Use of novel microbiome for preventing or treating gastric cancer

Abstract

According to the present invention, strains for preventing gastric cancer have been identified and, in particular, it has been identified that when a microbiome was administered together with H. felis, which is known to cause various gastrointestinal diseases by inducing gastric tissue damage and inflammatory responses, gastric tissue damage and inflammatory responses did not occur. Furthermore, it has been identified that the microbiome suppresses gastric tissue damage when administered even in cases already infected with H. felis. Therefore, the microbiome can be effectively used as a preventive or therapeutic agent for normalizing gastric intestinal metaplasia, which is a precancerous lesion, and for suppressing gastric carcinogenesis.

Description

Novel uses of the microbiome for preventing or treating gastric cancer

[0001] The present invention relates to innovative new drugs and personalized healthcare, and more particularly to the use of a novel microbiome for preventing or treating gastric cancer.

[0002]

[0003] In April 2023, the suppository 'rbx2660' (FDA approved on November 30, 2022) and the oral medication 'SER-109' were the first microbial therapeutics commercialized for the treatment of recurrent C. difficile enteritis (FDA approved on April 26, 2023). This marks the first microbiome-based drug treatment and presents limitless potential for microbial therapeutics utilizing the microbiome. However, the currently developed microbiome therapeutics are limited by their unclear mechanism of action, as they are a mixture of dozens of microorganisms. Meanwhile, there are 105 domestic microbiome LBP programs, rapidly growing through preclinical, phase 1, and 2 clinical trials, primarily targeting inflammatory bowel disease (IBD), asthma, psoriasis, and metabolic diseases.

[0004] Epidemiologically, gastric metaplasia is a typical precursor lesion of gastric cancer, and extensive research is being conducted as a target for treatment development and gastric cancer prevention. While anti-Helicobacter pylori eradication therapy can halt intestinal metaplasia, studies demonstrating its normalization (regression) are lacking. In this regard, dysbiosis has been identified as a key driver of gastrointestinal diseases, including cancer. Therefore, there is an urgent need to develop standardized and purified microbiome therapeutics, either in the form of a single species or a microbial community with a proven mechanism of action.

[0005] Meanwhile, Grand View Research predicted that the microbiome therapeutics market will grow from the current $114 million (approximately KRW 150 billion) to over $1.06 billion (KRW 1.43 trillion) by 2030, and the Ministry of Food and Drug Safety and international regulatory agencies are developing and revising guidelines for the development of clear microbiome therapeutics.

[0006] One object of the present invention is to provide a microbiome containing multiple strains and a pharmaceutical composition for preventing or treating gastric diseases or Helicobacter pylori infection, which comprises the microbiome as an active ingredient.

[0007] Another object of the present invention is to provide a microbiome containing multiple strains and a method for preventing or treating gastric disease or Helicobacter pylori infection comprising the microbiome as an active ingredient.

[0008] Another object of the present invention is to provide a microbiome containing multiple strains and a food composition for preventing or treating gastric diseases or Helicobacter pylori infection, which comprises the microbiome as an active ingredient.

[0009]

[0010] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below.

[0011]

[0012] Various embodiments of the present invention are described with reference to the drawings. In the following description, various specific details, such as specific configurations, compositions, and processes, are set forth to provide a thorough understanding of the present invention. However, certain embodiments may be practiced without one or more of these specific details, or in conjunction with other known methods and configurations. In other instances, well-known processes and manufacturing techniques are not described in specific detail so as not to unnecessarily obscure the present invention. Reference throughout this specification to an embodiment means that a particular feature, configuration, composition, or characteristic described in connection with the embodiment is included in one or more embodiments of the present invention. Thus, the appearance of an embodiment in various places throughout this specification does not necessarily indicate the same embodiment of the present invention. Additionally, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more embodiments.

[0013]

[0014] In one embodiment of the present invention, a microbiome is provided comprising at least one strain selected from the group consisting of Akkermansia muciniphila, Bifidobacterium animalis subsp. Lactis, Clostridium celatum, Romboutsia ilealis, and Blautia faecis.

[0015] The above strain may be isolated. As used herein, the term "isolated" means something that is artificially isolated and available rather than existing in nature.

[0016] In this specification, the term "culture medium" may be used interchangeably with "culture supernatant", "conditioned medium" or "conditioned medium", and may mean the entire medium including the strain, its metabolites, extra nutrients, etc., obtained by culturing the strain for a certain period of time in a medium capable of supplying nutrients so that the strain can grow and survive in vitro, selected from the group consisting of Akkermansia muciniphila, Bifidobacterium animalis subsp. Lactis, Clostridium celatum, Romboutsia ilealis and Blautia faecis. In addition, the culture medium may mean a culture medium obtained by removing the bacterial cells from the bacterial culture obtained by culturing the strain. Meanwhile, the liquid from the culture medium from which the bacterial cells have been removed is also called a "supernatant", and may be obtained by allowing the culture medium to stand still for a certain period of time and taking only the liquid from the upper layer excluding the portion that has settled to the lower layer, removing the bacterial cells through filtration, or centrifuging the culture medium to remove the sediment at the lower layer and taking only the liquid at the upper layer. The above "bacterial cells" refers to the strain of the present invention itself, and includes the strain itself selected by separating from a sample or the like, or the strain isolated from the culture medium by culturing the strain. The bacterial cells can be obtained by centrifuging the culture medium and taking the portion that has settled to the lower layer, or by allowing the bacterial cells to settle to the lower layer of the culture medium by gravity and then removing the upper liquid after leaving them still for a certain period of time.

[0017] In the present invention, the above “Akkermansia muciniphila” or “Akkermansia Muciniphila” is a type of microorganism that lives by feeding on intestinal mucin (a commensal bacterium that continuously resides in the human intestine from childhood to the elderly), and is known to continuously improve the intestinal environment and maintain intestinal health. Recently, as research results have been published that Akkermansia Muciniphila affects various metabolic functions such as obesity, diabetes, inflammation, immunity, and aging, various efforts and attempts have been made to utilize Akkermansia Muciniphila industrially. However, Akkermansia Muciniphila is difficult to mass-produce due to strict cultivation conditions, so it is true that commercialization of the strain itself is difficult at the current level.

[0018] In the present invention, “Bifidobacterium animalis subsp. lactis” or “Bifidobacterium animalis subsp. Lactis” is a type of probiotic (“good” bacteria) that lives in the intestines and produces lactic acid and acetic acid. “Good” bacteria such as B. lactis can help break down food, absorb nutrients, and fight “bad” organisms that can cause disease. B. lactis is a subspecies of B. animalis that is found in the human intestines and is used in probiotic supplements. B. lactis is used to treat or prevent colic, constipation, irritable bowel syndrome (IBS), and respiratory infections.

[0019] In the present invention, the “Clostridium celatum” is a gram-positive anaerobic bacterium of the genus Clostridium isolated from human feces.

[0020] In the present invention, the “Romboutsia ilealis” is a gram-positive bacterium.

[0021] In the present invention, “Blautia faecis” is a gram-positive bacterium.

[0022] In the present invention, the "microbiome" refers to the "sum of all microorganisms existing in a specific environment." Diversity includes alpha diversity, beta diversity, and gamma diversity. Alpha diversity determines the distribution of various microorganisms present in a single sample. Beta diversity indicates whether the diversity of samples is similar. Gamma diversity refers to the total diversity of all communities within a geographic region.

[0023] The microbiome provided in the present invention includes at least one strain selected from the group consisting of Akkermansia muciniphila, Bifidobacterium animalis subsp. Lactis, Clostridium celatum, Romboutsia ilealis, and Blautia faecis, and in other embodiments, it may be a microbiome including three or more, four or more, or all five of the above strains.

[0024] In another embodiment of the present invention, the Akkermansia muciniphila may be deposited under the accession number ATCC BAA-835, the Bifidobacterium animalis subsp. Lactis may be deposited under the accession number DSM 10140, the Clostridium celatum may be deposited under the accession number ATCC 27791, the Romboutsia ilealis may be deposited under the accession number DSM 25109, or the Blautia faecis may be deposited under the accession number DSM 27629, but is not limited thereto.

[0025] In another embodiment of the present invention, the concentration of strains in the microbiome is 1 x 10 4 1 x 10 10cells / mL, providing a microbiome. In another embodiment, 1 x 10 5 1 x 10 9 cells / mL, 5 x 10 5 5 x 10 9 cells / mL, 1 x 10 5 1 x 10 9 cells / mL, 5 x 10 6 5 x 10 8 cells / mL or 1 x 10 6 1 x 10 8 It may be, but is not limited to, cells / mL.

[0026] In the present invention, the strains were cultured under various culture conditions. However, the culture is not limited to the culture conditions specified in the examples, and any method capable of culturing the five strains described above using a method widely known in the relevant technical field is not particularly limited. In addition, in the microbiome, Clostridium celatum and Romboutsia ilealis were cultured in a liquid medium, and the remaining strains were cultured in a solid medium. However, any method capable of culturing the strains described above is not particularly limited.

[0027]

[0028] In one embodiment of the present invention, a pharmaceutical composition for preventing or treating gastrointestinal diseases comprising the microbiome as an active ingredient.

[0029] In another embodiment of the present invention, a pharmaceutical composition is provided, wherein the gastrointestinal disease is at least one selected from the group consisting of intestinal metaplasia, gastritis, atrophic gastritis, gastric spasm, gastric ulcer, and gastric cancer.

[0030] In the present invention, the stomach is a key digestive organ located just below the esophagus. When a person swallows food, it passes through the esophagus and reaches the stomach. It is the most elastic organ in the human body, expanding to hold approximately 1.5 liters of food in an adult human. Furthermore, the stomach primarily digests proteins. The gastric glands in the stomach wall secrete hydrochloric acid, a highly acidic substance with a pH of approximately 2, and pepsinogen, an inactive form of the digestive enzyme pepsin, which breaks down proteins into peptones. Furthermore, the strong acidity of the stomach also prevents ingested food from decaying within the body. Because this organ provides an extremely hostile environment for bacteria and viruses, most bacteria and viruses that may be mixed in food are killed here. Because the strong acidity of the stomach denatures proteins, the gastric glands secrete a mucus called mucin to protect the stomach wall from being dissolved by the stomach acid itself.

[0031] Therefore, if a microbiome protects tissues and alleviates inflammation even in an environment that denatures proteins, such as a strongly acidic environment of about pH 2, it will be obvious to those skilled in the art that it can also protect tissues and alleviate inflammation in other tissues.

[0032] In the present invention, "tissue" refers to a mass of cells of the same type within an organ in biology. Tissues are an important unit for studying physiology. Because cells are so small, molecular biology studies typically study humans and higher animals as tissue units. These tissues include animal tissues, such as connective tissue, muscle tissue, nervous tissue, and epithelial tissue, as well as plant tissues and permanent tissues.

[0033] Gastric cancer is classified as a cancer with a poor prognosis, second only to lung cancer, in Korea. The molecular pathophysiology of gastric cancer remains unknown. It is understood to be influenced by four major risk factors: genetic factors, Helicobacter pylori infection, environmental factors, and the microbiome. Chronic gastric mucosal infection leads to atrophic gastritis and intestinal metaplasia over time, which are important precancerous lesions. Currently, H. pylori eradication therapy is considered the most important treatment for gastric cancer prevention. However, the factors that can normalize the precancerous lesions, atrophic gastritis and intestinal metaplasia, remain unknown. Therefore, inducing normalization of intestinal metaplasia is the most effective treatment and prevention method for gastric cancer.

[0034] In another embodiment of the present invention, a pharmaceutical composition is provided, wherein the gastric disease is caused by cell hyperplasia, metaplasia, or dysplasia.

[0035] A composition according to one aspect of the present invention may be for preventing or treating a gastrointestinal disease.

[0036] The “treatment” of the present invention means any act of delaying, stopping or reversing the progression of a disease or condition, and for the purpose of the present invention, the composition means a composition that prevents or treats a gastrointestinal disease.

[0037] The pharmaceutical composition of the present invention may be characterized as being in the form of a capsule, tablet, granule, injection, ointment, powder or beverage, and the pharmaceutical composition may be characterized as being intended for humans.

[0038] The pharmaceutical composition of the present invention is not limited to these, but can be formulated and used in the form of oral formulations such as powders, granules, capsules, tablets, aqueous suspensions, external preparations, suppositories, and sterile injection solutions, each according to a conventional method.

[0039] The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may include a binder, a lubricant, a disintegrant, an excipient, a solubilizer, a dispersant, a stabilizer, a suspending agent, a coloring agent, a fragrance, etc. for oral administration. In the case of injections, a buffer, a preservative, an analgesic, a solubilizer, an isotonic agent, a stabilizer, etc. may be mixed and used. In the case of topical administration, a base, an excipient, a lubricant, a preservative, etc. may be used.

[0040] The pharmaceutical composition of the present invention can be prepared in various forms by mixing it with a pharmaceutically acceptable carrier as described above. For example, for oral administration, it can be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and for injections, it can be prepared in the form of unit dose ampoules or multiple doses. In addition, it can be formulated as a solution, suspension, tablet, capsule, sustained-release preparation, etc.

[0041] The composition of the present invention may be prepared in any one formulation selected from the group consisting of a suspension, syrup, emulsion, liposome, powder, granule, tablet, sustained-release formulation, eye drop, capsule, contact lens cleaner, and contact lens lubricant, but is not limited thereto.

[0042] Examples of carriers, excipients, and diluents suitable for the formulation of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, or mineral oil. In addition, fillers, anticoagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives, and the like may be additionally included.

[0043] The routes of administration of the pharmaceutical composition of the present invention include, but are not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal. Oral or parenteral administration is preferred. In the present invention, the "parenteral" includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, sublingual, rectal, intralesional, ocular (e.g., eye drops, etc.), and intracranial injection or infusion techniques.

[0044] The pharmaceutical composition of the present invention may vary depending on various factors including the activity of the specific compound used, age, body weight, general health, sex, dosage, administration time, administration route, excretion rate, drug combination, and severity of the specific disease to be prevented or treated, and the dosage of the pharmaceutical composition may vary depending on the patient's condition, body weight, degree of disease, form of drug, administration route, and period, but may be appropriately selected by those skilled in the art, and may be administered at 0.0001 to 50 mg / kg or 0.001 to 50 mg / kg per day. The administration may be administered once a day or divided into several times. The dosage does not limit the scope of the present invention in any way.

[0045]

[0046] In another embodiment of the present invention, a pharmaceutical composition for preventing or treating Helicobacter pylori infection is provided, comprising the microbiome as an active ingredient.

[0047] Meanwhile, Helicobacter pylori (H. pylori) colonization in the gastric mucosa causes acute and chronic inflammation, and bacterial virulence factors and host genetic factors determine the severity of inflammation and pathological sequelae. H+, K+-adenosine triphosphatase (H+, K+-ATPase) of parietal cells is the final step in gastric acid secretion and is affected by H. pylori. In the acute infection stage, H. pylori suppresses the activity of the promoter of the alpha subunit of H+, K+-ATPase, and several cytokines such as interleukin-1β (IL-1β; encoded by IL-1B) and tumor necrosis factor-α released during this process also suppress acid secretion. When H. pylori infection progresses to a chronic state, atrophy in the gastric corpus accelerates, gastric acid secretion decreases, and loss of parietal cells occurs.

[0048] Helicobacter pylori (H. pylori) primarily infects the gastric mucosa and is the main cause of gastritis, gastric ulcers, duodenal ulcers, gastric cancer, and gastric lymphoma. It is known that approximately 80% of Koreans are infected with Helicobacter pylori.

[0049] To treat Helicobacter pylori infection, a combination of two or more antibiotics and a potent acid suppressant is typically administered for one to two weeks. However, antibiotics often fail to reach the site of Helicobacter pylori infection, and repeated exposure to the same antibiotic class can easily lead to drug resistance.

[0050] In addition, representative diseases caused by Helicobacter pylori infection include gastritis (atrophic gastritis, chronic gastritis, chronic atrophic gastritis, superficial gastritis), and intestinal metaplasia including metaplasia is known as a representative disease lesion caused by Helicobacter pylori infection.

[0051] The term “prevention” as used herein means any action that inhibits or delays the proliferation of Helicobacter pylori by administering a composition according to an embodiment of the present invention.

[0052] The terms “treatment” and “improvement” as used herein mean any action that reduces or eradicates Helicobacter pylori and improves symptoms caused by Helicobacter pylori infection by administering a composition according to an embodiment of the present invention.

[0053]

[0054] In one embodiment of the present invention, a food composition for preventing or improving gastrointestinal diseases is provided, which comprises a microbiome as an active ingredient.

[0055] The term "health functional food" or "food composition" of the present invention encompasses both "functional food" and "health food." The term "functional food" is the same as "food for special health use (FoSHU)," and refers to a food with high medical or therapeutic effects that is processed to efficiently exhibit a bioregulatory function in addition to providing nutrition. In addition, the term "health food" refers to a food that has a more active health maintenance or promotion effect than general foods, and the term "health supplement food" refers to a food for the purpose of health supplementation. In some cases, the terms "functional food," "health food," and "health supplement food" may be used interchangeably.

[0056] The health functional food of the present invention can be manufactured in various forms such as tablets, capsules, powders, granules, liquids, and pills to obtain useful effects in improving and recovering dry eye syndrome.

[0057] The health functional food of the present invention can be manufactured in the form of a nutritional supplement, food additive, feed, etc., and can be consumed by humans or animals including livestock.

[0058] The health functional food of the present invention can be manufactured in various forms according to conventional methods known in the art. General foods include, but are not limited to, beverages (including alcoholic beverages), fruits and processed foods thereof (e.g., canned fruits, bottled fruits, jams, marmalades, etc.), fish, meats and processed foods thereof (e.g., ham, sausages, corned beef, etc.), breads and noodles (e.g., udon, buckwheat noodles, ramen, spaghetti, macaroni, etc.), fruit juices, various drinks, cookies, taffy, dairy products (e.g., butter, cheese, etc.), edible plant oils, margarine, vegetable proteins, retort foods, frozen foods, various seasonings (e.g., soybean paste, soy sauce, sauces, etc.), etc., which can be manufactured by adding anthocyanin oligomers.

[0059] When the health functional food of the present invention is manufactured as a nutritional supplement, anthocyanin oligomers may be added to the formulation of capsules, tablets, pills, etc., but are not limited thereto.

[0060] The health functional food of the present invention can be manufactured in the form of tea, juice, and drink, and can be manufactured by liquefying, granulating, encapsulating, and powdering so that it can be consumed. Here, when the health functional food is manufactured in the form of a drink, it can contain various flavoring agents or natural carbohydrates as additional ingredients like conventional beverages. The natural carbohydrates mentioned above can be monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; polysaccharides such as dextrin and cyclodextrin; and sugar alcohols such as xylitol, sorbitol, and erythritol. The sweetener can be a natural sweetener such as thaumatin and stevia extract; or a synthetic sweetener such as saccharin and aspartame. The proportion of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g, per 100 mL of the composition of the present invention.

[0061] The health functional food of the present invention may contain various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid, salts of pectic acid, alginic acid, salts of alginic acid, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, or carbonating agents. In addition, when manufactured in the form of a drink, it may contain fruit pulp for manufacturing fruit juice, fruit juice drinks, or vegetable drinks. The components of these additives may be used independently or in mixtures, and the ratio is not particularly important, but is generally selected from the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the active ingredient of the food composition of the present invention.

[0062] The “improvement” of the present invention means any act of improving or beneficially changing a disease or symptom state, and for the purpose of the present invention, the composition means any act of improving a gastrointestinal disease.

[0063] In one embodiment of the present invention, a microbial community is provided comprising at least one strain selected from the group consisting of Akkermansia muciniphila, Bifidobacterium animalis subsp. Lactis, Clostridium celatum, Romboutsia ilealis, and Blautia faecis.

[0064] In another embodiment of the present invention, the concentration of strains in the microbial community is 1 x 10 6 1 x 10 10 It provides a microbial community of cells / mL.

[0065] In one embodiment of the present invention, a pharmaceutical composition for preventing or treating a gastrointestinal disease is provided, comprising the microbial community as an active ingredient.

[0066] In another embodiment of the present invention, a pharmaceutical composition is provided, wherein the gastrointestinal disease is at least one selected from the group consisting of intestinal metpalsia, gastritis, atrophic gastritis, gastric spasm, gastric ulcer, and gastric cancer.

[0067] In another embodiment of the present invention, a pharmaceutical composition is provided, wherein the gastric disease is caused by cell hyperplasia, metaplasia, or dysplasia.

[0068] In one embodiment of the present invention, a pharmaceutical composition for preventing or treating Helicobacter pylori infection is provided, comprising the microbial community as an active ingredient.

[0069] In another embodiment of the present invention, a composition for protecting or preventing tissue damage is provided, which comprises the microbial community as an active ingredient.

[0070] In another embodiment of the present invention, a composition is provided wherein the tissue is at least one selected from the group consisting of connective tissue, muscle tissue, nerve tissue, and epithelial tissue.

[0071] In one embodiment of the present invention, a food composition for preventing or improving gastrointestinal diseases is provided, which comprises the microbial community as an effective ingredient.

[0072] In another embodiment of the present invention, a food composition is provided, wherein the gastrointestinal disease is at least one selected from the group consisting of intestinal metpalsia, gastritis, atrophic gastritis, gastric spasm, gastric ulcer, and gastric cancer.

[0073] In another embodiment of the present invention, a food composition is provided, wherein the gastrointestinal disease is caused by cell hyperplasia, metaplasia, or dysplasia.

[0074] In one embodiment of the present invention, a food composition for preventing or improving Helicobacter pylori infection is provided, which comprises the microbial community as an active ingredient.

[0075] In one embodiment of the present invention, a food composition for protecting or preventing tissue damage is provided, which comprises the microbial community as an active ingredient.

[0076] In another embodiment of the present invention, a food composition is provided, wherein the tissue is at least one selected from the group consisting of connective tissue, muscle tissue, nerve tissue, and epithelial tissue.

[0077]

[0078] The present invention identified gastric cancer-preventing strains and, in particular, confirmed that co-administration of the microbiome with H. felis, known to cause gastric tissue damage and inflammation, leading to various gastric diseases, prevented gastric tissue damage and inflammation. Furthermore, it was confirmed that administration of the microbiome inhibited gastric tissue damage even in cases already infected with H. felis.

[0079] Accordingly, the above microbiome can be usefully used as a preventive or therapeutic agent for normalizing gastric intestinal metaplasia, a precancerous lesion, and inhibiting gastric carcinogenesis.

[0080]

[0081] Figure 1 is a mouse experiment method of the microbiome provided in the present invention.

[0082] Figure 2a shows the gastric tissue and inflammatory response of the negative control group, as confirmed by H&E staining. No gastric tissue or inflammatory response was observed in the negative control group.

[0083] Figure 2b shows the H&E staining results of gastric tissue from mice inoculated with H. felis as a positive control. Gastric tissue damage and inflammatory response were observed in all mice inoculated with H. felis.

[0084] Figure 2c shows the results of H&E staining of gastric tissue from mice inoculated with the microbiome provided by the present invention. No gastric tissue damage or inflammatory response was observed in mice administered the microbiome provided by the present invention. Therefore, the safety of the microbiome provided by the present invention has been confirmed.

[0085] Figure 2d shows the results of H&E staining of gastric tissue from mice co-inoculated with H. felis and the microbiome provided by the present invention. In this case, gastric tissue damage and inflammatory responses were significantly reduced compared to the positive control group administered only with Helicobacter felis.

[0086] Figure 3a shows the H&E staining results of gastric tissues from mice administered with a negative control group, a positive control group (administered with H. felis alone), and a microbiome and H. felis together.

[0087] Figure 3b shows the immunochemical staining results of mice administered with H. felis alone, a negative control group, and a positive control group. Through Figure 3b, the loss of parietal cells in the gastric tissue (H + -K + It can be confirmed that the microbiome provided in the present invention prevents ATPase.

[0088] Figure 3c shows the immunochemical staining results of mice administered with the negative control group, the positive control group (H. felis alone), and the microbiome and H. felis together. As shown in Figure 3c, the expression level of the metaplasia marker (CD44v9) in mice administered with the microbiome and H. felis together was similar to that in the negative control group, confirming that the microbiome provided by the present invention prevented metaplasia.

[0089] Figure 3d shows the immunochemical staining results of mice administered with the negative control group, the positive control group (H. felis alone), and the microbiome and H. felis together. Figure 3d shows that the level of macrophages (F4 / 80), an indicator of gastric inflammation, in mice administered with the microbiome and H. felis together was similar to that in the negative control group, confirming that the microbiome provided by the present invention prevented gastric inflammation.

[0090] Figure 3e shows the immunofluorescence staining results of mice administered with the negative control group, the positive control group (administered with H. felis alone), and the microbiome and H. felis together.

[0091] Figure 4 shows the immunochemical staining results of mice administered with a negative control group, a positive control group (administered with H. felis alone), and a microbiome and H. felis together. When infected with the H. felis strain, the detection of H. felis decreases as the parietal cells of the gastric tissue are lost. However, when the microbiome provided by the present invention was administered together, it was confirmed that the loss of parietal cells of the gastric tissue was suppressed and H. felis was detected. This suggests that even in cases of H. felis infection, the microbiome provided by the present invention suppresses the damage to the gastric tissue caused by H. felis.

[0092]

[0093] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples, in accordance with the gist of the present invention.

[0094]

[0095] Example

[0096]

[0097] [Example 1] Production of a microbiome cocktail

[0098] To prepare a microbiome cocktail, a microbiome cocktail was prepared in an anaerobic chamber. First, strains were identified by 16S sequence in solid or liquid media. Among the strains, A. muciniphila, B. animalis subsp. Lactis, and B. faecis were harvested from solid media, resuspended in phosphate-buffered saline (PBS), and washed once with PBS. The cell number was then measured using a QUANTOM Tx Microbial cell counter (Logos biosystems). Similarly, among the strains, C. celatum and R. ilealis were harvested from liquid media by centrifugation, resuspended in phosphate-buffered saline (PBS), and washed once with PBS. Afterwards, the cell count was measured using QUANTOM Tx Microbial cell counter (Logos biosystems) in PBS. The final microbiome cocktail contained 5 x 10 each of the 5 strains. 8 A PBS solution containing 20% ​​glycerol was prepared to contain 10 cells / ㎖. The combination and culture conditions of the specific microbiome cocktail are shown in Table 1 below.

[0099] Microbiome strain combination and culture conditions Type of strains to be inoculated Culture medium Culture condition Concentration Injection method Injection medium Akkermansia muciniphila ATCC BAA‐835 BHI Agar (Brain heart infusion agar) Anaerobic 48-72 h 5 x 10 8 cells / mLTotal 2.5 x 10 9 cells / mL 200 μL Oral injection Mixed, 20% glycerol in PBS solution Bifidobacterium animalis subsp. Lactis DSM 10 140 BHI Agar (Brain heart infusion agar) Anaerobic 48-72 h 5 x 10 8 cells / mLClostridium celatumATCC 27791Chopped meat broth with carbohydratesAnaerobic48-72h5 x 10 8 cells / mLRomboutsia ilealisDSM 25109CMC mediumAnaerobic48-72h5 x 10 8 cells / mLBlautia faecisDSM 27629Trypticase soy agar 5% horse bloodAnaerobic48-72h5 x 10 8 cells / mL

[0100] [Example 2] Administration of microbiome cocktail to mice

[0101] The microbiome cocktail prepared in Example 1 was administered to mice after being stored in a deep freezer. Specifically, as shown in Fig. 1, a total of 15 8-week-old germ-free mice were prepared. Two 8-week-old germ-free mice were used as a negative control group, and mice that were administered H. felis three times in total on D+0, D+2, and D+4 were prepared as a positive control group. In addition, mice that were administered the microbiome cocktail prepared in Example 1 three times in total on D+0, D+2, and D+4 were prepared as an experimental group, and mice that were administered the microbiome cocktail prepared in Example 1 and H. felis three times in total on D+0, D+2, and D+4 were prepared as an experimental group. Eight weeks after the first administration, when all mice were 16 weeks old, gastric tissue damage and inflammatory responses were observed.

[0102]

[0103] [Example 3] Confirmation of gastric tissue damage and inflammatory response in the negative control group and positive control group (H. felis)

[0104] Before confirming the effect of the microbiome cocktail prepared in Example 1 above on protecting the gastric tissue damage and inflammatory response in mice, the gastric tissue damage and inflammatory response of the negative control group and the positive control group (H. felis) were confirmed.

[0105] Specifically, H&E staining was used to compare gastric tissue damage and inflammatory responses between the negative control group and the positive control group (H. felis). As shown in Fig. 2(a), no gastric tissue damage or inflammatory response was observed in the negative control group. However, as shown in Fig. 2(b), gastric tissue damage and inflammatory responses were observed in all positive control (H. felis) mice.

[0106] Therefore, additional experiments were conducted to determine whether the microbiome provided by the present invention can prevent or treat gastric tissue damage and inflammatory responses caused by H. felis.

[0107]

[0108] [Example 4] Safety verification of the microbiome cocktail

[0109] Before confirming the gastric tissue protective effect of the microbiome cocktail, to confirm safety, gastric tissue damage and inflammatory responses of mice administered only the microbiome cocktail prepared in Example 1 were observed.

[0110] Specifically, H&E staining was used to compare gastric tissue damage and inflammatory responses between the negative control group and mice administered the microbiome cocktail. As shown in Fig. 2(a), no gastric tissue damage or inflammatory response was observed in the negative control group. Similarly, as shown in Fig. 2(c), no gastric tissue damage or inflammatory response was observed in any mice administered the microbiome cocktail prepared in Example 1 alone. This confirmed the safety of the microbiome cocktail prepared in Example 1.

[0111]

[0112] [Example 5] Confirmation of the ability of a microbiome cocktail to suppress gastric tissue damage.

[0113] Gastric tissue damage and inflammatory responses were observed in mice administered with the microbiome cocktail prepared in Example 1 and H. felis, a positive control that induces gastric tissue damage and inflammatory responses.

[0114] Specifically, through H&E staining, the gastric tissue damage and inflammatory response of mice co-administered with the microbiome cocktail prepared in Example 1 and H. felis, a positive control group that induces gastric tissue damage and inflammatory response, were compared. As shown in Fig. 2(d), it was confirmed that the gastric tissue damage and inflammatory response of mice simultaneously treated with Helicobacter felis and the microbiome prepared in Example 1 were significantly reduced compared to the group administered only Helicobacter felis, the positive control group.

[0115]

[0116] [Example 6] Confirmation of the effect of a microbiome cocktail on preventing the loss of parietal cells in gastric tissue caused by infection.

[0117] Through immunohistochemical staining, the degree of parietal cell loss in the gastric tissue of mice administered with the negative control, positive control, and the microbiome cocktail prepared in Example 1 together with H. felis, a positive control that induces gastric tissue damage and inflammatory response, was compared.

[0118] As shown in Fig. 4, in the case of mice infected with the H. felis strain, the detection of H. felis decreases as the parietal cells of the gastric tissue are lost due to the infection. However, when the microbiome prepared in Example 1 was administered together, it was confirmed that the loss of parietal cells of the gastric tissue was suppressed and the detection of H. felis appeared. This shows that even in cases where infection with H. felis is already present, the microbiome strain prepared in Example 1 suppresses the damage to the gastric tissue caused by H. felis.

[0119]

[0120] [Comparative Example] Comparison with the negative control group, positive control group (H. felis alone), and experimental group (microbiome + H. felis)

[0121] We aimed to compare the gastric tissue damage and inflammatory responses of mice administered the negative control group, positive control group (H. felis alone), and experimental group (microbiome + H. felis).

[0122] Specifically, the gastric tissue damage and inflammatory response of mice administered with the microbiome cocktail prepared in Example 1 and H. felis, a positive control group that induces gastric tissue damage and inflammatory response, were compared through H&E staining, immunochemical staining, and immunofluorescence staining. As shown in Fig. 3(a) to Fig. 3(e), in the case of the positive control group mice administered with Helicobacter felis alone, the loss of parietal cells in the gastric tissue (H+-K+ ATPase), increased expression of metaplasia indicators (CD44v9), increased macrophages (F4 / 80), an inflammatory response indicator, and increased cell proliferation were observed, whereas in the case of the microbiome cocktail and H. felis, a positive control group that induces gastric tissue damage and inflammatory response, the loss of parietal cells in the gastric tissue (H+-K+ ATPase), increased expression of metaplasia indicators (CD44v9), increased macrophages (F4 / 80), and increased cell proliferation were observed. In mice co-administered with felis, no loss of parietal cells (H+-K+ ATPase), increased expression of metaplasia markers (CD44v9), increased macrophages (F4 / 80), an indicator of inflammatory response, or increased cell proliferation were observed.

[0123]

[0124] [conclusion]

[0125] In this way, since no gastric tissue damage or inflammatory response was observed in mice administered the microbiome, it was confirmed that the microbiome is safe. In addition, since no gastric tissue damage or inflammatory response was observed in mice administered the microbiome together with H. felis, which causes gastric tissue damage and inflammatory response, it was confirmed that the microbiome has a preventive and therapeutic effect on gastric diseases.

[0126]

[0127] While specific aspects of the present invention have been described in detail above, it should be apparent to those skilled in the art that these specific descriptions are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

[0128]

Claims

1. A microbial community comprising one or more strains selected from the group consisting of Akkermansia muciniphila, Bifidobacterium animalis subsp. Lactis, Clostridium celatum, Romboutsia ilealis, and Blautia faecis.

2. In paragraph 1, The concentration of strains in the above microbial community is 1 x 10 6 1 x 10 10 cells / mL, microbial community.

3. A pharmaceutical composition for preventing or treating gastrointestinal diseases, comprising a microbial community of any one of claims 1 to 2 as an active ingredient.

4. In paragraph 3, A pharmaceutical composition, wherein the above gastrointestinal disease is at least one selected from the group consisting of intestinal metaplasia, gastritis, gastric spasm, gastric ulcer, and gastric cancer.

5. In paragraph 4, A pharmaceutical composition wherein the above gastric disease is caused by hyperplasia, metaplasia, or dysplasia.

6. In paragraph 4, A pharmaceutical composition wherein the above gastritis is at least one selected from the group consisting of atrophic gastritis, chronic gastritis, chronic atrophic gastritis, and superficial gastritis.

7. In paragraph 4, A pharmaceutical composition wherein the above intestinal metaplasia is metaplasia.

8. A pharmaceutical composition for preventing or treating Helicobacter pylori infection, comprising a microbial community of any one of claims 1 to 2 as an active ingredient.

9. In paragraph 8, A pharmaceutical composition, wherein the above Helicobacter pylori infection is at least one selected from the group consisting of intestinal metaplasia, gastritis, gastric spasm, gastric ulcer, and gastric cancer.

10. In paragraph 9, A pharmaceutical composition wherein the above gastritis is at least one selected from the group consisting of atrophic gastritis, chronic gastritis, chronic atrophic gastritis, and superficial gastritis.

11. In paragraph 9, A pharmaceutical composition wherein the above intestinal metaplasia is metaplasia.

12. A composition for protecting or preventing tissue damage, comprising the microbial community of any one of claims 1 to 2 as an active ingredient.

13. In paragraph 12. A composition wherein the above tissue is at least one selected from the group consisting of connective tissue, muscle tissue, nervous tissue, and epithelial tissue.

14. A food composition for preventing or improving gastrointestinal diseases, comprising a microbial community according to any one of claims 1 to 2 as an effective ingredient.

15. In paragraph 14, A food composition, wherein the above gastrointestinal disease is at least one selected from the group consisting of intestinal metaplasia, gastritis, gastric spasm, gastric ulcer, and gastric cancer.

16. In paragraph 15, A food composition wherein the above gastritis is at least one selected from the group consisting of atrophic gastritis, chronic gastritis, chronic atrophic gastritis, and superficial gastritis.

17. In paragraph 5, The above intestinal metaplasia is a food composition of metaplasia.

18. A food composition for preventing or improving Helicobacter pylori infection, comprising a microbial community of any one of claims 1 to 2 as an active ingredient.

19. In paragraph 18, A food composition wherein the above Helicobacter pylori infection is at least one selected from the group consisting of intestinal metaplasia, gastritis, gastric spasm, gastric ulcer, and gastric cancer.

20. In paragraph 19, A food composition wherein the above gastritis is at least one selected from the group consisting of atrophic gastritis, chronic gastritis, chronic atrophic gastritis, and superficial gastritis.

21. In paragraph 19, The above intestinal metaplasia is a food composition of metaplasia.

22. A food composition for protecting or preventing tissue damage, comprising a microbial community according to any one of claims 1 to 2 as an active ingredient.

23. In paragraph 22. A food composition wherein the above tissue is at least one selected from the group consisting of connective tissue, muscle tissue, nervous tissue, and epithelial tissue.

24. A method for preventing or treating gastrointestinal diseases, comprising a microbial community of any one of paragraphs 1 and 2 as an effective ingredient.

25. A method for preventing or treating Helicobacter pylori infection, comprising a microbial community of any one of claims 1 to 2 as an active ingredient.

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