Composition for preventing, treating, or improving gastrointestinal diseases comprising Corynebacterium sp. strain and culture thereof
technical field
[1]
The present application relates to a composition for preventing, treating, or improving a gastrointestinal disease comprising a Corynebacterium sp. strain, a culture thereof, and threonine.
[2]
background
[3]
In gastric lesions, gastric ulcer accounts for the highest incidence, and is common in all livestock breeds worldwide. The incidence of gastric ulcer is increasing with the development of the livestock industry, and along with the decrease in growth rate due to decreased appetite, efforts to prevent the occurrence of gastric ulcer are being strengthened in terms of animal welfare as a symptom accompanied by pain.
[4]
Threonine (Thr) is a major amino acid (Amino acid, AA) constituting the intestinal epithelial protective material mucine (mucine). Mucin protein plays an important role in maintaining intestinal health by promoting protein absorption and protecting the digestive system from strong acidic digestive juices such as gastric juice. It is reported that Thr treatment in actual feed helps mucin synthesis in piglets and improves intestinal health (Non-Patent Document 1).
[5]
The production of Thr for feed or food is produced by the fermentation method by microorganisms, and the types of microorganisms mainly used at this time are Escherichia coli , E. coli ) and Corynebacterium glutamicum , C. glutamicum ), etc. Despite producing the same AA, these two strains are divided into gram-negative and gram-positive, respectively. Both gram-negative and gram-positive bacteria have cell walls composed of peptidoglycan (PG). However, in the case of Gram-negative bacteria, in addition to PG, there is an additional lipopolysaccharide (LPS) layer composed of lipoprotein and protein. It has characteristics that indicate Therefore, in the pharmaceutical industry, endotoxin has been released from parenteral preparations with deleterious biological activities such as pyrogenicity, lethality, Schwartzman reactivity, adjuvant activity and macrophage activation. It is essential to remove (Non-Patent Document 2).
[6]
The production of amino acids for feed in granule type by the simplification (or omission) of the advanced purification process due to the economical effectiveness of feed additives inevitably causes the bacteria used for fermentation to die in the form of dead cells (heat killed). incorporated into the product. Some studies have shown that the dead cells of lactic acid bacteria, a representative gram-positive bacteria, are stable in the environment due to strong resistance to acid and heat, and are easy to handle due to high concentration, and are used as food for beneficial bacteria that have settled in the intestines to provide immunity unique to lactic acid bacteria. It is reported to enhance the strengthening activity (Non-Patent Document 3).
[7]
Under this background, the present applicants have made diligent efforts to develop a pharmaceutical composition effective for the prevention and treatment of gastrointestinal diseases, as a result of which a composition comprising a Corynebacterium sp. strain, a culture thereof, and threonine prevents gastrointestinal diseases, It was confirmed that it was effective in treatment and improvement, and the present application was completed.
[8]
[9]
(비특허문헌 1) Law G. (2000) Threonine requirement and the effect of threonine on gut mucin characteristics in piglets receiving intragastric nutrition. Master thesis of university of Alberta. 1-143.
[10]
(비특허문헌 2) Miyamoto T., Okono S. and Kasai N (2009) Inactivation of Escherichia coli endotoxin by soft hydrothermal processing. Applied and Environmental Microbiology, 75(15), 5058-5063.
[11]
(Non-Patent Document 3) Lee IH (2018) The latest trends surrounding antibacterial agents and alternatives for animals. Pig & Consulting, 4, 70-73.
[12]
DETAILED DESCRIPTION OF THE INVENTION
technical challenge
[13]
The present application provides a feed composition for preventing or improving gastrointestinal diseases, including a Corynebacterium sp. strain, a culture thereof, and threonine.
[14]
The present application provides a pharmaceutical composition for preventing or treating gastrointestinal diseases, including a Corynebacterium sp. strain, a culture thereof, and threonine.
[15]
The present application provides a food composition for preventing or improving gastrointestinal diseases, including a Corynebacterium sp. strain, a culture thereof, and threonine.
[16]
The present application provides an antibacterial composition for Helicobacter pylori, including a Corynebacterium sp. strain, a culture thereof, and threonine.
[17]
means of solving the problem
[18]
One aspect may provide a composition for preventing, improving, or treating gastrointestinal diseases, including a Corynebacterium sp. strain, a culture thereof, and threonine.
[19]
In the present application, "prevention" refers to any action that suppresses or delays the onset of a disease by administration of the composition according to an embodiment, and "treatment" refers to an individual suspected and onset of a disease by administration of the composition according to an embodiment. means any action in which the symptoms are improved or beneficially changed, and "improvement" means any action that at least reduces a parameter related to a condition in which a disease is treated by administration of the composition according to an example, for example, at least the degree of the symptom can do. The disease may refer to a gastrointestinal disease.
[20]
[21]
One aspect may provide a feed composition for preventing or improving gastrointestinal diseases, including a Corynebacterium sp. strain, a culture thereof, and threonine.
[22]
In the present application, " Corynebacterium sp., Coryne sp. strain" may include all Corynebacterium sp. strains. Corynebacterium sp. strains are, for example, Corynebacterium glutamicum ( Corynebacterium glutamicum ), Corynebacterium ammoniagenes ), Corynebacterium crudilactis ( Corynebacterium crudilactis ), Corynebacter Leum deserti ( Corynebacterium deserti ), Corynebacterium efficiens ( Corynebacterium efficiens ), Corynebacterium callunae ( Corynebacterium callunae ), Corynebacterium station nis ( Corynebacterium stationis ), Corynebacterium singulare ( Corynebacterium singulare ), Corynebacterium halotolerans ( Corynebacterium halotolerans )), Corynebacterium striatum , Corynebacterium pollutisoli , Corynebacterium imitans , Corynebacterium testudinoris , and / or Corynebacterium flavescens may be, and more specifically, Corynebacterium glutamicum, Corynebacterium ammoniagenes, or a combination thereof (Corynebacterium glutamicum and Coryne). bacterium ammoniagenes).
[23]
In one example, the Corynebacterium sp. strain may have a threonine-producing ability. In the present application, having the ability to produce threonine means showing the ability to produce and accumulate threonine in and/or in the medium when the microorganism is cultured in the medium.
[24]
When the composition for preventing, improving, or treating gastrointestinal diseases according to an example includes Corynebacterium glutamicum, Corynebacterium sp. strains other than Corynebacterium glutamicum (for example, Corynebacterium ifficiens) may be superior in (1) prevention, improvement, or therapeutic effect of gastrointestinal diseases and/or (2) anti-Helicobacter pylori efficacy than a composition containing .
[25]
When the composition for preventing, improving, or treating gastrointestinal diseases according to an example includes Corynebacterium ammoniagenes, other types of Corynebacterium sp. strains other than Corynebacterium ammoniagenes (for example, Corynebacterium ifficiens) may be superior in (1) prevention, improvement, or therapeutic effect of gastrointestinal diseases and/or (2) anti-Helicobacter pylori efficacy than a composition containing .
[26]
When the composition for preventing, improving, or treating gastrointestinal diseases according to an embodiment includes Corynebacterium glutamicum and Corynebacterium ammoniagenes, Corynebacterium glutamicum and Corynebacterium ammoniac (1) prevention, improvement, or therapeutic effect of gastrointestinal diseases and/or (2) anti-H. The effect of pylori may be excellent.
[27]
In one example, that the composition has excellent anti-Helicobacter pylori efficacy means that it has excellent antibacterial activity against Helicobacter pylori strain or that it has excellent activity for preventing apoptosis of cells infected by Helicobacter pylori (eg, gastric mucosal cells). can mean
[28]
The Corynebacterium sp. strain may refer to concentrated cells after removing the culture medium from the culture medium, and may be subjected to centrifugation and/or filtration to recover only the concentrated cells from the culture.
[29]
In one example, the Corynebacterium sp. strain may be included in the form of dead cells. In the present application, the term "dead cells" is the opposite concept of live cells, and refers to a form in which live cells and metabolites obtained through fermentation are prevented from growing by heat treatment or the like. The dead cells may include cytoplasm, cell wall, antibacterial active substances such as bacteriocin, polysaccharide, and/or organic acid.
[30]
According to an example, the composition containing dead cells may have one or more characteristics selected from the group consisting of the following (1) to (6) rather than the composition containing live cells.
[31]
(1) good acid resistance;
[32]
(2) excellent heat resistance;
[33]
(2) can be concentrated to a high concentration;
[34]
(4) good stability;
[35]
(5) easy handling and storage; and
[36]
(6) Can be used as food for beneficial intestinal bacteria.
[37]
The Corynebacterium sp. strain may be killed by Tyndall and/or heat treatment. In one example, the Corynebacterium sp. strain and the sterilized culture solution thereof include dead cells of the strain. For example, the heat treatment may be performed at a temperature of 60 to 130° C. for 3 to 30 minutes. In addition, the heat treatment may be performed by 1 to 10 times of ultra high temperature sterilization autoclave, and/or hot air drying. The ultra-high temperature sterilization may be performed at a temperature of 110° C. to 130° C. for 3.0 to 10.0 seconds, for example, twice at 100° C. for 1.0 to 10 seconds, and once at 121° C. for 1.0 to 10.0 seconds. may be, and the autoclave may be performed at 120 to 125 ° C., or 121 ° C. for 10 to 30 minutes, 15 to 25 minutes, or 20 minutes, and the hot air drying is at a temperature of 60 to 70 ° C. may be performed in
[38]
In one embodiment, the composition comprising the dead cells of the Corynebacterium sp. strain, the culture of the Corynebacterium sp. strain, and threonine in vitro and/or in vivo (1) preventive or therapeutic effect of gastrointestinal diseases and/or (2) it was confirmed that the anti-H. pylori effect was excellent.
[39]
The composition according to one embodiment is the Corynebacterium sp. strain (or the dead cell of the strain) 20 to 40 OD (wave length 560 to 565 nm), 25 to 35OD (wave length 560 to 565 nm), or 30OD concentration (wave length) 560 to 565 nm).
[40]
The composition according to one embodiment is the Corynebacterium sp. strain (or the dead cell of the strain) 20 to 40 OD (wave length 560 to 565 nm), 25 to 35OD (wave length 560 to 565 nm), or 30OD concentration (wave length) 560 to 565 nm) to 1 to 100 g/L, 5 to 100 g/L, 10 to 100 g/L, 15 to 100 g/L, 20 to 100 g/L, 21 to 100 g/L, 1 to 80 g/L, 5 to 80 g /L, 10-80 g/L, 15-80 g/L, 20-80 g/L, 21-80 g/L, 1-60 g/L, 5-60 g/L, 10-60 g/L, 15-60 g/L , 20-60 g/L, 21-60 g/L, 1-50 g/L, 5-50 g/L, 10-50 g/L, 15-50 g/L, 20-50 g/L, 21-50 g/L, 1 to 30 g/L, 5 to 30 g/L, 10 to 30 g/L, 15 to 30 g/L, 20 to 30 g/L, 21 to 30 g/L, 1 to 25 g/L, 5 to 25 g/L, 10 to 25 g /L, 15 to 25 g/L, 20 to 25 g/L, or 21 to 25 g/L may be included.
[41]
The composition according to an example is 1 to 100 g/L, 5 to 100 g/L, 10 to 100 g/L, 15 to 100 g/L, 20 to 100 g/L of the Corynebacterium sp. , 21-100 g/L, 1-80 g/L, 5-80 g/L, 10-80 g/L, 15-80 g/L, 20-80 g/L, 21-80 g/L, 1-60 g/L, 5 to 60 g/L, 10 to 60 g/L, 15 to 60 g/L, 20 to 60 g/L, 21 to 60 g/L, 1 to 50 g/L, 5 to 50 g/L, 10 to 50 g/L, 15 to 50 g /L, 20-50 g/L, 21-50 g/L, 1-30 g/L, 5-30 g/L, 10-30 g/L, 15-30 g/L, 20-30 g/L, 21-30 g/L , 1 to 25 g / L, 5 to 25 g / L, 10 to 25 g / L, 15 to 25 g / L, 20 to 25 g / L, or may be included in a concentration of 21 to 25 g / L.
[42]
The composition according to an example contains 0.1 to 10% by weight of the Corynebacterium sp. strain (or dead cells of the strain), 0.1 to 8% by weight, 0.1 to 5% by weight, 0.1 to 4% by weight, 0.1 to 3.5% by weight, 0.1 to 3 wt%, 0.1 to 1 wt%, 1 to 10 wt%, 1 to 8 wt%, 1 to 5 wt%, 1 to 4 wt%, 1 to 3.5 wt%, 1 to 3 wt%, 2 to 10 wt%, 2 to 8 wt%, 2 to 5 wt%, 2 to 4 wt%, 2 to 3.5 wt%, 2-3 wt%, 3 to 10 wt%, 3 to 8 wt%, 3 to 5 wt%, 3 to 4 wt%, It may be included in an amount of 3 to 3.5 wt%, 3.5 to 10 wt%, 3.5 to 8 wt%, 3.5 to 5 wt%, or 3.5 to 4 wt%.
[43]
In one example, the Corynebacterium sp. strain may be included in the culture of the strain.
[44]
The "culture" refers to a product obtained after culturing the Corynebacterium sp. strain, and may include a fermented product. In one example, the culture is obtained by culturing the Corynebacterium sp. strain in a medium. It may be a fermented product. The “fermented product” refers to the result of enzymatic or metabolic decomposition of an organic material using microorganisms. In the present application, “fermentation” refers to enzymatic or metabolic degradation of an organic material using a microorganism. It may mean not any activity or in-process decay reaction involving
[45]
The culture (or ferment) may be a whole culture of a Corynebacterium sp. strain, a dilution thereof, a concentrate, a dried product, a lyophilisate, a lysate, and/or a fraction thereof, and the concentrate is the culture can be obtained by centrifugation or evaporation, and the dried product can be obtained by drying the culture using a dryer, etc., and the freeze-dried product can be obtained by freeze-drying the culture using a freeze dryer, etc. And, the lysate can be obtained by physically or ultrasonically treating the strain or culture, and the fraction can be obtained by applying the culture, lysate, etc. to methods such as centrifugation and chromatography.
[46]
The culture or fermented product may be in a solid (solid, for example, dry), liquid (liquid), or fluidized phase, but is not limited thereto.
[47]
In one example, the culture may refer to an entire medium including a cultured strain obtained by culturing a Corynebacterium sp. strain for a certain period, metabolites thereof, and/or extra nutrients.
[48]
In one example, the culture may refer to the remaining components except for the strain (cells) and/or threonine in the fermented product of the Corynebacterium sp. strain in a medium.
[49]
In one example, the culture may be one in which the Corynebacterium sp. strain is removed or not removed.
[50]
In one example, the culture may be a culture solution (or culture) in which the strain is removed from the culture solution of the Corynebacterium sp. strain in a medium. The culture solution (or culture) from which the strain is removed may be a cell-free culture solution (or culture) or a culture solution containing dead cells. supernatant), and/or a culture solution containing dead cells (or a dried product of the culture solution).
[51]
The composition according to an example comprises 1 to 80% by weight of the culture (or fermented product), 5 to 80% by weight, 10 to 80% by weight, 15 to 80% by weight, 20 to 80% by weight, 23 to 80% by weight , 25 to 80% by weight, 1 to 60% by weight, 5 to 60% by weight, 10 to 60% by weight, 15 to 60% by weight, 20 to 60% by weight, 23 to 60% by weight, 25 to 60% by weight, 1 to 50% by weight, 5 to 50% by weight, 10 to 50% by weight, 15 to 50% by weight, 20 to 50% by weight, 23 to 50% by weight, 25 to 50% by weight, 1 to 40% by weight, 5 to 40 wt%, 10-40 wt%, 15-40 wt%, 20-40 wt%, 23-40 wt%, 25-40 wt%, 1-30 wt%, 5-30 wt%, 10-30 wt% , 15-30 wt%, 20-30 wt%, 23-30 wt%, 25-30 wt%, 1-25 wt%, 5-25 wt%, 10-25 wt%, 15-25 wt%, 20 To 25% by weight, or 23 to 25% by weight may be included. According to one example, the composition containing the culture in the above range than the composition containing the culture in the content outside the above range (1) the prevention or treatment effect of gastrointestinal disease and / or (2) the anti-Helicobacter pylori efficacy is excellent. can
[52]
The culture may include a Corynebacterium sp. strain, and the amount of the Corynebacterium sp. strain in the culture is 0.1 to 10 wt%, 0.1 to 8 wt%, 0.1 to 5 wt%, 0.1 to 4 wt%, 0.1 to 3.5 wt., 0.1 to 3 wt., 0.1 to 1 wt., 1 to 10 wt.%, 1 to 8 wt.%, 1 to 5 wt.%, 1-4 wt., 1 to 3.5 wt.%, 1-3 wt., 2 to 10% by weight, 2 to 8% by weight, 2 to 5% by weight, 2 to 4% by weight, 2 to 3.5% by weight, 2-3% by weight, 3 to 10% by weight, 3 to 8% by weight, 3 to 5% by weight; It may be included in an amount of 3 to 4 wt%, 3 to 3.5 wt%, 3.5 to 10 wt%, 3.5 to 8 wt%, 3.5 to 5 wt%, or 3.5 to 4 wt%.
[53]
In one example, Corynebacterium sp. strain and its culture are heat-treated (or sterilized (eg, autoclave or hot air drying)) the prepared culture obtained by culturing the Corynebacterium sp. Or it may be a culture containing the dead cells of the Corynebacterium sp.
[54]
In the present application, "cultivation" refers to a series of actions in which microorganisms are appropriately grown in artificially controlled environmental conditions. The culture may use any culture conditions and culture methods known in the art. For example, the culture may be continuously performed in a known batch culture method, a continuous culture method, a fed-batch culture method, a batch process, or a fed batch or repeated fed batch process. . In this case, the culture conditions are not particularly limited thereto, but use a basic compound (eg, sodium hydroxide, potassium hydroxide or ammonia) or an acidic compound (eg, phosphoric acid or sulfuric acid) to an appropriate pH (eg, pH 5 to 9, pH 6). to 8, or pH 6.8). In one example, anti-foaming agents such as fatty acid polyglycol esters can be used to inhibit foaming and/or oxygen or oxygen-containing gas mixtures can be introduced into the culture to maintain aerobic conditions.
[55]
In one example, the culture temperature may be 20 to 45 ° C, 25 to 40 ° C, 30 to 40 ° C, 30 to 35 ° C, or 35 to 40 ° C, and the culture conditions are 100 to 500 rpm, 150 to 300 rpm, 150 to 250 rpm, or 200 rpm, and the incubation time (period) is 1 to 160 hours, 10 to 100 hours, 12 to 72 hours, 24 to 72 hours, 30 to 60 hours, 40 to 50 hours, 45 to 50 hours, or It may be 48 hours. The incubation period may be continued until useful substances (eg, L-threonine, threonine, threonine, Threonine) are obtained in a desired yield. In one example, the culturing may be performed at a culturing temperature in the above range for a culturing time in the above range.
[56]
The medium used for culturing must meet the requirements of a specific strain in an appropriate manner, and those skilled in the art can use it appropriately according to known content. According to one example, the medium for culturing the corynebacterium sp. strain may refer to known literature (eg, Manual of Methods for General Bacteriology. American Society for Bacteriology. Washington DC, USA, 1981), but this It is not limited.
[57]
In order to culture the Corynebacterium sp. strain, it is possible to meet the survival requirements of a specific strain in an appropriate way while controlling the temperature, pH, etc. under anaerobic conditions in a normal medium containing an appropriate carbon source, nitrogen source, amino acid, vitamin, etc. can Carbon sources that can be used in the medium include sugars and carbohydrates (eg, glucose, sucrose, lactose, fructose, maltose, molase, starch and cellulose), oils and fats (eg, , soybean oil, sunflower oil, peanut oil and coconut oil), fatty acids (such as palmitic acid, stearic acid and linoleic acid), alcohols (such as glycerol and ethanol) and organic acids (such as acetic acid), etc. may be used individually or in combination, but is not limited thereto. Nitrogen sources include nitrogen-containing organic compounds (e.g., peptone, yeast extract, broth, malt extract, corn steep liquor, soy meal and urea), or inorganic compounds (e.g., ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate) may be used individually or in combination, but is not limited thereto. As the phosphorus source, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium-containing salt corresponding thereto, etc. may be used individually or in combination, but the present invention is not limited thereto. In addition, the culture medium may contain other metal salts necessary for growth (eg, magnesium sulfate or iron sulfate) and/or may contain essential growth substances such as amino acids and vitamins, but is not limited thereto.
[58]
In one example, the medium may be a medium for threonine production (eg, no. 435 medium), and the Corybacterium sp. strain, and a culture thereof, are cultured in the Corynebacterium sp. strain in a threonine production medium. It may be a single culture, and threonine may be excreted into the culture medium or contained in cells.
[59]
In one example, the Corynebacterium sp. strain and/or its (the Corynebacterium sp. strain) culture may include threonine.
[60]
In one example, the Corynebacterium sp. strain and/or its culture may not contain threonine.
[61]
In the present application, "Threonine (Thr)" and "threonine" are hydroxy-α-amino acids, which are one of the essential amino acids that are not produced in the body and are of the formula HO 2 CCH (NH 2 )CH(OH)CH 3 means amino acids. The threonine may be an optical isomer L-form (L-Threonine (L-Thr)), D-form, or a combination thereof. Threonine is an essential amino acid and constitutes mucine, a substance that protects the intestinal epithelium, and its deficiency can cause growth arrest and weight loss.
[62]
According to an example, when a Corynebacterium sp. strain, a culture thereof, and/or threonine are mixed, a synergistically excellent (1) preventive or therapeutic effect of gastrointestinal disease and/or (2) anti-Helicobacter pylori efficacy appears. can
[63]
The composition according to an embodiment contains 50 to 90% by weight of threonine, 50 to 85% by weight, 50 to 80% by weight, 50 to 75% by weight, 50 to 70% by weight, 55 to 90% by weight, 55 to 85% by weight, 55 to 80% by weight, 55 to 75% by weight, 55 to 70% by weight, 60 to 90% by weight, 60 to 85% by weight, 60 to 80% by weight, 60 to 75% by weight, 60 to 70% by weight, 65 to 90 wt%, 65-85 wt%, 65-80 wt%, 65-75 wt%, 65-70 wt%, 70-90 wt%, 70-85 wt%, 70-80 wt%, 70-75 wt% %, or 70% by weight. A composition containing threonine within the above range may be superior to a composition containing threonine in an amount outside the above range than a composition containing threonine in (1) preventing or treating gastrointestinal diseases and/or (2) anti-Helicobacter pylori efficacy.
[64]
The threonine may be commercially available or may be produced using an extraction method, a fermentation method, an enzyme method, and/or a synthesis method. For example, it may be obtained by purification after obtaining a fermented product containing threonine using a coryneform strain, or biosynthesized through a threonine biosynthetic pathway, and/or chemically synthesized.
[65]
The threonine is (1) contained in the Corynebacterium sp. strain and/or its culture in the composition according to an example, or (2) is not included in the Corynebacterium sp. strain and/or its culture. It may be further included or (3) a combination thereof (for example, the Corynebacterium sp. strain and culture thereof included in the composition include threonine, and in addition, additionally include threonine).
[66]
Threonine included in the composition according to an embodiment,
[67]
(1) is included in the Corynebacterium sp. strain, its culture, or both (strain and culture);
[68]
(2) added separately;
[69]
(3) Included in the Corynebacterium sp. strain, its culture, or both (strain and culture), and may be additionally (separately) added thereto.
[70]
In the above, the separately added threonine may mean that purified threonine is additionally added in addition to the threonine contained in the strain and/or culture.
[71]
The threonine may be in powder and/or granular form. In the case of granular threonine, it may have a particle size of 100 to 1000 μm or 200 to 1000 μm.
[72]
The composition according to an example may include 0.1 to 5 parts by weight of the Corynebacterium sp. strain, 10 to 90 parts by weight of the culture, and 10 to 80 parts by weight of the threonine. Specifically, 0.2 to 5 parts by weight of a strain, 12 to 88 parts by weight of a culture, and 10.5 to 78 parts by weight of threonine, 0.3 to 5 parts by weight of a strain, 13 to 87 parts by weight of a culture and 11 to 77 parts by weight of threonine, 0.4 to 77 parts by weight of a strain 5 parts by weight, culture 14 to 86 parts by weight, and threonine 11.5 to 76 parts by weight, strain 0.5 to 5 parts by weight, culture 15 to 85 parts by weight and threonine 12 to 75 parts by weight, strain 0.6 to 5 parts by weight, culture 16 to 84 parts by weight and 12.5 to 74 parts by weight of threonine, 0.7 to 5 parts by weight of strain, 17 to 83 parts by weight of culture and 13 to 73 parts by weight of threonine, 0.8 to 5 parts by weight of strain, 18 to 82 parts by weight of culture and Threonine 13.5 to 72 parts by weight, strain 0.9 to 5 parts by weight, culture 19 to 81 parts by weight, and threonine 14 to 71 parts by weight, strain 0.9 to 4 parts by weight, culture 20 to 80 parts by weight, and threonine 14 to 70 parts by weight , 1 to 4 parts by weight of the strain, 21 to 79 parts by weight of the culture and 14 to 69 parts by weight of threonine, 1 to 3 parts by weight of the strain, 22 to 78 parts by weight of the culture and 14 to 68 parts by weight of threonine.
[73]
In the composition according to an embodiment, the weight ratio of the strain and its culture to threonine (weight of the strain and its culture: the weight of threonine) is 1:0.1 to 1:10, 1:0.1 to 1:5, 1:0.1 to 1:3, 1:0.2 to 1:10, 1:0.2 to 1:5, 1:0.2 to 1:3, 1:0.5 to 1:10, 1:0.5 to 1:5, 1:0.5 to 1: 3, 1:1 to 1:10, 1:1 to 1:5, 1:1 to 1:3, 1:3 to 1:10, 1:3 to 1:5, or 1:3.
[74]
The composition according to an embodiment may further include lignin sulfonate. When the composition according to an embodiment further includes lignin sulfonate, (1) the prevention or treatment effect of gastrointestinal diseases and/or (2) the anti-Helicobacter pylori efficacy may be improved.
[75]
The composition according to an embodiment may further include calcium lignosulfonate in order to adjust the threonine content included in the composition. According to one embodiment, the composition comprises 0.1 to 50% by weight of calcium ligninsulfonate, 0.1 to 30% by weight, 0.1 to 25% by weight, 0.1 to 20% by weight, 0.1 to 15% by weight, 0.1 to 10% by weight, 0.1 to 7 wt%, 1-50 wt%, 1-30 wt%, 1-25 wt%, 1-20 wt%, 1-15 wt%, 1-10 wt%, 1-7 wt%, 3-50 wt% , 3 to 30% by weight, 3 to 25% by weight, 3 to 20% by weight, 3 to 15% by weight, 3 to 10% by weight, 3 to 7% by weight, 5 to 50% by weight, 5 to 30% by weight, 5 to 25% by weight, 5 to 20% by weight, 5 to 15% by weight, 5 to 10% by weight, 5 to 7% by weight, 6.5 to 50% by weight, 6.5 to 30% by weight, 6.5 to 25% by weight, 6.5 to 20 Weight %, 6.5 to 15% by weight, 6.5 to 10% by weight, or 6.5 to 7% by weight may be included.
[76]
In one example, the composition may include the Corynebacterium sp. strain, and a culture thereof in a dried form (eg, a dried product of 'Corynebacterium sp. strain and a culture thereof'). The dried product may be prepared by drying a Corynebacterium sp. strain and a culture thereof, and the dried product may include threonine. In one example, the drying may be performed by one or more methods selected from the group consisting of vacuum drying, hot air drying, freeze drying, room temperature ventilation drying, thin film drying, and/or vacuum drying.
[77]
In one example, the composition comprises the Corynebacterium sp. strain, a culture thereof, and threonine in a dried form (eg, a dried product of 'Corynebacterium sp. strain and culture thereof' containing threonine) can do.
[78]
The formulation of the composition according to one embodiment may be in the form of a solution, suspension or emulsion in oil or an aqueous medium, or may be in the form of an extract, powder, granule, tablet, capsule, or gel (eg, hydrogel), dispersing or stable Additional topics may be included.
[79]
The composition according to one embodiment can be prepared by drying the Corynebacterium sp. strain and its culture (fermented product), and the composition includes threonine, Corynebacterium sp. strain (for example, in the form of dead cells). strain), and threonine and components of the culture other than the strain. In one example, the composition according to an example prepared by drying the Corynebacterium sp. strain and its culture may be in a granular form, which is better than a purified powdery threonine (1) for preventing or treating gastrointestinal diseases and/or (2) The anti-Helicobacter pylori efficacy may be excellent.
[80]
In the present application, "granule" means a state in which the powder is agglomerated to form 30 to 150 times larger particles, and "granule" means to induce the raw material by processing such as a process so that it becomes a granular state can do. In one example, in the process of drying the Corynebacterium sp. strain, its culture, and/or the composition containing threonine, the composition may exhibit a granular form.
[81]
In one example, the gastrointestinal disease may be caused by Helicobacter pylori infection.
[82]
In one example, the gastrointestinal disease may be one or more selected from the group consisting of gastritis, gastric ulcer, duodenal ulcer, peptic ulcer, and gastric cancer.
[83]
The gastritis refers to a state in which the lining of the stomach is inflamed, and the gastric ulcer may occur when the balance between a defense factor that protects the mucous membrane in the stomach and an aggressor factor that causes damage to the mucous membrane is broken. The attack factors that can be considered the cause of gastric ulcer include gastric acid, various digestive enzymes, bile, drugs taken, alcohol, Helicobacter pylori infection , nonsteroidal anti-inflammatory drugs, smoking, and the like.
[84]
When the composition according to an example is used as a feed composition, it may be prepared in the form of a feed composition in which the composition according to an example is added to a commercially available feed composition. The feed in which the composition according to an embodiment can be used is preferably in a powder or pellet formulation, or a liquid formulation, but is not limited thereto. In addition, the amount of the composition of the present application added to the feed does not require a special limitation.
[85]
In the present application, "feed" may mean any natural or artificial diet, meal meal, etc. or a component of the meal meal for or suitable for being eaten, ingested, and digested by an animal.
[86]
The type of feed is not particularly limited, and feed commonly used in the art may be used. Non-limiting examples of the feed include plant feeds such as grains, root fruits, food processing by-products, algae, fibers, pharmaceutical by-products, oils and fats, starches, gourds or grain by-products; and animal feeds such as proteins, inorganic materials, oils and fats, minerals, oils and fats, single cell proteins, zooplankton, or food. These may be used alone or in mixture of two or more.
[87]
The subject (animal) that can be fed the feed containing the composition according to one embodiment is not particularly limited, but may be mammals, fish, crustaceans and/or shellfish, for example, pigs, cattle, horses, goats, deer, It can be a sheep, chicken, duck, goose, turkey, dog, cat, rabbit, or fish.
[88]
The feed composition may further include excipients, diluents, and additives. In addition to the above components, the feed composition includes an ingredient effective for promoting animal growth, a nutrient ingredient, a nutritional supplement ingredient, a ingredient for enhancing storage stability, a coating ingredient, an amino acid agent for disease prevention, vitamins, an enzyme agent, and a non-protein nitrogen compound , silicate, buffer, extractant, probiotic; enzymes such as amylase and lipase; vitamins such as L-ascorbic acid, choline chloride, and inositol; minerals such as potassium chloride, iron citrate, magnesium oxide, and phosphates; amino acids such as lysine, alanine and methionine; organic acids or salts thereof, such as fumaric acid, butyric acid, and lactic acid; antioxidants such as vitamin C and vitamin E, and fungicides such as calcium propionate; emulsifiers such as lecithin and glycerin fatty acid ester; and/or may include a pigment and the like. Although not described above, in one example, the feed composition may further include other nutrients within the range that those of ordinary skill in the art would expect.
[89]
[90]
Another aspect may provide a pharmaceutical composition for preventing or treating gastrointestinal diseases, including a Corynebacterium sp. strain, a culture thereof, and threonine. Corynebacterium sp. strain, its culture, threonine, and/or gastrointestinal disease included in the pharmaceutical composition for prevention or treatment according to an example are the same as those described for the feed composition for preventing or improving gastrointestinal disease.
[91]
The pharmaceutical composition according to an embodiment may further include an appropriate carrier, excipient or diluent commonly used in the preparation of the pharmaceutical composition. Specifically, the pharmaceutical composition is formulated in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and sterile injection solutions according to conventional methods, respectively. can be used Carriers, excipients and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, it is prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders (powders), granules, capsules, etc., and these solid preparations include at least one excipient in the composition, for example, starch, calcium carbonate, It is prepared by mixing sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid formulations for oral administration include various excipients such as wetting agents, sweetening agents, fragrances, preservatives, in addition to simple diluents such as water and liquid paraffin, which are commonly used for suspensions, solutions, emulsions, syrups, etc. can Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, etc. can be used.
[92]
The pharmaceutical composition according to an embodiment may be administered in a pharmaceutically effective amount, and in the present application, the term "pharmaceutically effective amount" means to treat or prevent a disease at a reasonable benefit/risk ratio applicable to medical treatment or prevention. means an amount sufficient to The duration of treatment may be determined according to factors including drugs used in combination with or concurrently with the composition of the present application and other factors well-known in the medical field. The pharmaceutical composition according to an embodiment may be administered as an individual therapeutic agent or may be administered in combination with other therapeutic agents, and may be administered sequentially or simultaneously with a conventional therapeutic agent. and may be administered single or multiple. In consideration of all of the above factors, it is important to administer an amount capable of obtaining the maximum effect with a minimum amount without side effects.
[93]
The dosage of the pharmaceutical composition according to an embodiment may be, for example, about 0.0001 to 100 mg/kg, specifically 0.001 to 10 mg/kg, administered to a mammal for one day. The frequency of administration of the pharmaceutical composition of the present application is not particularly limited thereto, but may be administered once a day or administered several times by dividing the dose. The above dosage does not limit the scope of the present application in any way.
[94]
The pharmaceutical composition according to one embodiment is not limited in the administration method as long as it can reach the target tissue. For example, intra-articular injection, oral administration, arterial injection, intravenous injection or transdermal injection are included. In addition, the pharmaceutical composition may be administered by any device capable of transporting an active substance to a target cell.
[95]
[96]
Another aspect may provide a food composition for preventing or improving gastrointestinal diseases, including a Corynebacterium sp. strain, a culture thereof, and threonine. Corynebacterium sp. strain, its culture, threonine, and/or gastrointestinal disease included in the food composition for preventing or improving gastrointestinal disease is the same as described in the feed composition for preventing or improving gastrointestinal disease.
[97]
The above foods include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, vitamin complexes, health functions (Sex) There are food and health food, and it includes all food in the ordinary sense.
[98]
The functional food (functional food) is the same term as food for special health use (FoSHU), and in addition to nutritional supply, it is processed to efficiently exhibit bioregulatory functions and has high medical effects. means food. Here, "function (sex)" refers to obtaining useful effects for health purposes, such as regulating nutrients or physiological actions with respect to the structure and function of the human body. The food of the present application can be prepared by a method commonly used in the art, and during the preparation, raw materials and components commonly added in the art can be added. In addition, the formulation of the food may be prepared without limitation as long as it is a formulation recognized as a food. The composition for food of the present application can be prepared in various forms, and unlike general drugs, it has the advantage that there are no side effects that may occur when taking the drug for a long period of time using food as a raw material, and has excellent portability. Foods of can be consumed as a supplement to enhance the effect of preventing or improving gastric ulcer.
[99]
The health food means food having an active health maintenance or promotion effect compared to general food, and health supplement food means food for the purpose of health supplementation. In some cases, the terms health functional food, health food, and health supplement may be used interchangeably.
[100]
Specifically, the health functional food is a food prepared by adding the composition according to the egg to food materials such as beverages, teas, spices, gum, and confectionery, or encapsulating, powdering, suspension, etc. It means to bring a specific effect, but unlike general drugs, it has the advantage that there are no side effects that may occur when taking the drug for a long time using food as a raw material.
[101]
The food composition according to an example, because it is possible to ingest it on a daily basis, a high effect can be expected for the prevention or improvement of gastrointestinal diseases, and thus can be very usefully used.
[102]
The food composition may further include a physiologically acceptable carrier, the type of carrier is not particularly limited and any carrier commonly used in the art may be used.
[103]
In addition, the food composition may include additional ingredients that are commonly used in food compositions to improve odor, taste, vision, and the like. For example, vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, pantothenic acid, and the like may be included. In addition, it may include minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu), and chromium (Cr). In addition, it may include amino acids such as lysine, tryptophan, cysteine, and valine.
[104]
In addition, the food composition includes a preservative (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), a disinfectant (bleaching powder and high bleaching powder, sodium hypochlorite, etc.), an antioxidant (butylhydroxyanisole (BHA), butyl hydro Loxytoluene (BHT), etc.), coloring agents (tar pigments, etc.), coloring agents (sodium nitrite, sodium nitrite, etc.), bleach (sodium sulfite), seasonings (MSG sodium glutamate, etc.), sweeteners (dulcin, cyclemate, saccharin, etc.) , sodium, etc.), flavorings (vanillin, lactones, etc.), swelling agents (alum, D-potassium hydrogen tartrate, etc.), strengthening agents, emulsifiers, thickening agents (flavors), film agents, gum base agents, foam inhibitors, solvents, improving agents, etc. It may contain food additives. The additive may be selected according to the type of food and used in an appropriate amount.
[105]
The composition according to an example may be added as it is or used together with other foods or food ingredients, and may be appropriately used according to a conventional method. The mixed amount of the active ingredient may be appropriately determined according to the purpose of its use (prevention, health or therapeutic treatment). In general, in the production of food or beverage, the food composition of the present application may be added in an amount of 50 parts by weight or less, specifically 20 parts by weight or less with respect to the food or beverage. However, when consumed for a long period of time for health and hygiene purposes, it may contain an amount less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range.
[106]
As an example of the food composition, it may be used as a health drink composition, and in this case, it may contain various flavoring agents or natural carbohydrates as an additional component like a conventional drink. The above-mentioned natural carbohydrates include monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; polysaccharides such as dextrin and cyclodextrin; It may be a sugar alcohol such as xylitol, sorbitol, or erythritol. Sweeteners include natural sweeteners such as taumatine, stevia extract; A synthetic sweetener such as saccharin or aspartame may be used. The ratio of the natural carbohydrate may be generally about 0.01 to 0.04 g, specifically about 0.02 to 0.03 g per 100 mL of the health beverage composition of the present application.
[107]
In addition to the above, the health beverage composition includes various nutrients, vitamins, electrolytes, flavoring agents, colorants, pectic acid, pectic acid salts, alginic acid, alginic acid salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, It may contain alcohol, a carbonation agent, or the like. In addition, it may contain the pulp for the production of natural fruit juice, fruit juice beverage, or vegetable beverage. These components may be used independently or in combination. Although the ratio of these additives is not very important, it is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the health beverage composition of the present application.
[108]
The food composition according to an embodiment may include various weight % if it can exhibit the effect of preventing or improving gastrointestinal diseases, for example, the composition according to an embodiment is 0.00001 to 100% by weight, 0.01 compared to the total weight of the food composition to 80% by weight, or 10 to 50% by weight, but is not limited thereto.
[109]
[110]
Corynebacterium sp. strain according to an example, a culture thereof, and a composition comprising threonine Helicobacter pylori ( H. pylori ) Growth inhibition, Helicobacter pylori induced by, inflammation, oxidation It may be preventing, treating, or ameliorating a gastrointestinal disease (eg, gastric ulcer) by inhibiting stress, blood vessel growth, and/or apoptosis.
[111]
Corynebacterium sp. according to an embodiment , a culture thereof, and a composition comprising threonine promotes mucus synthesis and secretion, promotes tissue regeneration, inhibits gastric mucosal damage, promotes tissue regeneration, inhibits oxidative stress, It may be to prevent, treat, or ameliorate gastrointestinal diseases by inhibiting gastric mucosal cell apoptosis and/or inflammation.
[112]
Corynebacterium sp. strain in the composition according to an example; whether the Corynebacterium sp. strain is included as a dead cell; types of strains of the genus Corynebacterium; with or without culture; with or without threonine; And / or Corynebacterium sp. strain, it was confirmed that there is a difference in (1) prevention or treatment effect of gastrointestinal disease and / or (2) anti-Helicobacter pylori efficacy according to the combination of its culture and threonine, It was confirmed that the composition according to the method showed the most excellent effect.
[113]
[114]
Another aspect provides a method for preventing, improving, or treating gastrointestinal diseases, comprising administering a composition (feed composition, food composition, or pharmaceutical composition) for the prevention, improvement, or treatment of the gastrointestinal disease to an individual (patient) can do. Feed compositions, food compositions, pharmaceutical compositions, and gastrointestinal diseases are the same as described above.
[115]
According to an example, the method for preventing, improving, or treating gastrointestinal disease further includes the step of identifying (selecting) an individual (patient) in need of prevention, improvement, or treatment of the gastrointestinal disease prior to the administering step. may include
[116]
In the present application, the term "individual" may mean any animal, including humans, that has or is likely to develop a gastrointestinal disease.
[117]
The subject subject to which the method for preventing or treating gastrointestinal diseases is applied may be mammals including humans that have or may develop gastrointestinal diseases, for example, pigs, cattle, horses, goats, deer, sheep, chickens. , duck, goose, turkey, dog, cat, and/or.
[118]
In the present application, "administration" means introducing a composition for prevention, improvement, or treatment of the gastrointestinal disease to the subject by any suitable method, and the administration route is oral or parenteral (as long as it can reach the target tissue) For example, intravenous, subcutaneous, intramuscular, intraperitoneal or topical application) may be administered.
[119]
The prevention, improvement, or treatment method may be to administer a (pharmaceutically) effective amount of the feed composition, food composition, or pharmaceutical composition according to an embodiment. A suitable total daily amount may be determined by treatment within the scope of correct medical judgment, and may be administered once or divided into several doses. However, a specific therapeutically effective amount for a specific subject (patient) may depend on the type and extent of the response to be achieved, the specific composition including whether other agents are used in some cases, the age, weight, general health condition of the subject (patient), It can be applied differently depending on various factors including sex and diet, administration time, administration route and secretion rate of the composition, treatment period, drugs used with or concurrently with a specific composition, and similar factors well known in the pharmaceutical field.
[120]
[121]
Another aspect is the Corynebacterium genus ( Corynebacterium sp.) strain, a culture thereof, and Helicobacter pylori comprising a threonine, Helicobacter pylori ( Helicobacter pylori ) It can provide an antibacterial composition. The Helicobacter pylori ( Helicobacter pylori ) Corynebacterium sp. strain contained in the antibacterial composition, its culture, threonine, and/or gastrointestinal diseases are as described in the pharmaceutical composition for preventing or treating gastrointestinal diseases.
[122]
According to an example, when a Corynebacterium sp. strain, a culture thereof, and threonine are combined, the antibacterial effect against Helicobacter pylori may be synergistically excellent.
[123]
[124]
Another aspect may provide a composition for preventing, improving, or treating gastrointestinal diseases, including, Corynebacterium sp. strains, and cultures thereof, wherein the strains and cultures include threonine may be doing
[125]
Another aspect may provide a composition for preparing an active ingredient for preventing, improving, or treating gastrointestinal diseases, including a Corynebacterium sp. strain and a culture (fermented product) thereof, the Corynebacterium sp. The sp. strain or the culture (fermented product) may contain or contain threonine.
[126]
The composition for preparing an active ingredient for preventing or treating gastrointestinal diseases may include 0.1 to 5 parts by weight of the Corynebacterium sp. strain, 10 to 90 parts by weight of the fermented product, and 10 to 80 parts by weight of the threonine based on the total weight of the composition. can Specifically, 0.2 to 5 parts by weight of the strain, 12 to 88 parts by weight of the fermented product, and 10.5 to 78 parts by weight of threonine, 0.3 to 5 parts by weight of the strain, 13 to 87 parts by weight of the fermented product, and 11 to 77 parts by weight of threonine based on the total weight of the composition parts, strain 0.4 to 5 parts by weight, ferment 14 to 86 parts by weight and threonine 11.5 to 76 parts by weight, strain 0.5 to 5 parts by weight, ferment 15 to 85 parts by weight and threonine 12 to 75 parts by weight, strain 0.6 to 5 Parts by weight, 16 to 84 parts by weight of fermented product and 12.5 to 74 parts by weight of threonine, 0.7 to 5 parts by weight of strain, 17 to 83 parts by weight of fermented product and 13 to 73 parts by weight of threonine, 0.8 to 5 parts by weight of strain, and fermented product 18 to 82 parts by weight and 13.5 to 72 parts by weight of threonine, 0.9 to 5 parts by weight of strain, 19 to 81 parts by weight of ferment and 14 to 71 parts by weight of threonine, 0.9 to 4 parts by weight of strain, 20 to 80 parts by weight of ferment and threonine 14 to 70 parts by weight, strains 1 to 4 parts by weight, fermented products 21 to 79 parts by weight and threonine 14 to 69 parts by weight, strains 1 to 3 parts by weight, fermented products 22 to 78 parts by weight, and threonine 14 to 68 parts by weight. can, but is not limited thereto.
[127]
The composition for preparing an active ingredient for preventing or treating gastrointestinal diseases may further include calcium lignosulfonate in order to control the threonine content. The calcium lignin sulfonate is 0.1 to 50% by weight, 0.1 to 30% by weight, 0.1 to 25% by weight, 0.1 to 20% by weight, 0.1 to 15% by weight, 0.1 to 10% by weight, 0.1 to 50% by weight based on the total weight of the composition 7 wt%, 1-50 wt%, 1-30 wt%, 1-25 wt%, 1-20 wt%, 1-15 wt%, 1-10 wt%, 1-7 wt%, 3-50 wt% %, 3-30 wt%, 3-25 wt%, 3-20 wt%, 3-15 wt%, 3-10 wt%, 3-7 wt%, 5-50 wt%, 5-30 wt%, 5 to 25% by weight, 5 to 20% by weight, 5 to 15% by weight, 5 to 10% by weight, 5 to 7% by weight, 6.5 to 50% by weight, 6.5 to 30% by weight, 6.5 to 25% by weight, 6.5 to 20 wt%, 6.5-15 wt%, 6.5-10 wt%, 6.5-7 wt%, 0.1-18 wt%, 0.1-16 wt%, 0.1-14 wt%, 0.1-13 wt%, 0.1-12 wt% %, 0.1 to 11% by weight, 0.1 to 9% by weight, 0.1 to 8% by weight, or 0.1 to 7% by weight may be included, but is not limited thereto, and threonine in the composition is added without limitation to reach the desired content can do.
[128]
Another aspect may provide a pharmaceutical composition for preventing or treating gastrointestinal diseases, including a composition for preparing an active ingredient for preventing or treating gastrointestinal diseases.
[129]
Another aspect may provide a food composition for preventing or improving gastrointestinal disease, including a composition for preparing an active ingredient for preventing or treating gastrointestinal disease.
[130]
Another aspect may provide a feed composition for preventing or improving gastrointestinal diseases, including a composition for preparing an active ingredient for preventing or treating gastrointestinal diseases.
[131]
In another aspect, the granular Corynebacterium sp. strain and its culture (fermented product) for the prevention or treatment of gastrointestinal diseases comprising a composition for preparing an active ingredient for the prevention or treatment as an active ingredient A formulation for the treatment of gastrointestinal diseases provides
[132]
The Corynebacterium sp. strain and its fermented product, gastrointestinal disease, prevention, improvement, and treatment are as described above.
[133]
In the present application, the term "formulation" means convenient for preparation, preservation or use, and a therapeutic effect, mainly by physical manipulation, such as grinding, mixing, kneading, leaching (brewing) or evaporation, etc. without changing the essence of the drug. It refers to processing so that it exerts its full potential, and the product made by it is also called a medical preparation (Chemical Dictionary, 2001. 5. 20., Sehwa Editorial Department).
[134]
Additives used when formulating to prepare the formulation, that is, fillers, extenders, binders, wetting agents, disintegrants, diluents or excipients are as described above, and solid formulations for oral administration and additives included therein, oral Liquid formulations for administration and additives included therein or formulations for parenteral administration and additives added thereto are as described above.
[135]
The formulation may be a solid formulation, a liquid formulation, or a fluid formulation, but is not limited thereto, and any formulation exhibiting the effects of preventing, treating and improving gastric ulcer may be applied without limitation.
[136]
The formulation is the same as described above.
[137]
[138]
Another aspect includes the step of drying Corynebacterium sp. strains and cultures thereof, (1) prevention, improvement, or therapeutic effect of gastrointestinal diseases; And / or (2) Helicobacter pylori ( Helicobacter pylori ) It may provide a method for producing an active ingredient having an antibacterial effect.
[139]
The Corynebacterium sp. strain and its culture are as described above.
[140]
In an example, the drying may be performed by one or more methods selected from the group consisting of vacuum drying, hot air drying, freeze drying, room temperature ventilation drying, thin film drying, and/or vacuum drying.
[141]
In one example, the drying may be drying the composition in which threonine is separately added to the Corynebacterium sp. strain and its culture.
[142]
In one embodiment, the method for preparing the active ingredient may further include the step of separately adding threonine to the dried product prepared after the drying step.
[143]
Effects of the Invention
[144]
The composition according to the present application has been confirmed to have excellent anti-Helicobacter pylori efficacy in cell experiments, gastric ulcer improvement efficacy, gastric mucus synthesis efficacy, etc. in animal experiments, pharmaceutical composition for prevention and treatment of gastrointestinal diseases, food or feed for gastrointestinal disease improvement It can be applied as a composition.
[145]
Brief description of the drawing
[146]
1 shows the antibacterial activity against H. pylori strains of groups 1 to 8 .
[147]
2a to 2c show the results of measuring the expression of inflammatory mediators when RGM1 cells infected with H. pylori strain at an MOI of 100 for 6 hours were treated with groups 1 to 8; Specifically, Figure 2a shows the change in Cox-2 mRNA expression according to the treatment of groups 1 to 8 in RGM1 cells infected with H. pylori strain, and Figure 2b is iNOS according to the treatment of groups 1 to 8 in RGM1 cells infected with the H. pylori strain. shows the protein expression change of , and FIG. 2c shows the phosphorylated NF-κB p65 protein expression change according to the treatment of groups 1 to 8 in RGM1 cells infected with H. pylori strain. GAPDH and β-actin were used as internal controls for mRNA and protein, respectively.
[148]
2a to 6b, N (first row from the left) represents a negative control group ( H. pylori untreated RGM1 cells), Hp (second row from the left) represents a positive control group ( H. pylori -infected RGM1 cells), and Group 1 Figures to 8 show the results in cells treated with the compositions of Groups 1 to 8 in H. pylori -infected RGM1 cells.
[149]
Figure 3a shows the expression change of oxidative stress-related protein (HIF-1a) according to the treatment of groups 1 to 8 in RGM1 cells infected with H. pylori strain. β-actin was used as an internal control. Figure 3b shows the results of measuring the change in the concentration of active oxygen in the cells according to the treatment of groups 1 to 8 in RGM1 cells infected with H. pylori strain (DCF increase or decrease results).
[150]
Figure 4a shows the change in the expression of HO-1 mRNA according to the treatment of groups 1 to 8 in RGM1 cells infected with H. pylori strain , Figure 4b is the GST (groups 1 to 8) in RGM1 cells infected with H. pylori strain ( pi) and HO-1 protein expression changes. GAPDH and β-actin were used as internal controls.
[151]
5a and 5b show the apoptosis inhibitory efficacy according to the treatment of groups 1 to 8 in RGM1 cells infected with H. pylori strain. Specifically, Figure 5a shows the changes in the expression of Bax and Bcl-2 proteins according to the treatment of groups 1 to 8 in RGM1 cells infected with H. pylori strain, and β-actin was used as an internal control. Figure 5b shows apoptosis changes (TUNEL staining results) according to the treatment of groups 1 to 8 in RGM1 cells infected with H. pylori strain.
[152]
6a and 6b show the results of changes in angiogenesis and mucosal growth factors according to the treatment of groups 1 to 8 in RGM1 cells infected with H. pylori strain. Figure 6a shows the expression changes of TGF-β and VEGF protein according to the treatment of groups 1 to 8 in RGM1 cells infected with H. pylori strain, β-actin was used as an internal control. 6b shows the change in expression of β-catenin protein according to the treatment of groups 1 to 8 in RGM1 cells infected with H. pylori strain, and Lamin B was used as an internal control.
[153]
[154]
7a to 10, groups 1-4 refer to animal test groups 1-4 in Table 5.
[155]
7a and 7b show the improvement effect of stress-related mucosal disease (SRMD) by administration of the composition according to an example in the WIRS model animal experimental group. Specifically, FIG. 7A shows pathological pictures of the stomach of each experimental group, and the pictures in the right two columns are displayed at x40 magnification. Figure 7b is (A) lesion score (gross lesion index: upper left graph), (B) inflammation by administration of the composition according to an example in the WIRS model animal experimental group; Inflammation; pathologic score: upper right graph), (C) ulcer/erosion; pathologic score (Ulcer/erosin; pathologic score: lower left graph), (D) regeneration; Pathological score (Regeneration; pathologic score: lower right graph) is shown.
[156]
8A to 8D show changes in inflammation, angiogenesis, and signal transduction by administration of a composition according to an example in the WIRS model animal test group. (A) Figure 8a shows the changes in the expression of iNOS, TNF-α and IFN-γ mRNA in the animal test group 1-4, Figure 8b shows the change in the expression of PDGF mRNA in the animal test group 1-4, Figure 8c is the animal test group Changes in p-IκBα protein expression are shown in 1-4, and FIG. 8d shows protein changes in p-ERK and p-JNK in animal test groups 1-4. GAPDH and β-actin were used as internal controls for mRNA and protein, respectively.
[157]
9A to 9C are results showing apoptosis and cell cycle changes in the WIRS model animal test group by administration of a composition according to an example. Specifically, FIG. 9a is a TUNEL analysis result showing the level of apoptosis in the SRMD region of the gastric mucosa in animal test groups 1-4, and FIG. 9b is PARP-1, Bcl-2, Bax, Cleaved caspase-8 and Cleaved caspase- 3 shows the protein expression change, and FIG. 9c shows the protein expression change of CDK4 and Cyclin D1. β-actin was used as an internal control.
[158]
Figure 10 shows the content of mucin (Mucin) in the gastric tissue of animal test groups 1-4.
[159]
11 shows pathological photographs of the stomach of WIRS rats administered with samples containing various weight percent threonine. The compositions and contents of Samples 1 to 5 of FIG. 11 are shown in Table 4.
[160]
Modes for carrying out the invention
[161]
Hereinafter, to help the understanding of the present application, examples will be described in detail. However, the following examples are merely illustrative of the content of the present application, and the scope of the present application is not limited to the following examples. The examples of the present application are provided to more completely explain the present application to those of ordinary skill in the art.
[162]
[163]
Example 1. Corynebacterium sp. strain-containing composition and cell infection
[164]
Example 1-1. Preparation of a composition containing a strain of Corynebacterium sp.
[165]
Corynebacterium sp. In order to verify the efficacy difference between the anti-helicobacter, apoptosis inhibition and cytoprotective efficacy of the strain composition containing the Corynebacterium sp. strain and the Corynebacterium sp. strain, the compositions of groups 1 to 8 were used. prepared.
[166]
Corynebacterium glutamicum ( C. glutamicum ), Corynebacterium ammoniagenes ( C. ammoniagenes ), and Corynebacterium efficiens ( C. efficiens ) wild-type strains of CJ CheilJedang BIO R&D Center ( Blossom park) was provided and used, and each of the strains was inoculated into a threonine production broth and cultured at 35° C. for 48 hours at 200 rpm. Components and contents of the broth for threonine production are shown in Table 1 below.
[167]
[168]
[Table 1]
ingredient Concentration (per liter)
glucose 70g
KH 2 PO 4 1g
(NH 4 ) 2 SO 4 30g
MgSO 4 7H 2 O 1g
FeSO 4 · 7H 2 O 200mg
MnSO4·4H2O 100mg
biotin 1mg
yeast extract 2.5g
Calcium-pantothenic acid 1mg
Thiamine hydrochloride 1mg
calcium carbonate 30g
pH 6.8
[169]
[170]
Group 1 includes a supernatant of the fermentation broth prepared by culturing Corynebacterium glutamicum under the above conditions to remove cells by centrifugation, and Group 2 contains a buffer containing threonine at a concentration of 100 g/L ( Tris-HCl). Groups 3 to 5 were prepared by centrifuging the fermentation broth prepared by culturing the strain under the above conditions to collect the cells, and suspending the collected cells in a buffer (Tris-HCl). Groups 6 to 8 are fermentation broths in which each strain is cultured under the above conditions, and include each strain and a culture cultured in the medium (referred to as 'supernatant of fermentation broth' in Table 2). The composition of groups 3 to 8 includes each strain at a concentration of 30 OD (wave length 562 nm), and includes the strain in the form of dead cells through autoclave.
[171]
Groups 1 to 8 all contain threonine, and since there is a group containing threonine in each strain (including in the form of dead cells) or culture, the threonine content in the strain and/or culture was measured and in groups 1 to 8 Additional threonine (threonine produced through CJ BIO strain; purity >99%) was added so that the final threonine concentration of was 100 g/L.
[172]
The components of each group are listed in Table 2 below. In Table 2, the 'fermentation broth supernatant' of groups 1 and 6 means that the cells were removed from the culture prepared by culturing the ' C. glutamicum strain' under the above conditions, and the supernatant of the fermentation broth of groups 7 and 8 was ' C. ammoniagenes strain' and ' C. ammoniagenes', respectively. efficiens strain' means removing the cells from the culture prepared by culturing under the above conditions.
[173]
[Table 2]
group ingredients in the composition
group 1 Threonine + supernatant of fermentation broth
group 2 threonine
group 3 Threonine + C. glutamicum
group 4 Threonine + C. ammoniagenes
group 5 Threonine + C. efficiens
group 6 Threonine + C. glutamicum + supernatant of fermentation broth
group 7 Threonine + C. ammoniagenes + Supernatant of Fermentation Broth
group 8 Threonine + C. efficiens + supernatant of fermentation broth
[174]
Groups 1-8 described in Examples 2 to 7 and FIGS. 1 to 6B below refer to Groups 1-8 of Table 2 above.
[175]
[176]
Example 1-2. Rat gastric mucosal cell line culture
[177]
RGM1 cells, the gastric mucosal cell line of normal rats, were cultured in a mixed medium of DMEM (Dulbecco's modified essential medium) and Ham F12 containing 10% fetal bovine serum at 37°C in a cell incubator (95% air, 5% CO 2 ). ) was incubated. The RGM1 cell line was established by Professor Matsui of Tsukuba University, Japan, and was used after consent.
[178]
[179]
Examples 1-3. H. pylori strains and cell infection
[180]
Helicobacter pylori ( H. pylori) strain (cytotoxin-associated gene A [CagA] + strain, NCTC 11637) was purchased from ATCC (American Type Culture Collection, Rockville, MD). H. pylori strain was cultured with shaking at 10% CO 2 condition until 1Х10 8 CFU/ml (OD 600=1) in Brucella broth supplemented with 5% bovine calf serum and antibiotics. did
[181]
RGM1 cells were infected with H. pylori strain for 6 hours at a multiplicity of infection (MOI) of 100:1 (hereinafter, 100 MOI) .
[182]
[183]
Example 2. Anti-Helicobacter efficacy assay of strain composition containing Corynebacterium genus in vitro
[184]
[185]
Agar diffusion assay (Disc diffusion assay) was used to measure the growth inhibitory effect of H. pylori , ie, anti-Helicobacter pylori , on blood agar plates . Specifically, 40 g/L of TSA (Trypticase Soy Agar) was dissolved in purified water and then autoclaved (121° C., 20 min). After cooling to about 50°C, 5% sheep blood was added, and then antibiotics {trimethoprim (5mg/L), polymyxin B (2500U/L), vancomycin (10mg/L)} were added. After 20 to 25 ml of the medium was dispensed on a sterilized plate, it was stored at 4°C.
[186]
200 μl of the H. pylori strain solution was dispensed on the prepared blood agar plate and spread on a sterilized disk paper, and 40 μl of each of the compositions of Groups 1 to 8 prepared in Example 1-1 were absorbed into the disk paper, respectively. , 37 ℃ CO 2 Incubated for 48 hours in an incubator, and then the diameter of the growth inhibition ring around the disk was measured and shown in FIG. 1 . (Results of group 1 are not shown in the graph) 'Non-treated' in FIG. 1 means RGM1 cells not infected with H. pylori .
[187]
As shown in FIG. 1 , it was confirmed that the diameter values ​​of the growth-stopping rings in groups 6 and 7 were significantly higher than in the untreated group, so that groups 6 and 7 had significant anti-Helicobacter activity.
[188]
[189]
In addition, it was attempted to derive the appropriate concentration of groups 1-8 to be used in future experiments.
[190]
40 μl of each diluted solution obtained by diluting the compositions of Groups 1 to 8 prepared in Example 1-1 at 1/4, 1/40, or 1/100 was treated in H.pylori , and cell viability was measured. At a dilution of 1/4, many groups showed a survival rate of 0.5 or less, and at a dilution of 1/100, there was no significant difference in results between groups, so a dilution of 1/40 showing a significant difference in results compared to group 1 was used for later experiments.
[191]
[192]
Example 3. Corynebacterium sp. strain-containing composition, inflammatory mediator (mediator) and the NF-κB transcriptional change assay
[193]
As in Example 1-3, RGM1 cells infected with H. pylori (100 MOI) were treated with 40 μl of the compositions of Groups 1 to 8 diluted at 1/40 concentration for 6 hours, respectively. Then, in the RGM1 cells treated with the composition, the expression level of Cox-2 mRNA, an inflammatory mediator, was confirmed through RT-PCT (reverse transcription PCR) analysis, and the result is shown in FIG. 2A.
[194]
Specifically, RNA was extracted using TRIzol (Gibco BRL, Rockville, MD), and the extracted RNA was synthesized into cDNA using moloney murine leukemia virus reverse transcriptase (Perkin Elmer, Morrisville, NC). The nucleotide sequence of each primer used for PCR analysis is shown in Table 3 below, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal standard.
[195]
[Table 3]
Gene Forward Primer(5' →3') Reverse Primer(5' →3')
IL-1β CAG GCT CCG AGA TGA ACA ACA AA (SEQ ID NO: 1) TGG GGA ACT CTG CAG ACT CA (SEQ ID NO: 2)
IL-8 CAG ACA GTG GCA GGG ATT CA (SEQ ID NO: 3) TTG GGG ACA CCC TTT AGC AT (SEQ ID NO: 4)
Cox-2 GAA ATG GCT GCA GAG TTG AA (SEQ ID NO: 5) TCA TCT AGT CTG GAG TGG GA (SEQ ID NO: 6)
iNOS CTC ACT GGG ACT GCA CAG AA (SEQ ID NO: 7) TGT TGA AGG GTG TCG TGA AA (SEQ ID NO: 8)
HO-1 GAC AGC ATG TCC CAG GAT TT (SEQ ID NO: 9) GGT TCT GCT TGT TTC GCT CT (SEQ ID NO: 10)
HSP70 GAG TTG AGC GGC ATC CCG CC (SEQ ID NO: 11) GTC CTA GAT TCA CAC CTG GAG (SEQ ID NO: 12)
Gapdh GGT GCT GAG TAT GTC GTG GA (SEQ ID NO: 13) TTC AGC TCT GGG ATG ACC TT (SEQ ID NO: 14)
[196]
[197]
In addition, in the RGM1 cells treated with the composition, the expression level of the iNOS protein and the increase or decrease of NF-κB p65 phosphorylation transcribed therefrom were confirmed through Western blot analysis, and the results are shown in FIGS. 2B and 2C .
[198]
Specifically, after the composition-treated and cultured cells were washed with a PBS (phosphate buffered saline) solution, cell lysis buffer (150 mM NaCl, 0.5% Triton X-100, 50 mM tris-HCl, pH 7.4, 25) A protein lysate was prepared by dissolving it with mM NaF, 20 mM ethyleneglycol-bis (βN'-tetraacetic acid, 1 mM dithiothreitol, 1 mM Na 3 VO 4 , Protease Inhibitor Cocktail tablet [Boehringer, Manneim, Germany]). After electrophoresis by PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), the developed protein was transferred to a PVDF membrane (Gelman Sciences, Ann Arbor, MI), each primary antibody and secondary antibody After the reaction was analyzed using a chemoluminescence system (chemoluminescence system).
[199]
As shown in FIG. 2a , the mRNA of Cox-2 was significantly increased in RGM1 cells ( Hp ) infected with H. pylori compared to normal RGM1 cells (N), and the mRNA expression of Cox-2 increased by infection with H. pylori It was reduced most significantly by the addition of this group 6 composition.
[200]
As shown in FIGS. 2b and 2c , iNOS protein expression and phosphorylation of NF-κB p65 transcribed therefrom were significantly increased by H. pylori infection , but increased iNOS protein expression and NF- The phosphorylation of κB p65 was most significantly reduced by the addition of the composition of group 6. From the above results, it was found that the composition according to one example regulates the inflammation-mediated pathway by the Helicobacter strain.
[201]
[202]
Example 4. Efficacy of weakening oxidative stress by a strain composition containing Corynebacterium genus in vitro
[203]
As in Example 1-3, RGM1 cells infected with H. pylori (100 MOI) were treated with 40 μl of the compositions of Groups 1 to 8 diluted to 1/40 concentration for 6 hours, respectively. Thereafter, Western blot was performed in a manner similar to Example 3, and the change in the expression level of HIF-1α associated with oxidative stress was measured, and the results are shown in FIG. 3A.
[204]
As shown in FIG. 3a , HIF-1a was significantly increased by Helicobacter pylori infection, and HIF-1a expression was significantly reduced in groups 6 and 7.
[205]
In addition, the DCF enhancement reaction using the DCF-DA probe was analyzed with a confocal imager in order to measure the change in the concentration of active oxygen in the cell. DCF-DA (2',7'-dichlorofluorescein diacetate), which is a non-fluorescent material, is oxidized by reactive oxygen species (ROS) in the presence of peroxides in the cell to emit green fluorescence. can be directly quantified. Specifically, RGM-1 cells ( H. pylori untreated RGM1 cells; or H. pylori -infected (100 MOI) RGM1 cells (Example 1-3) at 5x10 5 cells/well in a 24-well plate ) ) and incubated overnight at 37° C. in a cell incubator (95% air, 5% CO 2 ), each of the compositions of Groups 1 to 8 was treated for a certain period of time. After washing the cells with DMEM, 10 μg/ml DCF-DA (2',7'-dichlorofluorescein diacetate, Sigma-Aldrich Co., St Louis, MO) was added to the medium and cultured in an incubator for 30 minutes. After incubation, cells washed with PBS at 4° C. were observed and photographed under a fluorescence microscope, and the results are shown in FIG. 3B .
[206]
As shown in FIG. 3b, the fluorescence intensity of DCF-DA increased after Helicobacter pylori infection was statistically significantly decreased in groups 1, 3, 6, and 8 treatment groups (P<0.05), and in the group 6 treatment group, the It was confirmed that the decrease was the greatest (P<0.01). From the above results, it was found that the composition according to one example could immediately cope with the oxidative stress or hypoxia response caused by the Helicobacter strain.
[207]
[208]
Example 5. Helicobacter pylori-associated antioxidant-mediated cytoprotective efficacy assay by strain composition containing Corynebacterium genus in vitro
[209]
As in Example 1-3, 40 μl of the compositions of Groups 1 to 8 diluted at a concentration of 1/40 for 6 hours were treated with H. pylori -infected (100 MOI) RGM1 cells, respectively. Thereafter, the change in the expression of HO-1 mRNA having an antioxidant function, which is a representative cytoprotective factor, was analyzed by the method (PCR) according to Example 3 and shown in FIG. 4a, HO-1 and glutathione-s- The change in transferase (pi)) protein expression was analyzed similarly to the method according to Example 3 (Western blot analysis), and is shown in FIG. 4B.
[210]
As a result, as shown in FIGS. 4a and 4b, a significant decrease in HO-1 mRNA and protein expression was observed in the control group after Helicobacter pylori infection, and the decreased HO-1 expression in the groups 6 to 8 treatment group was significantly increased, among them The expression of HO-1 was most significantly increased in the group 6 treatment group. In addition, as shown in FIG. 4b , the expression of GST(pi) was significantly reduced in groups 6 and 7, and among them, the expression of GST(pi) was most significantly decreased in the group 6 treated.
[211]
From the above results, it can be seen that the composition according to an example exhibits antioxidant activity and cell protection efficacy mainly based on the HO-1 response against the Helicobacter strain.
[212]
[213]
Example 6. Helicobacter pylori-associated apoptosis inhibitory efficacy assay by strain composition containing Corynebacterium genus in vitro
[214]
As in Example 1-3, RGM1 cells infected with H. pylori (100 MOI) were treated with 40 μl of the compositions of Groups 1 to 8 diluted at 1/40 concentration for 6 hours, respectively. Then, similarly to the method of Example 3, the expression levels of Bcl-2 and Bax were confirmed through western blot, and the results are shown in FIG. 5a, and apoptosis (apoptosis) was analyzed by TUNEL staining, and the results are shown in FIG. 5b shown in Specifically, for TUNEL staining, cells were aliquoted at 1x10 5 cells/chamber on a Lab-Tek chamber slide and then cultured in a 37° C. cell incubator (95% air, 5% CO 2 ). After each reagent was treated for a certain period of time, an apoptosis detection kit (Oncogene Research Products, Cambridge, Mass.) was used to perform according to the manufacturer's experimental method. The culture medium was removed, washed 3 times with PBS solution, and 4% PFA (paraformaldehyde) was added and fixed at room temperature for 20 minutes. After washing twice again with PBS, 0.1% Triton X-100 was treated at 4℃ for 5 minutes, and a solution made by mixing TdT (terminal deoxynucleotidyl transferase) and nucleotide mixture at 37℃ after blocking light for 60 minutes reacted. After washing with PBS, apoptosis was observed under a fluorescence microscope.
[215]
As shown in Fig. 5a, it was possible to observe a decrease in the expression of Bcl-2 and an increase in the expression of Bax in the cells ( Hp ) infected with H. pylori , and the significantly increased Bax expression in the groups 6 to 8 treated decreased and decreased. It was confirmed that the expression of Bcl-2 was increased, and among them, the effect was the most excellent in the group 6 treatment group.
[216]
Helicobacter pylori infection is known to cause mucosal damage and ulceration by a significant increase in apoptosis, and a statistically significant increase in apoptosis was observed by Helicobacter pylori infection in the TUNEL staining result as shown in FIG. 5b. However, apoptosis was significantly reduced in groups 6 to 8 treatment groups, and among them, apoptosis was reduced the most in group 6 treatment group.
[217]
[218]
Example 7. Assay of changes in Helicobacter pylori-associated cell growth, blood vessel growth, and growth factors by a strain composition containing Corynebacterium genus in vitro
[219]
As in Example 1-3, RGM1 cells infected with H. pylori (100 MOI) were treated with 40 μl of the compositions of Groups 1 to 8 diluted at 1/40 concentration for 6 hours, respectively. Thereafter, similar to the method of Example 3, changes in TGF-β and VEGF were analyzed by Western blot, and the results are shown in FIG. 6A .
[220]
As a result, as shown in Figure 6a, the increased TGF-β and VEGF after Helicobacter infection promotes indiscriminate mucosal proliferation or cancerous inflammation, whereas the expression of TGF-β and VEGF in the groups 6 to 8 treatment groups was significantly reduced. , in particular, the expression of TGF-β and VEGF was reduced the most in the group 6 treatment group. From this, the composition according to an example showed results capable of alleviating inflammation or cancer caused by Helicobacter pylori infection.
[221]
In addition, using nuclear fraction in Helicobacter pylori-infected cells, nuclear transfer of β-catenin was confirmed by Western blot, and the results are shown in FIG. 6b. As shown in Figure 6b, it was confirmed that the nuclear transfer (nuclear transfer) of β-catenin increased by Helicobacter pylori infection can be significantly inhibited by the treatment of group 6.
[222]
[223]
All experimental values ​​of Examples 1 to 7 were statistically verified by a T-test, and a case of p<0.05 or more was defined as statistically significant.
[224]
[225]
Example 8. Preparation of Samples Containing Threonine
[226]
Fermentation broth prepared by inoculating C. glutamicum wild-type strain (CJ CheilJedang BIO R&D Center (Blossom park)) into the broth for threonine production (Table 1), and culturing for 48 hours at 35°C and 200 rpm conditions. was purified and crystallized to prepare Sample 1. Sample 1 contained 99% by weight or more of purified threonine in powder form.
[227]
Sample 2 was prepared by directly drying the fermentation broth (including C. glutamicum ) obtained by culturing C. glutamicum in a medium for threonine production (hot air drying at 60 to 70° C.). In addition , calcium-lignosulfonate (calcium-lignosulfonate, Aladdin industrial Co., Shanghai, Chana) was added in various amounts to the fermentation broth obtained by culturing C. glutamicum under the above conditions, and the fermentation broth to which calcium ligninsulfonate was added ( C. glutamicum ) was dried (hot air drying at 60 to 70° C.), and samples 3 to 5 were prepared. Samples 2 to 5 contained C. glutamicum in the form of dead cells through hot air drying .
[228]
Samples 2 to 5 were prepared in the form of granules having a particle size of about 200 to 1000 μm. Components and contents of Samples 1 to 5 are shown in Table 4. In Table 4, Thr (wt%), calcium lignin sulfonate (wt%), and the rest of the fermented product components ( including C. glutamicum cells) (wt%) refer to the weight% in the dried product prepared by drying the fermentation broth. And, the fermented product of the dried product contains C. glutamicum strain. In addition, the content of the C. glutamicum strain contained in the fermented product was described in Table 4.
[229]
[Table 4]
Thr (wt%) Fermented product (wt%) C. glutamicum in fermented product (wt%) 1 Calcium ligninsulfonate (wt%)
sample 1 100.0 - - -
sample 2 75 25.0 4.0 0.0
sample 3 70 23.3 3.8 6.7
sample 4 65 21.7 3.5 13.3
sample 5 60 20.0 3.2 20.0
[230]
1 The value converted to the dry matter of the cell content in the fermented product
[231]
[232]
Example 9. Water immersion-restraint stress (WIRS) model preparation for SRMD (stress-related mucosal disease)
[233]
A total of 110 SD rats were purchased from Charles River (Osaka, Japan) and kept in an animal facility. Laboratory animals were handled in an accredited animal facility in accordance with AAALAC International Animal Care Policies. Animals were fasted 24 hours before exposure to WIRS, and water was provided ad libitum. Ten mice from each group were placed in cages for WIRS and immersed in water for 6 hours.
[234]
8 hours before WIRS treatment, samples 1 to 5 prepared in Example 8 were orally administered to rats so that the same threonine was administered (0.15 wt%/diet), and after application of WIRS for 6 hours, the animals were sacrificed and the stomach was enucleated. Then, the incision and contents were removed, and a photograph of the inner surface of the stomach was taken and shown in FIG. 11 . As shown in FIG. 11 , as a result of visually examining the gastric tissue of the rat, gastric bleeding occurred in the WIRS-induced group (second row in FIG. 11), and the degree of gastric bleeding in the order of Sample 1 < Sample 5 < Sample 4 < Sample 3 or Sample 2 decreased (excellent gastric ulcer treatment effect).
[235]
[236]
On the other hand, subsequent experiments were carried out by dividing into 4 animal test groups as shown in Table 5 below. After oral administration of saline, no treatment group (positive control group, animal test group 1), WIRS group applied with WIRS for 6 hours (negative control group, animal test group 2), sample 1 (Example) 8 hours before WIRS treatment 8) containing 0.15 wt% of feed (animal test group 3) and sample 3 (prepared in Example 8) containing 0.21 wt% of feed (animal test group 4) were orally treated with 30 mg/kg body, respectively. divided into experimental groups. The L-Thr (L-type-threonine) content in the feeds of animal test groups 3 and 4 was equally set at 0.15 wt%/diet.

[Claim 1]
Corynebacterium sp. strain, a culture thereof, and a feed composition for preventing or improving gastrointestinal diseases, including threonine.
[Claim 2]
According to claim 1, wherein the Corynebacterium sp. strain is Corynebacterium glutamicum ( Corynebacterium glutamicum ), Corynebacterium ammoniagenes ), or a combination thereof, the feed composition.
[Claim 3]
According to claim 1, wherein the Corynebacterium sp. strain is a dead cell body, feed composition.
[Claim 4]
The feed composition of claim 1, wherein the culture is a fermented product of a Corynebacterium sp. strain in a medium.
[Claim 5]
The feed composition according to claim 4, wherein the medium is a medium for threonine production.
[Claim 6]
The feed composition according to claim 1, comprising the threonine in an amount of 60 to 80% by weight.
[Claim 7]
The feed composition of claim 1, wherein the Corynebacterium sp. strain and its culture are included in a dried form.
[Claim 8]
The feed composition according to claim 1, wherein the gastrointestinal disease is caused by Helicobacter pylori infection.
[Claim 9]
The feed composition according to claim 1, wherein the gastrointestinal disease is at least one selected from the group consisting of gastritis, gastric ulcer, duodenal ulcer, peptic ulcer, and gastric cancer.
[Claim 10]
10. The method according to any one of claims 1 to 9, wherein the threonine is (1) contained in the Corynebacterium sp. strain, a culture thereof, or both; (2) added separately; (3) Included in the Corynebacterium sp. strain, its culture, or both, and further added thereto, the feed composition.
[Claim 11]
Corynebacterium genus ( Corynebacterium sp.) strain, its culture, and a pharmaceutical composition for preventing or treating gastrointestinal diseases, including threonine.
[Claim 12]
Corynebacterium genus ( Corynebacterium sp.) strain, a culture thereof, and a food composition for preventing or improving gastrointestinal diseases, including threonine.
[Claim 13]
Corynebacterium genus ( Corynebacterium sp.) strains, cultures thereof, and Helicobacter pylori containing threonine, antibacterial composition for Helicobacter pylori .