KR101644595B1 - Lactobacillus paracasei 1- 2- Composition for Preventing Improving or Treating of 1-mediated Immune Disease or 2-mediated Immune Disease Comprising Extracts from and Lactobacillus paracasei as an Active Ingredients - Google Patents

Abstract

The present invention relates to a composition for preventing, ameliorating or treating a Th1-mediated immune disease or a Th2-mediated immune disease comprising Lactobacillus paracasei as an active ingredient. The Lactobacillus paracase of the present invention activates regulatory T cells, increases IL-10 production, which is an anti-inflammatory cytokine, and inhibits the production of IL-12, an immunologically active cytokine, thereby inhibiting Th1-mediated immune disease or Th2- There is an advantage that it can be used as an anti-allergic composition and an anti-inflammatory composition that effectively control immune diseases.

Description

A composition for preventing, ameliorating or treating a Th1-mediated disease or a Th2-mediated immune disease comprising Lactobacillus paracasei as an active ingredient (Composition for Preventing, Improving or Treating of Th1-mediated Immune Disease or Th2 -mediated Immune Disease Comprising Extracts and Lactobacillus paracasei as an Active Ingredients}

The present invention relates to a composition for preventing, ameliorating or treating a Th1-mediated immune disease or a Th2-mediated immune disease comprising Lactobacillus paracasei as an active ingredient.

The incidence of asthma continues to increase in Korea. According to 2007 statistics, 2,023 patients were reported to die from asthma. In addition, the prevalence of pediatric allergic asthma in Korea has more than doubled from the early 1980s, and 10 out of 100 children have asthma, of which 50% have lifelong asthma. Therefore, the prevention of the onset of allergic diseases and effective control technology are urgently needed.

Existing allergic asthma treatment is not a fundamental treatment, and it is merely to alleviate or reduce the disease by using two or more of several medicines, and most of these medicines have serious side effects and have problems. In addition, antibody preparations such as Omalizumab have a problem of costly development and treatment.

Recently, Metagenome project to identify intestinal microflora in the US has been linked to various immune system diseases including distribution of intestinal microflora and allergy. Therefore, the distribution and diversity of intestinal bacteria and intestinal immunity are closely related to each other.

The market size of the probiotics market was US $ 144.3 million as of 2003. The market size was 11.2%, and the annual average growth rate was estimated at 13.0% by 2010. The market size in 2010 was about 394 million dollars Respectively. The market size of Europe increased 15.8% in 2003 to 40 million US dollars, and the average annual growth rate until 2010 is expected to be 16.7%. The expected 2010 market size is estimated at $ 137.9 million, and the domestic probiotic market is estimated to exceed 100 billion won.

The technology for detecting and evaluating immunity control probiotics such as allergy suppression has not yet been established. Therefore, it is expected that if the systematic in-vitro or in-vivo mimic screening system is developed and utilized well, the economic utility value will also be enormous.

It is a poor state of study of the fundamental mechanism of action , Bit . Although the ingestion of probiotics is carried out through the oral cavity, most of the research to date has been focused on in vitro experiments using cell lines, and these experimental methods can be replaced by studies on the functions that people may experience when ingesting probiotics There is a major drawback. In addition, the intestinal immune system (GALT) exposed to probiotics exhibits immune functions of probiotics due to the simultaneous presence of various immune cells, and in vitro experiments are different from the in vivo environment.

In this 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 effect of Lactobacillus paracasei was evaluated on them , 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. The ability of probiotics to increase the immune response to fight against diseases such as influenza virus or cancer, as well as areas that target inflammatory responses to inflammatory diseases such as type 1 diabetes, rheumatoid arthritis, degenerative arthritis, and ulcerative colitis Research is also actively underway. In addition, the role of probiotics and their functions are attracting much attention for the treatment of diseases such as allergy, asthma, and atopy.

Because most probiotics are derived from human intestines or foods, there is an advantage in that there is no major problem in proving the stability of other chemical agents when proven efficacy using animal models. Recently, the efficacy of probiotics as an immunomodulator has been verified through various clinical trials. Asthma, ulcerative colitis, and arthritis. Recently, clinical trials and studies on atopy have been accelerated.

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 is being actively carried out in the US and Japan recently. VSL3 # was developed by the VSL Phamaceutical company in Canada. It is currently active in the global market, and recently it has been marketed as an immunity regulator in the domestic market.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

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 paracasei 698 strain having antiallergic and anti-inflammatory activity was selected from kimchi, which is a traditional fermented food, and when the strain was treated, activation of regulatory T cells, The present inventors have completed the present invention by confirming that allergy and inflammation can be effectively controlled by increasing IL-12 production, which is a cytokine having cytokine, IL-12, and IL-12 production, which is a cytokine having antiinflammatory activity.

Accordingly, an object of the present invention is to provide a composition for preventing, ameliorating or treating a Th1-mediated immune disease or a Th2-mediated immune disease comprising Lactobacillus paracasei as an active ingredient.

Another object of the present invention is to provide a novel Lactobacillus paracase 698 ( Lactobacillus < RTI ID = 0.0 > ( Lactobacillus < / RTI > paracasei 698).

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, there is provided a composition for preventing, ameliorating or treating a Th1-mediated disease or a Th2-mediated immune disease comprising Lactobacillus paracasei as an active ingredient.

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 paracasei 698 strain having antiallergic and anti-inflammatory activity was selected from kimchi, which is a traditional fermented food, and when the strain was treated, activation of regulatory T cells, It was confirmed that allergy and inflammation can be effectively controlled by increasing the production of IL-12, which is a cytokine having anti-IL-12 production and anti-inflammatory activity.

Accordingly, the present invention relates to a composition for preventing, ameliorating or treating a Th1-mediated immune disease or a Th2-mediated immune disease comprising Lactobacillus paracasei as an active ingredient.

As used herein, the term " comprising as an active ingredient " refers to the following Lactobacillus paracase < / RTI > paracasei ). Since the Lactobacillus paracasei of the present invention is a microbial strain isolated from food and has no adverse effect on the human body even when administered in an excessive amount, the quantitative upper limit of the Lactobacillus paracasei contained in the composition of the present invention is And can be selected by a person skilled in the art within a suitable range.

According to one embodiment of the invention, the composition of the invention activates regulatory T cells. Activation of the regulatory T cells can be confirmed by increased expression of Foxp3, a factor that regulates the activity of regulatory T cells.

According to another embodiment of the present invention, the composition of the present invention is administered at a dose of 3-30 fold, 3-20 fold, 3-15 fold, 3-10 fold, 5-10 fold, Increase 5-8 times.

According to one embodiment of the invention, the composition of the present invention increases IL-10 production. IL-10 inhibits the secretion of macrophages and monocytes and T-cell lymphocyte replication and the secretion of inflammatory cytokines (IL-1, TNF-α, TGF-β, IL-6, IL-8 and IL-12) It is a powerful Th-2 cytokine produced by cells. IL-10 is an anti-inflammatory cytokine, and the inflammatory response is suppressed when IL-10 is increased.

According to one embodiment of the present invention, the composition of the present invention inhibits IL-12 production.

According to one embodiment of the present invention, the composition of the present invention causes the ratio of the amount of IL-10 production to the amount of IL-12 production to be 5-30. According to another embodiment of the present invention, the composition of the present invention causes the ratio of the amount of IL-10 production to the amount of IL-12 production to be 10-30 or 10-25. According to some embodiments of the present invention, the composition of the present invention is such that the ratio of the amount of IL-10 production to the amount of IL-12 production is 5-20, 10-20, 13-20 or 13-18.

The compositions of the present invention can be used for the prevention, amelioration or treatment of various ThI-mediated immune diseases, diseases or conditions.

As used herein, the term "Th1-mediated immune disorder" refers to a cytokine, such as IL-1β, IL-2, IL-12, IL-17, IFN- TNF-alpha, and the like.

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 synthesize IL-2 and IFN-y, but IL-4, IL-5, IL-10 and IL-13 do not synthesize cytokine expression patterns. Th1 cells are involved in cell-mediated immune responses, organ-specific autoimmune diseases and delayed hypersensitivity reactions to a variety of intracellular pathogens.

Th1-mediated immune diseases to which the composition of the present invention is applied are not particularly limited. For example, a Th1-mediated immune disorder to which the composition of the present invention is applied is selected from the group consisting of colitis, inflammatory bowel disease, type 1 diabetes, type 2 diabetes, rheumatoid arthritis, Reactive Arthritis, osteoarthritis, psoriasis, Sarcoidosis, lupus, hepatocellular carcinoma, myocarditis, endocarditis, pericarditis, cystic fibrosis, Hashimoto's thyroiditis, Graves' disease, leprosy, syphilis, Lyme disease, Borreliosis, Inflammatory bowel syndrome, Crohn's disease, Schwann's syndrome, fibromyalgia, chronic fatigue syndrome, chronic fatigue immunodeficiency syndrome, myalgic encephalomyelitis, myasthenia gravis, irritable bowel syndrome, Amyotrophic lateral sclerosis, Parkinson's disease, multiple sclerosis, autism spectrum disorders, attention deficit disorder, and attention deficit hyperactivity disorder It is not fixed. According to one embodiment of the present invention, it is applied to graft rejection, autoimmune disease or inflammatory diseases.

According to one embodiment of the present invention, the Th1-mediated immune disease to which the composition of the present invention is applied is an inflammatory disease, and according to another embodiment of the present invention, is selected from the group consisting of colitis, inflammatory bowel disease, type 1 diabetes, It is arthritis.

The compositions of the present invention may be used for the prevention or treatment of various Th2-mediated immune diseases, diseases or conditions.

As used herein, the term " Th2-mediated immune disease " refers to diseases in which IgE and mast cells are involved by the production and activity of allergen-specific Th2 cells.

As used herein, the term " Th2 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, Th2 cells exhibit IL-4, IL-5, IL-10 and IL-13 cytokine expression patterns. Th2 cells are involved in the humoral immune response.

The Th2-mediated immune disease to which the composition of the present invention is applied is not particularly limited, and according to one embodiment of the present invention, the Th2-mediated immune disease is an allergic disease.

As used herein, the term " allergy " refers to a variety of diseases, conditions, or abnormal conditions caused by hypersensitivity to certain substances in the body, i.e., excessive response of the body's immune system to substances introduced from the outside. 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. The initial reaction is to 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. According to some embodiments of the present invention, the allergy is asthma, allergic rhinitis, allergic dermatitis, allergic atopic dermatitis or food allergy.

According to one embodiment of the present invention, the lactobacillus paracase of the present invention is a strain isolated from kimchi.

According to one embodiment of the present invention, the Lactobacillus paracase Lactobacillus paracase strain 698 of the present invention (Deposit No. KFCC11569P).

The composition of the present invention is a pharmaceutical composition, a food composition, a functional food composition, a cosmetic composition or a feed composition.

The compositions of the present invention may be prepared with pharmaceutical compositions.

According to a preferred embodiment of the present invention, the composition of the present invention comprises (a) a pharmaceutically effective amount of the above-mentioned lactobacillus paracase of the present invention; And (b) a pharmaceutically acceptable carrier. As used herein, the term " pharmaceutically effective amount " means an amount sufficient to achieve efficacy or activity of the lactobacillus paracase described above.

When the composition of the present invention is manufactured from a pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. The pharmaceutically acceptable carriers to be contained in the pharmaceutical composition of the present invention are those conventionally used in the present invention and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, But are not limited to, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. It is not. The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations include, but are not limited to, Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention can be administered orally or parenterally, and is preferably applied by parenteral administration, more preferably topical application by application.

The appropriate dosage of the pharmaceutical composition of the present invention may vary depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate, . The dosage of Lactobacillus paracase, which is a physiologically active substance contained in the pharmaceutical composition of the present invention, is within the range of 0.001-1000 mg / kg on an adult basis.

The pharmaceutical composition of the present invention may be formulated into a unit dose form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container. The formulations may be in the form of solutions, suspensions, syrups or emulsions in oils or aqueous media, or in the form of excipients, powders, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

The composition of the present invention can be provided as a food composition.

When the composition of the present invention is prepared as a food composition, it contains not only Lactobacillus paracase as an active ingredient but also a component ordinarily added at the time of food production, for example, a protein, a carbohydrate, a fat, a nutrient, And flavoring agents. Examples of the above-mentioned carbohydrates are monosaccharides such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose, oligosaccharides and the like; And polysaccharides such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. Natural flavorings such as tau martin and stevia extract (e.g., rebaudioside A and glycyrrhizin) and synthetic flavorings (saccharine, aspartame, etc.) can be used as flavorings.

 For example, when the food composition of the present invention is prepared as a drink, it may further contain citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, juice, mulberry extract, jujube extract, licorice extract, .

The composition of the present invention can be prepared with a cosmetic composition.

When the anti-allergic composition comprising the active ingredient of the lactobacillus paracase strain of the present invention is prepared from a cosmetic composition, the lactobacillus paracase of the present invention may contain, in addition to the lactobacillus paracase strain, For example, conventional adjuvants such as stabilizers, 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 have.

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.

The composition of the present invention may be prepared from a feed composition.

When a composition for antiallergy comprising the active ingredient of the lactobacillus paracase according to the present invention is prepared from a feed composition, the lactobacillus paracase may be prepared as it is or by adding an additive usually added in the production of feed. 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,

According to another aspect of the present invention, the present invention provides Lactobacillus paracasei 698 (Accession No. KFCC 11569P) strain having a preventive, ameliorative or therapeutic activity of a Th1-mediated immune disease or a Th2-mediated immune disease.

The strain for the prevention, amelioration or treatment of a Th1-mediated immune disease or a Th2-mediated immune disease of the present invention is a strain for preventing, ameliorating or treating a Th1-mediated immune disease or a Th2- Therefore, in order to avoid the excessive complexity of the present specification, the description thereof will be omitted.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a composition for preventing, ameliorating or treating a Th1-mediated immune disease or a Th2-mediated immune disease comprising Lactobacillus paracasei as an active ingredient.

(b) The present invention provides a novel Lactobacillus paracasei 698 (Accession No. KFCC 11569P) strain having a preventive, ameliorative or therapeutic activity of a Th1-mediated immune disease or a Th2-mediated immune disease.

(c) The Lactobacillus paracase of the present invention activates regulatory T cells, increases IL-10, which is an anti-inflammatory cytokine, and inhibits IL-12 production, which is an immunologically active cytokine, There is an advantage that it can be used as an anti-allergic composition and an anti-inflammatory composition for effectively controlling mediated immune diseases.

FIG. 1 shows the results of measuring the expression level of Foxp3 according to co-culture of Lactobacillus paracasei 698 and a positive control group IRT5.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Experimental Methods and Materials

Isolation and Identification of Strain

Two strains were isolated from kimchi prepared by traditional method using trypticase soybean agar medium (MRS medium, BD236950, Difco, USA), and the isolated strains were identified by 16S rDNA sequencing. As a result of the identification, the 16s rDNA nucleotide sequence of the above strains (Sequence Listing 1 sequence) showed 99.88% homology with the conventional Lactobacillus paracase strain JCM1171.

The culture medium for the cultivation of these strains is MRS medium, the culture condition is pH 7.3 ± 0.2, the temperature is 37 ° C. and the incubation is for 48 hours. Oxygen requirement is aseptic anaerobic and can be preserved by lyophilization preservation or cell suspension freezing Do. This strain was transformed into Lactobacillus < RTI ID = 0.0 > paracasei ) 698, deposited at the Korean Microorganism Conservation Center (KCCM) on November 13, 2013, and assigned the deposit number KFCC11569P.

Experimental animal

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

Immune cell isolation

After the mesenteric lymph node of the mouse was removed, the immune cells (mostly lymphocytes) present in the lymph nodes were suspended in a single cell and used for the experiment. Because the probiotics present in the intestine are recognized by immune cells present in the intestinal immune system, appropriate immune cells are immune cells present in the mesenteric lymph nodes to characterize the probiotics. Therefore, the characteristics of Lactobacillus paracasei 698 were analyzed by immune cells isolated from the lymph nodes.

Co-culture co - culture )

The separated immune cells and Lactobacillus paracasei 698 were cultured in a 1:10 ratio, that is, 3x10 ⁶ of the immune cells and 3x10 ⁷ cfu of the strain for 72 hours in a 37 ° C incubator. At that time, the antibiotic gentamycin (11811-031, 15 μg / mL, GIBCO) was treated together to prevent the overgrowth of the test strain.

Enzyme immunoassay Enzyme - linked immunosorbent assay , ELISA )

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-Go, eBioscience) ELISA. The coating antibody for detecting each cytokine was treated in 96 wells, and the supernatant was added thereto. After allowing the reaction to proceed for a predetermined period of time, 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.

Flow cell  Analyzer ( Flow cytometry ) Through surface molecules ( 표면 molecule ) Expression measurement

A flow cytometer is a laser-based technology that detects specific signals of cells. Such signals are detected by an electrical device embedded in the analyzer, and thus the degree of biomolecular expression of living cells can be checked. In order to compare the degree of expression of surface molecules of cells, immune cells co-cultured with Lactobacillus paracase were analyzed by Foxp3 expression by measuring the GFP signal that is expressed when Foxp3 is expressed through this flow cytometer (COULTER Epics XL, BECKMAN) And the amount of expression was measured and compared.

Experiment result

Measuring antiallergic activity - Foxp3  Increased expression

Regulatory T cells are one of a subset of T cells that, unlike other T cells, play a role in suppressing excessive immune responses. Various studies have shown that these regulatory T cells inhibit allergic diseases. When the test strain and mesenteric lymph node cells are co-cultured, a strain capable of increasing the distribution of regulatory T cells is likely to be a probiotic with antiallergic efficacy. Foxp3 can be used to evaluate the distribution of regulatory T cells efficiently by using Foxp3 knock-in mice as a typical regulator of regulatory T cells. Therefore, in order to examine the interaction between test strains and regulatory T cells, mesenteric lymph node cells of Foxp3 knockout mice were isolated and mixed with test strain at a ratio of 1:10, followed by co-culture for 3 days. After 3 days, the cell co-cultured with the test strain was removed and the expression level of GFP expressed with Foxp3 was confirmed by flow cytometry. The expression level of GFP, a fluorescent protein, is an indirect indicator of the expression of Foxp3 expression, and thus the distribution of regulatory T cells can be confirmed.

As a result, the expression level of Foxp3 was 9.63% when Lactobacillus paracasei 698 strain was co-cultured with lactic acid mesentery cells. The positive control group IRT5 (HK Kwon, CG Lee, JS So, CS Chae, JS Hwang, .. A Sahoo, Nam JH, JH Rhee, Hwang KC, SH Im 2010. Generation of regulatory dendritic cells and CD4 + Foxp3 + T cells by administration probiotics suppresses immune disorders PANS 107;.. just slightly lower than 5 2159-2164) negative The expression level of T cells was significantly higher than that of the control group.

IL -10 and IL -12 Secretory function  Confirm

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 can be determined and lactic acid bacteria having anti- can do. In this experiment, mesenteric lymph node cells of Foxp3 knock-in mice were isolated and cultured after mixing 1:10 ratio with test strain. 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, lactobacillus paracasei 698 was found to have anti-inflammatory activity when lactobacillus paracasei 699 strain was co-cultured with lL-10 / IL-12 at 15.77, which was close to the positive control.

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

* IRT5: Can be used as a positive control with antiallergic activity considering 5 articles and patents published in 5 strains.

summary

The relative proportion of Foxp3 + cell distribution and IL-10 and IL-12 two cytokines were examined. As a result, Lactobacillus paracasei strain 698 was identified as a probiotic agent having antiallergic and anti-inflammatory activity through regulatory T cell activation and anti-inflammatory cytokine secretion .

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.

Korea Microorganism Conservation Center (Domestic) KFCC11569P 20131113

<110> KOREA FOOD RESEARCH INSTITUTE <120> Composition for Preventing, Improving or Treating of Th1-mediated          Immune Disease or Th2-mediated Immune Disease Comprising Extracts          from and Lactobacillus paracasei as an Active Ingredients <130> PN140380 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 1443 <212> DNA <213> Lactobacillus paracasei698 <400> 1 gggtgttaca aactctcatg gtgtgacggg cggtgtgtac aaggcccggg aacgtattca 60 acccgcggcg tgctgatccg cgattactag cgattccgac ttcgtgtagg cgagttgcag 120 cctacagtcc gaactgagaa tggctttaag agattagctt gacctcgcgg tctcgcaact 180 cgttgtacca tccattgtag cacgtgtgta gcccaggtca taaggggcat gatgatttga 240 cgtcatcccc accttcctcc ggtttgtcac cggcagtctt actagagtgc ccaactaaat 300 gctggcaact agtcataagg gttgcgctcg ttgcgggact taacccaaca tctcacgaca 360 cgagctgacg acaaccatgc accacctgtc attttgcccc cgaaggggaa acctgatctc 420 tcaggtgatc aaaagatgtc aagacctggt aaggttcttc gcgttgcttc gaattaaacc 480 acatgctcca ccgcttgtgc gggcccccgt caattccttt gagtttcaac cttgcggtcg 540 tactccccag gcggaatgct taatgcgtta gctgcggcac tgaagggcgg aaaccctcca 600 acacctagca ttcatcgttt acggcatgga ctaccagggt atctaatcct gttcgctacc 660 catgctttcg agcctcagcg tcagttacag accagacagc cgccttcgcc actggtgttc 720 ttccatatat ctacgcattt caccgctaca catggagttc cactgtcctc ttctgcactc 780 aagtttccca gtttccgatg cgcttcctcg gttaagccga gggctttcac atcagactta 840 aaaaaccgcc tgcgctcgct ttacgcccaa taaatccgga taacgcttgc cacctacgta 900 ttaccgcggc tgctggcacg tagttagccg tggctttctg gttggatacc gtcacgccga 960 caacagttac tctgccgacc attcttctcc aacaacagag ttttacgacc cgaaagcctt 1020 cttcactcac gcggcgttgc tccatcagac ttgcgtccat tgtggaagat tccctactgc 1080 tgcctcccgt aggagtttgg gccgtgtctc agtcccaatg tggccgatca acctctcagt 1140 tcggctacgt atcatcgcct tggtgagcca ttacctcacc aactagctaa tacgccgcgg 1200 gtccatccaa aagcgatagc ttacgccatc tttcagccaa gaaccatgcg gttcttggat 1260 ctatgcggta ttagcatctg tttccaaatg ttatccccca cttaagggca ggttacccac 1320 gtgttactca cccgtccgcc actcgttcca tgttgaatct cggtgcaagc accgatcatc 1380 aacgagaact cgttcgactt gcatgtatta ggcacgccgc cagcgttcat cctgagccat 1440 gat 1443

Claims (11)

delete delete delete delete delete delete delete delete delete delete Lactobacillus paracasei 698 (Accession No. KFCC 11569P) having an activity of preventing, ameliorating or treating an inflammatory disease or an allergic disease.

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