KR101862051B1 - Novel lactic acid bacteria having immunoregulatory activities derived from human digestive tract and use thereof - Google Patents

Abstract

According to the present invention, a specific lactobacillus strain, a specific bifidobacterium strain, a specific streptococcus strain, or a specific collinsella strain is separated from human excrement to have high stability, and have various biological activities such as an immune regulatory effect, an immune enhancement effect, an antiallergy effect, an antiinflammation effect, a colitis improvement effect, an arthritis improvement effect, an anti-obesity effect, an anti-stress effect, an anti-anxiety effect, an anti-depression effect, or an anti-schizophrenia effect. According to the present invention, the specific lactobacillus strain, the specific bifidobacterium strain, the specific streptococcus strain, or the specific collinsella strain is able to be used as a material for regulating immunity, enhancing immunity, suppressing an inflammation reaction, and relieving stress, and also, is able to be used as a functional food and drug material for preventing, improving, or treating allergy, inflammation, colitis, arthritis, obesity, anxiety, depression, or schizophrenia.

Description

Novel lactic acid bacteria having immunoregulatory activities derived from human digestive tract and use thereof

The present invention relates to novel lactic acid bacteria and the like, and more particularly, derived from the human digestive tract, and in more detail, immunomodulatory effect, immunity enhancing effect, anti-allergic effect, anti-inflammatory effect, colitis improvement effect, arthritis improvement effect, anti-obesity effect, anti-stress effect , Anti-anxiety effect, anti-depressant effect or anti-schizophrenia, it relates to a variety of dietary and pharmaceutical uses of novel lactic acid bacteria and new lactic acid bacteria having various physiological activities.

As mankind gradually develops into a prosperous society, the pattern of disease is changing significantly as lifestyle habits rapidly become westernized. In particular, for modern people, immunity decreases due to the westernized diet, irregular eating, excessive drinking, lack of exercise, excessive stress, and exposure to harmful environments, disturbance of the intestinal flora, colitis, arthritis, allergic diseases, and obesity. It is increasing rapidly.

Intestinal flora Disruption and immune cells

Many bacteria live in the digestive tract of our body. While there are about 10 trillion normal cells in our body, the number of bacteria is about 100 trillion, about 10 times more than that. Most of these bacteria make up the intestinal flora and influence the immune regulation of the human body through interaction with various immune cells present in the intestine. And these bacteria can be divided into beneficial bacteria that help the health of our intestines and harmful bacteria that are harmful to our health. Our bodies are Lactobacillus bacteria (Lactobacillus), Bifidobacterium (Bifidobacterium), Streptococcus (Streptococcus), current yuikgyun E. coli, such as Kono Stock (Leuconostoc), Peddie OKO Syracuse (Pediococcus), sports as Lactobacillus (Sporolactobacilius) ( E. coli ), Veillonella (Veillonella), Welch ( Clostridium perfringens ), and other harmful bacteria are dominant, and various digestive tract-inhabiting microorganisms can maintain health when maintaining a balance.

The immune cells of our body maintain health by controlling antigens (including microorganisms) invading from the outside and abnormal cells that occur inside. More than 70% of these immune cells are located in the digestive tract, and they control microorganisms already inhabiting the digestive tract or invading microorganisms from the outside. If there is a problem with the immune regulation function of the human body, immunity decreases or immune hypersensitivity reaction is caused, and as a result, viral infection, cancer, inflammatory disease, enteritis (ulcerative colitis, Crohn's disease, diverticulosis, etc.), allergic disease, atopy, arthritis, Diseases such as obesity and diabetes can be caused. Therefore, in order to effectively eliminate the occurrence of the above diseases, it is necessary to control the disturbance of the intestinal microflora and to make the immune system of the digestive tract operate normally.

colitis

Conventionally, the incidence of ulcerative colitis and Crohn's disease has been known to be high in Westerners, but recently, the number of patients in East Asia such as Korea is increasing rapidly due to changes in lifestyles such as eating habits. However, there are reasons for which the cause is unclear, so no fundamental treatment has been established. For this reason, it is not aimed at complete treatment, but a situation in which a drug that relieves symptoms and maintains such a condition for as long as possible is being used. As drugs for such symptomatic therapy, mainly aminosalicylic acid preparations, adrenal cortical steroids, immunosuppressants, and the like are used, but various side effects have been reported. For example, salazosulfapyridine, which is frequently used as an aminosalicylic acid, has been reported to have side effects such as nausea, vomiting, loss of appetite, rash, headache, liver injury, leukocyte reduction, abnormal red blood cells, proteinuria, and diarrhea. In addition, adrenocorticosteroids are generally used by oral administration of prednisolone, enema, suppositories, intravenous injections, etc., but have strong side effects such as gastric ulcer or necrosis of the femoral head due to long-term use. However, because discontinuation of medication recurs symptoms, these drugs are compelled to continue to be used. Accordingly, there is a need for development of a therapeutic agent for intestinal diseases such as ulcerative colitis and Crohn's disease that is excellent in effect and does not cause side effects. Irritable bowel syndrome (IBS) is likewise a chronic abdominal disease whose cause is not clear. Currently, there is no fundamental treatment for IBS, and symptomatic therapy is being performed for the purpose of alleviating each type of symptom. For example, for Hari-type IBS, anticholinergic drugs that have antispasmodic action to inhibit smooth muscle contraction are used, salt laxatives for constipation-type IBS, and drugs for replacement-type IBS are difficult to control. have.

Allergic disease

As society becomes more complex, environmental pollution and stress increase due to the development of industries and civilizations, and diet changes, the number of patients with allergic diseases is increasing every year. In 1980, patients with allergic diseases such as atopy, anaphylaxis, and asthma were less than 1%, but in the 2000s, the number of patients with allergic diseases increased to more than 5%, and it is estimated that more than 10% including potential patients. The cause of allergic disease is the excessive immune response of the living body as a result of the antigen-antibody reaction, and allergic diseases are generally classified as type 1-4 hypersensitivity reactions depending on the reaction time and the presence or absence of complement involvement. Type 1 hypersensitivity reactions include atopy, anaphylactic shock, bronchial asthma, urticaria, pollinosis, etc., and type 2 hypersensitivity reactions include inappropriate blood transfusion, autoimmune hemolytic anemia, hemolytic anemia caused by drugs, granulocytopenia, thrombocytopenia purpura Type 3 hypersensitivity reactions include erythema, lymphadenopathy, joint pain, arthritis, nephritis, and acute glomerulonephritis following streptococcal infection, and type 4 hypersensitivity reactions include chronic inflammation. In order to improve allergic diseases, it is recommended to first take a shower or bath to remove allergens (house dust, mites, etc.) from the skin and do not consume allergens. However, if the allergic disease does not improve, drugs such as steroids, antihistamines, and immunosuppressants are used.These drugs include skin atrophy, vasodilation, bleaching, purpura (steroid preparations), sleepiness (antihistamines), renal failure ( Side effects such as immunosuppressants) are likely to occur. Among the drugs developed so far, there is no drug that can cure allergies, and symptoms improvement is expected, but there is a problem that side effects are large.

obesity

Obesity is a metabolic disease caused by an imbalance between intake and consumption of calories, and morphologically, it is caused by an increase in the size of fat cells in the body (hypertrophy) or an increase in the number (hyperplasia). Obesity is not only the most common nutritional disorder in Western society, but also in Korea recently, the importance of treatment and prevention has been greatly emphasized in the trend of rapidly increasing the frequency of obesity due to the improvement of diet and the westernization of lifestyle due to economic development. have. Obesity is an important factor that not only atrophys individuals psychologically, but also increases the risk of developing various adult diseases socially. Obesity is known to be directly related to an increase in the prevalence of various adult diseases such as type 2 diabetes, hypertension, hyperlipidemia, and heart disease (Cell 87:377, 1999), and obesity-related diseases are grouped together to form metabolic syndrome or insulin resistance. Syndrome (insulin resistance syndrome), these have been found to be the cause of arteriosclerosis and cardiovascular disease. The obesity treatment agents known to date include Xenical (Roche Pharmaceuticals, Switzerland), Reductil (Ebot, USA), and Exolise (Atopama, France), and are largely appetite suppressants, energy consumption accelerators, and fat absorption. Classified as an inhibitor, most obesity drugs are appetite suppressants that suppress appetite by regulating neurotransmitters related to the hypothalamus. However, conventional treatments have side effects such as heart disease, respiratory disease, and nervous system disease, as well as low persistence of their efficacy, and thus further improved obesity treatments need to be developed, and products currently being developed have satisfactory therapeutic effects without side effects. Since eggplant has few treatments, the development of new obesity treatments is required.

Probiotics ( probiotics ) And lactobacilli

In the gastrointestinal tract of animals including humans, living microorganisms that have a beneficial effect on the health of the host by improving the intestinal microbial environment of the host are collectively referred to as probiotics. In order to be effective as a probiotic, most of them should have excellent acid resistance, bile resistance, and intestinal epithelial cell adhesion when taken orally. Lactobacillus is used as probiotics because it plays a role in making it an important nutrient by decomposing fiber and complex proteins while coexisting in the human digestive system. Lactobacillus has been reported to exhibit effects such as maintenance of the intestinal normal flora, improvement of the intestinal flora, antidiabetic and antihyperlipidemia effects, suppression of carcinogenesis, suppression of colitis, and non-specific activity of the host's immune system. Among them, Lactobacillus strain is a major member of the normal microbial community living in the human intestine, and has long been known to be important in maintaining a healthy digestive system and vaginal environment. guidelines), currently all Lactobacillus strains deposited with the US Strains Depository (ATCC) are classified as'Bio-safty Level 1', which is recognized as having no known potential risk of causing disease to humans or animals. have. On the other hand, kimchi lactic acid bacteria are lactic acid bacteria involved in kimchi fermentation and have been reported to have immunity enhancement, anti-microbial, antioxidant, anti-cancer effect, anti-obesity effect, anti-hypertension or constipation prevention effect [Hivak P, Odrska J, Ferencik M, Ebringer L, Jahnova E, Mikes Z.: One-year application of Probiotic strain Enterococcus facium M-74 decreases Serum cholesterol levels. : Bratisl lek Listy 2005; 106(2); 67-72; Agerholm-Larsen L. Bell ML. Grunwald GK. Astrup A.: The effect of a probiotic milk product on plasma cholesterol: a metaanalysis of short-term intervention studies; Eur J Clin Nutr. 2000; 54(11) 856-860; Renato Sousa, Jaroslava Helper, Jian Zhang, Strephen J Lewis and Wani O Li; Effect of Lactobacillus acidophilus supernants on body weight and leptin expression in rats; BMC complementary and alternative medicine. 2008; 8(5)1-8]. As various physiological activities of these lactic acid bacteria are known, research has been actively conducted to develop lactic acid bacteria strains that are safe for the human body and have excellent functions and apply them as pharmaceuticals or functional foods.

For example, Korean Patent Publication No. 10-1486999 discloses Lactobacillus plantarum CJLP133 KCTC 11403BP, which is useful for preventing and treating intestinal diseases and immune diseases. In addition, Korean Patent Publication No. 10-1500974 discloses Lactobacillus plantarum (Lactobacillus plantarum, which has an effect of improving metabolic diseases selected from the group consisting of anti-inflammatory and obesity, diabetes, hyperlipidemia, high cholesterol, arteriosclerosis, fatty liver and cardiovascular disease). Lactobacillus plantarum ) HAC01 strain (accession number: KCTC 12647BP) is disclosed. In addition, European Patent Publication No. 01941892 discloses a composition for treating allergies comprising Lactobacillus acidophilus PM-A0002 (accession number: M 207038) as an active ingredient. In addition, Korean Laid-Open Patent Publication No. 10-2005-0057259 contains Lactobacillus fermentum variant VRI003 (accession number NM02/31074) as an active ingredient, and irritable bowel syndrome, inflammatory bowel syndrome. Compositions for use in treating gastrointestinal diseases such as inflammatory bowel disease are disclosed. In addition, Japanese Patent Publication No. 5273695 discloses Bifidobacterium longum OLB6001 (Bifidobacterium longum OLB6001, accession number: FERM P-13610), Bifidobacterium longum OLB6290 ( Bifidobacterium longum OLB6290, accession number: NITE P-75) is disclosed for the prevention or treatment of allergies comprising a) as an active ingredient. In addition, US Patent Publication No. 20060177424 discloses a method of treating allergic diseases, irritable bowel syndrome, and the like by administering Streptococcus faecalis.

However, technology related to lactic acid bacteria that can comprehensively improve or treat inflammatory diseases, colitis, arthritis, allergic diseases, atopy, obesity, etc., which are increasing incidence in modern people through normalization of abnormalities of immune regulation and control disturbance of the intestinal flora, has not emerged. There is a need for screening of new strains with various functionalities and efficacy at the level of commercialization, and development of pharmaceuticals and functional foods through the same.

The present invention is derived from the conventional technical background, one object of the present invention is the effect of immunomodulating, enhancing immunity, inhibiting inflammatory response, improving colitis, arthritis, anti-obesity or anti-allergic effect, etc. It is to provide novel lactic acid bacteria having various physiological activities or functions necessary for the control of disturbances and normalization of immune regulation abnormalities. In addition, an object of the present invention is to provide a novel lactic acid bacteria having an anti-stress effect, an anti-anxiety effect, an anti-depressant effect or an anti-schizophrenia effect. In addition, another object of the present invention is to provide a variety of food and pharmaceutical uses of the novel lactic acid bacteria.

The inventors of the present invention screen a myriad of lactic acid bacteria from human feces, and among them, a specific Lactobacillus strain, a specific Bifidobacterium strain, a specific Streptococcus strain or a specific Cholin cella strain has excellent immunomodulatory efficacy, immunity enhancing efficacy, It was confirmed that it has anti-allergic effect, anti-inflammatory effect, colitis improvement effect, arthritis improvement effect, or anti-obesity effect, and the present invention was completed.

In order to achieve one object of the present invention, an example of the present invention is Lactobacillus plantarum (Lactobacillus plantarum) IM2, Lactobacillus acidophilus (Lactobacillus acidophilus) IM7, Lactobacillus fermentum (Lactobacillus fermentum) IM12 (consigned Number: KCCM 11806P), Bifidobacterium longum ( Bifidobacterium longum ) IM26, Bifidobacterium adolescentes ( Bifidobacterium adolescentis ) IM38 (Accession No.: KCCM 11807P), Streptococcus faecium IM45 and Collinsella aerofaciens IM50 As a lactic acid bacteria selected from the group consisting of, immunomodulatory efficacy, immune enhancing efficacy, anti-allergic It provides lactic acid bacteria having efficacy, anti-inflammatory effect, colitis improvement effect, arthritis improvement effect, anti-obesity effect, anti-stress effect, anti-anxiety effect, anti-depressant effect or anti-schizophrenia effect.

In order to achieve another object of the present invention, an example of the present invention includes Lactobacillus plantarum IM2, a culture thereof, a lysate or an extract thereof as an active ingredient, and prevents and improves obesity or colitis Or it provides a composition for use for treatment. In addition, an example of the present invention is Lactobacillus acidophilus (Lactobacillus acidophilus) IM7 or Bifidobacterium longum ( Bifidobacterium longum ) It provides a composition for use for preventing, improving or treating allergic diseases, comprising as an active ingredient a lactic acid bacterium selected from IM26, a culture thereof, a lysate or an extract thereof. In addition, an example of the present invention includes Lactobacillus fermentum IM12 (accession number: KCCM 11806P), a culture thereof, a lysate or an extract thereof as an active ingredient, and allergic diseases, colitis, arthritis or obesity It provides a composition for use in preventing, improving or treating. In addition, an example of the present invention provides a composition for immunomodulatory or anti-inflammatory comprising Lactobacillus fermentum IM12 (accession number: KCCM 11806P), a culture thereof, a lysate thereof, or an extract thereof as an active ingredient. . In addition, an example of the present invention includes Bifidobacterium adolescentis IM38 (accession number: KCCM 11807P), its culture, its lysate or its extract as an active ingredient, allergic diseases, colitis, arthritis Or it provides a composition for use in preventing, ameliorating or treating obesity. In addition, an example of the present invention is an immunomodulatory or anti-inflammatory composition comprising Bifidobacterium adolescentis IM38 (accession number: KCCM 11807P), its culture, its lysate or its extract as an active ingredient Provides. In addition, an example of the present invention provides a composition for stress relief comprising Bifidobacterium adolescentis IM38 (accession number: KCCM 11807P), a culture thereof, a lysate thereof, or an extract thereof as an active ingredient. . In addition, an example of the present invention is Bifidobacterium adolesentis ( Bifidobacterium adolescentis ) IM38 (Accession No.: KCCM 11807P), a culture thereof, a lysate thereof, or an extract thereof as an active ingredient, and provides a composition for use in preventing, improving or treating anxiety disorders, depression or schizophrenia. In addition, an example of the present invention is one or more selected from Bifidobacterium adolescentis IM38 (accession number: KCCM 11807P), a culture thereof, a lysate thereof, or an extract thereof; And bicuculine or N-[2-[4-(2-Methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinylcyclohexanecarboxamide hydrochloride (WAY-100635 hydrochloride; CAS registration number: 146714-97-8) It provides a composition for stress relief comprising at least one selected from as an active ingredient. In addition, an example of the present invention is one or more selected from Bifidobacterium adolescentis IM38 (accession number: KCCM 11807P), a culture thereof, a lysate thereof, or an extract thereof; And bicuculine or N-[2-[4-(2-Methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinylcyclohexanecarboxamide hydrochloride (WAY-100635 hydrochloride; CAS registration number: 146714-97-8) It contains one or more selected from as an active ingredient, and provides a composition for use in preventing, improving or treating anxiety disorders, depression or schizophrenia. In addition, an example of the present invention provides a composition for immunomodulatory or immunity enhancement comprising Streptococcus faecium IM45, a culture thereof, a lysate thereof, or an extract thereof as an active ingredient. In addition, an example of the present invention provides a composition for anti-inflammatory comprising as an active ingredient a Collinsella aerofaciens IM50, a culture thereof, a lysate thereof or an extract thereof.

Specific Lactobacillus strains, specific Bifidobacterium strains, specific Streptococcus strains, or specific Cholinsella strains according to the present invention are isolated from human feces and have high safety and immunomodulatory efficacy, immunity enhancing efficacy, and anti-allergic efficacy. , Anti-inflammatory effect, colitis improvement effect, arthritis improvement effect, anti-obesity effect, anti-stress effect, anti-anxiety effect, anti-depressant effect or anti-schizophrenia effect. A specific Lactobacillus strain according to the present invention, a specific Bifidobacterium strain, a specific Streptococcus strain, or a specific Cholin Cella strain may be used as a material for immune regulation, immunity enhancement, inflammatory response suppression, stress relief, It can also be used as a functional dietary material useful for preventing, improving or treating allergic diseases, inflammatory diseases, colitis, arthritis, obesity, anxiety disorders, depression or schizophrenia.

1 shows the amount of weight gain when lactic acid bacteria was administered to a mouse in which immunity was decreased by γ-radiation, and FIG. 2 is a spleen weight when lactic acid bacteria was administered to a mouse in which immunity was decreased by γ-radiation. 3 shows the amount of IFN-γ in the serum when lactic acid bacteria are administered to a mouse in which immunity is reduced by γ-radiation, and FIG. 4 is a diagram illustrating the decrease in immunity induced by γ-radiation. It shows the amount of IL-2 in the serum when lactic acid bacteria were administered to the mouse, and FIG. 5 shows the amount of TNF-α in the serum when lactic acid bacteria were administered to mice whose immunity was induced by γ-radiation. .
Figure 6 shows the cytotoxicity of NK cells in the spleen extracted after administering lactic acid bacteria to a mouse in which immunity is reduced by γ-radiation, and Figure 7 is a mouse in which immunity is reduced by γ-radiation It shows the cytotoxicity of cytotoxic T cells in the spleen extracted after administration of lactic acid bacteria to cancer cells.
Figure 8 shows the effect of a predetermined lactic acid bacteria on a model animal in which arthritis is induced by a collagen immunogen as an increase in the volume of the foot or myeloperoxidase (MPO) activity, and Figure 9 is an arthritis induced by a collagen immunogen. The effect of a predetermined lactic acid bacteria on a model animal is shown as an inflammation-related cytokine, and FIG. 10 shows the effect of a predetermined lactic acid bacteria on a model animal in which arthritis is induced by a collagen immunogen as an inflammatory response index material.
11 shows the effect of a predetermined lactic acid bacteria on the obesity-inducing model animal in terms of weight change, and FIG. 12 shows the effect of the predetermined lactic acid bacteria on the obesity-inducing model animal, and the appearance score of the large intestine and myeloperoxidase (Myeloperoxidase, MPO) activity, and FIG. 13 shows the effect of certain lactic acid bacteria on an obesity-inducing model animal as an inflammation-related cytokine.
14 is a Bifidobacterium adolescentis (Bifidobacterium) for a model animal in which anxiety symptoms were induced by binding stress. adolescentis ) The effect of IM38 was expressed as the ratio of the time spent in the open arm and the number of times the mouse entered the open arm. In addition, FIG. 15 shows Bifidobacterium adolesentis (Bifidobacterium) for model animals in which anxiety symptoms were induced by binding stress. adolescentis ) The effect of IM38 is expressed by the content of corticosterone, interleukin-6 (IL-6), and TNF-α (tumor necrosis factor-alpha).
16 is a Bifidobacterium adolescentis in a model animal in which anxiety symptoms were induced by binding stress (Bifidobacterium adolescentis ) The effect of flumagenil, bicuculine, or WAY-100635 on the anti-anxiety activity of IM38 was expressed as the ratio of the time spent in the open arm and the number of times the mouse entered the open arm.
17 is a Bifidobacterium adolescentis in a model animal in which anxiety symptoms were induced by binding stress (Bifidobacterium adolescentis ) The effects of flumagenil, bicuculine or WAY-100635 on the anti-anxiety activity of IM38 were affected by corticosterone, interleukin-6, IL-6, and TNF. It is expressed as the content of -α (tumor necrosis factor-alpha).
18 is a Bifidobacterium adolescentis (Bifidobacterium) for anxiety behaviors of model animals not subjected to binding stress. adolescentis ) IM38 or Bifidobacterium adolescentis (Bifidobacterium adolescentis) IM38 and flumagenil (Flumazenil) the effect of the combined use of the ratio of the time to stay in the open arm and the number of times the mouse enters the open arm.
19 is a Bifidobacterium adolescentis (Bifidobacterium) for anxiety behaviors of model animals without binding stress. adolescentis ) IM38 or Bifidobacterium adolescentis ) The effect of combined use of IM38 and flumagenil was expressed as the content of corticosterone, interleukin-6, IL-6, and TNF-α (tumor necrosis factor-alpha). will be.

Hereinafter, terms used in the present invention will be described.

The term "culture" used in the present invention means a product obtained by culturing microorganisms in a known liquid medium or solid medium, and is a concept including microorganisms.

In the present invention, the terms "pharmaceutically acceptable" and "food pharmaceutically acceptable" mean not significantly irritating the organism and not inhibiting the biological activity and properties of the administered active substance.

The term "prevention" as used in the present invention refers to any action that suppresses or delays the progression of a symptom of a specific disease by administration of the composition of the present invention.

The term "treatment" used in the present invention refers to any action that improves or beneficially alters the symptoms of a specific disease by administration of the composition of the present invention.

As used herein, the term "improvement" refers to any action that at least reduces or alleviates the severity of a parameter related to the condition being treated, such as a symptom.

As used herein, the term "administering" means providing a given composition of the present invention to a subject by any suitable method. At this time, the individual refers to all animals, such as humans, monkeys, dogs, goats, pigs, mice, etc., having diseases in which symptoms of a specific disease can be improved by administering the composition of the present invention.

In the present invention, the term "pharmaceutically effective amount" refers to an amount sufficient to treat a disease at a reasonable benefit or risk ratio applicable to medical treatment, which is the type, severity, activity of the drug, and Sensitivity, time of administration, route of administration and rate of excretion, duration of treatment, factors including drugs used concurrently, and other factors well known in the medical field.

Hereinafter, the present invention will be described in detail.

One aspect of the present invention relates to a novel lactic acid bacteria having various physiological activities.

New lactic acid bacteria according to an embodiment of the present invention are Lactobacillus plantarum IM2, Lactobacillus acidophilus IM7, Lactobacillus fermentum IM12 (accession number: KCCM 11806P), ronggeom Bifidobacterium (Bifidobacterium longum) IM26, Bifidobacterium Adolfo Les sentiseu (Bifidobacterium adolescentis ) IM38 (Accession No.: KCCM 11807P), Streptococcus faecium IM45 and Collinsella aerofaciens IM50 , selected from the group consisting of immunomodulatory efficacy, immune enhancing efficacy, anti-allergic efficacy, It has anti-inflammatory effect, colitis improvement effect, arthritis improvement effect, anti-obesity effect, anti-stress effect, anti-anxiety effect, anti-depressant effect or anti-schizophrenia effect.

The Lactobacillus plantarum IM2 is preferably D-ribose, D-galactose, D-glucose, D-fructose, D-mannose, mannitol, sorbitol, amygdaline, arbutin, esculin as a carbon source. ), Salicin, Cellobiose, Maltose, Lactose, Sucrose, Trehalose, Melezitose and D-Turanose are used. The Lactobacillus acidophilus IM7 is preferably D-galactose, D-glucose, D-fructose, D-mannose, N-acetyl-glucosamine, amigdaline, arbutin, esculin as a carbon source. , Salicin, Cellobiose, Maltose, Lactose, Sucrose, Trehalose, Raffinose, and Genthiobiose are used. The Lactobacillus fermentum IM12 (accession number: KCCM 11806P) is preferably 16S rDNA and contains the nucleotide sequence set forth in SEQ ID NO: 1, and more preferably, esculin is used. The Bifidobacterium longum IM26 is preferably L-arabinose, D-galactose, D-glucose, D-fructose, mannitol, sorbitol, esculin, maltose, lactose, as a carbon source. Melibiose, Melezitos, raffinose and Genthiobios are used. The Bifidobacterium adolescentis IM38 (accession number: KCCM 11807P) preferably contains the nucleotide sequence set forth in SEQ ID NO: 2 as 16S rDNA, more preferably D-xylose, D as a carbon source. -Galactose, D-glucose, D-fructose, D-mannose, maltose and Melibose are used. The Streptococcus faecium IM45 is preferably 16S rDNA containing the nucleotide sequence shown in SEQ ID NO: 3, more preferably as a carbon source D-ribose, D-fructose, dulcitol, sorbitol , Amigdaline, mellegitose, starch, glycogen, sodium pyruvate, hippuric acid, and esculin ferric citrate. The Collinsella aerofaciens IM50 is preferably D-galactose, D-glucose, D-fructose, D-mannose, N-acetyl-glucosamine, maltose, lactose, sucrose and D- as a carbon source. Use tagatose.

Another aspect of the present invention relates to various veterinary uses of the novel lactic acid bacteria. For the use of new lactic acid bacteria, an example of the present invention includes Lactobacillus plantarum IM2, a culture thereof, a lysate thereof or an extract thereof as an active ingredient, and for preventing, improving or treating obesity or colitis Provides a composition for use. In addition, an example of the present invention is Lactobacillus acidophilus (Lactobacillus acidophilus) IM7 or Bifidobacterium longum ( Bifidobacterium longum ) It provides a composition for use for preventing, improving or treating allergic diseases, comprising as an active ingredient a lactic acid bacterium selected from IM26, a culture thereof, a lysate or an extract thereof. In addition, an example of the present invention includes Lactobacillus fermentum IM12 (accession number: KCCM 11806P), a culture thereof, a lysate or an extract thereof as an active ingredient, and allergic diseases, colitis, arthritis or obesity It provides a composition for use in preventing, improving or treating. In addition, an example of the present invention provides a composition for immunomodulatory or anti-inflammatory comprising Lactobacillus fermentum IM12 (accession number: KCCM 11806P), a culture thereof, a lysate thereof, or an extract thereof as an active ingredient. . In addition, an example of the present invention includes Bifidobacterium adolescentis IM38 (accession number: KCCM 11807P), its culture, its lysate or its extract as an active ingredient, allergic diseases, colitis, arthritis Or it provides a composition for use in preventing, ameliorating or treating obesity. In addition, an example of the present invention is an immunomodulatory or anti-inflammatory composition comprising Bifidobacterium adolescentis IM38 (accession number: KCCM 11807P), its culture, its lysate or its extract as an active ingredient Provides. In addition, an example of the present invention provides a composition for stress relief comprising Bifidobacterium adolescentis IM38 (accession number: KCCM 11807P), a culture thereof, a lysate thereof, or an extract thereof as an active ingredient. . In addition, an example of the present invention is Bifidobacterium adolesentis ( Bifidobacterium adolescentis ) IM38 (Accession No.: KCCM 11807P), a culture thereof, a lysate thereof, or an extract thereof as an active ingredient, and provides a composition for use in preventing, improving or treating anxiety disorders, depression or schizophrenia. In addition, an example of the present invention is one or more selected from Bifidobacterium adolescentis IM38 (accession number: KCCM 11807P), a culture thereof, a lysate thereof, or an extract thereof; And bicuculine or N-[2-[4-(2-Methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinylcyclohexanecarboxamide hydrochloride (WAY-100635 hydrochloride; CAS registration number: 146714-97-8) It provides a composition for stress relief comprising at least one selected from as an active ingredient. In addition, an example of the present invention is one or more selected from Bifidobacterium adolescentis IM38 (accession number: KCCM 11807P), a culture thereof, a lysate thereof, or an extract thereof; And bicuculine or N-[2-[4-(2-Methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinylcyclohexanecarboxamide hydrochloride (WAY-100635 hydrochloride; CAS registration number: 146714-97-8) It contains one or more selected from as an active ingredient, and provides a composition for use in preventing, improving or treating anxiety disorders, depression or schizophrenia. In addition, an example of the present invention provides a composition for immunomodulatory or immunity enhancement comprising Streptococcus faecium IM45, a culture thereof, a lysate thereof, or an extract thereof as an active ingredient. In addition, an example of the present invention provides a composition for anti-inflammatory comprising as an active ingredient a Collinsella aerofaciens IM50, a culture thereof, a lysate thereof or an extract thereof. In the composition of the present invention, the Lactobacillus plantarum IM2, Lactobacillus acidophilus IM7, Lactobacillus fermentum ) IM12 (accession number: KCCM 11806P), Bifidobacterium Technical features of Bifidobacterium longum IM26, Bifidobacterium adolescentis IM38 (accession number: KCCM 11807P), Streptococcus faecium IM45 and Collinsella aerofaciens IM50 Since is the same as described above, a description thereof will be omitted.

The type of allergic disease is not limited as long as it is a disease caused by an excessive immune response in a living body, and is preferably selected from atopic dermatitis, asthma, pharyngitis, or chronic dermatitis. In addition, the colitis refers to a state in which inflammation has occurred in the large intestine due to bacterial infection or pathological fermentation of intestinal contents, and is a concept including infectious colitis and non-infectious colitis. Specific examples of colitis include inflammatory bowel disease or irritable colitis syndrome. In addition, inflammatory bowel disease includes ulcerative colitis, or Crohn's disease. In addition, the arthritis includes rheumatoid arthritis. In addition, the anti-inflammatory composition of the present invention may be used for suppressing an inflammatory reaction or preventing, improving, or treating an inflammatory disease. The type of the inflammatory disease is not limited as long as it is a disease caused by an inflammatory reaction, and includes both inflammatory diseases caused by bacterial infection and inflammatory diseases caused by viral infection.

In the present invention, the culture of lactic acid bacteria is a product obtained by culturing a predetermined strain in a medium, and the medium may be selected from a known liquid medium or solid medium, for example, MRS liquid medium, MRS agar medium, BL agar. It may be a medium.

In the present invention, the composition may be embodied as a pharmaceutical composition, a food additive, a food composition (especially a functional food composition) or a feed additive, depending on the purpose or aspect of use. In addition, the content of the active ingredient, such as lactic acid bacteria, may also be adjusted in various ranges according to the specific form of the composition and the purpose or aspect of use.

In the pharmaceutical composition according to the present invention, the content of the new lactic acid bacteria, cultures thereof, lysates thereof, or extracts thereof, which are active ingredients, is not limited, for example, 0.01 to 99% by weight, preferably 0.5 to, based on the total weight of the composition. It may be 50% by weight, more preferably 1 to 30% by weight. In addition, the pharmaceutical composition according to the present invention may further include additives such as a pharmaceutically acceptable carrier, excipient, or diluent in addition to the active ingredient. Carriers, excipients and diluents that may be included in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate. , Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oils. In addition, the pharmaceutical composition of the present invention is a novel lactic acid bacteria, its culture, its lysate or its extract, in addition to the immunomodulatory effect, immunity enhancing effect, anti-inflammatory effect, anti-stress effect, allergic disease, inflammatory disease, colitis, arthritis, obesity, anxiety It may further contain one or more known active ingredients having a preventive or therapeutic effect of disorder, depression or schizophrenia. The pharmaceutical composition of the present invention can be formulated into a dosage form for oral administration or a dosage form for parenteral administration by a conventional method, and when formulated, such as fillers, extenders, binders, wetting agents, disintegrants, surfactants, etc. It can be formulated using diluents or excipients. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and these solid preparations include at least one excipient in the active ingredient, such as starch, calcium carbonate, and sucrose. ), lactose, or gelatin. In addition, in addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, liquid solutions, emulsions, and syrups.In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as humectants, sweeteners, fragrances, and preservatives may be included. have. Formulations for parenteral administration may include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations, and suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used. Further, it can be preferably formulated according to each disease or component by an appropriate method in the art or by using a method disclosed in Remington's Pharmaceutical Science (latest edition), Mack Publishing Company, Easton PA. The pharmaceutical composition of the present invention may be orally or parenterally administered to mammals including humans according to a desired method, and parenteral administration methods include skin external use, intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, muscle Intra-chest injection or intrathoracic injection. The dosage of the pharmaceutical composition of the present invention is not largely limited as long as it is a pharmaceutically effective amount, and the range is according to the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and disease severity. Varies. A typical daily dosage of the pharmaceutical composition of the present invention is not limited, but is preferably 0.1 to 3000 mg/kg, more preferably 1 to 2000 mg/kg, once a day based on the active ingredient. Alternatively, it may be administered in several divided doses.

In addition, in the food composition according to the present invention, the content of new lactic acid bacteria, cultures thereof, lysates or extracts thereof, which are active ingredients in the food composition according to the present invention, is 0.01 to 99% by weight, preferably 0.1 to 50% by weight, more preferably based on the total weight of the composition. It is 0.5 to 25% by weight, but is not limited thereto. The food composition of the present invention includes the form of pills, powders, granules, needles, tablets, capsules, or liquids, and examples of specific foods include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, Others include noodles, gums, dairy products including ice cream, various soups, beverages, tea, functional water, drinks, alcoholic beverages and vitamin complexes, and all health foods in the usual sense are included. In addition to the active ingredient, the food composition of the present invention may contain a food pharmaceutically acceptable carrier, various flavoring agents, or natural carbohydrates as additional ingredients. In addition, the food composition of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols. , Carbonating agents used in carbonated beverages, and the like may be contained. In addition, the food composition of the present invention may contain pulp for the production of natural fruit juice, fruit juice beverage, and vegetable beverage. These ingredients may be used independently or in combination. The natural carbohydrates described above are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As the flavoring agent, natural flavoring agents such as taumatin and stevia extract, or synthetic flavoring agents such as saccharin and aspartame may be used.

Hereinafter, the present invention will be described in more detail through examples. However, the following examples are only intended to clearly illustrate the technical features of the present invention, and do not limit the protection scope of the present invention.

1. Isolation and identification of lactic acid bacteria

(1) Isolation of lactic acid bacteria from human feces

Human feces were placed in a GAM liquid medium (GAM broth; Nissui Pharmaceutical, Japan) and suspended. Thereafter, the supernatant was taken and transplanted into BL agar medium (Nissui Pharmaceutical, Japan), and after anaerobic culture at 37° C. for about 48 hours, a colony-forming Lactobacillus sp. ), Bifidobacterium sp. strain, Streptococcus sp., and Collinsella sp. were isolated.

(2) Identification of the selected lactic acid bacteria

The physiological characteristics and 16S rDNA sequence of the strains isolated from human feces were analyzed to determine the species of the strain, and the strain name was given. Table 1 below shows the management number and strain name of lactic acid bacteria isolated from human feces.

Control Number Strain name One Lactobacillus plantarum IM2 2 Lactobacillus acidophilus IM5 3 Lactobacillus acidophilus IM6 4 Lactobacillus acidophilus IM7 5 Lactobacillus fermentum IM12 6 Lactobacillus fermentum IM15 7 Lactobacillus johnsonii IM18 8 Lactobacillus johnsonii IM20 9 Bifidobacterium longum IM23 10 Bifidobacterium longum IM26 11 Bifidobacterium longum IM27 12 Bifidobacterium infantis IM30 13 Bifidobacterium infantis IM32 14 Bifidobacterium breve IM33 15 Bifidobacterium breve IM36 16 Bifidobacterium adolescentis IM38 17 Bifidobacterium adolescentis IM39 18 Bifidobacterium adolescentis IM42 19 Streptococcus faecium IM45 20 Streptococcus faecium IM46 21 Streptococcus faecium IM49 22 Collinsella aerofaciens IM50

Among the strains listed in Table 1, 16S rDNA was measured as a chemical taxonomic property of Lactobacillus fermentum IM12, and as a result, it was found to have the nucleotide sequence of SEQ ID NO: 1. Lactobacillus fermentum (Lactobacillus fermentum) As a result of identifying the 16S rDNA sequence of IM12 by BLAST search of Genebank (http://www.ncbi.nlm.nih.gov/), Lactobacillus fermentum having the same 16S rDNA sequence. Mentum ( Lactobacillus fermentum ) strain was not detected, Lactobacillus fermentum (Lactobacillus fermentum) strain showed 99% homology with the 16S rDNA sequence of CIP 102980.

In addition, 16S rDNA was measured as a chemical taxonomic characteristic of Bifidobacterium adolescentis IM38 among the strains described in Table 1, and as a result, it was found to have the nucleotide sequence of SEQ ID NO: 2. As a result of identifying the 16S rDNA sequence of Bifidobacterium adolescentis IM38 by BLAST search of Genebank (http://www.ncbi.nlm.nih.gov/), it has the same 16S rDNA sequence. Bifidobacterium adolescentis strain was not detected, and Bifidobacterium adolescentis strain showed 99% homology with the 16S rDNA sequence of BBMN23.

In addition, 16S rDNA was measured by the chemical taxonomic properties of Streptococcus faecium IM45 among the strains described in Table 1, and as a result, it was found to have the nucleotide sequence of SEQ ID NO: 3. The 16S rDNA sequence of Streptococcus faecium IM45 was identified by BLAST search of Genebank (http://www.ncbi.nlm.nih.gov/). As a result, Enterococcus faecium (Enterococcus) faecium ) showed 99% homology with the 16S rDNA sequence of strain Aus0004.

In addition, Lactobacillus Planta Rum (Lactobacillus plantarum) IM2, Lactobacillus also know pillar's (Lactobacillus acidophilus) IM7, Lactobacillus momentum spread (Lactobacillus fermentum) IM12, ronggeom Bifidobacterium (Bifidobacterium longum ) IM26, Bifidobacterium adolescentes ( Bifidobacterium adolescentis ) IM38, Streptococcus faecium (Streptococcus faecium) IM45 and Collinsella aerofaciens (Collinsella aerofaciens) Among the physiological properties of IM50, the carbon source availability was determined by API Kit (model name: API 50 CHL; manufacturer: BioMerieux's, USA). It was analyzed by fermentation test. Table 2 below shows the carbon source availability results of Lactobacillus plantarum IM2, Table 3 below shows the carbon source availability results of Lactobacillus acidophilus IM7, and Table 4 below shows the results of the carbon source availability of Lactobacillus acidophilus IM7. Bacillus fermentum (Lactobacillus fermentum) shows the carbon source utilization results of IM12, Table 5 below shows the carbon source utilization results of Bifidobacterium longum (Bifidobacterium longum) IM26, Table 6 below shows Bifidobacterium adolescence ( Bifidobacterium adolescentis ) shows the carbon source availability results of IM38, Table 7 below shows the carbon source availability results of Streptococcus faecium IM45, Table 8 below shows the carbon source availability of Collinsella aerofaciens IM50 It shows the results. In Tables 2 to 8 below, "+" indicates a case where carbon source availability is positive, "-" indicates a case where carbon source availability is negative, and "±" indicates a case where the carbon source availability is ambiguous. Table 2 to Lactobacillus Planta column (Lactobacillus plantarum), as shown in Table 8 IM2, Lactobacillus know also the filler's (Lactobacillus acidophilus) IM7, Lactobacillus buffer momentum (Lactobacillus fermentum) IM12, Bifidobacterium ronggeom (Bifidobacterium longum ) IM26, Bifidobacterium adolescentis IM38, Streptococcus faecium IM45, Collinsella aerofaciens IM50 are different from strains of the same species for some carbon sources. Showed. In particular, in the case of Streptococcus faecium IM45, mannitol was not used as a carbon source, and glycogen and starch were simultaneously used as a carbon source. Common Enterococcus Paesium (Enterococcus faecium ) strains are known to use mannitol as a carbon source and cannot use glycogen and starch as a carbon source at the same time.

Carbon source Strain name Carbon source Strain name Lactobacillus plantarum IM2 Lactobacillus plantarum IM2 glycerol - cellobiose + erythritol - maltose + D-arabinose - lactose + L-arabinose - melibiose - D-ribose + sucrose + D-xylose - trehalose + L-xylose - inulin - D-adonitol - melezitose + methyl-β-D-xylopyranoside - raffinose - D-galactose + starch - D-glucose + glycogen - D-fructose + xylitol - D-mannose + gentiobiose ± L-sorbose - D-turanose + L-rhamnose - D-lyxose - dulcitol - D-tagatose - inositol - D-fucose - mannitol + L-fucose - sorbitol + D-arabitol - α-methyl-D-mannoside ± L-arabitol - α-methly-D-glucoside - glucoNaTe ± N-acetyl-glucosamine + 2-keto-gluconate - amygdalin + 5-keto-gluconate - arbutin + sodium pyruvate esculin + hippuric acid salicin + esculin ferric citrate

Carbon source Strain name Carbon source Strain name Lactobacillus acidophilus IM7 Lactobacillus acidophilus IM7 glycerol - cellobiose + erythritol - maltose + D-arabinose - lactose + L-arabinose - melibiose - D-ribose - sucrose + D-xylose - trehalose + L-xylose - inulin - D-adonitol - melezitose - methyl-β-D-xylopyranoside - raffinose + D-galactose + starch - D-glucose + glycogen - D-fructose + xylitol - D-mannose + gentiobiose + L-sorbose - D-turanose ± L-rhamnose - D-lyxose - dulcitol - D-tagatose - inositol - D-fucose - mannitol - L-fucose - sorbitol - D-arabitol - α-methyl-D-mannoside - L-arabitol - α-methly-D-glucoside - glucoNaTe - N-acetyl-glucosamine + 2-keto-gluconate - amygdalin + 5-keto-gluconate - arbutin + sodium pyruvate esculin + hippuric acid salicin + esculin ferric citrate

Carbon source Strain name Carbon source Strain name Lactobacillus fermentum IM12 Lactobacillus fermentum IM12 glycerol - cellobiose - erythritol - maltose - D-arabinose - lactose - L-arabinose ± melibiose ± D-ribose ± sucrose ± D-xylose ± trehalose - L-xylose - inulin ± D-adonitol - melezitose - methyl-β-D-xylopyranoside - raffinose - D-galactose ± starch - D-glucose ± glycogen ± D-fructose ± xylitol - D-mannose ± gentiobiose - L-sorbose - D-turanose - L-rhamnose - D-lyxose - dulcitol - D-tagatose - inositol - D-fucose - mannitol - L-fucose - sorbitol - D-arabitol - α-methyl-D-mannoside - L-arabitol ± α-methly-D-glucoside ± glucoNaTe - N-acetyl-glucosamine - 2-keto-gluconate - amygdalin ± 5-keto-gluconate - arbutin - sodium pyruvate esculin + hippuric acid salicin - esculin ferric citrate

Carbon source Strain name Carbon source Strain name Bifidobacterium longum IM26 Bifidobacterium longum IM26 glycerol - cellobiose - erythritol - maltose + D-arabinose - lactose + L-arabinose + melibiose + D-ribose - sucrose ± D-xylose - trehalose ± L-xylose - inulin - D-adonitol - melezitose + methyl-β-D-xylopyranoside - raffinose + D-galactose + starch - D-glucose + glycogen - D-fructose + xylitol - D-mannose - gentiobiose + L-sorbose - D-turanose ± L-rhamnose - D-lyxose - dulcitol - D-tagatose - inositol - D-fucose - mannitol + L-fucose - sorbitol + D-arabitol - α-methyl-D-mannoside - L-arabitol - α-methly-D-glucoside - glucoNaTe - N-acetyl-glucosamine - 2-keto-gluconate - amygdalin - 5-keto-gluconate - arbutin - sodium pyruvate esculin + hippuric acid salicin - esculin ferric citrate

Carbon source Strain name Carbon source Strain name Bifidobacterium adolescentis IM38 Bifidobacterium adolescentis IM38 glycerol - cellobiose - erythritol - maltose + D-arabinose - lactose - L-arabinose - melibiose + D-ribose ± sucrose - D-xylose + trehalose - L-xylose - inulin - D-adonitol - melezitose - methyl-β-D-xylopyranoside - raffinose - D-galactose + starch - D-glucose + glycogen - D-fructose + xylitol - D-mannose + gentiobiose - L-sorbose - D-turanose - L-rhamnose - D-lyxose - dulcitol - D-tagatose - inositol - D-fucose - mannitol - L-fucose - sorbitol - D-arabitol - α-methyl-D-mannoside - L-arabitol - α-methly-D-glucoside - glucoNaTe ± N-acetyl-glucosamine - 2-keto-gluconate - amygdalin - 5-keto-gluconate - arbutin - sodium pyruvate esculin - hippuric acid salicin - esculin ferric citrate

Carbon source Strain name Carbon source Strain name Streptococcus faecium IM45 Streptococcus faecium IM45 glycerol cellobiose erythritol maltose D-arabinose lactose L-arabinose melibiose D-ribose + sucrose D-xylose trehalose L-xylose inulin D-adonitol melezitose + methyl-β-D-xylopyranoside raffinose D-galactose starch + D-glucose glycogen + D-fructose + xylitol D-mannose gentiobiose L-sorbose D-turanose L-rhamnose D-lyxose dulcitol + D-tagatose inositol D-fucose mannitol - L-fucose sorbitol + D-arabitol α-methyl-D-mannoside L-arabitol α-methly-D-glucoside glucoNaTe N-acetyl-glucosamine 2-keto-gluconate amygdalin + 5-keto-gluconate arbutin sodium pyruvate + esculin hippuric acid + salicin esculin ferric citrate +

Carbon source Strain name Carbon source Strain name Collinsella aerofaciens IM50 Collinsella aerofaciens IM50 glycerol - cellobiose - erythritol - maltose + D-arabinose - lactose + L-arabinose - melibiose - D-ribose - sucrose + D-xylose - trehalose - L-xylose - inulin - D-adonitol - melezitose - methyl-β-D-xylopyranoside - raffinose - D-galactose + starch - D-glucose + glycogen - D-fructose + xylitol - D-mannose + gentiobiose - L-sorbose - D-turanose - L-rhamnose - D-lyxose - dulcitol - D-tagatose + inositol - D-fucose - mannitol - L-fucose - sorbitol - D-arabitol - α-methyl-D-mannoside - L-arabitol - α-methly-D-glucoside - glucoNaTe - N-acetyl-glucosamine + 2-keto-gluconate - amygdalin - 5-keto-gluconate - arbutin - sodium pyruvate esculin - hippuric acid salicin - esculin ferric citrate

(3) Consignment information of lactic acid bacteria

The inventors of the present invention deposited a patent on Lactobacillus fermentum IM12 to the Korea Microbial Conservation Center (address: 45 Yurim Building, Hongjenae 2-ga-gil, Seodaemun-gu, Seoul, Korea) on January 20, 2016. You have been assigned an accession number. In addition, the inventors of the present invention patented Bifidobacterium adolescentis IM38 on January 20, 2016 to the Korea Microbial Conservation Center (Address: 45 Yurim Building, Hongje-nae 2-ga-gil, Seodaemun-gu, Seoul, Korea) on January 20, 2016. It was deposited and assigned the accession number of KCCM 11807P. The deposit of the lactic acid bacteria was made in compliance with the Budapest Treaty on the International Recognition of the Deposit of Microorganism for the Purposes of Patent Procedure.

2. Evaluation of the immunomodulatory efficacy of lactic acid bacteria

To evaluate the immunomodulatory efficacy of lactic acid bacteria isolated from feces, the effect of lactic acid bacteria on the immune response of macrophages and the effect of lactic acid bacteria on the immune response of splenocytes were measured.

2-1. Macrophage immune response experiment

(1) Isolation of macrophages

Six-week-old C57BL/6J male mice (20-23g) were purchased from Raun Bio. 2 ml of sterilized 4% thioglycolate was administered to the intraperitoneal cavity of the mice, and after 96 hours, the mice were anesthetized, and 8 ml of RPMI 1640 medium was again administered to the intraperitoneal cavity of the mice. (Including phagocytes) was extracted again, centrifuged for 10 minutes at 1000 rpm, and washed twice with RPMI 1640 medium. Macrophages were spread on a 24-well plate in ^{a number of 0.5×10 6} per well, and after incubation for 24 hours, non-adherent cells were removed and used.

(2) Lactobacillus enhances macrophage immune response

Lactic acid bacteria were added to the macrophage culture solution of (1) in ^{an amount of 1×10 4} CFU/ml and cultured for 24 hours, and then a supernatant was obtained. The expression level of TNF-α from the obtained supernatant was measured with an ELISA kit, and the results are shown in Table 9 below. In the following tables, "IM2" represents Lactobacillus plantarum IM2, "IM5" represents Lactobacillus acidophilus IM5, and "IM6" represents Lactobacillus acidophilus ( Lactobacillus acidophilus ) IM6, "IM7" represents Lactobacillus acidophilus IM7, "IM12" represents Lactobacillus fermentum ) IM12, and "IM15" represents Lactobacillus permanent ( Lactobacillus fermentum ) IM15, "IM18" is Lactobacillus johnsonii ) IM18, "IM20" is Lactobacillus johnsonii ) IM20, and "IM23" is Bifidobacterium longum ( Bifidobacterium longum ) IM23, "IM26" refers to Bifidobacterium longum (IM26), "IM27" refers to Bifidobacterium longum (Bifidobacterium longum) IM27, and "IM30" refers to Bifidobacterium Infantis "IM32" denotes the Bifidobacterium Infante Tees (Bifidobacterium infantis) IM32, "IM33 " denotes the Bifidobacterium breather bracket (Bifidobacterium breve) IM33, "IM36 " denotes the (Bifidobacterium infantis) IM30, the BP gaming Bifidobacterium breve IM36, "IM38" refers to Bifidobacterium adolescentis IM38, and "IM39" refers to Bifidobacterium adolescentis (Bifidobacterium adole scentis ) refers to IM39, and "IM42" refers to Bifidobacterium adolesentis ( Bifidobacterium adolescentis ) IM42, “IM45” indicates Streptococcus faecium IM45, “IM46” indicates Streptococcus faecium IM46, and “IM49” indicates Streptococcus faecium Represents IM49, and "IM50" represents Collinsella aerofaciens IM50.

Lactobacillus treated with macrophages TNF-α expression increase rate No treatment - IM2 + IM5 + IM6 + IM7 + IM12 + IM15 ++ IM18 + IM20 + IM23 + IM26 - IM27 - IM30 - IM32 - IM33 + IM36 - IM38 + IM39 + IM42 + IM45 +++ IM46 ++ IM49 ++ IM50 ++

* Growth rate: -, <10%; +, 10-50%; ++, 50-100%; +++, >100%

(3) Lactobacillus inhibiting macrophage inflammatory response

The macrophage culture solution of (1) was treated with lactic acid bacteria as a test substance and lipopolysaccharide (LPS) as an inflammatory reaction inducing substance for 2 hours or 24 hours, and then a supernatant and cells were obtained. At this time, the concentration of lactic acid bacteria treatment was 1×10 ⁴ CFU/ml. The obtained cells were placed in a buffer (Gibco) and homogenized. The expression level of TNF-α from the obtained supernatant was measured with an ELISA kit. In addition, the expression levels of p65 (NF-kappa B), p-p65 (phosphor-NF-kappa B) and β-actin from the obtained cells were measured by immunoblotting. Specifically, 50µg of the supernatant was taken and electrophoresed for 1 hour and 30 minutes on SDS 10% (w/v) polyacrylamide gel. The electrophoresed sample was transferred to nitrocellulose paper for 1 hour and 10 minutes under the conditions of 100 V and 400 mA. After blocking the nitrocellulose paper from which the sample was transferred with 5% skim milk for 30 minutes, washing with PBS-Tween three times for 5 minutes each, and the primary antibody (Santa Cruz Biotechnology, USA) at a ratio of 1:100 And reacted overnight. Thereafter, it was washed three times for 10 minutes each, and the secondary antibody (Santa Cruz Biotechnology, USA) was reacted for 1 hour and 20 minutes at a ratio of 1:1000. Thereafter, it was washed three times for 15 minutes each, developed after being fluorescently colored, and the intensity of the colored band was measured, and the results are shown in Table 10 below.

Lactobacillus treated with macrophages NF-κB activation inhibition rate (p-p65/p65) TNF-α expression inhibition rate No treatment - - IM2 ++ ++ IM5 ++ ++ IM6 ++ ++ IM7 +++ +++ IM12 +++ +++ IM15 + + IM18 + ++ IM20 + + IM23 + + IM26 ++ ++ IM27 + + IM30 + ++ IM32 ++ ++ IM33 ++ ++ IM36 ++ ++ IM38 +++ +++ IM39 + + IM42 ++ + IM45 - - IM46 + - IM49 - - IM50 +++ +++

* Inhibition rate: -, <10%; +, 10-30%; ++, 30-60%; +++, >60%

2-2. Splenocyte immune response experiment

The spleen of C56BL/6J mice was isolated, appropriately crushed, suspended in RPMI 1640 exclusion containing 10% FCS, and CD4 T cells were isolated using a CD4 T cell isolation kit (MiltenyiBiotec, Bergisch Gladbach, Germany). The isolated CD4 T cells were dispensed into a 12-well plate at 5×10 ⁵ counts per well. Here, anti-CD3, anti-CD28, IL-2, and IL-12 are used to induce differentiation of T cells into Th1 cells, and anti-CD3, anti-CD28, and anti-CD3, anti-CD28, to induce differentiation of T cells into Th2 cells. IL-2 and IL-4 were used to induce differentiation of T cells into Th17 cells, anti-CD3, anti-CD28, IL-6 and TGF-β were used to induce differentiation of T cells into Treg cells. CD3 and anti-CD28 were added and the cells were cultured while lactic acid bacteria were added in an amount of ^{1×10 5 CFU/ml per well and cultured for 4 days.}

Thereafter, the differentiation ability of T cells isolated from the spleen into Th1 cells, Th2 cells, Th17 cells and Treg cells was measured. Specifically, the cells of the culture medium were stained with anti-FoxP3 or anti-IL-17A antibody, and Th1 cells, Th2 were stained using a fluorescence-activated cell sorting (FACS) device (C6 Flow Cytometer® System, San Jose, CA, USA). The distribution of cells, Th17 cells and Treg cells were analyzed, and the results are shown in Table 11 below. In Table 11 below, lactic acid bacteria having a high rate of inhibition of differentiation of T cells into Th1 cells, Th2 cells, and Th17 cells and a high rate of increase in differentiation of T cells into Treg cells can effectively inhibit inflammatory reactions and effectively improve inflammatory diseases. .

Lactobacillus treated with spleen T cells Differentiation inhibition rate Differentiation increase rate Th1 cells Th2 cells Th17 cells Treg cells No treatment - - - - IM2 + ++ + + IM5 + ++ + + IM6 + ++ ++ + IM7 +++ + +++ +++ IM12 +++ + +++ +++ IM15 + + +++ + IM18 + + +++ ++ IM20 + + +++ + IM23 + + +++ + IM26 + +++ ++ ++ IM27 ++ ++ + + IM30 - + + - IM32 + ++ ++ + IM33 ++ + + ++ IM36 ++ + + + IM38 +++ +++ +++ + IM39 + ++ + + IM42 + ++ ++ + IM45 + + ++ + IM46 ++ ++ + + IM49 + ++ + + IM50 +++ + +++ ++

* Inhibition rate: -, <10%; +, 10-30%; ++, 30-60%; +++, >60%

* Growth rate: -, <10%; +, 10-50%; ++, 50-100%; +++, >100%

In addition, transcription factors and cytokine expression rates of Th1 cells, Th2 cells, Th17 cells, and Treg cells differentiated from splenic T cells were measured. Specifically, using qRT-PCR, T-bet, IFN-γ and IL-12 from Th1 cell differentiation induction culture medium, GATA3 and IL-5 from Th2 cell differentiation induction culture medium, RORγt and IL from Th17 cell differentiation induction culture medium For -17, the expression levels of Foxp3 and IL-10 were analyzed from the culture medium for inducing Treg cell differentiation, and the results are shown in Table 12 below.

Lactobacillus treated with spleen T cells Expression inhibition rate Expression increase rate T-bet IFN-γ GATA3 IL-5 RORγt IL-17 FOXp3 IL-10 No treatment - - - - - - - - IM2 ++ + + + + + - + IM5 + + + + + + + + IM6 + + + + + + + + IM7 ++ ++ + + +++ +++ ++ ++ IM12 +++ +++ + + + + ++ ++ IM15 - - + + + + + + IM18 + + + + + + + + IM20 + + ++ ++ + + + + IM23 + + + + ++ ++ + + IM26 + + +++ +++ ++ ++ ++ ++ IM27 + + + + + +++ ++ + IM30 + + + + + ++ + ++ IM32 + + + + + + + + IM33 ++ + + + + + + + IM36 + + + + ++ ++ ++ ++ IM38 +++ +++ + + +++ +++ +++ +++ IM39 + + + + + + + + IM42 + ++ + + + + + + IM45 + ++ + + + + + + IM46 ++ + + + - - + + IM49 ++ + + + - - + + IM50 ++ ++ + + +++ +++ + +

* Inhibition rate: -, <10%; +, 10-30%; ++, 30-60%; +++, >60%

* Growth rate: -, <10%; +, 10-50%; ++, 50-100%; +++, >100%

2-3. Lactobacillus immune enhancing activity measurement experiment

(1) Administration of experimental animals and experimental drugs

Immunity-enhancing activity of lactic acid bacteria was measured using mice whose immunity was induced by γ-radiation. Specifically, 5-week-old male C57BL/6 mice were supplied from Raon Bio Co., Ltd. and raised and acclimatized for 7 days at the Korea Hydro & Nuclear Power Research Institute. Then, one group was divided into 8 mice, and the mice were divided into 6 experimental groups. Thereafter, mice of 5 groups excluding the normal group were treated with γ-radiation at an intensity of 2.5 Gy/2 min. Thereafter, the experimental drug, Lactobacillus, ^{was administered orally at a dose of 2 × 10 9} CFU/mouse or the positive control drug, Levamisole hydrochloride, at a dose of 2.5 mg/kg, for 6 days every day from the next day of radiation treatment. In addition, only physiological saline was orally administered to Masu, which is a normal group and a negative control group. In addition, all experimental groups were allowed to freely eat food and water.

(2) Measurement of body weight, spleen weight, and blood cytokines of experimental animals

The weight of the mouse was measured before administration of the test drug, lactic acid bacteria, and after oral administration for 6 days, and after sacrifice, the spleen was separated and the weight of the spleen was measured. In addition, blood was isolated, and the amounts of IFN-γ, IL-2, TNF-α, and IL-10 were measured using an ELISA Kit.

1 shows the amount of weight gain when lactic acid bacteria was administered to a mouse in which immunity was decreased by γ-radiation, and FIG. 2 is a spleen weight when lactic acid bacteria was administered to a mouse in which immunity was decreased by γ-radiation. 3 shows the amount of IFN-γ in the serum when lactic acid bacteria are administered to a mouse in which immunity is reduced by γ-radiation, and FIG. 4 is a diagram illustrating the decrease in immunity induced by γ-radiation. It shows the amount of IL-2 in the serum when lactic acid bacteria were administered to the mouse, and FIG. 5 shows the amount of TNF-α in the serum when lactic acid bacteria were administered to mice whose immunity was induced by γ-radiation. . 1 to 5, "NOR" represents a normal group, "CON" represents a negative control group, and "IM45" represents a Streptococcus faecium IM45 administration group. As shown in FIGS. 1 to 5, Streptococcus faecium IM45 improved the weight loss of mice in which immunity was induced by γ-radiation, such as IFN-γ, IL-2, and TNF-α. The expression level of immune cytokines was increased, and its effect was superior to that of Levamisole hydrochloride, a commercial immune booster.

(3) Measurement of cytotoxicity on YAC-1 cells of NK cells and Cytotoxic T cells isolated from the spleen of experimental animals

After oral administration of the test drug for 6 days, the mice were sacrificed, the stomach was sterilized with a 70% ethanol aqueous solution, and the spleen was aseptically removed. Thereafter, NK cells were isolated from splenocytes using NK cell Isolation Kit (MACS), and Cytotoxic T cells were isolated using CD8+ T cell Isolation Kit (MACS), and the number of each cell was measured with a flow cytometer. In addition, NK cells or Cytotoxic T cells isolated from spleen cells and YAC-1 lymphoma cells (cancer cells) were cultured together to measure the cytotoxicity of NK cells and Cytotoxic T cells to cancer cells. Specifically, YAC-1 cells labeled with fluorescence using the Vybrant CFDA SE Cell Tracer kit were dispensed into a 96-well plate at 0.4×10 ⁵ cells per well, and NK cells and Cytotoxic T cells isolated thereto were 2× per well. After dispensing with the number of 10 ⁵ cells, the cells were cultured for 24 hours, and the number of YAC-1 cells was measured with a flow cytometer.

Figure 6 shows the cytotoxicity of NK cells in the spleen extracted after administering lactic acid bacteria to a mouse in which immunity is reduced by γ-radiation, and Figure 7 is a mouse in which immunity is reduced by γ-radiation It shows the cytotoxicity of cytotoxic T cells in the spleen extracted after administration of lactic acid bacteria to cancer cells. In FIGS. 6 and 7, "NOR" represents a normal group, "CON" represents a negative control group, and "IM45" represents a Streptococcus faecium IM45 administration group. As shown in FIGS. 6 and 7, when Streptococcus faecium IM45 was administered to a mouse in which immunity was induced by γ-radiation, the cancer cell killing effect of NK cells and Cytotoxic T cells in the spleen was strongly increased. .

3. Lactobacillus allergy Evaluation of improvement efficacy

(1) Measurement of inhibition rate of degranulation of lactic acid bacteria

RBL-2H3 cell line (rat mast cell line, Korea Cell Line Bank, Cat.No.22256) was prepared with DMEM (Dulbeccos' modified Eagle's medium, Sigma, 22256) containing 10% FBS (fetal bovine serum) and L-glutamine. Using a 37 ℃, humidified (humidified) 5% CO ₂ was cultured in an incubator. Cells contained in the culture medium were suspended using a trypsin-EDTA solution, separated and recovered, and used in the experiment. The recovered RBL-2H3 cells were dispensed into a 24-well plate in ^{an amount of 5×10 5} cells per well, and then 0.5 μg/ml of mouse monoclonal IgE was added and incubated for 12 hours to sensitize. The sensitized cells were washed with 0.5 ml of siraganian buffer (119mM NaCl, 5mM KCl, 0.4mM MgCl ₂ , 25mM PIPES, 40mM NaOH, pH 7.2), and then 0.16 ml of siraganian buffer (5.6 mM glucose, 1mM CaCl ₂ , 0.1% BSA was added) and incubated at 37°C for 10 minutes. Thereafter, the test drug, lactic acid bacteria, was added to the cell culture ^{solution at a concentration of 1×10 4} CFU/ml, or 0.04 ml of DSCG (disodium cromoglycate) was added to the cell culture solution, and 0.02 ml of antigen (DNP-BSA) was added after 20 minutes. 1 μg/ml) was activated at 37° C. for 10 minutes. Thereafter, the cell culture solution was centrifuged at 2000 rpm for 10 minutes to obtain a supernatant. 0.025 ml of the obtained supernatant was transferred to a 96-well plate, and 1 mM p-NAG as a substrate solution (a solution in which p-nitrophenyl-N-acetyl-β-D-glucosamined was dissolved at pH 4.5 in 0.1 M citrate buffer) After adding 0.025 ml, the mixture was reacted at 37°C for 60 minutes. Thereafter, _{0.2 ml of 0.1M Na 2} CO ₃ /NaHCO ₃ was added to the reaction solution to stop the reaction, and absorbance was measured with an ELISA analyzer at 405 nm.

(2) Measurement of inhibition rate of pruritus reaction of lactic acid bacteria

5 BALB/c mice were used as a group, and the test drug, lactic acid bacteria, ^{was orally administered once a day in an amount of 1×10 9} CFU for 3 days to the rest of the experimental groups excluding the normal group and the control group, or DSCG (disodium cromoglycate) as a control drug. Alternatively, Azelastine was orally administered once at a time of 0.2 mg/mouse for 3 days. After 1 hour after the last oral administration of the drug, the mouse was left in an observation box (24cm×22cm×24cm) for 10 minutes to acclimate to the environment, and then the hairs on the back of the head (the back of the neck) were removed. Then, normal group mice were injected with physiological saline, and other mice were injected with a pruritus inducing agent (50 µg of compound 48/80; Sigma, USA) using a 29 gauge needle. Thereafter, the mice were immediately quarantined in an observation box and recorded for 1 hour with an 8-mm video camera (SV-K80, Samsung) under unmanned conditions to observe the pruritus behavior. For the soyang action, the act of scratching the injection site with the hind paw was recognized, and other parts were not recognized.

(3) Experiment result

Table 13 below shows the results of measuring the degranulation inhibition rate and the pruritus reaction inhibition rate of lactic acid bacteria. As shown in Table 13 below, the lactic acid bacteria selected in the present invention strongly inhibited the degranulation and pruritus reaction of basophils, and thus atopy, asthma, sore throat or chronic dermatitis caused by allergies can be very effectively improved.

Treatment medication Inhibition rate (%) Degranulation Pruritus reaction none 0 2 IM7 43 42 IM12 47 46 IM26 49 52 IM38 54 49 IM45 48 32 IM50 40 38 DSCG (disodium cromoglycate) 65 25 Azelastine - 68

4. Evaluation of the efficacy of Lactobacillus to improve colitis (In vivo)

(1) Experimental animals

5-week-old C57BL/6 male mice (24-27g) were purchased from Orient Bio Co., Ltd., and humidity was 50±10%, temperature was 25±2℃, and the lighting was turned on for 12 hours and then turned off for 12 hours. It was reared for a while and used for the experiment. As feed, standard experimental feed (Samyang, Korea) was used, and drinking water was freely ingested. In all experiments, one group consisted of 6 animals.

(2) Induction of colitis and sample administration by TNBS

One of the experimental animals was a normal group, and 2,4,6-trinitrobenzenesulfonic acid (TNBS) was used to induce acute colitis in the other experimental animals. Specifically, after lightly anesthetizing the experimental animal with ether, a solution of 2.5 g of a TNBS (2,4,6-Trinitrobenzene sulfonic acid) solution mixed with 100 ml of 50% ethanol was used to enter the anus using a 1 ml syringe with a round tip. Through the intestine, 0.1 ㎖ each was administered and held vertically for 30 seconds to induce inflammation. On the other hand, 0.1 ml of physiological saline was orally administered to the normal group. Thereafter, the test sample, lactic acid bacteria, was suspended in physiological saline for 3 days once a day from the next day ^{and administered orally in an amount of 1.5×10 9} CFU. The day after the administration of the sample was completed, the experimental animal was killed by suffocation with carbon dioxide. The large intestine up to the area just before the anus was excised and used. In addition, only physiological saline instead of lactic acid bacteria was orally administered to the experimental animals of the normal group. In addition, the experimental animals of the negative control group were also orally administered only physiological saline instead of lactic acid bacteria after colitis induced by TNBS. In addition, sulfasalazine, a colitis treatment drug, was orally administered to the experimental animals of the positive control group in an amount of 50 mg/kg instead of lactic acid bacteria.

(3) Analysis of the appearance of the large intestine

The length and appearance of the extracted large intestine were observed, and the appearance was analyzed by scoring according to the criteria in Table 14 (Hollenbach et al. In the colon tissue, all the contents of the colon were removed, washed with physiological saline, and some were fixed with 4% formaldehyde fixative for use as a sample for pathological tissue, and the rest were frozen and stored at -80°C for molecular biological analysis. I did.

Macroscopic Score standard 0 No ulcers and inflammation found One Congestion is found without bleeding 2 Ulcers with congestion are found 3 Ulcers and sores found in only one place 4 Ulcers and inflammation found in more than one place 5 The ulcer is enlarged to more than 2cm

(4) Myeloperoxidase (MPO) activity measurement

200 µl of a 10 mM potassium phosphate buffer (pH 7.0) containing 0.5% hexadecyl trimethyl ammonium bromide was added to 100 mg of colon tissue and homogenized. Thereafter, centrifugation was performed at 4° C. and 10,000×g for 10 minutes to obtain a supernatant. 50 µl of the supernatant was added to 0.95 ㎖ of the reaction solution (containing 1.6mM tetramethyl benzidine and 0.1mM H ₂ O ₂ ), and the absorbance was measured over time at 650 nm while reacting at 37°C. Myeloperoxidase (MPO) activity was calculated as 1 unit of 1 μmol/ml of peroxide generated as a reactant.

(5) Measurement of inflammatory indicator substances

A supernatant was obtained in the same manner as in the experiment for measuring myeloperoxidase (MPO) activity. Thereafter, inflammation-related cytokines such as TNF-α and IL-17 were measured using an ELISA kit from the supernatant.

(6) Experiment result

Table 15 shows that when lactic acid bacteria were administered to model animals in which acute colitis was induced by TNBS, the amount of lactic acid bacteria gained weight, length of the large intestine, appearance score of the large intestine, myeloperoxidase (MPO) activity, and inflammation-related cytokines It shows the effect on the content change. As shown in Table 15 below, Lactobacillus acidophilus IM7, Lactobacillus fermentum IM12, Bifidobacterium adolecentis ( Bifidobacterium) adolescentis ) IM38 was more effective in improving colitis than sulfasalazine, a commercial colitis treatment.

Experimental group Weight gain(g) Colon length(cm) Macroscopic score MPO activity
(μU/mg) TNF-α
(pg/mg) IL-17
(pg/mg) Normal 0.6 5.5 0.14 0.36 22 9 Negative control -2.5 4.3 2.32 1.79 185 52 IM7 administration group -1.3 4.6 1.3 1.32 85 42 IM12 administration group -0.3 4.8 1.08 0.78 68 38 IM38 administration group 0.2 5.0 0.5 0.48 59 28 Positive control -0.54 4.7 1.43 0.98 75 42

5. Evaluation of the efficacy of lactic acid bacteria to improve arthritis (In vivo )

(1) Experiment method

To evaluate the arthritis improvement efficacy of lactobacilli, a total of 42 8-week-old male DBA/1J mice were divided into 6 groups (NOR, CIA, IM7, IM12, IM38, IB) of 7 mice, and the rest of the military CIA except the normal group, NOR group. In group, IM7 group, IM12 group, IM38 group, and IB group, bobbin type II collagen was used as an immunogen to induce arthritis. Specifically, 100 µg of collagen immunogen was injected intradermally to the proximal part of the mouse's tail for primary immunization, and on the 21st day after the first immunization, the same amount of collagen immunogen was intradermally injected in the same manner to perform secondary immunization. At this time, as a collagen immunogen, bobbin type II collagen was dissolved in 0.05M acetic acid and then emulsified by adding the same amount of Freund Complete Adjuvant was used.

From the day following the second immunization, vehicle (50mM sodium bicarbonate buffer containing 1% glucose) was orally administered daily for 20 days to the NOR group and the CIA group, and 2×10 ⁹ CFU/ The mice were orally administered daily for 20 days, and the IB group was orally administered with ibupropen at a dose of 50 mg/kg for 20 days, and the experiment was terminated the next day of the final administration.

(2) Analysis method

Arthritis severity and arthritis incidence of the foot were measured from the stage of inducing arthritis. The arthritis severity of the foot was evaluated by visually observing the foot and the periphery of the foot and calculating a macroscopic score in stages according to the criteria in Table 16 (Arii et al., 2008) below. The highest macroscopic appearance score that can be assigned to each mouse is 16.

step Appearance degree 0 normal One focal slight swelling and/or redness in one digit 2 moderate swelling and erythema 3 marked swelling and erythema of the limb 4 maximal swelling, erythema, deformity, and/or ankylosis

In addition, after measuring the increase in the volume of the feet of the experimental animals on the end of the experiment, the experimental animals were sacrificed and the paw joint tissue was collected and immediately frozen at -70°C and used as a sample. Myeloperoxidase (MPO) activity in the joint tissue of the foot was measured by the same method as the method used to evaluate the efficacy of improving colitis. In addition, the supernatant was obtained from the foot joint tissue using the same method as the myeloperoxidase (MPO) activity measurement experiment, and an inflammatory response index substance such as NO, PGE2 and TNF-α, IL-17 were used using an ELISA kit. Inflammation related cytokines were measured.

(3) Experiment result

Figure 8 shows the effect of a predetermined lactic acid bacteria on a model animal in which arthritis is induced by a collagen immunogen as an increase in the volume of the foot or myeloperoxidase (MPO) activity, and Figure 9 is an arthritis induced by a collagen immunogen. The effect of a predetermined lactic acid bacteria on a model animal is shown as an inflammation-related cytokine, and FIG. 10 shows the effect of a predetermined lactic acid bacteria on a model animal in which arthritis is induced by a collagen immunogen as an inflammatory response index material. As shown in FIGS. 8 to 10, Lactobacillus fermentum IM12 effectively improved arthritis.

6. Obesity improvement and anti-inflammatory effect evaluation of lactobacillus (In vivo )

(1) Experiment method

C57BL6/J mice were purchased from Raun Bio, and a total of 45 mice were acclimated for 1 week on a chow diet (Purina) under the conditions of a temperature of 20±2° C., humidity 50±10%, and a 12 hr light/12 hr dark cycle. Thereafter, the experimental animals were divided into 5 groups of 9 animals each (LFD, HFD, HFD+IM2, HFD+IM12, HFD+IM38), and the LFD group had a normal diet for 4 weeks (LFD, 10% of calories from fat; Research, NJ. , USA) and the HFD group, HFD+IM2 group, HFD+IM12 group, and HFD+IM38 group were supplied with a high fat diet (HFD, 60% of calories from; Research, NJ, USA) for 4 weeks. Then, the LFD group was supplied with a normal diet for 4 weeks and at the same time, orally administered PBS. In addition, a high-fat diet was supplied to the HFD group for 4 weeks and PBS was orally administered. In addition, HFD+IM2 group, HFD+IM12 group, and HFD+IM38 group were supplied with a high-fat diet for 4 weeks, and the lactic acid bacteria Lactobacillus plantarum IM2, Lactobacillus fermentum IM12 and Bifidobacterium adolesentis ( Bifidobacterium adolescentis ) IM38 was each suspended in PBS and administered orally in an amount of ^{2×10 9 CFU.}

(2) Analysis of anti-obesity and anti-inflammatory effects of lactic acid bacteria

The anti-obesity effect of Yoo-gyu was analyzed through weight change. In addition, the anti-inflammatory effect of lactic acid bacteria was analyzed using the same method as measured in a model animal experiment in which acute colitis was induced by TNBS.

(3) Experiment result

11 shows the effect of a predetermined lactic acid bacteria on the obesity-inducing model animal in terms of weight change, and FIG. 12 shows the effect of the predetermined lactic acid bacteria on the obesity-inducing model animal, and the appearance score of the large intestine and myeloperoxidase (Myeloperoxidase, MPO) activity, and FIG. 13 shows the effect of certain lactic acid bacteria on an obesity-inducing model animal as an inflammation-related cytokine. 11 to 13, Lactobacillus plantarum IM2, Lactobacillus fermentum IM12 and Bifidobacterium adolescentis IM38 are obesity due to a high fat diet. Increased body weight, increased colitis index, and increased myeloperoxidase (MPO) activity in induced model animals were greatly reduced, and the occurrence of colitis was suppressed. In addition, the lactic acid bacteria significantly inhibited the production of inflammatory cytokines and increased the production of IL-10, an anti-inflammatory cytokine, in model animals in which obesity was induced by a high fat diet.

7. Bifidobacterium Adolecentis ( Bifidobacterium adolescentis A) IM38 term stress, Anti-anxiety , Antidepressant or Anti-schizophrenia Effectiveness evaluation

In modern society, diseases such as anxiety disorder, depression, and schizophrenia are rapidly increasing due to stress. For example, when a person or animal is subjected to stress such as immobilization or forced swimming, corticosterone, interleukin-6 (IL-6), and TNF-α( Cytokines such as tumor necrosis factor-alpha) increase, and as a result, symptoms such as anxiety, depression, and schizophrenia appear. In this study, the anti-stress, anti-anxiety, anti-depressant or anti-schizophrenia effect of Bifidobacterium adolescentis IM38 was conducted in experimental animals by inducing stress and using an elevated plus-maze test. Was measured.

(1) Experiment method

C57BL/6 mice (male, 19-22g) were acclimated for 1 week with a chow diet (Purina) under the conditions of a temperature of 20±2° C., humidity 50±10%, and a 12 hr light/12 hr dark cycle. Thereafter, the experimental animals were divided into several groups, and physiological saline was orally administered to the normal group once a day for 3 days, and the remaining experimental groups were administered with lactic acid bacteria once daily for 3 days, an anti-anxiety drug buspirone, or dexamethasone, a corticosteroid drug. (dexamethasone) was administered. Specifically, lactic acid bacteria were suspended in a predetermined amount in physiological saline and administered orally, and the anti-anxiety drug buspirone and the corticosteroid dexamethasone were administered by intraperitoneal injection.

In addition, binding stress was applied to mice in the experimental group, excluding the normal group, to induce anxiety symptoms or depressive symptoms. Specifically, 1 hour after administration of the drug, the mouse is placed in a 500 ml falcon tube with a rounded tip, and cotton is inserted in the left, right, top, bottom, and back of the falcon tube to prevent the mouse from moving. The falcon tube was set up to go and left for 2 hours.

In addition, if necessary, 30 minutes before the final administration of the drug and applying binding stress, flumagenil, an antagonist of the benzodiazepine receptor, Bicuculine, an antagonist of the GABAA receptor, or WAY-100635, an antagonist of the 5-HT1A receptor ( N-[2-[4-(2-Methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinylcyclohexanecarboxamide hydrochloride; CAS registration number: 146714-97-8) was administered intraperitoneally.

Two hours after the final binding stress was applied to the mice, the anti-anxiety effect or antidepressant effect of the drug was measured using an elevated plus-maze device. The elevated plus-maze device consists of two open arms (30 cm long and 7 cm wide) facing each other among four aisles and two closed arms (30 cm long and 7 cm wide) surrounded by a wall with a height of 20 cm. 7cm), and each extends 7cm wide and 7cm long from the central platform. The labyrinth was made of black Plexiglas and was placed 50cm above the floor. A video camera was installed on the ceiling in the center of the maze to record the animal's behavior, and the brightness was adjusted to 20 lux. Mice naturally like to stay in the dark closed arm. So, when passing through the open arm, it shows a lot of anxiety. At the beginning of the experiment, the mouse was placed with its head facing the open arm in the middle and then allowed to freely navigate the maze. Thereafter, the behavior of the mouse was observed for 5 minutes, and the arm entry was assumed to contain all four feet. After each experiment, it was wiped clean with 70% alcohol to remove traces of all experimental animals so as not to affect the next experiment. The ratio of the time the mouse stayed in the open arm and the number of times the mouse entered the open arm were calculated by the following equation.

In the elevated plus-maze test, the decrease in anxiety was indicated by an increase in the percentage of time spent in the open arm and an increase in the percentage of number of entries into the open arm.

In addition, 1 hour after the elevated plus-maze test was completed, blood was collected from mice, and serum was separated, and then the cortisone was performed using an enzyme-linked immunosorbent assay (ELISA), an enzyme-linked immunospecific assay. The contents of corticosterone, interleukin-6, IL-6, and TNF-α (tumor necrosis factor-alpha) were measured.

(2) Experiment result

14 is a Bifidobacterium adolescentis (Bifidobacterium) for a model animal in which anxiety symptoms were induced by binding stress. adolescentis ) The effect of IM38 was expressed as the ratio of the time spent in the open arm and the number of times the mouse entered the open arm. In addition, FIG. 15 shows Bifidobacterium adolesentis (Bifidobacterium) for model animals in which anxiety symptoms were induced by binding stress. adolescentis ) The effect of IM38 is expressed by the content of corticosterone, interleukin-6 (IL-6), and TNF-α (tumor necrosis factor-alpha). In FIGS. 14 and 15, "NC" denotes a normal group in which only physiological saline was administered as a drug without applying binding stress, "IS" denotes an experimental group in which only physiological saline as a drug was administered with binding stress, and "SB" is It represents the experimental group in which buspirone was administered once daily for 3 days at a dose of 1 mg/kg mouse bw with binding stress, and "SD" represents dexamethasone as a drug along with binding stress for 3 days. It represents the experimental group administered once a day at a dose of 1 mg/kg mouse bw for a period of time, and "SIL" is a drug with binding stress and Bifidobacterium adolesentis (Bifidobacterium adolescentis ) Represents an experimental group in which IM38 was administered once daily for 3 days at a dose of 2×10 ⁸ CFU/mouse, and “SIM” is a drug with binding stress and Bifidobacterium adolescentis (Bifidobacterium adolescentis ) It represents the experimental group in which IM38 was administered once daily for 3 days at a dose of 1×10 ⁹ CFU/mouse, and "SIH" is a drug with binding stress and Bifidobacterium adolescentis IM38 3 Represents an experimental group administered once daily for a day at ^{a dose of 5×10 9 CFU/mouse.}

16 is a Bifidobacterium adolescentis in a model animal in which anxiety symptoms were induced by binding stress (Bifidobacterium adolescentis ) The effect of flumagenil, bicuculine, or WAY-100635 on the anti-anxiety activity of IM38 was expressed as the ratio of the time spent in the open arm and the number of times the mouse entered the open arm. In addition, Figure 17 is a model animal in which anxiety symptoms are induced by binding stress, Bifidobacterium adolescentis (Bifidobacterium adolescentis ) The effects of flumagenil, bicuculine or WAY-100635 on the anti-anxiety activity of IM38 were affected by corticosterone, interleukin-6, IL-6, and TNF. It is expressed as the content of -α (tumor necrosis factor-alpha). In FIGS. 16 and 17, "NC" represents a normal group in which only physiological saline was administered as a drug without applying binding stress, "IS" represents an experimental group in which only physiological saline was administered as a drug, and "SIM" is It represents the experimental group in which Bifidobacterium adolescentis IM38 was administered once daily for 3 days at a dose of 1×10 ⁹ CFU/mouse as a drug along with the binding stress, and "SIM+F" indicates the binding stress and Together with the drug, Bifidobacterium adolescentes ( Bifidobacterium adolescentis ) Represents an experimental group in which IM38 was administered once daily for 3 days at a dose of 1×10 ⁹ CFU/mouse, and flumagenil was administered at a dose of 3 mg/kg mouse bw after the final administration of lactic acid bacteria, "SIM+B" is a drug with binding stress. Bifidobacterium adolescentis IM38 is administered once daily for 3 days at ^{a dose of 1×10 9} CFU/mouse, and after the final administration of lactic acid bacteria, BQ kyulrin (Bicuculine) a represents a group administered at a dose of 0.5㎎ / ㎏ mouse bw, "SIM + W" is Te Solarium Adolfo bipyridinium gambling drug with straps stress LES sentiseu (Bifidobacterium adolescentis ) It represents an experimental group in which IM38 was administered once daily for 3 days at a dose of 1×10 ⁹ CFU/mouse, and WAY-100635 was administered at a dose of 0.5 mg/kg mouse bw after the final administration of lactic acid bacteria.

18 is a Bifidobacterium adolescentis (Bifidobacterium) for anxiety behaviors of model animals not subjected to binding stress. adolescentis ) IM38 or Bifidobacterium adolescentis (Bifidobacterium adolescentis) IM38 and flumagenil (Flumazenil) the effect of the combined use of the ratio of the time to stay in the open arm and the number of times the mouse enters the open arm. In addition, Figure 19 is a Bifidobacterium adolescentis (Bifidobacterium adolescentis) for the anxiety behavior of the model animal to which binding stress was not applied adolescentis ) IM38 or Bifidobacterium adolescentis ) The effect of combined use of IM38 and flumagenil was expressed as the content of corticosterone, interleukin-6, IL-6, and TNF-α (tumor necrosis factor-alpha). will be. In FIGS. 18 and 19, "NC" denotes a normal group to which only physiological saline was administered as a drug without applying binding stress, and "IL" denotes Bifidobacterium adolescence as a drug without applying binding stress. adolescentis ) It represents the experimental group in which IM38 was administered once daily for 3 days at a dose of 2×10 ⁸ CFU/mouse, and “IM” refers to Bifidobacterium adolescentis IM38 as a drug without applying binding stress. Represents the experimental group administered once daily for 3 days at a dose of 1×10 ⁹ CFU/mouse, and "IH" is a drug without binding stress, Bifidobacterium adolesentis (Bifidobacterium adolescentis ) Represents an experimental group in which IM38 was administered once daily for 3 days at a dose of 5×10 ⁹ CFU/mouse, and “IM+F” is a drug without binding stress and Bifidobacterium adolescentis It shows an experimental group in which IM38 was administered once daily for 3 days at a dose of 1×10 ⁹ CFU/mouse, and flumagenil was administered at a dose of 3 mg/kg mouse bw after the final administration of lactic acid bacteria.

As shown in FIGS. 15 to 19, Bifidobacterium adolescentis IM38 greatly alleviated anxiety symptoms or depressive symptoms in model animals regardless of the presence or absence of binding stress. In addition, Bifidobacterium adolescentis (Bifidobacterium adolescentis ) IM38 was administered and additionally, bicuculine, an antagonist of GABAA receptor, or WAY-100635, an antagonist of 5-HT1A receptor, (N-[2-[4-(2-Methoxyphenyl)-1-piperazinyl]ethyl]- When N-2-pyridinylcyclohexanecarboxamide hydrochloride; CAS registration number: 146714-97-8) was administered, the anti-anxiety effect or antidepressant effect was higher.

As described above, the present invention has been described through the above embodiments, but the present invention is not necessarily limited thereto, and various modifications may be possible without departing from the scope and spirit of the present invention. Accordingly, the scope of protection of the present invention should be construed as including all embodiments falling within the scope of the claims appended to the present invention.

Korea Microorganism Conservation Center (overseas) KCCM11806P 20160120 Korea Microorganism Conservation Center (overseas) KCCM11807P 20160120

<110> University-Industry Cooperation Group of Kyung Hee University <120> Novel lactic acid bacteria having immunoregulatory activities derived from human digestive tract and use thereof <130> ZDP-18-0125-D1 <150> KR 10-2016-0010761 <151> 2016-01-28 <160> 3 <170> KopatentIn 2.0 <210> 1 <211> 1442 <212> DNA <213> Unknown <220> <223> 16S rDNA of Lactobacillus fermentum IM12 <400> 1 gcaagtcgaa cgcgttggtc caattgattg atggtgcttg cacctgattg attttggtcg 60 ccaacgagtg gcggacgggt gagtaacacg taggtaacct gcccagaagc gggggacaac 120 atttggaaac agatgctaat accgcataac agcgttgttc gcatgaacaa cgcttaaaag 180 atggcttctc gctatcactt ctggatggac ctgcggtgca ttagcttgtt ggtggggtaa 240 cggcctacca aggcgatgat gcatagccga gttgagagac tgatcggcca caatgggact 300 gagacacggc ccatactcct acgggaggca gcagtaggga atcttccaca atgggcgcaa 360 gcctgatgga gcaacaccgc gtgagtgaag aagggtttcg gctcgtaaag ctctgttgtt 420 aaagaagaac acgtatgaga gtaactgttc atacgttgac ggtatttaac cagaaagtca 480 cggctaacta cgtgccagca gccgcggtaa tacgtaggtg gcaagcgtta tccggattta 540 ttgggcgtaa agagagtgca ggcggttttc taagtctgat gtgaaagcct tcggcttaac 600 cggagaagtg catcggaaac tggataactt gagtgcagaa gagggtagtg gaactccatg 660 tgtagcggtg gaatgcgtag atatatggaa gaacaccagt ggcgaaggcg gctacctggt 720 ctgcaactga cgctgagact cgaaagcatg ggtagcgaac aggattagat accctggtag 780 tccatgccgt aaacgatgag tgctaggtgt tggagggttt ccgcccttca gtgccggagc 840 taacgcatta agcactccgc ctggggagta cgaccgcaag gttgaaactc aaaggaattg 900 acgggggccc gcacaagcgg tggagcatgt ggtttaattc gaagctacgc gaagaacctt 960 accaggtctt gacatcttgc gccaacccta gagatagggc gtttccttcg ggaacgcaat 1020 gacaggtggt gcatggtcgt cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa 1080 cgagcgcaac ccttgttact agttgccagc attaagttgg gcactctagt gagactgccg 1140 gtgacaaacc ggaggaaggt ggggacgacg tcagatcatc atgcccctta tgacctgggc 1200 tacacacgtg ctacaatgga cggtacaacg agtcgcgaac tcgcgagggc aagcaaatct 1260 cttaaaaccg ttctcagttc ggactgcagg ctgcaactcg cctgcacgaa gtcggaatcg 1320 ctagtaatcg cggatcagca tgccgcggtg aatacgttcc cgggccttgt acacaccgcc 1380 cgtcacacca tgagagtttg taacacccaa agtcggtggg gtaaccttta ggagccagcc 1440 gc 1442 <210> 2 <211> 1358 <212> DNA <213> Unknown <220> <223> 16S rDNA of Bifidobacterium adolescentis IM38 <400> 2 aaggttgggc caccggcttc gggtgctacc cactttcatg acttgacggg cggtgtgtac 60 aaggcccggg aacgcattca ccgcggcgtt gctgatccgc gattactagc gactccgcct 120 tcatggagtc gggttgcaga ctccaatccg aactgagacc ggttttaagg gatccgctcc 180 acctcacagt gtcgcatccc gttgtaccgg ccattgtagc atgcgtgaag ccctggacgt 240 aaggggcatg atgatctgac gtcatcccca ccttcctccg agttgacccc ggcggtcccc 300 cgtgagttcc caccacgacg tgctggcaac acagggcgag ggttgcgctc gttgcgggac 360 ttaacccaac atctcacgac acgagctgac gacgaccatg caccacctgt gaacccgccc 420 cgaagggaga ccgtatctct acggctgtcg ggaacatgtc aagcccaggt aaggttcttc 480 gcgttgcatc gaattaatcc gcatgctccg ccgcttgtgc gggcccccgt caatttcttt 540 gagttttagc cttgcggccg tactccccag gcgggatgct taacgcgttg gctccgacac 600 ggagaccgtg gaatggtccc cacatccagc atccaccgtt tacggcgtgg actaccaggg 660 tatctaatcc tgttcgctcc ccacgctttc gctcctcagc gtcagtgacg gcccagagac 720 ctgccttcgc cattggtgtt cttcccgata tctacacatt ccaccgttac accgggaatt 780 ccagtctccc ctaccgcact caagcccgcc cgtacccggc gcggatccac cgttaagcga 840 tggactttca caccggacgc gacgaaccgc ctacgagccc tttacgccca ataattccgg 900 ataacgcttg caccctacgt attaccgcgg ctgctggcac gtagttagcc ggtgcttatt 960 cgaaaggtac actcaccccg aagggcttgc tcccagtcaa aagcggttta caacccgaag 1020 gccgtcatcc cgcacgcggc gtcgctgcat caggcttgcg cccattgtgc aatattcccc 1080 actgctgcct cccgtaggag tctgggccgt atctcagtcc caatgtggcc ggtcgccctc 1140 tcaggccggc tacccgtcga agccatggtg ggccgttacc ccgccatcaa gctgatagga 1200 cgcgacccca tcccataccg caaaatcttt cccagaggac catgcggtca actggagcat 1260 ccggcattac cacccgtttc caggagctat tccggtgtat ggggcaggtc ggtcacgcat 1320 tactcacccg ttcgccactc tcacccagga gcaagctc 1358 <210> 3 <211> 1001 <212> DNA <213> Unknown <220> <223> 16S rDNA of Streptococcus faecium IM45 <400> 3 cgcttctttt tccaccggag cttgctccac cggaaaaaga ggagtggcga acgggtgagt 60 aacacgtggg taacctgccc atcagaaggg gataacactt ggaaacaggt gctaataccg 120 tataacaatc gaaaccgcat ggttttgatt tgaaaggcgc tttcgggtgt cgctgatgga 180 tggacccgcg gtgcattagc tagttggtga ggtaacggct caccaaggcc acgatgcata 240 gccgacctga gagggtgatc ggccacattg ggactgagac acggcccaaa ctcctacggg 300 aggcagcagt agggaatctt cggcaatgga cgaaagtctg accgagcaac gccgcgtgag 360 tgaagaaggt tttcggatcg taaaactctg ttgttagaga agaacaagga tgagagtaac 420 tgttcatccc ttgacggtat ctaaccagaa agccacggct aactacgtgc cagcagccgc 480 ggtaatacgt aggtggcaag cgttgtccgg atttattggg cgtaaagcga gcgcaggcgg 540 tttcttaagt ctgatgtgaa agcccccggc tcaaccgggg agggtcattg gaaactggga 600 gacttgagtg cagaagagga gagtggaatt ccatgtgtag cggtgaaatg cgtagatata 660 tggaggaaca ccagtggcga aggcggctct ctggtctgta actgacgctg aggctcgaaa 720 gcgtggggag caaacaggat tagataccct ggtagtccac gccgtaaacg atgagtgcta 780 agtgttggag ggtttccgcc cttcagtgct gcagctaacg cattaagcac tccgcctggg 840 gagtacgacc gcaaggttga aactcaaagg aattgacggg ggcccgcaca agcggtggag 900 catgtggttt aattcgaagc aacgcgaaga accttaccag gtcttgacat cctttgacca 960 ctctagagat agagcttccc cttcgggggc aaagtgacag g 1001

Claims (4)

Lactobacillus fermentum IM12 (accession number: KCCM 11806P) having immunomodulating activity, immunostimulating activity, anti-allergic activity, anti-inflammatory activity, colitis improving activity, arthritis improving activity or anti-obesity activity.
Lactobacillus fermentum IM12 (accession number: KCCM 11806P), a culture thereof, a crushed product thereof or an extract thereof, as an active ingredient,
For the prevention, amelioration or treatment of allergic diseases, colitis, arthritis or obesity.
3. The composition of claim 2, wherein the allergic disease is selected from atopic dermatitis, asthma, sore throat or chronic dermatitis.
A composition for immunomodulatory or anti-inflammation comprising Lactobacillus fermentum IM12 (Accession No. KCCM 11806P), a culture thereof, a lysate thereof or an extract thereof as an active ingredient.

Images