WO2017026620A1 - Composition for preventing, alleviating, or treating th1-mediated immune diseases, th17-mediated immune diseases, or th2-mediated immune diseases, containing, as active ingredient, lactobacillus pentosus - Google Patents

Abstract

The present invention relates to: a Lactobacillus pentosus KF340 strain (deposit number KCCM 11675P); and a composition for preventing, alleviating, or treating Th1-mediated immune diseases, Th17-mediated immune diseases, or Th2-mediated immune diseases, containing the strain as an active ingredient. The novel Lactobacillus pentosus KF340 strain according to the present invention effectively controls allergies and inflammation by inducing regulatory T cells and regulatory B cells, controlling the ratio of an immune tolerant cytokine (IL-10; anti-inflammatory cytokine) to an immune activating cytokine (IL-12), inhibiting the generation of immunoglobulin E (IgE), inhibiting the generation of Th1-related cytokines, Th17-related cytokines, and Th2-related cytokines during immune hypersensitivity reactions, and promoting gut immune reactions according to the secretion of IgA. Therefore, the strain is expected to be usable as a composition for preventing, alleviating, or treating Th1-mediated immune diseases, Th17-mediated immune diseases, or Th2-mediated immune diseases and, more preferably, allergies or inflammatory diseases.

Description

Composition for the prevention, improvement, or treatment of Tｈ1-mediated immune disease, Tｈ17-mediated immune disease, or Tｈ2-mediated immune disease comprising Lactobacillus pentosus as an active ingredient

The present invention provides a novel Lactobacillus pentosus KF340 strain (Accession No. KCCM 11675P) and Th1-mediated immune disease, Th17-mediated immune disease, or Th2-mediated immune disease comprising the strain as an active ingredient. , To improve or treat compositions.

Recently, the metagenome project, which identifies the intestinal flora of the intestinal bacteria, mainly in the United States, reveals the relationship between the distribution of intestinal flora and various immune system diseases including allergies. As a result, the distribution and diversity of intestinal bacteria and intestinal immunity and the immune system are closely related.

Looking at the size of the probiotics market, the global probiotics market is estimated at KRW35 trillion in 2014, and is expected to grow at an annual average rate of 7.6% to KRW 57 trillion by 2020. The domestic probiotics market is small in size, with KRW 150 billion as of 2014, but the market is growing rapidly with 250% growth compared to 2013.

Probiotic discovery and evaluation techniques for immunocontrol, such as allergy suppression, have not yet been established, so it is economical to develop a systematic in vitro or in vivo experiment screening system and make good use of the techniques. The value is also expected to be huge.

There is little research on the fundamental mechanisms of probiotics and most of the research is done in vitro . In other words, although probiotics intake is done through the oral cavity, most of the previous studies have been conducted in vitro experiments using cell lines, and these methods cannot replace research on the functions that humans can exhibit when ingesting probiotics. There is a big disadvantage. In addition, the gut immune system (GALT) exposed to probiotics shows the immune action of probiotics by the presence of various immune cells at the same time and interact, but in vitro experiments differ from the in vivo environment.

Therefore, in the present invention, in order to evaluate the antiallergic activity of strains isolated from Korean traditional foods, various immune cells were directly isolated from mice, and then the effects of Lactobacillus pentosus on them were evaluated in the in vivo system. A close study was obtained.

Currently, the evaluation of immunomodulatory ability against probiotics abroad is applied to various disease animal models, and the achievement is also increasing at a steep rate every year. Probiotics that can boost the immune response to fight diseases such as the flu virus or cancer, as well as target the inflammatory response of inflammatory diseases such as type 1 diabetes, rheumatoid arthritis, degenerative arthritis, and ulcerative colitis Research is also actively conducted on the function of. In addition, the role and function of probiotics have attracted much attention for the prevention and treatment of diseases such as allergies, asthma and atopy.

Since most probiotics are derived from human intestines or foods, there is an advantage that there is no big problem in the demonstration of stability with other chemical agents if the efficacy using animal models is proven. In recent years, the efficacy of probiotics as an immunomodulator has been verified through various clinical trials, and many diseases such as asthma, ulcerative colitis, and arthritis have been tried. Recently, clinical trials and studies on atopy have been accelerated. There is a situation.

According to the analysis of industrialization trends, we are leading the development of probiotics in Europe, which is traditionally strong, and the development and commercialization of functional probiotics has recently been actively conducted in the US and Japan. As a representative product, VSL3 # was developed by VSL Pharmaceutical company in Canada, and is currently active in the global market, and recently, it is also sold in the domestic market as an individual immunomodulator.

The present inventors have made intensive research efforts to develop probiotics for preventing or treating allergic and inflammatory diseases caused by Th1, Th17, or Th2 immune responses. As a result, a novel Lactobacillus pentosus strain having anti-allergic and anti-inflammatory activity was selected from kimchi, a traditional fermented food, and when the strain was treated, regulatory T cell induction, regulatory B cell induction, and immune activity. Regulates the ratio of immune tolerant cytokines (IL-10; anti-inflammatory cytokines) to cytokines (IL-12), inhibits immunoglobulin E (IgE) production, Th1-related cytokines and Th17-related cytokines in immune hypersensitivity The present invention was completed by confirming that allergic and inflammation are effectively controlled by inhibition of the production of Caine, and Th2-related cytokines, and enhanced intestinal immune response.

Accordingly, the present invention provides a Lactobacillus pentosus KF340 strain (Accession No. KCCM 11675P) having a prophylactic, ameliorating, or therapeutic activity for Th1-mediated immune disease, Th17-mediated immune disease, or Th2-mediated immune disease. It aims to provide.

In addition, the present invention comprises the Lactobacillus pentosus KF340 strain (Accession No. KCCM 11675P) as an active ingredient, prevention of Th1-mediated immune disease, Th17-mediated immune disease, or Th2-mediated immune disease, It is another object to provide a composition for improvement or treatment.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the object of the present invention as described above, the present invention is a novel Lactobacillus ( Lactobacillus) having a prophylactic, ameliorating or therapeutic activity of Th1-mediated immune disease, Th17-mediated immune disease, or Th2-mediated immune disease. pentosus ) KF340 strain (Accession No. KCCM 11675P) is provided.

In addition, the present invention includes the Lactobacillus pentosus KF340 strain (Accession No. KCCM 11675P) as an active ingredient, preventing, ameliorating, or treating Th1-mediated immune disease, Th17-mediated immune disease, or Th2-mediated immune disease. It provides a composition for.

In one embodiment of the invention, the strain is characterized in that it is isolated from kimchi.

In another embodiment of the present invention, the strain may be live or dead.

In another embodiment of the present invention, the composition may induce regulatory T cells.

In another embodiment of the present invention, the composition may induce regulatory B cells.

In another embodiment of the present invention, the composition may allow the ratio of interleukin-10 (IL-10) production to interleukin-12 (IL-12) production to be 5-30.

In another embodiment of the present invention, the composition may inhibit the production of immunoglobulin E (IgE).

In another embodiment of the invention, the composition may inhibit Th1, Th17, or Th2 related cytokine production.

In another embodiment of the present invention, the composition may enhance intestinal immune response.

In another embodiment of the present invention, the Th1-mediated immune disease or Th17-mediated immune disease may be transplant rejection, autoimmune disease, or inflammatory disease.

In another embodiment of the present invention, the Th2-mediated immune disease may be an allergic disease. The allergic diseases include food allergies, bronchial asthma, allergic rhinitis (acute or chronic), atopic dermatitis, allergic conjunctivitis, allergic otitis media, urticaria and anaphylactic shock, contact hypersensitivity, allergic contact dermatitis, Bacterial allergies, fungal allergies, viral allergies, drug allergies, thyroid and allergic encephalitis.

In another embodiment of the present invention, the composition may be a pharmaceutical, food, nutraceutical, cosmetics, or feed composition.

In addition, the present invention comprises administering to a subject a composition comprising a Lactobacillus pentosus KF340 strain (Accession No. KCCM 11675P) as an active ingredient, a Th1-mediated immune disease, a Th17-mediated immune disease, or a Th2-mediated disease Provided is a method for preventing or treating an immune disease.

The present invention also provides a prophylactic or therapeutic use of a Th1-mediated immune disease, Th17-mediated immune disease, or Th2-mediated immune disease of a composition comprising Lactobacillus pentosus KF340 strain (Accession No. KCCM 11675P) as an active ingredient. do.

The novel Lactobacillus pentosus KF340 strain (Accession No. KCCM 11675P) according to the present invention is immune tolerant cytokine (IL-10; anti-inflammatory) for regulatory T cells and regulatory B cell induction, immune active cytokines (IL-12). Cytokine) ratio, inhibition of immunoglobulin E (IgE) production, inhibition of Th1-related cytokines, Th17-related cytokines, and Th2-related cytokines during immune hypersensitivity, and enhanced intestinal immune response following IgA secretion Effective control of allergy and inflammation can be usefully used as a composition for the prevention, amelioration, or treatment of Th1-mediated autoimmune diseases, Th17-mediated autoimmune diseases, or Th2-mediated autoimmune diseases, more preferably allergic or inflammatory diseases. It is expected to be able.

1 is a result of measuring the expression change of Foxp3 according to the co-culture of Lactobacillus pentosus KF340 strain and immune cells.

2A to 2D show the effects of atopic dermatitis improvement following oral administration of Lactobacillus pentosus KF340 strain in mice with atopic dermatitis, FIG. 2A shows ear thickness changes, and FIG. 2B shows erythema, keratin, and edema. 2C is a photograph of a mouse ear which is an atopic dermatitis lesion site, and FIG. 2D is a result of observing epithelial cell thickness and inflammatory cell infiltration by H & E or TB staining of the lesion site.

Figure 3a is a result of measuring the IL-4 secretion from lymphocytes isolated from each lymph node of mice after oral administration of Lactobacillus pentosus KF340 strain in mice with atopic dermatitis (PP: Payer's patch, mLN: mesenteric lymph nodes, dLN: draining lymph node), Figure 3b is the result of measuring the expression of GATA-3, a transcription factor of Th2 in the lesion tissue.

Figures 4a to 4d is a result showing the effect of atopic dermatitis improved by oral administration of live or dead Lactobacillus pentosus KF340 in mice induced atopic dermatitis, Figure 4a is a result of measuring the change in ear thickness, Figure 4b Figure 4c is a photograph of the mouse ear, atopic dermatitis lesion site, the result of measuring the amount of IL-17A secreted by Th17 (PBS: control, 340: live, HK340: dead), Figure 4d is Th17-related transcription It is the result of measuring the expression of RORγt which is a factor.

Figure 5a is a result showing the increase in IL-10 secretion by Lactobacillus pentosus KF340 strain in splenocytes, Figure 5b is a result showing the increase of B cell activity in the spleen by the strain treatment, Figure 5c is B220 + CD5 + CD1d + B10 cell population increase by strain treatment.

Figure 6 is the result of measuring the expression change of B220 + CD5 + CD1d + by administration of Lactobacillus pentosus KF340 strain in atopic dermatitis mouse model (SPL: spleen, PP: Payer's patch).

Figure 7 shows the results of increasing the frequency of dendritic cells (DC) in the Payer's patch, increased CD80 and CD86 expression of dendritic cells after administration of Lactobacillus pentosus KF340 strain in atopic dermatitis mouse model.

8a and 8b are to determine the direct effect on the dendritic cells (DC) of the Lactobacillus pentosus KF340 strain, Figure 8a is bone marrow dendritic cells (BMDC) after treatment with the strain IL- 10 and TGF-β mRNA and protein expression was measured, Figure 8b is a result of measuring the BAFF mRNA expression in dendritic cells (DC) or Payer's patch.

Figure 9a is a result of measuring the amount of IgA by recovering the mouse feces before the administration of Lactobacillus pentosus KF340 strain (0W) and the end of the experiment (8W) after administration to atopic dermatitis, Figure 9b is IL-6 This is the result of measuring the amount of.

10 is a diagram showing an experimental method and a schedule for producing a food allergic mouse model.

11A to 11C are results showing food allergic inhibitory activity of the Lactobacillus pentosus KF340 strain, FIG. 11A is a change in rectal temperature, FIG. 11B is a diarrhea response, and FIG. 11C is a result of measuring anaphylaxis.

12 is a result showing the change in ear thickness according to oral administration of Lactobacillus pentosus KF340 strain in the contact dermatitis mouse model.

The present inventors have made intensive research efforts to develop probiotics for preventing or treating allergic and inflammatory diseases, diseases or conditions caused by Th1 or Th2 immune responses. As a result, a novel Lactobacillus pentosus strain having anti-allergic and anti-inflammatory activity was selected from kimchi, a traditional fermented food, and when the strain was treated, regulatory T cell induction, regulatory B cell induction, and immune activity. Regulates the ratio of immune tolerant cytokines (IL-10; anti-inflammatory cytokines) to cytokines (IL-12), inhibits immunoglobulin E (IgE) production, Th1-related cytokines and Th17-related cytokines in immune hypersensitivity It has been confirmed that allergy and inflammation are effectively controlled by inhibition of the production of Cain, and Th2-related cytokines, and enhancement of intestinal immune response.

Accordingly, the present invention provides a novel Lactobacillus pentosus KF340 strain (Accession No. KCCM 11675P) having prophylactic, ameliorating or therapeutic activity for Th1-mediated immune disease, Th17-mediated immune disease, or Th2-mediated immune disease. To provide.

In addition, the present invention includes the Lactobacillus pentosus ( Lactobacillus pentosus ) KF340 strain (Accession No. KCCM 11675P) as an active ingredient Th1-mediated immune disease, Th17-mediated immune disease, or Th2-mediated immune disease prevention, improvement Or a therapeutic composition.

As used herein, the term "prevention" means any action that inhibits or delays the onset of Th1-mediated immune disease, Th17-mediated immune disease, or Th2-mediated immune disease by administration of a composition according to the present invention. do.

As used herein, the term " improvement " means any action that at least reduces the parameters associated with the condition being treated, such as the extent of symptoms. At this time, the composition may be used simultaneously or separately with a medicament for treatment before or after the onset of the disease for the prevention or improvement of Th1-mediated immune disease, Th17-mediated immune disease, or Th2-mediated immune disease. .

As used herein, the term “treatment” refers to any action in which symptoms for Th1-mediated immune disease, Th17-mediated immune disease, or Th2-mediated immune disease are ameliorated or beneficially altered by administration of a composition according to the present invention. it means.

As used herein, the term "comprising as an active ingredient" means to include an amount sufficient to achieve the efficacy or activity of the Lactobacillus pentosus KF340 strain. Since the Lactobacillus pentosus KF340 strain of the present invention is a microorganism strain isolated from food, preferably kimchi, there is no side effect on the human body even when the drug is administered in an excessive amount, so that the upper limit of the quantity contained in the composition of the present invention is within the appropriate range. You can choose from.

The Lactobacillus pentosus KF340 strain of the present invention is a strain isolated from kimchi, the strain may be live or dead bacteria.

According to one embodiment of the invention, the composition of the invention can induce regulatory T cells. The regulatory T cell induction can be confirmed by increasing the expression of Foxp3, a regulatory T cell transcription factor, as demonstrated in the following examples.

In one embodiment of the present invention, the composition of the present invention increases the activation of regulatory T cells by 2-20 fold, 5-20 fold, 5-15 fold, 5-10 fold or 7-9 fold as compared to the negative control. It confirmed (refer Example 3).

According to one embodiment of the invention, the composition of the invention can induce regulatory B cells. The regulatory B cell induction can be confirmed by increased expression of B220 + CD5 + CD1d + B cells (B10 cells) as demonstrated in the following examples. B10 cells, a subset of B cells, are known to have anti-inflammatory cytokine IL-10 production activity and play an important role in regulating inflammatory and autoimmune responses.

In one embodiment of the present invention, the Lactobacillus pentosus KF340 strain is induced to induce dendritic cells (DC) of the Payer's patch to be inhibited dendritic cells to promote the production of IL-10 and BAFF, or to the spleen through lymph nodes It was confirmed to migrate to directly induce regulatory B cells (see Example 6). The composition of the present invention increases the activation of regulatory B cells 2-20 fold, 2-10 fold, 2-8 fold, 3-7 fold or 4-6 fold compared to the negative control.

According to one embodiment of the present invention, the composition of the present invention may increase IL-10 production. IL-10 inhibits macrophage and monocytic (monocyte) and T-cell lymphocyte replication and secretion of inflammatory cytokines (IL-1, TNF-α, TGF-β, IL-6, IL-8 and IL-12) As an anti-inflammatory cytokine produced by inhibiting lymphoid cells, an increase in IL-10 inhibits the inflammatory response.

In one embodiment of the present invention, the co-culture of the mesenteric lymph node lymphocytes and Lactobacillus pentosus KF340 strain, it was confirmed that the IL-10 production in the lymphocytes is significantly increased by the strain. Therefore, the composition of the present invention may be a ratio of the IL-10 production amount to the IL-12 production amount to 5-30, more preferably the ratio of the IL-10 production amount to the IL-12 production amount is 10-30, 15 It may be -30 or 20-30 (see Example 3-1).

According to one embodiment of the present invention, the composition of the present invention can inhibit IgE production. IgE is immunoglobulin E, which has affinity for mast cells or basophils and causes an inflammatory reaction when the IgE antibody attached to them reacts with the corresponding antigen (allergen). That is, IgE is an antibody causing anaphylaxis or inflammatory reactions due to allergies. Therefore, it can be seen that allergic reactions or inflammatory reactions are suppressed when IgE production is inhibited.

According to one embodiment of the present invention, the composition of the present invention may inhibit Th1-related cytokine production. Accordingly, the compositions of the present invention can be used for the prevention, amelioration or treatment of various Th1-mediated immune diseases, diseases or conditions.

As used herein, the term "Th1 cell" refers to a subset of helper T cell lymphocytes that are characterized in terms of gene expression, protein secretion and functional activity. For example, Th1 cells exhibit cytokine expression patterns that produce IL-2 and IFN-γ but not IL-4, IL-5, IL-10 and IL-13. Th1 cells are involved in cell-mediated immune responses, organ-specific autoimmune diseases and delayed hypersensitivity to various intracellular pathogens.

As used herein, the term "Th1-mediated immune disease" refers to cytokines (Th1-associated cytokines) produced by the production and / or activity of Th1 cells, such as IL-1β, IL-2, IL-12, IFN means a disease involving -γ or TNF-α. The Th1-mediated immune disease may be transplant rejection, autoimmune disease, or inflammatory disease, and more specifically, colitis, inflammatory bowel disease, type 1 diabetes, type 2 diabetes, rheumatoid arthritis, and reactive arthritis. , Osteoarthritis, Psoriasis, Scleroderma, Osteoporosis, Atherosclerosis, Myocarditis, Endocarditis, Pericarditis, Cystic fibrosis, Hashimoto's thyroiditis, Graves' disease, Leprosy, Syphilis, Lyme disease, Borreliosis, Nervous-Borelliosis, Tuberculosis, Sarcoidosis, Lupus, Discus lupus, Alumnial Lupus, Lupus Nephritis, Systemic Lupus Erythematosus, Asthma, Macular Degeneration, Uveitis, Irritable Bowel Syndrome, Crohn's Disease, Gran Syndrome, Fibromyalgia, Chronic Fatigue Syndrome, Chronic Fatigue Fatigue immunodeficiency syndrome, myalgia encephalomyelitis, amyotrophic lateral sclerosis, Parkinson's disease, multiple sclerosis, autism spectrum disorder, attention deficit disorder, and attention deficit Hyperactivity Disorders include, but are not limited to.

According to one embodiment of the present invention, the composition of the present invention may inhibit Th17-associated cytokine production. Accordingly, the compositions of the present invention can be used for the prevention, amelioration or treatment of various Th17-mediated immune diseases, diseases or conditions.

As used herein, the term “Th17 cells” refers to a subset of helper T cell lymphocytes that are specified in terms of gene expression, protein secretion, and functional activity. For example, Th17 cells are induced by TGF-β, IL-6 or IL-21 to produce IL-17, IL-22 and IL-21, but not IL-2, IL-4, IL-5 and IL. -13 indicates a cytokine expression pattern that does not produce. Th17 cells play a direct role in inflammatory and autoimmune pathogenesis and have been reported to induce host defense or aberrant immune responses against pathogens.

As used herein, the term “Th17-mediated immune disease” refers to a disease involving cytokines (Th17-associated cytokines), such as IL-17, produced by the production and / or activity of Th17 cells. Preferably, the Th17-mediated immune disease may specifically be a transplant rejection, an autoimmune disease or an inflammatory disease, and more specifically, an inflammatory autologous disease such as rheumatoid arthritis, psoriasis, multiple sclerosis, colitis, inflammatory bowel disease, systemic lupus erythematosus, and the like. It may be an immune disease, but is not limited thereto.

According to one embodiment of the invention, the composition of the invention may inhibit Th2-related cytokine production. Accordingly, the compositions of the present invention can be used for the prevention, amelioration or treatment of various Th2-mediated immune diseases, diseases or conditions. According to another embodiment of the present invention, the composition of the present invention can inhibit cytokine production selected from the group consisting of IL-4 and IL-13.

As used herein, the term “Th2 cells” refers to a subset of helper T cell lymphocytes that are specified in terms of gene expression, protein secretion, and functional activity. For example, Th2 cells exhibit IL-4, IL-5 and IL-13 cytokine (Th2-related cytokine) expression patterns, and Th2 cells are involved in humoral immune responses.

As used herein, the term "Th2-mediated immune disease" refers to a disease involving IgE and mast cells by the production and activity of allergen-specific Th2 cells, preferably an allergic disease. More specifically, the allergic diseases include food allergy, bronchial asthma, allergic rhinitis (acute or chronic), atopic dermatitis, allergic conjunctivitis, allergic otitis media, urticaria and anaphylactic shock, contact hypersensitivity, allergic contact dermatitis, bacteria Allergies, fungal allergies, viral allergies, drug allergies, thyroid and allergic encephalitis.

In one embodiment of the present invention, Th1-associated cytokine was determined by oral administration of the Lactobacillus pentosus KF340 strain of the present invention to mice inducing atopic dermatitis and measuring cytokine changes from lymphocytes isolated from lymph nodes at the site of inflammation. It was confirmed that the production of IL-4 and IL-13, which mediate IgE secretion, was induced by inducing degranulation of mast cells with IFN-γ, Th17-associated cytokines IL-17, and Th2-associated cytokines (Example 5- 3).

According to one embodiment of the present invention, the composition of the present invention may enhance intestinal immune response.

In one embodiment of the present invention, the administration of day 0 or Lactobacillus pentosus KF340 strain in atopic dermatitis-induced mice and feces collected at week 8 resulted in increased IgA secretion, IL-inducing IgA secretion It was confirmed that the production of -6 also increased (see Example 7).

As used herein, the term “allergy” refers to a variety of diseases, diseases or abnormalities caused by hypersensitivity to certain substances in the human body, ie, excessive reaction of the body's immune system to substances from outside. The allergic diseases applied to the composition of the present invention are preferably type I immediate hypersensitivity reactions and type IV delayed hypersensitivity reactions. Type I immediate hypersensitivity reactions are bronchial asthma, allergic rhinitis, atopic dermatitis, allergic conjunctivitis, allergic otitis media, urticaria and anaphylatic shock, and type IV delayed hypersensitivity is contact hypersensitivity. It is allergic contact dermatitis, bacterial allergy, fungal allergy, viral allergy, drug allergy, thyroiditis and allergic encephalitis. Type I immediate hypersensitivity is divided into two stages, the first stage of which is a Th1 cell reaction that produces IL-12 and IFN-γ, which inhibits the release of IgE and IgG1 and increases the secretion of IgG2a by invasion of allergens. When Th2 cell responses that produce IL-4, IL-5 and IL-13 are inclined toward Th2, excessive immune response of Th2 secretes IL-4 and IL-13, and B cells IgE-specific antibodies produced are attached to the surface of mast cells and basophil to prepare for the development of allergy. It is said to be sensitized to allergens. The second stage of the onset of allergy is divided into early and late reactions, and the initial reaction is an allergen reinvading the body to stimulate mast cells and induce a degranulation reaction, which is released by histamine, lipid metabolites, cytokines, etc. Expansion, etc. occur, and the late response is invasion and activation of neutrophils, eosinophils, macrophages, Th2 cells, basophils, etc. in the tissue, causing inflammation to cause atopic dermatitis, rhinitis, asthma. Preferably the allergy of the present invention may be allergic contact dermatitis, allergic atopic dermatitis, or food allergy.

In one embodiment of the present invention, the atopic dermatitis, food allergy, contact dermatitis inhibitory activity of the Lactobacillus pentosus KF340 strain was confirmed, and not only live bacteria but also heat treated bacteria showed similar effects (Examples 5 and 8). , And 9).

Thus, the novel Lactobacillus pentosus KF340 strain of the present invention may be prepared as a pharmaceutical, health functional food, food, cosmetics, or feed composition.

The pharmaceutical composition of the present invention comprises (a) a pharmaceutically effective amount of the Lactobacillus pentosus of the present invention as described above; And (b) a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically effective amount” means an amount sufficient to achieve the efficacy or activity of the Lactobacillus pentosus described above.

The pharmaceutical composition according to the present invention comprises Lactobacillus pentosus KF340 strain as an active ingredient, it may further comprise a pharmaceutically acceptable carrier. Such pharmaceutically acceptable carriers are conventionally used in the preparation, and include, but are not limited to, saline solution, sterile water, Ringer's solution, buffered saline, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, liposomes, and the like. If necessary, other conventional additives such as antioxidants and buffers may be further included. In addition, diluents, dispersants, surfactants, binders, lubricants and the like may be additionally added to formulate injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Suitable pharmaceutically acceptable carriers and formulations can be preferably formulated according to the individual components using methods disclosed in Remington's literature. The pharmaceutical composition of the present invention is not particularly limited in formulation, but may be formulated as an injection, inhalant, or external skin preparation.

The pharmaceutical composition of the present invention may be administered orally or parenterally (eg, applied intravenously, subcutaneously, intraperitoneally, or topically) according to a desired method, but preferably may be administered orally. Depends on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the time of day, and may be appropriately selected by those skilled in the art.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, “pharmaceutically effective amount” means an amount sufficient to treat or diagnose a disease at a reasonable benefit / risk ratio applicable to medical treatment or diagnosis, and an effective dose level refers to a patient's disease type, severity, or drug. Can be determined according to the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and the rate of release, the duration of treatment, factors including the drug used concurrently and other factors well known in the medical field. The pharmaceutical compositions according to the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be single or multiple administrations. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects, which can be easily determined by those skilled in the art.

Specifically, the effective amount of the pharmaceutical composition of the present invention may vary depending on the age, sex, condition, weight of the patient, the absorption of the active ingredient in the body, the inactivation rate and excretion rate, the type of disease, the drug used in general 0.001 to 150 mg, preferably 0.01 to 100 mg per kg of body weight may be administered daily or every other day, or divided into 1 to 3 times a day. However, the dosage may be increased or decreased depending on the route of administration, the severity of obesity, sex, weight, age, etc., and the above dosage does not limit the scope of the present invention in any way.

When the composition of the present invention is prepared as a health functional food composition, the composition comprises a tablet, pill, powder, granule, powder, capsule, and liquid formulation, including one or more of a carrier, diluent, excipient, and additive. Characterized in that formulated as one selected from. Foods which can be added to the composition of the present invention include various foods, powders, granules, tablets, capsules, syrups, beverages, gums, teas, vitamin complexes, and health functional foods. As additives which may be further included in the present invention, natural carbohydrates, flavoring agents, nutrients, vitamins, minerals (electrolytes), flavoring agents (synthetic flavoring agents, natural flavoring agents, etc.), colorants, fillers, pactic acid and salts thereof, Alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH regulators, stabilizers, preservatives, antioxidants, glycerin, alcohols, carbonation agents and one or more components selected from the group consisting of pulp can be used. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As the flavoring agent, natural flavoring agents (tauumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. Compositions according to the invention include various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic and natural flavors, colorants and neutralizers, fact acids and their salts, alginic acids and their salts, organic acids, protective colloidal thickeners , pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, etc. In addition, the composition of the present invention may contain pulp for the production of natural fruit juices and vegetable drinks. These components may be used independently or in combination. Specific examples of the carrier, excipient, diluent, and additives include, but are not limited to, lactose, dextrose, Chromose, sorbitol, mannitol, erythritol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium phosphate, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, polyvinylpyrrolidone, methylcellulose, water, Preference is given to using at least one selected from the group consisting of sugar syrup, methyl hydroxy benzoate, propyl hydroxy benzoate, talc, magnesium stearate and mineral oil.

When the anti-allergic composition containing the active ingredient of the Lactobacillus pentosus KF340 strain of the present invention is prepared as a cosmetic composition, it contains components commonly used in cosmetic compositions in addition to Lactobacillus pentosus as an active ingredient, for example, a stabilizer. , Conventional adjuvants such as solubilizers, vitamins, pigments and flavorings, and carriers.

The cosmetic composition of the present invention may be prepared in any formulation commonly prepared in the art, for example, solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansing , Oils, powder foundations, emulsion foundations, wax foundations and sprays, and the like, but are not limited thereto. More specifically, it may be prepared in the form of a flexible lotion, nutrition lotion, nutrition cream, massage cream, essence, eye cream, cleansing cream, cleansing foam, cleansing water, pack, spray or powder.

When the formulation of the present invention is a paste, cream or gel, animal oils, vegetable oils, waxes, paraffins, starches, trachants, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicas, talc or zinc oxide may be used as carrier components. Can be.

When the formulation of the present invention is a paste, cream or gel, animal oils, vegetable oils, waxes, paraffins, starches, trachants, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicas, talc or zinc oxide may be used as carrier components. Can be.

When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used, in particular in the case of a spray, additionally chlorofluorohydrocarbon, propane Propellant such as butane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, solubilizer or emulsifier is used as the carrier component, such as water, ethanol, isopropanol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 Fatty acid esters of, 3-butylglycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan.

When the formulation of the present invention is a suspension, liquid carrier diluents such as water, ethanol or propylene glycol, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystals Soluble cellulose, aluminum metahydroxy, bentonite, agar or tracant and the like can be used.

When the formulation of the present invention is a surfactant-containing cleansing, the carrier component is an aliphatic alcohol sulfate, an aliphatic alcohol ether sulfate, a sulfosuccinic acid monoester, an isethionate, an imidazolinium derivative, a methyltaurate, a sarcosinate, a fatty acid amide. Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters and the like can be used.

In addition, when the anti-allergic composition containing the active ingredient Lactobacillus pentosus KF340 strain of the present invention is prepared as a feed composition, it can be prepared by adding Lactobacillus pentosus as it is, or additives commonly added during feed preparation. have. For example, various nutrients such as vitamins, amino acids and minerals, antioxidants, antibiotics, antibacterial agents and other additives. The shape includes powder, granules, pellets or suspensions and the like.

When the composition of the present invention is made of a feed composition, it may be supplied alone or mixed with the feed to land or aquatic animals. The feed of the present invention includes, but not limited to, powder feed, solid feed, moist pellet feed, dry pellet feed, EP (Extruder Pellet) feed, feeding.

Strains having the prophylactic, ameliorating or therapeutic activity of the Th1-mediated immune disease, Th17-mediated immune disease or Th2-mediated immune disease of the present invention include the above-mentioned Th1-mediated immune disease, Th17-mediated immune disease or Th2-mediated immune disease. Since the composition for preventing, ameliorating, or treating a compound and the target disease are common, common contents in relation to the composition are omitted in order to avoid excessive complexity of the present specification.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.

EXAMPLE

Example 1 Isolation and Identification of Strains

Strains were isolated from traditionally prepared kimchi using Lactobacillus MRS agar medium (MRS medium, BD288130, Diffcosa, USA), and the isolated strains were identified by 16S rDNA sequencing. As a result, the 16s rDNA sequence (SEQ ID NO: 1) of the strain showed 99.73% homology with the existing Lactobacillus pentosus JCM1558 (T) strain.

The medium composition for the cultivation of the strain is MRS medium, the culture conditions are pH 6.5 ± 0.2, temperature 37 ℃ and stationary culture for 48 hours, oxygen urinary tract is anaerobic, strain preservation through freeze-drying preservation or cell suspension freezing It is possible. The present inventors named the strain Lactobacillus pentosus KF340, and deposited it on March 6, 2015 to the Korea Microorganism Conservation Center (KCCM), an international microbial deposit institution, and was given accession number KCCM 11675P.

Example 2: Experiment Preparation and Methods

2-1. Laboratory animals

For this study, wild-type mice (system number-C57BL / 6, stock number-000664, the Jackson laboratory) with no genetic modifications were used. In addition, genetically modified mice (Foxp3-GFP knockout mice, line name-B6.Cg-Foxp3 ^tm2Tch / J, Stock No.-006772, Jackson laboratory) were used to relatively easily confirm the expression level of Foxp3. Since the mouse is based on a wild-type species, the overall genetic information is the same, but the expression of Foxp3 is expressed by enhanced expression of enhanced green fluorescence protein (EGFP), which is a molecule that can generate a fluorescent signal when Foxp3 is expressed. Since the degree represents the degree, the expression level of Foxp3, which is difficult to detect using the mouse, can be relatively easily identified without using an antibody.

2-2. Immune cell isolation and co-culture with Lactobacillus pentosus

After mesenteric lymph nodes (MLNs) of mice were extracted, immune cells (mostly lymphocytes) present in lymph nodes were suspended in single cells and used for experiments. Since probiotics present in the gut are recognized by immune cells present in the intestinal immune system, suitable immune cells for characterizing probiotics are immune cells present in the mesenteric lymph nodes. Therefore, the characteristics of Lactobacillus pentosus KF340 were analyzed by the immune cells isolated from the lymph nodes.

To this end, the immune cells and Lactobacillus pentosus KF340 isolated according to the above method was incubated in a 1:10 ratio, that is, immune cells 3 × 10 ⁶ and strain 3 × 10 ⁷ cfu at 37 ° C. in a CO ₂ incubator for 72 hours. At this time, the antibiotic gentamicin (11811-031, 15 ㎍ / mL, GIBCO) was treated together to prevent overgrowth of the test strain.

Example 3: Confirmation of anti-allergic and anti-inflammatory activity

3-1. Cytokine Measurement in Coculture

ELISA is one of the methods of measuring the amount of antigen or antibody using an antigen-antibody reaction using an enzyme as a marker. Co-culture of immune cells and lactic acid bacteria to measure the amount of cytokines present in the supernatant IL-10 (Mouse IL-10 ELISA Ready-SET-Go, eBioscience) and IL-12p70 (Mouse IL-12p70 ELISA Ready-SET -Go, eBioscience) ELISA was performed. After treatment with a coating antibody for detecting each cytokine in 96-well, the supernatant was added and allowed to react for a certain time, and then another detection antibody for detecting each cytokine. At this time, the detection antibody to be treated is bound to the enzyme (HRP), the color change occurs as a result of the enzyme-substrate reaction by adding the enzyme substrate (Tetramethylbenzidine Substrate Solution, eBioscience). This indirect method can be used to determine the absolute amount of cytokines.

IL-10 (interleukin 10) is a representative immune tolerant cytokine, and IL-12 (interleukin 12) is known as a representative immunologically active cytokine. Therefore, the lactic acid bacteria having antiallergic properties are selected by identifying the absolute / relative amounts of the two cytokines. can do. Thus, mesenteric lymph node cells of Foxp3 knock-in mice were isolated and mixed with each test strain in a ratio of 1:10 according to the method of Example 2-2, followed by co-culture. After incubation for 3 days, the culture supernatant was taken and the absolute amounts of the immunologically active cytokine IL-12p70 and anti-inflammatory cytokine IL-10 were measured by ELISA (Enzyme linked immunosorbent assay). Compared.

As a result, as shown in Table 1, when the mesenteric lymph node cells and Lactobacillus pentosus KF340 strains were co-cultured, the relative ratio (lL-10 / IL-12p70) of lL-10 and IL-12p70 secretion in lymphocytes was 24.17. As compared with the negative control group, it appeared to appear higher. It was found that the Lactobacillus pentosus KF340 strain has anti-inflammatory activity.

	IL-10 (pg / mL)	IL-12p70 (pg / mL)	IL10 / IL-12p70
PBS	241.68	0*	0*
Lactobacillus pentosus KF340	512.27	21.187	24.17

* Not detect, anti-inflammatory activity can not be expected because a small amount of deviation from the detection area is secreted.

3-2. Foxp3 expression measurement

Foxp3 is a transcription factor of regulatory T cells, which is a subset of T cells that suppress excessive immunity, and the distribution of regulatory T cells can be confirmed through the degree of Foxp3 expression.

The regulatory T cell is one of a subset of T cells and serves to suppress an excessive immune response unlike other T cells. Various studies have supported that these T cells suppress allergic diseases. Therefore, when co-culturing test strains and mesenteric lymph node cells, strains that can increase the distribution of regulatory T cells will become probiotics with antiallergic efficacy. Most likely.

Therefore, in order to compare the degree of surface molecule expression of mesenteric lymphocyte immune cells co-cultured with Lactobacillus pentosus KF340 strain, mesenchymal lymph node immune cells isolated according to the method of Example 3-1 were subjected to flow cytometry. The expression level of Foxp3 was measured and compared by measuring the GFP signal that co-expresses at the time of expression.

As a result, as shown in Figure 1, in the case of immune cells co-cultured with Lactobacillus pentosus KF340 strain, Foxp3 expression (12.48%) increased about 8.43 times compared to the control group (1.48%). It was confirmed that the Lactobacillus pentosus KF340 strain has regulatory T cell inducing activity, thereby suppressing allergic diseases.

Example 4: Confirmation of anti-allergic activity by Th2 cytokine inhibition

The purpose of this study was to evaluate whether Lactobacillus pentosus KF340 strain has anti-allergic activity through Th2 cytokine production inhibition.

To this end, a mixture of OVA (20 μg) and aluminium (aluminum hydroxide gel, 2 mg) was mixed for 30 minutes, and then injected into the 5-week old female Balb / c mice intraperitoneally twice at a weekly interval of 100 μl per horse. Induced. One week after immunization, the mouse spleens were extracted and single-celled and hemolyzed splenocytes were dispensed at 5 × 10 ⁶ cells / well in 96-well plates. At this time, the antigen (OVA, 100 μg / mL) and the Lactobacillus pentosus KF340 (5 × 10 ⁷ CFU / well) strain were added and the supernatant was recovered after incubation for 72 hours in a 37 ° C. CO ₂ incubator and a representative Th2-related cytokine The secretion amount of IL-4 was measured.

As a result, it was confirmed that IL-4 production is reduced when the Lactobacillus pentosus KF340 strain is treated as shown in Table 2 below. This means that Lactobacillus pentosus KF340 strain can inhibit the allergic reaction by inhibiting the production of IL-4.

OVA	Sample	IL-4 (%)
-	Control group 1	25.13 ± 10.18
+	Control 2	100.00
+	Lactobacillus pentosus KF340	50.26 ± 8.62

Control group 1: allergy-induced experimental group, control group 2: allergy-induced experimental group

Example 5: Anti-Atopic Activity Confirmation

5-1. Atopic Dermatitis Induction

Mice were subjected to oral administration of Lactobacillus pentosus KF340 strain at least 5 × 10 ⁸ CFU / day five times a week for eight weeks. Three weeks after the first oral administration, the ears of the mouse were removed using a surgical tape, followed by 1.0% DNCB (2,4-dinitrochlorobenzene) and 20 μl of house dust mite (10). mg / mL) was applied. After a week, atopic dermatitis was induced by repeating 5 weeks in a manner of alternately treating 1% DNCB and 20 μl (10 mg / mL) of ticks once a week.

5-2. Ear thickness and histological changes

In accordance with the method of Example 5-1, the Lactobacillus pentosus KF340 strain was administered to the house dust mite-induced atopic dermatitis mouse model for 8 weeks, and then histologic changes were measured to evaluate anti-atopic activity. At this time, the experiment was conducted under the same conditions using dexamethasone (dexamethasone), which is a steroid used to treat atopic dermatitis.

As a result of measuring the change of ear thickness, erythema, edema, and keratinogenesis in the mouse, as shown in FIG. 2A, unlike the negative control group (PBS control) administered PBS after atopic dermatitis, Lactobacillus pentosus In the case of the group 340 administered with the KF340 strain, the ear thickness was reduced to a similar degree to the positive control (Dex), and as shown in FIG. 2B, a negative control in which erythema, edema, keratin and dry scoring of the ear were severely occurred. In contrast, oral administration of Lactobacillus pentosus KF340 strain showed similar levels to the positive control.

In addition, as a result of visual observation, as shown in FIG. 2C, oral administration of Lactobacillus pentosus KF340 significantly improved atopic dermatitis. As a result of histological analysis of ear tissue as an inflammation site, the H & E and TB staining results of FIG. It was confirmed that the Lactobacillus pentosus KF340-administered group had reduced epithelial cell thickness and significantly inhibited the infiltration of inflammatory cells compared to the negative control group.

5-3. Cytokine Change Measurement

For cytological analysis, draining lymphocytes were isolated from the lymph nodes of the inflamed area of the mouse (surface lymph nodes, axillary lymph nodes, bronchial lymph nodes), and then stimulated with an antigen house dust mite. It was confirmed by ELISA method using a cytokine quantitative kit from Bioscience.

As a result, as shown in Table 3, in the experimental group continuously administered Lactobacillus pentosus KF340, respectively, the amount of IFN-γ and IL-17A, which are inflammatory cytokines that promote the development and exacerbation of atopic dermatitis, was decreased. In addition, the secretion of Th2-related cytokines IL-4 and IL-13 was also reduced, confirming that Lactobacillus pentosus KF340 had atopic dermatitis inhibitory activity.

Experimental group	IFN-γ (ng / mL)	IL-17A (ng / mL)	IL-4 (pg / mL)	IL-13 (ng / mL)
Normal control	0.01	0	0	0
Negative control group (PBS group)	3.31	38.16	69.75	2.57
Lactobacillus pentosus KF340	0.58	3.37	29.75	0.71

In addition, lymphocytes were isolated from Payer's patches (hereinafter referred to as PPs) and mesenteric lymph nodes (mLN) corresponding to gut-associated lymphoid tissue (GALT), and the Th2 cytokine IL-4 was isolated in the same manner. As a result, as shown in Figure 3a, it was confirmed that IL-4 secretion is significantly reduced, the expression of GATA-3, a transcription factor of Th2 in the inflamed ear as shown in Figure 3b significantly decreased.

5-4. Anti-Atopic Activity of Lactobacillus Pentosus KF340

Example 5-2 and 5-3 confirmed that the Lactobacillus pentosus KF340 live bacteria exhibited anti-atopic activity, it was intended to verify whether the bacterium also exhibits the same anti-atopic activity.

To this end, the atopic dermatitis mouse model was prepared in the same manner as in Example 5-1, and the live Lactobacillus pentosus KF340 microorganisms or the dead bacteria prepared by heat-treating them at 121 ° C. for 15 minutes were administered to the mice in the same manner.

As a result of measuring the ear thickness of the mouse by administration of Lactobacillus pentosus KF340 live or dead bacteria, as shown in FIG. 4A, it was confirmed that both the live bacteria and dead bacteria were alleviated by the increase in ear thickness compared to the control (PBS). In addition, as shown in Figure 4b. In the ear picture, the symptoms such as erythema, edema, and keratin were alleviated, and the anti-atopic activity was higher in live bacteria than in dead bacteria.

In addition, changes in Th17 cells and IL-17A following Lactobacillus pentosus KF340 live or dead administration were measured. Most T cell mediated diseases are thought to be caused by Th1 / Th2, but recently, Th17 cells secreting IL-17 have been reported to play an important role in skin immunity. IL-17A is a major cytokine produced by Th17 cells and is known to play an important role in tissue inflammatory response and to bring neutrophils into tissues.

As a result, as shown in Figure 4c, the production of IL-17 by the administration of the live or dead bacteria compared to the control group, as shown in Figure 4d through the flow cytometry to reduce the expression of RORγt, Th17-related transcription factor It confirmed that it was seen. The results showed that both live and dead bacteria of Lactobacillus pentosus KF340 strain had atopic dermatitis inhibitory activity through the inhibition of Th17 immune response.

Example 6 Anti-Atopic Activity Mechanism of Lactobacillus Pentosus KF340

6-1. in vitro  And in vivo in  B220 + CD5 + CD1d + Increase cell count

Since Example 3-1 confirmed that the Lactobacillus pentosus KF340 strain increased the production of IL-10, the strain affects cells that induce IL-10 secretion in addition to regulatory T cells, thereby inhibiting antiallergic activity. As determined, the cells secreting IL-10 were screened in vitro and in vivo using a flow cytometer (COULTER Epics XL, BECKMAN).

As a result of in vitro experiments, as shown in Figure 5a, the secretion of IL-10 in the spleen was significantly increased by treatment of Lactobacillus pentosus KF340 strain, in particular, the activity of the spleen B cells as shown in Figure 5b significantly It was found to increase. Furthermore, as shown in Figure 5c, it was confirmed that the population of B220 + CD5 + CD1d + cells in splenic B cells significantly increased by the strain treatment.

B10 cells (phenotype: B220 + CD5 + CD1d + or CD19 + CD5 + CD1d +) are a subset of regulatory B cells that have been reported to inhibit allergy by secreting a large number of IL-10 cytokines specifically in the spleen, It is known to play an important role in regulating inflammatory and autoimmune responses.

In order to prove the results, oral administration of Lactobacillus pentosus KF340 strain to the atopic dermatitis mouse model prepared in Example 5-1, the spleen of the mouse was isolated and unicellularized by flow cytometry B220 + CD5 + CD1d + Expression was measured.

As a result, as shown in Figure 6, in the Lactobacillus pentosus KF340 strain administration group 340 it was confirmed that the population of B220 + CD5 + CD1d + B cells significantly increased compared to the negative control group (PBS control). However, B220 + CD5 + CD1d + B cells in PPs do not increase, which may be due to the cystemic action of Lactobacillus pentosus KF340 strain.

6-2. Confirmation of DC Induction by Lactobacillus Pentosus KF340

As confirmed in the results of Example 6-1, the Lactobacillus pentosus KF340 strain increased the population of B220 + CD5 + CD1d + B cells in the spleen, but since it was very unlikely that it directly acted on the spleen, It is possible that it acted on the spleen as a secondary response. To demonstrate this, the oral administration of Lactobacillus pentosus KF340 strain to the atopic dermatitis mouse model prepared in Example 5-1 and observed the cell population in PPs.

As a result, as shown in FIG. 7, the group 340 administered the strain compared to the control group (PBS control) was found to have a higher frequency of dendritic cells (hereinafter referred to as DC) in the PPs. In addition, the increased DC showed a pattern showing a decrease in the expression of CD80, CD86.

6-3. Confirmation of the Promotion of BAFF Expression by Lactobacillus Pentosus KF340

Based on the results of Example 6-2, it was determined that the inhibitory DC may move from the lymphoid tissue to the spleen to affect the splenocytes, thereby inducing the induction of regulatory B cells by direct or indirect effects. The direct effect on the DC of the Lactobacillus pentosus KF340 strain was attempted.

To this end, after treating the strain with bone marrow dendritic cells (BMDC), mRNA and protein levels of BAFF, which are known to be important factors in inducing the related cytokines IL-10 and TGF-β and regulatory B cells, Was measured via Real-time PCR and Western blot. Each primer sequence used when performing the real-time PCR is shown in Table 4 below.

gene	direction	order	SEQ ID NO:
IL-10	forward	5′-ATAACTGCACCCACTTCCCA-3 ′	2
	reverse	5′-TCATTTCCGATAAGGCTTGG-3 ′	3
TGF-β	forward	5′- TGGAGCAACATGTGGAACTC-3 ′	4
	reverse	5′- TGCCGTACAACTCCAGTGAC-3 ′	5
BAFF	forward	5′- AGGCTGGAAGAAGGAGATGAG-3 ′	6
	reverse	5′- CAGAGAAGACGAGGGAAGGG-3 ′	7

As a result, as shown in Figure 8a, in the case of IL-10 mRNA expression and protein production significantly increased compared to the control group, TGF-β did not show a significant difference. However, as a result of measuring the mRNA expression level of BAFF, which is an important factor in the induction of regulatory B cells, as shown in Figure 8b, when treated with KF340 strain expression was significantly increased in DC compared to the control group, the same atopy It was confirmed that the expression of BAFF was increased in PPs of the dermatitis mouse model.

Through the above experimental results, the Lactobacillus pentosus KF340 strain exhibiting anti-atopic effect in the atopic dermatitis model increases IL-10 secretion by increasing the population of B220 + CD5 + CD1d + B10 cells in the spleen, a type of regulatory B cell. It was confirmed to have. This increase in B10 cells promotes the production of IL-10 and BAFF by inducing DCs of PPs to which the Lactobacillus pentosus KF340 strain is absorbed into inhibitory DCs, or migrates to the spleen through lymph nodes to directly induce B10 cells. It is considered to be.

Example 7 Confirmation of Intestinal Immunity Enhancing Activity of Lactobacillus Pentosus KF340

In order to evaluate the intestinal immunity of the Lactobacillus pentosus KF340 strain, atopic dermatitis was induced in mice in the same manner as in Example 5-1, and the Lactobacillus pentosus KF340 strain was orally administered and secreted according to the strain administration. The concentration of type IgA was measured. To this end, fecals were collected from mice on week 0 and 8 weeks after the end of the experiment, and then the amount of cytokine IL-6, which induces the secretion of IgA and IgA in feces, was measured using BD's mouse IgA quantitative kit.

As a result, as shown in Fig. 9a, the production of IgA was increased by administration of Lactobacillus pentosus KF340 strain in mice induced by atopic dermatitis. In addition, as a result of measuring IL-6, it was confirmed that IL-6 production, which induces IgA secretion, is increased in the payer's patch. The results show that the Lactobacillus pentosus KF340 strain induces the production of IgA and affects mucosal immunity enhancement.

Example 8: Food Allergy Inhibitory Activity

8-1. Induced food allergy and administration of Lactobacillus pentosus KF340

Experimental animals received 5 weeks old female BALB / c mice from Orient Bio Co., Ltd., and were used after breeding and purifying for one week in the experimental animal room. According to the experimental procedure illustrated in FIG. 10, 100 μl per horse was injected intraperitoneally with OVA (20 μg) and alum (2 mg) mixed solution, followed by primary immunization. Fourteen days after the second immunization, the Lactobacillus pentosus KF340 strain was administered daily at a concentration of 5 × 10 ⁸ CFU / day. At this time, the normal control group (Naive) was administered the same amount of suspension solvent PBS instead of the Lactobacillus pentosus KF340 suspension, the positive control group (PC (daxa)) was administered dexamethasone, an immunosuppressive agent. Allergenicity was induced by oral administration of OVA at a concentration of 50 mg / mice / day to all groups at 3 day intervals after 14 days of secondary immunity to induce food allergy. Anaphylaxis and diarrhea reactions, which are food allergens, were measured at 30-minute intervals after oral administration of OVA. Changes in rectal temperature were measured at 60-minute intervals for 20 minutes.

As a result, as shown in Figure 11a, when administered Lactobacillus pentosus KF340 strain, the rectal temperature was maintained higher than the sham group (sham), as shown in Figure 11b and 11c diarrhea reaction or anaphylaxis reaction also in the Siamese group Rather, it was confirmed that the Lactobacillus pentosus KF340 strain has a food allergy symptomatic activity.

8-2. Confirmation of Serum IgE Change

After completion of the experiment, the blood was collected through mouse orbital vein, and serum was separated by centrifugation at 3500 rpm for 10 minutes. Serum IgE content was measured using BD mouse IgE quantitative kit.

As a result, as shown in Table 5 below, the administration of Lactobacillus pentosus KF340 strain decreased IgE, which is closely related to allergic symptoms.

8-3. Check for cytokine changes

After extracting MLN and spleen from mice in each group, single cell suspension was seeded in 96-well at 5x10 ⁶ concentration, and treated with 20 ug of OVA to confirm antigen-specific reaction. Incubated for 72 hours. After incubation for 72 hours, the cytokines present in the supernatant were confirmed by an ELISA method using a cytokine quantitative kit from BD.

As a result, as shown in Table 5 below, the administration of Lactobacillus pentosus KF340 strain reduced IL-4 and IL-13 secretion that induce IgE production in the MLN culture supernatant, which is a food allergy of the Lactobacillus pentosus KF340 strain. Inhibitory activity was confirmed.

Experimental group	IL-4 (pg / mL)	IL-13 (pg / mL)	IgE (ng / mL)
Normal control (Naive)	8.01	46.58	1737.40
Sham	466.77	3049.87	27209.26
Lactobacillus pentosus KF340	331.61	2544	20422.95
Dexamethasone	8.71	62.875	17434.98

Example 9 Contact Dermatitis Inhibitory Activity

9-1. Induction of contact dermatitis and administration of Lactobacillus pentosus KF340

Five-week-old female BALB / c was allowed to acclimate for one week, followed by hair removal of the flanks of all experimental groups except Naive on day 0 (Day 0), followed by 5% TMA solution [acetone and isopropylmyristate. Using a 4: 1 (v / v) mixture as a solvent] 50 µl was applied for the first sensitization. After 5 days of sensitization, 10 μl of 5% TMA was secondarily sensitized to both ears, except for the normal control group (Naive), and then 10 μl of 2% TMA was applied to both ears once every 3 days. The Lactobacillus pentosus KF340 was administered at a concentration of 5 × 10 ⁸ CFU / horse daily after the first sensitization, and the Siamese group was administered the same amount of PBS used to suspend the Lactobacillus pentosus KF340. The experiment was performed by treating P).

TMA is a type of chemical hapten that induces allergic hypersensitivity of immune cells by invading the skin through the skin and invading the skin, and induces a strong Th2 response. Inflammation of the site and increased thickness of the tissue. As a result, as shown in FIG. 12, the TMA-treated ear thickness decreased with Lactobacillus pentosus KF340 compared to the negative control.

9-2. Confirmation of Serum IgE Change

After completion of the experiment, the blood was collected through mouse orbital vein, and serum was separated by centrifugation at 3500 rpm for 10 minutes. Serum IgE content was measured using BD mouse IgE quantitative kit.

As a result, as shown in Table 6, the IgE content showing high expression in allergic contact dermatitis patients was reduced compared to the Siamese group.

9-3. Check for cytokine changes

DLN and spleen were removed from each group of mice, and then cultured for 96 hours after 3x10 ⁶ cells after unicellular suspension. In this case, the cells were divided into two cases in which only 10 μg of mites were treated and only cells were cultured to confirm the antigen-specific reaction.The cytokines in the supernatant after 96 hours of cultivation were analyzed using the cytokine quantification kit of e-Bioscience. It was confirmed by the ELISA method.

 As a result, as shown in Table 6, the secretion amount of Th-4 related cytokine IL-4 in DLN was shown to be reduced, confirming that Lactobacillus pentosus KF340 has allergic contact dermatitis inhibitory activity.

Experimental group	IgE (ng / mL)	IL-4 (pg / mL)
Normal control (Naive)	242.25	10.30833
Sham	4729.47	196.974
Lactobacillus pentosus KF340	3820.30	164.33
Prednisolone (P: Prednisolone)	2264.40	101.8833

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Therefore, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

<210> 1

<211> 1532

<212> DNA

<213> Lactobacillus pentosus KF340 16S rDNA

<400> 1

cagagtttga tcatggctca ggacgaacgc tggcggcgtg cctaatacat gcaagtcgaa 60

cgaactctgg tattgattgg tgcttgcatc atgatttaca tttgagtgag tggcgaactg 120

gtgagtaaca cgtgggaaac ctgcccagaa gcgggggata acacctggaa acagatgcta 180

ataccgcata acaacttgga ccgcatggtc cgagtttgaa agatggcttc ggctatcact 240

tttggatggt cccgcggcgt attagctaga tggtggggta acggctcacc atggcaatga 300

tacgtagccg acctgagagg gtaatcggcc acattgggac tgagacacgg cccaaactcc 360

tacgggaggc agcagtaggg aatcttccac aatggacgaa agtctgatgg agcaacgccg 420

cgtgagtgaa gaagggtttc ggctcgtaaa actctgttgt taaagaagaa catatctgag 480

agtaactgtt caggtattga cggtatttaa ccagaaagcc acggctaact acgtgccagc 540

agccgcggta atacgtaagg tggcaagcgt tgtccggatt tattgggcgt aaagcgagcg 600

caggcggttt tttaagtctg atgtgaaagc cttcggctca accgaagaag tgcatcggaa 660

actgggaaac ttgagtgcag aagaggacag tggaactcca tgtgtagcgg tgaaatgcgt 720

agatatatgg aagaacacca gtggcgaagg cggctgtctg gtctgtaact gacgctgagg 780

cttcgaaaag tatgggtcgc aaacaggatt agataccctg gtagtccata ccgtaaacga 840

tgaatgctaa gtgttggagg gtttccgccc ttcagtgctg cagctaacgc attaagcatt 900

ccgcctgggg agtacggccg caaggctgaa actcaaagga attgacgggg gcccgcacaa 960

gcggtggagc atgtggttta attcgaagct acgcgaagaa ccttaccagg tcttgacata 1020

ctatgcaaat ctaagagatt agacgttccc ttcggggaca tggatacagg tggtgcatgg 1080

ttgtcgtcag ctcgtgtcgt gagatgttgg gttaagtccc gcaacgagcg caacccttat 1140

tatcagttgc cagcattaag ttgggcactc tggtgagact gccggtgaca aaccggagga 1200

aggtggggat gacgtcaaat catcatgccc cttatgacct gggctacaca cgtgctacaa 1260

tggatggtac aacgagttgc gaactcgcga gagtaagcta atctcttaaa gccattctca 1320

gttcggattg taggctgcaa ctcgcctaca tgaagtcgga atcgctagta atcgcggatc 1380

agcatgccgc gggtgaatac gttcccgggc cttgtacaca ccgcccgtca caccatgaga 1440

gtttgtaaca cccaaagtcg gtggggtaac cttttaggaa ccagccgcct aacgtgggac 1500

agatgattag ggtgaagtcg taacaaggta cc 1532

<210> 2

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> IL-10 Forward

<400> 2

ataactgcac ccacttccca 20

<210> 3

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> IL-10 Reverse

<400> 3

tcatttccga taaggcttgg 20

<210> 4

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> TGF-b Forward

<400> 4

tggagcaaca tgtggaactc 20

<210> 5

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> TGF-b Reverse

<400> 5

tgccgtacaa ctccagtgac 20

<210> 6

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> BAFF Forward

<400> 6

aggctggaag aaggagatga g 21

<210> 7

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> BAFF Reverse

<400> 7

cagagaagac gagggaaggg 20

Claims (26)

1. Lactobacillus pentosus KF340 strain (Accession No. KCCM 11675P) having prophylactic, ameliorating, or therapeutic activity for Th1-mediated immune disease, Th17-mediated immune disease, or Th2-mediated immune disease.
2. Pharmaceutical for the prevention or treatment of Th1-mediated immune disease, Th17-mediated immune disease, or Th2-mediated immune disease, comprising the Lactobacillus pentosus KF340 strain (Accession No. KCCM 11675P) of claim 1 as an active ingredient Composition.
3. The pharmaceutical composition of claim 2, wherein the strain is isolated from kimchi.
4. The pharmaceutical composition of claim 2, wherein the strain is live or dead.
5. The pharmaceutical composition of claim 2, wherein the composition induces regulatory T cells.
6. The pharmaceutical composition of claim 2, wherein the composition induces regulatory B cells.
7. According to claim 2, wherein the composition is characterized in that the ratio of the production of interleukin-10 (IL-10) to the production of interleukin-12 (IL-12) is 5-30, pharmaceutical composition.
8. The pharmaceutical composition of claim 2, wherein the composition inhibits immunoglobulin E (IgE) production.
9. The pharmaceutical composition of claim 2, wherein the composition inhibits Th1-related cytokine production.
10. The pharmaceutical composition of claim 2, wherein the composition inhibits Th17-related cytokine production.
11. The pharmaceutical composition of claim 2, wherein the composition inhibits Th2-related cytokine production.
12. The pharmaceutical composition of claim 2, wherein the composition enhances intestinal immunity.
13. The pharmaceutical composition of claim 2, wherein the Th1-mediated immune disease or Th17-mediated immune disease is transplant rejection, autoimmune disease, or inflammatory disease.
14. The pharmaceutical composition of claim 2, wherein the Th2-mediated immune disease is an allergic disease.
15. Claim 1 Lactobacillus pentosus ( Lactobacillus pentosus ) KF340 strain (Accession No. KCCM 11675P) comprising an active ingredient, Th1-mediated immune disease, Th17-mediated immune disease, or health functional food composition for improving the Th2-mediated immune disease .
16. The health functional food composition of claim 15, wherein the strain is isolated from kimchi.
17. The health functional food composition of claim 15, wherein the strain is live or dead.
18. The health functional food composition of claim 15, wherein the Th1-mediated immune disease or Th17-mediated immune disease is transplant rejection, autoimmune disease, or inflammatory disease.
19. The health functional food composition of claim 15, wherein the Th2-mediated immune disease is an allergic disease.
20. Claim 1 Lactobacillus pentosus ( Lactobacillus pentosus ) comprising a KF340 strain (Accession No. KCCM 11675P) as an active ingredient, a cosmetic composition for improving Th1-mediated immune disease, Th17-mediated immune disease, or Th2-mediated immune disease.
21. The cosmetic composition of claim 20, wherein the strain is isolated from kimchi.
22. The cosmetic composition according to claim 20, wherein the strain is live or dead.
23. The cosmetic composition according to claim 20, wherein the Th1-mediated immune disease or Th17-mediated immune disease is transplant rejection, autoimmune disease, or inflammatory disease.
24. The cosmetic composition according to claim 20, wherein the Th2-mediated immune disease is an allergic disease.
25. A Th1-mediated immune disease, Th17-mediated immune disease, or Th2, comprising administering to a subject a composition comprising the Lactobacillus pentosus KF340 strain (Accession No. KCCM 11675P) of claim 1 as an active ingredient. How to prevent or treat mediated immune diseases.
26. Use for preventing or treating a Th1-mediated immune disease, a Th17-mediated immune disease, or a Th2-mediated immune disease of the Lactobacillus pentosus KF340 strain (Accession No. KCCM 11675P) of claim 1.

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