阅读说明：本技术 用于预防或治疗幽门螺杆菌相关疾病的泡菜 (Kimchi for preventing or treating helicobacter pylori-associated diseases ) 是由 李东润 崔承惠 吴智暎 于 2019-12-27 设计创作，主要内容包括：本申请涉及能够有效地用于预防和治疗幽门螺杆菌相关疾病的含有乳酸菌的泡菜。(The present application relates to kimchi containing lactic acid bacteria that can be effectively used for the prevention and treatment of helicobacter pylori-associated diseases.)

1. A kimchi is prepared by salting vegetables, mixing with flavoring agent, and fermenting with lactobacillus,

the lactic acid bacteria include Leuconostoc mesenteroides CJLM119 (accession No. KCTC 11403BP) and Lactobacillus plantarum CJLP133 (accession No. KCTC 13043 BP).

2. The kimchi of claim 1, wherein the vegetables are selected from the group consisting of cabbage, radish, shallot, mustard leaf, cucumber, and combinations thereof.

3. The kimchi of claim 2, wherein the vegetable is Chinese cabbage.

4. The kimchi of claim 3, wherein the Chinese cabbage is a high lycopene content Chinese cabbage.

5. The kimchi according to claim 4, wherein the content of lycopene is 0.05 to 5mg per 100g of the kimchi.

6. The kimchi of claim 1, wherein the content of leuconostoc mesenteroides CJLM119 is 0.01 to 5 wt% based on the entire weight of the kimchi, and the content of lactobacillus plantarum CJLP133 is 0.01 to 5 wt% based on the entire weight of the kimchi.

7. A composition for ameliorating Helicobacter pylori (Helicobacter pylori) -associated diseases, the composition comprising the kimchi or the extract thereof of any one of claims 1 to 6.

8. The composition as set forth in claim 7, wherein the kimchi extract is an extract obtained by extracting the kimchi with water, an organic solvent or a mixed solvent thereof.

9. The composition of claim 8, wherein the organic solvent is selected from the group consisting of alcohol, hexane, ether, glycerin, propylene glycol, butylene glycol, ethyl acetate, methyl acetate, methylene chloride, chloroform, benzene, and a mixed solvent thereof.

10. The composition of claim 8, wherein the mixed solvent is selected from the group consisting of water, C₁-C₄Alcohol and C₁-C₄A mixed solvent of alcohol and water.

11. The composition of claim 10, wherein the mixed solvent is an aqueous methanol solution or an aqueous ethanol solution.

12. The composition as set forth in claim 7, wherein the kimchi extract is selected from the group consisting of an extract, a concentrate of the extract, a dried product of the concentrate, and a powder.

Technical Field

The present application relates to kimchi containing lactic acid bacteria that can be effectively used for the prevention and treatment of Helicobacter pylori (Helicobacter pylori) -associated diseases.

Background

In 1983, Marchall and Warren, microbiologists in Australia, first discovered Campylobacter bacteria in biopsy tissue of gastric mucosa from patients with type B gastritis, and then cultured the bacteria and identified it as a new species of bacteria related to Campylobacter (Campybacter genus). Since then, the correlation between helicobacter pylori and upper gastrointestinal diseases such as gastritis and peptic ulcer has been actively studied. Helicobacter pylori is a gram-negative bacterium known to have a flagellum surrounded by a membrane, release a large amount of urease, transmit through the fecal-oral route, and is identified as a carcinogen by the World Health Organization (WHO) in 1994, and thus is a pathogen that has been of interest worldwide.

Currently, the pathogenic mechanism of diseases caused by helicobacter pylori is known as follows: bacteria entering the stomach cavity together with food penetrate the gastric mucus layer through flagella having strong motility, and colonize the junction between the upper part of the gastric epithelial cell layer and the cells within the gastric mucus layer, and secrete a large amount of urease to change the mucus layer to alkaline, thereby abnormally excessively stimulating secretion of gastric acid by gastrin, and thus causing inflammation and ulcer. In addition, according to recent research reports that helicobacter pylori is an important risk factor for the pathogenesis of gastric adenocarcinoma, helicobacter pylori has been designated as a class I carcinogen by the world health organization.

In particular, it was reported that the incidence of H.pylori infection is high in developing countries including Korea, and according to the Korean epidemiological investigation of 5732 patients without upper gastrointestinal symptoms, the infection rate of H.pylori is 46.6%, the infection rate of adults over 16 years is 69.4%, and particularly, a high infection rate of 78.5% is confirmed in people over 40 years. Furthermore, according to the investigation of the helicobacter pylori infection rate in 1031 cases of peptic ulcer patients, 66% of patients with gastric ulcer and 79% of patients with duodenal ulcer were infected with helicobacter pylori, while 71% of patients with gastric and duodenal ulcer were infected with helicobacter pylori.

As a method for treating diseases caused by helicobacter pylori infection as described above, chemotherapy using antibiotics is mainly performed at present. However, the widespread use of this method may be effective in a short period of time, but this method has a problem of risk of occurrence of antibiotic-resistant strains due to the continuous use of antibiotics. In addition, it has been reported that continuous use of antibacterial therapy causes various side effects such as diarrhea, reflux esophagitis, inhibition of normal flora in the intestine, appearance of resistant strains, and the like.

Therefore, there is a need to develop an appropriate antibacterial substance or food capable of inhibiting helicobacter pylori while substituting for a pharmacotherapeutic agent that causes the side effects and resistance as described above in the human body.

In addition, fermented lactic acid bacteria of kimchi, which is a traditional fermented food, are GRAS (Generally Recognized As Safe) grade bacteria that humans ingest together with fermented foods for a long time, and development of functional foods or medicines using lactic acid bacteria has been receiving attention due to the advantages of the functionality and safety of such lactic acid bacteria. For example, the present applicant has reported leuconostoc mesenteroides CJLM119 strain having low gas production and a method of preparing kimchi using the same, lactobacillus plantarum CJLP133 strain and a composition for treating intestinal diseases and enhancing immunity including the same (patent documents 1 to 3). However, research on a method for preventing or treating diseases caused by helicobacter pylori infection by fermented foods that are frequently consumed, such as kimchi, has not been completed, and is still underway.

Documents of the prior art

(patent document 1) Korean registered patent publication No. 1,807,995

(patent document 2) korean registered patent publication No. 1,486,999

(patent document 3) Korean registered patent publication No. 1,406,168

Disclosure of Invention

Technical problem to be solved

The present application relates to kimchi containing a lactic acid bacterium having an inhibitory effect on helicobacter pylori as an active ingredient, use of the kimchi or an extract thereof for preventing and treating helicobacter pylori-associated diseases.

Technical solution

In order to solve the technical problems, an aspect of the present application provides kimchi and a kimchi preparation method including Leuconostoc mesenteroides (Leuconostoc mesenteroides) CJLM119 and Lactobacillus plantarum (Lactobacillus plantarum) CJLP133 as lactic acid bacteria derived from the kimchi.

Another aspect of the present application provides a composition for ameliorating helicobacter pylori-associated diseases, comprising the kimchi or the extract thereof.

Another aspect of the present application provides a method for preventing, treating or ameliorating helicobacter pylori-associated diseases using the kimchi or the extract thereof.

Effects of the invention

The kimchi or its extract containing leuconostoc mesenteroides CJLM119 and lactobacillus plantarum CJLP133 as lactic acid bacteria derived from kimchi according to the present application has an inhibitory activity against helicobacter pylori, and can prevent, treat or delay progression of atrophic gastritis and gastric ulcer caused by helicobacter pylori when administered for a long period of time, and also can be usefully used for preventing or treating gastric cancer caused by helicobacter pylori.

However, the effects of the present application are not limited to the above-described effects, and other effects not mentioned can be clearly understood from the following description by those skilled in the art to which the present invention pertains.

Drawings

Fig. 1 is a graph showing the inhibitory effect of kimchi of the present application on helicobacter pylori bacteria.

Fig. 2 is an electrophoresis photograph showing the effect of kimchi of the present application on the expression of various vascular growth factors.

Fig. 3 is an electrophoresis photograph showing the effect of kimchi of the present application on the expression of various inflammatory factors.

Fig. 4 is a graph showing the effect of kimchi of the present application on tight junctions of cells in a helicobacter pylori infection model.

Fig. 5 is a graph showing the effect of kimchi of the present application on the growth of a gastric cancer cell line.

Fig. 6 is a graph showing the effect of kimchi of the present application on the apoptosis of gastric cancer cell lines.

Fig. 7 is an electrophoresis photograph showing the effect of kimchi of the present application on the expression of various apoptosis factors of a gastric cancer cell line.

Fig. 8 is an electrophoresis photograph showing the effect of kimchi of the present application on the expression of cell cycle-related factors of a gastric cancer cell line.

Fig. 9 shows the design of animal experiments for confirming the effect of kimchi of the present application on helicobacter pylori-associated diseases.

Fig. 10 is a graph showing the effect of kimchi of the present application on the total Lesion Score (Gross loss Index Score) in an animal model infected with helicobacter pylori.

Fig. 11 is a photograph showing the effect of kimchi of the present application on the occurrence of gastric cancer in an animal model infected with helicobacter pylori.

Detailed Description

Hereinafter, the content of the present application will be specifically described.

1.Pickle containing Leuconostoc mesenteroides CJLM119 and Lactobacillus plantarum CJLP133 and preparation method thereof

The application provides pickles comprising leuconostoc mesenteroides CJLM119 and Lactobacillus plantarum CJLP133 and a preparation method thereof.

Leuconostoc mesenteroides CJLM119 is a microbial strain deposited at Korean Institute of Bioscience and Biotechnology Gene Bank (Korea Research Institute of Bioscience and Biotechnology Gene Bank) under accession number KCTC 13043BP, and a method for isolating and identifying the strain is specifically disclosed in Korean registered patent publication No. 1,807,995. In addition, lactobacillus plantarum CJLP133 was a microbial strain deposited at korean institute of bioscience and biotechnology gene bank with accession No. KCTC 11403BP, and a method for isolating and identifying the strain was specifically disclosed in korean registered patent publication No. 1,486,999.

In the present application, "kimchi" includes, but is not limited to, food made by salting vegetables, mixing seasonings, and fermenting. In one embodiment of the present application, the vegetables may use chinese cabbage, radish, shallot, mustard leaves, cucumber, etc., and specifically, chinese cabbage may be used, but any other vegetables may be used as a raw material of the kimchi that is the subject of the present application. In one embodiment of the present application, although the vegetable may be a vegetable having a high lycopene content, it is not limited thereto. In other embodiments of the present application, the content of lycopene may be 0.05 to 5mg per 100g of vegetables, and for example, may be 0.1 to 3mg, 0.2 to 2mg, 0.3 to 1mg, 0.5 to 0.8mg, but is not limited thereto.

The vegetable with high lycopene content can be Chinese cabbage, and specifically can be Chinese cabbage of Korean No. 1 (variety application publication No.: 2015-688, 2015 12 month and 15 days). When vegetables having a high lycopene content are fermented using Leuconostoc mesenteroides CJLM119 and Lactobacillus plantarum CJLP133, the decrease of the lycopene content can be prevented, and thus the cancer prevention function of lycopene can be maintained.

In one embodiment of the present application, the seasoning is prepared by mixing materials of chili powder, garlic, ginger, salted fish, etc., and in addition to these seasonings, the materials constituting the seasoning may be appropriately added or excluded as needed according to personal preference, fermentation method, fermentation period, etc.

In one embodiment of the present application, kimchi of the present application can be prepared by a conventional kimchi preparation method, i.e., a method of fermenting after coating seasonings on vegetables salted with salt (e.g., chinese cabbage salted with salt). Although it may be more appropriate to use a vegetable having a high lycopene content as the vegetable, it is not limited thereto, and the conventional vegetable may be used for the preparation of kimchi of the present application without limitation.

In other embodiments of the present application, the kimchi of the present application may additionally include known additives acceptable as food, and as the additives, the kimchi of the present application may include various natural spices of plum fragrance, lemon fragrance, pineapple fragrance, vanilla fragrance, etc., various natural pigments of natural fruit juice, chlorophyllin, flavonoids, etc., sweet components such as fructose, honey, sugar alcohol, white sugar, or acidulants such as citric acid, sodium citrate, etc., without limitation.

In one embodiment of the present application, in the kimchi of the present application, the content of the leuconostoc mesenteroides CJLM119 may be 0.01 to 5 wt%, specifically 0.02 to 3 wt%, and more specifically 0.05 to 2.0 wt%, based on the entire weight of the kimchi.

In other embodiments of the present application, in the kimchi of the present application, the content of the lactobacillus plantarum CJLP133 may be 0.01 to 5 wt%, specifically 0.02 to 3 wt%, and more specifically 0.05 to 2.0 wt% based on the entire weight of the kimchi.

In one embodiment of the present application, kimchi of the present application may be prepared by fermenting at a predetermined temperature during a predetermined period after adding 0.01 to 5 wt% (specifically, 0.02 to 3 wt%, and more specifically, 0.05 to 2.0 wt%) of the leuconostoc mesenteroides CJLM119 to a salted vegetable (e.g., a salted vegetable with a high lycopene content), and adding 0.01 to 5 wt% (specifically, 0.02 to 3 wt%, and more specifically, 0.05 to 2.0 wt%) of the lactobacillus plantarum CJLP 133.

The temperature at the time of fermentation is not particularly limited, but fermentation may be carried out, for example, at 1 ℃ to 30 ℃, specifically at 3 ℃ to 15 ℃, or at 5 ℃ to 10 ℃.

The fermentation cycle is not particularly limited, but may be performed, for example, during 1 day to 365 days, specifically, during 5 days to 180 days, 10 days to 90 days, 15 days to 60 days, or 20 days to 40 days.

In one embodiment of the present application, leuconostoc mesenteroides CJLM119 and lactobacillus plantarum CJLP133 strains themselves may be used, but cultures including the strains may also be used. In one embodiment of the present application, leuconostoc mesenteroides CJLM119 and lactobacillus plantarum CJLP133 strains may be used as viable bacteria, dead bacteria, or a mixture thereof.

In the present application, the term "culture" means a product of culturing Leuconostoc mesenteroides CJLM119 and Lactobacillus plantarum CJLP133 strains by inoculating the culture medium. For example, the culture may include the culture itself (i.e., may include leuconostoc mesenteroides CJLM119 and lactobacillus plantarum CJLP133 strains, metabolites of the culture medium or strains) in which leuconostoc mesenteroides CJLM119 and lactobacillus plantarum CJLP133 strains are cultured in a medium, a filtrate (e.g., centrifuged supernatant) from which the strains are removed by filtering or centrifuging the culture, and the like. In addition, the culture of the present application may also include, without limitation, a form in which it is powdered after being dried (e.g., freeze-dried).

In one embodiment of the present application, the culturing of the strain may be performed during 6 to 48 hours at 10 to 30 ℃, specifically 12 to 36 hours or 18 to 30 hours at 20 to 30 ℃, but is not limited thereto.

The medium may be used without limitation as long as it is a known medium for lactic acid bacteria. The medium may include a carbon source, which may include one or more of sucrose, glucose, fructose, etc., and a nitrogen source, which may include one or more of yeast extract, peptone, beef extract, malt extract, corn steep liquor, ammonium citrate, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc., but is not limited thereto. In addition, the medium may additionally comprise one or more of tween 80, sodium citrate, potassium phosphate, sodium acetate, manganese sulfate, magnesium sulfate, distilled water, and the like.

2.Use of kimchi or its extract for improving pyloric screwComposition for treating bacteria-related diseases

The present application provides a composition for improving helicobacter pylori-associated diseases, which includes kimchi or an extract thereof including leuconostoc mesenteroides CJLM119 and lactobacillus plantarum CJLP 133.

Pickled vegetables and preparation method thereof and pickled vegetables "1. Comprises Leuconostoc mesenteroides CJLM119 and Lactobacillus plantarum CJLP133 pickled vegetable and preparation method thereof"are the same as those mentioned above, and therefore, the description thereof is omitted.

In the present application, as helicobacter pylori-associated diseases, gastritis, gastric ulcer, gastric cancer, and the like are exemplified, but not limited thereto.

In the present application, "kimchi extract" means a substance extracted from kimchi as a raw material by any method, and is used in a meaning including, without limitation, such extracted extract, a concentrate obtainable from the extract, a dried product and powder of the concentrate, and the like.

When the extract is extracted and obtained from kimchi as a raw material, as an extraction method, a known conventional, common extraction method such as a solvent extraction method, an ultrasonic extraction method, a filtration method, a reflux extraction method, and the like may be used without limitation, and specifically, may be prepared by using the solvent extraction method or the reflux extraction method, but is not limited thereto. The extraction process may be repeated several times, and may be additionally followed by steps of concentration or freeze-drying. Specifically, the obtained kimchi extract can be concentrated under reduced pressure to obtain a concentrate, and a high-concentration kimchi extract powder is prepared using a pulverizer after freeze-drying the concentrate.

In one embodiment of the present application, the kimchi extract is used in the meaning including, without limitation, an extract obtained by extracting kimchi with water, an organic solvent or a mixed solvent thereof, a concentrate or powder of the extract, and the like. The organic solvent may be selected from alcohols (in particular C)₁-C₄Lower alcohols, e.g. methanol, ethanol, propanol or butanol, hexane (n-hexane), ethers, glycerol, propylene glycol, butylene glycol, ethyl acetate, methyl acetateAny one of ester, methylene chloride, chloroform, benzene and a mixed solvent thereof. Specifically, the extraction solvent can be selected from water, C₁-C₄Alcohol and C₁-C₄Any one of mixed solvents of alcohol and water, for example, the extraction solvent may be a methanol aqueous solution or an ethanol aqueous solution. The aqueous methanol or ethanol solution may be a 10% (v/v) to 99% (v/v) aqueous solution, specifically, may be a 20% (v/v) to 70% (v/v) aqueous solution, and more specifically, may be a 30% (v/v) to 60% (v/v) aqueous solution, but is not limited thereto.

In one embodiment of the present application, the extraction may be performed at a temperature of 5 ℃ to 100 ℃, specifically 20 ℃ to 95 ℃, and more specifically 50 ℃ to 90 ℃, but is not limited thereto. Further, the extraction may be performed during 1 hour to 20 hours, specifically may be performed during 2 hours to 16 hours, and more specifically may be performed during 3 hours to 12 hours, but is not limited thereto.

In one embodiment of the present application, the extraction may be performed 1 to 8 times, specifically 1 to 6 times, and more specifically 1 to 5 times, but is not limited thereto. The extract may be a single extract obtained in each extraction or a mixed extract of extracts obtained in each extraction, and specifically may be a mixed extract of extracts obtained in each extraction.

In the present application, the term "improvement" is used in a meaning that it means, without limitation, all actions of reducing at least a parameter (e.g., symptom degree) related to treatment due to the ingestion of kimchi or its extract of the present application. The improvement of helicobacter pylori-associated diseases of the present application may be the effects of leuconostoc mesenteroides CJLM119 and lactobacillus plantarum CJLP133 themselves, and may be the effects of the strains due to vegetable fermentation, but the mechanism is not limited thereto.

In the present application, the composition may be a health functional food composition.

A Health Functional Food (Functional Food), which is the same term as a specific Health Food (Food for Special Health Use, FoSHU), means a Food which is processed to have a high medical and medical effects of effectively exerting a body regulation function in addition to a nutrition supply. Here, the term "function" means to obtain a beneficial effect when performing nutrient regulation on the structure and function of the human body or health care use such as physiological action.

The health food composition can be prepared by a method commonly used in the art, and can be prepared by adding raw materials and ingredients commonly added in the art at the time of preparation. In addition, the formulation of the health food composition may include, without limitation, any formulation that is recognized as a food. The health food composition can be prepared into various forms of preparations, and unlike general medicines, because foods are used as raw materials, there is no advantage in that side effects and the like that may occur due to long-term intake of medicines do not occur, and because it is convenient to carry, the health food composition can be ingested as an adjuvant for promoting the ameliorating effect of symptoms of helicobacter pylori-related diseases.

The health food means a food having a health maintenance or promotion effect more positive than that of a general food, and the health supplementary food means a food for health supplementary purposes. The terms of health functional food, health supplementary food may be used interchangeably according to circumstances.

In the present application, the compositions of the present application may additionally comprise a physiologically acceptable carrier. The type of the carrier is not particularly limited, and any carrier may be used without limitation as long as it is a carrier commonly used in the art. In addition, the compositions of the present application may include additional ingredients that can be commonly used in food compositions to improve odor, taste, visual appearance, and the like. For example, the compositions of the present application may include vitamin a, vitamin C, vitamin D, vitamin E, vitamin B1, vitamin B2, vitamin B6, vitamin B12, niacin, biotin, folic acid, pantothenic acid, and the like. In addition, the composition of the present application may include minerals of zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu), chromium (Cr), etc., and amino acids of lysine, tryptophan, cysteine, valine, etc.

In addition, the compositions herein may include food additives such as preservatives, bactericides, antioxidants, colorants, fixatives, bleaches, flavorings, sweeteners, fragrances, swelling agents, enhancers, emulsifiers, thickeners, coatings, foam inhibitors, solvents, modifiers, and the like. The additives may be selected according to the type of food and used in an appropriate amount. The proportion of additives is not critical, but will generally be selected in the range of 0.01 to 5 parts by weight in 100 parts by weight of the composition herein.

The content of kimchi or its extract contained in the composition of the present application may be appropriately determined according to the purpose of use. In one embodiment of the present application, the content of kimchi or extract thereof contained in the composition of the present application may be, for example, 0.0001 to 20.0 wt%, and specifically may be 0.001 to 1.0 wt%, relative to the total weight of the composition, but is not limited thereto. In the case where the kimchi or extract thereof is contained in the above-mentioned amount, the improvement effect on the symptoms of helicobacter pylori-related disease can be obtained in an economical amount. However, in the case of long-term ingestion for the purpose of health and hygiene or for the purpose of health regulation, the amount may be below the above range, and in terms of safety, since there is no problem, the kimchi or extract thereof may also be used in an amount above the range.

The composition of the present application may additionally include ingredients, such as protein, carbohydrate, fat, nutrient, seasoning, flavoring agent, etc., which are generally added at the time of food preparation, in addition to the kimchi or the extract thereof of the present application. Examples of the carbohydrate may be monosaccharide such as glucose, fructose, etc., disaccharide such as maltose, sucrose, oligosaccharide, etc., and polysaccharide such as dextrin, cyclodextrin and sugar alcohol, and sugar alcohol such as xylitol, sorbitol, erythritol, etc. As the flavoring agent, natural flavoring agents such as thaumatin, stevia extract and synthetic flavoring agents can be used. For example, when the health food composition of the present application is prepared as a beverage, citric acid, fructose solution, white sugar, glucose, acetic acid, malic acid, fruit juice, natural extract, etc. may be additionally included in addition to the kimchi extract of the present application.

The type of the composition is not particularly limited. Examples of foods to which the kimchi or the extract thereof of the present application can be added may be meats, sausages, breads, chocolates, candies, snacks, cookies, pizza, noodles, other noodles, chewing gums, dairy products including ice cream, various soups, beverages, teas, beverages, alcoholic beverages, vitamin complexes, and the like, and may include all health foods in a general meaning.

The kimchi or extract thereof of the present application includes leuconostoc mesenteroides CJLM119 and lactobacillus plantarum CJLP133, which have characteristics of highly inhibiting the activity of helicobacter pylori, and thus the composition including the kimchi or extract thereof of the present application can be usefully used in the prevention or treatment of related diseases caused by helicobacter pylori.

3.Method for preventing, treating or ameliorating helicobacter pylori-associated diseases using kimchi or kimchi extract

The present application provides a method for preventing, treating or ameliorating a helicobacter pylori-associated disease, comprising the step of administering the kimchi or the extract thereof to an individual who suffered from the helicobacter pylori-associated disease or may suffer from the helicobacter pylori-associated disease.

The application provides the use of the kimchi or the extract thereof for preventing, treating, or improving helicobacter pylori-associated diseases.

The application provides application of the pickle or the extract thereof in preparing a composition for preventing, treating or improving helicobacter pylori related diseases.

Sauerkraut or its extract, and its preparation method "1. Comprises Leuconostoc mesenteroides CJLM119 and plants Pickled vegetable containing Lactobacillus plantarum CJLP133 and preparation method thereofAnd 2.Method for improving body fluid containing kimchi or its extract Composition for treating helicobacter pylori related diseases"are the same as those mentioned above, and therefore, the description thereof is omitted.

In the present application, the term "prevention" is used in a meaning including all behaviors that inhibit or delay the symptoms of helicobacter pylori-associated diseases by the administration of kimchi or an extract thereof of the present application.

Further, in the present application, the term "treatment" is used in a meaning including, without limitation, an action of ameliorating or favorably changing the symptoms of helicobacter pylori-associated diseases by the administration of kimchi or an extract thereof of the present application.

Further, in the present application, the term "administering" is used in the sense of introducing a predetermined substance into an individual by an appropriate method.

In addition, in the present application, the term "individual" is used in the meaning of all animals including rats, mice, livestock, and the like including humans. As a specific example, the subject may be a mammal, including a human. As another example, the subject may be a subject having or at risk of developing a helicobacter pylori-associated disease.

In the method of the present application, the route of administration and the mode of administration of the kimchi or extract thereof of the present application are not particularly limited, and any route of administration and mode of administration may be followed as long as the composition including the kimchi or extract thereof of the present application can reach a desired site. Specifically, the kimchi or the extract thereof can be administered by various routes of oral or non-oral administration, but is generally administered by oral administration.

In one embodiment of the present application, as a food composition for dietary modification of infection with helicobacter pylori, a component of kimchi for preventing cancer has been newly studied. In other embodiments of the present application, it is representatively assumed that helicobacter pylori-associated diseases such as gastritis, gastric ulcer, gastric cancer, etc. can be prevented, improved or treated by dietary adjustment of cancer-preventing kimchi using a mouse model. In the conventional kimchi preparation method, cancer-preventing kimchi is prepared by adding vegetables having high lycopene content, leuconostoc mesenteroides CJLM119 strain and lactobacillus plantarum CJLP133 strain, but not limited thereto. In one embodiment of the present application, kimchi or an extract thereof according to the present application exhibits excellent preventive and therapeutic effects on helicobacter pylori-associated diseases, particularly excellent effects in gastritis, gastric ulcer, and gastric cancer.

According to one embodiment of the present application, leuconostoc mesenteroides CJLM119 and lactobacillus plantarum CJLP133 strains, which are lactic acid bacteria used in the preparation of kimchi of the present application, exhibited significant inhibitory activity against helicobacter pylori as compared to other strains belonging to the same species.

According to another embodiment of the present application, kimchi including leuconostoc mesenteroides CJLM119 and lactobacillus plantarum CJLP133 strains exhibited inhibitory effects on helicobacter pylori at predetermined concentrations (see fig. 1).

According to another embodiment of the present application, kimchi of the present application can significantly inhibit the expression of vascular growth factors and inflammatory factors in normal gastric cells and can restore tight junctions of cells (see fig. 2 to 4).

According to another embodiment of the present application, kimchi of the present application can significantly inhibit the growth of gastric cancer cells, induce apoptosis of gastric cancer cells (see fig. 5 and 6), and inhibit the formation of gastric cancer in an animal model of helicobacter pylori infection (see fig. 10).

As described above, kimchi including leuconostoc mesenteroides CJLM119 and lactobacillus plantarum CJLP133 strains of the present application exhibits a significant inhibitory activity against helicobacter pylori, and thus can be usefully used in the prevention, treatment, or amelioration of various diseases (e.g., gastritis, gastric ulcer, and gastric cancer) that may be induced from helicobacter pylori.

Hereinafter, the present application will be described in detail by way of examples.

However, the following examples are merely to specifically illustrate the present application, and the content of the present application is not limited by the following examples.

First, a preparation process, an experimental method, statistics, and the like of kimchi according to the present application will be explained. Statistical analysis was performed using GraphPad Prism (GraphPad software, rahoya, ca, usa) and SPSS software (version 12.0, SPSS corporation, chicago, illinois, usa), statistical significance between groups was determined by the Mann-Whitney U test (Mann-Whitney U test), and statistical significance was recognized in p < 0.05.

Example 1: strain isolation with high growth inhibitory activity against helicobacter pylori

<1-1>Strain isolation and characterization

Leuconostoc mesenteroides CJLM119, which is a lactic acid bacterium derived from kimchi, used in the preparation of kimchi of the present application is a microbial strain deposited in the korean institute of bioscience and biotechnology gene bank under accession number KCTC 13043BP, and a method for isolating and identifying the strain is specifically disclosed in korean registered patent publication No. 10-1807995. In addition, lactobacillus plantarum CJLP133, which is a lactic acid bacterium derived from kimchi, used in the preparation of kimchi of the present application is a microbial strain deposited in the korean institute of bioscience and biotechnology gene bank under accession No. KCTC 11403BP, and a method of isolating and identifying the strain is specifically disclosed in korean registered patent publication No. 10-1486999. Therefore, the detailed description thereof is omitted.

In the present application, kimchi was prepared by a conventional preparation method using leuconostoc mesenteroides CJLM119 and lactobacillus plantarum CJLP133 strains.

<1-2>Screening of Strain having high inhibitory Activity against helicobacter pylori bacteria

The helicobacter pylori inhibitory activity was measured using the leuconostoc mesenteroides CJLM119 and lactobacillus plantarum CJLP133 strains isolated and identified in example <1-1>, as well as leuconostoc mesenteroides KCTC 3100/KCTC 3722, lactobacillus plantarum KCTC 3108/DSM17853 used as a control group.

The results showed that leuconostoc mesenteroides CJLM119 and lactobacillus plantarum CJLP133 strains exhibited significant inhibitory activity against helicobacter pylori bacteria, compared to other strains of the same species used as a control group.

Example 2: preparation of sauerkraut

Standard kimchi (hereinafter, there is a case indicated as "sbimchi" or "SK") as a control group and cancer-preventing kimchi (hereinafter, there is a case indicated as "CpKimchi" or "CPK") according to the present application were prepared in the following manner.

<2-1>Preparation of Standard kimchi (sKimchi)

The standard kimchi is prepared by fermenting at low temperature after applying seasonings including chili powder, garlic, ginger, anchovy sauce, radish juice, shallot, glutinous rice paste, and white sugar to salted Chinese cabbage based on the standardized kimchi formula of the world institute for kimchi.

<2-2>Preparation of sauerkraut (CpKimchi) for preventing cancer

The kimchi for preventing cancer is prepared by using a cabbage having a high lycopene content as a main material, purchasing seeds of "Korea 1" (variety application publication No.: 2015-688, published 2015 12 month and 15 days) which are a variety of a general cabbage having a high lycopene content, and making a planting contract with a cabbage grower to secure the cabbage, and the harvested cabbage is used after being pickled to meet the kimchi preparation process. Kimchi for preventing cancer was prepared in the same manner as the preparation of the standard kimchi, except that the chinese cabbage having a high lycopene content was used as the chinese cabbage and 2 kinds of lactic acid bacteria derived from leuconostoc mesenteroides CJLM119 and lactobacillus plantarum CJLP133 of kimchi isolated and identified in example <1-1> were added.

<2-3> preparation of concentrate of kimchi extract

The kimchi prepared in the examples <2-1> and <2-2> was pulverized and prepared into powder after freeze-drying. After 20 times by weight of methanol was added to the powder, it was extracted with a stirrer for 12 hours. The extracted supernatant was filtered through filter paper, and the remaining precipitate was extracted again by adding methanol for 12 hours. After the extract was concentrated by heating at a temperature of about 40 ℃ with a concentrator to prepare a concentrate, it was stored at 4 ℃ and used in further experiments.

Example 3: determination of growth inhibitory Effect of helicobacter pylori

<3-1> culture of helicobacter pylori strain

Helicobacter pylori ATCC43504 (American type culture Collection, cagA + and vacA s1-m1 strains) were incubated with 5% sheep blood in BBL Tryptic Soy (TS) agar medium (TSIII; BD bioscience, Franklin lake, N.J.) under microaerophilic conditions (BD GasPAK EZ gas Generation System, BD bioscience) at 37 ℃ for 3 days. Helicobacter pylori strains were collected in TS medium and centrifuged at 3000 × g for 5 minutes and finally at a concentration of 10⁹Colony Forming Units (CFU)/ml were resuspended in media. All experiments used cultures grown in TS agar medium for 72 hours.

<3-2> confirmation of inhibitory Activity of helicobacter pylori

In order to confirm the inhibition effect of helicobacter pylori of kimchi of the present application, the size of the inhibition loop was measured after treating and culturing the concentrate of kimchi extract prepared in example <2-3> at a concentration in the medium inoculated with the helicobacter pylori strain of example <3-1 >.

The results showed that the concentrate of kimchi extract of the present application was confirmed to have inhibitory effect on helicobacter pylori bacteria at a concentration of 500mg/ml or more (see fig. 1).

Example 4: determination of the Effect on helicobacter pylori-associated diseases in the Normal stomach cell line (RGM 1; Normal stomach mucosal cells)

<4-1>Determination of the inhibitory Capacity of angiogenic factors

To confirm the effect of kimchi on the production of angiogenic growth factors, the concentrate of kimchi extract prepared in example <2-3> was treated at a concentration of 5mg/ml for 6 hours after the treatment of helicobacter pylori at 50MOI on RGM1 cells of a normal gastric cell line. After treatment, the medium components were removed and the cells were washed 2 times with pbs (dulbecco). Subsequently, to perform RT-PCR, RiboEX (500. mu.l, GeneAll, Seoul, Korea) was added to the plate at 4 ℃ and incubated for 10 minutes. After RiboEX was recovered and placed in a 1.5ml tube, 100. mu.l of chloroform was added and mixing was performed slowly. After incubation in ice for 10 minutes, the samples were centrifuged at 10,000g for 30 minutes. The supernatant was extracted and mixed with 200. mu.l of isopropanol, and the mixture was incubated at 4 ℃ for 1 hour. After centrifugation at 13,000g for 30 minutes, the precipitate was washed with 70% (vol/vol) ethanol. After the ethanol was completely evaporated, the particles were dissolved in 100 μ l DEPC-treated water (invitrogen life technologies, carlsbad, california), and then PCR was performed and changes in the relevant genes were confirmed.

The results showed that the expression of VEGF and bFGF, which are indicators of increased angiogenic factors due to the treatment with helicobacter pylori, was confirmed to be reduced in the cancer-preventing kimchi treatment group (CPK) (fig. 2).

<4-2> comparison of anti-inflammatory Effect

To confirm the anti-inflammatory effect of kimchi of the present application, after helicobacter pylori treatment at 50MOI in RGM1 cells, the concentrate of kimchi extract prepared in example <2-3> was treated at a concentration of 5mg/ml, and the change of the relevant gene was determined according to the RT-PCR method of example <4-1 >.

The results showed that the expression of COX-2 and iNOS, which are indicators of increased inflammation by helicobacter pylori treatment, was confirmed to be reduced in the cancer-preventing sauerkraut treatment group (CPK) (FIG. 3).

<4-3> comparison of cell connecting Effect

To confirm the effect of kimchi of the present application on tight junction indicator of cells, after helicobacter pylori treatment at 100MOI in RGM1 cells, the concentrate of kimchi extract prepared in example <2-3> was treated at concentrations of 5g/ml and 10g/ml for 6 hours. At this time, after the upper layer portion of the monolayer which became a confluent state due to the proliferation of the cells was washed 3 times with 0.5ml of cold D-PBS (-), 0.5ml of cold HBSS was filled and transferred to a new well filled with 1.5ml of cold HBSS, and then the transepithelial resistance (TERR%) between the upper and lower layer portions was measured after leaving for 30 minutes.

The results showed that tight junctions of cells were restored upon treatment with high concentrations of the cancer preventing kimchi of the present application in a helicobacter pylori infection model (fig. 4).

Example 5: determination of Effect on helicobacter pylori-associated diseases in gastric cancer cell line (MKN 28; gastric cancer cells)

<5-1> measurement of growth inhibitory Effect of gastric cancer cell line

In order to confirm the growth inhibition effect of kimchi on the gastric cancer cell line of the present application, cell viability of each treatment group was comparatively analyzed after treating the concentrates of kimchi extracts obtained by preparing standard kimchi and cancer-preventive kimchi by the method in example <2-3> in MKN28 cells at concentrations of 10, 20, 50mg/ml, respectively, for 6 hours.

The results showed that it was confirmed that the cancer-preventing kimchi of the present application was able to inhibit the growth of the gastric cancer cell line more significantly than that on the standard kimchi in a concentration-dependent manner (fig. 5).

<5-2> measurement of apoptosis-inducing Effect of gastric cancer cell line

In order to confirm the apoptosis-inducing effect of kimchi treatment in preventing cancer for cancer cells having no apoptosis property, concentrates of kimchi extracts obtained by preparing standard kimchi and cancer-preventing kimchi by the method of example <2-3> to treat each cell at an appropriate concentration for 48 hours, and then recovered with 1X PBS added with 1% FBS. Each Cell recovered by concentration was mixed with annexin V and Dead Cell Reagent (annexin V & Dead Cell Reagent) at a ratio of 1: 1, reacting for 20 minutes under the condition of normal temperature and light protection, and measuring and analyzing the proportion of the cells inducing apoptosis.

The results showed that apoptosis of cells was induced in 70% or more of the cells when MKN28 gastric cancer cell line was subjected to the cancer preventing kimchi treatment of the present application (fig. 6).

<5-3> measurement of apoptosis regulators of gastric cancer cell lines

To confirm the effect of kimchi on the apoptosis regulator of the gastric cancer cell line of the present application, treatment was performed in MKN28 gastric cancer cell line at a concentration of 20mg/ml with the concentrate of kimchi extract prepared in example <2-3>, and then the medium components were removed and the cells were washed 2 times with pbs (dulbecco) solution. Cells were lysed with ice-cold cell lysis buffer (cell signaling technology, denver, massachusetts) containing 1mM PMSF (phenylmethylsulfonyl fluoride, sigma aldrich, st). After 20 minutes of reaction, each sample was centrifuged at 10,000 Xg for 10 minutes. Subsequently, a supernatant was obtained. Proteins of the lysate were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a polyvinylidene fluoride (PVDF) membrane, reacted with a primary antibody, followed by washing, and reacted with a peroxidase-conjugated secondary antibody, followed by washing again, and then confirmed by an ECL system (universal electro-medical, white kihan, uk).

The results showed that it was confirmed that the expression of cleaved caspase 3, cleaved caspase 8, and cleaved PARP involved in apoptosis of cells was significantly increased in the Cancer Prevention Kimchi (CPK) treatment group of the present application, compared to the standard kimchi treatment group (SK) (fig. 7).

<5-4>Determination of cell cycle-associated factors for gastric cancer cell lines

To confirm the effect of kimchi on cell cycle-related factors of gastric cancer cell lines of the present application, the concentrate of kimchi extract prepared in example <2-3> was treated at a concentration of 20mg/ml in MKN28 cell line, and then the medium components were removed and the cells were washed 2 times with pbs (dulbecco) solution. Cells were lysed with ice-cold cell lysis buffer (cell signaling technology, denver, massachusetts) containing 1mM PMSF (sigma aldrich, st louis). After 20 minutes of reaction, each sample was centrifuged at 10,000 Xg for 10 minutes. Subsequently, a supernatant was obtained. Proteins of the lysate were separated by SDS-PAGE, transferred to a PVDF membrane and reacted with a primary antibody, followed by washing, and reacted with a peroxidase-conjugated secondary antibody, followed by washing again, and then changes in expression of cell cycle-related proteins were confirmed by an ECL system (general electric medicine, Kyowa).

The results showed that the expression of cyclin D1, CDK4, CDK2, cyclin E and cyclin a involved in the cell cycle was confirmed to be significantly reduced in the cancer-preventing kimchi (CPK) treated group of the present application compared to the Standard Kimchi (SK) treated group, and thus it was found that the apoptosis mechanism of the gastric cancer cell line was dependent on cell cycle regulation (fig. 8).

Example 6: determining the Effect on helicobacter pylori-associated diseases in Long-term animal models infected with helicobacter pylori

To confirm the effect of kimchi of the present application in inhibiting pathological indexes in a long-term animal model infected with helicobacter pylori, 5-week-old female C57BL/6 mice (charles river, japan) were fed with sterile commercial granular diet (Biogenomics, korea) and sterile water ad libitum and were raised in a room adjusted to a temperature of 24 ℃. After 1 week of acclimation, pantoprazole (pantoprazole) at 20mg/kg was injected 3 times to lower the acidity of the stomach to promote helicobacter pylori colonization. Then, 0.3ml of clean TS medium and H.pylori suspension (containing 10 ml) were injected into the stomach of each animal using a gastrointestinal cannula needle⁹CFU/ml). The control group was injected with TS medium, and all experimental groups were injected with helicobacter pylori a total of 4 times within one week. Mice were fed a special pellet feed based on AIN76 with 7.5% NaCl to induce gastritis for up to 4 weeks.

Then, helicobacter pylori-positive mice were randomly divided into 4 groups (n ═ 10). Granulated feed AIN76 containing 7.5% NaCl was administered for up to 24 weeks to promote carcinogenesis caused by helicobacter pylori in all animals. The control and experimental groups are illustrated in fig. 9, respectively. The following groups 1: control, group 2: helicobacter pylori infection group, group 3: kimchi low-concentration diet group for helicobacter pylori infection and prevention of cancer, group 4: high concentration kimchi diet group for helicobacter pylori infection and prevention of cancer, group 5: helicobacter pylori infection was tested in the standard sauerkraut diet group.

The results showed that the total lesion total score was significantly increased in G2 as the control group, whereas significant decreases (p < 0.01) were shown in G3 and G4 as the cancer-preventing kimchi-treated group. In addition, a significant size of gastric cancer lesion was formed in G2 as a control group for helicobacter pylori infection, whereas the formation of gastric cancer was confirmed to be reduced in G3 and G4 as a kimchi-treated group for preventing cancer (fig. 10 and 11).

Although the preferred embodiments of the present application have been described above by way of example, the scope of the present application is not limited to the specific embodiments described above, and those skilled in the art can make appropriate changes within the scope described in the claims of the present application.