Limocylic Lactobacillus reuteri strain and its immune-enhancing use

Abstract

The present application relates to a rimocylic Lactobacillus reuteri strain and its immune-enhancing uses.

Description

[Technical Field]

[0001] The present application relates to a rimocylic Lactobacillus reuteri strain and its immune-enhancing uses. [Background technology]

[0002] Immunity is a defense mechanism in which the internal environment of the body recognizes various substances that have invaded from the outside as foreign bodies, and eliminates and metabolizes them. However, immune function can be impaired due to various causes, and there is a demand for various immune-enhancing agents to prevent or treat this.

[0003] The intestinal microbiota that inhabits the human gastrointestinal tract provides various health benefits through close interaction with the host. Recently, the demand for probiotics has increased significantly worldwide, spurring the development of health functional foods and drugs that use probiotics. Summary of the Invention [Problem to be solved by the invention]

[0004] In this context, the present inventors have demonstrated that Limosilactobacillus reuteri KBL346 strain has the effect of activating immune cells and enhancing immunity. Therefore, one example of the present application provides an immune-enhancing use of Limosilactobacillus reuteri KBL346 strain, which can be used for the prevention and treatment of immunosuppression.

[0005] Furthermore, the present inventors have demonstrated that the Limosilactobacillus reuteri KBL346 strain has the effects of suppressing weight loss caused by influenza virus, reducing mortality, and improving pulmonary inflammation. Therefore, another example of the present application provides the anti-influenza virus activity and immune-enhancing efficacy of the Limosilactobacillus reuteri KBL346 strain, which can be used to prevent and improve infectious respiratory immune diseases, including influenza virus infections. [Means for solving the problem]

[0006] One example of the present application relates to the Limosilactobacillus reuteri KBL346 strain having accession number KCTC15268BP, its use for immune enhancement, its use for the prevention or treatment of immunosuppression, its use as an anti-influenza virus agent, or its use for the prevention or treatment of influenza virus infection. The strain may have a 16S rRNA sequence of SEQ ID NO: 3.

[0007] The present application is described in more detail below. One example of the present application relates to a composition for immune enhancement, comprising at least one selected from the group consisting of the Limosilactobacillus reuteri KBL346 strain having accession number KCTC15268BP, a culture of the strain, a homogenate of the strain, and an extract of the strain. In one embodiment of the present application, the bacterial strain according to the example of the present application was administered to macrophages, resulting in activation of the macrophages. Therefore, the immune enhancement is achieved by activating macrophages, and the composition may be an immune stimulator. The bacterial strain may specifically be a Limosilactobacillus reuteri subsp. reuteri strain.

[0008] Specifically, the composition may have one or more properties selected from the group consisting of the following (1) to (10): (1) promotion of macrophage phagocytosis, for example, macrophage phagocytosis of 1.1 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; (2) promoting macrophage nitric oxide secretion, for example, increasing macrophage nitric oxide secretion by 1.5 times or more, 2 times or more, 2.5 times or more, 3 times or more, 4 times or more, 5 times or more, 6 times or more, 7 times or more, 8 times or more, 8.5 times or more, or 9 times or more compared to an untreated control group; (3) Promotion of COX-2 expression in macrophages (4) Promotion of PGE2 secretion by macrophages, for example, the amount of PGE2 secreted by macrophages is 2 times or more, 3 times or more, 4 times or more, 5 times or more, 8 times or more, 10 times or more, 15 times or more, 20 times or more, 22 times or more, 25 times or more, 26 times or more, 27 times or more, 28 times or more, 29 times or more, or 30 times or more, compared to an untreated control group; (5) promoting cytokine production by macrophages, for example, promoting TNF-α and / or IL-6 production; (6) Enhanced expression and phosphorylation of NF-κB and / or IκBα in macrophages (7) Promoting MAPK activation in macrophages (8) Promotion of recovery from viral infections, for example, promotion of recovery from influenza virus infections, (9) Preventing weight loss due to immune deficiency, and (10) Increased mononuclear cells in the blood.

[0009] Another example of the present application relates to a food composition containing the immune-improving composition. The food may be a functional health food, such as meat, bread, chocolate, candy, jelly, snacks, confectionery, kimchi, soy sauce, cheese, dairy products, powder, beverage, or vitamin complex.

[0010] Another example of the present application relates to a quasi-drug composition containing the immune-improving composition, which may be in a form selected from the group consisting of toothpaste, gargle, mouth spray, oral ointment, mouthwash, mouth freshener, adhesive bandage, and pest (mosquito, tick, etc.) repellent.

[0011] Another example of the present application relates to a composition for preventing or treating immunocompromise, comprising at least one selected from the group consisting of Limosilactobacillus reuteri KBL346 strain having accession number KCTC15268BP, a culture of the strain, a homogenate of the strain, and an extract of the strain.

[0012] The immunocompromise occurs when immune cells are unable to defend against external pathogens or cancer cells due to a lack of immunity, and can be, for example, an immunodeficiency syndrome, specifically, a secondary or acquired immunodeficiency.

[0013] The immunocompromised condition may be one or more selected from the group consisting of shingles, tuberculosis, meningitis, bronchiolitis, and bacterial or viral infections, and the viral infection may be, for example, influenza virus infection.

[0014] The influenza virus is the main cause of influenza epidemics, commonly known as the "flu," and is known to cause approximately 5 million severe cases each year due to its extremely high contagiousness compared to other respiratory viruses. Influenza virus infection is accompanied by symptoms such as fever, cough, runny nose, and phlegm, and in those with a weak immune system, it can cause complications and has a high mortality rate, so caution is required.

[0015] Influenza viruses invade the upper respiratory mucosa, causing respiratory diseases. Viral particles released from an infected patient's respiratory tract through coughing and sneezing are transmitted through the respiratory tract of other people, affecting their lungs and airways. In contrast, norovirus is transmitted through the ingestion of contaminated food or drink, infecting small intestinal cells and thus has a completely different infection route from influenza viruses. While previously known anti-norovirus effects are achieved through a mechanism that inhibits infection by physical contact with small intestinal epithelial cells or norovirus, physical contact is unlikely to produce antiviral effects against influenza viruses. Therefore, due to differences in the mechanism of action, it is difficult to predict whether a substance with antiviral efficacy against norovirus will also have antiviral efficacy against influenza viruses.

[0016] Another example of the present application relates to an anti-influenza virus composition comprising at least one selected from the group consisting of Limosilactobacillus reuteri KBL346 strain having accession number KCTC15268BP, a culture of the strain, a homogenate of the strain, and an extract of the strain. In an embodiment of the present application, administration of the strain according to an example of the present application to an influenza virus infection model significantly reduced the severity and mortality of influenza virus infection.

[0017] Specifically, the composition may have one or more properties selected from the group consisting of the following (1) to (4): (1) Prevention of weight loss due to influenza virus infection, for example, weight loss after influenza virus infection of 75% or more, 79% or more, 80% or more, 81% or more, 82% or more, 82.5% or more, 84% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, or 94% or more, based on 100% weight before influenza virus infection (here, the weight after influenza virus infection can be measured as the weight on the 7th day after influenza infection), (2) a reduction in the mortality rate due to influenza virus infection, for example, a survival rate of 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, or 90% or more (wherein the survival rate may be the survival rate 21 days after influenza virus infection); (3) Improvement of lung inflammation, and (4) Decreased blood immunoglobulin G (IgG), for example, blood immunoglobulin G levels of 0.9 times or less, 0.8 times or less, 0.7 times or less, or 0.65 times or less compared to untreated controls.

[0018] Another example of the present application relates to a composition for preventing or treating influenza virus infection, comprising at least one selected from the group consisting of Limosilactobacillus reuteri KBL346 strain having accession number KCTC15268BP, a culture of the strain, a homogenate of the strain, and an extract of the strain.

[0019] The influenza virus infection may be one or more selected from the group consisting of flu, pneumonia, Reye's syndrome, acute respiratory failure, myocarditis, bronchitis, otitis media, pharyngitis, sinusitis, empyema, Guillain-Barré syndrome, and encephalitis.

[0020] The symptoms of influenza virus infection may be one or more selected from the group consisting of fever, chills, nausea, muscle pain, fatigue, cough, dyspnea, shortness of breath, pneumonia, phlegm, sore throat, headache, hemoptysis, and diarrhea.

[0021] In the present application, the term "strain" may refer to live or heat-inactivated strains of a strain according to an example of the present application.

[0022] The term "strain culture" as used herein refers to a product obtained after culturing a strain according to an example of the present application. The culture may be a whole culture of a strain according to an example of the present application, a dilution, concentrate, dried product, lyophilized product, disrupted product, and / or a fraction thereof. 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. The disrupted product may be obtained by physically or sonicating the strain or culture. The fraction may be obtained by subjecting the culture or disrupted product to centrifugation, chromatography, or other methods. The culture may be in a solid (e.g., dried) state, a liquid (liquid), or a fluid state, but is not limited thereto. In one example, the culture may refer to a whole medium containing the cultured strain, its metabolites, and / or excess nutrients obtained by culturing a strain according to an example of the present application for a certain period of time. For example, the culture may or may not contain the strain according to an example of the present application. For example, the culture may refer to the remaining components obtained by culturing a strain according to an example of the present application in a medium, excluding the strain (bacterial cells). For example, the culture may be a culture broth (or culture product) obtained by culturing a strain according to an example of the present application in a medium and removing the strain (bacterial cells). The culture broth (or culture product) obtained by culturing a strain according to an example of the present application in a medium may be a cell-free culture broth (or culture product) or a culture broth containing dead cells, for example, a filtrate (supernatant after centrifugation) obtained by removing the strain, and / or a culture broth (or dried culture product) containing dead cells. Specifically, the culture may exhibit anti-inflammatory activity or preventive, ameliorative, or therapeutic activity for inflammatory diseases at a level equivalent to that exhibited by the strain according to an example of the present application.

[0023] The term "lysate of a bacterial strain" as used herein may refer to a product obtained by disrupting a bacterial strain according to an example of the present application using chemical or physical forces. Specifically, the lysate may exhibit anti-inflammatory activity or preventive, ameliorative, or therapeutic activity for inflammatory diseases at a level equivalent to that exhibited by the bacterial strain according to an example of the present application.

[0024] The term "extract" used herein refers to a strain according to an example of the present application, a culture of the strain, a homogenate of the strain, or a mixture thereof, regardless of the extraction method, extraction solvent, extracted component, or extract form. It is a broad concept that includes all substances obtained by processing or treating the extracted material in other ways. For example, the extract may be an extract of a strain according to an example of the present application, an extract of a culture of the strain, or an extract of a homogenate of the strain. Specifically, the extract may exhibit anti-inflammatory activity or preventive, ameliorative, or therapeutic activity for inflammatory diseases at a level equivalent to that exhibited by a strain according to an example of the present application, a culture of the strain, or a homogenate of the strain.

[0025] The composition according to one example of the present application contains 1.0×10 Limosilactobacillus reuteri KBL346 strain having accession number KCTC15268BP. 3 ~1.0×10 15 CFU, 1.0 × 10 3 ~1.0×10 14 CFU, 1.0 × 10 3 ~1.0×10 13 CFU, 1.0 × 10 3 ~1.0×10 12 CFU, 1.0 × 10 3 ~1.0×10 11 CFU, 1.0 × 10 3 ~1.0×10 10 CFU, 1.0 × 10 5 ~1.0×10 15 CFU, 1.0 × 10 5 ~1.0×10 14 CFU, 1.0 × 10 5 ~1.0×10 13 CFU, 1.0 × 105 ~1.0×10 12 CFU, 1.0 × 10 5 ~1.0×10 11 CFU, 1.0 × 10 5 ~1.0×10 10 CFU, 1.0 × 10 7 ~1.0×10 15 CFU, 1.0 × 10 7 ~1.0×10 14 CFU, 1.0 × 10 7 ~1.0×10 13 CFU, 1.0 × 10 7 ~1.0×10 12 CFU, 1.0 × 10 7 ~1.0×10 11 CFU, 1.0 × 10 7 ~1.0×10 10 CFU, 1.0 × 10 8 ~1.0×10 15 CFU, 1.0 × 10 8 ~1.0×10 14 CFU, 1.0 × 10 8 ~1.0×10 13 CFU, 1.0 × 10 8 ~1.0×10 12 CFU, 1.0 × 10 8 ~1.0×10 11 CFU, or 1.0 x 10 8 ~1.0×10 10 It may be contained at a CFU concentration.

[0026] As used herein, the term "prevention" means inhibiting or delaying the onset of a disease, disorder, or condition. Prevention can be considered complete if the onset of a disease, disorder, or condition is inhibited or delayed for a predetermined period of time.

[0027] As used herein, the term 'treating' means partially or completely alleviating, ameliorating, alleviating, inhibiting or delaying the symptoms of a particular disease, disorder and / or condition or disorder, or reducing the severity or occurrence of one or more symptoms or characteristics thereof.

[0028] The compositions of the present application, such as pharmaceutical compositions or food compositions, may contain one or more additional active ingredients that exhibit the same or similar functions in addition to the active ingredients.

[0029] Furthermore, compositions according to the present application, such as pharmaceutical compositions or food compositions, can be prepared in unit dose forms or in multi-volume containers by formulating them with pharmaceutically acceptable carriers according to a method that can be clearly implemented by a person skilled in the art to which the present invention pertains. In this application, the term 'carrier' refers to a compound that facilitates the addition of a compound into cells or tissues, and the term 'pharmaceutically acceptable' refers to a composition that is physiologically acceptable and does not normally cause gastrointestinal disorders, allergic reactions such as dizziness, or similar reactions when administered to humans.

[0030] The pharmaceutically acceptable carriers are those commonly used in pharmaceutical 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.

[0031] In addition, the composition according to the present application, for example, a pharmaceutical composition or a food composition, may further contain additives such as fillers, anti-agglomerating agents, lubricants, humectants, flavorings, emulsifiers, preservatives, etc. In the present application, the content of the additives contained in the composition is not particularly limited, and can be appropriately adjusted within the content range used in conventional formulations.

[0032] Compositions according to the present application, such as pharmaceutical or food compositions, can also be formulated into oral preparations. Non-limiting examples of oral preparations include tablets, troches, lozenges, aqueous suspensions, oily suspensions, prepared powders, granules, emulsions, hard capsules, soft capsules, syrups, and elixirs. To formulate pharmaceutical or food compositions according to the present application for oral administration, binders such as lactose, sucrose, sorbitol, mannitol, starch, amylopectin, cellulose, or gelatin; excipients such as dicalcium phosphate; disintegrants such as corn starch or sweet potato starch; magnesium stearate, calcium stearate, and sodium stearyl fumarate can be used. Sweeteners, flavorings, and syrups can also be used. Furthermore, in the case of capsules, liquid carriers such as fatty oils can be used in addition to the above-mentioned substances.

[0033] As used herein, the term 'excipient' refers to a substance, other than a therapeutic agent, that is used as a carrier or vehicle for delivery of a therapeutic agent or that is added to a pharmaceutical composition to improve handling and storage characteristics or to permit and facilitate the formation of a unit dose of the composition.

[0034] The compositions according to the present application, for example, pharmaceutical compositions, can be formulated into various forms such as oral dosage forms such as solutions, suspensions, powders, granules, tablets, capsules, pills, extracts, emulsions, syrups, aerosols, and sterile injectable solutions, according to conventional methods depending on the intended use, and can be administered orally or via various routes including intravenous, intraperitoneal, subcutaneous, rectal, topical, etc. In the present application, the term 'oral administration' means that the active substance is administered to the gastrointestinal tract for absorption, i.e., a substance prepared to be digested.

[0035] The preferred dosage of the composition according to the present application, for example, a pharmaceutical composition or a food composition, varies depending on the patient's condition and body weight, age, sex, health condition, specific dietary habits, properties of the formulation, severity of the disease, administration time of the composition, administration method, administration period or interval, excretion rate, and drug form, and can be appropriately selected by a person skilled in the art.

[0036] The term "effective dose" as used herein refers to an amount of a composition of an active ingredient sufficient to treat a specific symptom. This amount varies depending on the formulation method, mode, time, and / or route of administration of the pharmaceutical or food composition, the type and degree of response to be achieved by administration of the pharmaceutical or food composition, the type, age, weight, general health condition, symptoms and degree of disease, sex, diet, excretion, drugs or other components of the composition used simultaneously or concomitantly with the individual, and other similar factors well known in the medical field. A person of ordinary skill in the art can easily determine and prescribe an effective dose for the intended treatment.

[0037] The pharmaceutical or food composition of the present application can be administered once a day or in divided doses. The composition can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents. Taking all of the above factors into consideration, the composition can be administered in an amount that can achieve maximum effect with the minimum amount without side effects.

[0038] 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, 0.0 01~0.01mg, 0.01~10,000mg, 0.01~5,000mg, 0.01~1,000mg, 0.01~500mg, 0.01~300mg, 0.01~100mg, 0.01~50mg, 0.01 ~30mg, 0.01~10mg, 0.01~5mg, 0.01~1mg, 0.01~0.5mg, 0.01~0.1mg, 0.01~0.05mg, 0.1~10,000mg, 0.1~5,000mg, 0.1~ 1,000mg, 0.1~500mg, 0.1~300mg, 0.1~200mg, 0.1~100mg, 0.1~50mg, 0.1~30mg, 0.1~10mg, 0.1~5mg, 0.1~1mg, 0.1~0. 5mg, 1~10,000mg, 1~5,000mg, 1~1,000mg, 1~500mg, 1~300mg, 1~200mg, 1~100mg, 1~50mg, 1~10mg, 1~5mg, 10~10,000m The composition may be administered in a daily dose of, but not limited to, 10-5,000 mg, 10-1,000 mg, 10-500 mg, 10-300 mg, 10-200 mg, 10-100 mg, 10-50 mg, 10-40 mg, 10-30 mg, 10-20 mg, 100-10,000 mg, 100-5,000 mg, 100-1,000 mg, 100-500 mg, 100-300 mg, or 100-200 mg. For example, the daily oral dose of the composition of the present application may be 0.001-10 g / day, 0.001-5 g / day, 0.01-10 g / day, or 0.01-5 g / day for an adult patient. The total daily dose may also be divided and administered continuously or discontinuously as needed.

[0039] Another example of the present application relates to a method for enhancing immunity, comprising administering to a subject one or more selected from the group consisting of the Limosilactobacillus reuteri KBL346 strain having accession number KCTC15268BP, a culture of the strain, a homogenate of the strain, and an extract of the strain.

[0040] Another example of the present application relates to a method for preventing or treating influenza virus infection, comprising administering to a subject one or more selected from the group consisting of Limosilactobacillus reuteri KBL346 strain having accession number KCTC15268BP, a culture of the strain, a homogenate of the strain, and an extract of the strain. [Effects of the Invention]

[0041] The present application has confirmed that oral administration of live and heat-treated Lactobacillus reuteri KBL346 bacteria has the effect of reducing body weight and increasing survival rates in animals infected with influenza, and has also verified that it brings about changes in key inflammation- and recovery-related indicators. Furthermore, it has been verified that the strain according to one example of the present application has immune-enhancing activity. Therefore, the strain has strong immune-enhancing activity and anti-influenza effects, and can be widely used in the development of health functional foods and therapeutic agents for the prevention and treatment of influenza infectious diseases or immune-related diseases. [Brief explanation of the drawings]

[0042] [Figure 1] FIG. 1 is a diagram confirming the cytotoxicity of a strain according to an example of the present application. [Figure 2a] FIG. 1 is a diagram confirming that the phagocytic ability of macrophages was improved by a bacterial strain according to an example of the present application. [Figure 2b] This is a continuation of Figure 2a. [Figure 3a] FIG. 10 is a diagram confirming that the iNOS expression level in macrophages was increased by a bacterial strain according to an example of the present application. [Figure 3b] FIG. 1 is a diagram confirming that the amount of NO secreted in macrophages was increased by a bacterial strain according to an example of the present application. [Figure 4a] FIG. 10 is a diagram confirming that the expression level of COX-2 in macrophages was increased by a bacterial strain according to an example of the present application. [Figure 4b] FIG. 1 is a diagram confirming that the amount of PGE2 secreted in macrophages was increased by a bacterial strain according to an example of the present application. [Figure 5a] FIG. 1 is a diagram confirming that the bacterial strain according to an example of the present application increased cytokine production in macrophages. [Figure 5b] This is a continuation of Figure 5a. [Figure 6] FIG. 1 is a diagram confirming that the expression of NF-κB and the expression and phosphorylation levels of IκBα in macrophages were increased by a bacterial strain according to an example of the present application. [Figure 7] FIG. 10 is a diagram confirming that the phosphorylation levels of JNK, ERK, and p38 proteins in macrophages were increased by a bacterial strain according to an example of the present application. [Figure 8a] FIG. 1 is a graph showing changes in body weight in an influenza-infected animal model with a 0.5 median lethal dose administered with live bacteria of a strain according to an example of the present application. [Figure 8b] FIG. 1 is a graph showing the body weight on day 7 of an influenza-infected animal model administered with live bacteria of a strain according to an example of the present application at a median lethal dose of 0.5, compared with the body weight on the day of infection. [Figure 8c] FIG. 1 is a graph showing the survival rate of an influenza infection animal model in which live bacteria of a strain according to an example of the present application were administered at a median lethal dose of 0.5. [Figure 9a] FIG. 1 is a graph showing changes in body weight in an influenza-infected animal model with a 0.5 median lethal dose administered with heat-inactivated bacteria of a bacterial strain according to an example of the present application. [Figure 9b] FIG. 10 is a graph showing the body weight on day 7 of an influenza-infected animal model administered with heat-inactivated bacteria of a strain according to an example of the present application at a median lethal dose of 0.5, compared with the body weight on the day of infection. [Figure 9c]FIG. 1 is a graph showing the survival rate of an influenza-infected animal model with a 0.5 median lethal dose administered with heat-inactivated bacteria of a bacterial strain according to an example of the present application. [Figure 10a] FIG. 1 is a graph showing changes in body weight in an animal model of influenza infection with a median lethal dose administered with a strain according to an example of the present application. [Figure 10b] FIG. 10 is a graph showing the body weight on day 7 of an influenza infection animal model administered with a 4 median lethal dose of a bacterial strain according to an example of the present application, compared with the body weight on the day of infection. [Figure 10c] FIG. 1 shows the survival rate of an animal model of influenza infection with a 4 median lethal dose administered with a strain according to an example of the present application. [Figure 11a] FIG. 1 is a graph showing the level of IFN-γ expression in lung tissue of an influenza-infected animal model administered with a strain according to an example of the present application. [Figure 11b] FIG. 1 is a graph showing the level of TLR2 expression in lung tissue of an influenza-infected animal model administered with a strain according to one example of the present application. [Figure 11c] FIG. 1 is a graph showing the level of ADAMTS4 expression in lung tissue of an influenza-infected animal model administered with a strain according to an example of the present application. [Figure 12] FIG. 1 shows blood IgG concentrations in an influenza-infected animal model administered with a strain according to an example of the present application. [Figure 13] FIG. 1 shows the results of observing lung tissue of an influenza-infected animal model administered with a bacterial strain according to an example of the present application. [Figure 14a] FIG. 1 is a graph confirming the weight loss prevention effect of a bacterial strain according to an example of the present application in an immunocompromised animal model. [Figure 14b] FIG. 10 is a diagram confirming the blood mononuclear cell increasing effect of a bacterial strain according to an example of the present application in an immunocompromised animal model. DETAILED DESCRIPTION OF THE INVENTION

[0043] The present application will be described in more detail below with reference to the following examples, but these examples are for illustrative purposes only and do not limit the scope of the present application. [Example]

[0044] Example 1. Confirmation of cytotoxicity of Lactobacillus reuteri KBL346 strain Fecal samples from 3-month-old infants were provided through Samsung Medical Center, Seoul, and streak-inoculated onto selective medium (TOS-propionate agar medium; 43314 Transgalactosylated oligosaccharide agar medium, Fluka). The samples were then cultured at 37°C in an anaerobic environment for 48 hours, after which the bacteria were isolated by colony picking and pure culture.

[0045] The isolated strain was cultured in a CO2 incubator at 37°C under an anaerobic environment for one day, then centrifuged at 13,000 rpm at 4°C to obtain the isolated strain. A 0.2 ul pellet of the strain was then placed in 25 ul of lysis buffer (pH 10; 2.5 M NaCl, 100 mM EDTA, 10 mM Trizma base, 1% Triton X-100) and heat-treated at 95°C for 10 minutes to obtain a template. The V4 region of the 16S rRNA gene was then amplified by polymerase chain reaction using the G-Taq PCR kit to obtain a PCR product. The nucleotide sequences of the primers used are shown in Table 1. In the primer sequences below, "M" is selected from A or C, and "Y" is selected from C or T.

[0046] [Table 1]

[0047] The PCR products were purified using an Ultra Clean PCR clean-up Kit (Mobio Laboratories Inc.), and the strains were identified by requesting Macrogen Co., Ltd. for gene sequence analysis. The strains were confirmed to be Limosilactobacillus reuteri and Lacticaseibacillus rhamnosus, and named Limosilactobacillus reuteri KBL346 and Lacticaseibacillus rhamnosus KBL352, respectively. The 16S rRNA sequence of Limosilactobacillus reuteri KBL346 is shown in SEQ ID NO: 3, and the 16S rRNA sequence of Lacticaseibacillus rhamnosus KBL352 is shown in SEQ ID NO: 4.

[0048] The two strains were deposited at the Korean Collection for Type Culture, Korea Institute of Bioscience and Biotechnology, an international depository under the Budapest Treaty, and were assigned accession numbers KCTC15268BP and KCTC15267BP, respectively.

[0049] The results of examining cell viability when treating mouse macrophage cell line RAW264.7 with live Rimosylated Lactobacillus reuteri KBL346 bacteria at concentrations of 12.5 to 50 times are shown in Figure 1. As shown in Figure 1, treatment with up to 50 times the concentration of RAW264.7 cells did not affect macrophage viability, confirming that Rimosylated Lactobacillus reuteri KBL346 can be used without cytotoxicity.

[0050] Example 2. Effect of improving the phagocytic activity of macrophages The effect of a bacterial strain according to the present application on improving macrophage phagocytosis was analyzed using Candida albicans MYA-4788 (hereinafter referred to as C. albicans). Specifically, the macrophage cell line RAW264.7 was treated with live Lactobacillus reuteri KBL346 at a 50:1 ratio and co-cultured for 24 hours. The RAW264.7 cells were then washed twice with 1X PBS. As a positive control, RAW264.7 cells were treated with LPS at a concentration of 10 ng / mL.

[0051] The prepared RAW264.7 cells were treated with yeast-state C. albicans at a 10x concentration and incubated at 37°C for 1 hour. The RAW264.7 cells co-cultured with C. albicans were washed twice with 1X PBS, fixed with 2.5% glutaraldehyde, and stained with 0.5% methylene blue. The stained cells were observed under a microscope; phagocytosis was considered to have occurred if one or more C. albicans cells were visible within the macrophage. The microscopic observation results are shown in Figure 2a, and the phagocytic capacity of the macrophages was calculated as follows and shown in Figure 2b and Table 2: Phagocytic ability (%) = (number of cells that underwent phagocytosis) / (number of cells observed) × 100

[0052] [Table 2]

[0053] As shown in Figures 2a and 2b and Table 2, when live rimosyl Lactobacillus reuteri KBL346 cells were treated, a significant increase in the phagocytic ability of macrophages was confirmed.

[0054] Example 3. Effect of increasing iNOS expression and NO secretion in macrophages The mouse macrophage cell line RAW264.7 was treated with live Lactobacillus reuteri KBL346 bacteria at 6.3-50x concentrations, and the expression levels of iNOS, a protein involved in nitric oxide (NO) production, and the amount of NO secreted by macrophages were measured. As a positive control, LPS was treated at 10 ng / ml or 100 ng / ml, and the expression levels of the housekeeping protein β-actin were also monitored to ensure that the protein used was consistent across all samples. The iNOS expression levels are shown in Figure 3a, and the amount of NO secreted by macrophages is shown in Figure 3b and Table 3.

[0055] As shown in Figures 3a, 3b, and Table 3, the iNOS expression level and NO secretion amount increased depending on the level of treatment with the strain according to an example of the present application, which means that the strain according to an example of the present application is an immunostimulant that activates immune cells.

[0056] [Table 3]

[0057] Example 4. Confirmation of COX-2 expression and increased PGE2 secretion in macrophages The mouse macrophage cell line RAW264.7 was treated with a 3.1- to 50-fold gradient of live Lactobacillus reuteri KBL346 cells, and the expression levels of COX-2, a protein involved in PGE2 production, and the amount of PGE2 secreted by macrophages were measured. As a positive control, LPS was treated at 10 ng / ml or 100 ng / ml, and the expression levels of the housekeeping protein β-actin were also monitored to demonstrate that the protein used was consistent across all samples. The COX-2 expression levels are shown in Figure 4a, and the amount of PGE2 secreted by macrophages is shown in Figure 4b.

[0058] As shown in Figures 4a, 4b, and Table 4, COX-2 expression levels and PGE2 secretion levels increased depending on the level of treatment with the strain according to an example of the present application, which means that the strain according to an example of the present application is an immunostimulant that activates immune cells.

[0059] [Table 4]

[0060] Example 5. Increased cytokine production in macrophages The mouse macrophage cell line RAW264.7 was treated with live Lactobacillus reuteri KBL346 cells at 12.5-50 times the concentration, and the expression levels of TNF-α and IL-6 in the macrophages were measured. As a positive control, LPS was treated at a concentration of 10 ng / ml.

[0061] As shown in Figures 5a, 5b, and Table 5, the levels of TNF-α and IL-6 secretion in macrophages increased depending on the level of treatment with the strain according to an example of the present application, which means that the strain according to an example of the present application has the effect of enhancing immune function.

[0062] [Table 5]

[0063] Example 6. Effect of increasing expression and phosphorylation of NF-κB and IκBα in macrophages The mouse macrophage cell line RAW264.7 was treated with live Lactobacillus reuteri KBL346 cells at 10x or 50x concentrations, and the expression and phosphorylation of NF-κB and IκBα proteins were examined. As a control, LPS was treated at 10ng / mL or 100ng / mL, and the expression levels of the housekeeping protein β-actin were also monitored to ensure that the proteins used were consistent across all samples.

[0064] As shown in FIG. 6, it was confirmed that the treatment with the strain according to one example of the present application increased the expression of NF-κB and the expression and phosphorylation levels of IκBα, thereby activating immune competence.

[0065] Example 7. Effect of MAPKs activation in macrophages The mouse macrophage cell line RAW264.7 was treated with live Lactobacillus reuteri KBL346 bacteria to examine the expression and phosphorylation levels of MAPKs (p38, ERK, JNK, etc.), a signaling pathway important for immune cell activation through the promotion of NO production in immune cells and the production of cytokines such as TNF-α and IL-6. As a control, LPS was treated at 10 ng / mL or 100 ng / mL, and the expression level of the housekeeping protein β-actin was also observed to demonstrate that the protein used was consistent across all samples.

[0066] As shown in FIG. 7, treatment with the strain according to an example of the present application increased the phosphorylation levels of JNK, ERK, and p38 proteins, confirming that immune competence was activated.

[0067] Example 8.0.5 Improved Severity and Survival Rate in an Animal Model of Influenza Infection with a Median Lethal Dose (1) The freeze-dried powders of the culture broth of the Lactobacillus reuteri KBL346 strain obtained in Example 1 and the Lacticase Bacillus rhamnosus strain with accession number KCTC15267BP were prepared, and the bacterial cell count was verified by CFU assay. The concentration of each strain was 5.0 × 10 9 The freeze-dried culture powder was diluted with PBS to a concentration of CFU / ml.

[0068] Eight-week-old female Balb / c mice were inoculated with 1.0 × 10 9 The diluted solution was orally administered in 200 μL portions once a day until the end of the experiment to achieve a CFU concentration. After administration of the strain for one week, the mice were given a 0.5 median lethal dose (0.5LD) via the nasal cavity after respiratory anesthesia. 50 ) of PR8 influenza virus was administered to induce infection. After infection, each strain was administered at a concentration of 1.0 × 10 9 The animals were orally administered once daily at a CFU concentration.

[0069] After the start of administration of the strain, the mice were weighed daily, and the weight changes are shown in Figure 8a, with the weight on the day of influenza infection being 100%. The weight on the day of influenza infection and the weight on day 7 after infection are compared and shown in Figure 8b and Table 6. The survival rates of the mice after the end of the experiment are shown in Figure 8c and Table 7.

[0070] [Table 6]

[0071] [Table 7]

[0072] As shown in Figures 8a and 8b and Table 6, the strain according to an example of the present application was effective in preventing weight loss caused by influenza virus infection and reducing the severity of influenza virus infection. Furthermore, as shown in Figure 8c and Table 7, the strain according to an example of the present application significantly reduced the mortality rate caused by influenza virus infection.

[0073] Example 9.0.5 Improved Severity and Survival Rate in an Influenza Infection Animal Model with a Median Lethal Dose (2) The Lactobacillus reuteri KBL346 strain obtained in Example 1 was heat-inactivated at 55°C for 30 minutes, and then the same experiment as in Example 8 was performed using heat-inactivated (hi) cells or live cells. The weight change was calculated based on 100% of the weight on the day of influenza infection. Figure 9a shows the weight change on the 7th day after infection compared to the weight on the day of influenza infection. Figure 9b and Table 8 show the weight change. The survival rate of mice after the experiment is shown in Figure 9c and Table 9.

[0074] [Table 8]

[0075] [Table 9]

[0076] As shown in Figures 9a and 9b, and Table 7, the bacterial strain according to an example of the present application, even when heat-inactivated, prevented weight loss due to influenza virus infection and reduced the severity of influenza virus infection. In particular, when heat-inactivated, the bacterial strain maintained the same level of effectiveness as live bacteria. Therefore, it is not necessary to maintain the bacterial strain in a live state when used for immune enhancement or anti-influenza purposes, thereby reducing the cost of maintaining the live state. Furthermore, as shown in Figure 9c and Table 9, the heat-inactivated bacterial strain according to an example of the present application significantly reduced the mortality rate due to influenza virus infection.

[0077] Example 10.4: Improvement of severity and survival rate in a median lethal dose influenza infection-induced mouse model The same experiment as in Example 8 was carried out, and the strain was administered for one week, and the median lethal dose (4 LD 50 ) PR8 influenza virus was administered to induce infection, and the weight change, based on 100% of the weight on the day of influenza infection, is shown in Figure 10a. The weight comparison results on the 7th day after infection with the weight on the day of influenza infection are shown in Figure 10b and Table 10. The survival rate of mice after the end of the experiment is shown in Figure 10c and Table 11.

[0078] [Table 10]

[0079] [Table 11]

[0080] As shown in Figures 10a to 10c and Tables 10 to 11, the strain according to an example of the present application exhibited a significant effect of reducing the severity and mortality rate even when infected with influenza virus at four times the LD50.

[0081] Example 11. Effect of improving pulmonary inflammation In the influenza virus infection models with a 0.5 LD50 in Examples 8 and 9, lung tissues were collected on day 7 of infection and analyzed by qPCR to determine the expression levels of the inflammatory cytokine IFN-γ, the receptor TLR2 that mediates inflammatory responses, and the ADAMTS4 gene, which induces recovery during infection. The expression levels are shown in Figures 11a to 11c and Table 12 relative to the untreated control (PBS).

[0082] [Table 12]

[0083] As shown in Figures 8a to 8c and Table 12, it was confirmed that IFN-γ and TLR2 gene expression was suppressed and ADAMTS4 gene expression was induced in the groups administered live bacteria and heat-treated bacteria according to an example of the present application.

[0084] Example 12. Effect of reducing blood antibody (IgG) concentration In the 0.5 LD5 influenza infection model of Example 9, blood was collected from the heat-inactivated strain-administered animal model on day 7 of infection, and IgG concentrations were determined by ELISA. IgG specifically binds to the surface protein haemagglutinin (HA) of the PR8 influenza virus, and blood IgG concentrations represent the amount of influenza virus in plasma.

[0085] As shown in Figure 12 and Table 13, it was confirmed that the administration of the strain according to one example of the present application to an influenza infection model reduced blood IgG levels. Therefore, the strain according to one example of the present application has the effect of inducing rapid recovery from influenza virus infection.

[0086] [Table 13]

[0087] Example 13. Histopathological improvement effect In the 0.5 LD influenza virus infection models described in Examples 8 and 9, lung tissue was collected on day 7 of infection, fixed in 10% neutral buffered formalin, embedded (formalin fixed paraffin embedded (FPPE)), and then sliced ​​at 4 μm thickness for hematoxylin and eosin (H&E) staining. As a positive control, an animal model was administered oseltamivir at a concentration of 20 mg / kg starting one day before influenza virus infection. Lung tissue observation results are shown in Figure 13 (A: PBS-administered group; B: oseltamivir-administered group; C: L. reuteri-administered group; D: heat-inactivated L. reuteri-administered group).

[0088] As shown in Figure 13A, the PBS-treated group experienced excessive infiltration of immune cells, shown as blue dots, during lung infection, and histopathological symptoms such as rupture, bronchial epithelial necrosis, and atelectasis were observed. In contrast, as shown in Figure 13C and D, the group treated with a strain according to an example of the present application experienced relatively fewer of these symptoms, and lung tissue was well maintained at a level comparable to that of the group treated with oseltamivir (Figure 13B), a conventional influenza virus prophylactic and therapeutic agent.

[0089] Example 14. Immunity-boosting effect (1) Preparation of immunocompromised animal models Five-week-old male Balb / c mice were divided into groups and orally administered water, Lactobacillus reuteri KBL346 strain, or red ginseng concentrate once daily according to Table 14 until the end of the experiment. On days 15 and 16 of the experiment, cyclophosphamide was administered intraperitoneally to all mice except the naive group at a concentration of 150 mg / kg to induce immune suppression.

[0090] [Table 14]

[0091] (2) Prevents weight loss The body weight of the mice was measured once a day before oral administration from day 1 to the end of the experiment and is shown in Figure 14a. As shown in Figure 14a, the Lactobacillus reuteri KBL346 strain according to an example of the present application showed the effect of preventing weight loss due to immune depression.

[0092] (3) Increase in mononuclear cells After the experiment, blood was collected from the orbit of each mouse group, and the number of mononuclear cells in the blood was measured using a hematology analyzer. The results are shown in Figure 14b. As shown in Figure 14b, the number of mononuclear cells significantly increased in the group administered with the remosil Lactobacillus reuteri KBL346 strain according to an example of the present application, demonstrating an effect equal to or greater than that of red ginseng. Therefore, the remosil Lactobacillus reuteri KBL346 strain according to an example of the present application exhibits a significantly superior immune-boosting effect, inducing rapid recovery from immune depression. In Figure 14b, * indicates p<0.05; ** indicates p<0.01; *** indicates p<0.001; and **** indicates p<0.0001 compared to the control group. [Accession number]

[0093] Depository institution name: Korea Institute of Bioscience and Biotechnology Biological Resource Center Accession number: KCTC15268BP Date of acceptance: 20230104 TIFF2026507539000016.tif241158

[0094] Depository institution name: Korea Institute of Bioscience and Biotechnology Biological Resource Center Accession number: KCTC15267BP Date of acceptance: 20230104 TIFF2026507539000017.tif243159

Claims

1. A composition for immune enhancement comprising one or more selected from the group consisting of Limosilactobacillus reuteri KBL346 strain having accession number KCTC15268BP, a culture of said strain, a lysate of said strain, and an extract of said strain.

2. The composition of claim 1, wherein the immune enhancement is to activate macrophages.

3. The composition of claim 1 , wherein the composition is an immune stimulator.

4. The composition of claim 1 , wherein the bacterial strain is a live or heat-inactivated bacterial strain.

5. The composition (1) Promoting the phagocytic activity of macrophages, (2) Promoting nitric oxide secretion from macrophages, (3) promoting COX-2 expression in macrophages; (4) PGE of macrophages ２ secretion promotion, (5) promoting cytokine production by macrophages; (6) promoting the expression and phosphorylation of NF-κB and / or IκBα in macrophages; (7) promoting MAPKs activation in macrophages; (8) Promoting recovery from viral infections, (9) Prevention of weight loss due to immune deficiency, and (10) Increased mononuclear cells in the blood 10. The composition of claim 1, wherein the composition has one or more properties selected from the group consisting of:

6. A food composition comprising a composition according to any one of claims 1 to 5.

7. A quasi-drug composition comprising the composition according to any one of claims 1 to 5.

8. An anti-influenza virus composition comprising at least one selected from the group consisting of Limosilactobacillus reuteri KBL346 strain having accession number KCTC15268BP, a culture of said strain, a homogenate of said strain, and an extract of said strain.

9. The composition of claim 8, wherein the bacterial strain is a live or heat-inactivated bacterial strain.

10. The strain was added at 1.0 × 10 ３ ~1.0 x 10 １５ 9. The composition of claim 8, comprising a concentration of CFU.

11. The composition (1) Preventing weight loss caused by influenza virus infection, (2) Decrease in mortality rate due to influenza virus infection, (3) Improvement of lung inflammation, and (4) Decreased blood immunoglobulin G (IgG) 9. The composition of claim 8, wherein the composition has one or more properties selected from the group consisting of:

12. A composition for preventing or treating influenza virus infection, comprising one or more selected from the group consisting of Limosilactobacillus reuteri KBL346 strain having accession number KCTC15268BP, a culture of said strain, a homogenate of said strain, and an extract of said strain.

13. 13. The composition of claim 12, wherein the influenza virus infection is at least one selected from the group consisting of flu, pneumonia, Reye's syndrome, acute respiratory failure, myocarditis, bronchitis, otitis media, pharyngolaryngitis, sinusitis, empyema, Guillain-Barré syndrome, and encephalitis.

14. 13. The composition of claim 12, wherein the symptoms of influenza virus infection are one or more selected from the group consisting of fever, chills, nausea, muscle pain, fatigue, cough, difficulty breathing, shortness of breath, pneumonia, phlegm, sore throat, headache, hemoptysis, and diarrhea.

15. Limosilactobacillus reuteri KBL346 strain having accession number KCTC15268BP.

16. The strain of claim 15, wherein the strain has the 16S rRNA sequence of SEQ ID NO:3.

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