, an extract of the strain, and a combination thereof.

[0094] Another aspect of the present application provides a use for producing an immune-enhancing composition and / or an immune-enhancing food, wherein the composition is selected from the group consisting of Bifidobacterium longumsubsp.suillum KBL2967 strain having the accession number KCTC 15739BP, a culture of the strain, a lysate of the strain, an extract of the strain, and a combination thereof.

[0095] Another aspect of the present application provides a use for preventing, treating or improving one or more immune diseases selected from the group consisting of Bifidobacterium longumsubsp.suillum KBL2967 strain having accession number KCTC 15739BP, a culture of the strain, a lysate of the strain, an extract of the strain, and a combination thereof.

[0096] Another aspect of the present application provides a use for manufacturing a pharmaceutical composition for preventing or treating an immune disease and / or a food for preventing or improving an immune disease, wherein the composition is selected from the group consisting of Bifidobacterium longumsubsp.suillum KBL2967 strain having the accession number KCTC 15739BP, a culture of the strain, a lysate of the strain, an extract of the strain, and a combination thereof.

[0097] In the case of one or more of the uses selected from the group consisting of the Bifidobacterium longumsubsp.suillum strain KBL2967 having the accession number KCTC 15739BP of the present application, a culture of the strain, a lysate of the strain, an extract of the strain, and a combination thereof, the use for use in the manufacture of an immune-enhancing composition and / or an immune-enhancing food, the use for use in the prevention, treatment, or improvement of an immune disease, and the use for use in the manufacture of a pharmaceutical composition for the prevention or treatment of an immune disease and / or a food for the prevention or improvement of an immune disease, since the uses of the Bifidobacterium longumsubsp.suillum strain KBL2967 having the accession number KCTC 15739BP according to an example of the present application, a culture of the strain, a lysate of the strain, an extract of the strain, and a combination thereof, the overlapping contents are commonly referred to as It is applied, and its description is omitted to avoid excessive complexity of this specification.

[0098]

[0099] The present application relates to a Bifidobacterium longum subsp. suillum strain and its use for enhancing immunity. The Bifidobacterium longum subsp. suillum KBL2967 strain of the present application enhances immunity by increasing the expression of IL-6, TNF-α, and IL-1β, which are indicator cytokines of macrophage activity, and does not produce biogenic amines or have antibiotic resistance, so that it can be widely used as a safe probiotic for enhancing immunity in the development of food additives, health functional foods, and immune disease treatments.

[0100]

[0101] Figure 1a shows the gel data of the 16S rRNA gene of the Bifidobacterium longumsubsp.suillum KBL2967 strain of the present application compared with the 16S rRNA gene of Escherichia coli (DH5α) to prove that the sample used for genetic analysis of the strain was intact.

[0102] Figure 1b shows the results of analyzing the presence of the tyrosine decarboxylase gene in the KBL2967 strain of the present application.

[0103] Figure 1c shows the results of analyzing the presence of the histidine decarboxylase gene in the KBL2967 strain of the present application.

[0104] Figure 2a is a drawing confirming that IL-6 production in macrophages increased by the KBL2967 strain of the present application.

[0105] Figure 2b is a drawing confirming that TNF-α production in macrophages increased by the KBL2967 strain of the present application.

[0106] Figure 2c is a drawing confirming that IL-1β production in macrophages increased by the KBL2967 strain of the present application.

[0107] Hereinafter, the present application will be described in more detail through examples. These examples are intended solely to illustrate the present application more specifically, and it will be apparent to those skilled in the art that the scope of the present application is not limited by these examples, in accordance with the gist of the present application.

[0108]

[0109] Example

[0110]

[0111] (Throughout this specification, "%" used to indicate the concentration of a particular substance is (weight / weight) % for solid / solid, (weight / volume) % for solid / liquid, and (volume / volume) % for liquid / liquid, unless otherwise stated.)

[0112]

[0113] Example 1: Isolation and identification of Bifidobacterium longumsubsp.suillum strain KBL2967

[0114] This strain was isolated from a stool sample provided by a healthy Korean woman in her 30s in 2014. The stool was homogenized in PBS, and the original solution, 10-fold dilutions, and 100-fold dilutions were prepared, which were then spread on M2GSC agar plates. M2GSC agar medium was prepared with the following composition per 1 L: Casitone (can be substituted with peptone) 10.0 g, Yeast Extract 2.5 g, NaHCO3 (added after boiling) 4.0 g, Glucose 2.0 g, Cellobiose 2.0 g, Soluble Starch 2.0 g, Clarified Rumen Fluid 300 ml, Cysteine ​​(added after boiling) 1.0 g, K2HPO4 0.45 g, KH2PO4 0.45 g, (NH4)2SO4 0.9 g, NaCl 0.9 g, MgSO4* 7H20 0.09 g, CaCl2 0.09 g, Resazurin 1.0 mg, Agar 15 g. Sterilized M2GSC medium was poured into a petri dish, solidified, and 100 μL of homogenized fecal sample was plated on the plate and anaerobically cultured at 37°C for more than 48 hours. Colonies grown on the cultured medium were picked and streaked onto BL agar medium, and then anaerobically cultured again at 37°C for 24 to 48 hours. The composition of BL agar medium is as follows. Per 1 L: Beef Extract 3.0 g, Liver Extract 5.0 g, Yeast Extract 5.0 g, Proteose Peptone 10.0 g, Tryptone 5.0 g, Soy Peptone 3.0 g, Soluble Starch 0.5 g, Glucose 10.0 g, K2HPO4 1.0 g, KH2PO4 1.0 g, MgSO4 0.2 g, NaCl 0.01 g, MnSO4 0.0067 g, L-Cysteine ​​HCl * H2O 0.5 g, FeSO4 0.01 g, Polysorbate 80 1.0 g, Agar 15 g. A single colony grown on BL agar medium was picked and inoculated into 3 mL of BL liquid medium, and cultured under the same conditions of 37℃, 24 to 48 hours to obtain a single strain culture. 1 mL of the obtained culture was centrifuged at 12,000 RCF, 2 min to obtain a single strain pellet. The obtained single strain pellet was used to isolate the genomic DNA of the above lactic acid bacteria using the Promega Wizard® Genomic DNA Purification Kit according to the manufacturer's protocol.

[0115] The isolated genomic DNA was sequenced using the Oxford Nanopore Native Barcoding Kit 24 V14 according to the protocol, and the sequence analysis was performed by injecting the constructed library into a Flow Cell (R10.4.1). The analyzed genomic DNA sequence was analyzed for 16S rRNA sequence using the Geneious Prime genome analysis program. The determined base sequence was identified using the database of the Bacteria and Archaea: 16S ribosomal RNA project (ftp: / ftp.ncbi.nlm.nih.gov / refseq / TargetedLoci / Bacteria / ) and is shown in Table 1.

[0116] The 16S rRNA sequence of the above strain is shown in Table 1 below. The sequence was compared with the NCBI Bacteria and Archaea: 16S ribosomal RNA project DB, and the isolated strain was confirmed to be Bifidobacterium longumsubsp.suillum, and the strain was named Bifidobacterium longum subsp.suillum KBL2967 strain. The strain was deposited with the Korean Collection for Type Cultures, Korea Research Institute of Bioscience and Biotechnology, an international depository institution under the Budapest Treaty, and was assigned the accession number KCTC 15739BP.

[0117]

[0118]

[0119] Example 2: Analysis of the sugar decomposition capacity of the strain

[0120] In order to compare the sugar decomposition ability of the Bifidobacterium longumsubsp.suillum KBL2967 strain according to an example of the present application and the KCTC15605 strain as a type strain representing Bifidobacterium longumsubsp.suillum, a sugar fermentation test was performed using the API 20A kit (API 20A, BioMerieux's, USA), and the sugar decomposition ability analysis results are shown in Table 2 below. If there is a decomposition ability for the corresponding sugar, it is indicated by +, and if there is no decomposition ability, it is indicated by -.

[0121] SubstrateKBL2967KCTC15605L-Tryptophane--Urea--D-Glucose+-D-Mannitol--D-Lactose+-D-Saccharose+-D-Maltose+-Salicin--D-Xylose+-L-Arabinose+-Gelatin--Esculin--Glycerol-+D-Cellobiose-+D-Mannose++D-Melezitose-+D-Raffinose (D-Raffinose)++D-Sorbitol++D-Rhamnose-+D-Trehalose-+

[0122] As shown in Table 2, it was confirmed that the sugars that showed differences in decomposition ability between the strains were D-glucose, D-lactose, D-saccharose, D-maltose, D-xylose, L-arabinose, glycerol, D-cellobiose, D-melezitose, D-rhamnose, and D-trehalose. As such, it was found that the Bifidobacterium longum subsp. suilum KBL2967 strain according to an example of the present application had a different sugar decomposition ability compared to the conventional KCTC15605 strain.

[0123]

[0124] Example 3: Confirming the safety of the strain - Confirming antibiotic resistance

[0125] If a strain has an antibiotic resistance gene, there is a risk that the antibiotic resistance gene may be transmitted to opportunistic bacteria or pathogenic microorganisms residing in the intestines, which may result in the antibiotic not being effective in bacterial infections, so it is important to determine the antibiotic resistance of the strain in order to utilize it as a probiotic. Therefore, the antibiotic resistance of the Bifidobacterium longum subsp. suilum KBL2967 strain according to an example of the present application was confirmed using the following method.

[0126] In this experiment, eight kinds of antibiotics, including ampicillin (AM), vancomycin (VA), gentamycin (GM), streptomycin (SM), erythromycin (EM), clindamycin (CM), tetracycline (TC), and chloramphenicol (CL), were used, and the concentration at the point where the antibiotic strip meets the inhibition zone was defined as the minimum inhibitory concentration (MIC). Bifidobacterium longum subsp. suilum KBL2967 strain cultured on BL medium for 20 hours was washed twice with 1× PBS and then diluted to a 1.0 McFarland concentration in 1× PBS. The prepared cells were evenly spread on LSM (LAB susceptibility test medium) agar medium (90% IST broth, 10% MRS broth, 1.8% agar) using a sterilized cotton swab to form a lawn. The LSM agar medium on which the cells were spread was left at room temperature for 10-15 minutes to allow the inoculated cells to permeate well, and then each antibiotic strip to be tested (bioMerieux E-TEST®) was placed on the medium and incubated at 37°C for 48 hours. The values ​​(μg / mL) measured for the minimum inhibitory concentration (MIC) are shown in Table 3 below. The cut-off value was determined according to the European Food Safety Authority (EFSA) guidelines.

[0127] Minimum inhibitory concentration (MIC) (μg / mL) - EFS A Bifidobacterium KBL2967 Ampicillin <2 <0.125 Kanamycin nrnr Vancomycin <2 <0.5 Gentamicin <6 <4 Streptomycin <1 2 <8 Erythromycin <1 <0.02 3 Clindamycin <1 <0.016 Tetracycline <8 <0.75 Chloramphenicol <4 <0.75

[0128] *nr, not required

[0129] As shown in Table 3, the MIC value of the Bifidobacterium longum subsp. suilum KBL2967 strain was lower than the cut-off value, confirming that it did not exhibit resistance to the above eight antibiotics.

[0130]

[0131] Example 4: Verification of possession of biogenic amine producing genes

[0132] Biogenic amines, such as histamine and tyramine, cause allergic reactions such as itching and headaches. Histidine decarboxylase and tyrosine decarboxylase are enzymes that produce histamine and tyramine, respectively.

[0133] In order to verify whether the Bifidobacterium longumsubsp.suillum KBL2967 strain according to an example of the present application produces biogenic amines, the presence of genes encoding tyrosine decarboxylase and histidine decarboxylase in the genome of the strain was verified. The tyrosine decarboxylase and histidine decarboxylase genes were amplified using primer pairs of SEQ ID NOs: 2 to 5 in Table 4 below.

[0134] In order to verify that the genome sample of KBL2967 used as a template in the experiment to detect the tyrosine decarboxylase and histidine decarboxylase genes was intact, the entire region of 16S rRNA was amplified using the primer pair of SEQ ID NO: 6 (27F) and SEQ ID NO: 7 (1492R) in Table 4 below. The 16S rRNA gene is a gene of approximately 1,500 bp in size that is common to bacteria, and whether the genome is intact in the used sample can be determined based on whether the 16S rRNA gene is amplified. As a control, a genome sample of Escherichia coli (DH5α) was also used as a template, and it was confirmed that the sizes of the 16S rRNA genes amplified through the genome sample of KBL2967 and the genome sample of E. coli were identical.

[0135] Sequence nameSequence (5'→3')SEQ ID NOTyrosine decarboxylase forward primerACATAGTCAACCATRTTGAA2Tyrosine decarboxylase reverse primerCAAATGGAAGAAAGAAGTAGG3Histidine decarboxylase forward primerGATGGTATTGTTTCKTATGA4Histidine decarboxylase reverse primerCAAACACCAGCATCTTC527F primerAGAGTTGATYMTGGCTCAG61492R primerTACGGYTACCTTGTTACGACT7

[0136] The 16S rRNA gene and each biogenic amine gene were amplified in a PCR tube with the corresponding primer set. The PCR conditions were as follows: 98°C for 1 min, followed by 30 cycles of 98°C for 10 s, 60°C for 6 s for 16S rRNA, 58°C for 6 s, and 68°C for 5 s for biogenic amine genes, followed by a final extension at 68°C for 2 min. Enterococcus faecalis KCTC3206 genomic DNA and Lactobacillus sakei LTH 2076 sequence were used as positive controls for the tyrosine decarboxylase and histidine decarboxylase genes, respectively.

[0137] As shown in Fig. 1a, the 16S rRNA gene amplified using the KBL2967 genome sample as a template was successfully detected in the region of 1,500 bp, which is the expected size, similar to the control E. coli (DH5α), indicating that the KBL2967 genome sample used in the gene detection experiment was intact without any abnormalities.

[0138] As shown in Figs. 1b and 1c, the Bifidobacterium longum subsp. suillum KBL2967 strain did not possess the tyrosine decarboxylase and histidine decarboxylase genes, respectively. On the other hand, PCR products for these genes were detected in the genomic DNA of Enterococcus faecalis KCTC3206 and the sequence of Lactobacillus sakei LTH 2076, which were positive controls. Thus, since the genes encoding histidine decarboxylase and tyrosine decarboxylase were not detected in the genome of the Bifidobacterium longum subsp. suillum KBL2967 strain, it was confirmed that the KBL2967 strain does not produce biogenic amines.

[0139]

[0140] Example 5: Confirmation of the effect of increasing cytokine production in macrophages.

[0141] The animal cell line used in this study was the mouse-derived macrophage cell line RAW 264.7, obtained from ATCC. For general cell maintenance, cells were cultured in a medium containing 90% DMEM medium (Gibco, 11965118) and 10% FBS (Hyclone, SV30207.02), and passaged three times a week at a 1:6 ratio.

[0142] To confirm the increase in cytokine production by Bifidobacterium longumsubsp.suillum KBL 2967 strain in mouse macrophages, mouse macrophages were seeded at 2 × 10 per well in a 96-well plate. 4 100 μL was dispensed into each well. After 24 hours, 2 × 10 were seeded per well, so that the viable cells were 1:100 compared to the number of cells. 6 Cells were treated on macrophages and cultured at 37°C for 24 hours in the presence of 5% CO2.

[0143] After incubation, centrifugation was performed, and 180 μL of the supernatant was taken from each well. The amounts of IL-6, TNF-α, and IL-1β contained in the supernatant were measured using an ELISA Kit (555240, 555268, DY401).

[0144] As a negative control group, an untreated group was used, and as a positive control group, a group treated with LPS (Lipopolysaccharide, Sigma, L2630) at a concentration of 10 ng / mL was used. As a comparison group, the KCTC15605 strain was used at a ratio of 1:100 to the number of cells, i.e., 2 × 10 6 The group treated with cells was used.

[0145] As a result, as shown in FIGS. 2a to 2c, the expression levels of IL-6, TNF-α, and IL-1β in macrophages significantly increased when the KBL2967 strain according to an example of the present application was treated, thereby showing that the Bifidobacterium longum subsp. suilum KBL2967 strain according to an example of the present application has immune-enhancing activity.

[0146]

[0147] [Accession number]

[0148] Name of depositor: Korea Research Institute of Bioscience and Biotechnology, Biological Resource Center (KCTC)

[0149] Accession number: KCTC15739BP

[0150] Date of acceptance: 20231213

[0151]

Claims

1. Bifidobacterium longumsubsp.suillum strain KBL2967 with accession number KCTC 15739BP.

2. A strain according to claim 1, wherein the strain has a 16S rRNA sequence of sequence number 1.

3. In the first paragraph, the strain has one or more of the following (1) to (11) sugar decomposition characteristics: (1) D-Glucose degradation capacity; (2) D-Lactose decomposition capacity; (3) D-Saccharose decomposition capacity; (4) D-Maltose decomposition capacity; (5) D-Xylose resolution; (6) L-Arabinose degradation capacity; (7) Non-degradability of glycerol; (8) Non-degradability of D-Cellobiose; (9) D-Melezitose non-degradable; (10) D-Rhamnose non-degradability; and (11) Non-degradability of D-Trehalose.

4. A strain according to claim 1, wherein the strain has antibiotic sensitivity.

5. A strain according to claim 4, wherein the antibiotic susceptibility is susceptibility to at least one antibiotic selected from the group consisting of ampicillin, vancomycin, gentamycin, streptomycin, erythromycin, clindamycin, tetracycline, and chloramphenicol.

6. A strain according to claim 1, wherein the strain does not produce biogenic amine.

7. A strain according to claim 1, wherein the strain has immune-enhancing activity.

8. A strain according to claim 7, wherein the immune enhancement is due to activation of macrophages.

9. A strain according to claim 7, wherein the immune enhancement is due to increased cytokine production by macrophages.

10. An immune-enhancing composition comprising at least one selected from the group consisting of Bifidobacterium longumsubsp.suillum KBL2967 strain having accession number KCTC 15739BP, a culture of the strain, a lysate of the strain, an extract of the strain, and a combination thereof.

11. A composition for enhancing immunity in claim 10, wherein the enhancement of immunity is achieved by activation of macrophages.

12. A composition for enhancing immunity in claim 10, wherein the enhancement of immunity is due to increased cytokine production by macrophages.

13. A composition according to claim 10, wherein the strain is a live strain or a heat-inactivated strain.

14. A food comprising at least one selected from the group consisting of Bifidobacterium longumsubsp.suillum KBL2967 strain having accession number KCTC 15739BP, a culture of the strain, a lysate of the strain, an extract of the strain, and a combination thereof.

15. In paragraph 14, the food is a health functional food.

16. In paragraph 14, the food is meat, bread, chocolate, candy, jelly, snacks, confectionery, kimchi, soy sauce, cheese, dairy products, powder, beverage, vitamin complex, water-soluble film, or food additive.

17. A method for enhancing immunity, comprising administering to a subject a composition comprising at least one selected from the group consisting of Bifidobacterium longumsubsp.suillum KBL2967 strain having accession number KCTC 15739BP, a culture of the strain, a lysate of the strain, an extract of the strain, and a combination thereof.

18. A method for preventing or treating an immune disease, comprising administering to a subject a composition comprising at least one selected from the group consisting of Bifidobacterium longumsubsp.suillum KBL2967 strain having accession number KCTC 15739BP, a culture of the strain, a lysate of the strain, an extract of the strain, and a combination thereof.

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