WO2017047968A1

WO2017047968A1 - Novel lactobacillus having various functions, and use thereof

Info

Publication number: WO2017047968A1
Application number: PCT/KR2016/009994
Authority: WO
Inventors: 김동현; 한명주
Original assignee: 경희대학교 산학협력단
Priority date: 2015-09-15
Filing date: 2016-09-07
Publication date: 2017-03-23

Abstract

The present invention provides a novel Lactobacillus sp. strain, a novel Bifidobacterium sp. strain or a lactobacillus mixture thereof, which is isolated from kimchi or human feces. According to the present invention, the specific Lactobacillus sp. strain or the specific Bifidobacterium sp. strain is isolated from kimchi or human feces, thereby having high safety and various physiological activities such as an antioxidant activity, a β-glucuronidase inhibitory activity, a lipopolysaccharide (LPS) generation inhibitory activity or a tight junction protein expression-inducing activity. Therefore, according to the present invention, the specific Lactobacillus sp. strain, the specific Bifidobacterium sp. strain or a lactobacillus mixture thereof can be used as a functional food and drug material useful for preventing, alleviating or treating a bowel injury, a liver injury, allergies, inflammatory diseases or obesity.

Description

New lactic acid bacteria with various functionalities and uses thereof

The present invention relates to novel lactic acid bacteria, and more particularly, it is isolated from kimchi or human feces, antioxidant activity, beta-glucuronidase (β-glucuronidase) inhibitory activity, lipopolysaccharide (LPS) production inhibitory activity Or it relates to a novel lactic acid bacteria or a new mixed lactic acid bacteria having a variety of physiological activities such as tight junction protein expression inducing activity. The present invention also relates to various food and pharmaceutical uses of the novel lactic acid bacteria or new mixed lactic acid bacteria.

As mankind develops into an affluent society, lifestyles are rapidly westernized, and the pattern of disease is changing greatly. In particular, in modern people, the intestinal bacterial flora is disturbed, intestinal leak syndrome, colitis, liver disease, allergic disorders, obesity due to the eating and fat-oriented westernized diet, irregular diet, excessive drinking, lack of exercise, excessive stress, exposure to harmful environment, etc. The back is increasing rapidly.

Disruption of Intestinal Bacteria

Many bacteria live in the digestive tract of our bodies. There are about 10 trillion normal cells in our body, while the number of bacteria is about 100 trillion times more than that. And these bacteria can be divided into beneficial bacteria that help our gut health and harmful bacteria that are harmful to our health. Our bodies are Lactobacillus bacteria (Lactobacillus), Bifidobacterium (Bifidobacterium), Streptococcus (Streptococcus), current Kono Stock (Leuconostoc), Peddie yuikgyun the harmful bacteria, such as OKO Syracuse (Pediococcus), sports as Lactobacillus (Sporolactobacilius) The more prevalent bacteria live in the digestive tract and maintain their health. Otherwise, it can lead to diseases such as obesity, bowel leak syndrome, liver disease, aging, enteritis, and the like.

Intestinal Permeability Syndrome

The digestive tract of our body is composed of mucus and villi, which efficiently absorb nutrients and prevent the absorption of high molecular weight pathogens and their toxins. In addition, our body has an immune system that can protect against high molecular weight foreign antigens invading the body. However, the intestinal bacterial flora is disturbed by infections of many hospital microorganisms or toxins, excessive stress, food intake such as high-fat diets that can multiply harmful bacteria in the digestive tract, excessive alcohol intake, and abuse of drugs (eg antibiotics). The immune system in the digestive tract develops abnormally and the expression of tight junction proteins is suppressed. When the expression of tight junction proteins is suppressed, the dense bonds of the mesenteric membranes are loosened, and these loosened gaps and immune system abnormalities facilitate the invasion of macromolecules such as pathogens. Intestinal Permeability Syndrome (Leaky Gut Syndrome) is a leaky gut syndrome, and the tightly connected defense system of the epithelial cells that make up the gastrointestinal tract is not working well. It is a condition that continues to flow into the blood. When intestinal leak syndrome occurs, external antigens that are not absorbed into the body generally enter the body, so ulcerative colitis, Crohn's disease, liver damage, liver dysfunction, allergic diseases (including asthma), atopy, autoimmune diseases, lipodiasis, Digestive absorption disorders, acne, aging, endotoxins, intestinal infections, eczema, hypersensitivity syndrome, chronic fatigue syndrome, psoriasis, rheumatoid arthritis, pancreatic insufficiency, inflammatory joint disease, etc.

colitis

Conventionally, the incidence rate of ulcerative colitis and Crohn's disease has been known to Westerners, but recently, the number of patients has increased rapidly in the Asian countries such as Korea due to changes in lifestyles such as eating habits. However, there are some reasons why the cause is unclear, and no fundamental treatment is established. For this reason, it is not aimed at complete treatment, but the situation which uses the medicament which alleviates a symptom and maintains this state for as long as possible. As drugs for such symptomatic therapy, aminosalicylic acid agents, corticosteroids, immunosuppressants and the like are mainly used, but various side effects have been reported. For example, salazosulpapyridine, which is frequently used as an aminosalicylic acid agent, has been reported to have side effects such as nausea, vomiting, anorexia, rash, headache, liver injury, white blood cell reduction, abnormal red blood cells, proteinuria, and diarrhea. In addition, corticosteroids are generally used for oral administration of prednisolone, enemas, suppositories, and intravenous injections, but side effects such as femoral head necrosis due to gastric ulcer and long-term use are strong. However, because discontinuation of medication causes symptoms to recur, these drugs cannot be used continuously. Therefore, there is a need for development of a therapeutic agent for intestinal diseases such as ulcerative colitis, Crohn's disease, which is excellent in effect but safe and does not cause side effects. Irritable bowel syndrome (IBS) is also a chronic abdominal disease whose cause is unclear. Currently, there is no fundamental therapeutic agent for IBS, and symptomatic therapy for the purpose of alleviating symptoms of each type is being performed. For example, an anticholinergic agent that suppresses contraction of smooth muscle is used for hari-type IBS, and it is difficult to control with constipation-type IBS with salt laxatives, and alternative-type IBS with drugs. have.

Liver disease

The liver plays a role in energy metabolism (nutrient treatment, storage and waste excretion), toxin detoxification, synthesis of serum proteins, and smooth absorption of fat from the intestine through bile excretion. And vitamins are also important. However, liver diseases such as hepatitis, fatty liver or cirrhosis occur due to hepatitis virus infection and excessive intake of alcohol or high lipid diet. In addition, liver disease may also be caused by drugs (TB, aspirin, antibiotics, anesthetics, antihypertensives, oral contraceptives), congenital metabolic disorders, heart failure, and shock. Liver disease can develop from chronic hepatitis to acute hepatitis with fatigue, vomiting, diarrhea, loss of appetite, jaundice, right upper abdominal pain, fever, and muscle pain.

Allergic diseases

The complexity of society, the development of industry and civilization increase environmental pollution, stress, and dietary changes, increasing the number of allergic patients every year. In 1980, less than 1% of allergic patients, such as atopy, anaphylaxis, and asthma, increased to more than 5% in the 2000s, and more than 10% of potential patients were estimated. The cause of allergic disease is excessive immune response of the living body resulting from antigen antibody reaction, and allergic disease is generally classified into type 1-4 hypersensitivity reaction according to reaction time and presence of complement involvement. Type 1 hypersensitivity reactions include atopy, anaphylaxis shock, bronchial asthma, urticaria, hay fever, and type 2 hypersensitivity reactions include inadequate transfusion, autoimmune hemolytic anemia, hemolytic anemia by medications, granulocytopenia, thrombocytopenic purpura, etc. Type III hypersensitivity reactions include erythema, lymphadenopathy, arthralgia, arthritis, nephritis, acute glomerulonephritis after streptococcal infection, and type 4 hypersensitivity reactions include chronic inflammation. In order to improve allergic diseases, you should take a shower or bath to remove allergens (house dust, mites, etc.) on your skin, and do not eat allergens. However, if the allergic disease does not improve, drugs such as steroids, antihistamines, and immunosuppressants are used, such as skin atrophy, vasodilation, discoloration, purpura (steroid preparation), drowsiness (antihitamin), and kidney failure ( Side effects such as immunosuppressants) are likely to occur. None of the drugs that have been developed so far can cure allergies, and the symptoms are expected to improve, but side effects are large.

obesity

Obesity is a metabolic disease caused by an imbalance between calorie intake and consumption and is morphologically caused by hypertrophy or hyperplasia of fat cells in the body. Obesity is not only the most common malnutrition in Western society, but the importance of treatment and prevention has been highlighted in recent years in Korea, as the frequency of obesity is rapidly increasing due to the improvement of dietary life and the westernization of lifestyle. have. Obesity is an important factor that not only psychologically diminishes individuals but also increases the risk of developing various adult diseases. Obesity is known to be directly related to the increased prevalence of various adult diseases, such as type 2 diabetes, hypertension, hyperlipidemia, and heart disease (Cell 87: 377, 1999), and the combination of obesity-related disorders together with metabolic syndrome or insulin resistance It is called syndrome (insulin resistance syndrome), and these have been found to be the cause of atherosclerosis and cardiovascular disease. Known obesity treatments such as Xenical (Roche Pharmaceuticals, Switzerland), Reductil (Eboth, USA), Exolise (Atopama, France), etc. Classified as inhibitors, most anti-obesity agents are appetite suppressants that suppress appetite by regulating neurotransmitters associated with the hypothalamus. However, conventional treatments have low side effects such as heart disease, respiratory disease, and neurological disease, and thus have low sustainability. Therefore, development of an improved obesity treatment agent is needed, and currently developed products have satisfactory treatment effects without side effects. Since there are few therapeutic agents, development of new obesity agents is required.

In the gastrointestinal tract of animals including humans, living organisms that improve the host's intestinal microbial environment and have beneficial effects on the health of the host are collectively called probiotics. In order to be effective as a probiotic, when ingested orally, most of them must reach the small intestine and adhere to the intestinal surface. Therefore, the resistance to acid, bile and intestinal epithelial cells should be excellent. Lactic acid bacteria are used as probiotics because they play a role in breaking down fiber and complex proteins into important nutrients while living in the digestive system of the human body. Lactobacillus has been reported to show the effects of maintaining normal intestinal flora, improving intestinal flora, antidiabetic and antihyperlipidemic effects, inhibiting carcinogenesis, inhibiting colitis, and nonspecific activity of the host's immune system. Among them, the strain Lactobacillus is a major member of the normal microbial community in the intestine of the human body, which has long been known to be important for maintaining a healthy digestive system and the vaginal environment, and the US Public Health Service (US Public Health Service). guidelines) are currently classified as 'Bio-safty Level 1', where none of the Lactobacillus strains currently deposited with the American Strain Deposit Organization (ATCC) is known about the potential risk of causing disease in humans or animals. have. On the other hand, kimchi lactic acid bacteria are reported to have the effect of immuno-enhancing, anti-microbial, antioxidant, anti-cancer, anti-obesity, hypertension or constipation as a lactic acid bacteria involved in kimchi fermentation [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, researches to develop lactic acid bacteria strains which are safe for humans and excellent in function and apply them as pharmaceuticals or functional foods are being actively conducted. For example, Korean Laid-open Patent Publication No. 10-2009-0116051 discloses Lactobacillus brevis HY7401, which has the effect of treating and preventing colitis. In addition, Korean Patent Publication No. 10-2006-0119045 discloses Leuconostoc citreum) KACC91035, flow may Pocono stock mesen teroyi des subspecies mesen teroyi des (Leuconostoc mesenteroides subsp. mesenteroides) The KCTC 3100 and Lactobacillus brevis (Lactobacillus brevis) atopic lactic acid bacteria for dermatitis treatment or prevention, selected from the group consisting of KCTC 3498 discloses . In addition, Korean Patent Laid-Open No. 10-2013-0092182 discloses a health functional food for preventing or hangover of alcoholic liver disease, which contains Lactobacillus brevis HD-01 [Accession No .: KACC91701P], which has excellent alcohol resolution. Is disclosed. In addition, Korean Patent Laid-Open Publication No. 10-2010-0010015 discloses Lactobacillus johnsonii HFI 108 strain (KCTC 11356BP) having blood cholesterol concentration lowering and anti-obesity activity. In addition, Korean Unexamined Patent Publication No. 10-2014-0006509 describes obesity including strain Bifidobacterium longum CGB-C11 (Accession No. KCTC 11979BP), which produces conjugated linoleic acid, as an active ingredient. Prophylactic or therapeutic compositions are disclosed.

However, there are no technologies related to lactic acid bacteria that can collectively improve or treat the disturbance of enterobacteriaceae, intestinal leak syndrome, colitis, liver disease, allergic diseases, and obesity. Screening of strains and the development of pharmaceuticals, functional foods and the like through the same is necessary.

The present invention has been derived under such a conventional technical background, and an object of the present invention is to provide a novel lactic acid bacterium having various physiological activities or functions required as probiotics and a medicinal use thereof.

In addition, another object of the present invention to provide a novel lactic acid bacteria mixture and its pharmaceutical use that can maximize a variety of physiological activity or functionality.

The inventors of the present invention screen a myriad lactic acid bacteria from kimchi or human feces, the specific Lactobacillus strains, certain Bifidobacterium strains or mixed lactic acid bacteria thereof, liver damage such as intestinal leak syndrome, fatty liver and the like, The present invention was completed by confirming that it has an excellent improvement effect on allergic diseases such as atopic dermatitis, inflammatory diseases such as colitis, or obesity.

In order to achieve the object of the present invention, one example of the present invention is Lactobacillus brevis ( Lactobacillus brevis ) comprising the nucleotide sequence described in SEQ ID NO: 1 as 16S rDNA, comprising the nucleotide sequence described in SEQ ID NO: 3 as 16S rDNA Bifidobacterium longum , Lactobacillus plantarum comprising the nucleotide sequence of SEQ ID NO: 4 as 16S rDNA, Lactobacillus plantarum , Lactobacillus planta comprising the nucleotide sequence of SEQ ID NO: 5 as 16S rDNA Lactobacillus plantarum or Lactobacillus is selected from Bifidobacterium longum ( Bifidobacterium longum ) comprising the nucleotide sequence of SEQ ID NO: 7 in 16S rDNA. The Lactobacillus brevis ( Lactobacillus brevis ), Lactobacillus plantarum ( Lactobacillus plantarum ) or Bifidobacterium long gum ( Bifidobacterium) longum ) has antioxidant activity, beta-glucuronidase inhibitory activity, lipopolysaccharide (LPS) production inhibitory activity, or tight junction protein expression inducing activity. In addition, one embodiment of the present invention is Lactobacillus brevis ( Lactobacillus brevis ) comprising the nucleotide sequence of SEQ ID NO: 1 as 16S rDNA, Bifidobacterium long gum ( Bifidobacterium) comprising the nucleotide sequence of SEQ ID NO: 3 as 16S rDNA longum), Lactobacillus Planta room (Lactobacillus plantarum) comprising a base sequence described in Lactobacillus Planta room (Lactobacillus plantarum) comprising the nucleotide sequence set forth in SEQ ID NO: 4 with 16S rDNA, SEQ ID NO: 5 with 16S rDNA or 16S Bifidobacterium longgum containing the nucleotide sequence of SEQ ID NO: 7 as rDNA longum ) lactic acid bacteria selected from, lactic acid bacteria cultures, lysates of lactic acid bacteria or extracts of the lactic acid bacteria as an active ingredient, and for preventing or treating intestinal damage, liver damage, allergic diseases, inflammatory diseases, or obesity It provides a pharmaceutical composition. In addition, one embodiment of the present invention is Lactobacillus brevis ( Lactobacillus brevis ) comprising the nucleotide sequence of SEQ ID NO: 1 as 16S rDNA, Bifidobacterium long gum ( Bifidobacterium) comprising the nucleotide sequence of SEQ ID NO: 3 as 16S rDNA longum), Lactobacillus Planta room (Lactobacillus plantarum) comprising a base sequence described in Lactobacillus Planta room (Lactobacillus plantarum) comprising the nucleotide sequence set forth in SEQ ID NO: 4 with 16S rDNA, SEQ ID NO: 5 with 16S rDNA or 16S Bifidobacterium longgum containing the nucleotide sequence of SEQ ID NO: 7 as rDNA longum ) lactic acid bacteria, the culture of the lactic acid bacteria, lactic acid bacteria lysate or extract of the lactic acid bacteria as an active ingredient, and for preventing or improving intestinal damage, liver damage, allergic diseases, inflammatory diseases, or obesity It provides a food composition.

In order to achieve another object of the present invention, one example of the present invention is Lactobacillus brevis comprising the nucleotide sequence of SEQ ID NO: 1 as 16S rDNA, comprising the nucleotide sequence of SEQ ID NO: 3 as 16S rDNA Bifidobacterium longum , Lactobacillus plantarum comprising the nucleotide sequence of SEQ ID NO: 4 as 16S rDNA, Lactobacillus plantarum , Lactobacillus planta comprising the nucleotide sequence of SEQ ID NO: 5 as 16S rDNA Provided is a mixed lactic acid bacteria selected from the group consisting of Bifidobacterium longum ( Lactobacillus plantarum ) and Bifidobacterium longum comprising the nucleotide sequence of SEQ ID NO: 7 in 16S rDNA. The mixed lactic acid bacteria have antioxidant activity, beta-glucuronidase (β-glucuronidase) inhibitory activity, lipopolysaccharide (LPS) production inhibitory activity or tight junction protein expression inducing activity. In addition, one embodiment of the present invention is Lactobacillus brevis ( Lactobacillus brevis ) comprising the nucleotide sequence of SEQ ID NO: 1 as 16S rDNA, Bifidobacterium long gum ( Bifidobacterium) comprising the nucleotide sequence of SEQ ID NO: 3 as 16S rDNA longum), Lactobacillus Planta room (Lactobacillus plantarum), Lactobacillus Planta room (Lactobacillus plantarum comprising the nucleotide sequence set forth in SEQ ID NO: 5 as 16S rDNA) containing the nucleotide sequence shown in SEQ ID NO: 4 to the 16S rDNA and 16S BP gambling comprising the nucleotide sequence set forth in SEQ ID NO: 7 by rDNA Te Solarium ronggeom (Bifidobacterium longum ) comprises a mixed lactic acid bacteria selected from the group consisting of two or more, the culture of the mixed lactic acid bacteria, the shredded product of the mixed lactic acid or the extract of the mixed lactic acid bacteria as an active ingredient, intestinal damage, liver damage, allergic diseases, inflammatory diseases Or compositions for use in preventing or treating obesity. In addition, one embodiment of the present invention is Lactobacillus brevis ( Lactobacillus brevis ) comprising the nucleotide sequence of SEQ ID NO: 1 as 16S rDNA, Bifidobacterium long gum ( Bifidobacterium) comprising the nucleotide sequence of SEQ ID NO: 3 as 16S rDNA longum), Lactobacillus Planta room (Lactobacillus plantarum), Lactobacillus Planta room (Lactobacillus plantarum comprising the nucleotide sequence set forth in SEQ ID NO: 5 as 16S rDNA) containing the nucleotide sequence shown in SEQ ID NO: 4 to the 16S rDNA and 16S Bifidobacterium longgum containing the nucleotide sequence of SEQ ID NO: 7 as rDNA longum ) comprises a mixed lactic acid bacteria selected from the group consisting of two or more, the culture of the mixed lactic acid bacteria, the shredded product of the mixed lactic acid or the extract of the mixed lactic acid bacteria as an active ingredient, intestinal damage, liver damage, allergic diseases, inflammatory diseases Or a food composition for use in preventing or improving obesity.

Specific Lactobacillus strains or specific Bifidobacterium strains according to the present invention are isolated from kimchi or human feces, high safety, antioxidant activity, beta-glucuronidase inhibitory activity, lipopolysaccharide ( lipopolysaccharide (LPS) production inhibitory activity or tight junction protein (tight junction protein) expression inducing activity and the like have a variety of physiological activities. Accordingly, certain Lactobacillus strains, certain Bifidobacterium strains or mixed lactic acid bacteria thereof according to the present invention are useful in preventing, ameliorating or treating intestinal damage, liver damage, allergic diseases, inflammatory diseases, or obesity. It can be used as a food and pharmaceutical material.

1 is a graph showing changes in GOT values when lactic acid bacteria are administered to a model animal induced by liver damage by D-galactosamine in the first experiment of the present invention, and FIG. In the first experiment, when the lactic acid bacteria were administered to the model animal induced liver damage by D-galactosamine (D-Galactosamine) is a graph showing the change in the GPT value, Figure 3 in the first experiment of the present invention, D -It is a graph showing the change of MDA value when lactic acid bacteria were administered to a model animal in which liver damage was induced by galactosamine (D-Galactosamine).

4 is a graph showing the effect of lactic acid bacteria selected in the first experiment of the present invention on the inflammatory response of dendritic cells induced by LPS (lipopolysaccharide). 4 is a graph showing the effect of lactic acid bacteria on cells not treated with LPS (lipopolysaccharide), and the graph on the right is a graph showing the effect of lactic acid bacteria on cells treated with LPS (lipopolysaccharide).

5 is a graph showing the effect of Bifidobacterium longum CH57 on the inflammatory response of macrophage induced by LPS (lipopolysaccharide) in the first experiment of the present invention.

FIG. 6 shows the effect of Lactobacillus brevis CH23 on the differentiation of T cells isolated from the spleen into Th17 cells or Treg cells in the first experiment of the present invention using a Fluorescence-activated cell sorting (FACS) device. One result.

Figure 7 in the first experiment of the present invention, Lactobacillus brevis ( Lactobacillus brevis ) CH23, Bifidobacterium long gum ( Bifidobacterium) longum ) The result of analyzing the effect of CH57 or mixed lactic acid bacteria on the expression of ZO-1 protein in CaCO2 cells.

FIG. 8 shows Bifidobacterium longgum for a model animal induced by acute colitis by TNBS in a first experiment of the present invention. longum ) shows the effect of CH57 on the appearance of the large intestine or myeloperoxidase (MPO) activity, etc., Figure 9 is a non-gambling for the model animal induced acute colitis by TNBS in the first experiment of the present invention Te Solarium ronggeom (Bifidobacterium longum ) shows the effect of CH57 in the histological picture of the colon, Figure 10 shows the Bifidobacterium longum CH57 in a model animal induced by acute colitis by TNBS in the first experiment of the present invention The effect is indicated by inflammation related cytokines and the like.

FIG. 11 illustrates the effect of Lactobacillus brevis CH23 on model animals induced by acute colitis by TNBS in the first experiment of the present invention, such as the appearance of colon or myeloperoxidase (MPO) activity. 12 is a histological picture of the large intestine showing the effect of Lactobacillus brevis CH23 on a model animal induced by acute colitis by TNBS in the first experiment of the present invention. In the first experiment of the present invention, the effect of Lactobacillus brevis CH23 on model animals induced by acute colitis induced by TNBS is shown as a differentiation aspect of T cells, and FIG. 14 is a first experiment of the present invention. in, Lactobacillus brevis for an animal model of acute colitis is induced by TNBS (Lactobacillus brevis) influence on the CH23 It shows the inflammatory cytokines and the like.

15 is ronggeom Bifidobacterium (Bifidobacterium for an animal model of acute colitis is induced by the primary experiment according to the present invention, TNBS longum ) The effect of mixed lactic acid bacteria of CH57 and Lactobacillus brevis CH23 is shown by the appearance of colon or myeloperoxidase (MPO) activity, and FIG. 16 shows the effect of TNBS on the first experiment of the present invention. Bifidobacterium long gum ( Bifidobacterium) on model animals induced by acute colitis longum ) shows the effect of mixed lactic acid bacteria of CH57 and Lactobacillus brevis CH23 in the histological picture of the large intestine, Figure 17 shows in a model animal induced by acute colitis by TNBS in the first experiment of the present invention. ronggeom for Bifidobacterium (Bifidobacterium longum ) The effect of mixed lactic acid bacteria of CH57 and Lactobacillus brevis CH23 is shown by cytokines related to inflammation.

FIG. 18 shows the Bifidobacterium long gum for obesity-induced model animals in the first experiment of the present invention. longum) CH57 and Lactobacillus brevis (Lactobacillus brevis) will represented by such weight the effect of mixing lactic acid bacteria on the CH23 variation, Figure 19 is Bifidobacterium ronggeom for the primary test of the invention, the obesity induction model animal (Bifidobacterium longum ) The effect of mixed lactic acid bacteria of CH57 and Lactobacillus brevis CH23 is shown in the appearance of the large intestine, myeloperoxidase (MPO) activity, histological picture of the large intestine, and FIG. In the second experiment, the effects of mixed lactic acid bacteria of Bifidobacterium longum CH57 and Lactobacillus brevis CH23 on obesity-induced model animals are shown by inflammation related cytokines and the like. In the first experiment of Bifidobacterium longum CH57 on obesity-induced model animals And Lactobacillus brevis ( Lactobacillus brevis ) the effect of the mixed lactic acid bacteria of CH23 is shown as an indicator of inflammatory response.

22 shows the effect of lactic acid bacteria on the differentiation of T cells into Th17 cells in a model animal induced by acute colitis by TNBS in a second experiment of the present invention, and FIG. 23 is a second experiment of the present invention. In the present invention, the effect of lactic acid bacteria on TNBS-induced acute colitis is shown as a differentiation pattern of T cells into Treg cells.

FIG. 24 shows the effects of lactic acid bacteria on the model animals induced by acute colitis by TNBS in the second experiment of the present invention as inflammatory response indicators.

FIG. 25 is a photograph showing the effect of lactic acid bacteria on the gastric mucosa of the mouse induced by gastric ulcers in the second experiment of the present invention. FIG. 26 is a second experiment of the present invention. The effect of lactic acid bacteria on the gastric mucosa of the gastric ulcer induced by the gastric ulcer is shown by the gross gastric lesion score (Gross gastric lesion score), Figure 27 is a gastric ulcer by ethanol in the second experiment of the present invention The effect of lactic acid bacteria on the gastric mucosa of the induced mice (ulcer index) is shown by the ulcer index (ulcer index), Figure 28, in the second experiment of the present invention, gastric mucosa of the mice induced gastric ulcer by ethanol (Histological activity index) shows the effect of lactic acid bacteria on (stomach mucosa).

FIG. 29 shows the effect of lactic acid bacteria on the gastric mucosa of the mice induced by gastric ulcer in ethanol in the second experiment of the present invention as myeloperoxidase (MPO) activity.

FIG. 30 shows the effect of lactic acid bacteria on the gastric mucosa of the mice induced by gastric ulcers in the second experiment of the present invention by expression level of CXCL4, and FIG. 31 is a second experiment of the present invention. In the present invention, the effect of lactic acid bacteria on the gastric mucosa (stomach mucosa) of the gastric ulcer induced by ethanol is expressed as the expression level of TNF-α.

Hereinafter, the term used by this invention is demonstrated.

As used herein, the term "culture" means a product obtained by culturing a microorganism in a known liquid medium or a solid medium, and is a concept in which a microorganism is included.

As used herein, "pharmaceutically acceptable" and "food acceptable" means that they do not significantly stimulate the organism and do not inhibit the biological activity and properties of the administered active substance.

As used herein, the term "prevention" means any action that inhibits the symptoms or delays the progression of a particular disease by administration of a composition of the present invention.

As used herein, the term "treatment" means any action that improves or beneficially alters the symptoms of a particular disease by administration of a composition of the present invention.

The term "improvement" as used herein refers to any action that at least reduces the parameters associated with the condition being treated, for example, the extent of symptoms.

As used herein, the term "administration" means providing a subject with a composition of the present invention in any suitable manner. At this time, the subject refers to all animals, such as humans, monkeys, dogs, goats, pigs or mice having a disease that can improve the symptoms of a particular disease by administering the composition of the present invention.

As used herein, 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 refers to the type of disease, the severity, the activity of the drug, the drug, and the like. Sensitivity, time of administration, route of administration and rate of excretion, duration of treatment, factors including drug used concurrently, and other factors well known in the medical arts.

EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated in detail.

One aspect of the present invention relates to a novel lactic acid bacteria having a variety of physiological activity or a novel mixed lactic acid bacteria that can increase the biological activity.

The novel lactic acid bacteria according to an embodiment of the present invention is Lactobacillus brevis (16S rDNA) comprising the nucleotide sequence of SEQ ID NO: 1Lactobacillus brevis), Bifidobacterium longgum containing the nucleotide sequence of SEQ ID NO: 3 as 16S rDNA (Bifidobacterium longum), Lactobacillus plantarum comprising the nucleotide sequence of SEQ ID NO: 4 as 16S rDNA (Lactobacillus plantarum), Lactobacillus plantarum comprising the nucleotide sequence of SEQ ID NO: 5 as 16S rDNA (Lactobacillus plantarumOr Bifidobacterium longgum comprising the nucleotide sequence of SEQ ID NO: 7 as 16S rDNA (Bifidobacterium longum) Has antioxidant activity, beta-glucuronidase inhibitory activity, lipopolysaccharide (LPS) production inhibitory activity or tight junction protein expression inducing activity. Lactobacillus brevis comprising the nucleotide sequence of SEQ ID NO: 1 as the 16S rDNA (Lactobacillus brevis) Is an anaerobic bacillus isolated from kimchi, shows positive in Gram staining, can survive in a wide temperature range, low pH, high salt concentration, and produces glucosidase. In addition, Lactobacillus brevis containing the nucleotide sequence of SEQ ID NO: 1 as the 16S rDNA (Lactobacillus brevis) Is the carbon source of D-ribose, D-xylose, D-glucose, D-fructose, Esculin, Salicin, Maltose, Melibiose, 5-keto-gluconate (5 -keto-gluconate). In addition, Lactobacillus brevis containing the nucleotide sequence of SEQ ID NO: 1 as the 16S rDNA (Lactobacillus brevis) Is preferably Lactobacillus brevis (Lactobacillus brevis) CH23 (accession number: KCCM 11762P). Bifidobacterium long gum comprising the nucleotide sequence of SEQ ID NO: 3 as the 16S rDNA (Bifidobacterium longum) Is an anaerobic bacillus isolated from human feces, positive in Gram staining, and produces glucosidase. In addition, Bifidobacterium long gum comprising the nucleotide sequence of SEQ ID NO: 3 as the 16S rDNA (Bifidobacterium longum) Uses D-galactose, D-glucose, D-fructose as a carbon source. In addition, Bifidobacterium long gum comprising the nucleotide sequence of SEQ ID NO: 3 as the 16S rDNA (Bifidobacterium longum) Is preferably Bifidobacterium long gum (Bifidobacterium longum) CH57 (Accession No .: KCCM 11764P). Lactobacillus plantarum comprising the nucleotide sequence of SEQ ID NO: 4 as the 16S rDNA (Lactobacillus plantarum) Is an anaerobic bacillus isolated from Kimchi and shows positive gram staining. In addition, Lactobacillus plantarum comprising the nucleotide sequence of SEQ ID NO: 4 as the 16S rDNA (Lactobacillus plantarum) Is the carbon source of D-ribose, D-galactose, D-glucose, D-fructose, D-mannose, mannitol, sorbitol, N-acetyl-glucosamine, amigdaline, arbutin, esculin, salicycin, Cellobiose, maltose, melibiose, sucrose, trehalose, melezitose and the like are used. In addition, Lactobacillus plantarum comprising the nucleotide sequence of SEQ ID NO: 4 as the 16S rDNA (Lactobacillus plantarum) Is preferably Lactobacillus plantarum (Lactobacillus plantarum) LC5 (Accession No .: KCCM 11800P). Lactobacillus plantarum comprising the nucleotide sequence of SEQ ID NO: 5 as the 16S rDNA (Lactobacillus plantarum) Is an anaerobic bacillus isolated from Kimchi and shows positive gram staining. In addition, Lactobacillus plantarum comprising the nucleotide sequence of SEQ ID NO: 5 as the 16S rDNA (Lactobacillus plantarum) Is the carbon source of L-arabinose, D-ribose, D-glucose, D-fructose, D-mannose, mannitol, sorbitol, N-acetyl-glucosamine, amigdaline, arbutin, esculin, salicycin , Cellobiose, maltose, lactose, melibiose, sucrose, trehalose, melezitose and the like. In addition, Lactobacillus plantarum comprising the nucleotide sequence of SEQ ID NO: 5 as the 16S rDNA (Lactobacillus plantarum) Is preferably Lactobacillus plantarum (Lactobacillus plantarum) LC27 (Accession No .: KCCM 11801P). Bifidobacterium long gum comprising the nucleotide sequence of SEQ ID NO: 7 as the 16S rDNA (Bifidobacterium longum) Is an anaerobic bacillus isolated from human feces and shows positive gram staining. In addition, Bifidobacterium long gum comprising the nucleotide sequence of SEQ ID NO: 7 as the 16S rDNA (Bifidobacterium longum) Uses L-arabinose, D-xylose, D-glucose, D-fructose, esculin, maltose, lactose, melibios, sucrose and the like as carbon sources. In addition, Bifidobacterium long gum comprising the nucleotide sequence of SEQ ID NO: 7 as the 16S rDNA (Bifidobacterium longum) Is preferably Bifidobacterium long gum (Bifidobacterium longum) LC67 (Accession No .: KCCM 11802P).

Mixed lactic acid bacteria according to an embodiment of the present invention is Lactobacillus brevis ( Lactobacillus brevis ) comprising the nucleotide sequence of SEQ ID NO: 1 as 16S rDNA, Bifidobacterium long gum (SEQ ID NO: 3) Bifidobacterium longum), Lactobacillus Planta room (Lactobacillus plantarum) comprising the nucleotide sequence set forth in SEQ ID NO: 4 with 16S rDNA, Lactobacillus Planta room (Lactobacillus plantarum comprising the nucleotide sequence set forth in SEQ ID NO: 5 as 16S rDNA) and Bifidobacterium longgum comprising the nucleotide sequence of SEQ ID NO: 7 as 16S rDNA It is selected from the group consisting of two or more kinds longum). Mixed lactic acid bacteria according to an embodiment of the present invention is preferably Lactobacillus brevis ( Lactobacillus brevis ) comprising the nucleotide sequence of SEQ ID NO: 1 as 16S rDNA and bases described in SEQ ID NO: 3 as 16S rDNA It consists of a combination of Bifidobacterium longum containing sequences. In addition, the mixed lactic acid bacteria according to an embodiment of the present invention, when considering the synergistic action of the lactic acid bacteria, preferably in Lactobacillus plantarum ( Lactobacillus plantarum ) or 16S rDNA comprising the nucleotide sequence of SEQ ID NO: 4 in 16S rDNA One or more Lactobacillus genus bacteria selected from Lactobacillus plantarum comprising the nucleotide sequence of No. 5; And Bifidobacterium longum comprising the nucleotide sequence set forth in SEQ ID NO: 7 as 16S rDNA. The mixed lactic acid bacteria have a higher antioxidant activity, a beta-glucuronidase inhibitory activity than a single lactic acid bacterium by synergistic action of a specific Lactobacillus strain and a specific Bifidobacterium strain, and a lipopolysaccharide (LPS). A) has a production inhibitory activity or a tight junction protein expression inducing activity, and is more useful in terms of functional food and pharmaceutical materials. Lactobacillus brevis ( Lactobacillus brevis ) comprising the nucleotide sequence of SEQ ID NO: 1 as the 16S rDNA in the mixed lactic acid bacteria according to an embodiment of the present invention is Lactobacillus brevis CH23 (Accession Number: KCCM 11762P), 16S rDNA as Bifidobacterium ronggeom comprising the nucleotide sequence set forth in SEQ ID NO: 3 (Bifidobacterium longum) is ronggeom Bifidobacterium (Bifidobacterium longum ) CH57 (Accession No .: KCCM 11764P), Lactobacillus plantarum ( Lactobacillus plantarum ) LC5 (Accession No .: KCCM) containing the nucleotide sequence of SEQ ID NO: 4 as 16S rDNA Lactobacillus plantarum Lactobacillus plantarum LC27 (Accession No .: KCCM 11801P) comprising the nucleotide sequence of SEQ ID NO: 5 as 16S rDNA, and sequence as 16S rDNA BP gambling comprising the nucleotide sequence shown in No. 7 Te Solarium ronggeom (Bifidobacterium longum ) is preferably Bifidobacterium longum LC67 (Accession Number: KCCM 11802P).

Another aspect of the invention relates to various uses of the novel lactic acid bacteria or new mixed lactic acid bacteria. For the use of the novel lactic acid bacteria, the present invention is Lactobacillus brevis comprising the nucleotide sequence of SEQ ID NO: 1 as 16S rDNA, Bifidobacterium long gum comprising the nucleotide sequence of SEQ ID NO: 3 as 16S rDNA longum), Lactobacillus Planta room (Lactobacillus plantarum) comprising a base sequence described in Lactobacillus Planta room (Lactobacillus plantarum) comprising the nucleotide sequence set forth in SEQ ID NO: 4 with 16S rDNA, SEQ ID NO: 5 with 16S rDNA or 16S Bifidobacterium longgum containing the nucleotide sequence of SEQ ID NO: 7 as rDNA longum ) comprising a lactic acid bacteria, its culture, its crush or its extract as an active ingredient, and provides a composition for use in preventing, improving or treating intestinal damage, liver damage, allergic diseases, inflammatory diseases, or obesity do. In addition, for use of the novel mixed lactic acid bacteria, the present invention is Lactobacillus brevis comprising the nucleotide sequence of SEQ ID NO: 1 as 16S rDNA, Bifidobacterium comprising the nucleotide sequence of SEQ ID NO: 3 as 16S rDNA Long Gum ( Bifidobacterium) longum), Lactobacillus Planta room (Lactobacillus plantarum), Lactobacillus Planta room (Lactobacillus plantarum comprising the nucleotide sequence set forth in SEQ ID NO: 5 as 16S rDNA) containing the nucleotide sequence shown in SEQ ID NO: 4 to the 16S rDNA and 16S Bifidobacterium longgum containing the nucleotide sequence of SEQ ID NO: 7 as rDNA longum ) containing two or more selected lactic acid bacteria, its culture, its lysate or its extract as an active ingredient, and prevents, improves or treats intestinal damage, liver damage, allergic diseases, inflammatory diseases, or obesity A composition for use is provided. Lactobacillus brevis ( Lactobacillus brevis ), Lactobacillus plantarum ( Lactobacillus plantarum ) and Bifidobacterium long gum ( Bifidobacterium ) in the composition of the present invention longum ) technical features are the same as described above, and thus description thereof is omitted. The intestinal damage refers to a condition in which the function of the intestine (particularly the small intestine or the large intestine) is not normal due to disturbance of the intestinal bacterial flora, and preferably intestinal leak syndrome. In addition, the liver damage refers to a condition in which liver function is not normal due to external factors or internal factors, and is preferably selected from hepatitis, fatty liver, or liver cirrhosis. In addition, the hepatitis includes both nonalcoholic hepatitis and alcoholic hepatitis. In addition, the fatty liver includes both non-alcoholic fatty liver and alcoholic fatty liver. In addition, the allergic disease is not limited in kind as long as it is caused by an excessive immune response of the living body, and is preferably selected from atopic dermatitis, asthma, sore throat or chronic dermatitis. In addition, if the inflammatory disease is a disease caused by an inflammatory response, the type thereof is not particularly limited and is preferably selected from gastritis, gastric ulcer, arthritis or colitis. In addition, the arthritis includes rheumatoid arthritis. In addition, the colitis refers to a condition in which the intestine is inflamed due to bacterial infection or pathological fermentation of the intestinal contents, and is a concept including infective colitis and non-infectious colitis. Specific examples of colitis include inflammatory bowel disease or irritable colitis syndrome. Inflammatory bowel disease also includes ulcerative colitis, Crohn's disease, and the like.

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

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

In the pharmaceutical composition according to the present invention, the content of the novel lactic acid bacteria, new mixed lactic acid bacteria, its culture, its lysate or its extract is not particularly limited, for example, 0.01 to 99% by weight, based on the total weight of the composition, preferably Preferably 0.5 to 50% by weight, more preferably 1 to 30% by weight. In addition, the pharmaceutical composition according to the present invention may further include an additive 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 compositions of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In addition, the pharmaceutical composition of the present invention, in addition to the novel lactic acid bacteria, new mixed lactic acid bacteria, cultures thereof, crushed products or extracts thereof, known active ingredients having the effect of preventing or treating intestinal damage, liver damage, allergic diseases, inflammatory diseases or obesity It may contain 1 or more types. The pharmaceutical composition of the present invention may be formulated into a formulation for oral administration or a parenteral administration by a conventional method, and when formulated, such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc. Diluents or excipients may be used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations contain at least one excipient such as starch, calcium carbonate, sucrose in active ingredients. ), Lactose (Lactose) or gelatin can be prepared by mixing. In addition to simple excipients, lubricants such as magnesium styrate talc may also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups, and various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents, water and liquid paraffin. have. Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used. Furthermore, it may be preferably formulated according to each disease or component by an appropriate method in the art or using a method disclosed in Remington's Pharmaceutical Science (Recent Edition), Mack Publishing Company, Easton PA. The pharmaceutical composition of the present invention can be administered orally or parenterally to mammals including humans according to a desired method, and parenteral administration methods include external skin, intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, muscle Intra-injection or intrathoracic injection; The dosage of the pharmaceutical composition of the present invention is not particularly limited as long as it is a pharmaceutically effective amount, and the range thereof depends on the weight, age, sex, health condition, diet, time of administration, method of administration, excretion rate and severity of the disease. Varies. Typical daily dosages of the pharmaceutical compositions of the present invention are not particularly limited but are preferably 0.1 to 3000 mg / kg, more preferably 1 to 2000 mg / kg, once daily based on the active ingredient. Or divided into several doses.

In addition, the content of the novel lactic acid bacteria, new mixed lactic acid bacteria, its culture, its lysate or extract thereof as an active ingredient in the food composition according to the present invention is 0.01 to 99% by weight, preferably 0.1 to 50% by weight, based on the total weight of the composition. %, More preferably 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, acupuncture, tablets, capsules, or liquids, and examples of specific foods include meat, sausage, bread, chocolate, candy, snacks, confectionary, pizza, ramen, Other noodles, gums, dairy products, including ice cream, various soups, beverages, tea, functional water, drinks, alcoholic beverages and vitamin complexes, and includes all of the health food in the usual sense. In addition to the active ingredient, the food composition of the present invention may contain, as an additional component, a food acceptable carrier, various flavors, or natural carbohydrates. In addition, the food composition of the present invention is a variety of nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohols And carbonation agents used in carbonated beverages. In addition, the food composition of the present invention may contain a flesh for preparing natural fruit juice, fruit juice beverage and vegetable beverage. These components can be used independently or in combination. The above-mentioned natural carbohydrates are glucose, monosaccharides such as fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, sugar alcohols such as xylitol, sorbitol and erythritol. As the flavoring agent, natural flavoring agents such as taumartin, stevia extract, synthetic flavoring agents such as saccharin, aspartame, etc. may be used.

Hereinafter, the present invention will be described in more detail with reference to 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.

Ⅰ. 유산균 선별 및 효능 확인을 위한 1차 실험I. First experiment to screen for lactic acid bacteria and confirm efficacy

1. 유산균의 분리 및 동정1. Isolation and Identification of Lactic Acid Bacteria

(1) Isolation of Lactic Acid Bacteria from Kimchi

Chinese cabbage kimchi, radish kimchi, and green kimchi were respectively crushed, and the crushed liquid was suspended in MRS broth (MRS Broth; Difco, USA). Then, the supernatant was taken, transplanted into MRS agar medium (MRS agar medium; Difco, USA), and cultured anaerobicly at 37 ° C. for about 48 hours, and then colonies were formed.

(2) Isolation of Lactic Acid Bacteria from Human Feces

Human feces were suspended in GAM liquid medium (GAM broth; Nissui Pharmaceutical, Japan). Subsequently, the supernatant was taken and transplanted into BL agar medium (Nissui Pharmaceutical, Japan) and incubated anaerobicly at 37 ° C. for about 48 hours, after which colony-forming Bifidobacterium sp. .) Strains were isolated.

(3) Identification of selected lactic acid bacteria

Physiological characteristics and 16S rDNA sequences of strains isolated from kimchi or human feces were analyzed to determine the species of the strains and to give a strain name. Table 1 shows the control numbers and strain names of lactic acid bacteria isolated from Chinese cabbage kimchi, radish kimchi, green onion kimchi and human feces.

관리번호Control Number	균주명Strain name	관리번호Control Number	균주명Strain name
1One	Lactobacillus acidophilus CH1 Lactobacillus acidophilus CH1	3131	Lactobacillus sakei CH31 Lactobacillus sakei CH31
22	Lactobacillus acidophilus CH2 Lactobacillus acidophilus CH2	3232	Lactobacillus johnsonii CH32 Lactobacillus johnsonii CH32
33	Lactobacillus acidophilus CH3 Lactobacillus acidophilus CH3	3333	Lactobacillus sakei CH33 Lactobacillus sakei CH33
44	Lactobacillus brevis CH4 Lactobacillus brevis CH4	3434	Lactobacillus sakei CH34 Lactobacillus sakei CH34
55	Lactobacillus curvatus CH5 Lactobacillus curvatus CH5	3535	Lactobacillus plantarum CH35 Lactobacillus plantarum CH35
66	Lactobacillus brevis CH6 Lactobacillus brevis CH6	3636	Lactobacillus sanfranciscensis CH36 Lactobacillus sanfranciscensis CH36
77	Lactobacillus casei CH7 Lactobacillus casei CH7	3737	Bifidobacterium pseudocatenulatum CH37 Bifidobacterium pseudocatenulatum CH37
88	Lactobacillus planantrum CH8 Lactobacillus planantrum CH8	3838	Bifidobacterium pseudocatenulatum CH38 Bifidobacterium pseudocatenulatum CH38
99	Lactobacillus sakei CH9 Lactobacillus sakei CH9	3939	Bifidobacterium adolescentis CH39 Bifidobacterium adolescentis CH39
1010	Lactobacillus curvatus CH10 Lactobacillus curvatus CH10	4040	Bifidobacterium adolescentis CH40 Bifidobacterium adolescentis CH40
1111	Lactobacillus sakei CH11 Lactobacillus sakei CH11	4141	Bifidobacterium adolescentis CH41 Bifidobacterium adolescentis CH41
1212	Lactobacillus curvatus CH12 Lactobacillus curvatus CH12	4242	Bifidobacterium animalis CH42 Bifidobacterium animalis CH42
1313	Lactobacillus plantarum CH13 Lactobacillus plantarum CH13	4343	Bifidobacterium animalis CH43 Bifidobacterium animalis CH43
1414	Lactobacillus fermentum CH14 Lactobacillus fermentum CH14	4444	Bifidobacterium bifidum CH44 Bifidobacterium bifidum CH44
1515	Lactobacillus fermentum CH15 Lactobacillus fermentum CH15	4545	Bifidobacterium bifidum CH45 Bifidobacterium bifidum CH45
1616	Lactobacillus gasseri CH16 Lactobacillus gasseri CH16	4646	Bifidobacterium breve CH46 Bifidobacterium breve CH46
1717	Lactobacillus paracasei CH17 Lactobacillus paracasei CH17	4747	Bifidobacterium breve CH47 Bifidobacterium breve CH47
1818	Lactobacillus helveticus CH18 Lactobacillus helveticus CH18	4848	Bifidobacterium breve CH48 Bifidobacterium breve CH48
1919	Lactobacillus helveticus CH19 Lactobacillus helveticus CH19	4949	Bifidobacterium catenulatum CH49 Bifidobacterium catenulatum CH49
2020	Lactobacillus johnsonii CH20 Lactobacillus johnsonii CH20	5050	Bifidobacterium catenulatum CH50 Bifidobacterium catenulatum CH50
2121	Lactobacillus johnsonii CH21 Lactobacillus johnsonii CH21	5151	Bifidobacterium dentium CH51 Bifidobacterium dentium CH51
2222	Lactobacillus johnsonii CH22 Lactobacillus johnsonii CH22	5252	Bifidobacterium infantis CH52 Bifidobacterium infantis CH52
2323	Lactobacillus brevis CH23 Lactobacillus brevis CH23	5353	Bifidobacterium infantis CH53 Bifidobacterium infantis CH53
2424	Lactobacillus paracasei CH24 Lactobacillus paracasei CH24	5454	Bifidobacterium infantis CH54 Bifidobacterium infantis CH54
2525	Lactobacillus kimchi CH25 Lactobacillus kimchi CH25	5555	Bifidobacterium longum CH55 Bifidobacterium longum CH55
2626	Lactobacillus gasseri CH26 Lactobacillus gasseri CH26	5656	Bifidobacterium longum CH56 Bifidobacterium longum CH56
2727	Lactobacillus paracasei CH27 Lactobacillus paracasei CH27	5757	Bifidobacterium longum CH57 Bifidobacterium longum CH57
2828	Lactobacillus pentosus CH28 Lactobacillus pentosus CH28	5858	Bifidobacterium longum CH58 Bifidobacterium longum CH58
2929	Lactobacillus pentosus CH29 Lactobacillus pentosus CH29	5959	Bifidobacterium longum CH59 Bifidobacterium longum CH59
3030	Lactobacillus reuteri CH30 Lactobacillus reuteri CH30	6060	Bifidobacterium longum CH60 Bifidobacterium longum CH60

Among the strains described in Table 1, Lactobacillus brevis CH23 is an anaerobic bacilli that show positive gram staining, and shows viability even under aerobic conditions without forming spores. In addition, Lactobacillus brevis CH23 survived at 10-42 ° C. and was a stable acid resistant strain at pH 2 for 2 hours. In addition, Lactobacillus brevis CH23 survived in 2% sodium chloride solution and actively produced glucosidase. In addition, 16S rDNA was measured by the chemical classification of Lactobacillus brevis CH23, and as a result, it was found to have the nucleotide sequence of SEQ ID NO: 1. The 16S rDNA sequence of Lactobacillus brevis CH23 was identified by BLAST search of Genebank (http://www.ncbi.nlm.nih.gov/). As a result, Lactobacillus brevis ( Lactobacillus brevis ) having the same 16S rDNA sequence was identified. Lactobacillus brevis strain was not detected and showed 99% homology with the 16S rDNA subsequence of Lactobacillus brevis strain FJ004.

Among the strains described in Table 1, Lactobacillus johnsonii CH32 is an anaerobic bacilli that show positive gram staining, and shows viability even under aerobic conditions without forming spores. In addition, Lactobacillus johnsonii CH32 survived stably up to 45 ℃, was a stable acid resistant strain for 2 hours in H 2. In addition, Lactobacillus johnsonii CH32 actively produced glucosidase, but did not produce beta-glucuronidase. In addition, 16S rDNA was determined by the chemical classification of Lactobacillus johnsonii CH32, and as a result, it was found to have the nucleotide sequence of SEQ ID NO: 2. The Lactobacillus johnsonii CH32 16S rDNA sequence was identified by BLAST search of Genebank (http://www.ncbi.nlm.nih.gov/), and the Lactobacillus zone having the same 16S rDNA sequence was found. The Sony ( Lactobacillus johnsonii ) strain was not detected and showed 99% homology with the 16S rDNA subsequence of Lactobacillus johnsonii strain JCM 2012.

Among the strains described in Table 1, Bifidobacterium longum ( Bifidobacterium longum ) CH57 is an anaerobic bacilli that show positive gram staining, and did not form spores and had very low viability under aerobic conditions. In addition, Bifidobacterium longum CH57 was unstable to heat. In addition, Bifidobacterium longum CH57 actively produced glucosidase, but did not produce beta-glucuronidase. In addition, ronggeom Bifidobacterium (Bifidobacterium longum ) 16S rDNA was determined by chemical classification of CH57, and the result was found to have the nucleotide sequence of SEQ ID NO: 3. Ronggeom Bifidobacterium (Bifidobacterium longum ) 16S rDNA nucleotide sequence of CH57 was identified by BLAST search of Genebank (http://www.ncbi.nlm.nih.gov/) and Bifidobacterium long gum having the same 16S rDNA sequence longum ) was not detected and Bifidobacterium longgum longum ) showed 99% homology with the 16S rDNA subsequence of strain CBT-6.

In addition, the physiological characteristics of Lactobacillus brevis CH23, Lactobacillus johnsonii CH32, and Bifidobacterium longum CH57 were found in API Kit (Model: API 50 CHL; BioMerieux's, USA) for analysis of sugar fermentation. Table 2 shows the carbon source availability results of Lactobacillus brevis CH23, Table 3 below shows the carbon source availability results of Lactobacillus johnsonii CH32, and Table 4 shows the Bifidobacterium long gum. ( Bifidobacterium longum ) shows the results of carbon source availability of CH57. In Table 2, Table 3 and Table 4, "+" represents a case where the carbon source availability is positive, "-" represents a case where the carbon source availability is negative, and "±" represents a case where the availability of the carbon source is ambiguous. Lactobacillus brevis CH23, Lactobacillus johnsonii CH32 and Bifidobacterium long gum ( Bifidobacterium ) as shown in Tables 2, 3 and 4 below longum ) CH57 has different availability to some carbon sources than strains of the same species.

탄소원Carbon source	균주명Strain name		탄소원Carbon source	균주명Strain name
탄소원Carbon source	L. brevisL. brevis ^1)One)	L. brevis CH23 L. brevis CH23	탄소원Carbon source	L. brevisL. brevis ^1)One)	L. brevis CH23 L. brevis CH23
glycerolglycerol	--	--	salicinsalicin	++	++
erythritolerythritol	--	--	cellobiosecellobiose	++	--
D-arabinoseD-arabinose	--	--	maltosemaltose	++	++
L-arabinoseL-arabinose	++	--	lactoselactose	++	--
D-riboseD-ribose	++	++	melibiosemelibiose	--	++
D-xyloseD-xylose	++	++	sucrosesucrose	++	--
L-xyloseL-xylose	--	--	trehalosetrehalose	++	--
D-adonitolD-adonitol	--	--	inulininulin	++	--
methyl-β-D-xylopyranosidemethyl-β-D-xylopyranoside	--	--	melezitosemelezitose	++	--
D-galactoseD-galactose	++	--	raffinoseraffinose	--	--
D-glucoseD-glucose	++	++	starchstarch	--	--
D-fructoseD-fructose	++	++	glycogenglycogen	--	--
D-mannoseD-mannose	++	--	xylitolxylitol	--	--
L-sorboseL-sorbose	--	--	gentiobiosegentiobiose	++	--
L-rhamnoseL-rhamnose	--	--	D-turanoseD-turanose	++	--
dulcitoldulcitol	++	--	D-lyxoseD-lyxose	--	--
inositolinositol	--	--	D-tagatoseD-tagatose	++	--
mannitolmannitol	++	--	D-fucoseD-fucose	--	--
sorbitolsorbitol	++	--	L-fucoseL-fucose	--	--
α-methyl-D-mannosideα-methyl-D-mannoside	--	--	D-arabitolD-arabitol	--	--
α-methly-D-glucosideα-methly-D-glucoside	--	--	L-arabitolL-arabitol	--	--
N-acetyl-glucosamineN-acetyl-glucosamine	++	±±	gluconategluconate	++	±±
amygdalinamygdalin	++	--	2-keto-gluconate2-keto-gluconate	--	--
arbutinarbutin	++	--	5-keto-gluconate5-keto-gluconate	--	++
esculinesculin	++	++

1) Suriasih K., Aryanta WR, Mahardika G, Astawa NM. Microbiological and Chemical Properties of Kefir Made of Bali Cattle Milk. Food Science and Quality Management 2012; 6: 112-22.

탄소원Carbon source	균주명Strain name		탄소원Carbon source	균주명Strain name
탄소원Carbon source	L. johnsoniiL. johnsonii ²⁾²⁾	L. johnsonii CH32 L. johnsonii CH32	탄소원Carbon source	L. johnsoniiL. johnsonii ²⁾²⁾	L. johnsonii CH32 L. johnsonii CH32
glycerolglycerol	--	--	salicinsalicin	--	--
erythritolerythritol	--	--	cellobiosecellobiose	++	--
D-arabinoseD-arabinose	--	--	maltosemaltose	--	++
L-arabinoseL-arabinose	--	--	lactoselactose	--	++
D-riboseD-ribose	--	--	melibiosemelibiose	++	--
D-xyloseD-xylose	--	--	sucrosesucrose	++	++
L-xyloseL-xylose	--	--	trehalosetrehalose	++	--
D-adonitolD-adonitol	--	--	inulininulin	--	--
methyl-β-D-xylopyranosidemethyl-β-D-xylopyranoside	--	--	melezitosemelezitose	--	--
D-galactoseD-galactose	--	--	raffinoseraffinose	++	--
D-glucoseD-glucose	--	++	starchstarch	--	--
D-fructoseD-fructose	--	++	glycogenglycogen	--	--
D-mannoseD-mannose	++	++	xylitolxylitol	--	--
L-sorboseL-sorbose	--	--	gentiobiosegentiobiose	--	++
L-rhamnoseL-rhamnose	--	--	D-turanoseD-turanose	--	--
dulcitoldulcitol	--	--	D-lyxoseD-lyxose	--	--
inositolinositol	--	--	D-tagatoseD-tagatose	--	--
mannitolmannitol	--	--	D-fucoseD-fucose	--	--
sorbitolsorbitol	--	--	L-fucoseL-fucose	--	--
α-methyl-D-mannosideα-methyl-D-mannoside	--	--	D-arabitolD-arabitol	--	--
α-methly-D-glucosideα-methly-D-glucoside	--	--	L-arabitolL-arabitol	--	--
N-acetyl-glucosamineN-acetyl-glucosamine	++	++	gluconategluconate	--	--
amygdalinamygdalin	--	--	2-keto-gluconate2-keto-gluconate	--	--
arbutinarbutin	--	--	5-keto-gluconate5-keto-gluconate	--	--
esculinesculin	--	--

2) Pridmore RD, Berger B, Desiere F, Vilanova D, Barretto C, Pittet AC, Zwahlen MC, Rouvet M, Altermann E, Barrangou R, Mollet B, Mercenier A, Klaenhammer T, Arigoni F, Schell MA. The genome sequence of the probiotic intestinal bacterium Lactobacillus johnsonii NCC 533. Proc Natl Acad Sci U S A. 2004 Feb 24; 101 (8): 2512-7

탄소원Carbon source	균주명Strain name		탄소원Carbon source	균주명Strain name
탄소원Carbon source	B. longumB. longum ³⁾³⁾	B. longum CH57 B. longum CH57	탄소원Carbon source	B. longumB. longum ³⁾³⁾	B. longum CH57 B. longum CH57
glycerolglycerol	±±	--	salicinsalicin	±±	--
erythritolerythritol	--	--	cellobiosecellobiose	±±	±±
D-arabinoseD-arabinose	--	--	maltosemaltose	--	--
L-arabinoseL-arabinose	--	--	lactoselactose	--	--
D-riboseD-ribose	±±	--	melibiosemelibiose	--	--
D-xyloseD-xylose	--	--	sucrosesucrose	++	±±
L-xyloseL-xylose	--	--	trehalosetrehalose	±±	--
D-adonitolD-adonitol	--	--	inulininulin	--	--
methyl-β-D-xylopyranosidemethyl-β-D-xylopyranoside	--	--	melezitosemelezitose	--	--
D-galactoseD-galactose	++	++	raffinoseraffinose	--	--
D-glucoseD-glucose	++	++	starchstarch	--	--
D-fructoseD-fructose	++	++	glycogenglycogen	--	--
D-mannoseD-mannose	--	--	xylitolxylitol	--	--
L-sorboseL-sorbose	--	--	gentiobiosegentiobiose	--	--
L-rhamnoseL-rhamnose	--	--	D-turanoseD-turanose	--	--
dulcitoldulcitol	--	--	D-lyxoseD-lyxose	--	--
inositolinositol	--	--	D-tagatoseD-tagatose	--	--
mannitolmannitol	++	--	D-fucoseD-fucose	--	--
sorbitolsorbitol	--	--	L-fucoseL-fucose	--	--
α-methyl-D-mannosideα-methyl-D-mannoside	--	--	D-arabitolD-arabitol	--	--
α-methly-D-glucosideα-methly-D-glucoside	--	--	L-arabitolL-arabitol	--	--
N-acetyl-glucosamineN-acetyl-glucosamine	±±	--	gluconategluconate	±±	--
amygdalinamygdalin	--	--	2-keto-gluconate2-keto-gluconate	--	--
arbutinarbutin	±±	--	5-keto-gluconate5-keto-gluconate	--	--
esculinesculin	--	--

3) Lukacova D, Karovucova J, Greifova M, Greif G, Sovcikova A, Kohhajdova Z. In vitro testing of selected probiotic characteristics of Lactobacillus plantarum and Bifidobacterium longum. Journal of Food and Nutrition Research 2006; 45: 77-83.

(4) trust information of lactic acid bacteria

The inventors of the present invention deposited the Lactobacillus brevis CH23 on September 1, 2015 to the Korea Microorganism Conservation Center (address: Yurim Building, 45, 2 Ga-gil, Hongje-si, Seodaemun-gu, Seoul, Korea) on September 1, 2015 and consigned KCCM 11762P. I am assigned a number. In addition, the inventors of the present invention patented Lactobacillus johnsonii CH32 to the Korea Microorganism Conservation Center (Address: 45 Yurim Building, 45, 2ga-gil, Hongdae, Seodaemun-gu, Seoul, Korea) on September 1, 2015 KCCM You have been assigned an accession number of 11763P. In addition, the inventors of the present invention patented Bifidobacterium longum CH57 to the Korea Microbiological Conservation Center (address: 45 Yurim Building 45, Honggae, Seojemun-gu, Seoul, Korea) on September 1, 2015 The accession number of KCCM 11764P is given.

2. 유산균의 장 손상 또는 장 누수 개선 효능 평가2. Evaluation of Efficacy of Lactic Acid Bacteria for Intestinal Damage or Intestinal Leakage Improvement

In order to evaluate the intestinal damage or intestinal leakage of lactic acid bacteria isolated from kimchi or human feces, the antioxidant activity of lactic acid bacteria, the inhibitory activity of lipopolysaccharide (LPS) production, beta-glucuronidase (β-) Glucuronidase) inhibitory activity and tight junction protein expression induction activity were measured.

(1) Experimental method

* Antioxidant activity

DPPH (2,2-Diphenyl-1-picrylhydrazyl) was dissolved in ethanol to a concentration of 0.2 mM to prepare a DPPH solution. 0.1 mL of the DPPH solution was added to lactic acid bacteria suspension (1 × 10 ⁸ CFU / mL) or vitamin C solution (1 g / mL) and incubated at 37 ° C. for 20 minutes. The culture was centrifuged at 3000 rpm for 5 minutes to obtain a supernatant. Thereafter, the absorbance of the supernatant was measured at 517 nm, and the antioxidant activity of the lactic acid bacteria was calculated.

* Inhibitory activity of lipopolysaccharide (LPS) production

0.1 g of fresh human feces were suspended in 0.9 ml of sterile saline solution and diluted 100-fold with normal anaerobic medium to prepare fecal suspension. 0.1 ml of the fecal suspension and 0.1 ml of lactic acid bacteria (1 × 10 ⁴ or 1 × 10 ⁵ CFU) were implanted into 9.8 ml of sterile general anaerobic medium (Nissui Pharmaceutical, Japan) and incubated anaerobicly for 24 hours. Thereafter, the culture solution was sonicated for about 1 hour to destroy the outer membrane of bacteria, and centrifuged at 5000 × g to obtain a supernatant. Then, the content of the representative endotoxin LPS (lipopolysaccharide) present in the supernatant was measured by LAL (Limulus Amoebocyte Lysate) assay kit (manufacturer: Cape Cod Inc., USA). In addition, in order to evaluate the E. coli proliferation inhibitory activity of the lactic acid bacteria, the culture solution obtained through the same experiment was diluted 1000 times and 100,000 times, and cultured in DHL medium, and the number of E. coli was measured.

* Beta-glucuronidase (β-glucuronidase) inhibitory activity

0.1 ml of p-nitrophenyl-β-D-glucuronide solution at 0.1 mM concentration, 0.2 ml of 50 mM phosphate buffer solution and 5 ml of lactic acid bacteria suspension (lactic acid bacteria culture medium) After collection, 0.1 ml of the solution was prepared by suspending it in 5 ml of physiological saline. The reaction was performed in a reactor for 15 minutes, followed by beta-glucuronidase enzyme reaction, and 0.5 ml of 0.1 mM NaOH solution was added thereto. The reaction was stopped. Thereafter, the reaction solution was centrifuged at 3000 rpm for 5 minutes to obtain a supernatant. The absorbance of the supernatant was then measured at 405 nm.

* Induced activity of tight junction protein expression

Caco2 cells cultured in the Korean Cell Line Bank were incubated for 48 hours in RPMI 1640 medium, and then Caco2 cell cultures were dispensed in 12-well plates at an amount of 2 × 10 ⁶ cells per well. Thereafter, each well was treated with 1 μg of LPS (lipopolysaccharide) alone or 1 μg of LPS (lipopolysaccharide) and 1 × 10 ³ CFU were incubated together for 24 hours. Then, cells cultured from each well were scraped, and the expression level of tight junction protein ZO-1 was measured by immunoblotting.

(2) Experiment result

Antioxidative activity, inhibitory activity of lipopolysaccharide (LPS) production, β-glucuronidase inhibitory activity, and tight junction protein expression induction activity of lactic acid bacteria isolated from kimchi or human feces It measured and the result is shown in following Table 5 and 6. Table 5 and Lactobacillus ku Yerba As seen in Table 6 tooth (Lactobacillus curvatus) CH5, Lactobacillus four K (Lactobacillus sakei) CH11, Lactobacillus brevis (Lactobacillus brevis) CH23, Lactobacillus zone Sony (Lactobacillus johnsonii) CH32, Bifidobacterium Bifidobacterium pseudocatenulatum) CH38 ronggeom and Bifidobacterium (Bifidobacterium longum ) CH57 lactic acid bacteria have excellent antioxidant activity, strongly inhibited lipopolysaccharide (LPS) production and beta-glucuronidase activity, and strongly induced tight junction protein expression. It was. The lactic acid bacteria have an excellent inhibitory effect on the enzymatic activity of the enterobacteriaceae bacteria related to the antioxidant effect, inflammation and carcinogenesis, and also inhibit the production of endotoxin LPS (lipopolysaccharide) produced by the harmful bacteria of the intestinal flora, as well as tightly coupled proteins ( Induction of tight junction protein may improve intestinal permeability.

관리번호Control Number	균주명Strain name	항산화 활성Antioxidant activity	베타-글루쿠로니다제 저해 활성Beta-glucuronidase inhibitory activity	LPS 생성 억제 활성LPS production inhibitory activity	밀착연접단백질 발현 유도 활성Induced activity of tight junction protein expression
1One	Lactobacillus acidophilus CH1 Lactobacillus acidophilus CH1	++	++	--	--
22	Lactobacillus acidophilus CH2 Lactobacillus acidophilus CH2	++	++	++	--
33	Lactobacillus acidophilus CH3 Lactobacillus acidophilus CH3	++	++	++	--
44	Lactobacillus brevis CH4 Lactobacillus brevis CH4	++	++	--	--
55	Lactobacillus curvatus CH5 Lactobacillus curvatus CH5	++++++	++	++++++	++++
66	Lactobacillus brevis CH6 Lactobacillus brevis CH6	++	++	--	--
77	Lactobacillus casei CH7 Lactobacillus casei CH7	++	++	--	--
88	Lactobacillus planantrum CH8 Lactobacillus planantrum CH8	++	++	--	--
99	Lactobacillus sakei CH9 Lactobacillus sakei CH9	--	++	--	--
1010	Lactobacillus curvatus CH10 Lactobacillus curvatus CH10	--	++	--	--
1111	Lactobacillus sakei CH11 Lactobacillus sakei CH11	++++++	++	++++++	++++
1212	Lactobacillus curvatus CH12 Lactobacillus curvatus CH12	--	++	--	++
1313	Lactobacillus plantarum CH13 Lactobacillus plantarum CH13	--	++	--	--
1414	Lactobacillus fermentum CH14 Lactobacillus fermentum CH14	--	++	--	--
1515	Lactobacillus fermentum CH15 Lactobacillus fermentum CH15	++++++	++	++++	--
1616	Lactobacillus gasseri CH16 Lactobacillus gasseri CH16	++	++	--	--
1717	Lactobacillus paracasei CH17 Lactobacillus paracasei CH17	++	++	--	--
1818	Lactobacillus helveticus CH18 Lactobacillus helveticus CH18	++	++	--	--
1919	Lactobacillus helveticus CH19 Lactobacillus helveticus CH19	++	++	--	--
2020	Lactobacillus johnsonii CH20 Lactobacillus johnsonii CH20	++	++	--	++
2121	Lactobacillus johnsonii CH21 Lactobacillus johnsonii CH21	++	++	--	++
2222	Lactobacillus johnsonii CH22 Lactobacillus johnsonii CH22	++	++	--	++
2323	Lactobacillus brevis CH23 Lactobacillus brevis CH23	++++++	++	++++	++++
2424	Lactobacillus paracasei CH24 Lactobacillus paracasei CH24	++	++	--	--
2525	Lactobacillus kimchi CH25 Lactobacillus kimchi CH25	++	++	--	--
2626	Lactobacillus gasseri CH26 Lactobacillus gasseri CH26	++	++	--	--
2727	Lactobacillus paracasei CH27 Lactobacillus paracasei CH27	++	++	--	++
2828	Lactobacillus pentosus CH28 Lactobacillus pentosus CH28	++	++	--	--
2929	Lactobacillus pentosus CH29 Lactobacillus pentosus CH29	++	++	--	--
3030	Lactobacillus reuteri CH30 Lactobacillus reuteri CH30	++	--	--	--

관리번호Control Number	균주명Strain name	항산화 활성Antioxidant activity	베타-글루쿠로니다제 저해 활성Beta-glucuronidase inhibitory activity	LPS 생성 억제 활성LPS production inhibitory activity	밀착연접단백질 발현 유도 활성Induced activity of tight junction protein expression
3131	Lactobacillus sakei CH31 Lactobacillus sakei CH31	--	++	--	++
3232	Lactobacillus johnsonii CH32 Lactobacillus johnsonii CH32	++++++	++	++++	++++
3333	Lactobacillus sakei CH33 Lactobacillus sakei CH33	++	++	--	++
3434	Lactobacillus sakei CH34 Lactobacillus sakei CH34	++	++	--	++
3535	Lactobacillus plantarum CH35 Lactobacillus plantarum CH35	++	++	++	++
3636	Lactobacillus sanfranciscensis CH36 Lactobacillus sanfranciscensis CH36	++	++	++	++
3737	Bifidobacterium pseudocatenulatum CH37 Bifidobacterium pseudocatenulatum CH37	--	++	--	++
3838	Bifidobacterium pseudocatenulatum CH38 Bifidobacterium pseudocatenulatum CH38	++++++	++	++++	++++
3939	Bifidobacterium adolescentis CH39 Bifidobacterium adolescentis CH39	--	++	--	++
4040	Bifidobacterium adolescentis CH40 Bifidobacterium adolescentis CH40	--	++	++++++	++
4141	Bifidobacterium adolescentis CH41 Bifidobacterium adolescentis CH41	++	++	--	++
4242	Bifidobacterium animalis CH42 Bifidobacterium animalis CH42	++	++	--	--
4343	Bifidobacterium animalis CH43 Bifidobacterium animalis CH43	++	++	--	--
4444	Bifidobacterium bifidum CH44 Bifidobacterium bifidum CH44	++	++	--	--
4545	Bifidobacterium bifidum CH45 Bifidobacterium bifidum CH45	++	++	--	--
4646	Bifidobacterium breve CH46 Bifidobacterium breve CH46	++	--	--	--
4747	Bifidobacterium breve CH47 Bifidobacterium breve CH47	++	++	--	++
4848	Bifidobacterium breve CH48 Bifidobacterium breve CH48	++	++	--	++
4949	Bifidobacterium catenulatum CH49 Bifidobacterium catenulatum CH49	++	++	--	++++
5050	Bifidobacterium catenulatum CH50 Bifidobacterium catenulatum CH50	--	++	--	--
5151	Bifidobacterium dentium CH51 Bifidobacterium dentium CH51	++	--	--	--
5252	Bifidobacterium infantis CH52 Bifidobacterium infantis CH52	--	++	--	--
5353	Bifidobacterium infantis CH53 Bifidobacterium infantis CH53	--	++	--	--
5454	Bifidobacterium infantis CH54Bifidobacterium infantis CH54	++	++	--	--
5555	Bifidobacterium longum CH55 Bifidobacterium longum CH55	++	++	--	++
5656	Bifidobacterium longum CH56 Bifidobacterium longum CH56	++++++	++	++++	++
5757	Bifidobacterium longum CH57 Bifidobacterium longum CH57	++++++	++	++++++	++++
5858	Bifidobacterium longum CH58 Bifidobacterium longum CH58	++	++	++	++
5959	Bifidobacterium longum CH59 Bifidobacterium longum CH59	++	++	++	++
6060	Bifidobacterium longum CH60 Bifidobacterium longum CH60	++	--	++	++

* Final concentration of lactic acid bacteria in antioxidant activity measurement: 1 × 10 ⁴ CFU / mL; Graft concentration of lactic acid bacteria in measuring beta-glucuronidase inhibitory activity and lipopolysaccharide (LPS) production inhibitory activity: 1 × 10 ⁴ CFU / ml; Lactobacillus concentration when measuring tight junction protein expression-inducing activity: 1 × 10 ⁴ CFU / mL

* Standard for measuring various activities of lactic acid bacteria: very strongly (+++;> 90%); strongly (++;> 60-90%); weakly (+;> 20-60%); not or less than 20% (-; <20%)

3. 유산균의 간 손상 개선 효과 평가3. Evaluation of Lactobacillus Liver Damage Improvement Effect

Lactobacillus curvatus CH5, Lactobacillus sakei CH11, Lactobacillus fermentum CH15, Lactobacillus , Lactobacillus curvatus CH5, Lactobacillus curvatus CH5, Lactobacillus fermentum CH15 Lactobacillus brevis CH23, Lactobacillus johnsonii CH32, Bifidobacterium pseudocatenulatum CH38 and Bifidobacterium longum CH57 were selected and these alone or mixed lactic acid bacteria The liver damage improvement effect was evaluated using various liver damage model animals.

(1) Measurement of the liver damage improvement effect of lactic acid bacteria through a model animal experiment in which liver damage was induced by D-galactosamine

1) Experiment Method

Six mice (C57BL / 6, male) were used as a group, and liver damage was induced by intraperitoneal administration of D-galactosamine at a dose of 800 mg / kg to the experimental animals except the normal group. It was. D-galactosamine was intraperitoneally administered and 2 hours later, lactic acid bacteria were orally administered to the experimental animals of the group except for the normal group and the negative control group in an amount of 1 × 10 ⁹ CFU once a day for 3 days. In addition, the experimental animals of the positive control group was orally administered silymarin (silymarin) once daily for 3 days in an amount of 100 mg / kg instead of lactic acid bacteria. Six hours after the last dose of the drug, the heart was bled. The collected blood was left at room temperature for 60 minutes and centrifuged at 3,000 rpm for 15 minutes to separate serum. GPT (glutamic pyruvate transaminase) and GOT (glutamic oxalacetic transaminase) of the isolated serum were measured using a blood analysis kit (ALT & AST measurement kit; Asan Pharm. Co., South Korea).

In addition, the liver tissue of the experimental animal was removed and the amount of malondialdehyde (MDA) present in the liver tissue was measured. Malondialdehyde is an indicator of lipid peroxidation. Specifically, 16 g of RIPA solution (0.21 M Mannitol, 0.1 M EDTA-2Na, 0.07 M Sucrose, 0.01 M Trizma base) was added to 0.5 g of the liver tissue, and then homogenized using a homogenizer. . The homogenate was again centrifuged at 3,000 rpm for 10 minutes to obtain liver homogenate. 0.4 ml of 10% SDS was added to 0.5 ml of liver homogenate, incubated at 37 ° C. for 30 minutes, cooled, and then added 3 ml of 1% phosphate buffer and 1 ml of 0.6% TBA, followed by 45 minutes in a 100 ° C. water bath. It developed by heating. 4 ml of n-butanol was added to the developed sample solution, mixed, and centrifuged at 3000 rpm for 10 minutes to obtain a supernatant. The absorbance of the obtained supernatant was measured at 535 nm to quantify MDA. In addition, the calibration curve for MDA measurement was prepared using 1,1,3,3-tetraethoxypropane.

2) Experiment result

1 is a graph showing the change in GOT value when lactic acid bacteria are administered to a model animal induced liver damage by D-galactosamine (D-Galactosamine), Figure 2 is a D-galactosamine (D-Galactosamine) It is a graph showing the change of GPT value when the lactic acid bacteria were administered to a model animal that caused liver damage, and FIG. 3 is an MDA when lactic acid bacteria were administered to a model animal induced by liver damage by D-galactosamine. A graph showing the change in value. 1 to 3, "Nor" on the X axis represents a normal group, and "Con" represents a negative control group that is not administered a separate drug to a model animal in which liver damage is induced by D-galactosamine. "Ch11" represents the Lactobacillus sakei CH11 administration group, "ch15" represents the Lactobacillus fermentum CH15 administration group, and "ch23" represents the Lactobacillus brevis CH23 administration group. "Ch32" represents Lactobacillus johnsonii CH32 administration group, and "ch38" represents Bifidobacterium pseudocatenulatum ( Bifidobacterium). pseudocatenulatum) represents a group CH38, "ch57" is ronggeom Bifidobacterium (Bifidobacterium longum) denotes a group CH57, "ch57 + ch11" is ronggeom Bifidobacterium (Bifidobacterium longum ) represents the administration group of mixed lactic acid bacteria prepared by mixing the same amount of CH57 and Lactobacillus sakei CH11, "ch57 + ch23" Bifidobacterium longum CH57 and Lactobacillus brevis ( Lactobacillus brevis) ) Represents the administration group of the mixed lactic acid bacteria prepared by mixing the same amount of CH23, "ch57 + ch32" is a mixture made by mixing the same amount of Bifidobacterium longum CH57 and Lactobacillus johnsonii CH32 The group showing the administration of lactic acid bacteria, "SM" represents a positive control group administered silymarin (silymarin) instead of lactic acid bacteria.

Also in model animals GOT, GPT, MAD value increased by liver damage as shown in Figs. 1 to 3 Lactobacillus brevis (Lactobacillus brevis) CH23, Lactobacillus zone Sony (Lactobacillus johnsonii) CH32 and Bifidobacterium ronggeom (Bifidobacterium longum ) CH57 improved liver damage, especially Bifidobacterium long lum) Lactobacillus brevis CH23 mixed lactic acid bacteria or Bifidobacterium longum CH57 and Lactobacillus johnsonii CH32 when mixed lactic acid bacteria significantly improved liver damage. In addition, certain lactic acid bacteria or mixed lactic acid bacteria selected from them had better liver damage improvement effect than silymarin (silymarin) used as a drug for treating liver damage. From the above results, it can be seen that the specific lactic acid bacteria or mixed lactic acid bacteria selected therefrom are useful materials for improving fatty liver induced by alcohol and high fat diet or liver disease resulting from oxidative stress.

(2) Measurement of the liver damage improvement effect of lactic acid bacteria through a model animal experiment in which liver damage was induced by Tert-butylperoxide.

1) Experiment Method

Six rats (C57BL / 6, male) were used as a group, and tert-butylperoxide was dosed at a dose of 2.5 mmol / kg to experimental animals of the other groups except the normal group for liver damage. Induced. After 2 hours of tert-butylperoxide administration, lactic acid bacteria were orally administered to the experimental animals of the group except the normal group and the negative control group once a day for 3 days in an amount of 2 × 10 ⁹ CFU. In addition, the experimental animals of the positive control group was orally administered silymarin (silymarin) once daily for 3 days in an amount of 100 mg / kg instead of lactic acid bacteria. Six hours after the last dose of the drug, the heart was bled. The collected blood was left at room temperature for 60 minutes and centrifuged at 3,000 rpm for 15 minutes to separate serum. GPT (glutamic pyruvate transaminase) and GOT (glutamic oxalacetic transaminase) of the isolated serum were measured using a blood analysis kit (ALT & AST measurement kit; Asan Pharm. Co., South Korea).

2) Experiment result

Table 7 below shows the changes in GOT and GPT values when lactic acid bacteria were administered to a model animal induced by liver damage by Tert-butylperoxide. As shown in Table 7, Lactobacillus brevis CH23, Lactobacillus johnsonii CH32, and Bifidobacterium longum CH57 showed better liver damage improvement effect than silymarin, ronggeom Bifidobacterium (Bifidobacterium longum) CH57 and Lactobacillus brevis (Lactobacillus brevis) mixing lactic acid bacteria or Bifidobacterium ronggeom of CH23 (Bifidobacterium longum) mixing lactic acid bacteria and Lactobacillus CH57 John Sony (Lactobacillus johnsonii) CH32 was better improve liver damage effects.

실험군Experimental group	GOT(IU/L)GOT (IU / L)	GPT(IU/L)GPT (IU / L)
정상군Normal	36.136.1	26.326.3
음성 대조준Negative contrast	84.184.1	96.196.1
CH23 투여군CH23 administration group	58.058.0	74.274.2
CH32 투여군CH32 administration group	53.053.0	70.570.5
CH57 투여군CH57 administration group	57.657.6	71.271.2
CH57+CH23 투여군CH57 + CH23 administration group	48.648.6	64.364.3
CH57+CH32 투여군CH57 + CH32 administration group	51.251.2	68.468.4
실리마린(silymarin) 투여군Silymarin administration group	61.761.7	69.169.1

In Table 7 "CH23" is Lactobacillus brevis (Lactobacillus brevis) show a CH23, "CH32" is Lactobacillus zone Sony (Lactobacillus johnsonii) represents a CH32 "CH57" is ronggeom Bifidobacterium (Bifidobacterium longum) represents a CH57, "CH57 CH23 +" is ronggeom Bifidobacterium (Bifidobacterium longum ) Lactic acid bacteria prepared by mixing the same amount of CH57 and Lactobacillus brevis CH23 Represents, "CH57 + CH32" is ronggeom Bifidobacterium (Bifidobacterium longum ) represents a mixed lactic acid bacterium prepared by mixing the same amount of CH57 and Lactobacillus johnsonii CH32.

4. 유산균의 알러지 개선 효과 평가4. Evaluation of allergic effect of lactic acid bacteria

(1) Degranulation inhibition rate measurement of lactic acid bacteria

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

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

Five groups of BALB / c mice were used as a group, and the test drug lactic acid bacterium was orally administered once a day for 3 days in a quantity of 1 × 10 ⁹ CFU to the control group except the control group and the control group, or DSCG (disodium cromoglycate) as a control drug. Alternatively, Azelastine was orally administered once every 3 days in an amount of 0.2 mg / mouse. One hour after the last oral administration of the drug, the mice were left in the observation box (24cm × 22cm × 24cm) for 10 minutes to purify the environment, and the hairs on the back of the head (back of the neck) were removed. Subsequently, normal mice were injected with saline, and other experimental mice were injected with a pruritic inducer (50 μg of compound 48/80; Sigma, USA) using a 29 gauge needle. Thereafter, mice were immediately isolated into observation boxes one by one and recorded for 1 hour with an 8-mm video camera (SV-K80, Samsung) under unmanned conditions to observe the behavior. It was admitted that scratching the injection site with the hind paw was not permitted.

(3) Measurement of capillary permeability inhibition rate of lactic acid bacteria

It is known that capillary permeability increases at the site of pruritus. In this experiment, it was performed to see if the lactic acid bacteria according to the present invention can effectively inhibit capillary permeability induced by various compounds. Drugs were administered to the same mouse in the same manner as in the previous antipruritic activity measurement experiment. Subsequently, physiological saline was injected subcutaneously into the cervical back region of the normal group mouse, and a pruritic inducer (50 μg of compound 48/80; Sigma, USA) was injected into the cervical back region of another experimental group mouse. Thereafter, 0.2 ml of a 1% Evans blue solution (Sigma, USA) was administered intravenously and mice were killed one hour later. Subsequently, the skin of the subcutaneous injection site was cut and placed in 1 ml of 1N KOH, and then reacted at 37 ° C. the next day. Then, 4 ml of 0.6N phosphate-acetone (5:13) mixed solution was added and mixed, followed by mixing at 3000 rpm. After centrifugation for 15 minutes, the supernatant was taken and the absorbance was measured at 620 nm. Capillary permeability inhibition rate (%) was calculated by the following equation.

% Inhibition = {1- [absorbance at sites treated with drug and pruritus-absorbance at sites not treated with antipruritic] / [absorbance at sites treated with antipruritic-absorbance at sites not treated with antipruritic]} × 100

(4) Experiment result

Table 8 is a result of measuring the degranulation inhibition rate, pruritus inhibition rate and capillary permeability inhibition rate of lactic acid bacteria. In Table 8, "CH5" represents Lactobacillus curvatus CH5, "CH11" represents Lactobacillus sakei CH11, and "CH15" represents Lactobacillus fermentum CH15. "CH23" represents Lactobacillus brevis CH23, "CH32" represents Lactobacillus johnsonii CH32, and "CH38" represents Bifidobacterium pseudocatenulatum ( Bifidobacterium pseudocatenulatum ). represents a CH38, "CH57" is ronggeom Bifidobacterium (Bifidobacterium longum) represents a CH57, "CH57 CH11 +" is ronggeom Bifidobacterium (Bifidobacterium longum) CH57 and Lactobacillus four K (Lactobacillus sakei) represents a mixture of lactic acid bacteria prepared by mixing with an equal volume CH11, "CH57 CH23 +" is ronggeom Bifidobacterium (Bifidobacterium longum) CH57 and Lactobacillus brevis (Lactobacillus brevis) represents a mixture of lactic acid bacteria prepared by mixing with an equal volume CH23, "CH57 CH32 +" is ronggeom Bifidobacterium (Bifidobacterium longum ) represents a mixed lactic acid bacterium prepared by mixing the same amount of CH57 and Lactobacillus johnsonii CH32.

As shown in Table 8, Lactobacillus curvatus CH5, Lactobacillus brevis CH23, Lactobacillus johnsonii CH32 and Bifidobacterium longum CH57 were dephilized Bifidobacterium longum (CH57) inhibited pruritus and capillary permeability very strongly. In addition, lactic acid bacteria alone rather than a mixture of these lactic acid bacteria, especially Bifidobacterium ronggeom (Bifidobacterium longum) CH57 and Lactobacillus brevis (Lactobacillus brevis) CH23 mixing lactic acid bacteria or Bifidobacterium ronggeom (Bifidobacterium of longum ) showed higher degranulation inhibition, pruritus inhibition and capillary permeability inhibition in the mixed lactic acid bacteria of CH57 and Lactobacillus johnsonii CH32. Thus, the lactic acid bacteria or lactic acid bacteria mixture can very effectively improve atopic dermatitis, asthma, sore throat or chronic dermatitis caused by allergy.

처리 약물Treatment medication	저해율(%)% Inhibition
처리 약물Treatment medication	탈과립Degranulation	소양 반응Pruritic reaction	모세혈관 투과성 Capillary permeability

없음none	00	22	1One
CH5 CH5	5353	4646	4545
CH11CH11	4747	4646	4545
CH15CH15	4848	4242	4242
CH23CH23	5454	4747	4747
CH32CH32	5252	4545	4646
CH38CH38	4444	4545	4242
CH57CH57	5555	5555	5252
CH57+CH11CH57 + CH11	5959	5656	5454
CH57+CH23CH57 + CH23	6363	6262	6161
CH57+CH32CH57 + CH32	6161	5858	5656
DSCG(disodium cromoglycate)DSCo (disodium cromoglycate)	6262	2525	3737
AzelastineAzelastine	--	6565	6868

5. 유산균의 항염 및 장 누수 억제 효과 평가(in vitro)5. Evaluation of anti-inflammatory and intestinal leakage inhibitory effect of lactic acid bacteria (in vitro)

(1) Isolation of Dendritic Cells and Measurement of Inflammatory Indicators

Immune cells were isolated from the bone marrow of C57BL / 6 mice (male, 20-23 g) using RPMI 1640 containing 10% FBS, 1% antibiotics, 1% glutamax, and 0.1% mercaptoethanol, and treated with RBC lysis buffer. After washing, each well of the 24-well-plate was dispensed and treated with GM-CSF and IL-4 in a ratio of 1: 1000 and incubated. The culture medium was replaced with fresh medium on the fifth day of culture, and collected on the eighth day, and used as dendritic cells. Subsequently, the dendritic cells were laid in a 24-well plate at a number of 0.5 × 10 ⁶ per well, and the supernatant and the cells were obtained after treating the test substance lactic acid bacteria and the inflammatory response inducing substance LPS (lipopolysaccharide) for 2 hours or 24 hours. It was. The expression levels of IL-10 and IL-12 were measured from the obtained supernatant by immunoblotting.

Figure 4 is a graph showing the effect of the lactic acid bacteria selected in the present invention on the inflammatory response of dendritic cells induced by LPS (lipopolysaccharide). 4 is a graph showing the effect of lactic acid bacteria on cells not treated with LPS (lipopolysaccharide), and the graph on the right is a graph showing the effect of lactic acid bacteria on cells treated with LPS (lipopolysaccharide). In addition, in Fig. 4, "Nor" on the X-axis represents a case in which the test substance is not treated with lactic acid bacteria and an inflammatory response inducing substance LPS (lipopolysaccharide), and "LPS" is treated with an inflammatory response inducing substance LPS (lipopolysaccharide). "Ch11" represents the Lactobacillus sakei CH11 treatment group, "ch15" represents the Lactobacillus fermentum CH15 treatment group, and "ch23" represents the Lactobacillus brevis . CH23 treatment group, "ch32" represents Lactobacillus johnsonii CH32 treatment group, "ch38" represents Bifidobacterium pseudocatenulatum CH38 treatment group, "ch57" ronggeom Bifidobacterium (Bifidobacterium longum) denotes a group CH57 treatment, "ch57 + ch11" is ronggeom Bifidobacterium (Bifidobacterium longum) CH57 and Lactobacillus four K (Lactobacillus sakei) represents the group treated with a mixture of lactic acid bacteria prepared by mixing with an equal volume CH11, "ch57 + ch23" is ronggeom Bifidobacterium (Bifidobacterium longum) CH57 and Lactobacillus brevis (Lactobacillus brevis) represents the group treated in a mixture of lactic acid bacteria prepared by mixing with an equal volume CH23, "ch57 + ch32" is Bifidobacterium ronggeom (Bifidobacterium longum) CH57 and Lactobacillus zone Sony (Lactobacillus johnsonii ) shows a treatment group of mixed lactic acid bacteria prepared by mixing CH32 in the same amount.

As shown in FIG. 4, Lactobacillus sakei CH11, Lactobacillus brevis CH23, and Lactobacillus johnsonii CH32 induced IL-10 production of dendritic cells differentiated and differentiated from bone marrow It effectively inhibited the production of IL-12 induced by lipopolysaccharide (LPS), and Bifidobacterium longgum ( Bifidobacterium) longum ) The effect was increased by use with CH57. In particular, the mixed lactic acid bacteria of Bifidobacterium longum CH57 and Lactobacillus brevis CH23 had the best inhibitory effect on inflammatory response. Since regulating dendritic cells can effectively control Treg cells (Regulatory T cells), the lactic acid bacteria selected in the present invention can effectively improve chronic inflammatory diseases such as colitis, autoimmune diseases such as rheumatoid arthritis, etc. have.

(2) Isolation of macrophages and measurement of inflammatory markers

Six-week-old C57BL / 6J male mice (20-23 g) were purchased from Roundbio. 2 ml of sterile 4% thioglycolate was injected into the abdominal cavity of the mouse, anesthetized the mouse after 96 hours, and again 8 ml of RPMI 1640 medium was injected into the abdominal cavity of the mouse, and 5 to 10 minutes later, RPMI medium in the mouse abdominal cavity (large Phagocytic cells) were extracted again, centrifuged at 1000 rpm for 10 minutes, and washed twice with RPMI 1640 medium. Macrophages were placed in a 24-well plate at a number of 0.5 × 10 ⁶ per well, and supernatants and cells were obtained after treatment with lactic acid bacteria, a test substance, and LPS (lipopolysaccharide), an inflammatory response-inducing substance, for 2 hours or 24 hours. The obtained cells were placed in a buffer (Gibco) and homogenized. From the obtained supernatant, the expression levels of cytokines such as TNF-α and IL-1β were measured by 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, 50 ㎍ of the supernatant was electrophoresed for 1 hour and 30 minutes in SDS 10% (w / v) polyacrylamide gel. The electrophoretic sample was transferred to nitrocellulose paper for 1 hour and 10 minutes at 100 V and 400 kPa. The sample was blocked with 5% skim milk for 30 minutes after transferring the transferred nitrocellulose paper, and washed with PBS-Tween three times for 5 minutes and the primary antibody (Santa Cruz Biotechnology, USA) at a ratio of 1: 100. The reaction was overnight. Thereafter, the mixture was washed three times for 10 minutes, and reacted with a secondary antibody (Santa Cruz Biotechnology, USA) at a ratio of 1: 1000 for 1 hour and 20 minutes. Thereafter, the mixture was washed three times for 15 minutes, and developed after fluorescence.

5 is Bifidobacterium long gum ( Bifidobacterium) longum ) is a graph showing the effect of CH57 on the inflammatory response of macrophage induced by LPS (lipopolysaccharide). As shown in Figure 5 Bifidobacterium longum ( Bifidobacterium longum ) CH57 effectively inhibited the inflammatory response induced by LPS (lipopolysaccharide).

(3) Isolation of T Cells from the Spleen and Differentiation Capability into Th17 Cells or Treg Cells

Spleens of C56BL / 6J mice were isolated, crushed, suspended, suspended in RPMI 1640 exclusion with 10% FCS and CD4 T cells were isolated using a CD4 T cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Separated CD4 T cells were dispensed in 12-well plates at 5 × 10 ⁵ cells per well, containing anti-CD3 (1 μg / ml, Miltenyi Biotec, Bergisch Gladbach, Germany) and anti-CD28 (1 μg / ml, Miltenyi Biotec). , Bergisch Gladbach, Germany), anti-CD3 (1 μg / ml, Miltenyi Biotec, Bergisch Gladbach, Germany), anti-CD28 (1 μg / ml, Miltenyi Biotec, Bergisch Gladbach, Germany), recombinant IL-6 (20 ng) / Ml, MiltenyiBiotec, Bergisch Gladbach, Germany) and recombinant transforming growth factor beta (1 ng / ml, MiltenyiBiotec, Bergisch Gladbach, Germany), incubating the cells and adding lactic acid bacteria to 1 × 10 ³ or 1 × 10 ⁵ CFU per well. Incubated for 4 days. Subsequently, the cells of the culture were stained with anti-FoxP3 or anti-IL-17A antibody, and the distribution of Th17 and Tregs using a Fluorescence-activated cell sorting (FACS) device (C6 Flow Cytometer® System, San Jose, CA, USA). Was analyzed.

6 is a result of analyzing the effect of Lactobacillus brevis CH23 on the differentiation of T cells isolated from the spleen into Th17 cells or Treg cells with a Fluorescence-activated cell sorting (FACS) device. As shown in FIG. 6, Lactobacillus brevis CH23 inhibited the differentiation of T cells into Th17 cells (T helper 17 cells) and promoted the differentiation into Treg cells. Based on the above results, Lactobacillus brevis CH23 can effectively improve inflammatory diseases such as colitis and arthritis.

(4) Measurement of the Effect of Lactic Acid Bacteria on the Expression of ZO-1 Protein in CaCO2 Cells

Caco2 cells cultured in the Korean Cell Line Bank were incubated for 48 hours in RPMI 1640 medium, and then Caco2 cell cultures were dispensed in 12-well plates at an amount of 2 × 10 ⁶ cells per well. Then, each well was treated with 1 μg of LPS (lipopolysaccharide) alone or 1 μg of LPS (lipopolysaccharide) and lactic acid bacteria 1 × 10 ³ CFU or 1 × 10 ⁵ CFU and then incubated for 24 hours. Then, cells cultured from each well were scraped, and the expression level of tight junction protein ZO-1 was measured by immunoblotting.

7 is a result of analyzing the effect of Lactobacillus brevis CH23, Bifidobacterium longum CH57 or their mixed lactic acid bacteria on the expression of ZO-1 protein of CaCO2 cells. In Figure 7, "CH23" represents Lactobacillus brevis CH23, "CH57" represents Bifidobacterium longum CH57, and "mix" represents Bifidobacterium longum CH57 and Lactobacillus johnsonii represents a mixed lactic acid bacterium prepared by mixing the same amount of CH32. As shown in Figure 7 Lactobacillus brevis ( Lactobacillus brevis ) CH23 and Bifidobacterium long gum ( Bifidobacterium longum ) CH57 increased the expression of tight junction protein ZO-1 and treated with mixed lactic acid bacteria of Bifidobacterium longum CH57 and Lactobacillus johnsonii CH32. The expression of tight junction protein ZO-1 increased synergistically. Increasing the expression of tight junction proteins can block the entry of toxic substances into the body, preventing colitis, arthritis, and exacerbation of liver damage.

6. 유산균의 항염 및 대장염 개선 효과 평가(in vivo)6. Evaluation of anti-inflammatory and colitis improvement effects of lactic acid bacteria (in vivo)

(1) experimental animals

Five-week-old C57BL / 6 male mice (24-27 g) were purchased from Orient Bio Co., Ltd., with controlled humidity conditions of 50 ± 10%, temperature 25 ± 2 ° C., lighting for 12 hours, and turning off for 12 hours. After the breeding was used for the experiment. Feed was used as standard experimental feed (Samyang, Korea) and drinking water was freely consumed. In all experiments, one group was 6 animals.

(2) TNBS-induced colitis and sample administration

One group of test animals was normal, and the other group of test animals induced acute colitis with 2,4,6-trinitrobenzenesulfonic acid (TNBS). Specifically, the animal was lightly anesthetized with ether, and 2.5 g of TNBS (2,4,6-Trinitrobenzene sulfonic acid) solution was mixed with 100 ml of 50% ethanol. Inoculated into the large intestine by 0.1ml and maintained vertically for 30 seconds to cause inflammation. In contrast, the normal group was orally administered 0.1 ml of saline. Afterwards, the test sample was suspended in physiological saline once a day for 3 days from the next day, orally in the amount of 2.0 × 10 ⁹ CFU. The day after the administration of the sample, the animal was smothered with carbon dioxide and killed. A large intestine from the middle caecum to the area immediately before the anus was extracted and used. In addition, only normal saline was administered orally to physiological saline instead of lactic acid bacteria. In addition, the experimental animals of the negative control group was orally administered only saline instead of lactic acid bacteria after induction of colitis caused by TNBS. In addition, the experimental animals of the positive control group were orally administered sulfasalazine (sulfasalazine), a colitis treatment drug, in an amount of 50 mg / kg instead of lactic acid bacteria.

(3) appearance analysis of the large intestine

The length and appearance of the extracted colon were observed and analyzed by appearance according to the criteria of Table 9 below (Hollenbach et al., 2005 criteria for colitis). The colon tissues were removed from the colon contents, washed in physiological saline, and some were fixed with 4% formaldehyde fixative for use as pathological samples, and the rest were frozen and stored at minus 80 ° C for molecular biological analysis. It was.

외관 점수(Macroscopic Score) Macroscopic Score		기준standard
00	어떠한 궤양과 염증도 발견되지 않음No ulcers and inflammations found
1One	출혈이 없는 충혈이 발견됨Congestion without bleeding is found
22	충혈이 있는 궤양이 발견됨Ulcers with congestion found
33	한 곳에서만 궤양과 염증이 발견됨Ulcers and inflammations only found in one place
44	궤양과 염증이 2곳 이상에서 발견됨Ulcers and inflammations found in more than one place
55	궤양이 2㎝ 이상으로 확대되어 있음Ulcers that extend beyond 2 cm

(4) Determination of Myeloperoxidase (MPO) Activity

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

(5) Inflammatory index measurement

Western blotting was used to measure inflammatory response markers such as p-p65, p65, iNOS, COX-2, and β-actin. Specifically, the supernatant was obtained in the same manner as the myeloperoxidase (MPO) activity measurement experiment. Then, 50 ㎍ of the supernatant was electrophoresed for 1 hour and 30 minutes in SDS 10% (w / v) polyacrylamide gel. The electrophoretic sample was transferred to nitrocellulose paper for 1 hour and 10 minutes at 100 V and 400 kPa. The sample was blocked with 5% skim milk for 30 minutes after transferring the transferred nitrocellulose paper, and washed with PBS-Tween three times for 5 minutes and the primary antibody (Santa Cruz Biotechnology, USA) at a ratio of 1: 100. The reaction was overnight. Thereafter, the mixture was washed three times for 10 minutes, and reacted with a secondary antibody (Santa Cruz Biotechnology, USA) at a ratio of 1: 1000 for 1 hour and 20 minutes. Thereafter, the mixture was washed three times for 15 minutes, and developed after fluorescence.

In addition, inflammation-related cytokines such as TNF-α and IL-1β were measured using an ELISA kit.

(6) Experiment result

FIG. 8 shows Bifidobacterium longgum for model animals in which acute colitis is induced by TNBS.Bifidobacterium longum) The effect of CH57 is shown by the appearance of colon or myeloperoxidase (MPO) activity, etc., FIG. 9 shows Bifidobacterium longgum for a model animal induced by acute colitis by TNBS.Bifidobacterium longumThe effect of CH57 is shown in the histological picture of the large intestine.Bifidobacterium longum) The effects of CH57 are shown on cytokines related to inflammation. 8 to 10, "NOR" represents a normal group, "TNBS" represents a negative control, "CH57" represents a Bifidobacterium long gum (Bifidobacterium longum) CH57 administration group, "SS50" represents a sulfasalazine administration group. As shown in FIGS. 8 to 10, Bifidobacterium long gum (Bifidobacterium longumCH57 has been shown to effectively improve colitis based on the body weight, colitis index, colon length, myeloperoxidase (MPO) activity of model animals induced by acute colitis induced by TNBS, and more effective than sulfasalazine. Found to be excellent. In addition, Bifidobacterium longgum (Bifidobacterium longumCH57 inhibited inflammatory cytokine production and increased the production of the anti-inflammatory cytokine IL-10 in model animals in which acute colitis was induced by TNBS.

FIG. 11 illustrates the effect of Lactobacillus brevis CH23 on model animals induced by acute colitis induced by TNBS by the appearance of colon or myeloperoxidase (MPO) activity, and FIG. 12 shows TNBS. The effect of Lactobacillus brevis CH23 on model animals induced by acute colitis is shown by histological picture of the intestine. FIG. 13 shows Lactobacillus brevis (T. Lactobacillus brevis ) shows the effect of CH23 on the differentiation of T cells, Figure 14 shows the effect of Lactobacillus brevis (CH23) on model animals induced by acute colitis induced by TNBS to inflammation-related cytokines and the like. It is shown. 11 to 14, "N" represents a normal group, "TNBS" represents a negative control group, "CH23" represents a Lactobacillus brevis CH23 administration group, and "SS" represents a sulfasalazine administration group. Indicates. As shown in FIGS. 11 to 14, Lactobacillus brevis CH23 is based on body weight, colitis index, colon length, myeloperoxidase (MPO) activity, etc. of a model animal induced by acute colitis by TNBS. It has been shown to effectively improve colitis and is better than sulfasalazine. In addition, Lactobacillus brevis CH23 inhibited the differentiation of T cells into Th17 cells and induced the differentiation into Treg cells in model animals in which acute colitis was induced by TNBS. In addition, Lactobacillus brevis CH23 inhibited inflammatory cytokine production and increased production of the anti-inflammatory cytokine IL-10 in model animals induced by acute colitis by TNBS.

Figure 15 shows the effect of mixed lactic acid bacteria of Bifidobacterium longum CH57 and Lactobacillus brevis CH23 in acute colitis induced by TNBS in the appearance of colon or myeloperoxidase (Myeloperoxidase) , MPO) activity, and FIG. 16 shows Bifidobacterium longgum ( Bifidobacterium) for a model animal induced by acute colitis by TNBS. longum ) shows the effect of mixed lactic acid bacteria of CH57 and Lactobacillus brevis CH23 in the histological picture of the large intestine, Figure 17 Bifidobacterium long gum ( Bifidobacterium) for the model animal induced acute colitis by TNBS longum ) The effect of mixed lactic acid bacteria of CH57 and Lactobacillus brevis CH23 is shown by cytokines related to inflammation. 15 to 17, "NOR" represents a normal group, "TNBS" represents a negative control, "BL" is Bifidobacterium long gum ( Bifidobacterium longum ) represents the administration group of the mixed lactic acid bacteria prepared by mixing the same amount of CH57 and Lactobacillus brevis CH23, "SS50" represents a sulfasalazine administration group. Bifidobacterium long gum as shown in FIGS. 15 to 17 longum ) Lactobacillus brevis CH23 mixed lactobacillus was observed in reduced weight, increased colitis index, shortened colon length, and increased myeloperoxidase (MPO) activity in model animals induced by acute colitis induced by TNBS. And significantly improved colitis than sulfasalazine. In addition, Lactobacillus mixed with Bifidobacterium longum CH57 and Lactobacillus brevis CH23 significantly inhibited the production of inflammatory cytokines and suppressed anti-inflammatory cytokine in model animals induced by acute colitis by TNBS. The production of IL-10 has been dramatically increased.

7. 유산균의 비만 개선 및 항염 효과 평가(in vivo)7. Evaluation of anti-inflammatory and anti-inflammatory effects of lactic acid bacteria (in vivo)

(1) Experimental method

C57BL6 / J mice were purchased from Round Bio Co., Ltd. and a total of 24 mice were acclimated for one week with a chow diet (Purina) under conditions of temperature 20 ± 2 ° C., humidity 50 ± 10%, and 12 hr light / 12 hr dark cycle. Thereafter, the experimental animals were divided into three groups (LFD, HFD, HFD + BL) by eight animals, and the LFD group was fed a normal diet (LFD, 10% of calories from fat; Research, NJ, USA) for 4 weeks, and the HFD group. And HFD + BL group was fed high-fat diet (HFD, 60% of calories from; Research, NJ, USA) for 4 weeks. The LFD group was then fed orally with PBS for 4 weeks. In addition, the HFD group was fed oral PBS at the same time as a high-fat diet for 4 weeks. In addition, the HFD + BL group was fed a high-fat diet for 4 weeks, and the mixed lactic acid bacteria were suspended in PBS orally administered in an amount of 2 × 10 ⁹ CFU. Mixing lactic acid bacteria Bifidobacterium ronggeom (Bifidobacterium longum ) CH57 and Lactobacillus brevis CH23 are prepared by mixing the same amount.

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

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

(3) Experiment result

FIG. 18 illustrates the effects of the mixed lactic acid bacteria of Bifidobacterium longum CH57 and Lactobacillus brevis CH23 on the obesity-induced model animal by weight change amount, and FIG. 19. ronggeom for Bifidobacterium (Bifidobacterium longum ) The effect of the mixed lactic acid bacteria of CH57 and Lactobacillus brevis CH23 is shown by the appearance of the large intestine, myeloperoxidase (MPO) activity, histological picture of the large intestine, and FIG. The effect of mixed lactic acid bacteria of Bifidobacterium longum CH57 and Lactobacillus brevis CH23 is shown in relation to inflammation-related cytokines and the like. FIG. 21 shows Bifidobacterium on obesity-induced model animals. Long Gum ( Bifidobacterium) longum ) The effect of mixed lactic acid bacteria of CH57 and Lactobacillus brevis CH23 is shown as an indicator of inflammatory response. As shown in FIGS. 18 to 21, obesity induced by high fat diet increased body weight, increased colitis index, and increased myeloperoxidase (Myeloperoxidase (MPO)) activity and inhibited the occurrence of colitis. In addition, mixed lactic acid bacteria of Bifidobacterium longum CH57 and Lactobacillus brevis CH23 significantly inhibited the production of inflammatory cytokines and reduced anti-inflammatory cytokines in obese model animals induced by high fat diet. Increased the production of phosphorus IL-10.

Ⅱ. 유산균 선별 및 효능 확인을 위한 2차 실험II. Second experiment for screening lactic acid bacteria and confirming efficacy

(1) Isolation of Lactic Acid Bacteria from Kimchi

Chinese cabbage kimchi, radish kimchi, and green kimchi were respectively crushed, and the crushed liquid was suspended in MRS broth (MRS Broth; Difco, USA). Then, the supernatant was taken and transplanted into MRS agar medium (MRS agar medium; Difco, USA) and incubated anaerobically at 37 ° C. for about 48 hours, after which colony-forming strains were separated according to shape.

(2) Isolation of Lactic Acid Bacteria from Human Feces

Human feces were suspended in GAM liquid medium (GAM broth; Nissui Pharmaceutical, Japan). Subsequently, the supernatant was taken, transplanted into BL agar medium (Nissui Pharmaceutical, Japan), and incubated anaerobicly at 37 ° C. for about 48 hours, and then colony-forming Bifidobacterium longum ( Bifidobacterium longum) ) Strains were isolated.

(3) Identification of selected lactic acid bacteria

Gram staining, physiological characteristics, and 16S rDNA sequences of strains isolated from kimchi or human feces were analyzed to determine the species of the strain and to assign a strain name. Table 10 shows the control numbers and strain names of lactic acid bacteria isolated from Chinese cabbage kimchi, radish kimchi and leek kimchi, Table 11 shows the control numbers and strain names of lactic acid bacteria isolated from feces.

관리번호Control Number	균주명Strain name	관리번호Control Number	균주명Strain name
1One	Lactobacillus plantarum LC1 Lactobacillus plantarum LC1	2626	Lactobacillus plantarum LC26 Lactobacillus plantarum LC26
22	Lactobacillus plantarum LC2 Lactobacillus plantarum LC2	2727	Lactobacillus plantarum LC27 Lactobacillus plantarum LC27
33	Lactobacillus plantarum LC3 Lactobacillus plantarum LC3	2828	Lactobacillus plantarum LC28 Lactobacillus plantarum LC28
44	Lactobacillus plantarum LC4 Lactobacillus plantarum LC4	2929	Lactobacillus plantarum LC29 Lactobacillus plantarum LC29
55	Lactobacillus plantarum LC5 Lactobacillus plantarum LC5	3030	Lactobacillus plantarum LC30 Lactobacillus plantarum LC30
66	Lactobacillus plantarum LC6 Lactobacillus plantarum LC6	3131	Lactobacillus plantarum LC31 Lactobacillus plantarum LC31
77	Lactobacillus plantarum LC7 Lactobacillus plantarum LC7	3232	Lactobacillus plantarum LC32 Lactobacillus plantarum LC32
88	Lactobacillus plantarum LC8 Lactobacillus plantarum LC8	3333	Lactobacillus plantarum LC33 Lactobacillus plantarum LC33
99	Lactobacillus plantarum LC9 Lactobacillus plantarum LC9	3434	Lactobacillus plantarum LC34 Lactobacillus plantarum LC34
1010	Lactobacillus plantarum LC10 Lactobacillus plantarum LC10	3535	Lactobacillus plantarum LC35 Lactobacillus plantarum LC35
1111	Lactobacillus plantarum LC11 Lactobacillus plantarum LC11	3636	Lactobacillus plantarum LC36 Lactobacillus plantarum LC36
1212	Lactobacillus plantarum LC12 Lactobacillus plantarum LC12	3737	Lactobacillus plantarum LC37 Lactobacillus plantarum LC37
1313	Lactobacillus plantarum LC13 Lactobacillus plantarum LC13	3838	Lactobacillus plantarum LC38 Lactobacillus plantarum LC38
1414	Lactobacillus plantarum LC14 Lactobacillus plantarum LC14	3939	Lactobacillus plantarum LC39 Lactobacillus plantarum LC39
1515	Lactobacillus plantarum LC15 Lactobacillus plantarum LC15	4040	Lactobacillus plantarum LC40 Lactobacillus plantarum LC40
1616	Lactobacillus plantarum LC16 Lactobacillus plantarum LC16	4141	Lactobacillus plantarum LC41 Lactobacillus plantarum LC41
1717	Lactobacillus plantarum LC17 Lactobacillus plantarum LC17	4242	Lactobacillus plantarum LC42 Lactobacillus plantarum LC42
1818	Lactobacillus plantarum LC18 Lactobacillus plantarum LC18	4343	Lactobacillus plantarum LC43 Lactobacillus plantarum LC43
1919	Lactobacillus plantarum LC19 Lactobacillus plantarum LC19	4444	Lactobacillus plantarum LC44 Lactobacillus plantarum LC44
2020	Lactobacillus plantarum LC20 Lactobacillus plantarum LC20	4545	Lactobacillus plantarum LC45 Lactobacillus plantarum LC45
2121	Lactobacillus plantarum LC21 Lactobacillus plantarum LC21	4646	Lactobacillus plantarum LC46 Lactobacillus plantarum LC46
2222	Lactobacillus plantarum LC22 Lactobacillus plantarum LC22	4747	Lactobacillus plantarum LC47 Lactobacillus plantarum LC47
2323	Lactobacillus plantarum LC23 Lactobacillus plantarum LC23	4848	Lactobacillus plantarum LC48 Lactobacillus plantarum LC48
2424	Lactobacillus plantarum LC24 Lactobacillus plantarum LC24	4949	Lactobacillus plantarum LC49 Lactobacillus plantarum LC49
2525	Lactobacillus plantarum LC25 Lactobacillus plantarum LC25	5050	Lactobacillus plantarum LC50 Lactobacillus plantarum LC50

관리번호Control Number	균주명Strain name	관리번호Control Number	균주명Strain name
5151	Bifidobacterium longum LC51 Bifidobacterium longum LC51	7676	Bifidobacterium longum LC76 Bifidobacterium longum LC76
5252	Bifidobacterium longum LC52 Bifidobacterium longum LC52	7777	Bifidobacterium longum LC77 Bifidobacterium longum LC77
5353	Bifidobacterium longum LC53 Bifidobacterium longum LC53	7878	Bifidobacterium longum LC78 Bifidobacterium longum LC78
5454	Bifidobacterium longum LC54 Bifidobacterium longum LC54	7979	Bifidobacterium longum LC79 Bifidobacterium longum LC79
5555	Bifidobacterium longum LC55 Bifidobacterium longum LC55	8080	Bifidobacterium longum LC80 Bifidobacterium longum LC80
5656	Bifidobacterium longum LC56 Bifidobacterium longum LC56	8181	Bifidobacterium longum LC81 Bifidobacterium longum LC81
5757	Bifidobacterium longum LC57 Bifidobacterium longum LC57	8282	Bifidobacterium longum LC82 Bifidobacterium longum LC82
5858	Bifidobacterium longum LC58 Bifidobacterium longum LC58	8383	Bifidobacterium longum LC83 Bifidobacterium longum LC83
5959	Bifidobacterium longum LC59 Bifidobacterium longum LC59	8484	Bifidobacterium longum LC84 Bifidobacterium longum LC84
6060	Bifidobacterium longum LC60 Bifidobacterium longum LC60	8585	Bifidobacterium longum LC85 Bifidobacterium longum LC85
6161	Bifidobacterium longum LC61 Bifidobacterium longum LC61	8686	Bifidobacterium longum LC86 Bifidobacterium longum LC86
6262	Bifidobacterium longum LC62 Bifidobacterium longum LC62	8787	Bifidobacterium longum LC87 Bifidobacterium longum LC87
6363	Bifidobacterium longum LC63 Bifidobacterium longum LC63	8888	Bifidobacterium longum LC88 Bifidobacterium longum LC88
6464	Bifidobacterium longum LC64 Bifidobacterium longum LC64	8989	Bifidobacterium longum LC89 Bifidobacterium longum LC89
6565	Bifidobacterium longum LC65 Bifidobacterium longum LC65	9090	Bifidobacterium longum LC90 Bifidobacterium longum LC90
6666	Bifidobacterium longum LC66 Bifidobacterium longum LC66	9191	Bifidobacterium longum LC91 Bifidobacterium longum LC91
6767	Bifidobacterium longum LC67 Bifidobacterium longum LC67	9292	Bifidobacterium longum LC92 Bifidobacterium longum LC92
6868	Bifidobacterium longum LC68 Bifidobacterium longum LC68	9393	Bifidobacterium longum LC93 Bifidobacterium longum LC93
6969	Bifidobacterium longum LC69 Bifidobacterium longum LC69	9494	Bifidobacterium longum LC94 Bifidobacterium longum LC94
7070	Bifidobacterium longum LC70 Bifidobacterium longum LC70	9595	Bifidobacterium longum LC95 Bifidobacterium longum LC95
7171	Bifidobacterium longum LC71 Bifidobacterium longum LC71	9696	Bifidobacterium longum LC96 Bifidobacterium longum LC96
7272	Bifidobacterium longum LC72 Bifidobacterium longum LC72	9797	Bifidobacterium longum LC97 Bifidobacterium longum LC97
7373	Bifidobacterium longum LC73 Bifidobacterium longum LC73	9898	Bifidobacterium longum LC98 Bifidobacterium longum LC98
7474	Bifidobacterium longum LC74 Bifidobacterium longum LC74	9999	Bifidobacterium longum LC99 Bifidobacterium longum LC99
7575	Bifidobacterium longum LC75 Bifidobacterium longum LC75	100100	Bifidobacterium longum LC100 Bifidobacterium longum LC100

Lactobacillus plantarum LC5 shown in Table 10 is an anaerobic bacillus showing positive in Gram staining, and its 16S rDNA was shown to have the nucleotide sequence of SEQ ID NO: 4. The 16S rDNA sequence of Lactobacillus plantarum LC5 was identified by BLAST search of Genebank (http://www.ncbi.nlm.nih.gov/). As a result, Lactobacillus having the same 16S rDNA sequence Lactobacillus plantarum strain was not detected and showed 99% homology with the 16S rDNA sequence of Lactobacillus plantarum strain KF9. In addition, Lactobacillus plantarum LC27 shown in Table 10 is an anaerobic bacillus showing positive upon Gram staining, and its 16S rDNA was shown to have the nucleotide sequence of SEQ ID NO: 5. 16S rDNA sequencing of Lactobacillus plantarum LC27 was identified by BLAST search of Genebank (http://www.ncbi.nlm.nih.gov/). Lactobacillus plantarum strain was not detected and showed 99% homology with the 16S rDNA sequence of Lactobacillus plantarum strain JL18. In addition, Lactobacillus plantarum LC28 shown in Table 10 is an anaerobic bacillus that shows positive upon Gram staining, and its 16S rDNA was shown to have a nucleotide sequence of SEQ ID NO: 6. The 16S rDNA sequence of Lactobacillus plantarum LC28 was identified by BLAST search of Genebank (http://www.ncbi.nlm.nih.gov/). As a result, Lactobacillus having the same 16S rDNA sequence Lactobacillus plantarum strain was not detected and showed 99% homology with the 16S rDNA sequence of Lactobacillus plantarum strain USIM01.

Bifidobacterium longgum shown in Table 11 longum ) LC67 is an anaerobic bacilli that show positive gram staining, and its 16S rDNA was found to have the nucleotide sequence of SEQ ID NO: 7. Ronggeom Bifidobacterium (Bifidobacterium longum ) 16S rDNA sequence of LC67 was identified by BLAST search of Genebank (http://www.ncbi.nlm.nih.gov/), and Bifidobacterium longum having the same 16S rDNA sequence was obtained. No strain was detected, Bifidobacterium It showed 99% homology with the 16S rDNA sequence of longum strain CBT-6. In addition, Bifidobacterium long gum shown in Table 11 longum ) LC68 is an anaerobic bacilli that show positive gram staining, and its 16S rDNA was found to have the nucleotide sequence of SEQ ID NO: 8. Ronggeom Bifidobacterium (Bifidobacterium longum ) 16S rDNA nucleotide sequence of LC68 was identified by BLAST search of Genebank (http://www.ncbi.nlm.nih.gov/) and Bifidobacterium long gum having the same 16S rDNA sequence was identified. longum ) strain was not detected and Bifidobacterium 99% homology with the 16S rDNA sequence of longum strain IMAUFB067.

In addition, Lactobacillus Planta Room (Lactobacillus plantarum) LC5, Lactobacillus Planta Room (Lactobacillus plantarum) LC27, ronggeom Bifidobacterium (Bifidobacterium longum) LC67 ronggeom and Bifidobacterium (Bifidobacterium longum ) Carbon source availability among the physiological characteristics of LC68 was analyzed by sugar fermentation test by API Kit (Model: API 50 CHL; Manufacturer: BioMerieux's, USA). Table 12 is a Lactobacillus Planta room (Lactobacillus plantarum) LC5 and Lactobacillus Planta room (Lactobacillus plantarum) will showing the results of carbon source utilization LC27, Table 13 ronggeom Bifidobacterium (Bifidobacterium longum ) Carbon source availability results of LC67 and Bifidobacterium longum LC68 are shown. In Tables 12 and 13, "+" represents a case where the carbon source availability is positive, "-" represents a case where the carbon source availability is negative, and "±" represents a case where the availability of the carbon source is ambiguous. As shown in Table 12 and Table 13, Lactobacillus plantarum LC5, Lactobacillus plantarum LC27, Bifidobacterium longum LC67 and Bifidobacterium longgum ( Bifidobacterium longgum) longum ) LC68 showed different availability to known strains of the same species for some carbon sources.

탄소원Carbon source	균주명Strain name		탄소원Carbon source	균주명Strain name
탄소원Carbon source	L. plantarum LC5 L. plantarum LC5	L. plantarum LC27 L. plantarum LC27	탄소원Carbon source	L. plantarum LC5 L. plantarum LC5	L. plantarum LC27 L. plantarum LC27
glycerolglycerol	--	--	salicinsalicin	++	++
erythritolerythritol	--	--	cellobiosecellobiose	++	++
D-arabinoseD-arabinose	--	--	maltosemaltose	++	++
L-arabinoseL-arabinose	--	++	lactoselactose	--	++
D-riboseD-ribose	++	++	melibiosemelibiose	++	++
D-xyloseD-xylose	--	--	sucrosesucrose	++	++
L-xyloseL-xylose	--	--	trehalosetrehalose	++	++
D-adonitolD-adonitol	--	--	inulininulin	--	--
methyl-β-D-xylopyranosidemethyl-β-D-xylopyranoside	--	--	melezitosemelezitose	++	++
D-galactoseD-galactose	++	±±	raffinoseraffinose	±±	--
D-glucoseD-glucose	++	++	starchstarch	--	--
D-fructoseD-fructose	++	++	glycogenglycogen	--	--
D-mannoseD-mannose	++	++	xylitolxylitol	--	--
L-sorboseL-sorbose	--	--	gentiobiosegentiobiose	++	++
L-rhamnoseL-rhamnose	--	±±	D-turanoseD-turanose	--	++
dulcitoldulcitol	--	--	D-lyxoseD-lyxose	--	--
inositolinositol	--	--	D-tagatoseD-tagatose	--	--
mannitolmannitol	++	++	D-fucoseD-fucose	--	--
sorbitolsorbitol	++	++	L-fucoseL-fucose	--	--
α-methyl-D-mannosideα-methyl-D-mannoside	--	±±	D-arabitolD-arabitol	--	--
α-methly-D-glucosideα-methly-D-glucoside	--	--	L-arabitolL-arabitol	--	--
N-acetyl-glucosamineN-acetyl-glucosamine	++	++	gluconategluconate	--	--
amygdalinamygdalin	++	++	2-keto-gluconate2-keto-gluconate	--	--
arbutinarbutin	++	++	5-keto-gluconate5-keto-gluconate	--	--
esculinesculin	++	++

탄소원Carbon source	균주명Strain name		탄소원Carbon source	균주명Strain name
탄소원Carbon source	B. longum LC67 B. longum LC67	B. longum LC68 B. longum LC68	탄소원Carbon source	B. longum LC67 B. longum LC67	B. longum LC68 B. longum LC68
glycerolglycerol	--	--	salicinsalicin	--	--
erythritolerythritol	--	--	cellobiosecellobiose	--	--
D-arabinoseD-arabinose	--	--	maltosemaltose	++	++
L-arabinoseL-arabinose	++	++	lactoselactose	++	++
D-riboseD-ribose	--	--	melibiosemelibiose	++	++
D-xyloseD-xylose	++	±±	sucrosesucrose	++	±±
L-xyloseL-xylose	--	--	trehalosetrehalose	--	±±
D-adonitolD-adonitol	--	--	inulininulin	--	--
methyl-β-D-xylopyranosidemethyl-β-D-xylopyranoside	--	--	melezitosemelezitose	--	++
D-galactoseD-galactose	±±	++	raffinoseraffinose	++	++
D-glucoseD-glucose	++	++	starchstarch	--	--
D-fructoseD-fructose	++	++	glycogenglycogen	--	--
D-mannoseD-mannose	--	--	xylitolxylitol	--	--
L-sorboseL-sorbose	--	--	gentiobiosegentiobiose	++	±±
L-rhamnoseL-rhamnose	--	--	D-turanoseD-turanose	±±	±±
dulcitoldulcitol	--	--	D-lyxoseD-lyxose	--	--
inositolinositol	--	--	D-tagatoseD-tagatose	--	--
mannitolmannitol	±±	++	D-fucoseD-fucose	--	--
sorbitolsorbitol	±±	++	L-fucoseL-fucose	--	--
α-methyl-D-mannosideα-methyl-D-mannoside	--	--	D-arabitolD-arabitol	--	--
α-methly-D-glucosideα-methly-D-glucoside	±±	±±	L-arabitolL-arabitol	--	--
N-acetyl-glucosamineN-acetyl-glucosamine	--	--	gluconategluconate	--	--
amygdalinamygdalin	--	±±	2-keto-gluconate2-keto-gluconate	--	--
arbutinarbutin	--	--	5-keto-gluconate5-keto-gluconate	--	--
esculinesculin	++	++

(4) trust information of lactic acid bacteria

The inventors of the present invention patented Lactobacillus plantarum LC5 to the Korea Microbiological Conservation Center (Address: 45 Yurim Building, 45, Honggae, Hongje, Seodaemun-gu, Seoul, Korea) on January 11, 2016 KCCM 11800P Was given an accession number. In addition, the inventors of the present invention patented Lactobacillus plantarum LC27 to the Korea Microbiological Conservation Center (Address: 45 Yurim Building, 45, 2ga-gil, Hongdae, Seodaemun-gu, Seoul, Korea) on January 11, 2016. The accession number of KCCM 11801P is given. In addition, the inventors of the present invention patented Bifidobacterium longum LC67 to the Korea Microbiological Conservation Center (Address: 45 Yurim Building, 45, 2ga-gil, Hongje, Seodaemun-gu, Seoul, Korea) on January 11, 2016. The accession number of KCCM 11802P is given.

(1) Experimental method

* Antioxidant activity

* Inhibitory activity of lipopolysaccharide (LPS) production

* Beta-glucuronidase (β-glucuronidase) inhibitory activity

* Induced activity of tight junction protein expression

(2) Experiment result

Antioxidative activity, inhibitory activity of lipopolysaccharide (LPS) production, β-glucuronidase inhibitory activity, and tight junction protein expression induction activity of lactic acid bacteria isolated from kimchi or human feces It measured and the result is shown in following Table 14-16. Table 14 - Lactobacillus Planta room (Lactobacillus plantarum), as shown in Table 16 LC5, Lactobacillus Planta room (Lactobacillus plantarum) LC15, Lactobacillus Planta room (Lactobacillus plantarum) LC17, Lactobacillus Planta room (Lactobacillus plantarum) LC25, Lactobacillus Planta Room (Lactobacillus plantarum) LC27, Lactobacillus Planta Room (Lactobacillus plantarum) LC28, ronggeom Bifidobacterium (Bifidobacterium longum) LC55, ronggeom Bifidobacterium (Bifidobacterium longum ) LC65, Bifidobacterium longum ) LC67 and Bifidobacterium longum LC68 lactic acid bacteria have excellent antioxidant activity, strongly inhibited lipopolysaccharide (LPS) production and beta-glucuronidase activity, and adhered closely. Tight junction protein expression was strongly induced. In particular, ronggeom Bifidobacterium (Bifidobacterium longum ) LC67 was the best inducing activity of tight junction protein expression. The lactic acid bacteria have an excellent inhibitory effect on the enzymatic activity of the enterobacteriaceae bacteria related to the antioxidant effect, inflammation and carcinogenesis, and also inhibit the production of endotoxin LPS (lipopolysaccharide) produced by the harmful bacteria of the intestinal flora, as well as tightly coupled proteins ( Induction of tight junction protein may improve intestinal permeability.

관리번호Control Number	균주명Strain name	항산화 활성Antioxidant activity	베타-글루쿠로니다제 저해 활성Beta-glucuronidase inhibitory activity	LPS 생성 억제 활성LPS production inhibitory activity	밀착연접단백질 발현 유도 활성Induced activity of tight junction protein expression
1One	Lactobacillus plantarum LC1 Lactobacillus plantarum LC1	++++	++	++	--
22	Lactobacillus plantarum LC2 Lactobacillus plantarum LC2	++++	++++	++	--
33	Lactobacillus plantarum LC3 Lactobacillus plantarum LC3	++++++	++++	++	--
44	Lactobacillus plantarum LC4 Lactobacillus plantarum LC4	++++++	++++	++	++
55	Lactobacillus plantarum LC5 Lactobacillus plantarum LC5	++++++	++++++	++++	++++
66	Lactobacillus plantarum LC6 Lactobacillus plantarum LC6	++++	++++++	++	--
77	Lactobacillus plantarum LC7 Lactobacillus plantarum LC7	++++++	++++	++	--
88	Lactobacillus plantarum LC8 Lactobacillus plantarum LC8	++++	++++++	++	--
99	Lactobacillus plantarum LC9 Lactobacillus plantarum LC9	++++	++++	++	++
1010	Lactobacillus plantarum LC10 Lactobacillus plantarum LC10	++++	++++++	++	++
1111	Lactobacillus plantarum LC11 Lactobacillus plantarum LC11	++++	++++	++	--
1212	Lactobacillus plantarum LC12 Lactobacillus plantarum LC12	++++	++++++	++	++
1313	Lactobacillus plantarum LC13 Lactobacillus plantarum LC13	++++	++++	++	++
1414	Lactobacillus plantarum LC14 Lactobacillus plantarum LC14	++++	++++	++	--
1515	Lactobacillus plantarum LC15 Lactobacillus plantarum LC15	++++++	++++++	++++	++++
1616	Lactobacillus plantarum LC16 Lactobacillus plantarum LC16	++	++++++	++	--
1717	Lactobacillus plantarum LC17 Lactobacillus plantarum LC17	++++++	++++++	++++	++++
1818	Lactobacillus plantarum LC18 Lactobacillus plantarum LC18	++++	++++	++	++
1919	Lactobacillus plantarum LC19 Lactobacillus plantarum LC19	++++	++++++	++	++
2020	Lactobacillus plantarum LC20 Lactobacillus plantarum LC20	++++	++++	++	--
2121	Lactobacillus plantarum LC21 Lactobacillus plantarum LC21	++++	++++	++	--
2222	Lactobacillus plantarum LC22 Lactobacillus plantarum LC22	++++	++++++	--	--
2323	Lactobacillus plantarum LC23 Lactobacillus plantarum LC23	++++++	++++	--	--
2424	Lactobacillus plantarum LC24 Lactobacillus plantarum LC24	++++++	++	--	--
2525	Lactobacillus plantarum LC25 Lactobacillus plantarum LC25	++++++	++++++	++++	++++
2626	Lactobacillus plantarum LC26 Lactobacillus plantarum LC26	++++	++	++	++
2727	Lactobacillus plantarum LC27 Lactobacillus plantarum LC27	++++++	++++++	++++	++++
2828	Lactobacillus plantarum LC28 Lactobacillus plantarum LC28	++++++	++++++	++++	++++
2929	Lactobacillus plantarum LC29 Lactobacillus plantarum LC29	++++	++	--	--
3030	Lactobacillus plantarum LC30 Lactobacillus plantarum LC30	++++	++	++	--
3131	Lactobacillus plantarum LC31 Lactobacillus plantarum LC31	++++++	++++	++	--
3232	Lactobacillus plantarum LC32 Lactobacillus plantarum LC32	++++++	++++	++	--
3333	Lactobacillus plantarum LC33 Lactobacillus plantarum LC33	++++++	++++	++	--
3434	Lactobacillus plantarum LC34 Lactobacillus plantarum LC34	++++	++++	++	++
3535	Lactobacillus plantarum LC35 Lactobacillus plantarum LC35	++++	++++	++	++

관리번호Control Number	균주명Strain name	항산화 활성Antioxidant activity	베타-글루쿠로니다제 저해 활성Beta-glucuronidase inhibitory activity	LPS 생성 억제 활성LPS production inhibitory activity	밀착연접단백질 발현 유도 활성Induced activity of tight junction protein expression
3636	Lactobacillus plantarum LC36 Lactobacillus plantarum LC36	++++	++++	++++	--
3737	Lactobacillus plantarum LC37 Lactobacillus plantarum LC37	++++++	++++	++	++
3838	Lactobacillus plantarum LC38 Lactobacillus plantarum LC38	++++	++++	++	--
3939	Lactobacillus plantarum LC39 Lactobacillus plantarum LC39	++++	++	++	--
4040	Lactobacillus plantarum LC40 Lactobacillus plantarum LC40	++++++	++	--	--
4141	Lactobacillus plantarum LC41 Lactobacillus plantarum LC41	++++	++++	--	++
4242	Lactobacillus plantarum LC42 Lactobacillus plantarum LC42	++++++	++	--	++
4343	Lactobacillus plantarum LC43 Lactobacillus plantarum LC43	++++	++	--	++
4444	Lactobacillus plantarum LC44 Lactobacillus plantarum LC44	++++	++	--	++
4545	Lactobacillus plantarum LC45 Lactobacillus plantarum LC45	++++	++++	--	++
4646	Lactobacillus plantarum LC46 Lactobacillus plantarum LC46	++++	++	--	++
4747	Lactobacillus plantarum LC47 Lactobacillus plantarum LC47	++++++	++	--	++
4848	Lactobacillus plantarum LC48 Lactobacillus plantarum LC48	++++	++++	--	++
4949	Lactobacillus plantarum LC49 Lactobacillus plantarum LC49	++++	++++++	++	++
5050	Lactobacillus plantarum LC50 Lactobacillus plantarum LC50	++++++	++++	++	--
5151	Bifidobacterium longum LC51 Bifidobacterium longum LC51	++++	++++	++	--
5252	Bifidobacterium longum LC52 Bifidobacterium longum LC52	++++++	++++++	++	--
5353	Bifidobacterium longum LC53 Bifidobacterium longum LC53	++++	++++++	--	++
5454	Bifidobacterium longum LC54 Bifidobacterium longum LC54	++++++	++++	++	++
5555	Bifidobacterium longum LC55 Bifidobacterium longum LC55	++++++	++++++	++++	++++
5656	Bifidobacterium longum LC56 Bifidobacterium longum LC56	++++++	++++	++	++
5757	Bifidobacterium longum LC57 Bifidobacterium longum LC57	++++	++	--	++
5858	Bifidobacterium longum LC58 Bifidobacterium longum LC58	++++++	++	--	++
5959	Bifidobacterium longum LC59 Bifidobacterium longum LC59	++++	++	--	--
6060	Bifidobacterium longum LC60 Bifidobacterium longum LC60	++++++	++	--	--
6161	Bifidobacterium longum LC61 Bifidobacterium longum LC61	++++	++	++	--
6262	Bifidobacterium longum LC62 Bifidobacterium longum LC62	++++++	++	++	--
6363	Bifidobacterium longum LC63 Bifidobacterium longum LC63	++++	++++	++++	--
6464	Bifidobacterium longum LC64 Bifidobacterium longum LC64	++++++	++	--	--
6565	Bifidobacterium longum LC65 Bifidobacterium longum LC65	++++++	++++++	++++	++++
6666	Bifidobacterium longum LC66 Bifidobacterium longum LC66	++++	++	++	++
6767	Bifidobacterium longum LC67 Bifidobacterium longum LC67	++++++	++++++	++++	++++++
6868	Bifidobacterium longum LC68 Bifidobacterium longum LC68	++++++	++++++	++++	++++
6969	Bifidobacterium longum LC69 Bifidobacterium longum LC69	++++++	++	--	--
7070	Bifidobacterium longum LC70 Bifidobacterium longum LC70	++++	++	--	++

관리번호Control Number	균주명Strain name	항산화 활성Antioxidant activity	베타-글루쿠로니다제 저해 활성Beta-glucuronidase inhibitory activity	LPS 생성 억제 활성LPS production inhibitory activity	밀착연접단백질 발현 유도 활성Induced activity of tight junction protein expression
7171	Bifidobacterium longum LC71 Bifidobacterium longum LC71	++++	++	--	++
7272	Bifidobacterium longum LC72 Bifidobacterium longum LC72	++++++	++++	--	++
7373	Bifidobacterium longum LC73 Bifidobacterium longum LC73	++++	++++	++	--
7474	Bifidobacterium longum LC74 Bifidobacterium longum LC74	++++	++++++	++	--
7575	Bifidobacterium longum LC75 Bifidobacterium longum LC75	++++++	++	--	++
7676	Bifidobacterium longum LC76 Bifidobacterium longum LC76	++++	++	--	++
7777	Bifidobacterium longum LC77 Bifidobacterium longum LC77	++++	++++	++	++
7878	Bifidobacterium longum LC78 Bifidobacterium longum LC78	++++	++	++	++
7979	Bifidobacterium longum LC79 Bifidobacterium longum LC79	++++++	++	++	++
8080	Bifidobacterium longum LC80 Bifidobacterium longum LC80	++++	++	++	++
8181	Bifidobacterium longum LC81 Bifidobacterium longum LC81	++++	++	++	++
8282	Bifidobacterium longum LC82 Bifidobacterium longum LC82	++++	++++	--	++
8383	Bifidobacterium longum LC83 Bifidobacterium longum LC83	++++++	++	--	++
8484	Bifidobacterium longum LC84 Bifidobacterium longum LC84	++++	++++	--	--
8585	Bifidobacterium longum LC85 Bifidobacterium longum LC85	++++++	++++	--	++
8686	Bifidobacterium longum LC86 Bifidobacterium longum LC86	++++	++	++	--
8787	Bifidobacterium longum LC87 Bifidobacterium longum LC87	++++	++++	++	--
8888	Bifidobacterium longum LC88 Bifidobacterium longum LC88	++++	++++++	++	++
8989	Bifidobacterium longum LC89 Bifidobacterium longum LC89	++++	++++	++	++
9090	Bifidobacterium longum LC90 Bifidobacterium longum LC90	++++	++++	++	++
9191	Bifidobacterium longum LC91 Bifidobacterium longum LC91	++++++	++++++	++	++
9292	Bifidobacterium longum LC92 Bifidobacterium longum LC92	++++++	++++	++	++
9393	Bifidobacterium longum LC93 Bifidobacterium longum LC93	++++	++++	++	++
9494	Bifidobacterium longum LC94 Bifidobacterium longum LC94	++++	++++	--	++
9595	Bifidobacterium longum LC95 Bifidobacterium longum LC95	++++	++++++	--	--
9696	Bifidobacterium longum LC96 Bifidobacterium longum LC96	++++	++	--	--
9797	Bifidobacterium longum LC97 Bifidobacterium longum LC97	++++	++	--	--
9898	Bifidobacterium longum LC98 Bifidobacterium longum LC98	++++	++++	--	--
9999	Bifidobacterium longum LC99 Bifidobacterium longum LC99	++++	++++	--	--
100100	Bifidobacterium longum LC100 Bifidobacterium longum LC100	++++	++++	--	++

Lactobacillus plantarum LC5, Lactobacillus plantarum LC15, Lactobacillus plantarum LC17 Lactobacillus Planta Room (Lactobacillus plantarum) LC25, Lactobacillus Planta Room (Lactobacillus plantarum) LC27, Lactobacillus Planta Room (Lactobacillus plantarum) LC28, ronggeom Bifidobacterium (Bifidobacterium longum) LC55, ronggeom Bifidobacterium (Bifidobacterium longum ) LC65, Bifidobacterium longum LC67 and Bifidobacterium longum LC68 were selected. Thereafter, the liver damage improvement effect of the selected lactic acid bacteria alone or mixed lactic acid bacteria was evaluated using a model animal in which liver damage was induced by Tert-butylperoxide.

(1) Experimental method

Six rats (C57BL / 6, male) were used as a group, and tert-butylperoxide was dosed at a dose of 2.5 mmol / kg to experimental animals of the other groups except the normal group for liver damage. Induced. After 2 hours of tert-butylperoxide administration, lactic acid bacteria were orally administered to the experimental animals of the group except the normal group and the negative control group once a day for 3 days in an amount of 2 × 10 ⁹ CFU. In addition, the experimental animals of the positive control group was orally administered silymarin (silymarin) once daily for 3 days in an amount of 100 mg / kg instead of lactic acid bacteria. Six hours after the last dose of the drug, the heart was bled. The collected blood was left at room temperature for 60 minutes and centrifuged at 3,000 rpm for 15 minutes to separate serum. GPT (glutamic pyruvate transaminase) and GOT (glutamic oxalacetic transaminase) of the isolated serum were measured using a blood analysis kit (ALT & AST measurement kit; Asan Pharm. Co., South Korea). 1 g of liver tissue was added to physiological saline and homogenized using a homogenizer, and the supernatant was analyzed by ELISA kit to determine the amount of TNF-α.

(2) Experiment result

Table 17 shows the change in GOT, GPT, TNF-α values when lactic acid bacteria were administered to a model animal induced by liver damage caused by Tert-butylperoxide (Tert-butylperoxide). As shown in Table 17, Lactobacillus plantarum LC5, Lactobacillus plantarum LC27, Lactobacillus plantarum LC28, Bifidobacterium longum LC67 And Bifidobacterium longum LC68 showed superior liver damage improvement effect than silymarin, and their mixed lactic acid bacteria showed better liver damage improvement effect.

실험군Experimental group	GOT(IU/L)GOT (IU / L)	GPT(IU/L)GPT (IU / L)	TNF-α(pg/g)TNF-α (pg / g)
정상군Normal	42.442.4	6.26.2	140.4140.4
음성 대조군Negative control	103.1103.1	28.028.0	298.0298.0
LC5 투여군LC5 administration group	36.936.9	5.45.4	115.7115.7
LC15 투여군LC15 administration group	60.360.3	6.26.2	154.3154.3
LC17 투여군LC17 administration group	65.865.8	6.86.8	136.7136.7
LC25 투여군LC25 administration group	64.664.6	11.311.3	132.4132.4
LC27 투여군LC27 administration group	35.335.3	3.33.3	157.1157.1
LC28 투여군LC28 administration group	42.042.0	1.01.0	185.7185.7
LC55 투여군LC55 administration group	55.655.6	17.617.6	251.4251.4
LC65 투여군LC65 administration group	61.461.4	17.317.3	127.6127.6
LC67 투여군LC67 administration group	50.850.8	3.83.8	150.5150.5
LC68 투여군LC68 administration group	40.840.8	5.75.7	82.482.4
LC5+LC67 투여군LC5 + LC67 administration group	32.732.7	3.13.1	115.9115.9
LC5+LC68 투여군LC5 + LC68 administration group	36.836.8	5.65.6	105.4105.4
LC27+LC67 투여군LC27 + LC67 administration group	30.530.5	2.32.3	121.2121.2
LC27+LC68 투여군LC27 + LC68 administration group	35.435.4	3.23.2	112.8112.8
LC28+LC67 투여군LC28 + LC67 administration group	32.532.5	2.82.8	128.2128.2
실리마린(silymarin) 투여군Silymarin administration group	52.952.9	5.95.9	93.893.8

In Table 17, "LC5" represents Lactobacillus plantarum LC5, "LC15" represents Lactobacillus plantarum LC15, and "LC17" represents Lactobacillus plantarum. ) represents the LC17, "LC25" is Lactobacillus Planta Room (Lactobacillus plantarum) represents the LC25, "LC27" represents the Lactobacillus Planta Room (Lactobacillus plantarum) LC27, "LC28 " is Lactobacillus Planta Room (Lactobacillus plantarum) represents the LC28, "LC55" is ronggeom Bifidobacterium (Bifidobacterium longum) represents an LC55, "LC65" is ronggeom Bifidobacterium (Bifidobacterium longum ) LC65 and "LC67" refers to Bifidobacterium longum ) LC67 and "LC68" refers to Bifidobacterium longum ) LC68, "LC5 + LC67" refers to a mixed lactic acid bacteria prepared by mixing Lactobacillus plantarum LC5 and Bifidobacterium longum LC67 in the same amount "LC5 + LC68" represents a mixed lactic acid bacterium prepared by mixing Lactobacillus plantarum LC5 and Bifidobacterium longum LC68 in the same amount. "LC27 + LC67" indicates Lactobacillus plantarum LC27 and Bifidobacterium longgum ( Bifidobacterium) longum ) mixed lactic acid bacteria prepared by mixing the same amount of LC67 "LC27 + LC68" denotes Lactobacillus plantarum LC27 and Bifidobacterium longgum ( Bifidobacterium) longum ) mixed lactic acid bacteria prepared by mixing the same amount of LC67 "LC28 + LC67" indicates a mixed lactic acid bacterium prepared by mixing Lactobacillus plantarum LC28 and Bifidobacterium longum LC67 in the same amount. Indicates. The same symbols were used for single lactic acid bacteria or mixed lactic acid bacteria in the tables showing the experimental results below.

(1) Degranulation inhibition rate measurement of lactic acid bacteria

(2) Experiment result

Table 18 shows the results of measuring the degranulation inhibition rate of lactic acid bacteria. As shown in Table 18, Lactobacillus plantarum LC5, Lactobacillus plantarum LC27, Lactobacillus plantarum LC28, Bifidobacterium longgum ( Bifidobacterium) longum ) LC67, Bifidobacterium longum ) LC68 and their mixed lactic acid bacteria effectively inhibited degranulation of basophils. Thus, the lactic acid bacteria or lactic acid bacteria mixture can very effectively improve atopic dermatitis, asthma, sore throat or chronic dermatitis caused by allergy.

처리 약물Treatment medication	탈과립 저해율(%)Degranulation inhibition rate (%)
없음 none	00
LC5LC5	6565
LC15LC15	4545
LC17LC17	4343
LC25LC25	4848
LC27LC27	5252
LC28LC28	5454
LC55LC55	3838
LC65LC65	4242
LC67LC67	6565
LC68LC68	6161
LC5+LC67LC5 + LC67	6565
LC5+LC68LC5 + LC68	6060
LC27+LC67LC27 + LC67	6565
LC27+LC68LC27 + LC68	5959
LC28+LC67LC28 + LC67	6262
DSCG(disodium cromoglycate)DSCo (disodium cromoglycate)	6262

5. 유산균의 항염 및 면역조절 효과 평가(in vitro)5. Evaluation of anti-inflammatory and immunomodulatory effects of lactic acid bacteria (in vitro)

(1) Isolation of macrophages and measurement of inflammatory markers

Six-week-old C57BL / 6J male mice (20-23 g) were purchased from Roundbio. 2 ml of sterile 4% thioglycolate was injected into the abdominal cavity of the mouse, anesthetized the mouse after 96 hours, and again 8 ml of RPMI 1640 medium was injected into the abdominal cavity of the mouse, and 5 to 10 minutes later, RPMI medium in the mouse abdominal cavity (large Phagocytic cells) were extracted again, centrifuged at 1000 rpm for 10 minutes, and washed twice with RPMI 1640 medium. Macrophages were placed in a 24-well plate at a number of 0.5 × 10 ⁶ per well, and supernatants and cells were obtained after treatment with lactic acid bacteria, a test substance, and LPS (lipopolysaccharide), an inflammatory response-inducing substance, for 2 hours or 24 hours. At this time, the lactic acid bacteria treatment concentration was 1 × 10 ⁴ CFU / ml. The obtained cells were placed in a buffer (Gibco) and homogenized. The expression level of cytokines such as TNF-α was measured from the obtained supernatant by 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, 50 ㎍ of the supernatant was electrophoresed for 1 hour and 30 minutes in SDS 10% (w / v) polyacrylamide gel. The electrophoretic sample was transferred to nitrocellulose paper for 1 hour and 10 minutes at 100 V and 400 kPa. The sample was blocked with 5% skim milk for 30 minutes after transferring the transferred nitrocellulose paper, and washed with PBS-Tween three times for 5 minutes and the primary antibody (Santa Cruz Biotechnology, USA) at a ratio of 1: 100. The reaction was overnight. Thereafter, the mixture was washed three times for 10 minutes, and reacted with a secondary antibody (Santa Cruz Biotechnology, USA) at a ratio of 1: 1000 for 1 hour and 20 minutes. Thereafter, the mixture was washed three times for 15 minutes, fluoresced and developed, and the intensity of the color band was measured, and then the inhibition rate was calculated as follows. In the following formula, the normal group represents a group treated with physiological saline only to macrophages, the LPS treated group represents a group treated with LPS only to macrophages, and the lactic acid bacteria treated group represents a group treated with both LPS and lactic acid bacteria to macrophages.

Table 19 shows the effects of NF-kappaB activation inhibition level and TNF-α expression inhibition level when treated with lactic acid bacteria to macrophage cells induced inflammatory response by lipopolysaccharide (LPS). As shown in Table 19, Lactobacillus plantarum LC5, Lactobacillus plantarum LC27, Lactobacillus plantarum LC28, Bifidobacterium long gum ( Bifidobacterium) longum ) LC67, Bifidobacterium longum LC68 and their mixed lactic acid bacteria effectively inhibited the inflammatory response induced by lipopolysaccharide (LPS).

처리 유산균Treatment Lactobacillus	TNF-α 발현 저해율(%)% Inhibition of TNF-α expression	p-p65/p65 활성화 저해율(%)% inhibition of p-p65 / p65 activation
LC5LC5	7171	7373
LC15LC15	5454	5555
LC17LC17	6161	5555
LC25LC25	5252	6565
LC27LC27	7070	7272
LC28LC28	7474	7171
LC55LC55	6363	6262
LC65LC65	6565	6868
LC67LC67	7676	7777
LC68 LC68	7575	7171
LC5+LC67LC5 + LC67	7878	7272
LC5+LC68LC5 + LC68	7676	7272
LC27+LC67LC27 + LC67	8181	7575
LC27+LC68LC27 + LC68	7777	7373
LC28+LC67LC28 + LC67	7777	7373

(2) Isolation of T Cells from the Spleen and Differentiation Capability into Th17 Cells or Treg Cells

Spleens of C56BL / 6J mice were isolated, crushed, suspended, suspended in RPMI 1640 exclusion with 10% FCS and CD4 T cells were isolated using a CD4 T cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Separated CD4 T cells were dispensed in 12-well plates at 5 × 10 ⁵ cells per well, containing anti-CD3 (1 μg / ml, Miltenyi Biotec, Bergisch Gladbach, Germany) and anti-CD28 (1 μg / ml, Miltenyi Biotec). , Bergisch Gladbach, Germany), anti-CD3 (1 μg / ml, Miltenyi Biotec, Bergisch Gladbach, Germany), anti-CD28 (1 μg / ml, Miltenyi Biotec, Bergisch Gladbach, Germany), recombinant IL-6 (20 ng) / Ml, MiltenyiBiotec, Bergisch Gladbach, Germany) and recombinant transforming growth factor beta (1 ng / ml, MiltenyiBiotec, Bergisch Gladbach, Germany), incubating the cells and adding lactic acid bacteria to 1 × 10 ³ or 1 × 10 ⁵ CFU per well. Incubated for 4 days. Subsequently, the cells of the culture were stained with anti-FoxP3 or anti-IL-17A antibody and Th17 cells and Treg cells using a Fluorescence-activated cell sorting (FACS) device (C6 Flow Cytometer® System, San Jose, CA, USA). The distribution of was analyzed.

Table 20 below shows the levels of differentiation into Th17 cells when treated with lactic acid bacteria after treatment with anti-CD3, anti-CD28, IL-6 and TGF-β on T cells isolated from the spleen, and Table 21 below. After treatment with anti-CD3 and anti-CD28 on T cells isolated from, the level of differentiation into Treg cells was shown when treated with lactic acid bacteria. As shown in Table 20 and Table 21, Lactobacillus plantarum LC5, Lactobacillus plantarum LC27, Lactobacillus plantarum LC28, Bifidobacterium longgum ( Bifidobacterium longum ) LC67, Bifidobacterium longum ) LC68 and their mixed lactic acid bacteria inhibited the differentiation of T cells into Th17 cells (T helper 17 cells) and promoted the differentiation into Treg cells. Based on the results, the lactic acid bacteria or lactic acid bacteria mixture can effectively improve inflammatory diseases such as colitis and arthritis.

T 세포 처리 방법T cell treatment method		Th17 세포로의 분화율(%) % Differentiation into Th17 cells
anti-CD3, anti-CD28, IL-6 및 TGF-β를 처리 여부Whether anti-CD3, anti-CD28, IL-6 and TGF-β are processed	유산균 처리 여부Lactic acid bacteria treatment	Th17 세포로의 분화율(%) % Differentiation into Th17 cells
무처리No treatment	무처리No treatment	12.212.2
처리process	무처리No treatment	25.625.6
처리process	LC5 처리LC5 treatment	14.214.2
처리process	LC15 처리LC15 treatment	19.619.6
처리process	LC17 처리LC17 treatment	17.917.9
처리process	LC25 처리LC25 treatment	18.218.2
처리process	LC27 처리LC27 treatment	15.115.1
처리process	LC28 처리LC28 treatment	14.914.9
처리process	LC55 처리LC55 treatment	18.818.8
처리process	LC65 처리LC65 treatment	17.917.9
처리process	LC67 처리LC67 treatment	15.915.9
처리process	LC68 처리LC68 treatment	15.715.7
처리process	LC5+LC67 처리LC5 + LC67 Treatment	14.214.2
처리process	LC5+LC68 처리LC5 + LC68 Treatment	14.514.5
처리process	LC27+LC67 처리LC27 + LC67 Treatment	13.913.9
처리process	LC27+LC68 처리LC27 + LC68 Treatment	14.414.4
처리process	LC28+LC67 처리LC28 + LC67 Treatment	14.114.1

T 세포 처리 방법T cell treatment method		Treg 세포로의 분화율(%) Differentiation rate into Treg cells (%)
anti-CD3 및 anti-CD28 처리 여부Whether anti-CD3 and anti-CD28 are processed	유산균 처리 여부Lactic acid bacteria treatment	Treg 세포로의 분화율(%) Differentiation rate into Treg cells (%)
무처리No treatment	무처리No treatment	9.19.1
처리process	무처리No treatment	11.411.4
처리process	LC5 처리LC5 treatment	22.922.9
처리process	LC15 처리LC15 treatment	15.815.8
처리process	LC17 처리LC17 treatment	16.916.9
처리process	LC25 처리LC25 treatment	18.418.4
처리process	LC27 처리LC27 treatment	21.821.8
처리process	LC28 처리LC28 treatment	21.421.4
처리process	LC55 처리LC55 treatment	19.519.5
처리process	LC65 처리LC65 treatment	19.219.2
처리process	LC67 처리LC67 treatment	21.621.6
처리process	LC68 처리LC68 treatment	20.520.5
처리process	LC5+LC67 처리LC5 + LC67 Treatment	21.821.8
처리process	LC5+LC68 처리LC5 + LC68 Treatment	21.821.8
처리process	LC27+LC67 처리LC27 + LC67 Treatment	22.022.0
처리process	LC27+LC68 처리LC27 + LC68 Treatment	21.521.5
처리process	LC28+LC67 처리LC28 + LC67 Treatment	21.921.9

(1) experimental animals

(2) TNBS-induced colitis and sample administration

(3) appearance analysis of the large intestine

The length and appearance of the extracted colon were observed and analyzed by appearance according to the criteria of Table 22 (Hollenbach et al., 2005 criteria for colitis). The colon tissues were removed from the colon contents, washed in physiological saline, and some were fixed with 4% formaldehyde fixative for use as pathological samples, and the rest were frozen and stored at minus 80 ° C for molecular biological analysis. It was.

(4) Determination of Myeloperoxidase (MPO) Activity

(5) Inflammatory index measurement

In addition, inflammation-related cytokines such as TNF-α, IL-17, and IL-10 were measured using an ELISA kit.

(6) Analysis of immunomodulatory indicator

The isolated colon was washed twice with 2.5 mM EDTA solution. Thereafter, the colon was washed in RPMI medium containing collagenase type VIII (Sigma) at a concentration of 1 mg / ml, shaken at 30 ° C. for 20 minutes, and filtered to separate Lamina propria. Lamina propria was then treated with 30-100% Percol solution and centrifuged to separate T cells. Thereafter, the isolated T cells were stained with an anti-FoxP3 or anti-IL-17A antibody, and stained with Th17 and Tregs using a FACS (Fluorescence-activated cell sorting) device (C6 Flow Cytometer® System, San Jose, CA, USA). The distribution was analyzed.

(7) Experiment result

Table 23 shows the effects of lactobacillus on the weight of colon, the appearance of colon, myeloperoxidase (MPO) activity, and inflammation-related cytokine content when Lactobacillus was administered to model animals induced by acute colitis induced by TNBS. The effect is shown. As shown in Table 23, in the model animals induced by acute colitis induced by TNBS, weight, appearance index of colitis, colon length decreased, and MPO activity increased. However, all of these indicators were improved when lactic acid bacteria were administered to a model animal induced by acute colitis induced by TNBS. In particular, administration of Bifidobacterium longum LC67 alone or a mixed lactic acid bacterium of Bifidobacterium longum LC67 and Lactobacillus plantarum LC5 significantly improves colitis. Excellent. In addition, TNF-α and IL-17 were increased and IL-10 was decreased in model animals induced by acute colitis induced by TNBS. However, all of these indicators were improved when lactic acid bacteria were administered to a model animal induced by acute colitis induced by TNBS. In particular, Bifidobacterium ronggeom (Bifidobacterium longum) administered alone to LC67 or Bifidobacterium ronggeom (Bifidobacterium longum) LC67 and Lactobacillus Planta room (Lactobacillus plantarum) When administering a mixture of lactic acid bacteria of the LC5 TNF-α, IL The amount of -17 was greatly reduced and the amount of IL-10 was greatly increased.

실험군Experimental group	Weight gain(g)Weight gain (g)	Macroscopic scoreMacroscopic score	Colon length(㎝)Colon length (cm)	MPO 활성(μU/㎎)MPO activity (μU / mg)	TNF-α(pg/㎎)TNF-α (pg / mg)	IL-17(pg/㎎)IL-17 (pg / mg)	IL-10(pg/㎎)IL-10 (pg / mg)
정상군Normal	0.640.64	5.95.9	0.140.14	0.420.42	35.135.1	18.418.4	61.261.2
음성 대조군Negative control	-2.46-2.46	4.24.2	2.322.32	1.541.54	95.595.5	65.265.2	30.730.7
LC5 투여군LC5 administration group	-1.90-1.90	4.654.65	1.301.30	0.910.91	75.575.5	52.852.8	43.943.9
LC27 투여군LC27 administration group	-1.0-1.0	4.564.56	1.081.08	0.820.82	67.267.2	50.450.4	44.044.0
LC67 투여군LC67 administration group	-0.28-0.28	4.924.92	0.500.50	0.430.43	48.548.5	38.538.5	54.654.6
LC 68 투여군LC 68 administration group	-1.02-1.02	4.54.5	1.341.34	1.041.04	54.454.4	50.550.5	48.148.1
LC5+LC67 투여군LC5 + LC67 administration group	-0.3-0.3	5.085.08	0.840.84	0.420.42	45.145.1	37.337.3	55.355.3
LC27+LC68 투여군LC27 + LC68 administration group	-1.15-1.15	4.84.8	1.171.17	0.780.78	59.859.8	45.045.0	50.050.0
양성 대조군Positive control	-0.91-0.91	4.584.58	1.431.43	0.950.95	58.258.2	48.548.5	45.545.5

Figure 22 shows the effect of lactic acid bacteria to differentiation of T cells into Th17 cells induced by acute colitis induced by TNBS, Figure 23 shows the effect of lactic acid bacteria against model animals induced by acute colitis induced by TNBS The effect is shown in the differentiation pattern of T cells into Treg cells. 22 and 23, "NOR" represents a normal group, "TNBS" represents a negative control, "LC5" represents a Lactobacillus plantarum LC5 administration group, "LC27" represents a Lactobacillus planta Lactobacillus plantarum LC27 administration group, "LC67" represents the Bifidobacterium longum LC67 administration group, "LC68" represents the Bifidobacterium longum LC68 administration group, "LC5 + LC67 " Lactobacillus plantarum LC5 and Bifidobacterium longum LC67 represents a mixed lactobacillus administration group prepared by mixing the same amount," LC27 + LC68 "is Lactobacillus plantarum ( Lactobacillus plantarum ) LC27 ronggeom and Bifidobacterium (Bifidobacterium longum ) represents a mixed lactic acid bacteria administration group prepared by mixing the same amount of LC68, "SS" represents a sulfasalazine administration group. As shown in FIGS. 22 and 23, in the model animals in which acute colitis was induced by TNBS, the differentiation of T cells into Th17 cells was promoted and the differentiation of T cells into Treg cells was suppressed. However, when lactic acid bacteria were administered to TNBS-induced acute colitis, the differentiation of T cells into Th17 cells was inhibited and the differentiation of T cells into Treg cells was promoted. In particular, ronggeom Bifidobacterium (Bifidobacterium longum ) LC67 alone or Bifidobacterium Lactobacillus administration of longum ) LC67 and Lactobacillus plantarum LC5 significantly inhibited the differentiation of T cells into Th17 cells and greatly promoted the differentiation of T cells into Treg cells.

FIG. 24 shows the effects of lactic acid bacteria on model animals induced by acute colitis induced by TNBS as inflammatory response indicators. In Figure 24 "Nor" represents the control group, "T" indicates a negative control, "LC5" denotes a Lactobacillus Planta room (Lactobacillus plantarum) LC5 group, "LC27" is Lactobacillus Planta room (Lactobacillus plantarum ) represents an LC27 administration group, "LC67" represents a Bifidobacterium longum LC67 administration group, "LC68" represents a Bifidobacterium longum LC68 administration group, and "LC5 + LC67" represents a lactose. Lactobacillus plantarum LC5 and Bifidobacterium longum LC67 represents a mixed lactobacillus administration group prepared by mixing the same amount, "LC27 + LC68" is Lactobacillus plantarum LC27 and ronggeom Bifidobacterium (Bifidobacterium longum ) represents a mixed lactic acid bacteria administration group prepared by mixing the same amount of LC68, "SS" represents a sulfasalazine administration group. As shown in FIG. 24, NF-κB was activated (p-p65) and expression levels of COX-2 and iNOS were increased in the model animal induced by acute colitis induced by TNBS, but NF-κB was activated by administration of lactic acid bacteria ( p-p65) was inhibited and the increase in the expression level of COX-2 and iNOS was also reduced. In particular, ronggeom Bifidobacterium (Bifidobacterium longum ) LC67 alone or Bifidobacterium Lactobacillus ( Lactobacillus plantarum ) LC5 administration of longum ) LC67 and Lactobacillus plantarum LC5 was effective in inhibiting the activation (p-p65) and increased expression of COX-2 and iNOS.

7. 유산균의 알코올 유도성 위궤양 개선 효과 평가(in 7. Evaluation of the Lactic Acid Bacteria Improvement Effect of Alcohol-Induced Gastric Ulcer (in vivovivo ))

(1) experimental animals

(2) Induction of gastric ulcer by alcohol and sample administration

In one experimental group, Lactobacillus plantarum LC27 was suspended in physiological saline and orally administered once daily for 3 days in an amount of 1 × 10 ⁹ CFU, and one experimental group included Bifidobacterium long gum ( Bifidobacterium). longum ) LC67 was suspended in physiological saline and administered orally once daily for 3 days in an amount of 1 × 10 ⁹ CFU, and one experimental group included Lactobacillus plantarum LC27 and Bifidobacterium long gum ( Bifidobacterium). longum ) The mixed lactic acid bacteria prepared by mixing the same amount of LC67 was suspended in physiological saline and administered orally in an amount of 1 × 10 ⁹ CFU once daily for 3 days. In the positive control group, ranitidine, a commercial gastric ulcer drug, was orally administered once daily in an amount of 50 mg / kg for 3 days. In addition, the normal group and the negative control group was administered orally for 3 days in an amount of 0.2 ml once daily. After oral administration of the sample for 3 days, the experimental mice were fasted and watered for 18 hours. One hour after the administration of the sample or saline on the fourth day of the experiment, mice of all experimental groups except the normal group were orally administered with 0.2 ml of 99% pure ethanol to induce gastric ulcers. In addition, the normal group was orally administered 0.2 ml of saline instead of ethanol.

(3) measuring macroscopic indicators of gastric damage

Three hours after ethanol administration, the mice were sacrificed and the stomach tissues were removed, vertically divided and washed with PBS (phosphate buffer saline) solution. (Reference: Park, SW, Oh, TY, Kim, YS, Sim, H., et al., Artemisia asiatica extracts protect against ethanol-induced injury in gastric mucosa of rats. J. Gastroenterol. Hepatol. 2008, 23 , 976? 984.).

(4) Determination of Myeloperoxidase (MPO) Activity

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

(5) Inflammatory index measurement

Gastric tissue was purified by Qiagen RNeasy Mini Kit to isolate 2 μg of mRNA, and cDNA was prepared using Takara Prime Script Rtase. Then, quantitative real time polymerase chain reaction (Qiagn thermal cycler, Takara SYBER premix agent, Thermal cycling conditions: activation of DNA polymerase for 5 min at 95 ° C, followed by 40 cycles of amplification for 10 s at 95 ° C and for 45 s at 60 ° C) was used to measure the expression levels of CXCL4 [chemokine (CXC motif) ligand 4] and TNF-α (tumor necrosis factor-alpha). Table 24 below shows primer sequences used for quantitative real time polymerase chain reaction for each cytokine to be analyzed.

분석 대상 사이토카인Cytokines Analyzed	프라이머 종류Primer type	프라이머 염기서열Primer Sequence
TNF-αTNF-α	ForwardForward	5'-CTGTAGCCCACGTCGTAGC-3'5'-CTGTAGCCCACGTCGTAGC-3 '
TNF-αTNF-α	ReverseReverse	5'-TTGAGATCCATGCCGTTG-3'5'-TTGAGATCCATGCCGTTG-3 '
CXCL4CXCL4	ForwardForward	5'-AGTCCTGAGCTGCTGCTTCT-3'5'-AGTCCTGAGCTGCTGCTTCT-3 '
CXCL4CXCL4	ReverseReverse	5'-GATCTCCATCGCTTTCTTCG-3'5'-GATCTCCATCGCTTTCTTCG-3 '

(6) Experiment result

FIG. 25 is a photograph showing the effect of lactic acid bacteria on the gastric mucosa of the mouse induced by gastric ulcers in the second experiment of the present invention. FIG. 26 is a second experiment of the present invention. The effect of lactic acid bacteria on the gastric mucosa of the gastric ulcer induced by the gastric ulcer is shown by the gross gastric lesion score (Gross gastric lesion score), Figure 27 is a gastric ulcer by ethanol in the second experiment of the present invention The effect of lactic acid bacteria on the gastric mucosa of the induced mice (ulcer index) is shown by the ulcer index (ulcer index), Figure 28, in the second experiment of the present invention, gastric mucosa of the mice induced gastric ulcer by ethanol (Histological activity index) shows the effect of lactic acid bacteria on (stomach mucosa). In addition, in the second experiment of the present invention, the effect of lactic acid bacteria on the gastric mucosa (stomach mucosa) of the gastric ulcer induced mice by ethanol is shown as myeloperoxidase (MPO) activity. In addition, Figure 30 shows the effect of lactic acid bacteria on the gastric mucosa (stomach mucosa) of the ethanol-induced gastric ulcer mice in the second experiment of the present invention, the expression level of CXCL4, Figure 31 In the next experiment, the effect of lactic acid bacteria on the gastric mucosa of gastric ulcer-induced gastric mucosa was expressed as the expression level of TNF-α. In FIG. 30 and FIG. 31, CXCL4 expression levels and TNF-α expression levels of the test groups except the normal group were expressed as fold changes based on the expression level of the normal group. In FIGS. 25 to 31, "Nor" represents a normal group, "Ethanol" represents a negative control in which gastric ulcers are induced by ethanol and physiological saline is administered as a sample, and "Ethanol + Ranitidine" induces gastric ulcers by ethanol. And an experiment group in which ranitidine was administered as a sample, and "Ethanol + LC27" represents an experimental group in which gastric ulcer was induced by ethanol and Lactobacillus plantarum LC27 was administered as a sample, and "Ethanol + LC67 "The gastric ulcer is caused by ethanol and the Bifidobacterium long gum ( Bifidobacterium) longum ) represents an experimental group to which LC67 was administered, and "Ethanol + LC27 / LC67" is a gastric ulcer induced by ethanol and the same amount of Lactobacillus plantarum LC27 and Bifidobacterium longum LC67 as a sample. The experimental group to which the mixed lactic acid bacteria prepared by mixing was administered. As shown in Fig. 25 to 29, Bifidobacterium longum LC67, Lactobacillus plantarum LC27 or a mixed lactic acid bacteria thereof effectively improve the gastric ulcer or gastric ulcer caused by ethanol It was. In addition, Bifidobacterium long gum ( Bifidobacterium) as shown in Figure 30 and 31 longum ) LC67, Lactobacillus plantarum LC27, or a mixed lactobacillus thereof significantly reduced the level of inflammatory markers in mice induced by gastric ulcer or gastric ulcer.

8. 유산균의 알코올 유도성 간 손상 개선 효과 평가(in 8. Evaluation of the Lactic Acid Bacteria Improvement Effect of Alcohol-induced Liver Injury (in vivovivo ))

(1) experimental animals

(2) Induce liver damage by alcohol and sample administration

In one experimental group, Lactobacillus plantarum LC27 was suspended in physiological saline and orally administered once daily for 3 days in an amount of 1 × 10 ⁹ CFU, and one experimental group included Bifidobacterium long gum ( Bifidobacterium). longum ) LC67 was suspended in physiological saline and administered orally once daily for 3 days in an amount of 1 × 10 ⁹ CFU, and one experimental group included Lactobacillus plantarum LC27 and Bifidobacterium long gum ( Bifidobacterium). longum ) The mixed lactic acid bacteria prepared by mixing the same amount of LC67 was suspended in physiological saline and administered orally in an amount of 1 × 10 ⁹ CFU once daily for 3 days. In addition, the positive control group was orally administered silymarin (silymarin), a therapeutic agent for the treatment of liver damage, once daily in an amount of 50 mg / kg for 3 days. In addition, the normal group and the negative control group were orally administered with saline solution once daily in an amount of 0.1 ml for 3 days. Hepatic damage was induced by oral administration of a sample or saline solution for 3 days, and intraperitoneally administered ethanol in an amount of 6 ml / kg to all experimental groups except the normal group after 3 hours. In addition, the normal group was intraperitoneally administered physiological saline instead of ethanol in an amount of 6 ml / kg. Thereafter, the mice were fasted and watered for 12 hours, and sacrificed for heart blood collection.

(3) liver function indicator measurement and results

The collected blood was left at room temperature for 60 minutes and centrifuged at 3,000 rpm for 15 minutes to separate serum. GPT (glutamic pyruvate transaminase) and GOT (glutamic oxalacetic transaminase) of the isolated serum were measured using a blood analysis kit (ALT & AST measurement kit; Asan Pharm. Co., South Korea), and the results are shown in Table 25 below. As shown in Table 25, Bifidobacterium longum LC67, Lactobacillus plantarum LC27, or mixed lactobacilli thereof, effectively improved liver damage caused by ethanol, in particular ronggeom Bifidobacterium (Bifidobacterium longum ) LC67 was superior to silymarin, a commercial treatment for liver damage.

실험군 구분Experiment group	GOT(IU/L)GOT (IU / L)	GPT(IU/L)GPT (IU / L)
정상군Normal	52.152.1	42.242.2
음성 대조군Negative control	107.7107.7	156.3156.3
에탄올 및 LC27을 투여한 군Group administered ethanol and LC27	82.382.3	95.495.4
에탄올 및 LC67을 투여한 군Group administered ethanol and LC67	62.562.5	65.865.8
에탄올 및 LC27/LC67을 투여한 군Group administered ethanol and LC27 / LC67	71.471.4	78.378.3
에탄올 및 실리마린을 투여한 군Ethanol and silymarin	79.579.5	87.587.5

* LC27: Lactobacillus plantarum LC27

* LC67: ronggeom Bifidobacterium (Bifidobacterium longum ) LC67

* LC27 / LC67: Lactobacillus Planta Room (Lactobacillus plantarum) LC27 ronggeom and Bifidobacterium (Bifidobacterium longum ) Mixed lactic acid bacteria prepared by mixing LC67 in the same amount

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

Claims

Lactobacillus brevis comprising the nucleotide sequence of SEQ ID NO: 1 as 16S rDNA, and Bifidobacterium long gum comprising the nucleotide sequence of SEQ ID NO: 3 as 16S rDNA. longum), Lactobacillus Planta room (Lactobacillus plantarum) comprising a base sequence described in Lactobacillus Planta room (Lactobacillus plantarum) comprising the nucleotide sequence set forth in SEQ ID NO: 4 with 16S rDNA, SEQ ID NO: 5 with 16S rDNA or 16S Bifidobacterium longgum containing the nucleotide sequence of SEQ ID NO: 7 as rDNA longum ) is selected from lactic acid bacteria,

Lactic acid bacteria having antioxidant activity, beta-glucuronidase inhibitory activity, lipopolysaccharide (LPS) production inhibitory activity or tight junction protein expression inducing activity.

According to claim 1, Lactobacillus brevis ( Lactobacillus brevis ) comprising the nucleotide sequence of SEQ ID NO: 1 as the 16S rDNA is Lactobacillus brevis CH23 (Accession Number: KCCM 11762P), SEQ ID NO: 16S rDNA Bifidobacterium longgum containing the nucleotide sequence of claim 3 longum) is ronggeom Bifidobacterium (Bifidobacterium longum ) CH57 (Accession No .: KCCM 11764P), Lactobacillus plantarum ( Lactobacillus plantarum ) LC5 (Accession No .: KCCM) containing the nucleotide sequence of SEQ ID NO: 4 as 16S rDNA Lactobacillus plantarum Lactobacillus plantarum LC27 (Accession No .: KCCM 11801P) comprising the nucleotide sequence of SEQ ID NO: 5 as 16S rDNA, and sequence as 16S rDNA No. 7 Bifidobacterium ronggeom comprising a base sequence described in (Bifidobacterium longum) is ronggeom Bifidobacterium (Bifidobacterium longum ) Lactobacillus, characterized in that LC67 (Accession Number: KCCM 11802P).

Lactobacillus brevis comprising the nucleotide sequence of SEQ ID NO: 1 as 16S rDNA, and Bifidobacterium long gum comprising the nucleotide sequence of SEQ ID NO: 3 as 16S rDNA. longum), Lactobacillus Planta room (Lactobacillus plantarum), Lactobacillus Planta room (Lactobacillus plantarum comprising the nucleotide sequence set forth in SEQ ID NO: 5 as 16S rDNA) containing the nucleotide sequence shown in SEQ ID NO: 4 to the 16S rDNA and 16S Bifidobacterium longgum containing the nucleotide sequence of SEQ ID NO: 7 as rDNA longum ) is a mixed lactic acid bacteria selected from the group consisting of two or more,

Mixed lactic acid bacteria having antioxidant activity, beta-glucuronidase inhibitory activity, lipopolysaccharide (LPS) production inhibitory activity or tight junction protein expression inducing activity.

According to claim 3, Lactobacillus brevis ( Lactobacillus brevis ) comprising the nucleotide sequence of SEQ ID NO: 1 as the 16S rDNA is Lactobacillus brevis CH23 (Accession Number: KCCM 11762P), SEQ ID NO: 16S rDNA Bifidobacterium longgum containing the nucleotide sequence of claim 3 longum) is ronggeom Bifidobacterium (Bifidobacterium longum ) CH57 (Accession No .: KCCM 11764P), Lactobacillus plantarum ( Lactobacillus plantarum ) containing the nucleotide sequence of SEQ ID NO: 4 as 16S rDNA is Lactobacillus plantarum LC5 (Accession No .: KCCM Lactobacillus plantarum Lactobacillus plantarum LC27 (Accession No .: KCCM 11801P) comprising the nucleotide sequence of SEQ ID NO: 5 as 16S rDNA, and sequence as 16S rDNA No. 7 Bifidobacterium ronggeom comprising a base sequence described in (Bifidobacterium longum) is ronggeom Bifidobacterium (Bifidobacterium longum ) mixed lactic acid bacteria, characterized in that LC67 (Accession Number: KCCM 11802P).

Claim 1 or claim 2 of the lactic acid bacteria, its culture, its lysate or extract thereof as an active ingredient,

A pharmaceutical composition for use in preventing or treating intestinal damage, liver damage, allergic diseases, inflammatory diseases, or obesity.

6. The pharmaceutical composition of claim 5, wherein the bowel injury is bowel leak syndrome.

6. The pharmaceutical composition of claim 5, wherein the liver damage is selected from hepatitis, fatty liver or cirrhosis of the liver.

6. The pharmaceutical composition of claim 5, wherein the allergic disease is selected from atopic dermatitis, asthma, sore throat or chronic dermatitis.

The pharmaceutical composition according to claim 5, wherein the inflammatory disease is selected from gastritis, gastric ulcer, colitis or arthritis.

Claim 1 or claim 2 of the lactic acid bacteria, the culture of the lactic acid bacteria, lysate of the lactic acid bacteria or extracts thereof as an active ingredient

A food composition for use in preventing or ameliorating bowel damage, liver damage, allergic diseases, inflammatory diseases, or obesity.

The mixed lactic acid bacteria of claim 3 or 4, its culture, its lysate or its extract as an active ingredient,

The pharmaceutical composition of claim 11, wherein the intestinal injury is intestinal leak syndrome.

The pharmaceutical composition of claim 11, wherein the liver injury is selected from hepatitis, fatty liver or cirrhosis of the liver.

The pharmaceutical composition of claim 11, wherein the allergic disease is selected from atopic dermatitis, asthma, sore throat, or chronic dermatitis.

The pharmaceutical composition of claim 11, wherein the inflammatory disease is selected from gastritis, gastric ulcer, colitis or arthritis.