RU2806746C2 - Composition for the prevention, treatment or alleviation of gastrointestinal diseases, containing a corynebacterium strain and a product of its cultivation - Google Patents

Abstract

FIELD: pharmaceuticals.

SUBSTANCE: use of a food composition for preventing gastric disease or alleviating a condition in gastric disease, containing (1) an effective amount of a Corynebacterium sp. strain, (2) a product of its cultivation, selected from a fermented medium obtained by culturing the specified strain of Corynebacterium sp. in the medium, and said fermented medium from which microbial cells have been removed, and (3) threonine. The use of a pharmaceutical composition containing the above composition for the prevention or treatment of gastric disease, the use of a food composition containing the above composition for the prevention of gastric disease or the alleviation of gastric disease, the use of an antimicrobial composition containing the above composition for inhibition of Helicobacter pylori are proposed.

EFFECT: invention is effective against Helicobacter pylori in experiments on cells, effective in alleviating gastrointestinal diseases in animal experiments, and effective in relation to the synthesis of gastric mucus.

12 cl, 11 dwg, 6 tbl, 4 ex

Description

FIELD OF THE INVENTION

The present invention relates to a composition for the prevention, treatment or relief of gastric disease, containing a strain of Corynebacterium sp., a product of its cultivation and threonine.

BACKGROUND ART

Gastric ulcers are the cause of the highest incidence of gastric injuries and are common throughout the world in all nations. The incidence of gastric ulcers is increasing with the development of the livestock industry, and efforts to prevent the occurrence of gastric ulcers from an animal welfare perspective are intensifying because, along with a decrease in growth rate due to decreased appetite, these symptoms are accompanied by pain.

Threonine (Thr) is a basic amino acid (AA) constituting mucin, a protective substance for the intestinal epithelium. The protein mucin stimulates the absorption of proteins while protecting the digestive organs from highly acidic fluids of the digestive tract, such as gastric juice, and plays an important role in maintaining intestinal health. It has been reported that the addition of Thr to feed actually improves intestinal health by stimulating mucin synthesis in piglets (Non-Patent Document 1).

The production of feed or food Thr is carried out by a microorganism fermentation method, and the types of microorganisms that are mainly used include Escherichia coli (E. coli) and Corynebacterium glutamicum (C. glutamicum) and the like. Despite producing the same AA, these two strains are Gram-negative and Gram-positive bacteria, respectively. The cell wall of both Gram-negative and Gram-positive bacteria is composed of peptidoglycan (PG). However, in the case of Gram-negative bacteria, in addition to the PG, a lipopolysaccharide (LPS) layer consisting of lipoprotein and protein is present, and since Lipid A, which is a somatic antigen (O-antigen), is present in this LPS layer, it is toxic. Therefore, in the pharmaceutical industry, it is important to remove endogenous toxins with harmful biological activities such as pyrogenicity, lethality, Schwarzman phenomenon, adjuvant activity and macrophage activation from parenteral drugs (Non-Patent Document 2).

Recently, the production of amino acids for feed in the form of pellets by simplifying (or skipping) the deep purification process to improve the cost-effectiveness of nutritional supplements inevitably involves the introduction of bacteria used for fermentation into the product in the form of heat-killed bacteria. Several studies have shown that heat-killed bacteria of a representative gram-positive bacteria, lactic acid bacteria, are stable in the environment, have strong resistance to acid and heat, and are easy to handle due to the ability to obtain high concentrations, and are used as a food product in relation to beneficial bacteria that inhabit the intestines and therefore enhance the immune-enhancing activity unique to lactic acid bacteria (Non-Patent Document 3).

In this context, the present inventors set out to develop a pharmaceutical composition having the effect of preventing and treating gastric disease, and as a result confirmed that a composition containing a Corynebacterium sp. strain, its culture product and threonine is effective in preventing, treating and alleviating the condition for gastric disease, thereby creating the present invention.

BACKGROUND DOCUMENTS NON-PATENT DOCUMENTS

(Non-patent document 1) Law G. (2000) Threonin requirement and the effect of threonin on gut mucin characteristics in piglets receiving intragastric nutrition. Master thesis from university of Alberta. 1-143.

(Non-patent document 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.

(Non-Patent Document 3) Lee I.H. (2018) Latest trend surrounding animal antimicrobial agents and alternatives. Pig & Consulting, 4, 70-73.

DETAILED DESCRIPTION OF THE INVENTION TECHNICAL PROBLEM

The present invention provides a feed composition for preventing gastric disease or alleviating gastric disease, containing a strain of Corynebacterium sp., a product of its cultivation and threonine.

The present invention provides a pharmaceutical composition for the prevention or treatment of gastric disease, containing a strain of Corynebacterium sp., a product of its cultivation and threonine.

The present invention provides a food composition for preventing gastric disease or alleviating gastric disease, containing a strain of Corynebacterium sp., a product of its cultivation and threonine.

The present invention provides an antimicrobial composition against Helicobacter pylori, containing a strain of Corynebacterium sp., a product of its cultivation and threonine.

TECHNICAL SOLUTION

In one aspect, a composition is provided for the prevention, relief or treatment of a gastric disease containing a strain of Corynebacterium sp., a product of its cultivation and threonine.

In the present invention, the term "prevention" may mean any act that inhibits or delays the onset of a disease by administering a composition according to one example, and the term "treating" may mean any act by which the symptoms of subjects suspected of having a disease or with the onset of a disease are alleviated or change for the better upon administration of a composition according to one of the examples, and the term "alleviation" may mean any action that at least reduces parameters related to the condition for which the disease is being treated, such as the severity of symptoms, by administering a composition according to one of the examples examples. The disease may be a gastric disease.

In one aspect, a feed composition is provided for preventing gastric disease or alleviating gastric disease, comprising a Corynebacterium sp. strain, a culture product thereof, and threonine.

In the present invention, "Corynebacterium sp. strain (Coryne sp.)" may be any strain of Corynebacterium sp. Corynebacterium sp. strain may be, for example, Corynebacterium glutamicum, Corynebacterium ammoniagenes, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris and/or Corynebacterium flavescens and, more specifically, it may be Corynebacterium glutamicum, Corynebacterium ammoniagenes or a combination thereof (Corynebacterium glutamicum and Corynebacterium ammoniagenes).

In one example, a Corynebacterium sp. strain may have threonine productivity. In the present invention, having threonine productivity means exhibiting the ability to produce and accumulate threonine in a microorganism and/or in a medium when the cultured microorganism is cultured in the medium.

When the composition for preventing, alleviating or treating gastric disease according to one example contains Corynebacterium glutamicum, (1) the effect of preventing, alleviating or treating gastric disease and/or (2) the effectiveness against Helicobacter pylori may be superior to that of the composition containing any other type of strain of Corynebacterium sp. other than Corynebacterium glutamicum (for example, Corynebacterium efficiens).

When the composition for preventing, alleviating or treating gastric disease according to one example contains Corynebacterium ammoniagenes, (1) the effect of preventing, alleviating or treating gastric disease and/or (2) the effectiveness against Helicobacter pylori may be superior to that of the composition containing any other type of strain of Corynebacterium sp. other than Corynebacterium ammoniagenes (for example, Corynebacterium efficiens).

When the composition for preventing, alleviating or treating gastric disease according to one example contains Corynebacterium glutamicum and Corynebacterium ammoniagenes, (1) the effect of preventing, alleviating or treating gastric disease and/or (2) the effectiveness against Helicobacter pylori may be superior to a composition containing any other species of strains of Corynebacterium sp. other than Corynebacterium glutamicum and Corynebacterium ammoniagenes (for example, Corynebacterium efficiens).

In one example, superior efficacy of a composition against Helicobacter pylori may mean that the antimicrobial activity against a strain of Helicobacter pylori is superior, or the activity in preventing apoptosis of cells (eg, gastric mucosal cells) infected with Helicobacter pylori is superior.

The term "strain Corynebacterium sp." may mean a microbial cell concentrate in which the culture medium has been removed from the culture solution, and it may have been subjected to centrifugation and/or filtration processes to separate only the microbial cell concentrate from the culture product.

In one example, the Corynebacterium sp. strain may be present in the form of heat-killed bacteria. In the present invention, the expression "heat-killed bacteria" is the opposite of viable bacteria, meaning a form in which the growth of bacteria is prevented by heat treatment of viable bacteria and fermentation-derived metabolites and the like. Heat-killed bacteria may contain antimicrobial substances such as cytoplasm, cell wall, bacteriocin and the like, polysaccharides and/or organic acids and the like.

A composition containing heat-killed bacteria, according to one example, may have at least one characteristic selected from the group consisting of the following sub-items (1)-(6), as opposed to a composition containing viable cells:

(1) superior acid resistance;

(2) superior heat resistance;

(2) the ability to concentrate to high concentrations;

(4) superior stability;

(5) ease of handling and storage; And

(6) the possibility of use as a source of nutrition for beneficial bacteria of the gastrointestinal tract.

Bacteria of the Corynebacterium sp. strain can be killed by Tyndall treatment and/or heat treatment. In one example, a culture solution for sterilizing bacteria of the Corynebacterium sp. strain and its culture product contains heat-killed bacteria of this strain. For example, heat treatment can be performed at a temperature of 60 to 130°C for 3 to 30 minutes. Additionally, heat treatment can be performed by ultra-high temperature sterilization, autoclaving and/or hot air drying one to 10 times. Ultra-high temperature sterilization can be carried out at a temperature of 110°C to 130°C for 3.0 to 10.0 seconds, and for example, it can be carried out at 100°C for 1.0 second to 10 seconds twice and at 121°C for 1.0 to 10.0 seconds once, and autoclaving can be done at 120-125°C or at 121°C for 10 minutes to 30 minutes, 15 minutes to 25 minutes or 20 minutes, and hot air drying can be performed at a temperature of 60 to 70°C.

In one specific example, a composition containing heat-killed Corynebacterium sp. bacteria, a culture product of Corynebacterium sp., and threonine was confirmed to have: (1) superior action in preventing or treating gastric disease and/or (2) superior efficacy against Helicobacter pylori in vitro and/or in vivo.

According to one example, the composition may contain a Corynebacterium sp. strain (or heat-killed bacteria strain) at a concentration of 20-40 OD (wavelength 560-565 nm), 25-35 OD (wavelength 560-565 nm) or 30 OD (wavelength 560-565 nm).

According to one example, the composition may contain 1-100 g/l, 5-100 g/l, 10-100 g/l, 15-100 g/l, 20-100 g/l, 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- 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, 1-30 g/l, 5-30 g/l, 10-30 g/l, 15-30 g /l, 20-30 g/l, 21-30 g/l, 1-25 g/l, 5-25 g/l, 10-25 g/l, 15-25 g/l, 20-25 g/ l or 21-25 g/l Corynebacterium sp. strain (or heat-killed bacteria strain) at a concentration of 20-40 OD (wavelength 560-565 nm), 25-35 OD (wavelength 560-565 nm) or 30 OD (wavelength 560-565 nm).

According to one example, the composition may contain a Corynebacterium sp. strain (or heat-killed bacteria strain) at a concentration of 1-100 g/L, 5-100 g/L, 10-100 g/L, 15-100 g/L, 20 -100 g/l, 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, 5-60 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, 1-30 g/l, 5-30 g /l, 10-30 g/l, 15-30 g/l, 20-30 g/l, 21-30 g/l, 1-25 g/l, 5-25 g/l, 10-25 g/ l, 15-25 g/l, 20-25 g/l or 21-25 g/l.

According to one example, the composition may contain a Corynebacterium sp. strain (or heat-killed bacteria strain) in an amount of 0.1-10% by weight, 0.1-8% by weight, 0.1-5% by weight, 0. 1-4% by weight, 0.1-3.5% by weight, 0.1-3% by weight, 0.1-1% by weight, 1-10% by weight, 1-8% by weight, 1-5% by weight, 1-4% by weight, 1-3.5% by weight, 1-3% by weight, 2-10% by weight, 2-8% by weight, 2-5% by weight , 2-4% by weight, 2-3.5% by weight, 2-3% by weight, 3-10% by weight, 3-8% by weight, 3-5% by weight, 3-4% by weight by weight, 3-3.5% by weight, 3.5-10% by weight, 3.5-8% by weight, 3.5-5% by weight or 3.5-4% by weight.

In one example, a Corynebacterium sp. strain may be contained in a product of culturing the strain.

The term "culture product" means the products obtained after culturing a strain of Corynebacterium sp., and may contain fermented products. In one example, the culture product may be fermented products obtained by culturing a Corynebacterium sp. strain in a medium. The term "fermented foods" means products of the enzymatic or metabolic breakdown of organic matter using microorganisms. In the present invention, the term "fermentation" can mean any action or process, including enzymatic or metabolic destruction of organic matter using microorganisms, excluding putrefaction reactions.

The culture product (or fermented product) may be a total culture product of a Corynebacterium sp. strain, a diluted solution thereof, a concentrate, a dry matter, a lyophilisate and/or a lysate, and the like, and the concentrate may be obtained by centrifugation or evaporation of the culture product, and the dry the substance can be obtained by drying the culture product using a dryer and the like, and the lyophilisate can be obtained by lyophilizing the culture product using a lyophilizer and the like, and the lysate can be obtained by mechanical or ultrasonic treatment of the strain or culture product.

The culture product or fermented product may be in a solid phase (solid matter, such as dry matter), liquid phase (liquid), or fluidized bed, but is not limited to this.

In one example, the culture product may be any media containing a cultured strain, a metabolite thereof and/or nutrient residues and the like obtained by culturing a Corynebacterium sp. strain for a certain period of time.

In one example, the term "culture product" may refer to components other than strain (microbial cells) and/or threonine in a fermented product obtained by culturing a Corynebacterium sp. strain in a medium.

In one example, the culture product may be one from which the Corynebacterium sp. strain has or has not been removed.

In one example, the culture product may be a culture solution (or culture product) wherein the strain is removed from the culture solution in which the Corynebacterium sp. strain was cultured in the medium. The culture solution (or culture product) from which the strain has been removed may be a cell-free culture solution (or culture product) or a culture solution containing heat-killed bacteria and, for example, may be a filtrate from which the strain has been removed by centrifugation (centrifuged supernatant) and filtration, and/or a culture solution (or culture solution solids) containing heat-killed bacteria.

In one example, the composition may contain a cultured product (or fermented product) in an amount of 1-80% by weight, 5-80% by weight, 10-80% by weight, 15-80% by weight, 20-80% by weight , 23-80% by weight, 25-80% by weight, 1-60% by weight, 5-60% by weight, 10-60% by weight, 15-60% by weight, 20-60% by weight, 23-60% by weight, 25-60% by weight, 1-50% by weight, 5-50% by weight, 10-50% by weight, 15-50% by weight, 20-50% by weight, 23 -50% by weight, 25-50% by weight, 1-40% by weight, 5-40% by weight, 10-40% by weight, 15-40% by weight, 20-40% by weight, 23- 40% by weight, 25-40% by weight, 1-30% by weight, 5-30% by weight, 10-30% by weight, 15-30% by weight, 20-30% by weight, 23-30 % by weight, 25-30% by weight, 1-25% by weight, 5-25% by weight, 10-25% by weight, 15-25% by weight, 20-25% by weight or 23-25% by weight. In one example, a composition containing a culture product within the above range may have: (1) superior activity in preventing or treating gastric disease and/or (2) superior efficacy against Helicobacter pylori compared to a composition containing a culture product at a concentration beyond the above range.

The culture product may contain a Corynebacterium sp. strain and may include a Corynebacterium sp. strain in the culture product at a concentration of 0.1-10% by weight, 0.1-8% by weight, 0.1-5% by weight, 0.1 -4% by weight, 0.1-3.5% by weight, 0.1-3% by weight, 0.1-1% by weight, 1-10% by weight, 1-8% by weight, 1 -5% by weight, 1-4% by weight, 1-3.5% by weight, 1-3% by weight, 2-10% by weight, 2-8% by weight, 2-5% by weight, 2-4% by weight, 2-3.5% by weight, 2-3% by weight, 3-10% by weight, 3-8% by weight, 3-5% by weight, 3-4% by weight , 3-3.5% by weight, 3.5-10% by weight, 3.5-8% by weight, 3.5-5% by weight or 3.5-4% by weight.

In one example, the Corynebacterium sp. strain and its culture product may be a culture product comprising heat-killed bacteria of the Corynebacterium sp. strain obtained by heat treating (or sterilizing (e.g., autoclaving or hot air drying)) the culture product obtained by culturing strain Corynebacterium sp. in the environment for a certain period of time.

In the present invention, the term "cultivation" means a series of activities for the cultivation and development of a microorganism under environmental conditions artificially adjusted in a suitable manner. When culturing, any culture conditions and culture method known in the art can be used. For example, the cultivation can be performed continuously in a known batch culture method, a continuous culture method, a fed-batch culture method, a batch process or a fed-batch process, or a repeated fed-batch process. The culture conditions can then be adjusted to a suitable pH (eg pH 5-9, pH 6-8 or pH 6.8) using a basic compound (eg sodium hydroxide, potassium hydroxide or ammonium hydroxide) or an acidic compound (eg phosphoric acid or sulfuric acid), but not limited to this. In one example, bubble formation can be inhibited using an antifoam agent such as a polyglycol fatty acid ester, and/or aerobic conditions can be maintained by introducing oxygen or an oxygen-containing mixture of gases to the cultured product.

In one example, the culture temperature may be 20-45°C, 25-40°C, 30-40°C, 30-35°C, or 35-40°C, and the culture condition may be 100-500 rpm , 150-300 rpm, 150-250 rpm or 200 rpm, and the cultivation time (period) can be 1-160 hours, 10-100 hours, 12-72 hours, 24-72 hours, 30- 60 hours, 40-50 hours, 45-50 hours or 48 hours. The culture period can be continued until the desired substance (eg L-threonine, threonine) is obtained in the desired amount. In one example, the cultivation can be performed at a culture temperature in the above range for a culture time in the above range.

The medium used for cultivation must suit the conditions of a particular strain appropriately, and those skilled in the art can use it appropriately according to the known content. According to one example, a reference to a medium for culturing a strain of Corynebacterium sp. can be found in known publications (for example, Manual of Methods for General Bacteriology. American Society for Bacteriology. Washington D.C., USA, 1981), but not limited to this.

For cultivating a Corynebacterium sp. strain, the survival requirements of a particular strain can be suitably satisfied by adjusting temperature, pH, and the like, under anaerobic conditions in a normal environment containing suitable carbon sources, nitrogen sources, amino acids, vitamins, and the like. As carbon sources for use in the medium, saccharide and carbohydrate (for example, glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose), oil and fat (for example, soybean oil, sunflower oil, peanut oil and coconut oil) can be used. , fatty acid (eg palmitic acid, stearic acid and linoleic acid), alcohol (eg glycerol and ethanol) and organic acid (eg acetic acid) and the like, alone or in combination, but not limited to. The nitrogen source can be a nitrogen-containing organic compound (for example: peptone, yeast extract, meat broth, malt extract, corn extract, soybean meal and urea) or an inorganic compound (for example: ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate) and the like, separately or in combination, but not limited to. As the phosphate source, potassium dihydrogen orthophosphate, potassium hydrogen orthophosphate, their corresponding nitrogen-containing salts and the like, separately or in combination, can be used, but not limited to. Additionally, the culture medium may contain other metal salts (eg, magnesium sulfate or ferrous sulfate) necessary for growth, and/or contain substances necessary for growth, such as, but not limited to, amino acids and vitamins.

In one example, the medium may be a threonine production medium (e.g., the medium of 435), and a Corynebacterium sp. strain and a culture product thereof may be a culture product in which a Corynebacterium sp. strain is cultured in a threonine production medium, and the threonine may be released into the culture medium or contained in cells.

In one example, the Corynebacterium sp. strain and/or its culture product may contain threonine.

In one example, the Corynebacterium sp. strain and/or its culture product may not contain threonine.

In the present invention, "threonine (Thr)" is a hydroxy-a-amino acid and is one of the essential amino acids that are not produced in the body, meaning an amino acid having the chemical formula HO ₂ CCH(NH ₂ )CH(OH)CH ₃ . Threonine may be an optical isomer of type L (L-type threonine (L-Threonine, L-Thr)), type D, or a combination thereof. Threonine is an essential amino acid and is a component of mucin, a protective substance for the intestinal epithelium, and when not sufficient can cause growth retardation and weight loss.

According to one example, when a Corynebacterium sp. strain, its culture product, and/or threonine are mixed, there may be: (1) a synergistically superior effect in preventing or treating gastric disease and/or (2) a synergistically superior efficacy against Helicobacter pylori.

According to one example, the composition may contain threonine in an amount of 50-90% by weight, 50-85% by weight, 50-80% by weight, 50-75% by weight, 50-70% by weight, 55-90% by weight by weight, 55-85% by weight, 55-80% by weight, 55-75% by weight, 55-70% by weight, 60-90% by weight, 60-85% by weight, 60-80% by weight , 60-75% by weight, 60-70% by weight, 65-90% by weight, 65-85% by weight, 65-80% by weight, 65-75% by weight, 65-70% by weight, 70-90% by weight, 70-85% by weight, 70-80% by weight, 70-75% by weight or 70% by weight. A composition containing threonine in the above range may have (1) superior effect in preventing or treating gastric disease and/or (2) have superior efficacy against Helicobacter pylori compared to a composition containing threonine at a concentration outside the above range.

Threonine may be commercially available or may be obtained by an extraction method, a fermentation method, an enzymatic method and/or a synthesis method and the like. For example, it can be obtained by preparing a fermented product containing threonine using a Corune-type strain and then purifying it, or be synthesized through the threonine biosynthesis pathway, and/or synthesized chemically.

In one example, the composition may (1) contain threonine in a Corynebacterium sp. strain and/or a culture product thereof, or (2) additionally be present in a form that is not contained in a Corynebacterium sp. strain and/or a culture product thereof, or ( 3) contained in their combination (for example, a Corynebacterium sp. strain and its cultivation product contained in a composition containing threonine and, in addition, additionally containing threonine).

The threonine contained in the composition, according to one example, may:

(1) contained in a Corynebacterium sp. strain, its cultivation product, or both (strain and cultivation product); or

(2) be added separately; or

(3) contained in a Corynebacterium sp. strain, its cultivation product, or both (strain and cultivation product) and be added therein separately.

The case where threonine is added separately may mean additional addition of purified threonine in addition to the threonine contained in the strain and/or culture product.

Threonine may be presented in the form of powder and/or granules. Threonine in the form of granules can have a particle size of 100-1000 microns or 200-1000 microns.

According to one example, the composition may contain 0.1-5 parts by weight of a Corynebacterium sp. strain, 10-90 parts by weight of a culture product, and 10-80 parts by weight of threonine. In particular, it may contain 0.2-5 parts by weight of the strain, 12-88 parts by weight of the cultivation product and 10.5-78 parts by weight of threonine; 0.3-5 parts by weight of the strain, 13-87 parts by weight of the cultivation product and 11-77 parts by weight of threonine; 0.4-5 parts by weight of the strain, 14-86 parts by weight of the cultivation product and 11.5-76 parts by weight of threonine; 0.5-5 parts by weight of the strain, 15-85 parts by weight of the cultivation product and 12-75 parts by weight of threonine; 0.6-5 parts by weight of the strain, 16-84 parts by weight of the cultivation product and 12.5-74 parts by weight of threonine; 0.7-5 parts by weight of the strain, 17-83 parts by weight of the cultivation product and 13-73 parts by weight of threonine; 0.8-5 parts by weight of the strain, 18-82 parts by weight of the cultivation product and 13.5-72 parts by weight of threonine; 0.9-5 parts by weight of the strain, 19-81 parts by weight of the cultivation product and 14-71 parts by weight of threonine; 0.9-4 parts by weight of the strain, 20-80 parts by weight of the cultivation product and 14-70 parts by weight of threonine; 1-4 parts by weight of the strain, 21-79 parts by weight of the cultivation product and 14-69 parts by weight of threonine, or 1-3 parts by weight of the strain, 22-78 parts by weight of the cultivation product and 14-68 parts by weight of threonine.

According to one example, the ratio of the mass of the strain and the product of its cultivation to the mass of threonine (the mass of the strain and the product of its cultivation: the mass of threonine) in the composition can be 1:0.1-1:10; 1:0.1-1:5; 1:0.1-1:3; 1:0.2-1:10; 1:0.2-1:5; 1:0.2-1:3; 1:0.5-1:10; 1:0.5-1:5; 1:0.5-1:3; 1:1-1:10; 1:1-1:5; 1:1-1:3; 1:3-1:10; 1:3-1:5 or 1:3.

According to one example, the composition may further contain a lignosulfonate. When the composition according to one example further contains a lignosulfonate, (1) the effect of preventing or treating gastric disease and/or (2) the effectiveness against Helicobacter pylori can become superior.

In one example, the composition may further contain a lignosulfonate to regulate threonine content. According to one example, the composition may contain 0.1-50% by weight, 0.1-30% by weight, 0.1-25% by weight, 0.1-20% by weight, 0.1-15% by weight by weight, 0.1-10% by weight, 0.1-7% by weight, 1-50% by weight, 1-30% by weight, 1-25% by weight, 1-20% by weight, 1- 15% by weight, 1-10% by weight, 1-7% by weight, 3-50% by weight, 3-30% by weight, 3-25% by weight, 3-20% by weight, 3-15 % by weight, 3-10% by weight, 3-7% by weight, 5-50% by weight, 5-30% by weight, 5-25% by weight, 5-20% by weight, 5-15% by weight, 5-10% by weight, 5-7% by weight, 6.5-50% by weight, 6.5-30% by weight, 6.5-25% by weight, 6.5-20% by weight, 6.5-15% by weight, 6.5-10% by weight or 6.5-7% by weight calcium lignosulfonate. In one example, the composition may contain a Corynebacterium sp. strain and a culture product thereof in dried form (eg, a dry matter of a "Corynebacterium sp. strain and a culture product thereof"). The dry matter may be obtained by drying a Corynebacterium sp. strain and its culture product, and the dry matter may contain threonine. In one example, drying can be accomplished by at least one method selected from the group consisting of vacuum drying, hot air drying, freeze drying, open air drying, thin film drying, and/or vacuum drying.

In one example, the composition may contain a Corynebacterium sp. strain, a culture product thereof, and threonine in dried form (eg, a dry matter of a "Corynebacterium sp. strain and culture product thereof" containing threonine).

In one example, the composition may be in the form of an emulsion, suspension or solution in an oil or aqueous medium, or in the form of an extract, powder, granule, tablet, capsule or gel (eg hydrogel), and may further contain a dispersing agent or stabilizing agent.

The composition according to one specific example can be obtained by drying a Corynebacterium sp. strain and its culture product (fermented product), and this composition may contain threonine, a Corynebacterium sp. strain (for example, a strain in the form of heat-killed bacteria) and a culture product component, different from threonine and strain. In one example, the composition according to one example, obtained by drying a strain of Corynebacterium sp. and its culture product, may be in the form of granules, and it may: (1) have a superior effect in preventing or treating gastric disease and/or (2) have a superior effectiveness against Helicobacter pylori compared to purified threonine in powder form.

In the present invention, the term "granule" means a state in which the powder aggregates to form particles 30 to 150 times larger, and the term "granulation" may mean inducing such a process in which the starting material takes the form of granules upon processing and the like. In one example, during the drying process of a composition containing a Corynebacterium sp. strain, its culture product, and/or threonine, the composition may take the form of granules.

In one example, gastric disease may be caused by Helicobacter pylori infection.

In one example, the gastric disease may be at least one selected from the group consisting of gastritis, gastric ulcers, duodenal ulcers, peptic ulcers, and gastric cancer.

The term "gastritis" means a condition in which inflammation of the inner wall of the stomach is caused and gastric ulcers can occur because the balance of attacking factors that cause damage to the mucosa and protective factors that protect the mucosa in the stomach and intestines is disrupted. Attack factors that can be considered as causes of stomach ulcers include stomach acid, various types of digestive enzymes, bile, medications taken, alcohol, Helicobacter pylori infection, non-steroidal anti-inflammatory drugs, smoking and the like.

When the composition of one example is used as a feed composition, the feed composition can be prepared in a form in which the composition of one example is added to a commercially available feed composition. The food in which the composition according to one example can be used is preferably a composition in the form of a powder or pellet or a liquid composition, but is not limited to this. Additionally, the added amount of the composition of the present invention that is added to the feed does not require any special limitation.

In the present invention, the term "feed" may mean any prescribed natural or artificial diet, food, etc., or food component for animal nutrition, digestion by or suitable for animals.

The type of feed is not particularly limited, and feeds commonly used in the art can be used. A non-limiting example of feed may include plant foods such as grains, roots, food processing by-products, algae, fiber, pharmaceutical by-products, fats and oils, starch, peels or grain by-products or the like; and animal feed such as proteins, inorganic substances, fats and oils, minerals, fats and oils, unicellular proteins, zooplankton or food products or the like. They can be used individually or in combination of two or more.

The subject (animal) that can be provided with food containing the composition according to one example is not particularly limited, but may be animals, fish, crustaceans and/or molluscs, and for example, may be a pig, cow, horse, goat, deer , sheep, chicken, duck, goose, turkey, dog, cat, rabbit or fish.

The feed composition may further contain an excipient, diluent or additive. The feed composition may contain a component effective for promoting animal growth, a nutritional component, a food additive, a component that improves storage stability, a coating material component, an amino acid preparation for disease prevention, a vitamin preparation, an enzyme preparation, a non-protein nitrogenous compound, a silicate preparation, a buffer , extractant, probiotic; enzymes such as amylase, lipase and the like; vitamins such as L-ascorbic acid, choline chloride, inositol and the like; minerals such as potassium chloride, ferric citrate, magnesium oxide, phosphates and the like; amino acids such as lysine, alanine, methionine and the like; organic acids such as fumaric acid, butyric acid, lactic acid and the like or salts thereof; antioxidants such as vitamin C, vitamin E and the like; mold inhibiting agents such as calcium propionate and the like; emulsifiers such as lecithin, glycerol fatty acid esters and the like; and/or pigments and the like, in addition to components. Although not described above, in one example, the food composition may additionally contain other nutrients within a range that would be expected by one skilled in the art.

In another aspect, a pharmaceutical composition for the prevention or treatment of gastric disease may be provided, comprising a Corynebacterium sp. strain, a culture product thereof, and threonine. The Corynebacterium sp. strain, its culture product and threonine contained in the pharmaceutical composition for preventing or treating according to one example, and/or gastric disease are as described for a feed composition for preventing or alleviating gastric disease.

In one example, the pharmaceutical composition may further comprise a suitable carrier, excipient or diluent commonly used for the manufacture of pharmaceutical compositions. In particular, the pharmaceutical composition can be used by preparing in the form of an oral preparation such as powder, granule, tablet, capsule, suspension, emulsion, syrup, aerosol and the like, topical application, suppositories and sterile injectable solutions, each according to a conventional method . The carrier, excipient and diluent contained in the pharmaceutical composition may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. A diluent or excipient such as a filler, diluent, binder, wetting agent, disintegrant, surfactant and the like is used to formulate the formulation. Solid compositions for oral administration include tablet, pill, powder, granule, capsule and the like, and these solid compositions are prepared by mixing at least one excipient, for example starch, calcium carbonate, sucrose or lactose, gelatin and the like, with the composition. Moreover, in addition to the simple excipient, the composition may contain a lubricant such as magnesium stearate and talc. Liquid compositions for oral administration include suspension, oral liquid, emulsion, syrup and the like, and in addition to conventional simple diluents, water and liquid paraffin, may contain various excipients, for example, a humectant, sweetener, air freshener, preservative and the like. . Compositions for parenteral administration include sterilized aqueous solution, non-aqueous diluent, suspension, emulsion, lyophilized composition and suppositories. As the non-aqueous solvent and suspension, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable esters such as ethyl oleate and the like can be used. Witepsol, Macrogol, Tween 61, cocoa butter, laurinum, glycerogelatin and the like can be used as the base compound for suppositories.

According to one example, the pharmaceutical composition can be administered in a pharmaceutically effective amount, and in the present invention, the term "pharmaceutically effective amount" means an amount sufficient to treat or prevent a disease at an acceptable benefit/risk ratio applicable for medical treatment or prevention, and an effective dose level can be determined according to factors including the severity of the disease, the activity of the drug, the patient's age, body weight, health, gender, the patient's sensitivity to drugs, time of administration, route of administration and release rate of the composition of the present invention used, treatment period, drugs , used in combination or concurrently with the composition of the present invention and other factors known in the field of medicine. In one example, the pharmaceutical composition may be administered as a single therapeutic agent or administered in combination with other therapeutic agents, and may be administered sequentially or simultaneously with a conventional therapeutic agent. In addition, it can be administered once or repeatedly. It is important to administer an amount that allows the maximum effect to be achieved in the minimum amount without side effects, taking all factors into account.

According to one example, the dosage of the pharmaceutical composition may be about 0.0001-100 mg/kg, in particular 0.001-10 mg/kg, per mammal per day. The frequency of administration of the pharmaceutical composition of the present invention may be administered once a day or several times by dividing the dose, but is not specifically limited to this. The dosage does not limit the scope of the present invention in any aspect.

In one example, the pharmaceutical composition is not limited to the route of administration as long as it can reach the target tissue. For example, intra-articular injection, oral administration, intra-arterial administration, intravenous administration or transdermal injection or the like are included.

Additionally, the pharmaceutical composition can be administered using any device capable of transporting the active substance to the target cell.

In another aspect of the present invention, there is provided a food composition for preventing gastric disease or alleviating gastric disease, comprising a Corynebacterium sp. strain, a culture product thereof, and threonine. The Corynebacterium sp. strain, its culture product and threonine contained in the food composition for preventing gastric disease or alleviating gastric disease and/or gastric disease are the same as those described for the food composition for preventing gastric disease or alleviating gastric disease.

Food products include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, instant noodles, other noodles, chewing gum, dairy products including ice cream, various types of soup, beverages, tea, drinking beverages, alcoholic drinks, vitamin complexes, functional foods and health food products and the like, and include any food products in the usual sense.

The term "functional foods" means the same as "special foods for health benefits (FoSHU)" and refers to foods with significant medical and therapeutic effects that are processed to effectively exert a body regulatory function in addition to the nutritional function. . Here, "function (functional)" means obtaining a beneficial effect when used for health, such as regulating nutrients or physiological effects in accordance with the structure and function of the human body. The food products of the present invention can be prepared by a method conventional in the art, and can be prepared by adding a starting material and a commonly added component. In addition, the food composition can be prepared without restrictions, provided that its composition is recognized as food grade. The food composition of the present invention can be prepared in various formulation forms, and unlike conventional drugs, it has the advantage of being free of side effects that may occur when taking drugs for a long time, and has excellent tolerability, and therefore the food product is The present invention can be used as a supplement to enhance the prevention of stomach ulcers or relieve stomach ulcers.

The term “health food” means a food product that has an active health maintenance or enhancement effect over regular food, and the term “health food supplement” means a food product intended to supplement a health benefit. In some cases, the terms “functional foods,” “health foods,” and “health supplements” may be used interchangeably.

In particular, functional foods are food products in which the composition according to one example is added to food products such as beverages, tea, spices, chewing gum, confectionery and the like, or food products manufactured in the form of a capsule, powder , suspensions and the like, and are purported to have a certain health effect when taken orally, but unlike conventional drugs, they have the advantage of not having the side effects that may be present when taking drugs for a long time, when using food products as starting material.

According to one example, the nutritional composition can be ingested daily, and therefore can be expected to have a strong effect in preventing gastric disease or alleviating gastric disease, and thus can be used with high efficiency.

The food composition may further contain a physiologically acceptable carrier, and the type of carrier is not particularly limited, and any carrier commonly used in the art can be used.

In addition, the food composition may contain an additional component that can improve the smell, taste, appearance, and the like when generally used in the food composition. For example, it may contain vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, pantothenic acid and the like. In addition, it may contain minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu), chromium (Cr) and others. similar. In addition, it may contain amino acids such as lysine, tryptophan, cysteine, valine and the like.

Additionally, the food composition may contain food additives such as preservatives (potassium sorbate, potassium benzoate, salicylic acid, sodium dehydroacetic acid, etc.), disinfectant (bleach powder and high-efficiency bleach powder, sodium hypochlorite, etc.), antioxidant (butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and so on), dye (aniline dye and so on), dye (sodium nitrite, sodium nitrous acid and so on), bleaching agent (sodium sulfite), seasoning (monosodium glutamate MSG and so on further), sweetener (dulcine, cyclamate, saccharin, sodium and so on), flavoring (vanillin, lactones and so on), foaming agent (alum, potassium D-bitartrate and so on), hardening agent, emulsifier, thickening agent (paste), coating agent, base rubber, defoamer, solvent, improver and the like. Additives can be selected depending on the type of food product and used in suitable quantities.

According to one example, the composition can be added alone or used with other food products or food components, and can be used suitably in accordance with a conventional method. The amount of active ingredient in the mixture can be suitably determined depending on the purpose of its use (prevention, health promotion or therapeutic treatment). In general, when preparing a food or beverage, the food composition of the present invention can be added in an amount of 50 parts by weight or less, particularly 20 parts by weight or less, based on the weight of the food or drink. However, taking into account the objective of improving health and hygiene over a long period, contents beyond the lower limit of the above range may be provided, and since there are no problems from a safety point of view, the active ingredient can be used in an amount beyond the upper limit of the above range.

In one example, the food composition may be used as a composition in the form of a health drink, in which case, just like a conventional drink, it may contain additional components such as various flavors or natural carbohydrates or the like. The above-mentioned natural carbohydrates may be monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; polysaccharides such as dextrin and cyclodextrin; sugar alcohols such as xylitol, sorbitol, erythritol and the like. Natural sweeteners such as thaumatin and stevia extract can be used as a sweetener; artificial sweeteners such as saccharin and aspartame and the like can be used. The ratio of natural carbohydrates may typically be about 0.01-0.04 g, in particular about 0.02-0.03 g, per 100 ml of the health drink composition of the present invention.

Additionally, the health-promoting beverage composition may contain various nutrients, vitamins, electrolytes, flavorings, colorants, pectic acid, pectic acid salts, alginate, alginate salts, organic salts, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives , glycerin, alcohol or carbonation agents or the like. In addition, it may contain pulp for the preparation of natural fruit juice, fruit juice drink or vegetable drink. These components can be used independently or in combination. The ratio of these additives is not very important, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the health drink composition of the present invention.

According to one example, the content of the food composition may be different % by weight, provided that it exhibits the effect of preventing gastric disease or alleviating gastric disease, but for example, according to one example, the content of the composition may be an amount of 0.00001-100% by weight , 0.01-80% by weight or 10-50% by weight, based on the total weight of the food composition.

In one example, a composition containing a Corynebacterium sp. strain, its culture product, and threonine can prevent, treat, or alleviate gastric disease (e.g., gastric ulcers) by inhibiting the proliferation of Helicobacter pylori (H. pylori) and inhibiting inflammation, oxidative stress , blood vessel growth and/or apoptosis induced by Helicobacter pylori.

According to one example, a composition containing a strain of Corynebacterium sp., a product of its cultivation and threonine, can prevent, treat or alleviate gastric disease by stimulating the synthesis and release of mucus, stimulating tissue regeneration, inhibiting damage to the gastric mucosa, stimulating tissue regeneration, inhibiting oxidative stress, inhibition of apoptosis of gastric mucosal cells and/or inhibition of inflammation.

With respect to the composition according to one example, it is confirmed that there is a difference in (1) the effect of preventing or treating gastric disease and/or (2) the effectiveness against Helicobacter pylori depending on whether it contains a Corynebacterium sp. strain or not; whether it contains a strain of Corynebacterium sp. in the form of heat-killed bacteria; strain type Corynebacterium sp.; the presence or absence of a cultivation product; presence or absence of threonine; and/or a combination of a Corynebacterium sp. strain, its cultivation product and threonine, and the composition according to one example is confirmed to have the most superior effect.

In another aspect, a method of preventing, alleviating or treating a gastric disease is provided, comprising administering a composition (feed composition, nutritional composition or pharmaceutical composition) for preventing, alleviating or treating a gastric disease in a subject. The feed composition, food composition, pharmaceutical composition and gastric disease are as described above.

In one example, a method of preventing, alleviating or treating a gastric disease may further include confirming (selecting) a subject (patient) requiring prevention, alleviation or treatment of a gastric disease before administration.

In the present invention, the term "subject" can mean any animal, including a human, who has a gastric disease or is at risk of developing a gastric disease.

The target subject to which the method of preventing or treating gastric disease is applied may be a mammal, including a human, that has gastric disease or is at risk for developing gastric disease, and may, for example, be a pig, cow, horse, goat, reindeer , sheep, chicken, duck, goose, turkey, dog, cat, and/or.

In the present invention, the term "administration" means the administration of a composition for preventing, alleviating or treating a gastric disease to the target subject by any suitable route, and with respect to the route of administration, it can be administered by various routes of oral or parenteral administration (for example, intravenous, subcutaneous, intramuscular, intraperitoneal or topical application) so that it can reach the target tissue.

As a method of preventing, alleviating, or treating a condition, a feed composition, nutritional composition, or pharmaceutical composition according to one example may be administered at a (pharmaceutically) effective dose. The appropriate total amount for daily dosing can be determined by treatment within the correct medical definition range and can be administered in a single or multiple doses. However, the specific therapeutically effective dose for a particular subject (patient) may be otherwise determined depending on various factors, including the specific composition, age of the subject (patient), body weight, general health status, sex and diet, time of administration, route of administration and rate secretion of the composition, the period of treatment and the drugs used with the particular composition or used concomitantly, and similar factors well known in the pharmaceutical field, in addition to the type and extent of response to be achieved and whether other agents are used in some cases.

In another aspect, an antimicrobial composition against Helicobacter pylori is provided, containing a strain of Corynebacterium sp., a product of its cultivation and threonine. The Corynebacterium sp. strain, its culture product and threonine contained in the antimicrobial composition against Helicobacter pylori and/or gastric disease are as described for a pharmaceutical composition for the prevention or treatment of gastric disease.

By combining a Corynebacterium sp. strain, its culture product and threonine according to one example, the antimicrobial effect against Helicobacter pylori can be synergistically superior.

In another aspect, a composition is provided for preventing, alleviating or treating a gastric disease comprising a Corynebacterium sp. strain and a culture product thereof, and the strain and culture product thereof may contain threonine.

In another aspect, a composition is provided for producing an active ingredient for the prevention, relief or treatment of a gastric disease, comprising a Corynebacterium sp. strain and a culture product thereof (fermented product), and the Corynebacterium sp. strain or a culture product thereof (fermented product) may include or contain threonine

The composition for producing an active ingredient for the prevention, relief or treatment of gastric disease may contain 0.1-5 parts by weight of a Corynebacterium sp. strain, 10-90 parts by weight of a fermented product and 10-80 parts by weight of threonine, based on the total mass of the composition. In particular, it may contain 0.2-5 parts by weight of strain, 12-88 parts by weight of fermented product and 10.5-78 parts by weight of threonine; 0.3-5 parts by weight of strain, 13-87 parts by weight of fermented product and 11-77 parts by weight of threonine; 0.4-5 parts by weight of strain, 14-86 parts by weight of fermented product and 11.5-76 parts by weight of threonine; 0.5-5 parts by weight of the strain, 15-85 parts by weight of the fermented product and 12-75 parts by weight of threonine; 0.6-5 parts by weight of the strain, 16-84 parts by weight of the fermented product and 12.5-74 parts by weight of threonine; 0.7-5 parts by weight of the strain, 17-83 parts by weight of the fermented product and 13-73 parts by weight of threonine; 0.8-5 parts by weight of the strain, 18-82 parts by weight of the fermented product and 13.5-72 parts by weight of threonine; 0.9-5 parts by weight of the strain, 19-81 parts by weight of the fermented product and 14-71 parts by weight of threonine; 0.9-4 parts by weight of the strain, 20-80 parts by weight of the fermented product and 14-70 parts by weight of threonine; 1-4 parts by weight of strain, 21-79 parts by weight of fermented product and 14-69 parts by weight of threonine; 1-3 parts by weight of strain, 22-78 parts by weight of fermented product and 14-68 parts by weight of threonine, based on the total weight of the composition, but not limited to.

The composition for the preparation of an active ingredient for the prevention or treatment of gastric disease may further contain calcium lignosulfonate to regulate the threonine content. Calcium lignosulfonate may be contained in an amount of 0.1-50% by weight, 0.1-30% by weight, 0.1-25% by weight, 0.1-20% by weight, 0.1-15% by weight , 0.1-10% by weight, 0.1-7% by weight, 1-50% by weight, 1-30% by weight, 1-25% by weight, 1-20% by weight, 1-15 % by weight, 1-10% by weight, 1-7% by weight, 3-50% by weight, 3-30% by weight, 3-25% by weight, 3-20% by weight, 3-15% by weight, 3-10% by weight, 3-7% by weight, 5-50% by weight, 5-30% by weight, 5-25% by weight, 5-20% by weight, 5-15% by weight by weight, 5-10% by weight, 5-7% by weight, 6.5-50% by weight, 6.5-30% by weight, 6.5-25% by weight, 6.5-20% by weight by weight, 6.5-15% by weight, 6.5-10% by weight, 6.5-7% by weight, 0.1-18% by weight, 0.1-16% by weight, 0.1 -14% by mass, 0.1-13% by mass, 0.1-12% by mass, 0.1-11% by mass, 0.1-9% by mass, 0.1-8% by mass or 0.1-7% by weight, based on the total weight of the composition, but not limited to, and can be added without limitation so as to achieve the desired threonine content in the composition.

In another aspect, a pharmaceutical composition for preventing or treating a gastric disease is provided, comprising a composition for producing an active ingredient for preventing or treating a gastric disease.

In another aspect, a food composition for preventing or alleviating gastric disease is provided, comprising a composition for producing an active ingredient for preventing or treating gastric disease.

In another aspect, a feed composition for preventing or alleviating gastric disease is provided, comprising a composition for producing an active ingredient for preventing or treating gastric disease.

In another aspect, a composition for treating a gastric disease is provided, comprising a granular composition for producing an active ingredient for preventing or treating a gastric disease, containing a strain of Corynebacterium sp. and a culture product thereof (fermented product) as the active ingredient.

Corynebacterium sp. strain and its fermented product, gastric disease, prevention, relief and treatment are as described above.

In the present invention, the term "composition" refers to such processing, which provides convenience for manufacture, storage or use and provides the therapeutic effect to a sufficient extent, mainly through mechanical operations, for example, grinding, mixing, stirring, leaching (cooking) or evaporation or the like without changing the nature of the medicines, and additionally, the products obtained in this way are also called medicines (chemical unabridged dictionary, 2001. 5. 20., Sehwa editorial department).

The additives used in the preparation of the composition in the form of a preparation, that is, excipient, diluent, binder, wetting agent, disintegrant, solvent or excipient, are as described above, and solid compositions for oral administration and additives contained therein, liquid compositions for oral administration and the additives contained therein or parenteral compositions and the additives included therein are as described above.

The composition may be a solid composition, a liquid composition and a fluidized composition, but is not limited to these, and any composition exhibiting the effect of preventing, treating and relieving gastric ulcer can be used without limitation.

The compositions are as described above.

In another aspect, there is provided a method for producing an active ingredient having: (1) an effect in preventing, alleviating or treating gastric disease; and/or (2) an antimicrobial effect against Helicobacter pylori, including drying the Corynebacterium sp. strain and its cultivation product.

The Corynebacterium sp. strain and its culture product are as described above.

In one example, drying can be accomplished by at least one method selected from the group consisting of vacuum drying, hot air drying, freeze drying, open air drying, thin film drying, and/or vacuum drying.

In one example, drying may dry a composition in which threonine is separately added to a Corynebacterium sp. strain and its culture product.

In one example, the method of producing the active ingredient may further comprise adding threonine to the dry matter obtained after drying.

BENEFITIVE EFFECTS

It is confirmed that the composition of the present invention is superior in effectiveness against Helicobacter pylori in cell experiments and in the effectiveness of gastric ulcer relief, gastric mucus synthesis efficiency and the like in animal experiments, and therefore can be used as a pharmaceutical composition for the prevention or treatment of gastric disease, or as a food or feed composition for the prevention of gastric disease or relief of gastric disease.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

In FIG. 1 shows antimicrobial activity against H. pylori for Groups 1-8.

In FIG. 2a - Fig. Figure 2c shows the results of measuring the expression of inflammatory mediators when treated with compositions of Groups 1-8 RGM1 cells infected at 100 MOI for 6 hours using the H. pylori strain. In particular, in FIG. 2a shows the change in Cox-2 mRNA expression according to Groups 1-8 treatment in RGM1 cells infected with H. pylori strain, and FIG. 2b shows the change in iNOS protein expression according to Groups 1-8 treatment in RGM1 cells infected with H. pylori strain, and FIG. Figure 2c shows the change in the expression of phosphorylated NF-κB p65 protein in accordance with the treatment of Groups 1-8 in RGM1 cells infected with the H. pylori strain. GAPDH and β-actin were used as internal controls for mRNA and protein, respectively.

In FIG. 2a - Fig. 6b, N (first position from the left) represents the negative control group (non-H. pylori-treated RGM1 cells), and H.p (second position from the left) represents the positive control group (H. pylori-treated RGM1 cells), and Groups 1-8 represent the results in RGM1 cells infected with H. pylori treated with compositions of Groups 1-8.

In FIG. 3a shows the change in oxidative stress related protein (HIF-1a) expression according to Groups 1-8 treatment in H. pylori-infected RGM1 cells. β-actin was used in the internal control group. In FIG. Figure 3b shows the result of measuring the change in the concentration of intracellular active oxygen in accordance with the treatment of Groups 1-8 in RGM1 cells infected with H. pylori (result of increasing and decreasing DCF).

In FIG. 4a shows the change in HO-1 mRNA expression according to Groups 1-8 treatment in H. pylori-infected RGM1 cells, and FIG. Figure 4b shows the change in the expression of GST(pi) and HO-1 proteins in accordance with the treatment of Groups 1-8 in H. pylori-infected RGM1 cells. GAPDH and β-actin were used in the internal control group.

In FIG. Figure 5 shows the apoptosis inhibition efficacy of Groups 1-8 treatment in H. pylori infected RGM1 cells. In particular, in FIG. Figure 5a shows the change in the expression of Bax and Bcl-2 proteins according to the treatment of Groups 1-8 in H. pylori-infected RGM1 cells, and p-actin was used in the internal control group. In FIG. Figure 5b shows the change in apoptosis (TUNEL staining results) according to Groups 1-8 treatment in H. pylori infected RGM1 cells.

In FIG. 6 shows the result of changes in angiogenesis and mucosal proliferation growth factors according to Groups 1-8 treatment in H. pylori-infected RGM1 cells. In FIG. 6a shows the change in protein expression of TGF-β and VEGF according to the treatment of Groups 1-8 in H. pylori-infected RGM1 cells, and β-actin was used in the internal control group. In FIG. Figure 6b shows the change in β-catenin protein expression according to the treatment of Groups 1-8 in H. pylori-infected RGM1 cells, and lamin B was used in the internal control group.

In FIG. 7a - Fig. 10, Groups 1-4 mean experimental groups of animals 1-4 of Table 5.

In FIG. 7 shows the effectiveness of alleviation of SRMD (stress-related mucosal disease) when administered the composition according to one example in the experimental group of animals of the WIRS model. In particular, in FIG. Figure 7a shows photographs of the gastric pathologies of each experimental group, and the photographs in the two rows on the right are presented at a magnification of ×40. In FIG. 7b shows (A) Macroscopic damage index (top graph on the left), (B) Inflammation; pathology score (top graph on the right), (B) Ulcer/erosion; pathology indicator (lower graph on the left), (D) Regeneration; pathology indicator (lower graph on the right) when the composition was administered according to one of the examples in the experimental group of animals of the WIRS model.

In FIG. 8 shows changes in inflammation, angiogenesis and signaling pathways upon administration of the composition according to one example in the experimental group of the WIRS animal model. (A) In FIG. 8a shows the change in the expression of iNOS, TNF-α and IFN-γ mRNA in experimental animal groups 1-4, and FIG. 8b shows the change in PDGF mRNA expression in experimental animal groups 1-4, and FIG. 8c shows the change in p-IκBα protein expression in experimental groups of animals 1-4, and FIG. 8 g shows the change in the expression of p-ERK and p-JNK proteins in experimental groups of animals 1-4. GAPDH and β-actin were used in the internal mRNA and protein control group, respectively.

In FIG. 9 presents a result showing the change in apoptosis and cell cycle when the composition was administered according to one example in the experimental group of animals of the WIRS model. In particular, in FIG. 9a shows the result of TUNEL analysis showing the level of apoptosis in the SRMD region of the gastric mucosa in experimental animal groups 1-4, and FIG. 9b shows the change in protein expression of PARP-1, Bc1-2, Bax, cleaved caspase-8 and cleaved caspase-3, and FIG. Figure 9c shows changes in the expression of CDK4 and cyclin D1 proteins. β-actin was used as an internal control group.

In FIG. Figure 10 shows the mucin content in the stomach tissue of experimental animal groups 1-4.

In FIG. Figure 11 shows photographs of the pathologies of the stomach of WIRS rats that were injected with samples containing threonine in various % by weight. Composition and content of Samples 1-5 in Fig. 11 are disclosed in Table 4.

DETAILED DESCRIPTION OF EMBODIMENTS

Below, the invention will be described in more detail by way of examples for a better understanding of the present invention. However, the following examples illustrate the contents of the present invention, and the scope of the present invention is not limited to the following examples. Examples of the present invention are provided to describe the present invention in more detail to those skilled in the art.

Example 1. Composition containing a strain of Corynebacterium sp. and infection of cells

Example 1-1. Preparation of a composition containing a strain of Corynebacterium sp.

To test the differences in effectiveness against Helicobacter pylori, inhibition of apoptosis and strain cell protection of a composition containing a Corynebacterium sp. strain, and effectiveness between Corynebacterium sp. strains, Group 1-Group 8 compositions were prepared.

Wild type strains of Corynebacterium glutamicum (C. glutamicum), Corynebacterium ammoniagenes (C. ammoniagenes) and Corynebacterium efficiens (C. efficiens) were obtained from CJ CheilJedang BIO R&D Center (Blossom park) and used, and the strains were inoculated into threonine production medium, respectively , and cultured at 35°C for 48 hours at 200 rpm. The components and contents of the threonine production medium are disclosed in the following Table 1.

Group 1 contains the supernatant of the fermentation medium from which microbial cells have been removed by centrifugation of the fermentation medium obtained by culturing Corynebacterium glutamicum under the above conditions, and Group 2 is a buffer (Tris-HCl) containing threonine at a concentration of 100 g/L. Groups 3-5 were obtained by centrifuging the fermentation medium obtained by culturing the strain under the above conditions to collect microbial cells, and resuspending the collected microbial cells in buffer (Tris-HCl). Groups 6-8 represent the fermentation medium in which each strain was cultured under the above conditions, and which contains each strain and the culture product in which each strain was cultured in the medium (referred to as "fermentation medium supernatant" in Table 2). Groups 3-8 compositions contain each strain at a concentration of 30 OD (wavelength 562 nm) and contain the strain in the form of bacteria killed by heat by autoclaving.

All Groups 1-8 contain threonine, and there is a group containing threonine in each strain (contained in the form of heat-killed bacteria) or culture product, and therefore threonine (threonine produced by CJ BIO strains; purity greater than 99%) additionally added so that the final concentration of threonine in Groups 1-8 was 100 g/l when measuring the threonine content of the strain and/or culture product.

The components of each group are disclosed in the following Table 2. In Table 2, the expression "fermentation medium supernatant" of Group 1 and Group 6 means that the microbial cells are removed from the culture product obtained by culturing the "C. glutamicum strain" under the above conditions, and the expression "supernatant fermentation medium" Groups 7 and 8 means that microbial cells have been removed from the culture product obtained by cultivating the "C. ammoniagenes strain" and the "C. efficiens strain" under the above conditions.

Groups 1-8, disclosed in the following Examples 2-7 and FIGS. 1 - Fig. 6b mean Groups 1-8 of Table 2.

Example 1-2. Cultivation of a rat gastric mucosal cell line

RGM1 cells from the gastric mucosal cell line of normal rats were cultured in Ham F12 and DMEM (Dulbecco's modified essential medium) mixed medium containing 10% fetal bovine serum at 37°C in a cell incubator (95% air, 5% CO ₂ ). The RGM1 cell line was established by Professor Matsui from Japan Tsukuba University, and used with his consent.

Example 1-3. H. pylori strain and cell infection

Helicobacter pylori (H. pylori) strain (cytotoxin-associated gene A [CagA]+strain, NCTC 11637) was purchased from ATCC (American Type Culture Collection, Rockville, MD). The H. pylori strain was cultured with shaking under 10% CO ₂ conditions to obtain 1X10 ⁸ CFU/ml (OD 600=1) in Brucella medium supplemented with 5% calf serum and antibiotic.

RGM1 cells were infected with the Hpylori strain for 6 hours at a multiplicity of infection (MOI) of 100:1 (hereinafter 100 MOI).

Example 2. Testing the effectiveness of a composition containing a strain of Corynebacterium sp. against Helicobacter in vitro

The potency to inhibit the growth of H. pylori, ie the potency against Helicobacter, was measured on blood agar plates using the disk diffusion method. In more detail, 40 g/L TSA (Trypticase™ Soy Agar) was dissolved in purified water and then autoclaved (121°C, 20 min). After cooling to approximately 50°C, 5% sheep blood was added and then antibiotic {trimethoprim (5 mg/L), polymyxin B (2500 U/L), vancomycin (10 mg/L)} was added. The medium was divided into 20-25 ml aliquots into sterile dishes and then stored at 4°C.

200 μl of the H. pylori strain solution was aliquoted and spread on a blood agar plate, and 40 μl of each composition of Groups 1-8 obtained in Example 1-1 was impregnated on paper disks, and cultured in a CO ₂ incubator at 37°C C for 48 hours, and then the diameter of the clear area around the disc was measured and is shown in FIG. 1 (the result for Group 1 is not shown in the graph). In FIG. 1 “untreated” means RGM1 cells not infected with H. pylori.

As shown in FIG. 1, Groups 6 and 7 were confirmed to exhibit significant activity against Helicobacter, since the numerical value of the clean zone diameter was significantly higher in Groups 6 and 7 compared with the untreated group.

Additionally, the appropriate concentration of Groups 1-8 was determined for use in the following experiments.

Diluted solutions in which the compositions of Groups 1-8 obtained in Example 1-1 were diluted 1/4, 1/40 or 1/100 were treated with 40 μl of H. pylori, respectively, and cell viability was measured. Since at 1/4 dilution a viability of 0.5 or less was demonstrated in many groups, and at 1/100 dilution there was no obvious difference in results between groups, the 1/40 dilution solution for which a significant difference in result was shown compared with Group 1, were used in the following experiments.

Example 3. Testing for changes in an inflammatory mediator and NF-κB, which activates the transcription of this mediator, using a composition containing a strain of Corynebacterium sp.

As in the method of Example 1-3, RGM1 cells infected with H. pylori (100 MOI) were treated with compositions of Groups 1-8, diluted 1/40, 40 μl, respectively, for 6 hours. Then, in RGM1 cells treated with these compositions, the expression level of the inflammatory mediator Cox-2 mRNA was confirmed by RT-PCR (reverse transcription-PCR) analysis, and the result is shown in FIG. 2a.

In more detail, RNA was extracted using TRIzol (Gibco BRL, Rockville, MD), and cDNA was synthesized from the extracted RNA template using Moloney murine leukemia virus reverse transcriptase (Perkin Elmer, Morrisville, NC). The nucleic acid sequence of each primer used for the PNR assay is shown in the following Table 3, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control.

Additionally, in RGM1 cells treated with these compositions, the increase and decrease in the expression level of iNOS protein and phosphorylation of NF-κB p65, which activates its transcription, were confirmed by Western blot analysis, and the result is shown in FIG. 2b and Fig. 2c.

In more detail, cells cultured under composition treatment conditions were washed with PBS (phosphate buffered saline) solution and then dissolved in cell lysis buffer (150 mM NaCl, 0.5% Triton X-100, 50 mM Tris-HCl, pH 7 ,4, 25 mM NaF, 20 mM ethylene glycol bis(βN'tetraacetic acid, 1 mM dithiothreitol, 1 mM Na ₃ VO ₄ , protease inhibitor cocktail tablet [Boehringer, Manneim, Germany]) to obtain a protein lysate. After separation of these proteins By SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis), unfolded proteins were transferred to a PVDF membrane (Gelman Sciences, Ann Arbor, MI) to react with primary antibodies and secondary antibodies, respectively, and then analyzed with using a chemiluminescence system.

As shown in FIG. 2a, compared with normal RGM1(N) cells, in RGM1 cells infected with H. pylori (Hp), the amount of Cox-2 mRNA was significantly increased, and the increased expression of Cox-2 mRNA caused by H. pylori infection was the most significant reduced when adding a Group 6 composition.

As shown in FIG. 2b and Fig. 2c, iNOS protein expression and phosphorylation of NF-κB p65, which activates its transcription, were significantly increased during H. pylori infection, but iNOS protein expression and phosphorylation of NF-κB p65, which activates its transcription, were most significantly decreased with the addition of the Group 6 composition. From These results show that the composition according to one example regulated the transmission pathway of inflammation caused by a Helicobacter strain.

Example 4: Testing the effectiveness of a composition containing a Corynebacterium sp. strain in reducing oxidative stress in vitro

As in the method of Example 1-3, RGM1 cells infected with H. pylori (100 MOI) were treated with compositions of Groups 1-8, diluted 1/40, 40 μl, respectively, for 6 hours. Western blotting was then performed in the same manner as in Example 3 and the expression level of oxidative stress-associated HIF-1α was measured, and the result is shown in FIG. 3a.

As shown in FIG. 3a, the level of HIF-1a was significantly increased in Helicobacter pylori infection, and the expression of HIF-1a was significantly decreased in Groups 6 and 7.

Additionally, to measure the change in intracellular reactive oxygen concentration, the DCF amplification response was analyzed using the DCF-DA probe using a confocal imaging device. A non-fluorescent substance, DCF-DA (2',7'-dichlorofluorescein diacetate) is oxidized by reactive oxygen species (ROS) when peroxides are present in the cell and exhibits green fluorescence, and therefore the amount of reactive oxygen species can be directly quantified by the fluorescence intensity at a certain wavelength. Specifically, RGM-1 cells (RGM1 cells untreated with H. pylori; or RGM1 cells infected with H. pylori (100 MOI) (Example 1-3)), 5×10 ⁵ cells/well, were aliquoted into a 24-well plate and cultured in an incubator at 37°C (95% air, 5% CO ₂ ) overnight, and then treated with the compositions of Groups 1-8 for a certain time, respectively. Cells were washed with DMEM and then 10 μg/ml DCF-DA (2',7'-dichlorofluorescein diacetate, Sigma-Aldrich Co., St. Louis, MO) was added to the medium and incubated for 30 minutes. After incubation, cells washed with PBS at 4°C were examined visually and photographed under a fluorescence microscope, and the result is shown in FIG. 3b.

As shown in FIG. 3b, it was confirmed that the fluorescence intensity of DCF-DA increased after Helicobacter pylori infection was statistically significantly decreased in Treatment Groups 1, 3, 6 and 8 (P<0.05), and in Treatment Group 6, the degree of reduction was the most significant ( P<0.01). From these results it is clear that the composition according to one example can quickly cope with oxidative stress or hypoxic reaction caused by a strain of Helicobacter pylori.

Example 5. Testing the effectiveness of a composition containing a Corynebacterium sp. strain in protecting cells from oxidative stress associated with Helicobacter pylori in vitro

As in the method of Example 1-3, RGM1 cells infected with H. pylori (100 MOI) were treated with compositions of Groups 1-8, diluted 1/40, 40 μl, respectively, for 6 hours. Then, the change in the expression of HO-1 mRNA with antioxidant function, a representative cytoprotective factor, was analyzed by the method in accordance with Example 3 (PCR) and as shown in FIG. 4a, and the change in protein expression of HO-1 and GST (glutathione-s-transferase(pi)) was analyzed in the same way as in Example 3 (Western blot analysis), and the result is shown in FIG. 4b.

As a result, as shown in FIG. 4a and Fig. 4b, in the control group, a significant decrease in the amount of HO-1 mRNA and protein was observed after Helicobacter pylori infection, and in Treatment Groups 6-8, the decreased expression of HO-1 was significantly increased, and among them, in Treatment Group 6, the expression of HO-1 was most significantly increased . Additionally, as shown in FIG. 4b, in Groups 6 and 7, the expression of GST(pi) was significantly reduced, and among them, in Treatment Group 6, the expression of GST(pi) was most significantly reduced.

From these results, it can be seen that in one example, the composition exhibits anti-oxidation and cell protection efficacy based primarily on the HO-1 response of the Helicobacter pylori strain.

Example 6. Testing the effectiveness of a composition containing a Corynebacterium sp. strain in inhibiting apoptosis associated with Helicobacter pylori in vitro

As in the method of Example 1-3, RGM1 cells infected with H. pylori (100 MOI) were treated with compositions from Groups 1-8, diluted 1/40, 40 μl, respectively, for 6 hours. Then, using a method similar to that of Example 3, the expression of Bcl-2 and Bax was confirmed by Western blotting, and the result is shown in FIG. 5a, and apoptosis was analyzed by TUNEL staining, and the result is shown in FIG. 5 B. Specifically, TUNEL staining was performed by aliquoting cells into a Lab-Tek slide chamber at 1 x 10 ⁵ cells/chamber and then culturing them in an incubator at 37°C (95% air, 5% CO ₂ ). After treatment with each reagent for a specified time, it was then performed according to the manufacturer's experimental method using an apoptosis detection kit (Oncogene Research Products, Cambridge, MA). The culture solution was removed and washed with PBS solution 3 times and then 4% PFA (paraformaldehyde) was added and fixed at room temperature for 20 minutes. After repeated washing with PBS twice, treatment with 0.1% Triton X-100 was carried out at 4°C for 5 minutes, light was blocked and then the solution obtained by mixing TdT (terminal deoxynucleotidyl transferase) and a mixture of nucleotides was reacted at 37°C for 60 minutes. After washing with PBS, apoptosis was examined visually under a fluorescence microscope.

As shown in FIG. 5a, a decrease in the expression of Bcl-2 and an increase in the expression of Bax could be observed in cells infected with H. pylori (H.p), and it was confirmed that the significantly increased expression of Bax was reduced and the decreased expression of Bcl-2 was increased in Treatment Groups 6-8. and among these, Treatment Group 6 had the most significant effect.

Helicobacter pylori infection is known to cause mucosal injury and ulceration by significantly increasing apoptosis, and as shown in FIG. 5b, as a result of TUNEL staining, a statistically significant increase in apoptosis was observed during Helicobacter pylori infection (P<0.01), but in Treatment Groups 6-8, apoptosis was significantly reduced, and among them, in Treatment Group 6, the reduction in apoptosis was the most significant.

Example 7: Testing changes in cell growth, blood vessel growth and proliferation factor associated with Helicobacter pylori using a composition containing a Corynebacterium sp. strain in vitro

As in the method of Example 1-3, RGM1 cells infected with H. pylori (100 MOI) were treated with compositions from Groups 1-8 diluted 1/40 to 40 μl, respectively, for 6 hours. Then, using a method similar to that of Example 3, changes in TGF-β and VEGF were analyzed by Western blotting, and the result is shown in FIG. 6a.

As a result, as shown in FIG. 6a, TGF-β and VEGF, increased as a result of Helicobacter pylori infection, stimulated indiscriminate mucosal proliferation or malignant inflammation, but in Treatment Groups 6-8, the expression of TGF-β and VEGF was significantly reduced, and in particular in Treatment Group 6, TGF expression -β and VEGF decreased most significantly. Based on this, this result demonstrated that, according to one example, the composition can reduce inflammation or malignant transformation in Helicobacter pylori infection.

Additionally, nuclear translocation of β-catenin was confirmed using the nuclear fraction of Helicobacter pylori-infected cells by Western blotting, and the result is shown in FIG. 6b. As shown in FIG. 6b, it was confirmed that nuclear translocation of β-catenin, increased during Helicobacter pylori infection, could be significantly inhibited by Group 6 treatment.

All experimental values from Examples 1-7 were statistically verified by Student's test, and a p value of <0.05 or more was determined to be statistically significant.

Example 8: Preparation of a sample containing threonine

Fermentation medium prepared by inoculating wild type C. glutamicum strain (CJ CheilJedang BIO R&D Center (Blossom park)) into threonine production medium (Table 1) and culturing it at 35°C and 200 rpm for 48 hours, purified and crystallized to obtain Sample 1. Sample 1 contained 99% or more by weight of purified threonine in powder form.

The fermentation medium (containing C. glutamicum) obtained by culturing C. glutamicum in threonine production medium was directly dried (hot air drying at 60-70°C) to obtain Sample 2. In addition to the fermentation medium obtained by culturing C. glutamicum under the above conditions, calcium lignosulfonate (Aladdin industrial Co., Shanghai, Chana) was added in various concentrations, and the fermentation medium (containing C. glutamicum) to which calcium lignosulfonate was added was dried (hot air drying at 60-70°C) with obtaining Samples 3-5. Samples 2-5 contained C. glutamicum in the form of bacteria killed by heat by drying with hot air.

Samples 2-5 were obtained in the form of granules having a particle size of approximately 200-1000 microns. The components and contents of Samples 1-5 are disclosed in Table 4. In Table 4, Thr (% by weight), calcium lignosulfonate (% by weight) and other components of the fermented product (containing microbial cells of C. glutamicum) (% by weight) mean % by weight in the dry matter obtained by drying the fermented medium, and the fermented product in the dry matter contains the C. glutamicum strain. Additionally, the content of the C. glutamicum strain contained in the fermented product is disclosed in Table 4.

Example 9: Derivation of a WIRS (Water Immersion Stress) Model for SRMD (Stress-Related Mucosal Disease)

A total of 110 SD rats were purchased from Charles River (Osaka, Japan) and maintained in an animal facility. Experimental animals were handled in an authorized animal facility in accordance with AAALAC International Animal Care Policies. Animals were fasted for 24 hours before WIRS and water was freely available. 10 rats from each group were placed in a WIRS cage and immersed in water for 6 hours.

Samples 1-5 obtained in Example 8 were administered to rats orally 8 hours before WIRS, so that the same amount of threonine was administered (0.15% by weight/diet), and after WIRS for 6 hours, the animals were sacrificed, removed the stomach was dissected, the contents were removed, and an image of the inner surface of the stomach was obtained, which is shown in FIG. 11. As shown in FIG. 11, as a result of observing the stomach tissue of rats with the naked eye, it was confirmed that in the WIRS-induced group (second row in Fig. 11), gastrorrhagia was induced, and the degree of gastrorrhagia was reduced (excellent therapeutic effect on gastric ulcer) in the order Sample 1<Sample 5<Pattern 4<Pattern 3 or Pattern 2.

On the other hand, the following experiment was conducted by dividing the animals into 4 experimental groups, as shown in the following Table 5. They were divided into an untreated group that did not receive any treatment after oral administration of saline (positive control group, experimental animal group 1); WIRS group in which WIRS was performed for 6 hours (negative control group, experimental animal group 2) and experimental groups of animals that were orally administered food containing 0.15% by weight of Sample 1 (obtained in Example 8) (experimental animal group 3), and food containing 0.21% by weight of Sample 3 (obtained in Example 8) (experimental animal group 4), 30 mg/kg body weight, respectively, 8 hours before WIRS. The content of L-Thr (L-threonine) in the feed of experimental groups of animals 3 and 4 was set to 0.15% by weight/diet equally.

All experimental animals (rats) were sacrificed with a high dose of anesthetic (sodium thiopental, 50 mg/kg) after 6 hours of WIRS. Experimental animal groups 1-4 disclosed in the following Examples 10-12 mean experimental animal groups 1-4 of Table 5, and in FIG. 7a - Fig. 10 Groups 1-4 mean experimental animal groups 1-4 in Table 5.

Example 10 Testing Efficacy for Relieving SRMD (Stress-Related Mucosal Disease)

The stomach of the rats sacrificed in Example 9 was cut out and opened along the greater curvature of the stomach and then washed with a cold PBS solution. After determining the number and size of erosions or ulcers from enlarged photographs, half were opened for anatomical observations. The excised stomach was spread on a plastic surface and fixed with 10% buffered formaldehyde for 4 hours and used to obtain a paraffin section of tissue, and the other half was stored in a tank of liquid nitrogen for molecular biology studies. Additionally, a homogenate of the mucous membrane of animals of the same experimental group, obtained by mixing, was stored.

The stomach samples were examined using a microscope with the naked eye (Fig. 7a), and the macroscopic damage index, inflammation, was calculated in the control group and each experimental group (experimental animal groups 1-4); pathology coefficient, ulcer/erosion; pathology coefficient, regeneration; pathology coefficient, and they are presented in Fig. 7b. As shown in FIG. 7a, all rats in Experimental Animal Group 2 showed a significant incidence of SRMD, such as erosions, bleeding and ulcers, but SRMD was significantly alleviated in animals in Experimental Group 3 and Experimental Group 4. As shown in FIG. 7b, index of macroscopic damage, inflammation; pathology coefficient, ulcer/erosion; pathology coefficient, regeneration; pathology coefficient were significantly (p<0.05) improved in experimental animal groups 3 and 4, and in particular, the macroscopic damage index in experimental animal group 4 was significantly lower than in experimental animal group 3, and regeneration; the pathology coefficient was significantly improved than that of experimental animal group 3.

Example 11 Anti-Inflammatory Efficacy Testing

Since SRMD is physiologically related to ischemic reperfusion injury, the mRNA expression of angiogenic growth factors, including PDGF (platelet-derived growth factor), and the mRNA expression of inflammatory mediators, iNOS, TNF-α, IFN-γ and PDGF, were measured in animal groups 1-4, and the results are presented in Fig. 8a and Fig. 8b.

Specifically, total RNA was extracted using RNeasy Mini kit (Qiagen Korea, Seoul, Korea). The primers used to measure the expression of inflammatory cytokines and mediators are shown in the following Table 6. Amplification was analyzed using 10x reaction buffer (Promega Korea, Seoul, Korea), 1.5 mM/L MgCl ₂ , 200 mM/L deoxynucleotide triphosphates (dNTPs) , 1 mM/L of each primer and 2.5 units of Taq DNA polymerase (Promega) using a Perkin-Elmer GeneAmp PCR System 2400. Each cycle consisted of denaturation at 95°C for 1 minute, annealing at 55°C for 45 minutes and amplification at 72°C for 45 seconds.

Additionally, in each experimental group of animals 1-4, the protein levels of p-IκBα, p-ERK and p-JNK were measured and are shown in FIG. 8c and Fig. 8g. Specifically, to perform Western blotting to measure protein levels, the collected gastric mucosal material was homogenized in ice-cold 20 mM Tris-HCl buffer (pH 7.5) containing 2 mM EDTA, 0.5 mM EGTA. acid), 300 mM sucrose and 2 mM PMSF (phenylmethylsulfonyl fluoride) in a tissue homogenizer. After 30 μg of protein was electrophoresed on an 8% SDS-PAGE gel, it was transferred to a PVDF membrane using a semi-dry transfer system (Hoefer, Holliston, MA, USA). Nonspecific binding was blocked by incubation with 5% nonfat dry milk. The membrane was thermostatically incubated in a 500-fold dilution of primary antibodies at 4°C overnight and then incubated with HRP (horseradish peroxidase)-linked secondary antibodies diluted 1:1000. The immune complex was determined using the ECL detection kit (Amersham Biosciences Korea, Seoul, Korea) and the X-ray film was exposed automatically. The following antibodies were used for Western blotting. Antibodies to β-actin, iNOS (nitric oxide synthase), COX-2 (cyclooxygenase), P-JNK (phosphorylated JNK), P-ERK (phosphorylated ERK), Bcl-2 (B-cell lymphoma 2), Bax (X β-cell lymphoma-associated protein-2) and cyclin D1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). p-IκBα (phosphorylated inhibitor of kappa B alpha), P-STAT3 (phosphorylated signal transporter and activator of transcription 3), PARP (poly(ADP-ribose) polymerase), CDK4 (cyclin-dependent kinase 4) and CDK2 (cyclin-dependent kinase 2) purchased from Cell Signaling Technology (Beverly, MA). Antibodies to HO-1 (heme oxygenase-1) were purchased from Enzo life Sciences (Farmingdale, NY).

As shown in FIG. 8a, in experimental animal group 2 (SRMD induced by WIRD), the mRNA expression of iNOS, TNF-α and interferon gamma (IFN-γ) was significantly increased, but the increased expression of inflammatory mediators (iNOS, TNF-α, IFN-γ) was significantly was reduced in experimental animal group 3 or experimental animal group 4 (P<0.01 or P<0.05, Fig. 8a), and the expression of TNF-α and iFN-γ was significantly reduced, especially in experimental animal group 4 compared with experimental group of animals 3 (P<0.05, Fig. 8a).

As shown in FIG. 8b, the expression of angiogenic growth factor PDGF was significantly increased in experimental animal group 2, but it was significantly decreased in experimental animal group 3 or experimental animal group 4 (P<0.01, Fig. 8b), and in particular in experimental group animals 4, the degree of reduction was more significant.

As shown in FIG. 8c, as a result of measuring p-IκBα expression at the protein level, p-IκBα expression was significantly reduced in WIRS in experimental group 2 animals, and the decrease in p-IκBα expression was associated with transcriptional activation of NF-κB. Meanwhile, the decreased expression of p-IκBα was significantly increased (P<0.01, Figure 8c), and the expression of p-IκBα was significantly increased in experimental animal group 4 compared with experimental animal group 3 (P<0.01, Fig. 8c), and increased expression of p-IκBα induced redox inhibition of NF-kB.

On the other hand, with respect to signal transmission related to ischemia and inflammation, as shown in FIG. 8d, in the experimental group of animals 2 activation of p-ERK and p-JNK was shown, and in the experimental group of animals 4 the signal transmission was completely inactivated, and in the experimental group of animals 3 it was partially inactivated (P < 0.01).

Example 12 Testing the effectiveness of protecting the gastric mucosa. Since SRMD causing erosions or ulcers showed an increase in apoptosis in the corresponding area, apoptosis in the gastric mucosa was measured according to the experimental group of animals. Specifically, apoptosis was visualized using the TdT FragEL DNA fragmentation detection kit (Oncogene Research Products, Cambridge, MA) and the TUNEL (terminal deoxynucleotidyl transferase-mediated single-strand break insertion and labeling) method. To measure AI (apoptotic index) by detecting the region in which apoptosis occurs to a large extent (apoptotic hot spot), in each experimental group, TUNEL-immunostained section (terminal deoxynucleotidyl transferase dUTP insertion of single-strand breaks and labeling (TUNEL)-immunostained slice) was magnified 100 times and scanned. The number of TUNEL-positive cells was then counted in 400 AI recording fields. The average ratio was determined by selecting at least 5 hot spots randomly in the fragments, including necrotic or ulcerative lesions, and the result is presented in Fig. 9a, and this result is expressed as the average percentage of the total number of cells. As shown in FIG. 9a, the level of apoptosis was significantly increased in experimental animal group 2 compared with experimental animal group 1, and the increased level of apoptosis was significantly reduced in experimental animal group 3 and experimental animal group 4 compared with experimental animal group 2 (P<0.01 , Fig. 9a), and in the experimental group of animals 4 a significantly lower level of apoptosis was observed compared to the experimental group of animals 3 (P < 0.01, Fig. 9a).

To verify the results of TUNEL, Western blot analysis of the expression of Bcl-2, apoptosis prevention protein, and cleaved PARP, Bax, cleaved caspase-8, and cleaved caspase-3, which induce apoptosis, was carried out by the method of Example 11, and the result is shown in FIG. . 9b.

As shown in FIG. 9b, PARP cleavage, expression of Bax, cleavage caspase-8 and cleavage caspase-3 were significantly increased in experimental animal group 2, and elevated PARP cleavage, expression of Bax, cleavage caspase-8 and cleavage caspase-3 were significantly decreased in experimental animal groups 3 and 4, and were observed at a significantly lower level, especially in experimental animal group 4 compared to experimental animal group 3. On the other hand, the expression of Bcl-2, an apoptosis prevention protein, was significantly increased in experimental animal group 4 ( Fig. 9b).

The activity of CDK4 and cyclin D1, cell cycle regulating genes, was confirmed at the protein level by the method of Example 11, and the result is shown in FIG. 9th century As shown in FIG. 9c, the expression of CDK4 and cyclin D1 was significantly increased in experimental animal group 2 (P<0.01), and the increased expression of CDK4 and cyclin D1 was significantly reduced in experimental animal groups 3 and 4. In particular, the expression of CDK4 and cyclin D1 was significantly lower in experimental animal group 4 compared to experimental animal group 3 (Fig. 9c, P < 0.01 ~ 0.05).

In the stomach tissue of experimental animal groups 1-4, the mucin content was confirmed by PAS staining, and the result is presented in Fig. 10. As shown in FIG. 10, experimental group 2 animals showed significantly lower levels of gastric mucin (P<0.01), and gastric mucus production was maintained to a significant extent in experimental animal groups 3 and 4, and mucus production was significantly higher in experimental group 4 animals. compared with group 3 (P<0.01).

From the above results it is clear that WIRS compromises the integrity of the mucosa, but this danger is alleviated by pre-treatment with the composition (sample) according to one example.

Results from Examples 8-11 are presented as mean ±SD. Data were analyzed by one-way analysis of variance (ANOVA), and statistical significance between groups was determined using Duncan's multiple rank test. Statistical significance converged to P < 0.05.

From the foregoing description, one skilled in the art to which the present invention relates will understand that the present invention can be embodied in other specific forms without changing its technical concept or essential characteristics. In this regard, it should be understood that the above examples are illustrative in all respects and not limiting. The scope of the present invention is to be construed as the meaning and scope of the appended claims rather than the foregoing detailed description, and all modifications or modified forms derived from equivalent concepts are included within the scope of the present invention.

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<110>CJ CheilJedang Corporation

<120> Composition for prevention, treatment or improvement of

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thereof

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<213> Artificial Sequence

<220>

<223> IL-8_Reverse

<400> 16

cagtgtactt gtggcgtgga

20

<210> 17

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> iNos_Direct

<400> 17

ttttcccagg caaccagacg

20

<210> 18

<211> 19

<212> DNA

<213> Artificial Sequence

<220>

<223> iNos_Reverse

<400> 18

gtagcggggt tcagaatgg

19

<210> 19

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> IFN-gamma_Direct

<400> 19

atccatgagt gctacacgcc

20

<210> 20

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> IFN-gamma_Reverse

<400> 20

tctgtgggtt gttcacctcg

20

<210> 21

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> HIF-1alpha_Direct

<400> 21

tatcactgga cttcggcagc

20

<210> 22

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> HIF-1alpha_Reverse

<400> 22

gctgccgaag tccagtgata

20

<210> 23

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> PDGF_Direct

<400> 23

aggaagccat tcccgcagtt

20

<210> 24

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> PDGF_Reverse

<400> 24

ctaacctcac ctggacctct

20

<210> 25

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> VEGF_Direct

<400> 25

caatgatgaa gccctggagt

20

<210> 26

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> VEGF_Reverse

<400> 26

gatttcttgc gctttcgttt

20

<210> 27

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> GAPDH_Direct

<400> 27

ggtgctgagt atgtcgtgga

20

<210> 28

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> GAPDH_Reverse

<400> 28

ttcagctctg ggatgacctt

20

<210> 29

<211> 26

<212> DNA

<213> Artificial Sequence

<220>

<223> TNF-alpha_Direct

<400> 29

ccctcacact cagatcatct tctcaa

26

<210> 30

<211> 27

<212> DNA

<213> Artificial Sequence

<220>

<223> TNF-alpha_Reverse

<400> 30

tctaaggtac ttgggcaggt tgacctc

27

<---

Claims (22)

1. The use of a feed composition containing (1) an effective amount of a Corynebacterium sp strain, (2) a product of its cultivation selected from a fermented medium obtained by cultivating said Corynebacterium sp strain. in a medium, and said fermented medium from which microbial cells have been removed, and (3) threonine, for the prevention of gastric disease or the relief of gastric disease, wherein the gastric disease is at least one selected from the group consisting of gastritis, gastric ulcer, duodenal ulcer, peptic ulcer and gastric cancer, and where the strain Corynebacterium sp . is at least one selected from the group consisting of Corynebacterium glutamicum , Corynebacterium ammoniagenes and Corynebacterium efficiens . 2. Application according to claim 1, where the strain of Corynebacterium sp . is Corynebacterium glutamicum , Corynebacterium ammoniagenes , or a combination thereof. 3. Use according to claim 1, where the strain of Corynebacterium sp . represents bacteria killed by heat. 4. Use according to claim 1, wherein the culture product is a fermented product, which is a product of enzymatic or metabolic destruction of organic matter, obtained by cultivating said strain of Corynebacterium sp . in the environment. 5. Use according to claim 4, where the medium is a medium for the production of threonine. 6. Application according to claim 1, where threonine is contained in an amount from 60 to 80% by weight. 7. Use according to claim 1, where the strain of Corynebacterium sp . and the product of its cultivation are contained in dried form. 8. Use according to claim 1, where the gastric disease is caused by Helicobacter pylori infection. 9. Application according to any one of paragraphs. 1-8, where threonine: (1) is contained in a strain of Corynebacterium sp ., a product of its cultivation, or both, or (2) added separately, or (3) is contained in and additionally added to a strain of Corynebacterium sp ., a product of its cultivation, or both. 10. Use of a pharmaceutical composition containing (1) a pharmaceutically effective amount of a Corynebacterium sp . strain, (2) a product of its cultivation selected from a fermented medium obtained by cultivating said Corynebacterium sp . strain. in a medium, and said fermented medium from which microbial cells have been removed, and (3) threonine, for the prevention or treatment of gastric disease, wherein the gastric disease is at least one selected from the group consisting of gastritis, gastric ulcer, duodenal ulcer, peptic ulcer and gastric cancer, and where the strain Corynebacterium sp . is at least one selected from the group consisting of Corynebacterium glutamicum , Corynebacterium ammoniagenes and Corynebacterium efficiens . 11. Use of a food composition containing (1) an effective amount of a strain of Corynebacterium sp ., (2) a product of its cultivation selected from a fermented medium obtained by cultivating said strain of Corynebacterium sp . in a medium, and said fermented medium from which microbial cells have been removed, and (3) threonine, for the prevention of gastric disease or the relief of gastric disease, wherein the gastric disease is at least one selected from the group consisting of gastritis, gastric ulcer, duodenal ulcer, peptic ulcer and gastric cancer, and where the strain Corynebacterium sp . is at least one selected from the group consisting of Corynebacterium glutamicum , Corynebacterium ammoniagenes and Corynebacterium efficiens . 12. The use of an antimicrobial composition containing (1) an effective amount of a strain of Corynebacterium sp ., (2) a product of its cultivation selected from a fermented medium obtained by cultivating said strain of Corynebacterium sp . in the medium, and said fermented medium from which microbial cells have been removed, and (3) threonine, to inhibit Helicobacter pylori , and where the strain Corynebacterium sp . is at least one selected from the group consisting of Corynebacterium glutamicum , Corynebacterium ammoniagenes and Corynebacterium efficiens .