RU2790297C2 - Lactobacillus paracasei strain and its use - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: strain of Lactobacillus paracasei KBL382 with an identification number KCTC13509BP, having immunity enhancing activity, is proposed. A pharmaceutical composition, a food composition, and a food additive composition, containing the specified strain or its culture as an active ingredient, are also proposed. The specified strain or a culture of the specified strain, as well as a pharmaceutical composition containing a strain or its culture are used for the treatment of intestinal diseases, allergic diseases, autoimmune diseases, as well as inflammatory diseases.

EFFECT: invention provides a therapeutic effect relatively to intestinal diseases, allergic, autoimmune, as well as inflammatory diseases.

21 cl, 7 dwg, 1 tbl, 7 ex

Description

Technical field

[1] The present invention relates to strains of Lactobacillus paracasei and their use. More specifically, the present invention relates to a functional health product composition for alleviating allergies, improving immunoregulation, alleviating symptoms of inflammation, alleviating atopic dermatitis, and improving bowel health; and a pharmaceutical composition for the treatment or prevention of allergic diseases, autoimmune diseases, inflammatory diseases, atopic dermatitis and/or intestinal diseases, containing an effective amount of at least one component from the group consisting of Lactobacillus paracasei strain KBL382, Lactobacillus paracasei strain KBL384, Lactobacillus paracasei strain KBL385, cells of microorganisms of the specified strain, cultures of the specified strain, lysates of the specified strain and extracts of the specified strain.

[2]

State of the art

[3] Probiotics refers to microorganisms and products derived from them that have antibacterial activity and enzymatic activity that help maintain the balance of intestinal microorganisms. In addition, probiotics, by definition, are live bacteria in the form of one or more strains that improve the intestinal microflora when administered to humans or animals in the form of dry cells or fermentation products. Probiotics must inhabit the human gut, be non-pathogenic and non-toxic, and survive for a sufficiently long time before entering the gut. In addition, probiotics must be viable and active prior to dietary intake, be sensitive to antibiotics used to prevent infections, and be free of antibiotic resistance plasmids. In addition, probiotics must be resistant to acids, enzymes and bile in the environment present in the intestines.

[4] Such probiotics may include, for example, Bacillus sp., which has a remarkable ability to produce hydrolyzing enzymes, such as amylase, protease, lipase, cellulase, and phosphatase, Lactobacillus sp., which produce lactic acid, and photosynthetic bacteria, which prevent malodor from through the use of malodorous substances (for example, ammonia, hydrogen sulfide and amines) present in livestock feces in metabolic processes.

[5] In particular, it is known that Bacillus sp. and Lactobacillus sp. are extremely useful probiotics because they include strains that produce various antibacterial substances. These lactic acid bacteria produce antimicrobial peptides called bacteriocins, which have an antibacterial mechanism of action unrelated to antibiotic resistance mechanisms. Bacteriocins have polymorphic characteristics, their molecular weight, biochemical properties and ranges and mechanisms of antimicrobial action in relation to the host vary greatly. Klaenhammer defines bacteriocin as a protein or protein complex that has direct antibacterial activity against microbial species closely related to bacteriocin-producing bacteria.

[6] Wherein, irritable bowel syndrome (IBS) is a symptom characterized by abdominal pain and/or irritation associated with changes in intestinal motility or stool, and such symptoms cannot be explained by anatomical or biochemical pathologies. Common symptoms of IBS also include an urge to urinate, bloating, and a feeling of incomplete defecation. Accordingly, IBS can be classified as functional gastrointestinal disorders, including conditions such as functional bloating, non-cardiogenic chest pain, non-ulcer dyspepsia, and chronic constipation or diarrhea. In particular, since the symptoms associated with IBS affect both the well-being and the functional aspects of the condition of patients, this disease has a pronounced impact on morbidity and quality of life, even without taking into account pain and discomfort in the abdominal cavity.

[7] Inflammatory bowel disease (IBD) is a condition in which chronic inflammation develops and recurs in the intestine, including but not limited to all inflammatory bowel diseases such as Crohn's disease, ulcerative colitis or Behcet's disease. Many researchers are working in the field of drug development for the treatment of IBS and IBD. In this regard, various antidepressants are widely used, despite the fact that their effectiveness in clinical trials is moderate, and clinical applicability is limited due to significant side effects. In addition, serotogenic drugs have been shown to be effective in reversing the common symptoms of IBS. However, the use of these drugs is limited in various ways due to some problems with their safety. Accordingly, there is a growing interest in the development of a new therapeutic agent for the treatment of IBS.

[8] Allergy is a biochemical phenomenon characterized by a unique distorted response to a foreign substance (antigen, allergen). The foreign substance that causes symptoms is called an allergen, and the diseases in which these symptoms develop are called allergic diseases. Allergy is a pathological process in the body, the cause of which is the "antigen-antibody" reaction. In general, there are four types of allergy, depending on the time period during which the reaction occurs and the involvement of the complement. Type 1 allergy is an anaphylactic (immediate) type reaction in which the target organs are mainly the digestive system, skin and lungs, and common symptoms include gastrointestinal allergies, urticaria, atrophic dermatitis, allergic rhinitis, bronchial asthma, etc. d. It is known that the pathological mechanisms of type 1 allergy develop as follows: upon contact of antigens with IgE antibodies attached to the surface of mast cells and basophils, target cells are activated and secrete chemical mediators, for example, histamine, leukotrienes and PAF, followed by contraction of blood vessels and smooth muscles. This mechanism can often be seen in combination with type 4 (delayed) allergy. In other words, such an anaphylactic and allergic reaction may occur due to various changes in mast cells, etc. The activation of mast cells leading to their degranulation is due to the binding of antigen, anti-IgE antibody, lectin, etc. with Fc receptors, stimulation by anaphylotoxin, etc. or other drugs, such as calcium ionophore, compound 48/80, codeine and synthetic adrenocorticotropin.

[9] It is known that mast cells and blood basophils are the main cells of the body that cause various allergic diseases, such as allergic rhinitis, allergic dermatitis, asthma, food allergies and anaphylactic shock. These cells contain on their surface receptors (FcRI) for IgE, which are antibodies that cause allergies; these cells are stimulated by allergy-causing substances (antigens, allergens) and secrete their own allergy-causing substances (Kim K et al, Eur J Pharmacol, 581:191-203, 2008).

[10] Among allergic diseases, atopic dermatitis is a well-known chronic relapsing skin disease that occurs in newborns or children and may persist into adulthood. Like asthma or allergic rhinitis, atopic dermatitis is an inflammatory skin disease associated with local infiltration of T-lymphocytes producing IL-4 and IL-5. IL-4 controls the development of T-helper 2 phenotype (Th2), which leads to excessive production of immunoglobulins (Ig) and eosinophilia, as well as an increase in serum IgE levels. Atopic dermatitis is found in 80-90% of subjects with positive skin test results for food and inhalant allergens.

[11] There are various agents for the treatment or prevention of allergic diseases, including atopic dermatitis, but an effective method of treatment has not yet been found. Some drug therapy options are known, however, even short-term use of a drug can lead to the development of resistance, and long-term use can cause serious side effects, and therefore such drug therapy options for allergic diseases have recently been avoided. In these circumstances, in the absence of treatment options that provide an absolute demonstrable effect, attempts to improve the symptoms of irritation, such as itching and hyperemia in allergies, often do not work.

[12] WO 96/29083 and EP 554418 describe two types of colony-forming Lactobacillus strains in the intestine, ie. Lactobacillus plantarum 299v (DSM 6595) and Lactobacillus casei ssp. rhamnosus 271 (DSM 6594), etc. EP 415941 describes a method for preparing a nutrient composition comprising treating oatmeal with enzymes followed by mixing with lactobacilli. US Pat. No. 7,195,906 describes a strain of Bifidobacterium isolated from resected and lavaged human gastrointestinal tract for the treatment of inflammatory diseases, especially gastrointestinal inflammation, eg, IBD and IBS.

[13] At the same time, no strain has yet been found that has exceptional properties in improving the condition of the intestine, for example, treating IBD and IBS, and in relieving allergy symptoms, and many research institutions are working to find strains with such properties. .

[14] Under these circumstances, the present inventors screened various strains on the basis that the health benefits of probiotics are not generic and species-specific, but rather strain-specific (Report of the Joint FAO/WHO Working Group on the Development of Guidelines for the Evaluation of Probiotics in Foods , London, Ontario, Canada, 2002), and found a new strain having an excellent effect on immunoregulation, allergy alleviation, bowel improvement and treatment of intestinal diseases, and anti-inflammatory activity. Then, the present invention was completed, confirming the excellent properties of these strains.

[15]

[16] Summary of the Invention

[17] The purpose of the present invention is to provide a new strain that can be used to improve bowel health, treat or prevent bowel diseases, improve immunoregulation, treat or prevent autoimmune diseases, alleviate allergy symptoms, alleviate inflammation symptoms, alleviate and treat atopic dermatitis.

[18] To achieve this object, the present invention provides a strain of Lactobacillus paracasei KBL382 (identification number KCTC13509BP), a strain of Lactobacillus paracasei KBL384 (identification number KCTC13510BP) or a strain of Lactobacillus paracasei KBL385 (identification number KCTC13511BP).

[19] In addition, the present invention provides a food composition or a dietary supplement composition comprising an effective amount of at least one component selected from the group consisting of Lactobacillus paracasei strain KBL382 (identification number KCTC13509BP), Lactobacillus paracasei strain KBL384 (identification number KCTC13510BP ) or Lactobacillus paracasei strain KBL385 (identification number KCTC13511BP), cultures of said strain, lysates of said strain, and extracts of said strain.

[20] In addition, the present invention provides a pharmaceutical composition for the treatment or prevention of intestinal diseases, containing an effective amount of at least one component selected from the group consisting of a strain of Lactobacillus paracasei KBL382 (identification number KCTC13509BP), a strain of Lactobacillus paracasei KBL384 (identification number KCTC13510BP) or Lactobacillus paracasei strain KBL385 (identification number KCTC13511BP), cultures of said strain, lysates of said strain, and extracts of said strain.

[21] In addition, the present invention provides a pharmaceutical composition for the treatment or prevention of allergic diseases, containing an effective amount of at least one component selected from the group consisting of a strain of Lactobacillus paracasei KBL382 (identification number KCTC13509BP), a strain of Lactobacillus paracasei KBL384 (identification number KCTC13510BP) or Lactobacillus paracasei strain KBL385 (identification number KCTC13511BP), cultures of said strain, lysates of said strain, and extracts of said strain.

[22] In addition, the present invention provides a pharmaceutical composition for the treatment or prevention of autoimmune diseases or inflammatory diseases, containing an effective amount of at least one component selected from the group consisting of a strain of Lactobacillus paracasei KBL382 (identification number KCTC13509BP), a strain of Lactobacillus paracasei KBL384 (ID KCTC13510BP) or Lactobacillus paracasei strain KBL385 (ID KCTC13511BP), cultures of said strain, lysates of said strain, and extracts of said strain.

[23] In addition, the present invention provides a method for treating bowel diseases, comprising administering a therapeutically effective amount of at least one component selected from the group consisting of a strain of Lactobacillus paracasei KBL382 (identification number KCTC13509BP), a strain of Lactobacillus paracasei KBL384 (identification number KCTC13510BP ) or Lactobacillus paracasei strain KBL385 (ID KCTC13511BP), cultures of said strain, lysates of said strain, and extracts of said strain into a subject in need thereof.

[24] In addition, the present invention provides a method for treating allergic diseases, comprising administering a therapeutically effective amount of at least one component selected from the group consisting of a strain of Lactobacillus paracasei KBL382 (identification number KCTC13509BP), a strain of Lactobacillus paracasei KBL384 (identification number KCTC13510BP ) or Lactobacillus paracasei strain KBL385 (ID KCTC13511BP), cultures of said strain, lysates of said strain, and extracts of said strain into a subject in need thereof.

[25] In addition, the present invention provides a method for treating autoimmune diseases or inflammatory diseases, comprising administering a therapeutically effective amount of at least one component selected from the group consisting of a strain of Lactobacillus paracasei KBL382 (identification number KCTC13509BP), a strain of Lactobacillus paracasei KBL384 ( identification number KCTC13510BP) or Lactobacillus paracasei strain KBL385 (identification number KCTC13511BP), cultures of said strain, lysates of said strain, and extracts of said strain into the body of a subject in need thereof.

[26] In addition, the present invention provides a composition for use in the prevention or treatment of intestinal diseases, containing at least one component selected from the group consisting of a strain of Lactobacillus paracasei KBL382 (identification number KCTC13509BP), a strain of Lactobacillus paracasei KBL384 (identification number KCTC13510BP) or Lactobacillus paracasei strain KBL385 (ID KCTC13511BP), cultures of said strain, lysates of said strain, and extracts of said strain.

[27] In addition, the present invention provides a composition for use in the prevention or treatment of allergic diseases, containing at least one component selected from the group consisting of a strain of Lactobacillus paracasei KBL382 (identification number KCTC13509BP), a strain of Lactobacillus paracasei KBL384 (identification number KCTC13510BP) or Lactobacillus paracasei strain KBL385 (ID KCTC13511BP), cultures of said strain, lysates of said strain, and extracts of said strain.

[28] In addition, the present invention provides a composition for use in the prevention or treatment of autoimmune diseases or inflammatory diseases, containing at least one component selected from the group consisting of a strain of Lactobacillus paracasei KBL382 (identification number KCTC13509BP), a strain of Lactobacillus paracasei KBL384 (ID KCTC13510BP) or Lactobacillus paracasei strain KBL385 (ID KCTC13511BP), cultures of said strain, lysates of said strain, and extracts of said strain.

[29] In addition, the present invention provides the use of a composition containing at least one component selected from the group consisting of a strain of Lactobacillus paracasei KBL382 (identification number KCTC13509BP), a strain of Lactobacillus paracasei KBL384 (identification number KCTC13510BP) or a strain of Lactobacillus paracasei KBL385 (identification number KCTC13511BP), cultures of the specified strain, lysates of the specified strain and extracts of the specified strain, to obtain a drug for the prevention or treatment of intestinal diseases.

[30] In addition, the present invention provides the use of a composition containing at least one component selected from the group consisting of a strain of Lactobacillus paracasei KBL382 (identification number KCTC13509BP), a strain of Lactobacillus paracasei KBL384 (identification number KCTC13510BP) or a strain of Lactobacillus paracasei KBL385 (identification number KCTC13511BP), cultures of the specified strain, lysates of the specified strain and extracts of the specified strain, to obtain a drug for the prevention or treatment of allergic diseases.

[31] In addition, the present invention provides the use of a composition containing at least one component selected from the group consisting of a strain of Lactobacillus paracasei KBL382 (identification number KCTC13509BP), a strain of Lactobacillus paracasei KBL384 (identification number KCTC13510BP) or a strain of Lactobacillus paracasei KBL385 (identification number KCTC13511BP), cultures of the specified strain, lysates of the specified strain and extracts of the specified strain, to obtain a drug for the prevention or treatment of autoimmune diseases or inflammatory diseases.

[32]

Brief description of the drawings

[33] FIG. 1 shows a result confirming the control effect of inflammatory cytokines of the KBL382 strain, KBL384 strain, and KBL385 strain of the present invention in MICs.

[34] FIG. 2 shows a result confirming the effect of controlling the expression of T cell differentiation marker genes by KBL382 strain, KBL384 strain and KBL385 strain according to the present invention in PBMC.

[35] FIG. 3 shows the result of observing the change in the expression level of cytokines, (A) IL-2, (B) IFN-γ, (C) IL-4, (D) IL-13, (E) IL-17A and (F) IL- 10 according to the introduction of KBL382 strain, KBL384 strain and KBL385 strain of the present invention into PBMC cell lines by activating T cells by adding an anti-CD3 antibody.

[36] FIG. 4 shows the result confirming the effect on the relief of enteritis by (A) change in body weight, (B) change in colon length, (C) change in colon length and change in caecal weight, (D) H&E staining of colon tissues, and (E ) changes in the level of MPO (myeloperoxidase) in the tissues of the colon after the introduction of each of the strain KBL382, strain KBL384 and strain KBL385 according to the present invention mouse models with induced enteritis.

[37] FIG. 5 shows the result of monitoring the tight junction enhancement effect of the KBL382 strain and the KBL385 strain of the present invention by changing the expression level of the gut wall tight junction genes, (A) ZO1, (B) claudin 3, and (C) MUC4.

[38] FIG. 6 shows the result of comparing the effects of (A) restoring colon length and (B) improving body weight loss using the KBL382 strain of the present invention and infliximab, a commercially available enteritis treatment antibody, to confirm the effect of alleviating enteritis using the KBL382 strain of the present invention. invention.

[39] FIG. 7 shows the observation result of (A) dermatitis scoring measurement, (B) itch relief effect, (C) skin thickness thinning effect, and (D) blood IgE concentration reduction effect when KBL382 strain of the present invention was administered to animal models of induced atopic dermatitis. in order to confirm the effect of alleviating the symptoms of atopic dermatitis when using strain KBL382.

[40]

[41] Detailed Description of the Invention and Best Modes for Carrying Out the Invention

[42] Unless otherwise indicated, all technical and scientific terms used in this application have the same meaning as generally accepted among specialists in this field of technology. In general, the nomenclature used in this application is well known and widely used in the art.

[43]

[44] The present invention confirmed the immunoregulatory effect of microorganisms derived from the human body, and found that Lactobacillus paracasei strains having excellent immunoregulatory effect, i.e. KBL382 (identification number KCTC13509BP), KBL384 (identification number KCTC13510BP) and strain KBL385 (identification number KCTC13511BP). Analysis of the 16s rDNA of said strain demonstrates that said strain is a new strain that has never been known to the public.

[45]

[46] In one embodiment, the present invention relates to a novel probiotic strain of Lactobacillus paracasei KBL382, Lactobacillus paracasei KBL384 or Lactobacillus paracasei KBL385, and these strains are characterized by the presence of 16s rDNA sequences of SEQ ID NO: 1-3, respectively, as shown below.

[47] <SEQ ID NO: 1> 16s rDNA sequence of Lactobacillus paracasei strain KBL382

[48] <SEQ ID NO: 2> 16s rDNA sequence of Lactobacillus paracasei strain KBL384

[49] <SEQ ID NO: 3> 16s rDNA sequence of Lactobacillus paracasei strain KBL385

[50] The present invention also confirmed that each of KBL382 strain, KBL384 strain, and KBL385 strain has a firming action on intestinal wall tight junctions in addition to anti-inflammatory and immunoregulatory effects, and that administration of KBL382 strain, KBL384 strain, and KBL385 strain to animal models with induced enteritis significantly attenuated weight loss and shortening of the colon due to enteritis. In addition, administration of strain KBL382, among the above strains, to animal models of induced atopic dermatitis was confirmed to significantly improve dermatitis score, alleviate pruritus symptoms, reduce skin thickness, and markedly improve blood IgE concentration in allergic reaction.

[51]

[52] Accordingly, in another embodiment, the present invention relates to a food or dietary supplement composition comprising an effective amount of at least one component selected from the group consisting of microbial cells of strain KBL382, strain KBL384 or strain KBL385, cultures of said strain, lysates of said strain; and extracts of said strain.

[53] Said food composition or dietary supplement composition can be used as a food product that effectively improves the intestinal condition and promotes the prevention of intestinal diseases, for example, as the main or minor ingredients of food, nutritional supplements, composition of functional health products or functional drinks, but not limited to them.

[54] Said food composition or dietary supplement composition can be used as a food product effectively alleviating allergy symptoms, such as, but not limited to, as the main or minor ingredients of food, nutritional supplements, functional health product composition or functional drinks.

[55] In addition, said food composition or dietary supplement composition can be used as a food product effectively alleviating autoimmune diseases or inflammatory diseases, for example, as a main or minor ingredients of food, food supplements, functional health product composition or functional drinks, but not limited to them.

[56] The term "food" refers to a natural or artificial product containing at least one nutrient, and more preferably refers to a product that has been made edible as a result of certain processing, and usually covers all foods, nutritional supplements, functional health products and functional drinks.

[57] A food product that may contain the specified food composition according to the present invention as an additive may include, for example, various types of foods, drinks, chewing gum, tea, vitamin complex and functional foods. In addition, foods of the present invention include specialty fortified foods (e.g., modified milk, baby food), processed meats, fish products, tofu, flour, noodles (e.g., ramen noodles, Asian noodles), baked goods, organically active food additives, seasonings (e.g. soy sauce, soy paste, red pepper paste, mixed paste), sauces, confectionery (e.g. snack foods), candy, chocolate, chewing gums, ice cream, dairy products (e.g. fermented milk , cheese), other processed foods, kimchi, salty foods (e.g. various types of kimchi, pickled foods), drinks (e.g. fruit juice, vegetable juice, soy milk, fermented drinks) and natural condiments (e.g. broth ramen noodle powder), but not limited to. Said foodstuffs, beverages or nutritional supplements can be obtained by conventional methods.

[58] The term "functional health products" is a group of foods whose effectiveness is enhanced by the ability to manifest and express their function for a predetermined purpose using physical, biochemical or bioengineering techniques, or processed foods designed to the regulating functions of the corresponding food composition in vivo, for example, the function of regulating the defecation rhythm in disease prevention and recovery, were sufficiently exhibited. Such a functional food product may contain nutritionally acceptable food additives and may additionally contain suitable carriers, excipients and diluents commonly used in their manufacture.

[59] In the present invention, the term "functional drinks" collectively refers to drinks intended to quench thirst or to enjoy the taste. There are no specific restrictions on this term, except that these drinks contain a composition for improving or preventing the indicated symptoms of bowel disease as the main ingredients in the indicated ratio, and, like regular drinks, may contain various flavors or natural carbohydrates as additional ingredients.

[60] In addition to the above, a food product containing a nutritional composition according to the present invention for improving the symptoms of said bowel disease or preventing it may contain various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic and natural flavors. , colorants and fillers (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, colloidal thickeners, pH adjusting agents, stabilizing agents, preservatives, glycerin, alcohol, carbonizing agents used in carbonated drinks, and the like, and each of the above ingredients can be used alone or in combination with each other.

[61] The food product containing the food composition according to the present invention may contain the composition according to the present invention in an amount of from 0.001 wt.% to 100 wt.%, preferably from 1 wt.% to 99 wt.%, based on the total weight of the food ; in the case of beverages, it can be from 0.001 g to 10 g, preferably from 0.01 g to 1 g per 100 ml. However, with long-term use for recreational or hygienic purposes or in order to maintain a state of health, the amount of the product may be lower than the above range; and since the effective ingredients do not have problems in terms of the safety profile, they can be used in an amount exceeding the specified range, and their amount is not limited to the above range.

[62] The nutritional composition of the present invention may contain the KBL382 strain, the KBL384 strain, or the KBL385 strain alone or in combination with a suitable carrier, or may be formulated into a composition suitable for human or animal consumption. Thus, said composition can be added to a food product that does not contain probiotic bacteria or a pair of probiotic bacteria. For example, microorganisms that can be used in combination with the strain of the present invention in the preparation of the food of the present invention should be suitable for human or animal consumption and have probiotic activity in suppressing pathogenic, harmful bacteria or improving the balance of microflora in the intestinal tract of mammals when taken inside, but not limited to. Such probiotic microorganisms may include, for example, yeasts, such as Saccharomyces, Candida, Pichia, and Torulopsis, fungi, such as Aspergillus, Rhizopus, Mucor, and Penicillium, and bacteria of the genera Lactobacillus, Bifidobacterium, Leuconostoc, Lactococcus, Bacillus, Streptococcus, Propionibacterium, Enterococcus and Pediococcus. Подходящие конкретные пробиотические микроорганизмы могут включать, например, Saccharomyces cerevisiae, Bacillus coagulans, Bacillus licheniformis, Bacillus subtilis, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium longum, Enterococcus faecium, Enterococcus faecalis, Lactobacillus acidophilus, Lactobacillus alimentarius, Lactobacillus casei, Lactobacillus curvatus, Lactobacillus delbruckii , Lactobacillus johnsonii, Lactobacillus farciminus, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillus sakei, Lactococcus lactis, or Pediococcus acidilactici. The nutritional composition according to the present invention may further preferably contain a mixture of probiotic microorganisms having a pronounced probiotic activity and an excellent immune-enhancing action, further enhancing their effects. Carriers that may be included in the food composition of the present invention may include, for example, bulking agents, high fiber supplements, encapsulating agents, and lipids well known in the art. The strain of Lactobacillus paracasei according to the present invention may be in lyophilized or encapsulated form, or in the form of suspensions or dry culture powders.

[63]

[64] The composition according to the present invention can also be presented in the form of a feed additive or feed containing the specified strain.

[65] The feed additive according to the present invention may be in the form of a dry or liquid composition and, in addition, contain other non-pathogenic microorganisms in addition to the specified strain KBL382, KBL384 or KBL385. Microorganisms that can be added to the feed additive may include, for example, Bacillus subtilis, which can produce protease, lipase, and carbohydrate converting enzymes, a strain of Lactobacillus, which has physiological activity and the ability to degrade organic compounds under anaerobic conditions, such as in the stomach of a cow , filamentous fungi such as Aspergillus oryzae showing effects on livestock weight gain, milk yield and feed digestibility (Slyter, L. L. J. Animal Sci. 1976, 43. 910-926), and yeasts such as Saccharomyces cerevisiae (Johnson, D. E et al., J. Anim. Sci., 1983, 56, 735-739; Williams, P. E. V. et al, 1990, 211).

[66] The feed additive of the present invention may further contain at least one enzymatic agent in addition to said Lactobacillus paracasei strain KBL382, Lactobacillus paracasei strain KBL384 or Lactobacillus paracasei strain KBL385. Additional enzymatic agents may be in dry or liquid form and may include, for example, steatolytic enzymes, for example, lipase, phytase, which forms phosphate and inositol phosphate upon decomposition of phytic acid, amylase, i.e. an enzyme for the hydrolysis of the α-1,4-glycosidic bond in the composition of, for example, starch and glycogen, phosphatase, i.e. an enzyme for the hydrolysis of an organic ester of phosphoric acid, a carboxymethyl cellulase for the decomposition of cellulose, a xylase for the decomposition of xylose, a maltase for the hydrolysis of maltose into two molecules of glucose, and carbohydrate-producing enzymes, such as invertase, which form a mixture of glucose and fructose upon hydrolysis of sucrose.

[67] When using Lactobacillus paracasei strain KBL382, Lactobacillus paracasei strain KBL384 or Lactobacillus paracasei strain KBL385 according to the present invention, feed raw materials such as peanut, pea, beetroot, cake, grain processing products, animal gut powder and fish can be used as feed additives. flour, as well as various grains and soy protein. They may be processed or unprocessed and can be used without restriction. Processing may include a process in which the feedstock is loaded and forced under pressure through a predetermined opening, and for proteins, extrusion may preferably be used, in which the proteins are degenerated to increase their availability. Extrusion allows proteins to be denatured by heat treatment and destruction of anti-enzyme factors. In addition, the digestibility of soy proteins can be improved by extrusion to inactivate feed depleting substances, such as trypsin inhibitor, one of the protease inhibitors present in soy. In addition, extrusion can improve protease hydrolyzability, which increases the nutritional value of soy proteins.

[68]

[69] In yet another embodiment, the present invention relates to a pharmaceutical composition for the treatment or prevention of intestinal diseases, containing an effective amount of at least one component selected from the group consisting of microorganism cells of Lactobacillus paracasei KBL382 strain, Lactobacillus paracasei KBL384 strain or Lactobacillus strain paracasei KBL385, cultures of said strain, lysates of said strain and extracts of said strain.

[70] In yet another embodiment, the present invention relates to a pharmaceutical composition for the treatment or prevention of allergic diseases, containing an effective amount of at least one component selected from the group consisting of microorganism cells of Lactobacillus paracasei KBL382 strain, Lactobacillus paracasei KBL384 strain or Lactobacillus strain paracasei KBL385, cultures of said strain, lysates of said strain and extracts of said strain.

[71] In yet another embodiment, the present invention relates to a pharmaceutical composition for the treatment or prevention of autoimmune diseases or inflammatory diseases, containing an effective amount of at least one component selected from the group consisting of microorganism cells of Lactobacillus paracasei strain KBL382, Lactobacillus paracasei strain KBL384 or a strain of Lactobacillus paracasei KBL385, cultures of said strain, lysates of said strain, and extracts of said strain.

[72] The pharmaceutical composition of the present invention may be in the form of live bacteria, a dry strain, cultures of said strain, lysates of said strain, or a composition in combination with a pharmaceutically acceptable carrier or medium. Carriers or media that can be used in the present invention may include a solvent, dispersant, coating, enhancer, controlled release formulation (i.e., sustained release formulation), or at least one inert excipient, including starch, polyol , granules, fine cellulose, microcrystalline cellulose, eg Celphere, Celphere granules, diluent, lubricant, binder, disintegrant, and the like. The tablet of the above composition can optionally be coated using standard aqueous or non-aqueous techniques. Examples of a pharmaceutically acceptable carrier and excipient for use as a pharmaceutically acceptable inert carrier and these additional ingredients may include, for example, a binder, a filler, a disintegrant, a lubricant, an antimicrobial agent, and a coating agent.

[73] The present invention may be characterized in that said bowel diseases are selected from the group consisting of flatulence, abdominal discomfort, pathogenic infectious diarrhea, gastroenteritis, inflammatory bowel disease, neurogenic intestinal syndrome, irritable bowel syndrome, microbial overgrowth. small intestine and intestinal food diarrhea, and these diseases also include diseases caused by impaired intestinal barrier function.

[74] Inflammatory bowel disease (IBD) may include Crohn's disease, intestinal lesions associated with Behçet's disease, ulcerative colitis, hemorrhagic rectal ulcer, and reservoir ileitis, and belongs to the group of diseases including Crohn's disease and ulcerative colitis. Ulcerative colitis only affects the large intestine. The inflammation and ulcers in ulcerative colitis are limited to the two inner layers (mucosal and submucosal) of the four layers of the colon. Inflammation and ulcers in Crohn's disease can spread through all layers of the wall of both the small and large intestines.

[75] Irritable bowel syndrome is a chronic condition characterized not only by persistent recurrent abdominal discomfort and pain, such as bloating, but also by stool changes, such as diarrhea or constipation. These symptoms may be complicated by psychological factors or stressful social circumstances.

[76] In the present invention, allergic diseases may include, but are not limited to, atopic dermatitis, urticaria, allergic rhinitis, allergic conjunctivitis, asthma, food allergies, and anaphylactic shock.

[77] In the present invention, autoimmune diseases may include, for example, rheumatoid arthritis, lupus, systemic scleroderma, atopic dermatitis, psoriasis, psoriatic arthritis, asthma, Guillain-Barré syndrome, myasthenia gravis, dermatomyositis, polymyositis, multiple sclerosis, autoimmune encephalomyelitis, nodular periarteritis, Hashimoto's thyroiditis, multiple sclerosis, temporal arteritis, juvenile diabetes, alopecia areata, pemphigus, aphthous stomatitis, autoimmune hemolytic anemia, Wegener's granulomatosis, Sjögren's syndrome, Addison's disease, Crohn's disease, and Behçet's disease.

[78] In addition, the inflammatory diseases of the present invention collectively refer to conditions in which inflammation is the main pathological change, and may include, for example, edema, conjunctivitis, periodontitis, rhinitis, otitis media, chronic sinusitis, pharyngolaryngitis, tonsillitis, bronchitis, pneumonia, gastric ulcer, gastritis, colitis, gout, eczema, acne, atopic dermatitis, contact dermatitis, seborrheic dermatitis, ankylosing spondylitis, rheumatism, fibromyalgia, osteoarthritis, psoriatic arthritis, rheumatoid arthritis, periarthritis of the shoulder joint, tendinitis, tendosynovitis, myositis , hepatitis, cystitis, nephritis, Sjögren's syndrome, myasthenia gravis, sepsis, vasculitis, bursitis, temporal arteritis, and multiple sclerosis, but are not limited to.

[79] It is known that the prevention or treatment of autoimmune diseases can be based on the activation of regulatory T cells (Treg) or differentiation into T cells. Tregs, a type of CD4+ T cell, play a role in regulating the immune system and controlling autoimmune diseases by maintaining tolerance to self antigens. The transcription factor called Foxp3 is particularly involved in the development, maintenance and function of regulatory T cells that regulate the immune response by secreting the immunosuppressive cytokines IL-10 and TGF-β.

[80] In addition, it is known that if the frequency of T cell differentiation into Th1 cells, Th2 cells and Th17 cells is low and the frequency of T cell differentiation into Treg cells is high, the inflammatory response can be effectively suppressed.

[81] Th1 cells, Th2 cells, and Th17 cells differentiated from spleen T cells are known to be associated with the expression of the following transcription factors and cytokines: In the case of Th1 cells, T-bet, IFN-γ, and IL- 12; in the case of Th2 cells, GATA3 and IL-5; in the case of Th17 cells - RORγt, IL-17.

[82]

[83] The present invention may be characterized in that the effects of alleviating, treating or preventing autoimmune diseases or inflammatory diseases caused by the strain of the present invention can be induced by at least one mechanism selected from:

[84] i) suppression or expression of at least one factor selected from the group consisting of inflammatory cytokines IL-6, TNF, IL-1b, IL-8, IL-2, IFN-γ, IL-4, IL- 13 and IL-17A;

[85] ii) increased expression or secretion of the anti-inflammatory and immunoregulatory cytokine IL-10; And

[86] iii) increased expression of Tregs involved in anti-inflammatory activity and immunoregulation.

[87]

[88] The term "treatment" refers to the relief or alleviation of diseases or conditions used in conjunction with the term, or one or more of their symptoms, the suppression of their progression or their prevention, unless it is used in another sense. The term "therapy" in the present invention refers to the act of "treating" as defined above. Accordingly, treatment or therapy regimens for intestinal diseases, allergic diseases, autoimmune diseases, or inflammatory diseases in mammals may include one or more of the following:

[89] (1) Suppression of the development of intestinal diseases, allergic diseases, autoimmune diseases or inflammatory diseases,

[90] (2) Prevention of the spread of intestinal diseases, allergic diseases, autoimmune diseases or inflammatory diseases,

[91] (3) Relief of intestinal diseases, allergic diseases, autoimmune diseases or inflammatory diseases,

[92] (4) Prevention of recurrence of bowel disease, allergic disease, autoimmune disease or inflammatory disease, and

[93] (5) Palliative treatment of symptoms of bowel disease, allergic disease, autoimmune disease, or inflammatory disease

[94]

[95] The composition according to the present invention for the prevention or treatment of intestinal diseases, allergic diseases, autoimmune diseases or inflammatory diseases may contain a pharmaceutically effective amount of a strain of Lactobacillus paracasei KBL382, a strain of Lactobacillus paracasei KBL384, a strain of Lactobacillus paracasei KBL385 alone or in combination with at least one of the pharmaceutically acceptable carriers, excipients or diluents.

[96] In the present invention, the term "effective amount" means an amount high enough to cause the desired effect, but low enough to prevent serious side effects, as judged by physicians. The number of microorganisms introduced into the body with the composition according to the present invention can be adjusted according to the route of administration and the purpose of administration.

[97] The composition according to the present invention can be administered to the subject one or more times a day. Dosage form means a physically discrete unit suitable for standard administration to human subjects and other mammals, each unit containing a suitable pharmaceutical carrier and a predetermined amount of microorganisms according to the present invention, providing a therapeutic effect. The dosage form for oral administration to an adult patient preferably contains 0.001 g or more of the microorganism according to the present invention, and the dosage of the composition according to the present invention for oral use is about 0.001 g to 10 g, preferably from 0.01 g to 5 g per dose. A pharmaceutically effective amount of the microorganism according to the present invention is from 0.01 g to 10 g/day. At the same time, the dosage depends on the severity of the disease of the patient, the microorganism and the jointly used auxiliary effective ingredients. In addition, the total daily dosage can be divided into several times and continuously administered as needed. Accordingly, the above dosage ranges do not limit the scope of the present invention in any way.

[98] In addition, the term "pharmaceutically acceptable" as used above refers to a composition that is physiologically acceptable and does not cause an allergic reaction, such as gastrointestinal upset, dizziness, or the like, when administered to a human.

[99] the Composition according to the present invention can be formulated using methods known in the art, providing rapid, delayed or delayed release of the active ingredients after administration to a mammal. Dosage forms may be powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions or sterile powders. In addition, the composition according to the present invention for the prevention or treatment of diseases of the intestine can be administered in various ways, including oral, transdermal, subcutaneous, intravenous or intramuscular administration. The dosage of active ingredients can be selected depending on various factors, for example, the route of administration, age, sex, body weight of the patient and the severity of his condition. The composition according to the present invention for the prevention or treatment of diseases of the gastrointestinal tract can be administered in combination with a known compound capable of preventing or treating symptoms of intestinal diseases.

[100] The pharmaceutical composition according to the present invention can be presented, in particular, in the form of a dosage form for oral use in the composition with an enteric coating. As used herein, the term "enteric coating" includes any pharmaceutically acceptable coating that can persist in the stomach without being degraded by acidic gastric juice and can be sufficiently broken down in the intestine to release the active ingredients there. "Enteric coating" according to the present invention refers to a coating that can be preserved for 2 hours or more when in contact with artificial gastric juice, for example, HCl solution with pH 1 at 36-38°C, and can be further destroyed, preferably in artificial intestinal juice, for example, KH ₂ PO ₄ buffer solution with pH 6.8 for 30 minutes.

[101] The enteric coating of the present invention is applied to one core in an amount of about 16 mg to 30 mg, preferably 16 mg to 20 mg or 25 mg or less. With a thickness of the enteric coating according to the present invention of 5 µm to 100 µm, preferably 20 µm to 80 µm, satisfactory results can be obtained. The enteric coating material can be selected from known polymeric materials. Suitable polymeric materials are listed in a number of known documents [L. Lachman et al., The Theory and Practice of Industrial Pharmacy, ^3rd ed., 1986, pp. 365~373; H. Sucker et al., Pharmazeutische Technologie, Thieme, 1991, pp. 355-359; Hagers Handbuchder pharmazeutischen Praxis, ^4th ed., Vol. 7, pp. 739 ~ 742, and 766 ~ 778, (SpringerVerlag, 1971); and Remington's Pharmaceutical Sciences, ^13th ed., pp. 1689 ~ 1691 (Mack Publ., Co., 1970)], and they may include cellulose ester derivatives, cellulose ethers, an acrylic resin-methyl acrylate copolymer, and a copolymer of maleic acid and phthalic acid derivatives.

[102] The enteric coating of the present invention can be prepared using a conventional enteric coating method in which an enteric coating solution is sprayed onto a core. Suitable solvents used in the enteric coating process are alcohols, eg ethanol, ketones, eg acetone, halogenated hydrocarbon solvents, eg dichloromethane (CH ₂ Cl ₂ ), and mixtures of these solvents. A plasticizer, such as di-n-butyl phthalate or triacetylglycerol, is added to the coating solution in a ratio of about 1 : 0.05 to about 1 : 0.3 (coating material : plasticizer). considering the conditions of coating application. The atomization pressure can be adjusted in various ways, and in general, satisfactory results can be obtained at a spray pressure of from about 1 bar to about 1.5 bar.

[103] In another embodiment, the present invention relates to the use of said strain or composition for the prevention or treatment of intestinal diseases, allergic diseases, autoimmune diseases, or inflammatory diseases, and the use of said strain or composition for the preparation of a therapeutic agent for the treatment of intestinal diseases, allergic diseases, autoimmune diseases or inflammatory diseases.

[104] Specifically, the present invention relates to a composition for use in the prevention or treatment of intestinal diseases, containing at least one component selected from the group consisting of a strain of Lactobacillus paracasei KBL382 (identification number KCTC13509BP), a strain of Lactobacillus paracasei KBL384 (identification number KCTC13510BP ) or Lactobacillus paracasei strain KBL385 (identification number KCTC13511BP), cultures of said strain, lysates of said strain, and extracts of said strain.

[105] In addition, the present invention relates to a composition for use in the prevention or treatment of allergic diseases, containing at least one component selected from the group consisting of a strain of Lactobacillus paracasei KBL382 (identification number KCTC13509BP), a strain of Lactobacillus paracasei KBL384 (identification number KCTC13510BP) or Lactobacillus paracasei strain KBL385 (ID KCTC13511BP), cultures of said strain, lysates of said strain, and extracts of said strain.

[106] In addition, the present invention relates to a composition for use in the prevention or treatment of autoimmune diseases or inflammatory diseases, containing at least one component selected from the group consisting of a strain of Lactobacillus paracasei KBL382 (identification number KCTC13509BP), a strain of Lactobacillus paracasei KBL384 (ID KCTC13510BP) or Lactobacillus paracasei strain KBL385 (ID KCTC13511BP), cultures of said strain, lysates of said strain, and extracts of said strain.

[107] In addition, the present invention relates to the use of a composition containing at least one component selected from the group consisting of a strain of Lactobacillus paracasei KBL382 (identification number KCTC13509BP), a strain of Lactobacillus paracasei KBL384 (identification number KCTC13510BP) or a strain of Lactobacillus paracasei KBL385 (identification number KCTC13511BP), cultures of the specified strain, lysates of the specified strain and extracts of the specified strain, to obtain a drug for the prevention or treatment of intestinal diseases.

[108] In addition, the present invention relates to the use of a composition containing at least one component selected from the group consisting of a strain of Lactobacillus paracasei KBL382 (identification number KCTC13509BP), a strain of Lactobacillus paracasei KBL384 (identification number KCTC13510BP) or a strain of Lactobacillus paracasei KBL385 (identification number KCTC13511BP), cultures of the specified strain, lysates of the specified strain and extracts of the specified strain, to obtain a drug for the prevention or treatment of allergic diseases.

[109] In addition, the present invention relates to the use of a composition containing at least one component selected from the group consisting of a strain of Lactobacillus paracasei KBL382 (identification number KCTC13509BP), a strain of Lactobacillus paracasei KBL384 (identification number KCTC13510BP) or a strain of Lactobacillus paracasei KBL385 (identification number KCTC13511BP), cultures of the specified strain, lysates of the specified strain and extracts of the specified strain, to obtain a drug for the prevention or treatment of autoimmune diseases or inflammatory diseases.

[110] As used herein, the term "prevention" refers to the prevention, delay, inhibition, or slowing down of diseases in order to reduce their impact.

[111] As used herein, the term "treatment" refers to caring for a subject suffering from a disease in order to alleviate, cure, or lessen the symptoms of the disease, or lessen or stop the progression of the disease.

[112]

[113] In another embodiment, the present invention provides a method for the prevention or treatment of intestinal diseases, allergic diseases, autoimmune diseases or inflammatory diseases, comprising the introduction of a pharmaceutically effective amount of the specified strain or the specified composition in the body of a subject in need of the prevention or treatment of these diseases or in mitigation of the condition of the intestine, allergic reactions, reduced immunity or inflammatory reactions.

[114] Specifically, the present invention provides a method for treating bowel diseases, comprising administering a therapeutically effective amount of at least one component selected from the group consisting of Lactobacillus paracasei strain KBL382 (identification number KCTC13509BP), Lactobacillus paracasei strain KBL384 (identification number KCTC13510BP) or a strain of Lactobacillus paracasei KBL385 (identification number KCTC13511BP), cultures of said strain, lysates of said strain, and extracts of said strain into a subject in need thereof.

[115] In addition, the present invention provides a method for treating allergic diseases, comprising administering a therapeutically effective amount of at least one component selected from the group consisting of a strain of Lactobacillus paracasei KBL382 (identification number KCTC13509BP), a strain of Lactobacillus paracasei KBL384 (identification number KCTC13510BP ) or Lactobacillus paracasei strain KBL385 (ID KCTC13511BP), cultures of said strain, lysates of said strain, and extracts of said strain into a subject in need thereof.

[116] In addition, the present invention provides a method for treating autoimmune diseases or inflammatory diseases, comprising administering a therapeutically effective amount of at least one component selected from the group consisting of a strain of Lactobacillus paracasei KBL382 (identification number KCTC13509BP), a strain of Lactobacillus paracasei KBL384 ( identification number KCTC13510BP) or Lactobacillus paracasei strain KBL385 (identification number KCTC13511BP), cultures of said strain, lysates of said strain, and extracts of said strain into the body of a subject in need thereof.

[117] Since a pharmaceutical composition used in a method for the prevention or treatment of intestinal diseases, allergic diseases, autoimmune diseases, or inflammatory diseases, as well as a method of administration thereof, are described above, overlapping elements of the composition and method will not be included in this document in order to avoid unnecessary complexity of the present description. .

[118] The specified subject, which you can enter the composition for the prevention or treatment of these diseases of the intestine, allergic diseases, autoimmune diseases or inflammatory diseases, includes all animals, including humans. For example, the subject may be an animal, such as a dog, cat, or mouse.

[119]

[120] The present invention will be described in detail hereinafter by way of examples. These examples are provided for the sole purpose of illustrating the present invention, and those skilled in the art will appreciate that the spirit of the present invention should not be considered to be limited to these examples.

[121]

[122] Example 1. Screening of probiotic strains with immunoregulatory function

[123] Screening for probiotic strains with immunoregulatory function was performed using the THP-1 monocytic leukemia cell line and peripheral blood mononuclear cells (PBMC). Strains derived from human intestine or vagina, respectively, were seeded in these two cell lines so that the cell line:strain ratio was 1:100, and the ratio of IL-10, a major cytokine indicative of inflammation control, to IL- 6, the major cytokine indicative of an inflammatory response (IL-10/IL-6), and the ratio of IL-10 to IFN-γ, the major cytokine indicative of an autoimmune response (IL-10/IFN-γ). A total of 23 strains were used for screening: two strains of Lactobacillus gasseri, one strain of Lactobacillus reuteri, five strains of Lactobacillus rhamnosus, two strains of Lactobacillus fermentum, four strains of Lactobacillus paracasei, four strains of Lactobacillus salivarius, one strain of Lactobacillus plantarum, two strains of Lactobacillus acidophilus and two strains of Lactococcus lactis.

[124] As a result of screening, the maximum values of IL-10/IL-6 were demonstrated for three strains of Lactobacillus paracasei KBL382, KBL384 and KBL385. In particular, among these three Lactobacillus paracasei strains, KBL382 was characterized by the maximum IL-10/IL-6 value of 2.22 in the THP-1 cell line, and the IL-10/IFN-γ value in peripheral blood mononuclear cells was 9, which indicated an extremely high immunoregulatory effect. Table 1 below shows the obtained values of IL-10/IL-6 and IL-10/IFN-γ, measured for three strains of Lactobacillus paracasei from twenty-three probiotic strains used in the screening of the present invention. Accordingly, the three strains of Lactobacillus paracasei KBL382, KBL384, and KBL385 were predicted to have a pronounced effect on improving intestinal health, and further experiments were performed using these strains.

[125]

[126] [Table 1]

[127]

[128] Example 2 Testing the effect of Lactobacillus paracasei strains KBL382, KBL384 and KBL385 on the expression of inflammatory cytokines and the regulation of T-cell differentiation (in vitro test)

[129] To test the immunoregulatory effect of these strains of Lactobacillus paracasei KBL382, KBL384 and KBL385, the ability to generate cytokines involved in the regulation of the immune system was confirmed in THP-1 cells treated with Lactobacillus paracasei strains KBL382, KBL384 and KBL385, while the presence of T-cell differentiation marker gene expression was confirmed in PBMCs treated with Lactobacillus paracasei strains KBL382 and KBL385.

[130] First, THP-1 cells were seeded in each well of a 24-well plate in the amount of 1 × 10 ⁵ cells, differentiated into mature macrophages, and then the culture medium solution was replaced. After 3 hours, one well was treated with LPS at a concentration of 1 μg/ml as a positive control, and the other wells were treated with Lactobacillus paracasei strains KBL382, KBL384 or KBL385, 1 × 10 ⁷ cells based on the number of viable bacteria; after 24 hours, the culture medium solution was collected and the amount of each cytokine was measured using the BD Cytometric Bead Array (CBA) Human Inflammation Diagnosis Kit (catalog # 551811) according to the manufacturer's instructions. A well treated with PBS buffer according to the same experimental procedure was used as a negative control (Cont). As a result, as shown in FIG. 1, the measurement demonstrated that the THP-1 cell line group treated with Lactobacillus paracasei strains KBL382 and KBL385 had a significantly lower amount of inflammatory cytokines IL-6, TNF, IL-1b and IL-8 compared to the LPS-treated group. In the case of treatment with strain KBL384, it was confirmed that the amount of IL-6 was significantly lower compared to the LPS-treated experimental group, however, there were no significant differences in the levels of TNF, IL-1b and IL-8 compared to the LPS-treated group. In the groups treated with KBL382, KBL384 and KBL385, no significant increase or decrease in IL-10 was observed compared to the LPS-treated group, however, an overall increase in IL-10 was observed compared to the negative control group. Thus, it was concluded that Lactobacillus paracasei strains KBL382 and KBL385 effectively and significantly suppressed the production of inflammatory cytokines in THP-1 cells, and further experiments were performed to test the pattern of T cell differentiation.

[131] To confirm the effect of strains KBL382 and KBL385 on T-cell differentiation, PBMC cells were seeded in each well of a 24-well plate, 5 × 10 ⁵ cells, and then Lactobacillus paracasei strain KBL382 or KBL385 was added, 5 × 10 ⁶ cells. As a control, E. coli (E. coli 0157 EC4115) was added to each well at 5 x 10 ⁶ cells based on the number of viable cells, or LPS was added at a concentration of 500 ng/ml. E. coli is known to increase the expression of the T-bet, GATA3 and RORγt genes, which are markers of effector T cells related to the inflammatory response, while LPS increases the expression of the FOXP3 gene, a marker of Treg cells related to regulation of inflammation. An experimental group of PBMCs obtained under the above conditions were incubated for 5 days, and then cells were obtained and harvested. In order to confirm the level of gene expression, the easy-spin™ (DNA free) Total RNA Extraction Kit (Intron) was first used to extract RNA, and then the High Capacity RNA-to-RNA cDNA extraction kit was used. cDNA Kit (ThermoFisher) for cDNA synthesis. Real-time PCR was performed on the synthesized cDNA using the Rotor-Gene SYBR Green PCR kit (Qiagen) and Rotor-Gene® Q equipment (Qiagen) according to the manufacturer's instructions, and the expression of T-bet mRNA, which is a Th1 marker gene (marker effector cells), GATA3, which is a marker gene for Th2, RORγt, which is a marker gene for Th17, and FOXP3, which is a marker gene for Treg cells. At the same time, the levels of B2M gene expression were measured as an internal control to correct for the relative level of gene expression between the experimental groups. The base sequence of the primer used to confirm the expression of each gene is shown below.

[132]

[133] B2M

[134] Direct: 5'-CCA GCA GAG AAT GGA AAG TC-3'' (SEQ ID NO. 4)

[135] Reverse: 5'-GAT GCT TCT TAC ATG TCT CG-3' (SEQ ID NO. 5)

[136]

[137] T-bet

[138] Direct: 5'-CCC CAA GGA ATT GAC AGT TG-3' (SEQ ID NO. 6)

[139] Reverse) 5'-GGG AAA CTA AAG CTC ACA AAC-3' (SEQ ID NO. 7)

[140]

[141] GATA3

[142] Direct: 5'-CTG CAA TGC CTG TGG GCT C-3' (SEQ ID NO. 8)

[143] Reverse: 5'-GAC TGC AGG GAC TCT CGC T-3' (SEQ ID NO. 9)

[144]

[145] RORγt

[146] Direct: 5'-AAG ACT CAT CGC CAA AGC AT-3' (SEQ ID NO. 10)

[147] Reverse: 5'-TCC ACA TGC TGG CTA CAC A-3' (SEQ ID NO. 11)

[148]

[149] FOXP3

[150] Direct: 5'-TCA AGC ACT GCC AGG CG-3' (SEQ ID NO. 12)

[151] Reverse: 5'-CAG GAG CCC TTG TCG GAT-3' (SEQ ID NO. 13)

[152]

[153] As a result, as shown in FIG. 2 confirmed that the Lactobacillus paracasei KBL382 and KBL385 strains, unlike E. coli, maintained the expression of the T-bet, GATA3 and RORγt genes, which are markers of effector inflammatory T cells, at a significantly low level, but significantly increased the amount of FOXP3 mRNA, Treg cell marker, maintaining immune homeostasis similar to LPS.

[154]

[155] Example 3 Testing the effect of Lactobacillus paracasei strains KBL382, KBL384 and KBL385 on T-cell immunoregulation (in vitro test)

[156] In order to test the immunoregulatory effect of T cells due to strains of Lactobacillus paracasei KBL382, KBL384 and KBL385, the level of expression of inflammatory cytokines was checked when T cells were activated in the composition of the IPC, followed by treatment with the indicated strains of Lactobacillus paracasei KBL382, KBL384 and KBL385.

[157] PBMC cells were seeded in each well of a 96-well plate, 2 × 10 ⁵ cells with anti-CD3 antibody (1 μg/ml, ebioscience) to activate T cells, and then Lactobacillus paracasei strain KBL382, KBL384 or KBL385 was added according to 1 × 10 ⁷ cells based on the ratio “bacteria : MIC = 50 : 1” based on the number of viable cells. As a control, Escherichia coli K12 (ATCC 10798) was added to each well at 1 x 10 ⁷ cells based on the number of viable cells. After incubating an experimental group of PBMCs with activated T cells obtained under the above conditions, the supernatant was collected for 3 days and the amount of each cytokine was measured using the BD cytometric Bead Array (CBA) Human Inflammation Kit (CBA) (cat. no. 551811) according to with manufacturer's instructions.

[158] As a result, as shown in FIG. 3, in PBMC cell lines with T-cells activated by the addition of an anti-CD3 antibody, all strains of Lactobacillus paracasei KBL382, KBL384 and KBL385 significantly reduced the expression of IL-2, which is a Th1 cytokine, IL-4 and IL-13, which are cytokines of IFNγ and Th2 , respectively, and IL-17A, which is a Th17 cytokine, compared with the experimental group treated only with the addition of the E. coli strain and PBS, while the level of the anti-inflammatory cytokine IL-10 was markedly increased with all three strains of Lactobacillus paracasei used in experiment, compared with the experimental group treated with PBS alone; the experiment confirmed that strain KBL 382 among the three strains in particular significantly increased the level of IL-10 compared to the E. coli supplemented group. The above experimental results led to the conclusion that all strains of Lactobacillus paracasei KBL382, KBL384 and KBL385 significantly reduced the level of inflammatory T-cell cytokines and increased the level of anti-inflammatory cytokines.

[159]

[160] Example 4 Testing the effect of Lactobacillus paracasei strains KBL382, KBL384 and KBL385 on mitigating enteritis (in vivo test)

[161] This example was to confirm the presence of effects that improve bowel function in strains of Lactobacillus paracasei KBL382, KBL384 and KBL385, including in vivo. For this purpose, C57BL/6 mice were divided into groups of 10 mice, and then given tap water with 2% DSS solution for 9 days, thereby causing them enteritis. At the same time, mice in the control group were orally injected with 200 μl of PBS once a day, and mice in the experimental group were orally administered once a day with 200 μl of each of Lactobacillus paracasei strains KBL382, KBL384 or KBL385 diluted with PBS to a concentration of 2 × 10 ¹⁰ cfu/ ml, so that the daily input amount could be set as 4×10 ⁹ CFU. Then, for 9 days when enteritis was induced by DSS, body weight changes of mice in the control group and experimental group were measured daily, and on day 9 after DSS administration, mice were subjected to autopsy to measure the length of the colon.

[162] As a result, as shown in FIG. 4, with respect to body weight changes, it was confirmed that all groups treated with strains KBL382, KBL384 and KBL385 showed a significant effect in terms of alleviating body weight loss compared with the untreated control group; in relation to changes in the length of the colon in all groups treated with three strains, there was a significant improvement in the degree of reduction in the length of the colon compared to mice in the control group.

[163] In addition, performed H&E-staining to measure the degree of inflammation in the experimental groups of strains KBL382 and KBL385 and measured the levels of myeloperoxidase (MPO) in tissues. After autopsy, to evaluate colonic lesions by H&E staining, the distal colon was fixed in 10% neutral formalin solution, and then 5 μm thick paraffinized tissue samples stained with hematoxylin and eosin were obtained and examined under an optical microscope. Then, to measure MPO, colon tissue was first placed in RIPA buffer to which a protease inhibitor was added, and then minced with a homogenizer. After centrifuging the disrupted tissue at 4° C. and 15,000×g for 10 minutes, MPO was measured in the supernatant according to the Hycult Biotech Solid Phase ELISA kit protocol, mouse MPO, catalog # HK210-02).

[164] As a result, as shown in FIG. 4, the H&E staining experiment confirmed that the experimental groups treated with the two strains KBL382 and KBL385 were characterized by mitigation of inflammation compared with the control group, and the MPO level was slightly reduced in the KBL385 experimental group and significantly reduced in the KBL382 experimental group. The colon tissues thus obtained were used to confirm the effect of strengthening tight junctions in the wall of the intestinal tract.

[165]

[166] Example 5 Effect of Lactobacillus paracasei strains KBL382 and KBL385 on intestinal wall tight junction strengthening

[167] To test the effect of Lactobacillus paracasei strains KBL382 and KBL385 on intestinal wall tight junction strengthening, comparison and measurement of mRNA expression levels of genes involved in intestinal wall tight junctions in cells isolated from mouse colon tissue in Example 4 was performed. In order to quantify gene expression, RNA was first isolated from tissues using the easy-spin™ (DNA free) Total RNA Extraction Kit (Intron), and then cDNA was synthesized using the High Capacity RNA cDNA Extraction Kit -to-cDNA Kit (ThermoFisher). The Rotor-gene SYBR green PCR kit (Qiagen) and Rotor-Gene ® Q (Qiagen). As a control for normalization by the relative level of gene expression between the experimental groups, the expression level of the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene was measured. The primer used to validate expression was designed to bind specifically to each gene as follows.

[168]

[169] Zo-1

[170] Direct: 5'-ACC CGA AAC TGA TGC TGT GGA TAG-3' (SEQ ID No. 14)

[171] Reverse: 5'-AAA TGG CCG GGC AGA ACT TGT GTA-3' (SEQ ID No. 15)

[172]

[173] Claudine-3

[174] Direct: 5'-CAG ACG TCC GTC AGT TTT CG-3' (SEQ ID No. 16)

[175] Reverse: 5'-CAT GGC TGC TGG ACT TGA AC-3' (SEQ ID No. 17)

[176]

[177] MUC-4

[178] Direct: 5'-GTC TCC CAT CAC GGT TCA GT-3' (SEQ ID No. 18)

[179] Reverse: 5'-TGT CAT TCC ACA CTC CCA GA-3' (SEQ ID No. 19)

[180]

[181] HPRT

[182] Straight: 5'-TTA TGG ACA GGA CTG AAA GAC-3'' (SEQ ID No. 20)

[183] Reverse: 5'-GCT TTA ATG TAA TCC AGC AGG T-3'' (SEQ ID No. 21)

[184]

[185] As a result, as shown in FIG. 5, it was found that the levels of three genes measured in mice treated with strains KBL382 and KBL385 were increased compared to the control group treated with PBS alone, thereby restoring tight intestinal wall junctions; accordingly, these strains can be used to treat diseases such as IBD and IBS.

[186]

[187] Example 6 Comparison of the effects of a therapeutic antibody for the treatment of enteritis and KBL382 in terms of alleviating enteritis

[188] Infliximab (product name Remicade) is a therapeutic recombinant antibody drug used by injection for autoimmune diseases such as rheumatoid arthritis, ankylosing spondylitis, ulcerative colitis, childhood Crohn's disease, psoriasis, and psoriatic arthritis. The aim of the present study was to confirm the differences in the in vivo effect of alleviating bowel disease symptoms between KBL 382 strain and infliximab.

[189] To compare these effects, after dividing C57BL/6 mice into groups of eight mice, they were given tap water with 2% DSS solution for 9 days, causing them to enteritis. At the same time, mice in the control group were orally administered 200 μl of PBS once a day, while mice in the experimental group were orally administered 200 μl of strain KBL382 diluted with PBS to a concentration of 2 × 10 ¹⁰ cfu/ml once a day, so that daily the input amount could be given as 4×10 ⁹ cfu. In the therapeutic antibody group, the antibody infliximab was administered once on day 3 at a dose of 5 mg/kg per mouse.

[190] Then, for 9 days when enteritis was induced by DSS, changes in body weight of mice in the control group and experimental group were measured daily, and on day 9 after DSS administration, mice were subjected to autopsy to measure the length of the colon.

[191] As a result, as shown in FIG. 6(A), with respect to the change in the length of the colon, a significant improvement in the degree of reduction in the length of the colon was confirmed in the KBL382 and infliximab treated groups compared with mice in the control group. The experiment confirmed that the effect of colon length improvement in the KBL382 group was similar or even higher than in the infliximab group.

[192] As shown in FIG. 6(B), with regard to body weight change, a significant improvement in the effect on weight loss was confirmed in the KBL382 and infliximab treated groups compared to the untreated control mice. The study confirmed that the effect of alleviating weight loss within five days after administration was higher in the infliximab group compared to the KBL382 group, however, the beneficial effect of infliximab decreased further, while the effect on weight loss in the KBL382 strain group increased, showing similar effects between the two groups up to day 9. Accordingly, with respect to the use of this strain for the treatment of inflammatory bowel disease and irritable bowel syndrome, the study confirmed the possibility of obtaining an effect similar to that of commercially available therapeutic antibodies.

[193]

[194] Example 7. The effect of KBL382 on the mitigation of atopic conditions

[195] To test the mitigating effect of strain KBL382 on atopic conditions, NC/Nga mice, an animal model of atopic skin disease, were used.

[196] After dividing the NC/Nga mice into groups of nine mice, hair was removed on the back of each mouse from the bottom of the ear to the top of the tail and the mice were left for 24 hours. An ointment containing Dermatophagoides farinae extract (DFE) extract, house dust mite antigen (HDM) was then applied to the dehaired area once or twice a week for seven weeks, so that each mouse could receive 100 mg of DFE, thereby causing them to become atopic. dermatitis. From the third week of induction of dermatitis, mice in the control group received 200 μl of PBS orally once a day; mice in the experimental group were orally administered each of strains KBL382 and Lactobacillus rhamnosus KBL365 diluted with 200 μl of PBS to a concentration of at least 1 x 10 ⁹ cfu/mouse once a day in an amount of 200 μl. Then, during four weeks of bacteria administration, the dermatitis score of mice in the control and experimental groups was measured weekly, and at 4 weeks after bacteria administration, mice scratching time, skin thickness, and blood IgE concentration after autopsy were measured.

[197]

[198] 7-1. Performing dermatitis scoring

[199] To evaluate skin lesions caused by DFE, the dermatitis score was measured as follows. The condition of the skin was monitored by taking pictures for 4 weeks at weekly intervals, starting from 3 weeks after the introduction of the strain. Checked four indicators - dryness, swelling, erythema/bleeding and erosion/separation of the skin. The overall score was 0 for no lesions, 1 for mild, 2 for moderate, and 3 for severe.

[200] As a result, as shown in FIG. 7(A), the dermatitis score was significantly reduced in the KBL382 strain group compared to the control group (negative control) where atopic dermatitis was induced and the KBL365 strain group. As a result, the effect of taking the KBL382 strain on the atopic dermatitis mitigation score was confirmed.

[201]

[202] 7-2 Effect on mitigation of itch symptoms

[203] In order to test the mitigating effect on itching when the KBL382 strain was administered to mouse models suffering from DFE-induced atopic dermatitis, the number of mouse scratches was measured within 10 minutes 4 weeks after strain administration.

[204] As a result, as can be seen in FIG. 7(B), it turned out that the amount of scratching was significantly reduced in the experimental group treated with KBL382 compared with the group treated with PBS, which confirmed the mitigation of the symptoms of itching in atopic dermatitis due to the introduction of the KBL382 strain.

[205]

[206] 7-3. Reduction in skin thickness

[207] In order to test the effect of skin thickness reduction after administration of KBL382 to mouse models suffering from DFE-induced atopic dermatitis, mouse ear thickness and dorsal skin thickness were measured with calipers four weeks after strain administration, and a decrease in symptoms of edema due to atopic dermatitis was observed. As a result, as can be seen in FIG. 7(C), in the experimental group receiving KBL382, a significant decrease in the thickness of the ear and skin was observed compared with the control group and the group receiving KBL365.

[208]

[209] 7-4. Decrease in the concentration of IgE in the blood

[210] It was found that the concentration of IgE in patients with atopic dermatitis generally increased with increasing clinical severity of atopic dermatitis (Matsumoto M, J. Immunol. 1999). Therefore, the concentration of IgE, a typical hematological factor appearing in atopic dermatitis, was measured by collecting blood three weeks after strain administration, separating the serum and using a mouse IgE ELISA kit (catalog # 555248, BD OptEIA™). As a result, as shown in FIG. 7(D) found that the concentration of IgE in the blood was significantly reduced in the experimental group receiving oral KBL382, indicating the effect of treating atopic dermatitis after taking KBL382.

[211]

[212] Specific aspects of the present invention are described in detail above, and for those skilled in the art it is obvious that these specific aspects are only specific implementation options that do not limit the scope of the present invention. On the contrary, the scope of the present invention is largely defined by the following claims with the equivalents of the claims.

[213]

[214] Depositor Name: Korea Research Institute of Bioscience & Biotechnology

[215] ID number: KCTC13509BP

[216] Access date: 20180417

[217]

[218] Depositor Name: Korea Research Institute of Bioscience & Biotechnology

[219] ID number: KCTC13510BP

[220] Access date: 20180417

[221]

[222] Depositor name: Korea Research Institute of Bioscience & Biotechnology

[223] ID number: KCTC13511BP

[224] Access date: 20180417

[225]

Possibility of industrial application

[226] The strains of Lactobacillus paracasei KBL382 (identification number KCTC13509BP), Lactobacillus paracasei KBL384 (identification number KCTC13510BP) and Lactobacillus paracasei KBL385 (identification number KCTC13511BP) according to the present invention have excellent anti-inflammatory and immunomodulatory functions, have a pronounced strengthening effect on the intestinal tract wall tight junctions , suppress weight loss and shortening of the colon caused by enteritis, thereby exhibiting a therapeutic effect on enteritis, attenuate allergic cellular response, and significantly alleviate the symptoms of atopic dermatitis, thereby providing improvement in allergic diseases. Thus, a single strain can provide enhanced anti-inflammatory effects, enhanced immunity, improved gut health, and alleviated allergic diseases, and thus can be used as a probiotic material.

[227]

Sequence list text

[228] An electronic sequence listing file is attached

--->

<110> Ko BioLabs, Inc.

< 120> Lactobacillus paracasei strain and its use

<130> PP-B2198

<150> KR 10-2018-0053279

<151> 05/19/2018

<160> 21

<170> KoPatentIn 3.0

<210> 1

<211> 1472

<212> DNA

<213> Lactobacillus paracasei

<400> 1

gcaggtggcg ggtgctatac atgcagtcga cgagttctcg ttgatgatcg gtgcttgcac 60

cgagattcaa catggaacga gtggcggacg ggtgagtaac acgtgggtaa cctgccctta 120

agtgggggat aacatttgga aacagatgct aataccgcat agatccaaga accgcatggt 180

tcttggctga aagatggcgt aagctatcgc ttttggatgg acccgcggcg tattagctag 240

ttggtgaggt aatggctcac caaggcgatg atacgtagcc gaactgagag gttgatcggc 300

cacattggga ctgagacacg gcccaaactc ctacgggagg cagcagtagg gaatcttcca 360

caatggacgc aagtctgatg gagcaacgcc gcgtgagtga agaaggcttt cgggtcgtaa 420

aactctgttg ttggagaaga atggtcggca gagtaactgt tgtcggcgtg acggtatcca 480

accagaaagc cacggctaac tacgtgccag cagccgcggt aatacgtagg tggcaagcgt 540

600

cctcggctta accgaggaag cgcatcggaa actgggaaac ttgagtgcag aagaggacag 660

tggaactcca tgtgtagcgg tgaaatgcgt agatatatgg aagaacacca gtggcgaagg 720

cggctgtctg gtctgtaact gacgctgagg ctcgaaagca tgggtagcga acaggattag 780

ataccctggt agtccatgcc gtaaacgatg aatgctaggt gttggagggt ttccgccctt 840

cagtgccgca gctaacgcat taagcattcc gcctggggag tacgaccgca aggttgaaac 900

tcaaaggaat tgacgggggc ccgcacaagc ggtggagcat gtggtttaat tcgaagcaac 960

gcgaagaacc ttaccaggtc ttgacatctt ttgatcacct gagagatcag gtttcccctt 1020

cgggggcaaa atgacaggtg gtgcatggtt gtcgtcagct cgtgtcgtga gatgttgggt 1080

taagtcccgc aacgagcgca acccttatga ctagttgcca gcatttagtt gggcactcta 1140

gtaagactgc cggtgacaaa ccggaggaag gtggggatga cgtcaaatca tcatgcccct 1200

tatgacctgg gctacacacg tgctacaatg gatggtacaa cgagttgcga gaccgcgagg 1260

tcaagctaat ctcttaaagc cattctcagt tcggactgta ggctgcaact cgcctacacg 1320

aagtcggaat cgctagtaat cgcggatcag cacgccgcgg tgaatacgtt cccgggcctt 1380

gtacacaccg cccgtcacac catgagagtt tgtaacaccc gaagccggtg gcgtaaccct 1440

ttagggagcg agcgtctaag tggctcacgc ct 1472

<210> 2

<211> 1479

<212> DNA

<213> Lactobacillus paracasei

<400> 2

gccagtgggg ggggtgctat acatgcagtc gaacgagttc tcgttgatga tcggtgcttg 60

caccgagatt caacatggaa cgagtggcgg acgggtgagt aacacgtggg taacctgccc 120

ttaagtgggg gtaacattt ggaaacagat gctaataccg catagatcca agaaccgcat 180

ggttcttggc tgaaagatgg cgtaagctat cgcttttgga tggacccgcg gcgtattagc 240

tagttggtga ggtaatggct caccaaggcg atgatacgta gccgaactga gaggttgatc 300

ggccacattg ggactgagac acggcccaaa ctcctacgggg aggcagcagt agggaatctt 360

ccacaatgga cgcaagtctg atggagcaac gccgcgtgag tgaagaaggc tttcgggtcg 420

taaaactctg ttgttggaga agaatggtcg gcagagtaac tgttgtcggc gtgacggtat 480

ccaaccagaa agccacggct aactacgtgc cagcagccgc ggtaatacgt aggtggcaag 540

600

agccctcggc ttaaccgagg aagcgcatcg gaaactggga aacttgagtg cagaagagga 660

cagtggaact ccatgtgtag cggtgaaatg cgtagatata tggaagaaca ccagtggcga 720

aggcggctgt ctggtctgta actgacgctg aggctcgaaa gcatgggtag cgaacaggat 780

tagataccct ggtagtccat gccgtaaacg atgaatgcta ggtgttggag ggtttccgcc 840

cttcagtgcc gcagctaacg cattaagcat tccgcctggg gagtacgacc gcaaggttga 900

aactcaaagg aattgacggg ggcccgcaca agcggtggag catgtggttt aattcgaagc 960

aacgcgaaga accttaccag gtcttgacat cttttgatca cctgagagat caggtttccc 1020

cttcgggggc aaaatgacag gtggtgcatg gttgtcgtca gctcgtgtcg tgagatgttg 1080

ggttaagtcc cgcaacgagc gcaaccctta tgactagttg ccagcattta gttgggcact 1140

ctagtaagac tgccggtgac aaaccgggagg aaggtgggga tgacgtcaaa tcatcatgcc 1200

ccttatgacc tgggctacac acgtgctaca atggatggta caacgagttg cgagaccgcg 1260

aggtcaagct aatctcttaa agccattctc agttcggact gtaggctgca actcgcctac 1320

acgaagtcgg aatcgctagt aatcgcggat cagcacgccg cggtgaatac gttcccggggc 1380

cttgtacaca ccgcccgtca caccatgaga gtttgtaaca cccgaagccg gtggcgtaac 1440

1479

<210> 3

<211> 1473

<212> DNA

<213> Lactobacillus paracasei

<400> 3

gcagttgggg gggagctata catgcagtcg acgagttctc gttgatgatc ggtgcttgca 60

ccgagattca acatggaacg agtggcggac gggtgagtaa cacgtgggta acctgccctt 120

aagtggggga taacatttgg aaacagatgc taataccgca tagatccaag aaccgcatgg 180

ttcttggctg aaagatggcg taagctatcg cttttggatg gacccgcggc gtattagcta 240

gttggtgagg taatggctca ccaaggcgat gatacgtagc cgaactgaga ggttgatcgg 300

ccacattggg actgagacac ggcccaaact cctacgggag gcagcagtag ggaatcttcc 360

acaatggacg caagtctgat ggagcaacgc cgcgtgagtg aagaaggctt tcgggtcgta 420

aaactctgtt gttggagaag aatggtcggc agagtaactg ttgccggcgt gacggtatcc 480

aaccagaaag ccacggctaa ctacgtgcca gcagccgcgg taatacgtag gtggcaagcg 540

ttatccggat ttattgggcg taaagcgagc gcaggcggtt ttttaagtct gatgtgaaag 600

ccctcggctt aaccgaggaa gcgcatcgga aactgggaaa cttgagtgca gaagaggaca 660

gtggaactcc atgtgtagcg gtgaaatgcg tagatatatg gaagaacacc agtggcgaag 720

gcggctgtct ggtctgtaac tgacgctgag gctcgaaagc atgggtagcg aacagatta 780

gataccctgg tagtccatgc cgtaaacgat gaatgctagg tgttggaggg tttccgccct 840

tcagtgccgc agctaacgca ttaagcattc cgcctgggga gtacgaccgc aaggttgaaa 900

ctcaaaggaa ttgacgggggg cccgcacaag cggtggagca tgtggtttaa ttcgaagcaa 960

1020

tcgggggcaa aatgacaggt ggtgcatggt tgtcgtcagc tcgtgtcgtg agatgttggg 1080

ttaagtcccg caacgagcgc aacccttatg actagttgcc agcatttagt tgggcactct 1140

agtaagactg ccggtgacaa accggaggaa ggtggggatg acgtcaaatc atcatgcccc 1200

ttatgacctg ggctacacac gtgctacaat ggatggtaca acgagttgcg agaccgcgag 1260

gtcaagctaa tctcttaaag ccattctcag ttcggactgt aggctgcaac tcgcctacac 1320

1380

tgtacacacc gcccgtcaca ccatgagagt ttgtaacacc cgaagccggt ggcgtaaccc 1440

tttagggagc gagccgtcta agtgtacaaa gtt 1473

<210> 4

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 4

ccagcagaga atggaaagtc 20

<210> 5

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 5

gatgcttctt acatgtctcg 20

<210> 6

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 6

ccccaaggaa ttgacagttg 20

<210> 7

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 7

gggaaactaa agctcacaaa c 21

<210> 8

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 8

ctgcaatgcc tgtgggctc 19

<210> 9

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 9

gactgcaggg actctcgct 19

<210> 10

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 10

aagactcatc gccaaagcat 20

<210> 11

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 11

tccacatgct ggctacaca 19

<210> 12

<211> 17

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 12

tcaagcactg ccaggcg 17

<210> 13

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 13

caggagccct tgtcggat 18

<210> 14

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 14

acccgaaact gatgctgtgg atag 24

<210> 15

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 15

aaatggccgg gcagaacttg tgta 24

<210> 16

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 16

cagacgtccg tcagttttcg 20

<210> 17

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 17

catggctgct ggacttgaac 20

<210> 18

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 18

gtctcccatc acggttcagt 20

<210> 19

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 19

tgtcattcca cactcccaga 20

<210> 20

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 20

ttatggacag gactgaaaga c 21

<210> 21

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 21

gctttaatgt aatccagcag gt 22

<---

Claims (1)

1. Lactobacillus paracasei strain KBL382 with identification number KCTC13509BP having immune enhancing activity. 2. The strain according to claim 1, characterized in that said strain contains the 16s rDNA sequence of SEQ ID NO: 1. 3. A food composition containing an effective amount of at least one component selected from the group consisting of a strain according to claim 1 or 2 and cultures of said strain, and one or more edible excipients. 4. A dietary supplement composition containing an effective amount of at least one component selected from the group consisting of a strain according to claim 1 or 2 and cultures of said strain, and one or more edible excipients. 5. A food composition according to claim 3, characterized in that said composition is a functional health food composition for improving intestinal health. 6. A pharmaceutical composition for the treatment or prevention of intestinal diseases, containing an effective amount of at least one component selected from the group consisting of a strain according to claim 1 or 2, and cultures of the specified strain, and one or more pharmaceutically acceptable excipients. 7. Pharmaceutical composition according to claim 6, characterized in that said bowel diseases are selected from the group consisting of flatulence, abdominal discomfort, infectious diarrhea caused by pathogenic microorganisms, gastroenteritis, inflammatory bowel disease, neurogenic intestinal syndrome, irritable bowel syndrome, overgrowth microorganisms of the small intestine and intestinal food diarrhea. 8. Food composition according to claim 3, characterized in that said composition is a functional health food composition for improving allergy symptoms. 9. Pharmaceutical composition for the treatment or prevention of allergic diseases, containing an effective amount of at least one component selected from the group consisting of a strain according to claim 1 or 2, and cultures of the specified strain, and one or more pharmaceutically acceptable excipients. 10. Pharmaceutical composition according to claim 9, characterized in that said allergic disease is atopic dermatitis, urticaria, allergic rhinitis, allergic conjunctivitis, asthma, food allergy or anaphylactic shock. eleven. A food composition according to claim 3, characterized in that said composition is a functional health food composition for alleviating autoimmune diseases or inflammatory diseases. 12. Pharmaceutical composition for the treatment or prevention of autoimmune diseases, containing an effective amount of at least one component selected from the group consisting of a strain according to claim 1 or 2, and cultures of the specified strain, and one or more pharmaceutically acceptable excipients. 13. Pharmaceutical composition for the treatment or prevention of inflammatory diseases, containing an effective amount of at least one component selected from the group consisting of a strain according to claim 1 or 2, and cultures of the specified strain, and one or more pharmaceutically acceptable excipients. 14. A method for treating bowel diseases, comprising administering a therapeutically effective amount of at least one component selected from the group consisting of a strain according to claim 1 or 2, and cultures of said strain, to a subject in need thereof. 15. A method for treating allergic diseases, comprising administering a therapeutically effective amount of at least one component selected from the group consisting of a strain according to claim 1 or 2, and cultures of said strain, to a subject in need thereof. 16. A method for treating autoimmune diseases, comprising administering a therapeutically effective amount of at least one component selected from the group consisting of a strain according to claim 1 or 2, and cultures of said strain, to a subject in need thereof. 17. A method for treating inflammatory diseases, comprising administering a therapeutically effective amount of at least one component selected from the group consisting of a strain according to claim 1 or 2, and cultures of said strain, to a subject in need thereof. 18. The use of a pharmaceutical composition containing an effective amount of at least one component selected from the group consisting of a strain according to claim 1 or 2, and cultures of said strain, and one or more pharmaceutically acceptable excipients, for the preparation of a medicinal product for the prevention or treatment of diseases intestines. 19. The use of a pharmaceutical composition containing an effective amount of at least one component selected from the group consisting of a strain according to claim 1 or 2, and cultures of the specified strain, and one or more pharmaceutically acceptable excipients, for the production of a drug for the prevention or treatment of allergic diseases. 20. The use of a pharmaceutical composition containing an effective amount of at least one component selected from the group consisting of a strain according to claim 1 or 2, and cultures of the specified strain, and one or more pharmaceutically acceptable excipients, for the production of a drug for the prevention or treatment of autoimmune diseases. 21. The use of a pharmaceutical composition containing an effective amount of at least one component selected from the group consisting of a strain according to claim 1 or 2, and cultures of the specified strain, and one or more pharmaceutically acceptable excipients, for the production of a drug for the prevention or treatment of inflammatory diseases.

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