Immunity enhancement use of lactobacillus fermentum strain

Abstract

The present application relates to a use of a Lactobacillus fermentum strain for enhancing immunity.

Description

Immune-boosting uses of Lactobacillus fermentum strains

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

[0002] This application claims the benefit of priority from Republic of Korea Patent Application No. 10-2024-0050977, dated April 16, 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 the use of a Lactobacillus fermentum strain for enhancing immunity.

[0005] The immune system is the body's defense system against external substances. Under normal conditions, the body maintains immune homeostasis by eliminating antigens from pathogens and external pollutants and suppressing abnormal inflammatory responses to altered self-antigens. However, when immune homeostasis is disrupted due to various factors, such as irregular lifestyle habits, antibiotic misuse, stress, and environmental hormones, a weakened immune system can lead to harmful bacterial infections, as well as chronic diseases such as autoimmune diseases, allergies, and inflammatory diseases. The prevalence of chronic diseases caused by immune system imbalances continues to increase, but treatments are limited and side effects are reported. Consequently, the importance of preventive approaches, in addition to therapeutic approaches, has been highlighted in recent years.

[0006]

[0007] Against this backdrop, the inventors of the present invention have discovered that the Lactobacillus fermentum KBL375 strain, having the accession number KCTC 13381BP, exhibits a remarkably superior immune-enhancing effect. Accordingly, one embodiment of the present application provides a use for the Lactobacillus fermentum KBL375 strain, having the accession number KCTC 13381BP, for enhancing immunity, which can be used for the prevention and treatment of immunodeficiency.

[0008]

[0009] An example of the present application relates to an immune-enhancing composition comprising at least one selected from the group consisting of Lactobacillus fermentum KBL375 strain having the deposit number KCTC 13381BP, a culture of the strain, a lysate of the strain, and an extract of the strain.

[0010] Another example of the present application relates to a composition for improving an intestinal microbiome, comprising at least one selected from the group consisting of a Lactobacillus fermentum KBL375 strain having the deposit number KCTC 13381BP, a culture of the strain, a lysate of the strain, and an extract of the strain.

[0011] Another example of the present application relates to a pharmaceutical composition for preventing or treating immunodeficiency, comprising at least one selected from the group consisting of Lactobacillus fermentum KBL375 strain having the deposit number KCTC 13381BP, a culture of the strain, a lysate of the strain, and an extract of the strain.

[0012] Another example of the present application relates to a food comprising a composition according to an example of the present application.

[0013] Another example of the present application relates to a health functional food comprising a composition according to an example of the present application.

[0014] Another example of the present application relates to a pharmaceutical product comprising a composition according to an example of the present application.

[0015] Another example of the present application relates to a cosmetic comprising a composition according to an example of the present application.

[0016]

[0017] Hereinafter, the present application will be described in more detail. Furthermore, each description and embodiment disclosed in this application may also be applied to each other. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. Furthermore, the scope of this application is not limited by the specific descriptions set forth below. Furthermore, those skilled in the art will recognize or be able to ascertain, through routine experimentation alone, numerous equivalents to the specific embodiments of this application described herein. Furthermore, such equivalents are intended to be encompassed by this application.

[0018]

[0019] Cells that mediate the innate immune response include natural killer cells (NK cells), macrophages, and dendritic cells. Among these, NK cells detect and eliminate abnormal cells through various receptors expressed on the cell surface, and macrophages rapidly respond to external antigens and induce immune responses such as phagocytosis and cytokine secretion. In order to evaluate the immune-enhancing efficacy of the Lactobacillus fermentum KBL375 strain having the accession number KCTC 13381BP according to an example of the present application, the effects of the strain on NK cell cytotoxicity, NK cell surface protein marker expression, and cytokine secretion were evaluated using mouse splenocytes and macrophages in the examples of the present application. As a result, the strain according to an example of the present application activated immune cells, enhanced the target cell killing ability of immune cells, and promoted cytokine production by immune cells. Accordingly, a composition comprising at least one selected from the group consisting of a strain according to an example of the present application, a culture of the strain, a lysate of the strain, and an extract of the strain is useful for enhancing immunity.

[0020] In the present application, the term 'immune enhancement' means enhancing the strength of an immune response by promoting the activity and function of immune cells. Accordingly, 'immune enhancement' in the present application may include, but is not limited to, activation or maturation of immune cells (e.g., NK cells and / or macrophages), promotion of proliferation of immune cells, promotion of cytokine production by immune cells, restoration of immune function in an immunocompromised state (e.g., prevention of relative weight loss of the spleen due to immunocompromise, increase in blood immunoglobulin concentration, etc.), and / or strengthening of immunity through improvement of the intestinal microbiome, unlike the immune regulation process that suppresses excessive immune responses through suppression of inflammatory cytokines or increase in anti-inflammatory cytokines.

[0021] A composition according to an example of the present application may be administered to a subject with a weakened immune system.

[0022] In this application, 'immunocompromise' refers to a physiological or pathological state in which the immune function of a living body is reduced below the normal range due to congenital or acquired factors. The congenital factors include, but are not limited to, immunocompromise due to genetic defects or primary immunodeficiency (PID), an immature immune system in infancy or immediately after birth, and a condition in which genetically specific immune cells (e.g., NK cells, T cells, B cells) are deficient or have impaired function. The acquired factors include, but are not limited to, immunocompromise due to the use of anticancer drugs or immunosuppressants, immunocompromise after organ transplantation, immunocompromise due to aging, immunocompromise due to lifestyle-related causes, immunocompromise due to long-term steroid use, or immunocompromise due to bacterial or viral infection.

[0023] In the present application, the anticancer agent may refer to a chemotherapeutic agent used to inhibit the proliferation of tumor cells or induce death, and may be, for example, at least one selected from the group consisting of cyclophosphamide, doxorubicin, cisplatin, paclitaxel, 5-fluorouracil (5-FU), methotrexate, vincristine, carboplatin, etoposide, docetaxel, gemcitabine, irinotecan, and oxaliplatin, but is not limited thereto.

[0024] In the present application, the immunosuppressant refers to a drug that inhibits the activity of the immune system or suppresses an immune response, and may be at least one selected from the group consisting of, for example, cyclosporin A, tacrolimus, sirolimus, everolimus, azathioprine, mycophenolate mofetil, methotrexate, leflunomide, corticosteroids (e.g., prednisolone, dexamethasone), and biological agents such as anti-TNF antibodies, anti-IL-6R antibodies, and anti-CD20 antibodies, but is not limited thereto. The immunosuppressant may be used in the treatment of autoimmune diseases, suppression of rejection after organ transplantation, etc., but is not limited thereto.

[0025] The above immunocompromised subject may have a reduced spleen weight due to immunocompromise, a reduced activity of NK cells, a reduced level of immunoglobulin in the blood, and / or a reduced proliferation capacity of T cells and B cells in the spleen, but is not limited thereto.

[0026] In one example, the subject may be, but is not limited to, a mammal. In one example, the subject may be a human. In one example, the subject may be a non-human mammal (e.g., a dog, a cat, a cow, a horse, a pig, a donkey, a goat, a camel, a mouse, a rat, a guinea pig, a sheep, a llama, a monkey, a gorilla, or a chimpanzee).

[0027] A composition according to one embodiment of the present invention may activate immune cells. Activating immune cells may include enhancing the target cell death rate of immune cells, promoting the expression of apoptosis-inducing proteins in immune cells (specifically, NK cells), promoting cytokine production in immune cells, and / or promoting nitric oxide (NO) production in immune cells (specifically, macrophages).

[0028] Specifically, the composition may promote the activity of immune cells in a normal subject and / or an immunocompromised subject, thereby increasing the target cell death rate of the immune cells. For example, the composition may improve the target cell (e.g., Yac-1 cell) death rate of immune cells by 1.1 times or more, 1.2 times or more, 1.3 times or more, 1.35 times or more, 1.4 times or more, 1.45 times or more, 1.5 times or more, 1.55 times or more, 1.6 times or more, 1.7 times or more, 1.8 times or more, 1.9 times or more, 2 times or more, 2.1 times or more, 2.2 times or more, 2.3 times or more, 2.4 times or more, 2.45 times or more, 2.5 times or more, 2.6 times or more, 2.65 times or more, 2.7 times or more, 2.8 times or more, or 2.8 times or more compared to an untreated normal control. For example, the composition may enhance the target cell (e.g., Yac-1 cell) killing rate of immune cells (e.g., NK cells) by at least 1.1 times, at least 1.15 times, or at least 1.2 times compared to an untreated control group with compromised immunity.

[0029] Specifically, the composition may promote the activity of immune cells, thereby promoting the expression of an apoptosis-inducing protein in immune cells (specifically, NK cells). The apoptosis-inducing protein may include perforin and / or granzyme B (GzmB). For example, the composition may enhance the perforin expression amount of immune cells by 1.5 times or more, 2 times or more, 3 times or more, 4 times or more, 5 times or more, or 6 times or more compared to an untreated control group, and / or enhance the granzyme B expression amount of immune cells by 1.1 times or more, 1.2 times or more, 1.3 times or more, 1.4 times or more, or 1.5 times or more compared to an untreated control group.

[0030] Specifically, the composition may promote the activity of immune cells, thereby promoting cytokine production by the immune cells. The cytokine may include one or more of IFN-γ, IL-6, and TNF-α. For example, the composition may enhance IFN-γ production of immune cells (specifically, NK cells or macrophages) by at least 5 times, at least 10 times, at least 15 times, at least 20 times, or at least 23 times compared to an untreated control, IL-6 production by at least 1.1 times, at least 1.2 times, at least 1.25 times, at least 1.5 times, at least 2 times, at least 2.5 times, at least 3 times, or at least 3. times compared to an untreated control, and / or TNF-α production by at least 1.1 times, at least 1.2 times, at least 1.25 times, at least 1.3 times, at least 1.5 times, at least 2 times, at least 2.5 times, at least 2.6 times, or at least 3 times compared to an untreated control.

[0031] Specifically, the composition may promote the activity of immune cells, thereby promoting NO production by immune cells. For example, the composition may improve the nitric oxide production of immune cells (specifically, macrophages) by 0.1% or more, 0.2% or more, 0.3% or more, 0.35% or more, 0.4% or more, 0.5% or more, 1% or more, 2% or more, 3% or more, 3.5% or more, 4% or more, 5% or more, 10% or more, 15% or more, 16% or more, 17% or more, 18% or more, 19% or more, or 19.5% or more compared to an untreated control group.

[0032] A composition according to one example of the present application may mature immune cells. Specifically, the maturation of the immune cells may promote NKp46 expression in the immune cells. For example, the composition may increase the proportion of NKp46-expressing immune cells by 5% or more, 6% or more, 7% or more, 8% or more, 8.5% or more, 9% or more, 10% or more, 15% or more, 20% or more, or 24% or more compared to an untreated control group.

[0033] A composition according to an example of the present application may promote the proliferation of immune cells in an immunocompromised subject. For example, the composition may enhance the proliferation capacity of immune cells (specifically, T cells and / or B cells) by 1.1 times or more, 1.15 times or more, 1.2 times or more, 1.3 times or more, 1.4 times or more, or 1.45 times or more compared to an untreated control group (negative control group) with compromised immunity. In particular, according to an example of the present application, the composition had a superior effect of promoting the proliferation of immune cells compared to red ginseng, which is widely used as a conventional immune enhancer.

[0034] A composition according to an example of the present application may prevent a relative weight loss of the spleen due to immunosuppression in an immunocompromised subject. For example, the composition may improve the relative weight of the spleen (spleen index) of the immunocompromised subject by at least 1.1 times, at least 1.15 times, or at least 1.16 times compared to an untreated control group (negative control group) with immunosuppression. In particular, according to an example of the present application, the composition was more effective in preventing a relative weight loss of the spleen due to immunosuppression than red ginseng, which is widely used as a conventional immune enhancer.

[0035] A composition according to an example of the present application may increase the concentration of immunoglobulins in the blood in an immunocompromised subject. For example, the composition may increase the concentration of immunoglobulins (e.g., IgA) in the blood of an immunocompromised subject by 1.1 times or more, 1.2 times or more, 1.3 times or more, 1.4 times or more, 1.5 times or more, 1.6 times or more, or 1.7 times or more compared to an untreated control group (negative control group) with compromised immunity. In particular, according to an example of the present application, the composition had a superior effect of increasing the concentration of immunoglobulins in the blood compared to red ginseng, which is widely used as a conventional immune enhancer.

[0036] A composition according to one example of the present application may improve the intestinal microbiome of a subject. Specifically, the composition may increase the relative abundance of strains of the genus Bifidobacterium and / or the genus Lactobacillus, which are beneficial bacteria in the subject's intestine. Specifically, the composition may decrease the relative abundance of strains of the genus Bacteroides in the subject's intestine. The subject may be, but is not limited to, an immunocompromised subject.

[0037] Accordingly, another example of the present application relates to a composition for improving an intestinal microbiome, comprising at least one selected from the group consisting of a Lactobacillus fermentum KBL375 strain having the deposit number KCTC 13381BP, a culture of the strain, a lysate of the strain, and an extract of the strain.

[0038] Another example of the present application relates to a food product comprising a composition according to an example of the present application. The food product may be, for example, meat, bread, chocolate, candy, jelly, snacks, confectionery, kimchi, soy sauce, cheese, dairy products, powders, beverages, or vitamin complexes. The food product may be a health functional food.

[0039] The above food may contain not only the 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 monosaccharides (e.g., glucose, fructose, etc.), disaccharides (e.g., maltose, sucrose, oligosaccharides, etc.), and polysaccharides (e.g., conventional sugars such as dextrin, cyclodextrin, etc., and sugar alcohols such as xylitol, sorbitol, erythritol, etc.). The flavoring agents may include natural flavoring agents (thaumatin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.).

[0040] For example, when a food according to an example 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.

[0041] Foods according to one example of the present application include processed forms of all natural ingredients, such as 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 their processed foods (e.g., canned fruits, bottled fruits, jams, marmalades, etc.), fish, meats and their processed foods (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.

[0042] Another example of the present application relates to a quasi-drug composition comprising a composition according to an example of the present application. The quasi-drug composition may be in a form selected from the group consisting of toothpaste, mouthwash, oral spray, oral ointment, mouthwash, mouthwash, bandage, and pest repellent (e.g., mosquito, tick, etc.).

[0043] Another example of the present application relates to a cosmetic comprising a composition according to an example of the present application. The cosmetic may be formulated in a form selected from the group consisting of a cream, ointment, lotion, skin, mist, shampoo, detergent, cleansing foam, cleansing cream, cleansing water, and feminine wash.

[0044] Another example of the present application relates to a pharmaceutical composition for preventing or treating immunodeficiency, comprising at least one selected from the group consisting of Lactobacillus fermentum KBL375 strain having the deposit number KCTC 13381BP, a culture of the strain, a lysate of the strain, and an extract of the strain.

[0045] The above immunodeficiency is caused by a decrease in the body's immune function below the normal range due to congenital or acquired factors, and may be one or more selected from the group consisting of, but is not limited to, immunodeficiency (e.g., primary immunodeficiency (PID), secondary immunodeficiency (Secondary immunodeficiency), or acquired immunodeficiency syndrome (AIDS)), immunodeficiency due to use of anticancer drugs or immunosuppressants, leukopenia, neutropenia, lymphopenia, nutritional deficiency, diabetes, immunodeficiency after organ transplantation, immunodeficiency due to aging, immunodeficiency due to long-term steroid use, primary immunodeficiency disease, shingles, tuberculosis, meningitis, septic shock, and bacterial or viral infection.

[0046] Another example of the present application relates to a food for preventing or improving immunodeficiency, comprising at least one selected from the group consisting of Lactobacillus fermentum KBL375 strain having the deposit number KCTC 13381BP, a culture of the strain, a lysate of the strain, and an extract of the strain. The food may be a health functional food.

[0047] Another example of the present application relates to a pharmaceutical product for preventing or improving immunodeficiency, comprising at least one selected from the group consisting of Lactobacillus fermentum KBL375 strain having the deposit number KCTC 13381BP, a culture of the strain, a lysate of the strain, and an extract of the strain.

[0048] Another example of the present application relates to a cosmetic for preventing or improving immunodeficiency, comprising at least one selected from the group consisting of Lactobacillus fermentum KBL375 strain having the deposit number KCTC 13381BP, a culture of the strain, a lysate of the strain, and an extract of the strain.

[0049] In a pharmaceutical composition for preventing or treating immunodeficiency according to an example of the present application, or a food, quasi-drug, or cosmetic for preventing or improving immunodeficiency, the contents regarding activation of immune cells, maturation of immune cells, promotion of proliferation of immune cells, prevention of relative weight loss of the spleen due to immunodeficiency, increase in blood immunoglobulin concentration, improvement of intestinal microbiome, administration target of the composition, immunodeficiency, food, quasi-drug, cosmetic, etc. are as described above.

[0050]

[0051] In the present application, 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, etc. to a method such as centrifugation, chromatography, etc. 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 example, 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 one example of the present application for a certain period of time. In one example, the culture may be one in which the strain according to one example of the present application is removed or not removed. In one example, the culture may refer to the remaining components excluding the strain (bacterial cells) in the culture obtained by culturing the strain according to one example of the present application in a medium. In one example, the culture may be a culture (or culture) obtained by removing the strain (bacterial cells) from the culture solution obtained by culturing the strain according to one 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, or an activity for preventing, improving, or treating immunodeficiency.

[0052] The term "strain fragment" in this application may refer to a product obtained by disrupting a strain according to an example of this application using chemical or physical force. Specifically, the fragment may exhibit anti-inflammatory activity equivalent to the activity exhibited by the strain according to an example of this application, or activity to prevent, improve, or treat inflammatory diseases.

[0053] In the present application, 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 the culture of the strain, or an extract of the lysate of the strain. Specifically, the extract may exhibit an immune-enhancing activity equivalent to the activity exhibited by the strain according to an example of the present application, a culture of the strain, or a lysate of the strain, or an activity for preventing, improving, or treating immunodeficiency.

[0054] The term "prevention" in this application refers to the suppression or delay of the onset of a disease, disorder, or condition. Prevention may be considered complete if the onset of the disease, disorder, or condition is suppressed or delayed for a predetermined period of time.

[0055] The term 'treatment' in this application means partially or completely alleviating, improving, palliating, inhibiting or delaying the symptoms of a specific disease, disorder and / or condition or condition, reducing the severity or reducing the occurrence of one or more symptoms or characteristics.

[0056] In this application, the term 'improvement' means any act that reduces the symptoms associated with a specific disease, disorder and / or condition or condition.

[0057] A composition, food, over-the-counter drug, cosmetic, etc. according to an example of the present application may additionally include one or more types of effective ingredients exhibiting the same or similar function in addition to the above effective ingredients.

[0058] In addition, the composition, food, quasi-drug, cosmetic, etc. according to an example of the present application can be manufactured in the form of a unit dose or manufactured by introducing into a multi-dose container by formulating using a pharmaceutically acceptable carrier according to a method that can be clearly performed by a person having ordinary skill in the art to which the present invention pertains. The term 'carrier' in the present application means a compound that facilitates the addition of a compound into a cell or tissue, and the term 'pharmaceutically acceptable' means a composition that is physiologically acceptable and does not typically cause an allergic reaction such as gastrointestinal disorder or dizziness or a similar reaction when administered to a human.

[0059] The pharmaceutically acceptable carriers mentioned above are those commonly used in formulations, and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0060] In addition, compositions, foods, over-the-counter drugs, cosmetics, etc. according to an example of the present application may additionally include additives such as fillers, anticoagulants, lubricants, wetting agents, fragrances, emulsifiers, and preservatives in addition to the above ingredients. In the present application, the content of the additives is not particularly limited and may be appropriately adjusted within the content range used in conventional formulations.

[0061] In addition, the composition, food, etc. according to the present application can be formulated as an oral preparation. Non-limiting examples of the oral preparation include tablets, troches, lozenges, aqueous suspensions, oily suspensions, prepared powders, granules, emulsions, hard capsules, soft capsules, syrups, or elixirs, etc. In order to formulate the pharmaceutical composition or food composition according to the present application for oral administration, a binder such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose, or gelatin; an excipient such as dicalcium phosphate; a disintegrant such as corn starch or sweet potato starch; magnesium stearate, calcium stearate, sodium stearyl fumarate, etc.; and a sweetener, a flavoring agent, a syrup, etc. can also be used. In addition, in the case of capsules, in addition to the above-mentioned substances, a liquid carrier such as fatty oil may be additionally used.

[0062] The term "excipient" in this application refers to any substance other than a therapeutic agent, used as a carrier or medium for delivering the therapeutic agent, or added to a pharmaceutical composition, thereby improving handling and storage characteristics or facilitating and / or facilitating the formation of a unit dosage of the composition.

[0063] The composition according to the present application, for example, a pharmaceutical composition, may be formulated and used in various forms, such as oral formulations such as liquids, suspensions, powders, granules, tablets, capsules, pills, extracts, emulsions, syrups, aerosols, and injections of sterile injectable solutions, according to conventional methods according to each intended use, and may be administered orally or through various routes, including intravenous, intraperitoneal, subcutaneous, rectal, and topical administration. The term 'oral administration' in the present application means that the active substance is administered to a substance prepared to be digested, i.e., to the gastrointestinal tract for absorption.

[0064] The preferred dosage of the composition or food according to 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.

[0065] The term "effective dosage" in this application means the amount of a composition of an active ingredient sufficient to treat a specific symptom. This may vary depending on the formulation method of the pharmaceutical composition or food composition, the administration method, the administration time, and / or the administration route, and may vary depending on various factors including the type and degree of the response to be achieved by administering the pharmaceutical composition or food composition, the type, age, weight, general health condition, symptoms or degree of the disease, sex, diet, excretion, drugs used simultaneously or at the same time in the subject, other components of the composition, and similar factors well known in the medical field. A person of ordinary skill in the art can easily determine and prescribe an effective dosage for the desired treatment.

[0066] The composition or food according to the present application may be administered once daily or divided into several doses. The composition may be administered as an individual treatment or in combination with another treatment, and may be administered sequentially or simultaneously with conventional treatments. Taking all of the above factors into consideration, the composition may be administered in an amount that achieves maximum effect with minimal side effects.

[0067] 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 The daily dosage may 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. For example, the daily dosage of the composition according to the present application may be 0.001 to 10 g / day, 0.001 to 5 g / day, 0.01 to 10 g / day, or 0.01 to 5 g / day based on oral administration to an adult patient. In addition, the total daily dosage may be divided and administered continuously or discontinuously as needed.

[0068] Another example of the present application relates to a method for enhancing immunity, comprising administering to a subject at least one selected from the group consisting of Lactobacillus fermentum KBL375 strain having the deposit number KCTC 13381BP, a culture of the strain, a lysate of the strain, and an extract of the strain.

[0069] The above-mentioned immune enhancement may be at least one selected from the group consisting of, but not limited to, activating or maturing immune cells, promoting proliferation of immune cells, promoting cytokine production by immune cells, restoring immune function in an immunocompromised state (e.g., preventing relative weight loss of the spleen due to immunocompromise, increasing blood immunoglobulin concentration, etc.), and strengthening immunity through improvement of the intestinal microbiome.

[0070] Another example of the present application relates to a method for improving an intestinal microbiome, comprising administering to a subject at least one selected from the group consisting of a Lactobacillus fermentum KBL375 strain having the deposit number KCTC 13381BP, a culture of the strain, a lysate of the strain, and an extract of the strain.

[0071] Another example of the present application relates to a method for preventing or treating immunodeficiency, comprising administering to a subject at least one selected from the group consisting of Lactobacillus fermentum KBL375 strain having the deposit number KCTC 13381BP, a culture of the strain, a lysate of the strain, and an extract of the strain.

[0072] The method for enhancing immunity, the method for improving intestinal microbiome, and / or the method for preventing or treating immunodeficiency of the present application are methods using at least one selected from the group consisting of the Lactobacillus fermentum KBL375 strain having the deposit number KCTC 13381BP according to an example of the present application described above, a culture of the strain, a lysate of the strain, and an extract of the strain, and therefore, overlapping contents are commonly applied, and their description is omitted to avoid excessive complexity of the present specification.

[0073] Another example of the present application relates to an immune-enhancing use of at least one selected from the group consisting of Lactobacillus fermentum KBL375 strain having the deposit number KCTC 13381BP, a culture of the strain, a lysate of the strain, and an extract of the strain.

[0074] Another example of the present application relates to the use for the preparation of one or more immune-enhancing compositions selected from the group consisting of Lactobacillus fermentum KBL375 strain having the deposit number KCTC 13381BP, a culture of the strain, a lysate of the strain, and an extract of the strain.

[0075] Another example of the present application relates to a use for preventing or treating immunodeficiency, at least one selected from the group consisting of Lactobacillus fermentum KBL375 strain having the deposit number KCTC 13381BP, a culture of the strain, a lysate of the strain, and an extract of the strain.

[0076] Another example of the present application relates to the use for the manufacture of a pharmaceutical composition for preventing or treating immunodeficiency, selected from the group consisting of the Lactobacillus fermentum KBL375 strain having the deposit number KCTC 13381BP, a culture of the strain, a lysate of the strain, and an extract of the strain.

[0077] In the case of one or more uses selected from the group consisting of the Lactobacillus fermentum KBL375 strain having the deposit number KCTC 13381BP of the present application, a culture of the strain, a lysate of the strain, and an extract of the strain, the use for use in the preparation of an immune-enhancing composition, the use for use in the prevention or treatment of immunodeficiency, and / or the use for use in the preparation of a pharmaceutical composition for the prevention or treatment of immunodeficiency, since it is one or more uses selected from the group consisting of the Lactobacillus fermentum KBL375 strain having the deposit number KCTC 13381BP according to an example of the present application, a culture of the strain, a lysate of the strain, and an extract of the strain, overlapping contents are commonly applied, and the description thereof is omitted in order to avoid excessive complexity of the present specification.

[0078]

[0079] According to an example of the present application, the Lactobacillus fermentum KBL375 strain has an excellent immune-enhancing effect, such as activating NK cell activity and NKp46 expression in the spleen, increasing the secretion amount of IFN-γ, and increasing the secretion amounts of IL-6 and TNF-α in macrophages.

[0080]

[0081] Figure 1 is a drawing showing the NK cell activation effect of a strain according to an example of the present application.

[0082] Figure 2 is a drawing showing the immune cell activation effect of a strain according to an example of the present application.

[0083] Figures 3a and 3b are drawings showing the effect of increasing the expression of apoptosis-inducing proteins in immune cells by a strain according to an example of the present application.

[0084] Figure 4 is a drawing showing the effect of promoting cytokine production of immune cells by a strain according to an example of the present application.

[0085] Figures 5a to 5c are drawings showing the effect of macrophage activation by a strain according to an example of the present application.

[0086] Figures 6a to 6e are drawings showing the effect of restoring immunity in a subject with weakened immunity by a strain according to an example of the present application.

[0087] Figures 7a to 7c are drawings showing the effect of improving the intestinal microbiome by a strain according to an example of the present application.

[0088]

[0089] Hereinafter, the present application will be described in more detail with reference to the following examples. However, these examples are merely intended to illustrate the present application, and the scope of the present application is not limited by these examples.

[0090]

[0091] Example 1. Immune cell activation effect of Lactobacillus fermentum KBL375 strain

[0092] NK cells, a representative immune cell of innate immunity, detect and directly attack abnormal cells, such as cancer cells and virus-infected cells. The spleen is the primary immune organ responsible for the immune response to blood-borne antigens and is home to various immune cells, including T cells, B cells, and NK cells. Therefore, we evaluated the apoptotic activity of Yac-1 cells, a target cell of NK cells isolated from the spleen.

[0093] The spleen of a 5-week-old BALB / c male mouse was removed and kept in RPMI-1640 medium containing 10% FBS and 1% penicillin and kept cold. The ground suspension of the removed spleen was centrifuged at 400 x g for 4 minutes. The supernatant was removed, and 1 mL of red blood cell lysis buffer was added and reacted in a cold state for 3 minutes. The reaction was terminated by adding 10 mL of medium, and the splenocytes collected by centrifugation were suspended in the medium and passed through a 30 μm cell strainer once more. After measuring the cell number using a cell counter, 4 × 10 spleen cells were seeded in a V-bottom 96-well plate. 5 cells / well, 8 × 10 5 100 μL was dispensed per cell / well.

[0094] Lactobacillus fermentum KBL375 strain with accession number KCTC 13381BP was provided by GoBioLab Co., Ltd. The Lactobacillus fermentum KBL375 strain to spleen cell number ratio was prepared as 1:1, 10:1, or 20:1, and 100 μL was dispensed per well and cultured with spleen cells for 24 hours at 37°C and 5% CO2.

[0095] Yac-1 cells, which are target cells of NK cells, were centrifuged (400 × g, 4 min), the supernatant was removed, and the cells were suspended in a medium to create a cell suspension, and the cell number was measured. 5 × 10 6Yac-1 cells were washed at 400 × g for 4 min, resuspended in 1 mL DPBS, and mixed well with 1 mL of 10 uM CTV. After incubation for 15 min in a cell incubator at 37°C and 5% CO2 in a light-shielded state, 10 mL of medium was added, and the cells were collected by centrifugation (400 × g for 4 min). After staining, Yac-1 cells were suspended in a medium containing mouse recombinant IL-15 (NK cell-stimulating cytokine) diluted to 10 ng / mL, and the cell number was measured to be 2 × 10 5 It was prepared at cell / mL.

[0096] Splenocytes cultured with Lactobacillus fermentum KBL375 strain (accession number KCTC 13381BP) for 24 hours were centrifuged at 1,800 rpm for 3 minutes to remove the supernatant. 200 μL of CTV-stained Yac-1 cell suspension was aliquoted and co-cultured in a cell incubator (37°C, 5% CO2) for 4 hours. The supernatant was then removed by centrifugation at 1,800 rpm for 3 minutes. 100 μL of flow cytometry staining buffer was dispensed for washing, and 0.4 μL of 7-AAD (A9400, Sigma) was dispensed per well. 5 minutes of reaction in a darkened environment was followed by measuring the degree of Yac-1 cell death caused by NK cells using a flow cytometer. Statistical significance was verified using two-way ANOVA and Tukey's multiple comparisons test.

[0097] Table 1 and Figure 1 show the results of measuring the degree of Yac-1 cell death after treating splenocytes with the Lactobacillus fermentum KBL375 strain at a ratio of 10 times and 20 times (relative to the number of splenocytes) and co-culturing Yac-1 cells at a ratio of 1 / 10 and 1 / 20 relative to the number of splenocytes. As shown in Table 1 and Figure 1, in the results of co-culturing splenocytes and target cells at a ratio of 10:1 and 20:1, the activity of NK cells increased in a concentration-dependent manner depending on the strain treatment according to an example of the present application, confirming the excellent immune-enhancing effect of the Lactobacillus fermentum KBL375 strain according to an example of the present application.

[0098] KBL375 and splenocyte ratios, splenocyte and Yac-1 cell ratios, 10:120:1, control (Con) 13.24±0.39, 23.62±1.40, 1:132.50±1.70, 31.89±1.70, 10:135.18±1.47, 34.68±2.10, 20:138.12±0.77, 35.81±1.43

[0099]

[0100] Example 2. Effect of promoting NKp46 expression in NK cells

[0101] NKp46 (CD335) is one of the natural cytotoxicity receptors (NCRs), which are activating receptors of NK cells. NK cells with high expression of NCRs have been reported to have higher cytotoxic capacity than cells with low expression. Therefore, after treating spleen cells with the Lactobacillus fermentum KBL375 strain, the level of NKp46 expression in NK cells was evaluated.

[0102] Splenocytes extracted from mice were prepared as described above, and the number of cells in the suspension was measured, and then 2 × 10 6100 μL was dispensed into a V-bottom 96-well plate so that 100 cells / well was obtained. Lactobacillus fermentum KBL375 strain was diluted to strain-to-spleen cell ratios of 1:1, 10:1, and 20:1, dispensed into each well at 100 μL, and cultured in a cell incubator (37°C, 5% CO2) for 24 h. After centrifugation at 1,800 rpm for 3 minutes, the supernatant was removed, and 0.8 μL of Fc block was diluted in 20 μL of flow cytometry staining buffer and reacted. Afterwards, 0.2 μL each of PE anti-mouse CD335 (NKp46) Antibody and Brilliant Violet 510™ anti-mouse CD3 Antibody (BIoLegend) were diluted in 20 μL of flow cytometry staining buffer, dispensed, and reacted at room temperature for 30 minutes in a light-shielded state. Afterwards, 100 μL each of flow cytometry staining buffer was added, centrifuged at 1800 rpm for 3 minutes, the supernatant was removed, and the cells were resuspended in 100 μL of flow cytometry staining buffer. The expression of NK cell surface receptors was measured using a flow cytometer, and the proportion (%) of CD3-NKp46+ cells among total spleen cells was calculated. Statistical significance was verified using one-way ANOVA and Tukey's multiple comparisons test.

[0103] As shown in Table 2 and Figure 2, the proportion of CD3-NKp46+ cells significantly increased when the Lactobacillus fermentum KBL375 strain was treated. Accordingly, the Lactobacillus fermentum KBL375 strain according to an example of the present application had the effect of significantly activating NK cells.

[0104] KBL375 and spleen cell ratio CD3-NKp46+ cells ratio (%) Control group (Con) 3.24±0.13 1:13.52±0.03 10:13.90±0.11 20:14.02±0.11

[0105]

[0106] Example 3. Effect of increasing expression of apoptosis-inducing proteins

[0107] A key mechanism by which NK cells kill target cells involves the penetration of granular proteins, such as perforin and granzymes, secreted by NK cells. Perforin, secreted by NK cells, punctures the target cell wall, attracting granzymes to the cell, inducing apoptosis. Granzyme B, in particular, has been reported to have the strongest target cytotoxicity.

[0108] Single-celled splenocytes were suspended in a medium containing 10 ng / mL of mouse recombinant IL-15, a NK cell-stimulating cytokine. The cell number was measured and 2.5 × 10 were seeded in a 12-well plate. 6Cells / well were dispensed, and Lactobacillus fermentum KBL375 strain with accession number KCTC 13381BP was diluted to strain-to-spleen cell ratios of 1:1, 10:1, and 20:1, dispensed 100 μL per well, and cultured in a cell incubator (37℃, 5% CO2) for 24 h. RNA was extracted and quantified using an RNA Extraction Kit (BIONEER), and cDNA was synthesized using a qPCR RT master Mix (TOYOBO). The synthesized cDNA was mixed with primers specific for GAPDH, Perforin (Prf), and Granzyme B (GzmB), RNA-free water, and SYBR Green Realtime PCR Master Mix (TOYOBO), and qRT-PCR was performed, and quantified using the ΔΔCt method. Each gene expression was corrected for the value measured by GAPDH expression of the same sample, and statistical significance was verified using One-way ANOVA and Dunnett's multiple comparisons test.

[0109] As shown in Table 3, Figures 3a and 3b, the expression of Prf1 and GzmB increased at all concentrations treated with the strain according to an example of the present application, confirming the excellent NK cell activation effect of the Lactobacillus fermentum KBL375 strain according to an example of the present application.

[0110] KBL375 and NK cell ratio Relative Prf1 mRNA level Relative GzmB mRNA level Control group (Con) 1.00±0.25 1.00±0.07 1:16.30±0.88 1.43±0.08 10:13.37±0.5 11.42±0.06 20:14.21±0.77 1.54±0.15

[0111]

[0112] Example 4. Effect of promoting cytokine production by immune cells

[0113] The spleen contains various immune cells, including T lymphocytes, B lymphocytes, and macrophages. Therefore, the effect of Lactobacillus fermentum KBL375 strain on stimulating immune cell cytokine production was evaluated.

[0114] Single-celled spleen cells were seeded at 5 × 10 in a 96-well plate. 5 After dispensing 100 μL of the Lactobacillus fermentum KBL375 strain with the accession number KCTC 13381BP, the strain to the number of splenocytes was diluted to a ratio of 1:1, 10:1, and 20:1, and 100 μL was dispensed per well. After culturing for 72 hours in a cell incubator (37°C, 5% CO2), the amount of IFN-γ in the supernatant was measured using an ELISA Kit (BD). Statistical significance was verified using One-way ANOVA and Tukey's multiple comparisons test.

[0115] As shown in Table 4 and Figure 4, IFN-γ production was promoted at all concentrations treated with the strain according to an example of the present application, confirming the excellent immune enhancement effect of the Lactobacillus fermentum KBL375 strain according to an example of the present application.

[0116] KBL375 and NK cell ratio IFN-γ production (pg / mL) Control (Con) 7.62±5.8 21:118 6.61±20.80 10:118 2.32±23.4 220:117 8.21±49.04

[0117]

[0118] Example 5. Macrophage activation effect

[0119] Macrophages activated by external stimuli produce excessive amounts of nitric oxide (NO) and other substances, while also promoting the production of pro-inflammatory cytokines such as IL-6 and TNF-α, which in turn promote the immune response of other immune cells. Excessive NO kills pathogens, IL-6 promotes cell-mediated immune responses and the production of additional anti-inflammatory cytokines, and TNF-α functions as an important cytokine in the initial response to viral infection. Accordingly, the effect of the Lactobacillus fermentum KBL375 strain according to an example of the present application on promoting macrophage cytokine production was confirmed using the mouse-derived macrophage RAW 264.7 cell line.

[0120] RAW 264.7 cells purchased from the American Type Culture Collection (ATCC) were cultured in a cell incubator (37°C, 5% CO2) using a medium containing DMEM (LM001-05, Welgene), 10% FBS (S1480, Biowest), and 1% penicillin-streptomycin (LS202-02, Welgene). 2 × 10 RAW 264.7 cells were seeded in a 48-well plate. 5After dispensing 500 μL at cells / mL, it was stabilized in a cell incubator (37℃, 5% CO2) for 24 hours. After removing the existing culture medium, Lactobacillus fermentum KBL375 strain with accession number KCTC 13381BP was diluted to strain to RAW 264.7 cell ratios of 1:1, 10:1, and 20:1, and then cultured in a cell incubator (37℃, 5% CO2) for 72 hours. Afterwards, the culture supernatant was recovered, and the amounts of IL-6 and TNF-α were measured using an ELISA Kit (BD). An equal amount of Griess reagent (Sigma-Aldrich) was added to 100 uL of the culture supernatant, and the reaction was carried out at room temperature for 10 minutes in a light-shielded manner. The NO content was measured by comparing the absorbance at 540 nm with the standard solution. Statistical significance was verified using one-way ANOVA and Dunnett's multiple comparisons test as a post hoc test.

[0121] As shown in Table 5, FIG. 5a, FIG. 5b and FIG. 5c, when the Lactobacillus fermentum KBL375 strain according to an example of the present application was treated, the secretion of NO and cytokines from macrophages increased in a concentration-dependent manner, confirming the excellent macrophage activation effect of the Lactobacillus fermentum KBL375 strain according to an example of the present application.

[0122] KBL375 and NK cell ratio NO (μM) IL-6 (pg / mL) TNF-α (pg / mL) Control (Con) 5.28±0.09 5 1.94±1.8 12 10.95±7.2 1 1:15.30±0.09 6 6.75±1.92 2 6 5.56±8.80 10:15.49±0.09 1 3 7.78±7.5 7 5 5 4.60±33.2 7 20:16.33±0.09 1 8 3.25±7.9 3 6 5 1.75±4.70

[0123]

[0124] Example 6. Effect of improving immune function in an immunocompromised animal model

[0125] (1) Establishment of an immunocompromised animal model

[0126] To verify the immune function-enhancing effects of the Lactobacillus fermentum KBL375 strain, a cyclophosphamide (CP)-induced immunosuppressed mouse model was established, and the Lactobacillus fermentum KBL375 strain was administered to verify whether immune function was enhanced. This animal experiment was approved by the Animal Experiment Committee of the Korea Food Research Institute (KFRI-M-23014) and conducted in accordance with the standard operating procedures of the Korea Food Research Institute.

[0127] Four-week-old male BALB / c mice purchased from Orient Bio were acclimatized for a week and then divided into four groups of 10-15 mice each [Naive, negative control (CP), Lactobacillus fermentum KBL375 administration group (hereinafter referred to as KBL375 administration group), positive control (RG)]. Samples and sterilized drinking water were orally administered daily from 14 days before CP treatment until the day before sacrifice. During the experimental period, the mice were raised in an environment at 24℃ and 60% humidity, and drinking water and feed were provided in the form of free-feeding. The Naive and CP groups were administered 200 μL of sterilized drinking water each, and the RG group was administered 200 mg / kg of red ginseng concentrate. The red ginseng concentrate used was a product of Daedong Korea Sam Co., Ltd., containing 12.7 mg / g of the sum of ginsenosides Rg1, Rb1, and Rg3. The KBL375 administration group was administered 10 Lactobacillus fermentum KBL375 strain with the deposit number KCTC 13381BP. 9 CFU / head were diluted in sterilized drinking water and administered in 200 μL portions. All groups except the Naive group were administered CP (150 mg / kg / day) intraperitoneally on days 15 and 16 from the start of oral administration, and sacrificed on days 6 and 9 from the last CP administration. All mice were sacrificed by isoflurane overdose, and blood, spleen, thymus, and mesenteric lymph node (MLN) tissues were extracted for analysis.

[0128]

[0129] (2) Analysis of relative weight of the spleen

[0130] Spleens removed from mice were washed with saline, dried with filter paper, and weighed. The measured value was divided by the body weight of each individual to express it as a relative weight (Mathematical Formula 1).

[0131] [Mathematical Formula 1]

[0132] Relative weight = tissue weight (mg) / body weight (g)

[0133] As shown in Table 6 and Fig. 6a, the relative weight of the spleen to body weight was recovered in the group administered the Lactobacillus fermentum KBL375 strain according to an example of the present application due to CP treatment, and in particular, the group administered the Lactobacillus fermentum KBL375 strain according to an example of the present application showed a significantly higher effect. This was an excellent effect even compared to the RG group, which is a positive control group.

[0134] GroupSpleen index(mg / g)Splenic NK cell activity(% of control)Serum IgA production(ng / mL)Naive3.11±0.2117.01±0.725,381.33±555.45CP2.15±0.1213.70±2.381,192.67±1 50.32CP+KBL3752.51±0.4016.85±0.582,057.33±568.79CP+RG2.13±0.0717.63±0.671,792.00±184.31

[0135]

[0136] (3) NK cell activity analysis

[0137] NK cells are cells that rapidly detect and eliminate abnormal cells in the innate immune response, and it is known that NK cell activity also decreases in the CP-induced immunosuppressed model. Therefore, to check the NK cell activity in each group, the spleen removed from the mouse was ground and passed through a 70 μm cell strainer, and then red blood cell lysis buffer was added and reacted in a cold state for 3 minutes. After passing it through a 30 μm cell strainer once more, the number of single cells was measured using a cell counter, and 2 × 10 spleen cells were seeded in a V-bottom 96-well plate. 5 100 μL was dispensed per well to ensure a cell / well ratio. After CTV staining of Yac-1 cells, which are target cells of NK cells, 2 × 10 5 After dispensing 100 μL per well, the cells were co-cultured for 4 hours in a cell incubator (37°C, 5% CO2). The supernatant was removed by centrifugation at 1,800 rpm for 3 minutes. After washing with 100 μL of flow cytometry staining buffer, 7-AAD (A9400, Sigma) was dispensed per well at 0.4 μL, and the cells were incubated for 5 minutes in a light-shielded environment. The degree of Yac-1 cell death due to NK cells was measured using a flow cytometer. Statistical significance was verified using one-way ANOVA and Dunnett's multiple comparisons test.

[0138] As shown in Table 6 and FIG. 6b, the activity of NK cells increased in the KBL375 administration group according to an example of the present application, confirming the excellent immune-enhancing effect of the Lactobacillus fermentum KBL375 strain according to an example of the present application.

[0139]

[0140] (4) Analysis of immunoglobulins in serum

[0141] Immunoglobulin (IG) is a key protein in humoral immunity and plays a role in protecting against infection. Therefore, the level of immunoglobulin in serum can be used to assess immune function activation. Accordingly, after administering the Lactobacillus fermentum KBL375 strain according to an example of the present application, the amount of IgA, a representative immunoglobulin, in serum was measured.

[0142] Mice were anesthetized using isoflurane inhalation, and blood samples were collected from the orbit using vacuum-assisted blood collection tubes. The collected blood was centrifuged (3,000 rpm, 4°C, 10 min) to separate serum. The amount of IgA in the serum was then measured using the IgA Mouse Uncoated ELISA Kit (Invitrogen).

[0143] As shown in Table 6 and Figure 6c, the amount of IgA in the CP group was significantly reduced compared to the control group, but it was confirmed to have recovered in the group administered the Lactobacillus fermentum KBL375 strain according to an example of the present application.

[0144]

[0145] (5) Increased proliferation of T cells and B cells in the spleen

[0146] A decline in the proliferation of T and B cells in the spleen is a representative indicator of immunodeficiency. Therefore, the proliferation of T and B cells was analyzed by treating spleen cells with the Lactobacillus fermentum KBL375 strain according to an example of the present application.

[0147] Single-celled spleen cells were seeded at 5 × 10 in an F-bottom 96-well plate. 5100 μL was dispensed to each well so that there were 1 cell / well. 100 μL of ConA, a T cell stimulator, and lipopolysaccharide (LPS), a B cell stimulator, were dispensed to each well so that the final concentration was 1 μg / mL. After culturing for 48 hours in a cell incubator (37°C, 5% CO2), the supernatant was collected, and 200 μL / well of a 10:1 mixture of medium and water-soluble tetrazolium salt (WST) reagent was dispensed to the remaining cells, and the cells were incubated in a light-shielded incubator (37°C, 5% CO2) for 2 hours, and the absorbance was measured at 450 nm.

[0148] As shown in Table 7, Figures 6d and 6e, the proliferation capacity of T cells and B cells in the spleen was decreased in the CP group compared to the normal group, but it was confirmed that it was significantly recovered in the group administered the Lactobacillus fermentum KBL375 strain according to an example of the present application.

[0149] GroupT cell proliferation(% of control)B cell proliferation(% of control)Naive100.00±7.65100.00±3.23CP49.72±1.9573.63±6.56CP+KBL37573.73±3.2089.76±3.86

[0150]

[0151] Example 7. Effect of improving the intestinal microbiome

[0152] Among the animal models of Example 6, mouse feces were collected on the day of completion of administration of the Lactobacillus fermentum KBL375 strain having the deposit number KCTC 13381BP, and DNA (final elution volume = 100 μL) was extracted using a QIAamp PowerFecal Pro DNA Kit (Qiagen), and stored at -20°C until use.

[0153] According to the 16S metagenomic sequencing library preparation Illumina protocol (part no. 15044223 rev. B) of Illuminia, a library was constructed for sequencing the V3-V4 region of the 16S ribosomal RNA-coding gene (16S rRNA gene), and sequencing was performed using the Illumina system (Illuminia). The sequencing data of the 16S rRNA gene (V3-V4 region) were preprocessed and presented as a feature table using the DADA2 pipeline of QIIME2 (QIIME 2 development team). Taxonomic classification was performed using the V3-V4 fragment classifier of Greengenes ver. 13_8 (99% operational taxonomic units).

[0154] Differences in relative abundance of genus and amplicon sequence variant (ASV) by group were analyzed using LEfSe (The Huttenhower Lab). Additionally, differences in individual genus groups were confirmed using the Kruskal-Wallis test and Dunn's multiple comparisons test. The correlation between the relative abundance of ASV and major biomarkers was confirmed using Spearman correlation.

[0155] The relative abundance of the average intestinal microbiome by group after administration of the strain according to an example of the present application is as shown in Fig. 7a. In particular, in the group administered the strain according to an example of the present application, the abundance of Lactobacillus and Bifidobacterium significantly increased, and the abundance of Bacteroides decreased. In addition, as shown in Fig. 7b, the LEfSe analysis results confirmed a significant increase in Bifidobacterium in the group administered the strain according to an example of the present application compared to the other groups.

[0156] In addition, as shown in Fig. 7c, when the difference in relative abundance between groups at the ASV level was compared using LEfSe analysis, an increase in the relative abundance of some Lactobacillus ASV and Bifidobacterium ASV was confirmed in the strain administration group according to an example of the present application. On the other hand, an increase in Bacteroides_0003 ASV, etc. was confirmed in the CP treatment group. As a result of confirming the correlation between the relative abundance of ASV showing significant differences between groups and major biomarkers, Bacteroides_0003 showed a negative correlation with spleen index, thymus index, and blood IgA. On the other hand, a pattern of decreasing Bacteroides_0003 ASV was confirmed in the strain administration group according to an example of the present application, confirming that administration of the strain according to an example of the present application has the effect of improving the intestinal microbiome, such as inducing an increase in Lactobacillus and Bifidobacterium, which are major beneficial bacteria, and reducing intestinal microorganisms that may have a negative effect on enhancing the host's immunity.

[0157]

[0158] [Accession number]

[0159] Name of depositor: Korea Research Institute of Bioscience and Biotechnology, Biological Resource Center (KCTC)

[0160] Accession number: KCTC13381BP

[0161] Date of acceptance: 20171023

[0162]

[0163]

Claims

1. An immune-enhancing composition comprising at least one selected from the group consisting of Lactobacillus fermentum KBL375 strain having the deposit number KCTC 13381BP, a culture of the strain, a lysate of the strain, and an extract of the strain.

2. A composition according to claim 1, which activates or matures immune cells or promotes proliferation of immune cells.

3. A composition according to claim 2, wherein the immune cells include natural killer cells and / or macrophages.

4. A composition according to claim 2, wherein maturation of the immune cells promotes NKp46 expression of the immune cells.

5. A composition according to claim 2, wherein activating the immune cells promotes the expression of apoptosis-inducing protein of natural killer cells.

6. A composition according to claim 5, wherein the cell death-inducing protein of the natural killer cell is perforin and / or granzyme B (GzmB).

7. A composition according to claim 2, wherein activating the immune cells promotes cytokine production by the immune cells.

8. A composition according to claim 2, wherein activating the immune cells promotes nitric oxide (NO) production by macrophages.

9. A composition according to claim 1, wherein the composition prevents a relative weight loss of the spleen due to immunosuppression.

10. A composition according to claim 1, wherein the composition increases the concentration of immunoglobulin in the blood.

11. A composition according to claim 1, wherein the composition improves the intestinal microbiome.

12. A composition according to claim 1, wherein the composition increases the relative abundance of strains of the genus Bifidobacterium and / or the genus Lactobacillus in the intestine.

13. A composition according to claim 1, wherein the composition reduces the relative abundance of strains of the genus Bacteroides in the intestine.

14. A composition for improving the intestinal microbiome, comprising at least one selected from the group consisting of Lactobacillus fermentum KBL375 strain having the deposit number KCTC 13381BP, a culture of the strain, a lysate of the strain, and an extract of the strain.

15. A food comprising a composition according to any one of claims 1 to 14.

16. A health functional food comprising a composition according to any one of claims 1 to 14.

17. A quasi-drug comprising a composition according to any one of claims 1 to 14.

18. A cosmetic comprising a composition according to any one of claims 1 to 14.

19. A pharmaceutical composition for preventing or treating immunodeficiency, comprising at least one selected from the group consisting of Lactobacillus fermentum KBL375 strain having the deposit number KCTC 13381BP, a culture of the strain, a lysate of the strain, and an extract of the strain.

20. In claim 19, the immunodeficiency is at least one selected from the group consisting of immunodeficiency due to use of an anticancer agent or immunosuppressant, leukopenia, neutropenia, lymphopenia, nutritional deficiency, diabetes, immunodeficiency after organ transplantation, immunodeficiency due to aging, immunodeficiency due to lifestyle-related causes, immunodeficiency due to long-term steroid use, primary immunodeficiency disease, herpes zoster, tuberculosis, meningitis, septic shock, and bacterial or viral infection.

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