KR20170020204A - Composition for Preventing, Improving, or Treating of Th1-mediated Immune Disease, Th17-mediated Immune Disease, or Th2-mediated Immune Disease Comprising Extracts from Lactobacillus pentosus as an Active Ingredients - Google Patents

Abstract

The present invention relates to a Lactobacillus pentosus KF340 strain (donation number: KCCM 11675P), and to a composition for preventing, alleviating, or treating a Th1-mediated immune disease, a Th17-mediated immune disease, or a Th2-mediated immune disease, wherein the composition includes the strain as an active ingredient. The novel Lactobacillus pentosus KF340 strain according to the present invention effectively controls allergy and inflammation by inducing regulator T cells and regulator B cells, adjusting the rates of immunologic tolerance cytokine (IL-10, anti-inflammatory cytokine) with respect to immunoactive cytokine (IL-12), inhibiting the generation of immunoglobulin E (IgE), inhibiting the generation of Th1-related cytokine, Th17-related cytokine, and Th2-related cytokine during a hypersensitivity reaction, and improving mucosal immunity according to the secretion of IgA. Thus, it is expected that the strain can be useful as a composition for preventing, alleviating, or treating a Th1-mediated immune disease, a Th17-mediated immune disease, or a Th2-mediated immune disease, more preferably, allergy or an inflammatory disease.

Description

Composition for Preventing, Improving, or Treating Th1-mediated Immune Disease, Th17-Mediated Immune Disease, or Th2-Mediated Disease comprising Lactobacillus pentosus as an Active Ingredient Immune Disease, Th17-mediated Immune Disease, or Th2-mediated Immune Disease Comprising Extracts from Lactobacillus pentosus as Active Ingredients}

The present invention relates to a novel Lactobacillus pentosus KF340 strain (Accession No. KCCM 11675P) and a method for the prevention of a Th1-mediated immune disease, a Th17-mediated immune disease, or a Th2-mediated immune disease , ≪ / RTI >

Recently, the meta genome project to identify intestinal microflora in the United States has revealed the correlation between the distribution of intestinal microflora and various immune system diseases including allergies. Thus, the distribution and diversity of intestinal bacteria and intestinal immunity are closely related to the immune system.

Looking at the market size of probiotics, the world probiotic market size is estimated to be 35 trillion won by 2014 and 57 trillion won by 2020, which is expected to show an annual average growth rate of 7.6%. The domestic probiotic market is expected to reach 150 billion won by 2014, but the market is growing rapidly, with a 250% growth rate from 2013.

Since the technology for detecting and evaluating immune control probiotics such as allergy suppression has not yet been established, it is expected that if a systematic in vitro experiment or an in vivo experimental mimic screening system is developed and its technology is used well, The value is also expected to be enormous.

Studies on the fundamental mechanism of probiotics are inadequate, and most of the research has been done in vitro . In other words, despite the oral ingestion of probiotics, most of the previous studies have been focused on in vitro experiments using cell lines, and these experimental methods have not been able to replace the research on the functions that can be shown by people taking probiotics There are big disadvantages. In addition, the intestinal immune system (GALT) exposed to probiotics exhibits immune function of probiotics by the simultaneous presence of various immune cells and interaction, but the in vitro experiment is different from the in vivo environment.

Thus, in the present invention, the in vivo system by evaluating the effect of Lactobacillus pento suspension (Lactobacillus pentosus) of these were directly isolated from the mice to different immune cells in order to evaluate the anti-allergic activity of the bacterial strains isolated from Korean traditional food A close study result was derived.

Currently, the evaluation of immunity control for overseas probiotics is applied to various disease animal models, and the results are also increasing at a rapid pace every year. In addition to targeting inflammatory response inhibition of inflammatory diseases such as Type 1 diabetes, rheumatoid arthritis, degenerative arthritis and ulcerative colitis, a probiotic that can increase the immune response to fight diseases such as influenza virus or cancer Research is actively underway on the function of In addition, the role of probiotics and their functions are attracting much attention for the prevention and treatment of diseases such as allergy, asthma and atopy.

Since most probiotics are derived from human intestines or foods, there is an advantage in that there is no major problem in proving stability with other chemical agents once the efficacy has been demonstrated using animal models. In recent years, the therapeutic effect of probiotics as an immunomodulatory agent has been verified through various clinical trials. Many diseases such as asthma, ulcerative colitis, and arthritis have been tried, and clinical trials and studies on atopy have been accelerated In fact.

According to industrialization trend analysis, it is leading the development of probiotics in Europe, which is traditionally strong, and the development and commercialization of functional probiotics are being actively carried out in the US and Japan recently. VSL3 # was developed by VSL Pharmaceutical company in Canada and is currently active in the global market. Recently, VSL3 # has been marketed as an immunity regulator in the domestic market.

The present inventors have made extensive efforts to develop probiotics for the prevention or treatment of allergic and inflammatory diseases caused by Th1, Th17, or Th2 immune responses. As a result, a novel Lactobacillus pentosus strain having antiallergic and anti-inflammatory activity was selected from kimchi, which is a traditional fermented food, and when the strain was treated, regulatory T cell induction, regulatory B cell induction, (Th1-related cytokine, Th17-related cytokine (IL-10), and anti-inflammatory cytokine) in the regulation of the ratio of immunotolerant cytokine (IL-10; antiinflammatory cytokine) to cytokine Inhibiting the production of cytokines, cytokines, and Th2-related cytokines, and enhancing intestinal immune responses, thereby effectively controlling allergy and inflammation.

Accordingly, the present invention provides a Lactobacillus pentosus KF340 strain (Accession No. KCCM 11675P) having a preventive, ameliorating, or therapeutic activity of a Th1-mediated immune disease, a Th17-mediated immune disease or a Th2- The purpose is to provide.

The present invention also relates to the use of the Lactobacillus pentosus KF340 strain (Accession No. KCCM 11675P) as an active ingredient for the prevention of a Th1-mediated immune disease, a Th17-mediated immune disease, or a Th2- The present invention provides a composition for improving, treating, or ameliorating a disease or disorder.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

According to an aspect of the present invention as described above, the present invention Th1- mediated immune diseases, Th17--mediated immune diseases, or Th2- mediated prevention of autoimmune diseases, the novel Lactobacillus pento suspension having an improved or therapeutic activity (Lactobacillus pentosus KF340 strain (Accession No. KCCM 11675P).

The present invention also provides a method of preventing, ameliorating, or treating a Th1-mediated immune disease, a Th17-mediated immune disease, or a Th2-mediated immune disease, comprising the above-mentioned Lactobacillus pentosus KF340 strain (Accession No. KCCM 11675P) ≪ / RTI >

In one embodiment of the present invention, the strain is characterized by being isolated from the kimchi.

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

In another embodiment of the invention, the composition is capable of inducing regulatory T cells.

In another embodiment of the invention, the composition is capable of inducing regulatory B cells.

In another embodiment of the present invention, the composition may make the ratio of IL-10 production to IL-12 production 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 present invention, the composition may inhibit Th1, Th17, or Th2 related cytokine production.

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

In another embodiment of the present invention, the ThI-mediated disease or Th17-mediated immune disease may be a 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 above-mentioned allergic diseases are specifically exemplified by 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, virus allergies, drug allergies, thyroid and allergic encephalitis.

In another embodiment of the present invention, the composition may be a pharmaceutical, food, health functional food, cosmetic, or feed composition.

The present invention also relates to a method of treating or preventing a Th1-mediated immune disease, a Th17-mediated immune disease, or a Th2-mediated disease, comprising administering to a subject a composition comprising an Lactobacillus pentosus KF340 strain (Accession No. KCCM 11675P) Thereby providing a method for preventing or treating immune diseases.

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

The novel Lactobacillus pentosus KF340 strain (Accession No. KCCM 11675P) according to the present invention is a novel strain of Lactobacillus pentosus KF340 (Accession No. KCCM 11675P) in accordance with the present invention is an immunostimulatory cytokine (IL-10; antiinflammatory) against regulatory T cell and regulatory B cell induction, immunostimulatory cytokine Cytokine), suppression of the production of immunoglobulin E (IgE), suppression of Th1-related cytokine, Th17-related cytokine, and Th2-related cytokine production during immune hypersensitivity reaction, and enhancement of intestinal immune response by IgA secretion Allergic and inflammatory diseases, and is useful as a composition for preventing, improving or treating a Th1-mediated immune disease, a Th17-mediated immune disease, or a Th2-mediated immune disease, more preferably an allergic or inflammatory disease It is expected to be possible.

FIG. 1 shows the results of measurement of the expression of Foxp3 by co-culture of Lactobacillus pentosus KF340 and immune cells.
FIGS. 2A to 2D are the results showing the effect of oral administration of Lactobacillus pentosus KF340 on the atopic dermatitis in mice induced atopic dermatitis. FIG. 2A shows changes in ear thickness, FIG. 2B shows changes in ear thickness, FIG. 2c is a photograph of a mouse ear, which is a lesion of atopic dermatitis, and FIG. 2d is a result of observing epithelial cell thickness and inflammatory cell infiltration by staining the lesion with H & E or TB.
FIG. 3A shows the results of IL-4 secretion from lymphocytes isolated from each lymph node after oral administration of Lactobacillus pentosus strain KF340 in a mouse induced atopic dermatitis (PP: Payer's patch, mLN: mesenteric lymph node, dLN: draining lymph node), and FIG. 3B shows the result of measuring the expression of GATA-3, a transcription factor of Th2, in the lesion tissue.
4A to 4D are graphs showing the effect of oral administration of Lactobacillus pentosus KF340 live bacteria or dead bacteria in a mouse causing atopic dermatitis. Fig. 4A is a result of measurement of change in ear thickness, (PBS: control group, 340: live cells, HK340: dead cells). FIG. 4 (c) shows the results of measurement of the amount of IL-17A secreted by Th17 And the expression of ROR gamma t.
FIG. 5A shows the increase in IL-10 secretion by Lactobacillus pentosus KF340 in splenocytes, FIG. 5B shows the increase in B cell activity in spleen by the strain treatment, FIG. Indicating the increase in the population of B220 + CD5 + CD1d + B10 cells by the strain treatment.
FIG. 6 shows the results of measurement of the expression of B220 + CD5 + CD1d + by Lactobacillus pentosus KF340 in a mouse model of atopic dermatitis (SPL: spleen, PP: Payer's patch).
FIG. 7 is a graph showing the frequency of dendritic cells (DCs) in the Payer's patch after administration of the Lactobacillus pentosus KF340 strain in the atopic dermatitis mouse model and the decreased expression of CD80 and CD86 in dendritic cells.
8a and 8b show the direct effect of Lactobacillus pentosus KF340 on dendritic cells (DCs). FIG. 8a shows the effect of IL-12 on bone marrow dendritic cells (BMDC) 10 and TGF-?, And FIG. 8b shows the results of measurement of BAFF mRNA expression in dendritic cells (DC) or Payer's patch.
FIG. 9A shows the result of measuring the amount of IgA in the mice that had induced atopic dermatitis before the administration of the Lactobacillus pentosus KF340 strain (0 W) and the end of the experiment (8 W) after the administration, and FIG. The results are shown in Fig.
FIG. 10 is a diagram showing an experimental method and schedule for producing a food allergy mouse model. FIG.
Figs. 11A to 11C show the results of inhibiting the food allergic activity of Lactobacillus pentosus KF340 strain. Fig. 11A is a rectal temperature change, Fig. 11B is a diarrhea reaction, and Fig. 11C is an anaphylactic measurement result.
FIG. 12 shows the results of oral administration of Lactobacillus pentosus KF340 in the contact dermatitis mouse model.

The present inventors have made extensive efforts to develop probiotics for the prevention or treatment of allergy and inflammatory diseases, diseases or conditions induced by Th1 or Th2 immune responses. As a result, a novel Lactobacillus pentosus strain having antiallergic and anti-inflammatory activity was selected from kimchi, which is a traditional fermented food, and when the strain was treated, regulatory T cell induction, regulatory B cell induction, (Th1-related cytokine, Th17-related cytokine (IL-10), and anti-inflammatory cytokine) in the regulation of the ratio of immunotolerant cytokine (IL-10; antiinflammatory cytokine) to cytokine Inhibition of the production of cytokines, cytokines, and Th2-related cytokines, and enhancement of the intestinal immune response, thereby effectively controlling allergies and inflammation.

Accordingly, the present invention provides a novel Lactobacillus pentosus KF340 strain (Accession No. KCCM 11675P) having a preventive, ameliorative or therapeutic activity against a Th1-mediated immune disease, a Th17-mediated immune disease or a Th2- Lt; / RTI >

The present invention also relates to a method for the prevention and / or amelioration of a Th1-mediated immune disease, a Th17-mediated immune disease, or a Th2-mediated immune disease comprising the above-mentioned Lactobacillus pentosus KF340 strain (Accession No. KCCM 11675P) Or a therapeutic composition.

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

As used herein, the term "improvement" means all actions that at least reduce the degree of symptom associated with the condition being treated. Wherein the composition can be used simultaneously or separately with a medicament for treatment before or after the onset of the disease for the prevention or amelioration of a Th1-mediated immune disease, a Th17-mediated immune disease, or a Th2-mediated immune disease .

The term " treatment ", as used herein, refers to any action by the administration of a composition according to the present invention that alleviates or alleviates symptoms of a Th1-mediated disease, a Th17-mediated immune disease, or a Th2- it means.

As used herein, the term " comprising as an active ingredient " is meant to include an amount sufficient to achieve efficacy or activity of the above-mentioned Lactobacillus pentosus KF340 strain. Since the Lactobacillus pentosus KF340 strain of the present invention is a microorganism strain isolated from a food, preferably Kimchi, there is no adverse effect on the human body even when administered in an excessive amount, and the quantitative upper limit of the strain of the present invention is within the appropriate range As shown in FIG.

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

According to one embodiment of the invention, the composition of the present invention is capable of inducing regulatory T cells. The regulatory T cell induction can be confirmed by increased Foxp3 expression, a regulatory T cell transcription factor, as demonstrated in the Examples below.

In one embodiment of the invention, the composition of the present invention increases 2-20, 5-20, 5-15, 5-10, or 7-9 fold activation of regulatory T cells compared to negative control (See 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 increasing the expression of B220 + CD5 + CD1d + B cells (B10 cells) as demonstrated in the following examples. B10 cells are a subset of B cells known to have an anti-inflammatory cytokine, IL-10 producing activity, and play an important role in regulating inflammatory and autoimmune responses.

In one embodiment of the present invention, induction of dendritic cells (DCs) of Payer's patch in which Lactobacillus pentosus KF340 is absorbed into inhibitory dendritic cells promotes the production of IL-10 and BAFF, And directly induced regulatory B cells (see Example 6). The composition of the present invention increases 2-20 fold, 2-10 fold, 2-8 fold, 3-7 fold, or 4-6 fold activation of regulatory B cells compared to negative control.

According to one embodiment of the present invention, the composition of the present invention may increase IL-10 production. IL-10 secretes macrophages and monocytes and T-cell lymphocyte replication and inflammatory cytokines (IL-1, TNF-α, TGF-β, IL-6, IL-8 and IL-12) An increase in IL-10 as an anti-inflammatory cytokine produced by inhibitory lymphoid cells inhibits the inflammatory response.

In one embodiment of the present invention, co-culture of mesenteric lymph node lymphocyte and Lactobacillus pentosus KF340 strain showed that the amount of IL-10 produced by the strain was significantly increased in the lymphocytes. Therefore, the composition of the present invention can make the 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, -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 an immunoglobulin E, which has an affinity for mucous cells or basophils in the blood, and causes an inflammatory reaction when IgE antibodies attached thereto and the corresponding antigens (allergens) react with each other. That is, IgE is an antibody that induces anaphylactic or inflammatory reaction due to allergy. If the IgE production is inhibited, allergic reaction or inflammatory reaction is suppressed.

According to one embodiment of the present invention, the composition of the present invention can inhibit Th1-associated cytokine production. Thus, 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 specified in terms of gene expression, protein secretion, and functional activity. For example, Th1 cells express a cytokine expression pattern that produces IL-2 and IFN-y 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 reactions to a variety of intracellular pathogens.

The term "Th1-mediated immune disorder" as used herein refers to cytokines (Th1-associated cytokines) produced by the production and / or activity of Th1 cells such as IL-1β, IL-2, -γ or TNF-α. The Th1-mediated immune disorder may be a transplant rejection, an autoimmune disease, or an inflammatory disease, and more particularly, a disease selected from the group consisting of colitis, inflammatory bowel disease, type 1 diabetes, type 2 diabetes, rheumatoid arthritis, reactive arthritis, Myocarditis, endocarditis, pericarditis, cystic fibrosis, Hashimoto's thyroiditis, Graves' disease, leprosy, syphilis, Lyme disease, Borreliosis, neurogenic-borrelia, Inflammatory bowel syndrome, Crohn's disease, granulomatous disease, fibromyalgia, chronic fatigue syndrome, chronic inflammatory bowel disease, chronic obstructive pulmonary disease, chronic obstructive pulmonary disease, chronic obstructive pulmonary disease, Fatigue immunodeficiency syndrome, myalgic encephalomyelitis, amyotrophic lateral sclerosis, Parkinson's disease, multiple sclerosis, autistic spectrum disorders, attention deficit disorder, and attention deficit disorder Hyperactivity disorder, and the like.

According to one embodiment of the present invention, the composition of the present invention can inhibit Th17-related cytokine production. Thus, 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 cell" 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- [beta], IL-6 or IL-21 to produce IL-17, IL-22 and IL-21, but not IL-2, IL-4, -13 does not produce a cytokine expression pattern. Th17 cells have been reported to play a direct role in inflammation and autoimmune pathogenesis and to induce host defense or an abnormal immune response to the pathogen.

The term "Th17-mediated immune disease" as used herein means a disease involving a cytokine (Th17-related cytokine) such as IL-17 produced by the production and / or activity of Th17 cells. Preferably, the Th17-mediated immune disease may be a transplant rejection, an autoimmune disease or an inflammatory disease. More specifically, the Th17-mediated immune disease may be an inflammatory disease such as rheumatoid arthritis, psoriasis, multiple sclerosis, colitis, inflammatory bowel disease, But is not limited to, an immune disorder.

According to one embodiment of the present invention, the composition of the present invention can inhibit Th2-related cytokine production. Thus, 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 may inhibit cytokine production selected from the group consisting of IL-4 and IL-13.

The term " Th2 cell " as used herein 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 express IL-4, IL-5 and IL-13 cytokines (Th2-related cytokines) expression patterns, and Th2 cells are involved in humoral immune responses.

As used herein, the term " Th2-mediated immune disease " means a disease involving IgE and mast cells due to the production and activity of allergen-specific Th2 cells, preferably an allergic disease. More specifically, the above-mentioned allergic diseases are selected from food allergies, bronchial asthma, allergic rhinitis (acute or chronic), atopic dermatitis, allergic conjunctivitis, allergic otitis media, urticaria and anaphylactic shock, contact hypersensitivity, , Bacterial allergy, fungal allergy, virus allergy, drug allergy, thyroid and allergic encephalitis.

In one embodiment of the present invention, cytotoxicity of the Lactobacillus pentosus KF340 strain of the present invention to the mouse causing atopic dermatitis was measured and the cytokine changes were measured from lymphocytes isolated from inflammatory lymph nodes. As a result, the Th1-related cytokine IL-4 and IL-13, which mediate IgE secretion, were induced by inducing degranulation of mast cells with IFN-y, Th17-related cytokines IL-17 and Th2-related cytokines (Example 5- 3).

According to one embodiment of the present invention, the composition of the present invention can promote a glandular immune response.

In one embodiment of the present invention, when mice were infected with atopic dermatitis at day 0 or when Lactobacillus pentosus KF340 strain was administered and the feces were collected at week 8, the amount of IgA secretion increased, and IL -6 was also increased (see Example 7).

The term " allergy " as used herein refers to various diseases, diseases or abnormal conditions caused by hypersensitivity to any substance in the human body, i.e., excessive reaction of the body's immune system to an exogenous substance. The allergic diseases to be applied to the composition of the present invention are preferably Type I immediate hypersensitivity reaction and Type IV delayed hypersensitivity reaction. Type I immediate hypersensitivity reactions are bronchial asthma, allergic rhinitis, atopic dermatitis, allergic conjunctivitis, allergic otitis media, urticaria and anaphylatic shock, Type IV delayed hypersensitivity reactions are contact hypersensitivity , Allergic contact dermatitis, bacterial allergies, fungal allergies, viral allergies, drug allergies, thyroiditis and allergic encephalitis. The type I immediate-type hypersensitivity reaction is divided into two stages. The first step is to suppress the secretion of IgE and IgG1 by the invasion of the allergen and the Th1 cell reaction to produce IL-12 and IFN-γ which increase secretion of IgG2a And IL-4, IL-5, and IL-13, the IL-4 and IL-13 are secreted by the excessive immune response of Th2, The IgE-specific antibodies produced are attached to the surface of mast cells and basophils, so that allergic inflammation is prepared. It has been sensitized to allergens. The second stage of the allergic manifestation is divided into an initial reaction and a late reaction. Allergens re-enter the body to stimulate mast cells and induce a degranulation reaction, which causes release of histamine, lipid metabolites, cytokines, And eosinophil, eosinophil, macrophage, Th2 cell, and basophil are infiltrated into the affected tissue to induce inflammation, resulting in atopic dermatitis, rhinitis, asthma, and the like. Preferably, the allergies of the present invention can be allergic contact dermatitis, allergic atopic dermatitis, or food allergies.

In one embodiment of the present invention, the activity of inhibiting atopic dermatitis, food allergy and contact dermatitis of Lactobacillus pentosus KF340 was confirmed, and it was observed that not only live cells but also heat-treated dead cells exhibited similar effects (Examples 5 and 8 , And 9).

Accordingly, the novel Lactobacillus pentosus KF340 strain of the present invention can be manufactured from a pharmaceutical, health functional food, food, cosmetic, or feed composition.

The pharmaceutical composition of the present invention comprises (a) a pharmaceutically effective amount of the above-mentioned Lactobacillus pentosus of the present invention; 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 aforementioned Lactobacillus pentosus.

The pharmaceutical composition according to the present invention comprises Lactobacillus pentosus KF340 strain as an active ingredient, and may further comprise a pharmaceutically acceptable carrier. Such pharmaceutically acceptable carriers are those conventionally used in the field of application and include, but are not limited to, saline, sterile water, Ringer's solution, buffered saline, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, And may further contain other conventional additives such as antioxidants and buffers as needed. It may also be formulated into injectable formulations, pills, capsules, granules or tablets, such as aqueous solutions, suspensions, emulsions and the like, with the addition of diluents, dispersants, surfactants, binders and lubricants. Suitable pharmaceutically acceptable carriers and formulations can be suitably formulated according to the respective ingredients using the methods disclosed in Remington's reference. The pharmaceutical composition of the present invention is not particularly limited to a formulation, but may be formulated into injections, inhalants, external skin preparations, and the like.

The pharmaceutical composition of the present invention can be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically) according to the intended method, but preferably can be administered orally, Depends on the condition and the weight of the patient, the degree of the disease, the type of the drug, the administration route and time, but can 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, the term "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 the effective dose level will depend on the type of disease, severity, The activity of the compound, the sensitivity to the drug, the time of administration, the route of administration and the rate of release, the duration of the treatment, factors including co-administered drugs, and other factors well known in the medical arts. The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, sequentially or concurrently with conventional therapeutic agents, and may be administered singly or in multiple doses. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal 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, body weight, the degree of absorption of the active ingredient in the body, the rate of inactivation and excretion, the type of disease, 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 one to three divided doses per day. However, the dosage may be varied depending on the route of administration, the severity of obesity, sex, weight, age, etc. Therefore, the dosage is not limited to the scope of the present invention by any means.

When the composition of the present invention is manufactured from a health functional food composition, the composition may be in the form of tablets, pills, powders, granules, powders, capsules, and liquid formulations, including one or more of carriers, diluents, excipients, ≪ / RTI > Examples of foods that can be added to the composition of the present invention include various foods, powders, granules, tablets, capsules, syrups, drinks, gums, tea, vitamin complexes, and health functional foods. Examples of the additive that can be further included in the present invention include natural carbohydrates, flavors, nutrients, vitamins, minerals (electrolytes), flavors (synthetic flavors, natural flavors and the like), colorants, fillers, At least one component selected from the group consisting of alginic acid and its salts, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, antioxidants, glycerin, alcohols, carbonating agents and fats 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 polysaccharides such as dextrins, cyclodextrins and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. As the above-mentioned flavors, natural flavors (tautatin, stevia extract (for example, rebaudioside A and glycyrrhizin) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used. The composition according to the present invention can be used in various forms such as flavorings such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, colorants and heavies, factic acid and its salts, alginic acid and its salts, , pH adjusting agents, stabilizers, antiseptics, glycerin, alcohols, carbonating 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 ingredients may be used independently or in combination. Specific examples of the carrier, excipient, diluent, and additive include, but are not limited to, lactose, dextrose, But are not limited to, calcium carbonate, calcium carbonate, alginate, gelatin, calcium phosphate, calcium silicate, microcrystalline cellulose, It is preferable to use at least one selected from the group consisting of sugar syrup, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

When the antiallergic composition comprising an active ingredient of the Lactobacillus pentosus KF340 strain of the present invention is prepared from a cosmetic composition, it contains components commonly used in cosmetic compositions in addition to Lactobacillus pentosus as an active ingredient, , Customary adjuvants such as solubilizers, vitamins, pigments and flavoring agents, and carriers.

The cosmetic composition of the present invention can be prepared into any of the formulations conventionally produced in the art and can be used as a solution, a suspension, an emulsion, a paste, a gel, a cream, a lotion, a powder, a soap, , Oil, powder foundation, emulsion foundation, wax foundation and spray, but is not limited thereto. More specifically, it can be manufactured in the form of a soft lotion, a nutritional lotion, a nutritional cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray or a powder.

When the formulation of the present invention is a paste, cream or gel, an animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as the carrier component .

When the formulation of the present invention is a paste, cream or gel, an animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as the carrier component .

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 as a carrier component. In the case of a spray, in particular, / Propane or dimethyl ether.

When the formulation of the present invention is a solution or an emulsion, a solvent, a dissolving agent or an emulsifying agent is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, , 3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan fatty acid esters.

In the case where the formulation of the present invention is a suspension, a carrier such as water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Cellulose, aluminum metahydroxide, bentonite, agar or tracant, etc. may be used.

When the formulation of the present invention is an interfacial active agent-containing cleansing, the carrier component may include aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives, or ethoxylated glycerol fatty acid esters.

When a composition for anti-allergy comprising the active ingredient of the Lactobacillus pentosus KF340 strain of the present invention is prepared from a feed composition, it may be prepared by directly adding Lactobacillus pentosus or an additive usually added in the production of feed have. For example, various nutrients such as vitamins, amino acids and minerals, antioxidants, antibiotics, antibacterial agents and other additives. The shape includes powders, granules, pellets or suspensions, and the like.

When the composition of the present invention is prepared from a feed composition, it may be supplied to the land or aquatic animals singly or in a feed mixture. The feed of the present invention includes, but is not limited to, powdered feed, solid feed, moist pellet feed, dry pellet feed, extruder pellet (EP) feed,

A strain having the preventive, ameliorating, or therapeutic activity of the Th1-mediated immune disease, Th17-mediated disease or Th2-mediated immune disease of the present invention is a Th1-mediated immune disease, a Th17-mediated immune disease or a Th2- The present invention is not limited to the composition for preventing, ameliorating, or treating the disease, and the subject disease is common, so that the common content in relation to the composition is omitted in order to avoid the excessive complexity of the present specification.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

[ Example ]

Example  1: Isolation and Identification of Strain

The strain was isolated from a kimchi prepared by conventional methods using Lactobacillus MRS agar medium (MRS medium, BD288130, Difco, USA), and the isolated strain was identified through 16S rDNA sequencing. As a result of the identification, the 16s rDNA sequence (the first sequence of the sequence listing) of the strain showed 99.73% homology with the conventional Lactobacillus pentosus JCM1558 (T) strain.

The culture medium for the culture of the strain is MRS medium, the culture conditions are pH 6.5 ± 0.2, temperature incubation at 37 ° C. for 48 hours, oxygen requirement is uniform anaerobic, and preservation of the strain through freeze-drying preservation or cell suspension freezing It is possible. The present inventors named this strain as Lactobacillus pentosus KF340 and deposited it on March 6, 2015 with the deposit number KCCM 11675P at the Korean Microorganism Conservation Center (KCCM), an international microorganism depository institution.

Example  2: Preparation and method of experiment

2-1. Experimental animal

For this study, wild-type mice (strain No. -C57BL / 6, stock number -000664, the Jackson laboratory) without any genetic modification were used. In addition, genetically modified mice (Foxp3-GFP knock-in mouse, strain name-B6.Cg-Foxp3 ^tm2Tch / J, STOCK No. -006772, Jackson laboratory) were used to comparatively easily detect the expression level of Foxp3. Since the mouse was constructed based on a wild-type species, the overall genetic information is the same, but when Foxp3 is expressed, EGFP (enhanced green fluorescence protein), which is a molecule capable of emitting a fluorescent signal, is expressed together with the expression of Foxp3 Therefore, the expression level of Foxp3, which is difficult to detect using the mouse, can be comparatively easily detected without using the antibody.

2-2. Immune cell isolation and Lactobacillus With Pentosus  Co-culture

After removal of the mesenteric lymph node (MLN) from the mouse, immune cells (mostly lymphocytes) present in the lymph nodes were suspended in single cells and used for the experiment. Because the probiotics present in the intestine are recognized by immune cells present in the intestinal immune system, immune cells suitable for analyzing the characteristics of probiotics are immune cells present in mesenteric lymph nodes. Therefore, the characteristics of Lactobacillus pentosus KF340 were analyzed with the immune cells isolated from the lymph nodes.

For this, immune cells isolated according to the above method and Lactobacillus pentosus KF340 were cultured in a 1:10 ratio, that is, 3x10 ⁶ of immune cells and 3x10 ⁷ cfu of the strain for 72 hours at 37 ° C in a CO ₂ incubator. At that time, the antibiotic gentamicin (11811-031, 15 ㎍ / mL, GIBCO) was treated together to prevent the overgrowth of the test strain.

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

3-1. Cytokine measurement in co-culture solution

ELISA is a method of measuring the amount of an antigen or an antibody using an antigen-antibody reaction using an enzyme as a marker. (Mouse IL-10 ELISA Ready-SET-Go, eBioscience) and IL-12p70 (Mouse IL-12p70 ELISA Ready-SET) were used to measure the amount of cytokine present in the supernatant after co- -Go, eBioscience) ELISA. The antibody for coating, which detects each cytokine, was treated in 96-wells, and then the supernatant was added to allow the reaction to proceed for a predetermined period of time. Then, another detection antibody for detecting each cytokine was treated. At this time, the antibody for detection is bound to the enzyme (HRP), and a color change occurs as a result of an enzyme-substrate reaction by adding an enzyme substrate (Tetramethylbenzidine Substrate Solution, eBioscience). The absolute amount of cytokine can be measured by this indirect method.

IL-10 (interleukin 10) is a representative immune tolerance cytokine. Since IL-12 (interleukin 12) is known as a representative immunologically active cytokine, the absolute / relative amount of two cytokines is determined and lactic acid bacteria having anti- can do. Thus, mesenteric lymph node cells of Foxp3-knockout mice were isolated according to the method of Example 2-2, mixed with each test strain at a ratio of 1:10, and co-cultured. After culturing for 3 days, the culture supernatant was taken and the absolute amount of IL-12p70, an immunologically active cytokine, and IL-10, an anti-inflammatory cytokine, were measured by ELISA (enzyme linked immunosorbent assay) Respectively.

As a result, when the mesenteric lymph node cells and the Lactobacillus pentosus KF340 strain were co-cultured, the relative ratio (lL-10 / IL-12p70) of the amount of lL-10 and IL-12p70 secretion in the lymphocytes was 24.17 As compared with the negative control group. This indicates that Lactobacillus pentosus KF340 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

* It is not detectable, and anti-inflammatory activity can not be expected because it secretes a small amount to escape from the detection area.

3-2. Foxp3  Expression measurement

Foxp3 is a transcription factor of regulatory T cells, a subset of T cells that inhibit excessive immunity. Foxp3 expression can be used to determine the distribution of regulatory T cells.

The regulatory T cell is one of a subset of T cells and plays a role in suppressing excessive immune responses unlike other T cells. Since these regulatory T cells inhibit allergic diseases, it is supported by various studies. Therefore, when the test strain and mesenteric lymph node cells are co-cultured, a strain capable of increasing the distribution of regulatory T cells becomes a probiotic having an antiallergic effect There is a high possibility.

Therefore, in order to compare the expression level of surface molecules of mesenteric lymphocytes co-cultured with Lactobacillus pentosus KF340 strain, Foxp3 was assayed by flow cytometry on the mesenteric lymph node cells isolated according to the method of Example 3-1 The expression level of Foxp3 was measured and compared by measuring the expression of GFP signaling when expressed.

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

Example  4: Th2  Identification of antiallergic activity by cytokine inhibition

And to evaluate whether Lactobacillus pentosus KF340 exhibits antiallergic activity through inhibition of Th2 cytokine production.

For this purpose, a mixture of OVA (20 μg) and aluminum hydroxide gel (2 mg) was mixed for 30 minutes, and then 100 μl per mouse of 5-week-old female Balb / c mice were intraperitoneally injected twice . The mouse spleen after immunization 1 weeks were removed to dispense single cells Chemistry and 5 × 10 to hemolysis by spleen cells in a 96-well plate at ^{106 cells} / well. At this time, strains of antigen (OVA, 100 ㎍ / mL) and Lactobacillus pentosus KF340 (5 × 10 ⁷ CFU / well) were added and cultured in a 37 ° C. CO ₂ incubator for 72 hours. The supernatant was recovered and the representative Th2- The amount of IL-4 secretion was measured.

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

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

Control group 1: allergen-free experimental group, control group 2: allergen-induced experimental group

Example  5: Anti-atopic  Verify Active

5-1. Induction of atopic dermatitis

Lactobacillus pentosus KF340 strain was orally administered to the mouse at 5 x 10 ⁸ CFU / day for 5 weeks or more for 8 weeks. Three weeks after the first oral administration, the ear of the mouse was peeled off with a surgical tape, and 20 μl of 10% DNCB (2,4-dinitrochlorobenzene) and house dust mite mg / mL). One week later, atopic dermatitis was induced by repeating the treatment with 1% DNCB and 20 ㎕ (10 mg / mL) mites once a week for 5 consecutive weeks.

5-2. Ear thickness and histological changes

After the administration of Lactobacillus pentosus KF340 strain to a house dust mite-induced atopic dermatitis mouse model according to the method of Example 5-1, histological changes were measured to evaluate anti-atopic activity. At this time, dexamethasone, a steroid preparation used as a treatment for atopic dermatitis, was used as a positive control.

As a result of measuring the degree of ear thickness change, erythema, edema, and keratinization of the mouse, as shown in FIG. 2A, unlike the PBS control in which PBS was administered after inducing atopic dermatitis, the Lactobacillus pentosus In the case of the group administered with KF340 strain (340), the thickness of the ear was reduced to a level similar to that of the positive control (Dex), and a negative control group , The oral administration group of Lactobacillus pentosus KF340 strain showed a level similar to that of the positive control group.

In addition, as a result of visual observation, it was observed that atopic dermatitis was greatly improved in the group administered with Lactobacillus pentosus KF340 as shown in FIG. 2C. Histological analysis of ear tissue, which is an inflammation site, The epithelial cell thickness of Lactobacillus pentosus KF340 treated group was decreased and the infiltration of inflammatory cells was significantly suppressed compared to the negative control group.

5-3. Cytokine change measurement

For cytological analysis, draining lymphocytes were isolated from draining lymph nodes (surface lymph nodes, axillary lymph nodes, bronchial lymph nodes) of the inflammatory site of the mouse, stimulated with house dust mites, and then expressed as e - ELISA using a cytokine kit from Bioscience.

As a result, as shown in Table 3 below, in the experimental group in which Lactobacillus pentosus KF340 was continuously administered, the amounts of IFN-γ and IL-17A, which are inflammatory cytokines promoting the onset and aggravation of atopic dermatitis, , And secretion of IL-4 and IL-13, which are Th2-related cytokines, was also decreased, confirming that Lactobacillus pentosus KF340 inhibits atopic dermatitis.

Experimental group IFN-y (ng / mL) IL-17A (ng / mL) IL-4 (pg / mL) IL-13 (ng / mL) Normal control group 0.01 0 0 0 Negative controls (PBS treated 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 patch (PPs) and mesenteric lymph nodes (GALT) corresponding to gut-associated lymphoid tissue (GALT) and the Th2 cytokine IL-4 As a result, as shown in FIG. 3A, it was confirmed that the amount of IL-4 secretion decreased remarkably, and the expression of GATA-3, a transcription factor of Th2, was significantly decreased in the inflamed ear as shown in FIG. 3B.

5-4. Lactobacillus Pentosus  KF340 Dead bacteria Anti-atopic  Verify Active

The Lactobacillus pentosus KF340 live bacteria showed anti-atopic activity through Examples 5-2 and 5-3, and it was verified that the dead bacteria of the strain also had the same anti-atopic activity.

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

As a result of measurement of changes in glans of the mice caused by administration of Lactobacillus pentosus KF340 live or dead cells, it was confirmed that the increase in ear thickness was alleviated in all of the live cells and dead cells as compared with the control (PBS) as shown in FIG. Further, as shown in FIG. It was also observed that erythema, edema, keratin and other symptoms were alleviated through ear photographs. It was found that anti-atopic activity was higher in live bacteria than in dead bacteria.

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

As shown in FIG. 4C, the amount of IL-17 produced by the above-mentioned live cells or dead cells was decreased as compared with the control group, and the expression of RORγt, a Th17-related transcription factor, I confirmed it. From the above results, it was found that the live bacteria and dead bacteria of the Lactobacillus pentosus KF340 strain had atopic dermatitis inhibitory activity through the inhibition of the Th17 immune response.

Example  6: Lactobacillus Pentosus  Of KF340 Anti-atopic  Active mechanism

6-1. in vitro  And in vivo in  B220 + CD5 + CD1d + Cell increase confirmation

Since the Lactobacillus pentosus strain KF340 increased the production of IL-10 through the above Example 3-1, the strain affected the cells inducing IL-10 secretion in addition to the regulatory T cells, and thus the anti-allergic activity The cells secreted IL-10 were screened in vitro and in vivo using a flow cytometer (COULTER Epics XL, BECKMAN).

As shown in FIG. 5A, in the in vitro test, the secretion of IL-10 was significantly increased in the spleen by treatment with Lactobacillus pentosus KF340 strain, and the activity was significantly increased in B cells of spleen as shown in FIG. 5B . Furthermore, as shown in FIG. 5C, it was confirmed that the population of B220 + CD5 + CD1d + cells in splenic B cells was significantly increased by the strain treatment.

It has been reported that B10 cells (phenotype: B220 + CD5 + CD1d + or CD19 + CD5 + CD1d +) secrete many IL-10 cytokines specifically in the spleen as a subset of regulatory B cells, It is known to play an important role in regulating inflammatory and autoimmune responses.

In order to verify the above result, the mouse model of atopic dermatitis produced in Example 5-1 was orally administered to a strain of Lactobacillus pentosus KF340, and the spleen of the mouse was isolated and monoclonalized and analyzed by flow cytometry to obtain B220 + CD5 + CD1d + Were measured.

As a result, as shown in FIG. 6, the population of B220 + CD5 + CD1d + B cells was significantly increased in the group administered with Lactobacillus pentosus KF340 strain (340) as compared with the negative control (PBS control). However, B220 + CD5 + CD1d + B cells in PPs did not increase, suggesting that this was due to the systemic action of Lactobacillus pentosus KF340.

6-2. Lactobacillus Pentosus  DC induction confirmation by KF340

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

As a result, as shown in FIG. 7, it was found that the frequency of dendritic cells (hereinafter, DC) in PPs was increased in the group (340) in which the strain was administered compared to the control group (PBS control). Also, it was confirmed that the increased DC showed a pattern in which expression of CD80 and CD86 was decreased.

6-3. Lactobacillus Pentosus  By KF340 BAFF  Confirmation of expression promotion

The results of Example 6-2 above indicate that inhibitory DCs migrate from lymphoid tissue to the spleen and affect spleen cells, thus direct or indirect effect may have helped induce regulatory B cells, The direct effect of Lactobacillus pentosus KF340 on DC was investigated.

To this end, the above-mentioned strains were treated with bone marrow dendritic cells (BMDC), and the levels of IL-10 and TGF-β, which are related cytokines, and mRNA and protein levels of BAFF, Were measured by Real-time PCR and Western blotting. The primer sequences used in the real-time PCR are shown in Table 4 below.

gene direction order SEQ ID NO: IL-10 forward 5'-ATAACTGCACCCACTTCCCA-3 ' 2 reverse 5'-TCATTTCCGATAAGGCTTGG-3 ' 3 TGF-beta 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 FIG. 8A, mRNA expression and protein production were significantly increased in IL-10 compared with the control group, but no significant difference was observed in TGF-β. However, when BAFF mRNA expression level, which is an important factor in the induction of regulatory B cells, was measured, as shown in Fig. 8B, the expression of KF340 strain was significantly increased in DC compared to the control, The expression of BAFF was also increased in the PPs of the dermatitis mouse model.

Lactobacillus pentosus KF340, which exhibited an anti-atopic effect in the atopic dermatitis model, increased IL-10 secretion by increasing the population of spleen B220 + CD5 + CD1d + B10 cells, a kind of regulatory B cells Respectively. The increase of B10 cells promotes the production of IL-10 and BAFF by inducing DC of PPs absorbed by Lactobacillus pentosus strain KF340 into inhibitory DC, or induces direct B10 cell through lymph node through the splenic junction ≪ / RTI >

Example  7: Lactobacillus Pentosus  Confirming KF340's immunity enhancing activity

In order to evaluate whether or not the Lactobacillus pentosus KF340 strain promoted intestinal immunity, mice were treated with atopic dermatitis in the same manner as in Example 5-1, and Lactobacillus pentosus KF340 strain was orally administered, 0.0 > IgA < / RTI > For this, we measured the amount of IL-6, a cytokine that induces the secretion of IgA and IgA in the feces, using mouse IgA quantitation kit from BD after collecting the feces of the mice at the 0th day of the experiment and at the end of the experiment (week 8) Respectively.

As a result, as shown in FIG. 9A, the amount of IgA produced by the administration of Lactobacillus pentosus KF340 strain in the mouse induced atopic dermatitis was increased. In addition, IL-6 measurement revealed that IL-6 production, which induces IgA secretion in the payer's patch, is increased. These results indicate that Lactobacillus pentosus KF340 induces the production of IgA and thus affects mucosal immunity enhancement.

Example  8: Food allergy inhibitory activity

8-1. Food allergy  Induction and Lactobacillus Pentosus  Administration of KF340

The experimental animals were fed with 5-week-old magnetic BALB / c mice from Orient Bio Inc., and were used for breeding and refining for one week in an experimental animal room. According to the experimental procedure shown in Fig. 10, 100 μl of OVA (20 μg) and Alum (2 mg) mixed solution was intraperitoneally injected first by immunization, and then 14 days later, secondary immunization was carried out by the same method. After 14 days of the second immunization, Lactobacillus pentosus KF340 was administered at a daily dose of 5 × 10 ⁸ CFU / day. At this time, the normal control group (Naive) was administered the same amount of PBS as the suspension solvent instead of the Lactobacillus pentosus KF340 suspension, and the positive control group [PC (daxa)] was administered the immunosuppressant dexamethasone. To induce food allergy, OVA at 50 mg / day / day was orally administered to all groups at intervals of 3 days from the 14th day after the second immunization. After an oral administration of OVA, the anaphylactic reaction and diarrhea reaction, which are food allergy inducing indicators, were observed at intervals of 30 minutes. Changes in rectal temperature were measured at intervals of 20 minutes for 60 minutes.

As a result, as shown in FIG. 11A, when the Lactobacillus pentosus strain KF340 was administered, the rectal temperature remained higher than that of the sham group, and diarrhea and anaphylactic reactions were also observed in the sham group as shown in FIGS. 11B and 11C And Lactobacillus pentosus strain KF340 was found to be effective in alleviating the symptoms of food allergy.

8-2. serum IgE  Confirm change

After the end of the experiment, blood was drawn through the mouse orbit, and the serum was separated by centrifugation at 3500 rpm for 10 minutes. The IgE content in the separated serum was measured using a mouse IgE quantitative kit from BD.

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

8-3. Identification of cytokine changes

After the MLN and spleen were removed from each group of mice, a single cell suspension was seeded at a concentration of 5 × 10 ⁶ in 96-wells, and 20 μg of OVA was treated with cells to confirm the antigen-specific response , And cultured for 72 hours. After culturing for 72 hours, the cytokine present in the supernatant was confirmed by ELISA using a BD cytokine quantitation kit.

As a result, as shown in the following Table 5, when the Lactobacillus pentosus KF340 strain was administered, the secretion of IL-4 and IL-13 inducing IgE production in the MLN culture supernatant was decreased, and the food allergy of Lactobacillus pentosus KF340 Inhibitory activity.

Experimental group IL-4 (pg / mL) IL-13 (pg / mL) IgE (ng / mL) The 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. Contact dermatitis induction and Lactobacillus Pentosus  Administration of KF340

Five week old female BALB / c was applied for 1 week and then hair was removed from all the experimental groups except Naive at 0 (Day 0), and 5% TMA solution [acetone and isopropylmyristate] Of 4: 1 (v / v) as a solvent] was applied to perform the first sensitization. After 5 days of sensitization, 10 μl of 5% TMA was sensitized to both ears in all experimental groups except for the normal control (Naive), and 10 μl of 2% TMA was applied to both ears at 3 days. The Lactobacillus pentosus KF340 was administered at a concentration of 5 × 10 ⁸ CFU / ml daily after the first sensitization, and only the PBS used to suspend Lactobacillus pentosus KF340 was administered in the same amount as the Shamung group. The positive control group was prednisolone P) was used for the experiment.

TMA is a type of chemical hapten that induces allergic hypersensitivity of immune cells by binding to protein of skin tissue after penetrating into the skin through skin envelope and inducing Th2 reaction strongly and inducing repeated treatment of TMA Site inflammation and tissue thickness increase. As a result of the experiment, as shown in Fig. 12, when the Lactobacillus pentosus KF340 was administered, the thickness of the ear treated with TMA was decreased as compared with the negative control

9-2. serum IgE  Confirm change

After the end of the experiment, blood was drawn through the mouse orbit, and the serum was separated by centrifugation at 3500 rpm for 10 minutes. The IgE content in the separated serum was measured using a mouse IgE quantitative kit from BD.

As a result, as shown in Table 6 below, the IgE content exhibiting a high expression in allergic contact dermatitis patients was decreased as compared with the Sham group.

9-3. Identification of cytokine changes

DLN and spleen were removed from each group of mice, and then cultured for 96 hours at 3 × 10 ⁶ cells after single cell suspension. In order to confirm the antigen-specific reaction, the cells were divided into two groups, ie, culturing only the cells and treating 10 μg of the mites. After culturing for 96 hours, the cytokines in the supernatant were quantified using a cytokine quantitative kit of e-Bioscience And confirmed by ELISA method.

 As a result, the secretion amount of IL-4, which is a Th2-related cytokine, in DLN decreased, as shown in Table 6, and Lactobacillus pentosus KF340 was found to have an allergic contact dermatitis inhibitory activity.

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

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

<110> KOREA FOOD RESEARCH INSTITUTE <120> Composition for Preventing, Improving, or Treating of          Th1-mediated Immune Disease, Th17-mediated Immune Disease, or          Th2-mediated Immune Disease Comprising Extracts from          Lactobacillus pentosus as an Active Ingredients <130> MP16-217 <150> KR 10-2015-0114938 <151> 2015-08-13 <160> 7 <170> KoPatentin 3.0 <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 (24)

Lactobacillus pentosus KF340 strain (Accession No. KCCM 11675P) having a preventive, ameliorative, or therapeutic activity of Th1-mediated immune disease, Th17-mediated immune disease, or Th2-mediated immune disease.
A medicament for the prophylaxis or treatment of a Th1-mediated immune disease, a Th17-mediated immune disease, or a Th2-mediated immune disease, which comprises the Lactobacillus pentosus KF340 strain (Accession No. KCCM 11675P) Gt;
3. The pharmaceutical composition according to claim 2, wherein the strain is isolated from the kimchi.
The pharmaceutical composition according to claim 2, wherein the strain is a live or dead organism.
3. The pharmaceutical composition according to claim 2, wherein the composition induces regulatory T cells.
3. The pharmaceutical composition according to claim 2, wherein said composition induces regulatory B cells.
The pharmaceutical composition according to claim 2, wherein the composition is such that the ratio of IL-10 production to interleukin-12 (IL-12) production is 5-30.
3. The pharmaceutical composition according to claim 2, wherein the composition inhibits the production of immunoglobulin E (IgE).
3. The pharmaceutical composition according to claim 2, wherein the composition inhibits Th1-associated cytokine production.
3. The pharmaceutical composition of claim 2, wherein the composition inhibits Th17-associated cytokine production.
3. The pharmaceutical composition of claim 2, wherein said composition inhibits Th2-associated cytokine production.
3. The pharmaceutical composition according to claim 2, wherein the composition enhances intestinal immunity.
3. The pharmaceutical composition according to claim 2, wherein said Th1-mediated immune disease or Th17-mediated immune disease is transplant rejection, autoimmune disease, or inflammatory disease.
3. The pharmaceutical composition according to claim 2, wherein the Th2-mediated immune disease is an allergic disease.
A Th1-mediated immune disease, a Th17-mediated immune disease, or a health functional food composition for improving a Th2-mediated immune disease, comprising the Lactobacillus pentosus KF340 strain (Accession No. KCCM 11675P) .
16. The health functional food composition according to claim 15, wherein the strain is isolated from kimchi.
16. The health functional food composition according to claim 15, wherein the strain is a live or dead organism.
16. The health functional food composition according to claim 15, wherein said Th1-mediated immune disease or Th17-mediated immune disease is transplant rejection, autoimmune disease, or inflammatory disease.
16. The health functional food composition according to claim 15, wherein the Th2-mediated immune disease is an allergic disease.
A Th1-mediated immune disease, a Th17-mediated immune disease, or a Th2-mediated immune disease improvement cosmetic composition comprising the Lactobacillus pentosus KF340 strain (Accession No. KCCM 11675P) of claim 1 as an active ingredient.
21. The cosmetic composition according to claim 20, wherein the strain is isolated from the kimchi.
21. The cosmetic composition according to claim 20, wherein the strain is live cells or dead cells.
21. The cosmetic composition according to claim 20, wherein the Th1-mediated immune disease or Th17-mediated immune disease is a transplant rejection, autoimmune disease, or inflammatory disease.
21. The cosmetic composition according to claim 20, wherein the Th2-mediated immune disease is an allergic disease.

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