KR102038695B1 - Composition for Preventing or Treating Alcoholic Intestinal Damage Comprising Probiotics as Effective Ingredients - Google Patents

Abstract

The present invention relates to a composition for preventing or treating alcohol-induced intestine injury, comprising probiotics as an effective ingredient. The composition of the present invention comprises lactobacillus bulgaricus CKDB001 (accession number: KCTC13669B), lactobacillus helveticus CKDB001 (accession number: KCTC13670BP), lactobacillus plantarum CKDB008 (accession number: KCTC13673BP), bifidobacterium bifidum CKDB001 (accession number: KCTC13114BP), or mixed strains thereof, wherein the lactobacillus bulgaricus CKDB001, the lactobacillus helveticus CKDB001, the lactobacillus plantarum CKDB008, and the bifidobacterium bifidum CKDB001 are novel isolated lactic acid strains. The composition has an excellent effect of reducing inflammation of intestinal cells caused by alcoholic stimulation. Thus, the composition can be widely used as food or a therapeutic agent to prevent and relieve alcohol-induced intestine injury.

Description

Composition for Preventing or Treating Alcoholic Intestinal Damage Comprising Probiotics as Effective Ingredients}

The present invention relates to a composition for preventing or treating alcoholic intestinal injury, including probiotics.

Alcohol is a favorite product that has been consumed for a long time to relieve stress or socialize. Ingestion in small amounts may be beneficial to the blood circulation, which may be beneficial to your health, but chronic intake of excessive amounts may lead to liver diseases such as hepatitis and cirrhosis. However, the liver is not the only organ affected by excessive alcohol intake. Since more than 80% of the alcohol consumed is absorbed into the body through the small intestine, the effect of alcohol on the small intestine can never be overlooked.

The intestine is largely divided into small and large intestine, and the small intestine can be divided into duodenum, jejunum, and president. The effects of alcohol vary in different parts of the intestine. In the duodenum, bleeding and inflammatory reactions due to damage to the intestinal mucosa are most prominent, nutrient absorption is impaired in the jejunum, and diarrhea occurs in the ileum with increased contraction of villi.

Although alcohol is the best way to prevent intestinal damage from alcohol, eating foods that prevent intestinal damage can be a good alternative. However, most of the products on the market are for preventing liver damage and no products are available for preventing intestinal damage. In addition, although there are patents (KR10-1782848, KR10-1772810, KR10-2013-0036939, and KR10-1819026) for the prevention or treatment of alcoholic gastrointestinal diseases, all are inventions using natural extract as an active ingredient, and probiotics as an active ingredient. The invention is not yet developed. Therefore, the present inventors have sought to discover functional probiotic strains that can prevent or treat alcoholic intestinal damage, which is applicable to food and medicine.

Republic of Korea Patent No. 10-1782848 (Registered September 22, 2017) Republic of Korea Patent Registration No. 10-1772810 (Registered Aug. 23, 2017) Republic of Korea Patent Publication No. 10-2013-0036939 (published April 15, 2013) Republic of Korea Patent Registration No. 10-1819026 (January 10, 2018 registration)

The present inventors have made diligent research efforts to find functional probiotic strains that can prevent or treat alcoholic intestinal damage that can be applied to foods and medicines. As a result, a total of 530 species of lactic acid bacteria, which were identified as safety, stability, and industrial potential, were selected first, and then 12 kinds of lactic acid bacteria having excellent intestinal adhesion were selected. Among them, alcohol viability was reduced. Seven possible strains were selected secondarily. Finally, four strains that effectively lowered the inflammatory cytokine expression increased due to alcohol were selected. It was confirmed that the selected four strains could effectively prevent the inflammation of enterocytes caused by alcohol than each single strain.

The four strains of the present invention are Lactobacillus bulgaricus ( Lactobacillus bulgaricus ) CKDB001 (Accession No .: KCTC13669B), Lactobacillus helveticus ( Lactobacillus helveticus ) CKDB001 (Accession No .: KCTC13670BP), Lactobacillus plantarum plant (Lumumactobacillus plantarum) CKDB008 (Accession No .: KCTC13673BP), Bifidobacterium Bifidobacterium bifidum ) CKDB001 (accession number KCTC13114BP).

The present inventors have confirmed that each of the four selected strains is excellent in the prevention or treatment of alcoholic intestinal damage, in particular mixed strains of these can effectively prevent inflammation of the intestinal cells caused by alcohol than each single strain By clarifying, the present invention has been completed.

Accordingly, it is an object of the present invention to provide a lactic acid bacterium composition comprising each single strain selected or a mixed strain thereof.

Probiotics are microorganisms that benefit health when ingested at the right amount, protecting the intestinal mucosa surface from various pathogens and foreign substances. Recently, as probiotics have been shown to regulate intestinal mucosal defense and intestinal permeability in chronic inflammatory bowel disease, various studies have been conducted based on the hypothesis that they will be involved in intestinal cell protection.

The present inventors selected probiotics having an alcohol protection effect on epithelial cell lines of organs of the small intestine, and developed a mixed strain of probiotics capable of covering all damage to each small intestine.

In one embodiment, the invention provides lactic acid bacteria composition comprising the Lactobacillus bacteria (Lactobacillus) in lactic acid bacteria, Bifidobacterium (Bifidobacterium) in lactic acid bacteria, or a mixture thereof.

According to one embodiment of the present invention, the Lactobacillus genus Lactobacillus is Lactobacillus bulgaricus ( Lactobacillus bulgaricus ), Lactobacillus helveticus ( Lactobacillus helveticus ), Lactobacillus plantarum ( Lactobacillus plantarum ), or a mixture thereof.

In a specific embodiment of the present invention, the Lactobacillus genus Lactobacillus Lactobacillus bulgaricus ( Lactobacillus bulgaricus ) CKDB001 (Accession No .: KCTC13669B), Lactobacillus helveticus ( Lactobacillus helveticus ) CKDB001 (Accession No .: KCTC13silus flu), Lactobacillus Lactobacillus plantarum CKDB008 (Accession Number: KCTC13673BP), or mixtures thereof.

According to another embodiment of the present invention, the lactic acid bacteria of the Bifidobacterium spp. Bifidobacterium ( Bifidobacterium) bifidum ), Bifidobacterium lactis , or mixtures thereof.

In a specific embodiment of the present invention, the Bifidobacterium spp. Lactic acid bacterium is Bifidobacterium bifidum CKDB001 (Accession No. KCTC13114BP).

In the most specific embodiment of the present invention, the lactic acid bacteria is Lactobacillus bulgaricus ( Lactobacillus bulgaricus ) CKDB001 (Accession No .: KCTC13669BP), Lactobacillus helveticus ( Lactobacillus helveticus ) CKDB001 (Accession No .: KCTC13670BP), Lactobacillus Planta ( Lactobacillus plantarum ) CKDB008 (Accession Number: KCTC13673BP), Bifidobacterium Bifidobacterium ( Bifidobacterium bifidum ) CKDB001 (Accession No. KCTC13114BP), or mixed strains thereof.

The Lactobacillus Bulgaricus CKDB001 strain of the present invention was deposited on the microbial resource center (Korea Collection for Type Culture, KCTC) of October 23, 2018, and was given the accession number KCTC 13669BP.

In addition, the Lactobacillus helbetticus CKDB001 strain of the present invention was deposited on the microbial resource center (Korean Collection for Type Culture, KCTC) Korea Research Institute of Biotechnology on October 23, 2018, and was given the accession number KCTC 13670BP.

In addition, the Lactobacillus plantarum CKDB008 strain of the present invention was deposited on the microbial resource center (Korean Collection for Type Culture, KCTC) Korea Research Institute of Bioscience and Biotechnology on October 23, 2018, was given the accession number KCTC 13673BP.

Finally, the Bifidobacterium bifiderm CKDB001 strain of the present invention was deposited on September 23, 2016 to the Korea Biotechnology Research Institute Microbial Resources Center (KCTC), and was given accession number KCTC13114BP.

The four strains were Gram-positive, bacillus, hemolytic, spore-forming, and catalase negative. The sugar availability of the strain was analyzed using the API 50 CHL kit (Table 1), and gene identification was performed by analyzing the 16s rDNA sequence for identifying the strain (Table 2).

Source L. bulgaricus
CKDB001 L. helveticus
CKDB001 L. plantarum
CKDB008 B.bifidum
CKDB001 Temoin 0 + + + - Glycerol One - - - - Erythritol 2 - - - - D-Arabinose 3 - - - - L-Arabinose 4 - - + - D-Ribose 5 - - + w D-Xylose 6 - - - - L-Xylose 7 - - - - D-Adonitol 8 - - - -

D-XylopyranosideMethyl-

D-Xylopyranoside 9 - - - - D-Galactose 10 - + + + D-Glucose 11 + + + + D-Fructose 12 + + w + D-Mannose 13 + + + - L-Sorbose 14 - - - - L-Rhamnose 15 - - + - Dulcitol 16 - - - - Inositol 17 - - - - D-Mannitol 18 - - + - D-Sorbitol 19 - - - - Methyl-αD-Mannopyranoside 20 - - + - Methyl-αD-Glucopyranoside 21 - - - - N-Acetylglucosamine 22 + + + + Amygdaline 23 - - + - Arbutine 24 - - + - Esculine, citrate de fer 25 - - - - Salicine 26 - - + - D-Cellobiose 27 - - + - D-Maltose 28 w + + - D-Lactose 29 + + + + D-Melibiose 30 - - + - D-Saccharose 31 + - + - D-Trehalose 32 + + + - Inuline 33 - - - - D-Melezitose 34 - - + - D-Raffinose 35 - - + - Amidon 36 - - - - Glycogene 37 - - - - Xylitol 38 - - - - Gentiobiose 39 - - + + D-Turanose 40 - - + - D-Lyxose 41 - - - - D-Tagatose 42 - - - - D-Fucose 43 - - - - L-Fucose 44 - - - - D-Arabitol 45 - - + - L-Arabitol 46 - - - - Potassium GlucoNaTe 47 - - + - Potassium 2-CetoGluconate 48 - - - - Potassium 5-CetoGluconate 49 - - - w

Strains sequence L. bulgaricus CKDB001 GTTTGAATCATGGCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGAGCTGAATTCAAAGATCCCTTCGGGGTGATTTGTTGGACGCTAGCGGCGGATGGGTGAGTAACACGTGGGCAATCTGCCCTAAAGACTGGGATACCACTTGGAAACAGGTGCTAATACCGGATAACAACATGAATCGCATGATTCAAGTTTGAAAGGCGGCGCAAGCTGTCACTTTAGGATGAGCCCGCGGCGCATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCAATGATGCGTAGCCGAGTTGAGAGACTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGCAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGTCTTCGGATCGTAAAGCTCTGTTGTTGGTGAAGAAGGATAGAGGCAGTAACTGGTCTTTATTTGACGGTAATCAACCAGAAAGTCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGAATGATAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAACTGCATCGGAAACTGTCATTCTTGAGTGCAGAAGAGGAGAGTGGAACTCCATGTGTAGCGGTGGAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTCTCTGGTCTGCAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAGCGCTAGGTGTTGGGGACTTTCCGGTCCTCAGTGCCGCAGCAAACGCATTAAGCGCTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGG TCTTGACATCCTGCGCTACACCTAGAGATAGGTGGTTCCCTTCGGGGACGCAGAGACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCTTTAGTTGCCATCATTAAGTTGGGCACTCTAAAGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGGCAGTACAACGAGAAGCGAACCCGCGAGGGTAAGCGGATCTCTTAAAGCTGCTCTCAGTTCGGACTGCAGGCTGCAACTCGCCTGCACGAAGCTGGAATCGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGAAGTCTGCAATGCCCAAAGTCGGTGAGATAACCTTTAT L. helveticus CKDB001 GCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGAGCAGAACCAGCAGATTTACTTCGGTAATGACGCTGGGGACGCGAGCGGCGGATGGGTGAGTAACACGTGGGGAACCTGCCCCATAGTCTGGGATACCACTTGGAAACAGGTGCTAATACCGGATAAGAAAGCAGATCGCATGATCAGCTTATAAAAGGCGGCGTAAGCTGTCGCTATGGGATGGCCCCGCGGTGCATTAGCTAGTTGGTAAGGTAACGGCTTACCAAGGCAATGATGCATAGCCGAGTTGAGAGACTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGCAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTGGTGAAGAAGGATAGAGGTAGTAACTGGCCTTTATTTGACGGTAATCAACCAGAAAGTCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGAAGAATAAGTCTGATGTGAAAGCCCTCGGCTTAACCGAGGAACTGCATCGGAAACTGTTTTTCTTGAGTGCAGAAGAGGAGAGTGGAATTCCATGTGTAGCGGTGGAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGCAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAGTGCTAAGTGTTGGGAGGTTTCCGCCTCTCAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATC TAGTGCCATCCTAAGAGATTAGGAGTTCCCTTCGGGGACGCTAAGACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTTATTAGTTGCCAGCATTAAGTTGGGCACTCTAATGAGACTGCCGGTGATAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGTACAACGAGAAGCGAGCCTGCGAAGGCAAGCGAATCTCTGAAAGCTGTTCTCAGTTCGGACTGCAGTCTGCAACTCGACTGCACGAAGCTGGAATCGCTAGTAATCGCGGATCAGAACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGAAGTCTGCAATGCCCAAAGCCGGTGGCCTAACCTTCGGGAAGGAGCCGTCTAAGCAGGCAGATGACTGGGGTG L. plantarum CKDB008 GCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAACGAACTCTGGTATTGATTGGTGCTTGCATCATGATTTACATTTGAGTGAGTGGCGAACTGGTGAGTAACACGTGGGAAACCTGCCCAGAAGCGGGGGATAACACCTGGAAACAGATGCTAATACCGCATAACAACTTGGACCGCATGGTCCGAGCTTGAAAGATGGCTTCGGCTATCACTTTTGGATGGTCCCGCGGCGTATTAGCTAGATGGTGGGGTAACGGCTCACCATGGCAATGATACGTAGCCGACCTGAGAGGGTAATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAACTCTGTTGTTAAAGAAGAACATATCTGAGAGTAACTGTTCAGGTATTGACGGTATTTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCTTCGGCTCAACCGAAGAAGTGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGTATGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCATACCGTAAACGATGAATGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACA TACTATGCAAATCTAAGAGATTAGACGTTCCCTTCGGGGACATGGATACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTATCAGTTGCCAGCATTAAGTTGGGCACTCTGGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAACGAGTTGCGAACTCGCGAGAGTAAGCTAATCTCTTAAAGCCATTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCAAAGTCGGTGGGGTAACCTTT B. bifidum CKDB001 GCTCAGGATGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAACGGGATCCATCGGGCTTTGCTTGGTGGTGAGAGTGGCGAACGGGTGAGTAATGCGTGACCGACCTGCCCCATGCTCCGGAATAGCTCCTGGAAACGGGTGGTAATGCCGGATGTTCCACATGATCGCATGTGATTGTGGGAAAGATTCTATCGGCGTGGGATGGGGTCGCGTCCTATCAGCTTGTTGGTGAGGTAACGGCTCACCAAGGCTTCGACGGGTAGCCGGCCTGAGAGGGCGACCGGCCACATTGGGACTGAGATACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCGACGCCGCGTGAGGGATGGAGGCCTTCGGGTTGTAAACCTCTTTTGTTTGGGAGCAAGCCTTCGGGTGAGTGTACCTTTCGAATAAGCGCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGCGCAAGCGTTATCCGGATTTATTGGGCGTAAAGGGCTCGTAGGCGGCTCGTCGCGTCCGGTGTGAAAGTCCATCGCTTAACGGTGGATCTGCGCCGGGTACGGGCGGGCTGGAGTGCGGTAGGGGAGACTGGAATTCCCGGTGTAACGGTGGAATGTGTAGATATCGGGAAGAACACCGATGGCGAAGGCAGGTCTCTGGGCCGTCACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGACGCTGGATGTGGGGCACGTTCCACGTGTTCCGTGTCGGAGCTAACGCGTTAAGCGTCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGAAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGCGGATTAATTCGATGCAACGCGAAGAACCTTACCTGGGCTTGACATGTTCCCGACGACGCCAGAGATGGCGTTTCCCTTCGGGGCGG GTTCACAGGTGGTGCATGGTCGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGCCCCGTGTTGCCAGCACGTTATGGTGGGAACTCACGGGGGACCGCCGGGGTTAACTCGGAGGAAGGTGGGGATGACGTCAGATCATCATGCCCCTTACGTCCAGGGCTTCACGCATGCTACAATGGCCGGTACAGCGGGATGCGACATGGCGACATGGAGCGGATCCCTGAAAACCGGTCTCAGTTCGGATCGGAGCCTGCAACCCGGCTCCGTGAAGGCGGAGTCGCTAGTAATCGCGGATCAGCAACGCCGCGGTGAATGCGTTCCCGGGCCTTGTACACACCGCCCGTCAAGTCATGAAAGTGGGCAGCACCCGAAGCCGGTGGCCTAACCCCTTGTGGGATGGAGCCGTCTAAG

Lactobacillus bulgaricus CKDB001 (Accession No .: KCTC13669BP), Lactobacillus helveticus ( Lactobacillus helveticus ) CKDB001 (Accession No .: KCTC13670BP), Lactobacillus plantarum plant (Lactoba), which is an active ingredient of the composition of the present invention. CKDB008 (Accession No .: KCTC13673BP), Bifidobacterium Bifidobacterium bifidum ) CKDB001 (Accession No. KCTC13114BP) was selected among 17 probiotic strains to most effectively prevent intestinal damage caused by alcohol.

The lactic acid bacteria as an active ingredient of the composition of the present invention is Lactobacillus rhamnosus GG strain, which is a commercial strain having the ability to adhere to the small intestine (ileum) and colonic epithelial cells. It is superior to strain, LGG) and has very good intestinal adhesion regardless of the intestinal region.

In addition, despite the ethanol treatment of small intestine (duodenum and ileum) epithelial cells, the lactic acid bacteria exhibit a very high cell viability similar to normal cells as well as the commercial LGG strain.

In addition, the lactic acid bacteria greatly reduce the expression of inflammatory cytokines (IL-1β and TNFα) in comparison with the commercial strain LGG despite ethanol treatment on small intestine (duodenum and ileum) epithelial cells. Therefore, the lactic acid bacteria of the present invention shows a very good anti-inflammatory effect on the small intestine.

In particular, the four types of lactic acid bacteria deposited in the final selection and deposit institutions of the present invention are excellent in intestinal adhesion capacity, cell viability, and anti-inflammatory effect of each strain, but when used as a mixed strain mixed these than the anti-inflammatory effect of each single strain It is characteristic in that it has a better anti-inflammatory effect (synergy effect). Therefore, the lactic acid bacteria of the present invention is more useful when used as a mixed strain.

The strain, or a culture thereof, as an active ingredient of the compositions of the present invention is a culture comprising the cells in addition to the cells isolated and / or purified from the strain, extracts of the cells, culture supernatants, concentrates thereof, concentrates, It is a dry thing and also a dilution liquid, dilution liquid, etc. as needed, and the thing of all the states obtained by processing a culture liquid and a culture is included.

The cell culture method, extraction method, separation method, concentration method, drying method, dilution method and the like are not particularly limited.

As a medium for culturing the cells, milk protein such as skim milk, whey, casein, sugars, yeast extract, and the like are usually included. As a culture method, various aerobic or anaerobic methods can be suitably used.

For example, the culture temperature may be set to 35 to 45 ° C., and the culture medium may be neutralized using an alkali such as sodium hydroxide to maintain the pH of the medium from neutral to acidic, for example, pH 5-6. It may be. In addition to the neutralization culture method, any culture method such as a batch culture method can be used, and after culturing, the culture or its supernatant can be concentrated, dried, diluted, or the like as necessary.

In addition, the supernatant and the cells of the culture may be separated by centrifugation or membrane separation to recover the cells in a concentrated state. The cells may be subjected to sonication, enzyme treatment, or the like to extract the components in the cells, or to dry the culture, the supernatant, the cells, or the extract. These can be used as an active ingredient of the composition of the present invention.

According to one embodiment of the present invention, lactic acid bacteria as an active ingredient of the composition of the present invention exhibits excellent cell viability and anti-inflammatory effect on the intestinal epithelial cytotoxicity of the above-mentioned alcohol. Therefore, the composition of the present invention has the purpose of preventing, ameliorating or treating alcoholic intestinal injury.

As demonstrated in the specific embodiments of the present invention, the lactic acid bacteria of the present invention exhibit the above-mentioned beneficial effects on various parts of the small intestine (duodenum, ileum) and large intestine. Thus, the "gut" of the intestine damage means the small intestine and / or the large intestine, and more specifically the "small intestine" means the duodenum, jejunum, and / or ileum. Most specifically, the small intestine means duodenum and / or ileum.

As demonstrated in specific embodiments of the present invention, the lactic acid bacteria of the present invention significantly reduce the expression of inflammatory cytokines in the intestine. Therefore, the composition of the present invention has the purpose of preventing, ameliorating or treating enteritis or inflammatory bowel disease.

The enteritis in the present invention includes bacterial enteritis, viral enteritis, and alcoholic enteritis.

In the present invention, the inflammatory bowel disease is selected from the group consisting of Crohn's disease, Behcet's disease, and ulcerative colitis.

The lactic acid bacterium composition of the present invention may be prepared as a food composition or a pharmaceutical composition having the above-mentioned use.

When the composition of the present invention is made of a food composition, it may include not only the lactic acid bacteria as an active ingredient, but also components normally added during food production. Such additives include, for example, proteins, carbohydrates, fats, nutrients, seasonings and flavoring agents. Such carbohydrates include monosaccharides (eg, glucose, fructose, etc.), disaccharides (eg, maltose, sucrose, oligosaccharides, etc.) and polysaccharides (eg, dextrins, cyclodextrins, etc.). Sugars and sugar alcohols such as xylitol, sorbitol, erythritol, etc. As flavoring agents, natural flavoring agents (tauumatin, stevia extract (e.g., rebaudioside A, glycyrginine, etc.)) and synthetic flavoring agents (saccharin, aspart) Tom, etc.) can be used.

For example, when the food composition of the present invention is prepared with a drink, in addition to the strain which is the active ingredient of the present invention, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, jujube extract or licorice extract may be further included. have.

The food composition of the present invention includes processed forms of all natural materials such as food, functional food, nutritional supplement, health food and food additives. Food compositions of this type can be prepared in various forms according to conventional methods known in the art.

For example, the health food may be prepared by drinking the lactic acid bacteria itself in the form of tea, juice and drinks, or may be ingested by granulation, encapsulation and powdering. In addition, foods include beverages (including alcoholic beverages), fruits and processed foods (e.g. canned fruit, canned foods, jams, marmalade, etc.), fish, meat and processed foods (e.g. ham, sausage cornbeans, etc.). ), Breads and noodles (e.g. udon, soba, ramen, spaghetti, macaroni, etc.), fruit juices, various drinks, cookies, syrups, dairy products (e.g. yogurt, fermented milk, butter, cheese, etc.), edible vegetable oil, Vegetable protein, retort food, frozen food, various seasonings (eg, miso, soy sauce, sauce, etc.) can be prepared by adding the lactic acid bacteria of the present invention. In addition, in order to use the lactic acid bacteria of the present invention in the form of food additives can be prepared in powder or concentrate form.

When the composition of the present invention is made into a pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention are those commonly used in the preparation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like. It doesn't happen.

In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention may be administered orally or parenterally, and preferably applied by oral administration. The pharmaceutical composition of the present invention may be formulated in various oral or parenteral dosage forms as follows, but is not limited thereto.

Suitable dosages of the pharmaceutical compositions of the present invention vary depending on factors such as the formulation method, mode of administration, age, weight, sex, morbidity, food, time of administration, route of administration, rate of excretion and reaction sensitivity of the patient, Usually an experienced physician can easily determine and prescribe a dosage (pharmaceutically effective amount) effective for the desired treatment or prevention. According to a preferred embodiment of the present invention, the daily dose of the pharmaceutical composition of the present invention is 0.0001-100 mg / kg. As used herein, the term “pharmaceutically effective amount” means an amount sufficient to prevent or treat the aforementioned diseases.

As used herein, the term "treatment" refers to the reduction, inhibition, sedation or eradication of the disease state, and "prevention" means to inhibit the exacerbation of the disease state so that the disease state does not manifest, and includes the treatment.

Formulations for oral administration include, for example, tablets, pills, hard / soft capsules, solutions, suspensions, emulsifiers, syrups. Granules, elixirs, and the like, these formulations may be used one or more diluents or excipients, such as fillers, extenders, wetting agents, disintegrants, lubricants, binders, surfactants, etc. that are commonly used in addition to the active ingredient. As a disintegrant, agar, starch, alginic acid or its sodium salt, calcium monohydrogen phosphate anhydride, etc. may be used, and as lubricant, silica, talc, stearic acid or its magnesium salt or calcium salt, polyethylene glycol, etc. may be used. As the binder, magnesium aluminum silicate, starch paste, gelatin, tragacanth, methyl cellulose, sodium carboxymethyl cellulose, polyvinylpyrrolidine, low-substituted hydroxypropyl cellulose, and the like may be used. In addition to lactose, dextrose, sucrose, mannitol, sorbitol, cellulose. Glycine or the like may be used as a diluent, and in some cases, generally known boiling mixtures, absorbents, colorants, flavors, sweeteners, and the like may be used together.

The composition may be sterile or contain preservatives, stabilizers, hydrating or emulsifiers, salts for controlling osmotic pressure, buffers and other auxiliaries and other therapeutically useful materials, which are conventional methods of mixing, granulating or coating methods. It can be formulated accordingly.

The pharmaceutical compositions of the present invention may be prepared in unit dosage form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Or may be prepared by incorporation into a multi-dose container. The formulation may be in the form of solutions, suspensions, syrups or emulsions in oils or aqueous media, or in the form of extracts, powders, powders, granules, tablets or capsules, and may further comprise dispersants or stabilizers.

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

(a) The present invention provides a composition for preventing or treating alcoholic intestinal injury, which comprises probiotics as an active ingredient. The composition of the present invention is a novel isolated lactic acid strain Lactobacillus bulgaricus CKDB001 (Accession Number: KCTC13669B), Lactobacillus helveticus ( Lactobacillus helveticus ) CKDB001 (Accession Number: KCTC13670BP), Lactobacillus plantarum plant ( Lactobacillus plantarum) CKDB008 (Accession No .: KCTC13673BP), Bifidobacterium Bifidobacterium bifidum ) CKDB001 (Accession No. KCTC13114BP), or mixed strains thereof.

(b) The present invention is excellent in reducing inflammation of the intestinal cells due to alcohol stimulation, and thus may be usefully used as a food or therapeutic agent for preventing and improving alcoholic intestinal damage.

1a and 1b is a diagram showing the intestinal epithelial cell adhesion of the probiotic strain of the present invention.
Figure 2a is a diagram showing the protective effect of alcoholic cell damage to the duodenal cell line (HUTU-80) of the probiotic strain of the present invention.
Figure 2b is a diagram showing the protective effect of alcoholic cell damage to the ileal cell line (IEC-18) of the probiotic strain of the present invention.
Figure 3a is a diagram showing the mRNA expression inhibitory effect of the inflammatory cytokine IL-1β in the duodenal cell line (HUTU-80) of the probiotic strain of the present invention.
Figure 3b is a diagram showing the mRNA expression inhibitory effect of the inflammatory cytokine TNFα in the duodenal cell line (HUTU-80) of the probiotic strain of the present invention.
Figure 4a is a diagram showing the mRNA expression inhibitory effect of the inflammatory cytokine IL-1β in the ileal cell line (IEC-18) of the probiotic strain of the present invention.
Figure 4b is a diagram showing the mRNA expression inhibitory effect of the inflammatory cytokine TNFα in the ileal cell line (IEC-18) of the probiotic strain of the present invention.
Figure 5a is a diagram showing the effect of inhibiting inflammatory cytokine expression in the ileal cell line (IEC-18) of the mixed strain of the present invention.
Figure 5b is a diagram showing the inhibitory effect of inflammatory cytokine expression in the duodenal cell line (HUTU-80) of the mixed strain of the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example

Throughout this specification, "%" used to indicate the concentration of a particular substance is (weight / weight)% solids / solids, (weight / volume)%, unless otherwise indicated, and Liquid / liquid is (volume / volume)%.

1. Experimental Materials

1.1 Isolation and Screening of Probiotic Strains

1.1.1. New microbial isolation

The probiotics used in this experiment were isolated from Chong Kun Dang Bio Co., Ltd. (CKDBiO, Korea) and were isolated from infant feces, crude milk and other fermented foods. The collected samples were suspended in sterile anaerobic water, and after dilution in decimal, were plated on MRS (ManRogosa Sharpe) solid medium and BL (Blood-liver) solid medium. After incubation in the anaerobic chambers maintained at 37 ℃ to 48 hours _{5% CO 2, 10% H} 2, 85% N 2 atmosphere during composition, and form a separate pure by selecting different colonies (colony). As a result, a total of 530 strains of lactic acid bacteria were isolated. Subsequently, the 530 strains of the isolated lactic acid bacteria were tested for hemolysis, gelatinase production, urease production, and bioamine production. Were screened.

1.1.2 Comparative evaluation of resistance to digestive fluids (acidic pH and bile salts)

For each strain of the lactic acid bacteria selected in 1.1.1 was compared and analyzed for resistance to pH and bile salts.

In order to confirm the resistance to the acidic pH of the selected lactic acid bacteria, an MRS or BL liquid medium adjusted to pH 2.5 using hydrochloric acid was prepared. Each strain was inoculated at 1% in the medium, and then diluted in an anaerobic dilution (pH 6.2) to measure the initial bacterial count, followed by 1 ml aliquots, plated using the same solid medium, and incubated at 37 ° C. for 48 hours. . MRS liquid medium and BL liquid medium at pH 2.5 inoculated with the strain were incubated for 2 hours in a 37 ℃ incubator and then cultured in the same solid medium in the same manner as above. Colonies that appeared after incubation were counted to determine resistance to pH.

To confirm the bile resistance of the selected lactic acid bacteria, 1% of the secondary passaged lactic acid bacteria was added by adding 0.3% oxal (Sigma, USA) to the MRS or BL liquid medium using the method of Gilliland and Walker (1990). Inoculation. As a control, lactic acid bacteria were inoculated in MRS or BL liquid medium without 0.3% oxal. All samples were incubated in a 37 ° C. incubator and after 2 hours, incubated in each lactic acid bacteria growing solid medium to measure the number of microorganisms according to the conventional resistance measurement method (Equation 1).

Equation 1

Survival rate (%) = [(CFU / ml _120min ) / (CFU / ml _0min )] x100

As a result, a total of 128 strains were selected by selecting the top 30% of strains having high acid resistance and bile resistance.

1.1.3 Preparation of Lactic Acid Bacteria Raw Materials

The high-density medium and culture conditions optimized for each strain were cultured for the production of high-density powder of the selected 128-week lactic acid bacteria, and then the cell recovery and freeze-drying process parts were rapidly replaced by conventional methods (60 ° C. freezer after centrifugation). Frozen and then lyophilized under operating conditions between 0 ° C. and 45 ° C.). After cell recovery, maltodextrin 5-50% (volume / weight)% or trehalose 5-50% (volume / weight)% or cellulose 5-50% (volume / weight)% of the concentrate was added, and lyophilized and ground to prepare a lactic acid bacteria powder. It was. As a result, 17 species (Table 3) were finally selected, which showed the highest survival rate by species and the longest storage stability and the highest possibility of industrialization. Powdered strains were suspended in animal cell culture media.

List of Probiotic Strains Used in the Study Serial number Strain name Survival rate
(%) Abbreviation One Lactobacillus helveticus CKDB001 60 LH 2 Lactobacillus gasseri CKDB026 50 LG 3 Lactobacillus reuteri CKDB016 67 LU 4 Lactobacillus plantarum CKDB008 82 LP 5 Lactobacillus acidophilus CKDB007 29 LA 6 Lactobacillus bulgaricus CKDB001 72 LB 7 Lactobacillus casei CKDB007 75 LC 8 Lactobacillus fermentum CKDB004 74 LF 9 Lactobacillus rhamnosus CKDB 80 LR 10 Lactobacillus salivarius CKDB001 75 LS 11 Lactobacillus paracasei CKDB005 68 LPARA 12 Lactococcus lactis CKDB007 81 LCL 13 Streptococcus thermophilus CKDB021 70 ST 14 Bifidobacterium lactis CKDB005 35 BL 15 Bifidobacterium longum CKDB004 40 BO 16 Bifidobacterium bifidum CKDB001 27 BF 17 Bifidobacterium breve CKDB002 38 BB

1.2 Cell Culture

HUTU-80 (duodenal epithelial cell line, Human), IEC-18 (ileal epithelial cell line, Rat), HT-29 (colon epithelial cell line, Human) cells were distributed by the Korea Cell Line Bank (KCLB, Seoul, Korea). -80 and IEC-18 used Dulbeco's Modified Eagle's Media (DMEM) medium, HT-29 used Rowell Park Memorial Institue (RPMI) 1640 medium, and 1% Penicillin / Streptomycin (P / S) Fetal Bovine Serum (FBS) 10% was added to each culture using a culture medium at 37 ℃, 5% CO ₂ conditions.

1.3 Statistical Processing

The significant difference test for the experimental results was analyzed by ANO (Anova: analysis of variance) in the Prism software and then multiplexed at p <0.05.

2. of the present invention Probiotic  Intestinal epithelial cells Adhesion  Measure

The intestinal tract lives with numerous enterobacteria competing and interacting with each other. Intestinal adhesion is essential for probiotics to exert such functions as inhibiting harmful bacteria, enhancing barrier function, and regulating immune function. Therefore, the present inventors measured the adhesion ability to ileal epithelial cells (IEC-18) and colon epithelial cells (HT-29) to confirm the intestinal adhesion of the probiotic strains of the present invention.

2.1 Determination of the Adhesion of Probiotic Strains to the Isolation Epithelial Cell Line (IEC-18)

IEC-18 was dispensed in a 12 well cell culture dish at a concentration of 1Х10 ⁵ cell / mL and incubated in a cell incubator (37 ° C., 5% CO _{2 for} about 2 days) until monolayer. Probiotic strains were diluted in antibiotic free cell media DMEM at 1Х10 ⁹ CFU / mL. When IEC-18 achieves confluence, dispense 1 mL of each fungal solution into the cells and incubate in the cell culture for 90 minutes. Then washed twice with PBS and treated with Trypsin-EDTA to detach the cells. The separated cells are diluted in steps and inoculated on an MRS agar plate to measure viable cells after 18 hours of incubation at 37 ° C. The comparative group used a commercial strain Lactobacillus rhamnosus GG, the intestinal adhesion rate was calculated as shown in Equation 2.

Equation 2

Intestinal adhesion rate (%) = (viable cell count / initial viable cell number after 90 min incubation) x 100

As a result, 17 strains of CKDBiO showed better intestinal adhesion than L. GG (0.02%) in IEC-18. B. bifidum CKDB001 (0.18%), L. plantarum CKDB008 (0.11%), S. thermophilus CKDB021 (0.08%), L. salivarius CKDB001 (0.05%), L. bulgaricus CKDB001 (0.04%), L. helveticus CKDB001 (0.02%), and L. fermentum Seven species of CKDB004 (0.02%) were identified (FIG. 1A).

2.2 Determination of the Probiotic Strain Adhesion to the Colonic Epithelial Cell Line (HT-29)

HT-29 was dispensed in a 12 well cell culture dish at a concentration of 1Х10 ⁵ cell / mL and cultured in a cell culture (37 ° C., 5% CO ₂ ) for about 5 days until a monolayer was obtained. Each probiotic strain was diluted to 1Х10 ⁹ CFU / mL in RPMI 1640 without antibiotics. When HT-29 achieved confluency, each bacterial solution was dispensed into cells by 1 mL and incubated in a cell incubator for 90 minutes. Then washed twice with PBS and treated with Trypsin-EDTA to detach the cells. The detached cells were diluted in steps and inoculated on an MRS agar plate and cultured at 37 ° C. for 18 hours, and the number of viable cells was measured. As a comparative group, a commercial strain Lactobacillus rhamnosus GG was used, and the intestinal adhesion rate was calculated as in Equation 2 in the same manner as in 2.1.

Equation 2

Intestinal adhesion rate (%) = (viable cell count / initial viable cell number after 90 min incubation) x 100

L. helveticus strains superior in intestinal adhesion to LGG (2.23%) in HT-29 CKDB001 (8.17%), L. reuteri CKDB016 (4.88%), L. plantarum CKDB008 (4.41%), L. acidophilus CKDB007 (3.43%), L. bulgaricus CKDB001 (3.39%), L. casei Nine species of CKDB007 (3.32%), B. lactis CKD005 (3.22%), B. longum CKD004 (3.11%), and L. fermentum CKD004 (2.37%) were identified (FIG. 1B).

2.3. Sintered

In consideration of the strain having excellent intestinal adhesion to both cell lines, a total of 12 of 17 species were selected. Among them, BBF and LH were found to be significantly superior intestinal adhesion ability compared to LGG, which is well known as a strain with excellent intestinal adhesion ability.

3. of the present invention Probiotic  Alcohol Intestinal Cell Damage Protection Effect-Measurement of Cell Viability MTT  assay)

MTT assay was performed to evaluate the toxicity of alcohol to the intestinal epithelial cells and the prophylactic effects of it. MTT assay was performed with 12 strains that were superior to LGG in the previous intestinal adhesion test.

3.1 Experimental Method

First, HUTU-80 and IEC-18 were dispensed at a concentration of 1Х10 ⁵ cell / mL in a 96 well cell culture dish and cultured in a cell culture (37 ° C., 5% CO ₂ ) until a single layer was obtained. Probiotic strains were diluted to 1Х10 ⁶ CFU / mL in antibiotic free cell media MEM. When the cells achieved confluence, each bacterial solution was dispensed into the cells by 200 μL and incubated in the cell incubator for 90 minutes. Then, washed twice with PBS, the cell medium containing ethanol was dispensed and reacted for 90 minutes. After the reaction was completed, the medium was removed, MTT solution was added thereto, and incubated for 3 hours in a cell incubator. Dimethyl sulfoxide (DMSO) was added to terminate the reaction. DMSO was reacted for 10 minutes and cell viability was calculated by measuring absorbance at 540 nm. The control group was treated only with ethanol, and the comparative group used a commercial strain Lactobacillus rhamnosus GG. Cell viability was calculated as shown in Equation 3 below.

Expression 3

Cell survival rate (%) = (Experimental absorbance value / control absorbance value) x 100

3.2 Protective effect on duodenal epithelial cell line (HUTU-80)

In the duodenal epithelial cell line (HUTU-80), the cell survival rate was 33% compared to the normal group in the ethanol-only control group. Pretreatment with LGG, a comparative bacterium, increased cell viability to 82%. Two of 12 strains showed higher cell viability than LGG. L. plantarum CKDB008 is 95%, L. salivarius CKDB001 showed 88% cell viability and showed a significant difference from the control group as well as similar cell viability to normal cells (FIG. 2A).

3.3 Protective effect against ileal epithelial cell line (IEC-18)

In the ileal epithelial cell line (IEC-18), the cell survival rate was reduced to 59% in the ethanol-only control group. Pretreatment with LGG, a comparative bacterium, increased cell viability by 77%. Five out of 12 strains showed higher cell viability than LGG. Ie B. bifidum CKDB001 , L. bulgaricus CKDB001 , L. fermentum CKDB004 , L. helveticus CKDB001 , and L. acidophilus CKDB007 showed more than 80% cell viability and showed a significant difference from the control group (FIG. 2B).

4. Of the present invention Probiotic  Inhibitory Effect of Intestinal Inflammatory Cytokines

RT-PCR was performed to confirm whether the probiotic strain of the present invention inhibits the mRNA expression of inflammatory cytokines induced by alcohol. Inflammatory cytokine mRNA expression was confirmed by seven strains that were superior to LGG in the previous intestinal adhesion and MTT assay.

4.1 Experimental Method

First, divide the duodenal epithelial cell line (HUTU-80) and ileal epithelial cell line (IEC-18) into a 6 well cell culture dish at a concentration of 1Х10 ⁵ cell / mL until the cell becomes monolayer (37 ° C, 5% CO _2). Incubated). Probiotics were diluted to 1Х10 ⁹ CFU / mL in antibiotic free cell media DMEM. When the cells achieved confluence, each bacterial solution was dispensed into the cells by 3 mL and incubated in the cell incubator for 90 minutes. Then, washed twice with PBS, the cell medium containing ethanol was dispensed and reacted for 90 minutes. After the reaction, the cells were detached by treatment with Trypsin-EDTA. RNA was extracted from each cell line and cDNA was synthesized. And RT-PCR confirmed the mRNA expression of inflammatory cytokines. Genomic base sequences used for PCR analysis are described in Table 4.

Genome sequence used for PCR analysis Cell line dielectric Forward Reverse HUTU-80
(Human) GAPDH ACCCACTCCTCCACCTTTGA CTGTTGCTGTAGCCAAATTCGT IL-1β CCGACCACCACTACAGCAAG GGGCAGGGAACCAGCATCTT TNFα CTGGGCAGGTCTACTTTGGG CTGGAGGCCCCAGTTTGAAT IEC-18
(Rat) GAPDH AGTGCCAGCCTCGTCTCATA GATGGTGATGGGTTTCCCGT IL-1β CACCTCTCAAGCAGAGCACAG GGGTTCCATGGTGAAGTCAAC TNFα GGCTTTCGGAACTCACTGGA CCCGTAGGGCGATTACAGTC

4.2 Effect of Inflammatory Cytokine mRNA on the Duodenal Epithelial Cell Line (HUTU-80)

In the duodenal epithelial cell line, the IL-1β mRNA expression and TNFα expression increased by 385% and 357% in the ethanol-only control group. Pretreatment with LGG, a comparative bacterium, reduced IL-1β to 235% and TNFα to 257%. B. bifidum has a lower IL-1β expression level than LGG. One species of CKDB001 was reduced to 155%. B. bifidum was found to have lower TNFα expression than LGG. CKDB001, L. plantarum Two CKDB008 species were reduced to 130% and 142%, respectively. Therefore, two of the seven candidate strains were able to more effectively reduce the inflammatory response of the duodenal cells due to alcohol than the comparison group LGG (Fig. 3a and 3b).

4.3 Effect of Reduction of Inflammatory Cytokine mRNA Expression in the Isolate Epithelial Cell Line (IEC-18)

In the duodenal epithelial cell line, the ethanol-only control group showed an increase of 320% in IL-1β mRNA and a 289% increase in TNFα expression. Pretreatment with LGG, a comparative bacterium, reduced IL-1β to 201% and TNFα to 223%. L. bulgaricus strains with lower IL-1β expression than LGG CKDB001 , L. helveticus CKDB001 , L. plantarum Three types of CKDB008 were reduced to 145%, 166% and 182%, respectively. L. helveticus strains with lower TNFα expression than LGG One species of CKDB001 was reduced to 191%. Therefore, three of the seven candidate strains were found to be able to more effectively reduce the inflammatory response of the ileal epithelial cells due to alcohol than the comparative group LGG (Figs. 4a and 4b).

4.4 Sintering

B. bifidum CKDB001 has been shown to effectively reduce inflammatory cytokine expression in duodenal cell lines, but relatively ineffective in ileal cell lines. L. helveticus CKDB001 showed the opposite trend. Therefore, when these strains are mixed, they may complement each other and have anti-inflammatory effects all around the small intestine.

5. Verification of the Inhibitory Effect of Intestinal Inflammatory Cytokines of Mixed Strains of the Present Invention

The inventors of the present invention, in order to determine whether the strain mixture efficacious for damage to each small intestine region selected in the above 4 is more effective than a single strain, the mixed strain of the present invention (4 kinds of excellent strain inhibiting the inflammatory cytokine mRNA expression level: B. bifidum CKDB001, L. helveticus CKDB001, L. plantarum CKDB008, L. bulgaricus CKDB001) demonstrated the efficacy of inhibiting the expression of intestinal inflammatory cytokines. Experimental method was carried out in the same manner as described above 4 except that the four mixed strains were set as the experimental group.

5.1 Effect of Reduction of Inflammatory Cytokine mRNA Expression in the Isolate Epithelial Cell Line (IEC-18)

In the control group that reacted only 5% ethanol in the ileal cell line, IL-1β mRNA expression was increased to 481% compared to normal, and decreased to 220% when pretreated with the comparative strain LGG. L. helveticus CKDB001, L. plantarum CKDB008 and L. bulgaricus CKDB001 reduced IL-1β mRNA levels to 110%, 202%, and 132%, respectively, and were more effective than LGG. It was significantly lowered to%, similar to normal.

In the case of TNFα, 5% ethanol treatment increased the expression level to 343% compared to normal, and decreased to 150% when pretreatment of the comparative bacteria LGG. L. helveticus CKDB001 was shown to be more effective than LGG by reducing the amount of TNFα mRNA to 104%, and when mixed with four bacteria, it was lowered to 105%.

5.2 Effect of Reduced Inflammatory Cytokine mRNA Expression in the Duodenal Epithelial Cell Line (HUTU-80) of Mixed Strains

In the duodenal cell line, the control group that reacted with only 2.5% ethanol increased IL-1β mRNA expression to 427% compared to normal, and decreased to 217% when pretreated LGG. B. bifidum CKDB001, L. plantarum CKDB008 lowered IL-1β mRNA expression level to 168% and 212%, respectively, and showed more effective than LGG. When pretreatment with 4 strains, IL-1β mRNA expression level was 150%, showing synergistic effect. Appeared.

In the case of TNFα, 2.5% ethanol treatment increased the expression to 411% compared to normal, and the pretreatment of LGG, a comparative group, decreased to 266%. B. bifidum CKDB001, L. plantarum CKDB008 was shown to be more effective than LGG by lowering the mRNA expression level to 143% and 150%, respectively, and the pretreatment of 4 strains showed the lowest TNFα mRNA expression level of 138% (Fig. 5b). .

5.3 Sintering

From the above results, the present inventors confirmed that the mixed strain of the present invention was more effective in inhibiting inflammatory cytokine expression compared to a single strain in both ileal and duodenal cell lines.

6. Conclusion

In order to develop a probiotic mixture effectively preventing bowel damage caused by alcohol, each chapter was subdivided to verify the efficacy of the strain for each site. A total of 17 strains were identified from the total of 530 types of lactic acid bacteria that confirmed safety, stability, and industrialization. After that, 12 species having excellent intestinal adhesion were selected, and among them, seven strains were selected to improve cell viability lowering due to alcohol.

Finally, four strains that effectively lowered the inflammatory cytokine expression increased due to alcohol were selected. It was confirmed that the selected four strains could effectively prevent the inflammation of enterocytes caused by alcohol than each single strain. This mixed strain can be said to be an excavation in that it shows an excellent effect than the LGG bacteria, which have been proven to be effective in conventional intestinal adhesion and anti-inflammatory.

In addition, most existing studies have been used to prevent and treat liver damage caused by alcohol, and have used routine methods of measuring blood alcohol levels, inflammatory responses in the liver and histological results. In this study, the study was conducted to prevent alcoholic intestinal damage as well as all the intestinal tracts by confirming the degree of damage and alleviating the inflammatory response.

Korea Research Institute of Bioscience and Biotechnology KCTC13669BP 20181023 Korea Research Institute of Bioscience and Biotechnology KCTC13670BP 20181023 Korea Research Institute of Bioscience and Biotechnology KCTC13673BP 20181023 Korea Research Institute of Bioscience and Biotechnology KCTC13114BP 20160923

<110> CKD Bio Corp <120> Composition for Preventing or Treating Alcoholic Intestinal          Damage Comprising Probiotics as Effective Ingredients <130> PN180323 <160> 16 <170> KoPatentIn 3.0 <210> 1 <211> 1465 <212> RNA <213> Artificial Sequence <220> <223> 16s rRNA of L. bulgaricus CKDB001 (KCTC13669BP) <400> 1 gtttgaatca tggctcagga cgaacgctgg cggcgtgcct aatacatgca agtcgagcga 60 gctgaattca aagatccctt cggggtgatt tgttggacgc tagcggcgga tgggtgagta 120 acacgtgggc aatctgccct aaagactggg ataccacttg gaaacaggtg ctaataccgg 180 ataacaacat gaatcgcatg attcaagttt gaaaggcggc gcaagctgtc actttaggat 240 gagcccgcgg cgcattagct agttggtggg gtaaaggcct accaaggcaa tgatgcgtag 300 ccgagttgag agactgatcg gccacattgg gactgagaca cggcccaaac tcctacggga 360 ggcagcagta gggaatcttc cacaatggac gcaagtctga tggagcaacg ccgcgtgagt 420 gaagaaggtc ttcggatcgt aaagctctgt tgttggtgaa gaaggataga ggcagtaact 480 ggtctttatt tgacggtaat caaccagaaa gtcacggcta actacgtgcc agcagccgcg 540 gtaatacgta ggtggcaagc gttgtccgga tttattgggc gtaaagcgag cgcaggcgga 600 atgataagtc tgatgtgaaa gcccacggct caaccgtgga actgcatcgg aaactgtcat 660 tcttgagtgc agaagaggag agtggaactc catgtgtagc ggtggaatgc gtagatatat 720 ggaagaacac cagtggcgaa ggcggctctc tggtctgcaa ctgacgctga ggctcgaaag 780 catgggtagc gaacaggatt agataccctg gtagtccatg ccgtaaacga tgagcgctag 840 gtgttgggga ctttccggtc ctcagtgccg cagcaaacgc attaagcgct ccgcctgggg 900 agtacgaccg caaggttgaa actcaaagga attgacgggg gcccgcacaa gcggtggagc 960 atgtggttta attcgaagca acgcgaagaa ccttaccagg tcttgacatc ctgcgctaca 1020 cctagagata ggtggttccc ttcggggacg cagagacagg tggtgcatgg ctgtcgtcag 1080 ctcgtgtcgt gagatgttgg gttaagtccc gcaacgagcg caacccttgt ctttagttgc 1140 catcattaag ttgggcactc taaagagact gccggtgaca aaccggagga aggtggggat 1200 gacgtcaagt catcatgccc cttatgacct gggctacaca cgtgctacaa tgggcagtac 1260 aacgagaagc gaacccgcga gggtaagcgg atctcttaaa gctgctctca gttcggactg 1320 caggctgcaa ctcgcctgca cgaagctgga atcgctagta atcgcggatc agcacgccgc 1380 ggtgaatacg ttcccgggcc ttgtacacac cgcccgtcac accatggaag tctgcaatgc 1440 ccaaagtcgg tgagataacc tttat 1465 <210> 2 <211> 1490 <212> RNA <213> Artificial Sequence <220> <223> 16s rRNA of L. helveticus CKDB001 (KCTC13670BP) <400> 2 gctcaggacg aacgctggcg gcgtgcctaa tacatgcaag tcgagcgagc agaaccagca 60 gatttacttc ggtaatgacg ctggggacgc gagcggcgga tgggtgagta acacgtgggg 120 aacctgcccc atagtctggg ataccacttg gaaacaggtg ctaataccgg ataagaaagc 180 agatcgcatg atcagcttat aaaaggcggc gtaagctgtc gctatgggat ggccccgcgg 240 tgcattagct agttggtaag gtaacggctt accaaggcaa tgatgcatag ccgagttgag 300 agactgatcg gccacattgg gactgagaca cggcccaaac tcctacggga ggcagcagta 360 gggaatcttc cacaatggac gcaagtctga tggagcaacg ccgcgtgagt gaagaaggtt 420 ttcggatcgt aaagctctgt tgttggtgaa gaaggataga ggtagtaact ggcctttatt 480 tgacggtaat caaccagaaa gtcacggcta actacgtgcc agcagccgcg gtaatacgta 540 ggtggcaagc gttgtccgga tttattgggc gtaaagcgag cgcaggcgga agaataagtc 600 tgatgtgaaa gccctcggct taaccgagga actgcatcgg aaactgtttt tcttgagtgc 660 agaagaggag agtggaattc catgtgtagc ggtggaatgc gtagatatat ggaagaacac 720 cagtggcgaa ggcgactctc tggtctgcaa ctgacgctga ggctcgaaag catgggtagc 780 gaacaggatt agataccctg gtagtccatg ccgtaaacga tgagtgctaa gtgttgggag 840 gtttccgcct ctcagtgctg cagctaacgc attaagcact ccgcctgggg agtacgaccg 900 caaggttgaa actcaaagga attgacgggg gcccgcacaa gcggtggagc atgtggttta 960 attcgaagca acgcgaagaa ccttaccagg tcttgacatc tagtgccatc ctaagagatt 1020 aggagttccc ttcggggacg ctaagacagg tggtgcatgg ctgtcgtcag ctcgtgtcgt 1080 gagatgttgg gttaagtccc gcaacgagcg caacccttgt tattagttgc cagcattaag 1140 ttgggcactc taatgagact gccggtgata aaccggagga aggtggggat gacgtcaagt 1200 catcatgccc cttatgacct gggctacaca cgtgctacaa tggacagtac aacgagaagc 1260 gagcctgcga aggcaagcga atctctgaaa gctgttctca gttcggactg cagtctgcaa 1320 ctcgactgca cgaagctgga atcgctagta atcgcggatc agaacgccgc ggtgaatacg 1380 ttcccgggcc ttgtacacac cgcccgtcac accatggaag tctgcaatgc ccaaagccgg 1440 tggcctaacc ttcgggaagg agccgtctaa gcaggcagat gactggggtg 1490 <210> 3 <211> 1455 <212> RNA <213> Artificial Sequence <220> <223> 16 s rRNA of L. plantarum CKDB008 (KCTC13673BP) <400> 3 gctcaggacg aacgctggcg gcgtgcctaa tacatgcaag tcgaacgaac tctggtattg 60 attggtgctt gcatcatgat ttacatttga gtgagtggcg aactggtgag taacacgtgg 120 gaaacctgcc cagaagcggg ggataacacc tggaaacaga tgctaatacc gcataacaac 180 ttggaccgca tggtccgagc ttgaaagatg gcttcggcta tcacttttgg atggtcccgc 240 ggcgtattag ctagatggtg gggtaacggc tcaccatggc aatgatacgt agccgacctg 300 agagggtaat cggccacatt gggactgaga cacggcccaa actcctacgg gaggcagcag 360 tagggaatct tccacaatgg acgaaagtct gatggagcaa cgccgcgtga gtgaagaagg 420 gtttcggctc gtaaaactct gttgttaaag aagaacatat ctgagagtaa ctgttcaggt 480 attgacggta tttaaccaga aagccacggc taactacgtg ccagcagccg cggtaatacg 540 taggtggcaa gcgttgtccg gatttattgg gcgtaaagcg agcgcaggcg gttttttaag 600 tctgatgtga aagccttcgg ctcaaccgaa gaagtgcatc ggaaactggg aaacttgagt 660 gcagaagagg acagtggaac tccatgtgta gcggtgaaat gcgtagatat atggaagaac 720 accagtggcg aaggcggctg tctggtctgt aactgacgct gaggctcgaa agtatgggta 780 gcaaacagga ttagataccc tggtagtcca taccgtaaac gatgaatgct aagtgttgga 840 gggtttccgc ccttcagtgc tgcagctaac gcattaagca ttccgcctgg ggagtacggc 900 cgcaaggctg aaactcaaag gaattgacgg gggcccgcac aagcggtgga gcatgtggtt 960 taattcgaag ctacgcgaag aaccttacca ggtcttgaca tactatgcaa atctaagaga 1020 ttagacgttc ccttcgggga catggataca ggtggtgcat ggttgtcgtc agctcgtgtc 1080 gtgagatgtt gggttaagtc ccgcaacgag cgcaaccctt attatcagtt gccagcatta 1140 agttgggcac tctggtgaga ctgccggtga caaaccggag gaaggtgggg atgacgtcaa 1200 atcatcatgc cccttatgac ctgggctaca cacgtgctac aatggatggt acaacgagtt 1260 gcgaactcgc gagagtaagc taatctctta aagccattct cagttcggat tgtaggctgc 1320 aactcgccta catgaagtcg gaatcgctag taatcgcgga tcagcatgcc gcggtgaata 1380 cgttcccggg ccttgtacac accgcccgtc acaccatgag agtttgtaac acccaaagtc 1440 ggtggggtaa ccttt 1455 <210> 4 <211> 1436 <212> RNA <213> Artificial Sequence <220> <223> 16s rRNA of B. bifidum CKDB001 (KCTC13114BP) <400> 4 gctcaggatg aacgctggcg gcgtgcttaa cacatgcaag tcgaacggga tccatcgggc 60 tttgcttggt ggtgagagtg gcgaacgggt gagtaatgcg tgaccgacct gccccatgct 120 ccggaatagc tcctggaaac gggtggtaat gccggatgtt ccacatgatc gcatgtgatt 180 gtgggaaaga ttctatcggc gtgggatggg gtcgcgtcct atcagcttgt tggtgaggta 240 acggctcacc aaggcttcga cgggtagccg gcctgagagg gcgaccggcc acattgggac 300 tgagatacgg cccagactcc tacgggaggc agcagtgggg aatattgcac aatgggcgca 360 agcctgatgc agcgacgccg cgtgagggat ggaggccttc gggttgtaaa cctcttttgt 420 ttgggagcaa gccttcgggt gagtgtacct ttcgaataag cgccggctaa ctacgtgcca 480 gcagccgcgg taatacgtag ggcgcaagcg ttatccggat ttattgggcg taaagggctc 540 gtaggcggct cgtcgcgtcc ggtgtgaaag tccatcgctt aacggtggat ctgcgccggg 600 tacgggcggg ctggagtgcg gtaggggaga ctggaattcc cggtgtaacg gtggaatgtg 660 tagatatcgg gaagaacacc gatggcgaag gcaggtctct gggccgtcac tgacgctgag 720 gagcgaaagc gtggggagcg aacaggatta gataccctgg tagtccacgc cgtaaacggt 780 ggacgctgga tgtggggcac gttccacgtg ttccgtgtcg gagctaacgc gttaagcgtc 840 ccgcctgggg agtacggccg caaggctaaa actcaaagaa attgacgggg gcccgcacaa 900 gcggcggagc atgcggatta attcgatgca acgcgaagaa ccttacctgg gcttgacatg 960 ttcccgacga cgccagagat ggcgtttccc ttcggggcgg gttcacaggt ggtgcatggt 1020 cgtcgtcagc tcgtgtcgtg agatgttggg ttaagtcccg caacgagcgc aaccctcgcc 1080 ccgtgttgcc agcacgttat ggtgggaact cacgggggac cgccggggtt aactcggagg 1140 aaggtgggga tgacgtcaga tcatcatgcc ccttacgtcc agggcttcac gcatgctaca 1200 atggccggta cagcgggatg cgacatggcg acatggagcg gatccctgaa aaccggtctc 1260 agttcggatc ggagcctgca acccggctcc gtgaaggcgg agtcgctagt aatcgcggat 1320 cagcaacgcc gcggtgaatg cgttcccggg ccttgtacac accgcccgtc aagtcatgaa 1380 agtgggcagc acccgaagcc ggtggcctaa ccccttgtgg gatggagccg tctaag 1436 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of human GAPDH <400> 5 acccactcct ccacctttga 20 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer of human GAPDH <400> 6 ctgttgctgt agccaaattc gt 22 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of human IL-1 beta <400> 7 ccgaccacca ctacagcaag 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer of human IL-1 beta <400> 8 gggcagggaa ccagcatctt 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of human TNF alpha <400> 9 ctgggcaggt ctactttggg 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer of human TNF alpha <400> 10 ctggaggccc cagtttgaat 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of rat GAPDH <400> 11 agtgccagcc tcgtctcata 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer of rat GAPDH <400> 12 gatggtgatg ggtttcccgt 20 <210> 13 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of rat IL-1 beta <400> 13 cacctctcaa gcagagcaca g 21 <210> 14 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer of rat IL-1 beta <400> 14 gggttccatg gtgaagtcaa c 21 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of rat TNF alpha <400> 15 ggctttcgga actcactgga 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer of rat TNF alpha <400> 16 cccgtagggc gattacagtc 20

Claims (10)

In Lactobacillus bacteria (Lactobacillus) in lactic acid bacteria, Bifidobacterium (Bifidobacterium) in lactic acid bacteria, or alcoholic bowel injury, colitis, or inflammatory bowel disease preventing or treating composition of the lactic acid bacteria, including mixtures thereof,
The Lactobacillus genus Lactobacillus is Lactobacillus bulgaricus CKDB001 (Accession No .: KCTC13669B), Lactobacillus helveticus ( Lactobacillus helveticus ) CKDB001 (Accession No .: KCTC13670BP), or a combination thereof; And
The lactic acid bacteria of the Bifidobacterium genus Bifidobacterium bifidum ( Bifidobacterium bifidum ) CKDB001 (Accession No. KCTC13114BP), lactic acid bacteria composition.
delete According to claim 1, The Lactobacillus genus Lactobacillus Lactobacillus plantarum ( Lactobacillus plantarum ) CKDB008 (Accession No .: KCTC13673BP), Lactic acid bacteria composition that will further comprise.
delete delete According to claim 1, The composition is Lactobacillus bulgaricus CKDB001 (Accession No .: KCTC13669B), Lactobacillus helveticus ( Lactobacillus helveticus ) CKDB001 (Accession No .: KCTC13670BP), Lactobacillus plantarum plant (Lumactus plantarum CKDB008 (Accession No .: KCTC13673BP), Bifidobacterium Bifidobacterium bifidum ) CKDB001 (Accession No. KCTC13114BP), or a mixed strain thereof, wherein the lactic acid bacteria composition.
delete According to claim 1, wherein the intestine is a small intestine, lactic acid bacteria composition.
delete According to claim 1, The inflammatory bowel disease is selected from the group consisting of Crohn's disease, Behcet's disease, ulcerative colitis, lactic acid bacteria composition.

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