KR102555685B1 - Composition for preventing or treating gastric cancer containing a mixed strain of probiotics as an active ingredient - Google Patents

Abstract

The present invention relates to a composition for preventing or treating gastric cancer containing a mixed probiotic strain as an active ingredient, and specifically, to a composition for preventing, alleviating or treating gastric cancer containing mixed parabiotics of Bifidobacterium bifidum, Lactobacillus reuteri, and Lactobacillus rhamnosus. The composition according to the present invention is found to be excellent in activating apoptosis-related signaling molecules in gastric cancer cells and inhibiting the growth of tumors derived from gastric cancer cells, and also the composition can be usefully used in various industrial fields such as medicine and food for purposes such as preventing, alleviating, or treating stomach cancer.

Description

Composition for preventing or treating gastric cancer containing a mixed strain of probiotics as an active ingredient}

The present invention relates to a composition for preventing or treating gastric cancer comprising a mixed strain of probiotics as an active ingredient, and specifically, for treating gastric cancer comprising a mixed dead cell of Bifidobacterium bifidum, Lactobacillus reuteri and Lactobacillus rhamnosus. It relates to a composition for prevention, improvement or treatment.

Para-biotics are dead lactic acid bacteria that do not have survival activities, and are also called names such as dead lactic acid bacteria, tindalized lactic acid bacteria, and heat-treated lactic acid bacteria. Although it does not survive in the body, it contains enzymes, glycoproteins, short-chain fatty acids, DNA components, and other microbial cell components that can help health. there is.

First of all, in that most of the abnormal reactions that may occur after ingestion of lactic acid bacteria, such as an increase in the concentration of lactic acid in the blood or induction of gas, are caused by the survival activity of viable bacteria, parabiotics is free from these adverse reactions because it is the intake of dead lactic acid bacteria. it's bad Therefore, parabiotics have a high stability compared to probiotics represented by probiotic products, and thus have the greatest advantage of being able to consume hundreds of billions to trillions of high concentrations. In addition, it has excellent heat resistance and high stability to the external environment, so it is easier to store than existing probiotic products and the shelf life can be extended.

Based on these advantages, it is showing rapid growth in fields such as health functional foods, food additives, and pharmaceuticals to which existing lactic acid bacteria have been applied. In addition, since regulations on the use of antibiotics in the pharmaceutical field are being strengthened, they have usefulness as an alternative and there are only a handful of companies that have entered the production of parabiotics products in earnest, so the marketability and growth potential are great.

Although these dead cells of lactic acid bacteria are more advantageous for industrial use, studies on the mechanism of action in cells and the human body for these physiological effects or more diverse physiological functions are not currently being conducted not only in Korea but also around the world. The situation is.

Under the above technical background, the object of the present invention is to treat Bifidobacterium bifidum MG731 ( Bi. bifidum MG731, accession number: KCTC13452BP), Lactobacillus reuteri MG5346 ( L. reuteri MG5346, accession number: KCTC14507BP) and Lactobacillus rhamnosus MG5200 ( nosus MG5200, accession number: KCTC14424BP) to provide a composition for preventing, improving or treating gastric cancer, including a mixed strain or a culture thereof, and a method for preparing the same.

The problem to be solved by the present invention is not limited to the above-mentioned problem (s), and another problem (s) not mentioned will be clearly understood by those skilled in the art from the following description.

In order to solve the above problems, the present invention provides Bifidobacterium bifidum MG731 ( Bi. bifidum MG731, accession number: KCTC13452BP), Lactobacillus reuteri MG5346 ( L. reuteri MG5346, accession number: KCTC14507BP) and Lactobacillus rhamnosus MG5200 ( L. rhamnosus MG5200, accession number: KCTC14424BP) provides a pharmaceutical composition for preventing or treating gastric cancer, including a mixed strain or culture thereof.

The strain may be heat-killed parabiotics.

The heat killing may be heat treatment at a temperature of 70 ° C to 130 ° C for 10 minutes to 60 minutes.

The Bifidobacterium bifidum MG731, Lactobacillus reuteri MG5346, and Lactobacillus rhamnosus MG5200 may be included in a cell count ratio of 1:1 to 2:1 to 2.

In addition, the present invention relates to Bifidobacterium bifidum MG731 ( Bi. bifidum MG731, accession number: KCTC13452BP), Lactobacillus reuteri MG5346 ( L. reuteri MG5346, accession number: KCTC14507BP) and Lactobacillus rhamnosus MG5200 ( L. rhamnosus MG5200, Accession number: It provides a health functional food composition for preventing or improving gastric cancer, including a mixed strain or a culture thereof including KCTC14424BP).

The strain may be heat-killed parabiotics.

In addition, the present invention relates to Bifidobacterium bifidum MG731 ( Bi. bifidum MG731, accession number: KCTC13452BP), Lactobacillus reuteri MG5346 ( L. reuteri MG5346, accession number: KCTC14507BP) and Lactobacillus rhamnosus MG5200 ( L. rhamnosus MG5200, Accession number: It provides a feed additive composition for preventing or improving gastric cancer, including a mixed strain or culture thereof, including KCTC14424BP).

The strain may be heat-killed parabiotics.

In addition, the present invention comprises the steps of mixing heat-killed cells; Including, the cell body is Bifidobacterium bifidum MG731 ( Bi. bifidum MG731, accession number: KCTC13452BP), Lactobacillus reuteri MG5346 ( L. reuteri MG5346, accession number: KCTC14507BP) and Lactobacillus rhamnosus MG5200 ( L. rhamnosus MG5200, accession number: KCTC14424BP), which provides a method for preparing a composition for preventing, improving or treating gastric cancer.

The manufacturing method comprises the steps of lyophilizing and powdering the heat-killed cell mixture; may further include.

The Bifidobacterium bifidum MG731, Lactobacillus reuteri MG5346, and Lactobacillus rhamnosus MG5200 may be included in a cell count ratio of 1:1 to 2:1 to 2.

The composition according to the present invention has been found to be effective in activating apoptosis-related signaling molecules in gastric cancer cells and inhibiting the growth of gastric cancer cell-derived tumors. It can be usefully used in various industrial fields.

The effects of the present invention are not limited to the above effects, and should be understood to include all effects that can be inferred from the detailed description of the present invention or the configuration of the invention described in the claims.

Figure 1 is a diagram showing the cell viability of MKN1 cells treated with 11 kinds of parabiotics, respectively (mean ± SD, *: p <0.05).
Figure 2 is a diagram showing apoptosis (Apoptosis) of MKN1 cells treated with each of 11 kinds of parabiotics (mean ± SD, *: p <0.05). a: Percentage distribution by Annexin V-FITC and PI staining reactions, b: Total apoptosis % comparison.
Figure 3 is a diagram comparing the tumor size of the control group and the 3MIX parabiotics experimental group in human gastric cancer xenograft animal models.
Figure 4 is a diagram comparing the tumor suppression effect of the control group and the 3MIX parabiotics experimental group in human gastric cancer xenograft animal models (mean ± SD, n = 3, *: p <0.05). a: Tumor growth curve at 3 weeks, b: Comparison of tumor weight, c: Weight change at 3 weeks.
5 is a diagram showing a comparison of apoptosis signaling molecule activity between a control group and a 3MIX parabiotics experimental group in a human gastric cancer xenograft animal model (mean ± SD, *: p<0.05). a: p-AKT/AKT expression level, b: p53/GAPDH expression level, c: Bax/GAPDH expression level, d: Bak/GAPDH expression level.
6 is a diagram showing a comparison of caspase activity between a control group and a 3MIX parabiotics experimental group in a human gastric cancer xenograft animal model (mean ± SD, *: p<0.05). a: Western blot data, b: Comparison of cleaved caspse-9/caspase-9 expression, c: Comparison of cleaved caspse-3/caspase-3 expression, d: Comparison of cleaved PARP/PARP expression.
7 is a diagram showing the apoptosis signal molecule activity of the 3MIX parabiotics experimental group in a human gastric cancer xenograft animal model as an immunohistochemical (IHC) staining image.

Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning in the context of the related art, and unless explicitly defined in this application, it should not be interpreted in an ideal or excessively formal meaning. don't In addition, in describing the present invention, if it is determined that a detailed description of a related known technology may unnecessarily obscure the subject matter of the present invention, the detailed description will be omitted.

The present inventors identified the molecular mechanism of antitumor activity against gastric cancer of a mixed strain composed of heat-killed Bifidobacterium bifidum, Lactobacillus reuteri and Lactobacillus rhamnosus, and found a mixed parasite showing efficacy in preventing or treating gastric cancer. Invented biotics.

Specifically, the present invention relates to Bifidobacterium bifidum MG731 ( Bi. bifidum MG731, accession number: KCTC13452BP), Lactobacillus reuteri MG5346 ( L. reuteri MG5346, accession number: KCTC14507BP) and Lactobacillus rhamnosus MG5200 ( L. rhamnosus MG5200, accession number: KCTC14424BP) relates to a pharmaceutical composition for preventing or treating gastric cancer, including a mixed strain or culture thereof.

In the present invention, “gastric cancer” includes all malignant tumors occurring in the stomach, specifically gastric adenocarcinoma, lymphoma, gastric submucosal tumor, leiomyoma, etc. can

The composition according to the present invention has tumor growth inhibitory activity or cancer cell apoptosis inducing activity. Apoptosis-inducing activity and tumor growth inhibitory activity of mixed parabiotics composed of Leeum bifiderm MG731, Lactobacillus reuteri MG5346 and Lactobacillus rhamnosus MG5200 were confirmed, and the mixed strain was utilized as a preventive or therapeutic agent for gastric cancer. It was suggested that it can.

The Bifidobacterium bifidum MG731 was patented at the Center for Biological Resources on January 4, 2018 and received accession number KCTC13452BP.

The Lactobacillus reuteri MG5346 was patented at the Center for Biological Resources on March 22, 2021 and was given accession number KCTC14507BP.

The Lactobacillus rhamnosus MG5200 was patented at the Center for Biological Resources on January 05, 2021 and received accession number KCTC14424BP.

The strain may be heat-killed parabiotics.

In the present invention, "Para-biotics" is a form of preventing the growth of bacteria by methods such as heat treatment, chemical treatment, radiation treatment, etc. after culturing viable bacteria under certain conditions. is a decomposition product That is, it is characterized in that it contains metabolites, cytoplasm, cell wall, bacteriocin, polysaccharide, organic acid, etc., in a dead state. Most of the physiologically active components of lactic acid bacteria are concentrated in the cell walls of lactic acid bacteria, and these cell walls are absorbed by intestinal M cells after the lactic acid bacteria die, so dead cells are called parabiotics in the sense that they play a role similar to live cells. . In the present invention, parabiotics is meant to include all of the dead cells of lactic acid bacteria, dead cell cultures, dead cell extracts, and dead cells cell components.

In the present invention, "mixed parabiotics" means a mixture of parabiotics, and may be a mixture in which each lactic acid bacteria is killed after treatment or a mixture in which each lactic acid bacteria is killed and treated after mixing, and is not particularly limited.

The mixed parabiotics according to the present invention is preferably a mixed strain of parabiotics consisting of Bifidobacterium bifidum MG731, Lactobacillus reuteri MG5346 and Lactobacillus rhamnosus MG5200 or Bifidobacterium bifidum MG731 parabiotics, It may be a mixture of Lactobacillus reuteri MG5346 parabiotics and Lactobacillus rhamnosus MG5200 parabiotics.

Parabiotics according to the present invention may be obtained by killing lactic acid bacteria cells by heat treatment, radiation treatment, chemical treatment, etc., and preferably by heat treatment. It may be heat-killed for 10 minutes to 60 minutes at a temperature.

In the present invention, the mixed parabiotics may include Bifidobacterium bifidum MG731, Lactobacillus reuteri MG5346, and Lactobacillus rhamnosus MG5200 at a cell number ratio of 1: 1 to 2: 1 to 2 and then be killed or treated. Bifidobacterium bifidum MG731, Lactobacillus reuteri MG5346 and Lactobacillus rhamnosus MG5200 may be included in a cell count ratio of 1:1 to 2:1 to 2.

According to an embodiment of the present invention, three types of parabiotics, Bifidobacterium bifidum MG731, Lactobacillus reuteri MG5346, and Lactobacillus rhamnosus MG5200, having excellent cytotoxicity and apoptosis inducing activity against gastric cancer cells, were identified And, when administered at a concentration of 1×10 ⁹ CFU/mouse or 3×10 ⁹ CFU/mouse to gastric cancer-causing animal models with a mixture of the parabiotics, the tumor growth inhibitory activity and apoptosis inducing activity were improved in a concentration-dependent manner. It was confirmed that the effect was shown. In addition, it was confirmed that the mixed parabiotics were involved in an apoptosis-related signal pathway, and thus the inhibition of tumor growth in the animal model was not tumor necrosis, but the antitumor activity by induction of apoptotic signals of the mixed parabiotics. Therefore, the composition containing the mixed parabiotics according to the present invention can be provided as a pharmaceutical composition for preventing or treating gastric cancer.

The pharmaceutical composition of the present invention may be administered to a patient diagnosed with gastric cancer or confirmed to be at risk of developing gastric cancer. Prevention or treatment of gastric cancer using the composition may further have an activity of reducing metastasis of cancer cells in addition to an activity of delaying and/or suppressing tumor formation or tumor growth of cancer cells.

The composition may be used alone or in other therapies, such as surgery, radiation therapy, gene therapy, immunotherapy (eg, targeted antibody immunotherapy, CAR-T cell therapy), oncolytic viruses, bone marrow transplantation, stem cell transplantation, hormone therapy, It can be performed in combination with targeted therapy, cryotherapy, ultrasound therapy, photodynamic therapy, chemotherapy, and the like. Additionally, persons at high risk of developing a proliferative disease may receive treatment to inhibit and/or delay the onset of the disease. The anti-cancer activity of the composition according to the present invention may be effective when combined with more direct anti-cancer agents. Thus, in certain embodiments, the present invention may provide a composition comprising dead mixed cells and an anti-cancer agent.

Mixed strains or mixed parabiotics composed of Bifidobacterium bifidum MG731, Lactobacillus reuteri MG5346 and Lactobacillus rhamnosus MG5200, which are active ingredients of the composition according to the present invention, are 0.00001 to 100% by weight, 0.001% by weight, of the total anticancer composition. It may be included in weight % to 99.9 weight %, 0.1 weight % to 99 weight %, and more preferably 1 weight % to 50 weight %.

In the present invention, the pharmaceutical composition may be administered to mammals including humans through various routes. The administration method may be any method commonly used, and may be administered by, for example, oral, dermal, intravenous, intramuscular or subcutaneous routes, preferably orally.

The composition may include a pharmaceutically acceptable excipient or carrier. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical arts. Examples of suitable carriers include lactose, starch, glucose, methyl cellulose, magnesium stearate, mannitol, sorbitol and the like. Examples of suitable diluents include ethanol, glycerol and water. The choice of pharmaceutical carrier, excipient or diluent may be selected with reference to the intended route of administration and standard pharmaceutical practice. The pharmaceutical composition may include any suitable binder, lubricant, suspending agent, coating agent, solubilizing agent, as or in addition to a carrier, excipient or diluent. The carriers, excipients, and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitone, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, mineral oil, and the like.

When formulating or formulating the pharmaceutical composition, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations contain at least one excipient such as starch, calcium carbonate, sucrose, lactose, It is prepared by mixing gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.

Liquid preparations for oral use include suspensions, solutions for oral use, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included. . Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, suppositories, and the like. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As a base for the suppository, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogeratin and the like may be used. The components may be added in combination with active ingredients, that is, mixed strains, cultures thereof, extracts thereof, cell components thereof, or mixed parabiotics.

In addition, it may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories and sterile injection solutions according to conventional methods.

A suitable dosage of the pharmaceutical composition according to the present invention is variously prescribed by factors such as formulation method, administration method, patient's age, weight, sex, morbid condition, food, administration time, administration route, excretion rate and response sensitivity. It may be administered once or several times a day at regular time intervals according to the judgment of the doctor or pharmacist. For example, the daily dosage may be 0.1 to 10,000 mg/kg, preferably 1 to 2,000 mg/kg, based on the active ingredient content. The above dosage is an example of an average case, and the optimal dosage to be administered can be determined by those skilled in the art, and the type of disease, the severity of the disease, the content of active ingredients and other ingredients contained in the composition, the type of formulation, And it can be adjusted by an expert in the art according to various factors including the patient's age, weight, general health condition, sex and diet, administration time, administration route and secretion rate of the composition, treatment period, and concurrently used drugs.

In addition, the present invention relates to Bifidobacterium bifidum MG731 ( Bi. bifidum MG731, accession number: KCTC13452BP), Lactobacillus reuteri MG5346 ( L. reuteri MG5346, accession number: KCTC14507BP) and Lactobacillus rhamnosus MG5200 ( L. rhamnosus MG5200, Accession number: KCTC14424BP) can provide a health functional food composition for preventing or improving gastric cancer comprising a mixed strain or a culture thereof.

The strain may be heat-killed parabiotics. Therefore, the composition may preferably include mixed parabiotics consisting of Bifidobacterium bifidum MG731, Lactobacillus reuteri MG5346 and Lactobacillus rhamnosus MG5200.

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

The functional foods include beverages, fruits and their processed foods, fish, meat and their processed foods, breads and noodles, fruit juices, various drinks, cookies, taffy, dairy products, edible vegetable oils, margarine, vegetable proteins, retort foods, frozen foods , It can be prepared in the form of various seasonings, etc.

The health functional food (health functional food) is the same term as food for special health use (FoSHU), and in addition to supplying nutrients, it is processed to efficiently display bioregulatory functions, and has high medical effects. means food. Here, 'function (sex)' means obtaining useful effects for health purposes such as regulating nutrients for the structure and function of the human body or physiological functions. Health food refers to food that has a more active health maintenance or promotion effect than general food, and health supplement food refers to food for the purpose of supplementing health. In some cases, the terms health functional food, health food, and health supplement food may be used interchangeably.

Specifically, the health functional (sex) food is a food manufactured by encapsulation, powdering, suspension, etc., and means that when ingested, it brings a specific effect on health, but unlike general medicines, it is a medicine using food as a raw material. It has the advantage of not having side effects that may occur when taking it for a long time.

Preferred content of the food composition of the present invention is not limited thereto, but may be, for example, 0.01 to 80% by weight of the finally prepared food, preferably 0.01 to 50% by weight of the finally prepared food.

The food composition of the present invention can be prepared by a method commonly used in the art, and can be prepared by adding raw materials and components commonly added in the art. In addition, the food composition can be prepared without limitation in various types of formulations as long as they are recognized as food. In addition, the food composition may further include a physiologically acceptable carrier, the type of carrier is not particularly limited, any carrier commonly used in the art may be used. In addition, the food composition may include additional ingredients that are commonly used in food compositions and can improve smell, taste, and vision. For example, vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, panthotenic acid, and the like may be included. In addition, minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu), and chrome (Cr); and amino acids such as lysine, tryptophan, cysteine, and valine. In addition, the food composition may include preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), bactericides (bleaching powder, high bleaching powder, sodium hypochlorite, etc.), antioxidants (butylhydroxyanisole (BHA), butyl hydroxy Loxytoluene (BHT), etc.), coloring agents (tar color, etc.), coloring agents (sodium nitrite, sodium nitrite, etc.), bleaching agents (sodium sulfite, etc.), seasonings (MSG sodium glutamate, etc.), sweeteners (dulcin, cyclemate, saccharin) , sodium, etc.), flavoring (vanillin, lactones, etc.), expanding agent (alum, D-potassium hydrogen stannate, etc.), strengthening agent, emulsifier, thickener (thickener), coating agent, gum base agent, foam inhibitor, solvent, improver, etc. food May contain food additives. The additives may be selected according to the type of food and used in an appropriate amount.

In addition, the present invention relates to Bifidobacterium bifidum MG731 ( Bi. bifidum MG731, accession number: KCTC13452BP), Lactobacillus reuteri MG5346 ( L. reuteri MG5346, accession number: KCTC14507BP) and Lactobacillus rhamnosus MG5200 ( L. rhamnosus MG5200, Accession number: KCTC14424BP) may provide a feed additive composition for preventing or improving gastric cancer comprising a mixed strain or a culture thereof.

The strain may be heat-killed parabiotics. Therefore, the composition may preferably include mixed parabiotics consisting of Bifidobacterium bifidum MG731, Lactobacillus reuteri MG5346 and Lactobacillus rhamnosus MG5200.

The feed to which the composition is added may be provided to companion animals or livestock that are diagnosed with gastric cancer or are identified as having a risk of developing gastric cancer for the purpose of preventing cancer. Mixed strains or mixed parabiotics, which are raw materials of the composition, may be used in combination with conventionally known feed additives or feeds, and may also include various excipients added to known feed additives or feeds. The composition may further include trace nutrients such as nutrients, preservatives, preservatives, antibiotics, and vitamins commonly used in animal feed. The nutrients, preservatives, preservatives, antibiotics, trace nutrients, etc. may be arbitrarily selected and used by those skilled in the art in an amount commonly added to feed.

In another aspect of the present invention, mixing heat-killed cells; Including, the cell body is Bifidobacterium bifidum MG731 ( Bi. bifidum MG731, accession number: KCTC13452BP), Lactobacillus reuteri MG5346 ( L. reuteri MG5346, accession number: KCTC14507BP) and Lactobacillus rhamnosus MG5200 ( L. rhamnosus MG5200, accession number: KCTC14424BP), it relates to a method for preparing a composition for preventing, improving or treating gastric cancer.

The heat killing process may be a heat treatment of the lactic acid bacteria culture, preferably at a temperature of 70 ° C. to 130 ° C. for 10 minutes to 60 minutes, more preferably at 90 ° C. to 110 ° C. for 20 minutes to 40 minutes.

The method for preparing the composition may further include lyophilizing the heat-killed cells to powder. The dead cells may be coated with cryoprotectants such as trehalose, maltodextrin, starch and skim milk powder before lyophilization.

The powdered Bifidobacterium bifidum MG731, Lactobacillus reuteri MG5346 and Lactobacillus rhamnosus MG5200 were mixed at a cell count ratio of 1:1 to 2:1 to 2, or Bifidobacterium bifidum MG731 and Lactobacillus reuteri MG5346 and Lactobacillus rhamnosus MG5200 may be mixed at a cell number ratio of 1:1 to 2:1 to 2, killed by heating, and then pulverized.

The present invention will be described in more detail through examples or experimental examples below. These examples or experimental examples are only for exemplifying the present invention, and the scope of the present invention is not construed as being limited thereto.

Example 1: Selection of parabiotics exhibiting antitumor effects

In order to evaluate the antitumor efficacy of the heat-killed strain through human gastric cancer cell xenograft animal model, an experimental group was formed in the following manner.

Preparing Parabiotics

The strains used in the experiment were provided by Mediogen Co., Ltd. (Jecheon, Korea) and used (Table 1).

NO. strain name origin One Bifidobacterium bifidum (B. bifidum) MG731 Infant feces 2 Bifidobacterium breve (B. breve) MG729 3 Lactobacillus rhamnosus (L. rhamnosus ) MG316 4 Streptococcus thermophilus (S. thermophiles ) MG5140
Fermented food
5 Lactobacillus bulgaricus ( L. bulgaricus ) MG515 6 Lactobacillus casei (L. casei ) MG311 7 Lactobacillus rhamnosus (L. rhamnosus ) MG5200 8 Lactobacillus casei (L. casei ) MG4584 Human 9 Lactobacillus gasseri (L. gasseri ) MG4514 10 Lactobacillus plantarum (L. plantarum ) MG4215 11 Lactobacillus reuteri (L. reuteri ) MG5346 Food

Each of the strains in Table 1 was inoculated into MRS medium, incubated at 37 ° C for 18 hours, centrifuged (5,000 × g, 4 ° C, 5 minutes), and the cells were washed three times with distilled water. After adding sterile distilled water to the washed cells to prepare a suspension, heat treatment was performed at 100 ° C. for 30 minutes. Then, parabiotics powder was obtained through rapid lyophilization and grinding at -80 ° C and stored in a refrigerator until use in experiments.

cell culture

Human gastric cancer MKN1 cells were purchased from the Korean Cell Line Bank (Seoul, Korea). MKN1 cells were cultured in RPMI 1640 (Roswell Park Memorial Institute Medium 1640) supplemented with 1% penicillin/streptomycin and 10% inactivated fetal bovine serum and then incubated at 5% CO ₂ , 37°C.

Cytotoxicity assay

The cytotoxic effect of the heat-killed strain on human gastric cancer MKN1 cells was analyzed.

MKN1 cells were treated with each of the 11 types of parabiotics prepared at 1×10 ⁹ cells/mL for 24 hours, and cell viability was quantified using an MTT assay. A graph is shown in FIG. 1 . Referring to Figure 1, the survival of MKN1 cells treated with parabiotics was significantly reduced, Bifidobacterium bifidum ( B. bifidum ) MG731, Lactobacillus reuteri ( L. reuteri ) MG5346 and Lactobacillus rhamnosus ( L rhamnosus ) MG5200-treated cells showed low cell viability of 25.77%, 57.33% and 56.84%, respectively, showing high cytotoxic effect on MKN1 cells compared to other strains.

Apoptosis assay

The apoptosis effect of the heat-killed strain on human gastric cancer MKN1 cells was analyzed.

The prepared 11 parabiotics were treated with MKN1 cells at 1×10 ⁹ cells/mL for 24 hours, and the results using flow cytometry are shown in FIG. 2 . The sum of the percentages of early apoptosis (%) and late apoptosis (%) found in the Annexin V-FITC and PI staining quadrants (a) was defined as total apoptosis (%) and quantified (b). Looking at Figure 2 (b), 11 parabiotics showed a significantly higher apoptotic effect than the control group (4.6%), among which Bifidobacterium bifidum ( B. bifidum ) MG731, Lactobacillus reuteri ( L. reuteri ) MG5346, L. rhamnosus MG316 and Lactobacillus rhamnosus MG5200 showed high apoptosis inducing effects of 13.3%, 24.6%, 13.4% and 11.4%, respectively.

in-vivo Composition of experimental group

Bifidobacterium bifidum ( B. bifidum ) MG731, L. reuteri ( L. reuteri ) MG5346 and L. rhamnosus ( L. rhamnosus ) MG5200 showing high effects on cytotoxicity and apoptosis induction for human gastric cancer MKN1 cells 3MIX parabiotics administration group mixed with was composed of an experimental group for gastric cancer xenograft animal models.

The drinking water administration group was used as a control group, and Bifidobacterium bifidum ( B. bifidum ) MG731, Lactobacillus reuteri ( L. reuteri ) MG5346 and Lactobacillus rhamnosus ( L. rhamnosus ) MG5200 were mixed in equal proportions to obtain 1 × It was composed of experimental group 1 (3Mix-1) administered at a concentration of ^{10 9} CFU/mouse and experimental group 2 (3Mix-3) administered at a concentration of 3 × 10 9 CFU/mouse, and was used to analyze the antitumor effect according to administration by concentration. .

Example 2: Anti-gastric cancer efficacy of 3MIX parabiotics

2-1. Anti-tumor efficacy of 3MIX parabiotics

Animal experiments were conducted with the approval of the Animal Care and Use Committee of Duksung Women's University (permit number: 2021-011-015).

BALB/c rats (5-week-old females) were purchased from Raon Bio (Seoul, Korea). Rats were acclimatized for one week prior to the experiment in a controlled, pathogen-free environment (45 ± 5% humidity and 23-27 °C, 12-h day/12-h night cycle) with standard laboratory chow and water ad libitum. Human gastric cancer MKN1 cells (5 × 10 ⁶ cells/mouse) were subcutaneously injected into the back of the right hind leg of rats, and then randomly assigned to the three groups shown in Table 2 below.

Group Sample composition control group Conrtol drinking water experimental group 1 3Mix-1 MG731 + MG5346 + MG5200
(1:1:1 mix, total 1 × 10 ⁹ CFU/mouse) experimental group 2 3Mix-3 MG731 + MG5346 + MG5200
(1:1:1 mix, total 3 × 10 ⁹ CFU/mouse)

For 3 weeks, 0.1 mL of drinking water was orally administered to the control group daily, and 3Mix-1 and 3Mix-3 dissolved in drinking water were orally administered to experimental groups 1 and 2, respectively.

Tumors were identified and measured every two days using standard calipers. Tumor volume was calculated as [tumor length (mm) x tumor width (mm)2]/2. When the tumor volume reached 2000 mm ³ , the rats were euthanized and the tumors harvested.

Looking at Figure 3, which compares the appearance of the harvested tumor by photographing it, it can be seen with the naked eye that the size of the tumor is significantly reduced in the experimental group receiving 3MIX parabiotics compared to the control group.

For specific comparison, the volume and weight of tumors collected from three groups of rats were measured and shown in FIG. 4 . As shown in Figure 4a, compared to the control group in which the volume of the tumor increased rapidly, the experimental group administered with 3MIX parabiotics showed an effect of significantly inhibiting tumor growth.

Specifically, in the control group, which received only drinking water, tumor growth increased rapidly from the 5th day of administration, and the tumor volume recorded 318 ± 17.14 mm ³ on the 19th day of administration, whereas in the 3MIX parabiotics-administered group 1 and In experimental group 2, the tumor volume was 177 ± 27.09 mm ³ and 122 ± 30.00 mm ^{3 ,} respectively, on the 19th day of administration, and the tumor volume was 45% and 62%, respectively, compared to the control group, confirming that tumor growth was excellently inhibited. In particular, in Experimental Group 2, tumor growth was significantly inhibited from the beginning of administration, and it was confirmed that tumor growth was reduced by more than 27% compared to the control group when 3Mix-3 was orally administered 5 times.

Looking at FIG. 4B, which shows the weight of tumor tissue measured, the tumor weight of the control group was recorded as 78.12 ± 13.31 mg, whereas the experimental group 1 was 44.22 ± 10.08 mg and the experimental group 2 was 36.32 ± 6.18 mg, which was found to be 36.32 ± 6.18 mg in the case of experimental group 2 compared to the control group. was less than half the weight of

On the other hand, in order to confirm whether oral administration of 3MIX parabiotics affects the weight change of rats, the results of measuring the body weight every 2 days during the experimental period are shown in FIG. 4c. Looking at this, it was confirmed that the weight change between the control group and the experimental group did not show a significant difference until the 19th day after oral administration was completed.

2-2. Analysis of apoptosis signal protein expression of 3MIX parabiotics

The effect of 3MIX parabiotics on the expression of apoptosis target proteins in an animal model in which human gastric cancer MKN1 cells were xenotransplanted was confirmed through Western blot analysis.

The AKT-p53 signaling mechanism is involved in cancer cell proliferation regulation and apoptosis, and plays a role in activating downstream transcription factors such as Bax and Bak that promote cancer cell apoptosis.

Referring to FIG. 5, the p-AKT/AKT expression level (FIG. 5a) showed a decreasing effect in both experimental group 1 (3Mix-1) and experimental group 2 (3Mix-3) compared to the control group. The p53/GAPDH expression level (Fig. 5b) increased 1.70 and 2.48 times in Experimental Group 1 (3Mix-1) and Experimental Group 2 (3Mix-3), respectively, compared to the control group, and the Bax/GAPDH expression level (Fig. 5c) was higher than that of the control group. It increased significantly by 1.53 and 2.14 times in Experimental Group 1 (3Mix-1) and Experimental Group 2 (3Mix-3), respectively. In addition, the amount of Bak/GAPDH expression (Fig. 5d) increased by 1.17 and 2.49 times in Experimental Group 1 (3Mix-1) and Experimental Group 2 (3Mix-3), respectively, compared to the control group. confirmed to be stimulated.

Caspase (Cysteine aspartic acid proteases), as a major regulator of apoptosis, is an inactive form in cells, but is cleaved and activated, and induces apoptosis by degrading proteins such as PARP and DNA fragmentation factors.

The amount of activated caspase expression involved in the endogenous apoptosis mechanism was confirmed and shown in FIG. 6a and quantified and shown in b to d.

The expression level of cleaved caspase-9/caspase-9 in FIG. 6b increased by 1.70 and 2.09 times in experimental group 1 (3Mix-1) and experimental group 2 (3Mix-3), respectively, compared to the control group, and the activation level of caspase-3 in FIG. 6c Compared to the control group, both Experimental Group 1 and Experimental Group 2 increased more than 1.5 times. In addition, the expression level of Cleaved PARP/PARP in FIG. 6d increased by 1.73 and 1.78 times in Experimental Group 1 (3Mix-1) and Experimental Group 2 (3Mix-3), respectively, compared to the control group, suggesting that the apoptosis promoting function by administration of 3MIX parabiotics It was confirmed that the antitumor effect was exhibited by significantly increasing the apoptosis of cancer cells.

2-3. Immunohistochemical examination of tumor tissue

Immunohistochemical analysis (IHC) was performed to confirm the expression of specific antigens involved in apoptosis by 3MIX parabiotics in tumor tissue recovered from an animal model in which human gastric cancer MKN1 cells were xenotransplanted, and the results are shown in FIG. showed

As shown in FIG. 7, it can be seen that the expression level of p-AKT was decreased in both experimental group 1 and experimental group 2 in which 3MIX parabiotics was orally administered compared to the control group. Expression levels of P53, Bax, cleaved caspase-9, cleaved caspase-3, and cleaved PARP were increased in both Experimental Group 1 and Experimental Group 2 compared to the control group, and were especially distributed in large quantities in Experimental Group 2 (3Mix-3).

Through the results of the Western blot and IHC, it was confirmed that 3Mix parabiotics can exhibit antitumor effects by inducing cancer cell death.

Korea Research Institute of Bioscience and Biotechnology Biological Resources Center (KCTC) KCTC13452BP 20180104 Korea Research Institute of Bioscience and Biotechnology Biological Resources Center (KCTC) KCTC14507BP 20210322 Korea Research Institute of Bioscience and Biotechnology Biological Resources Center (KCTC) KCTC14424BP 20210105

Claims (11)

Heat-killed Bifidobacterium bifidum MG731 ( Bi. bifidum MG731, accession number: KCTC13452BP), heat-killed Lactobacillus reuteri MG5346 ( L. reuteri MG5346, accession number: KCTC14507BP) and heat-killed Lactobacillus rhamnosus MG5200 ( L. rhamnosus MG5200, accession number: KCTC14424BP) comprising a mixed strain or a culture thereof, a pharmaceutical composition for preventing or treating gastric cancer.
delete The pharmaceutical composition for preventing or treating gastric cancer according to claim 1, wherein the heat killing is performed at a temperature of 70°C to 130°C for 10 minutes to 60 minutes.
The method of claim 1, wherein the Bifidobacterium bifidum MG731, Lactobacillus reuteri MG5346 and Lactobacillus rhamnosus MG5200 are included in a cell number ratio of 1: 1 to 2: 1 to 2, for preventing or treating gastric cancer. pharmaceutical composition.
Heat-killed Bifidobacterium bifidum MG731 ( Bi. bifidum MG731, accession number: KCTC13452BP), heat-killed Lactobacillus reuteri MG5346 ( L. reuteri MG5346, accession number: KCTC14507BP) and heat-killed Lactobacillus rhamnosus MG5200 ( L. rhamnosus MG5200, accession number: KCTC14424BP) containing a mixed strain or a culture thereof, a health functional food composition for preventing or improving gastric cancer.
delete Heat-killed Bifidobacterium bifidum MG731 ( Bi. bifidum MG731, accession number: KCTC13452BP), heat-killed Lactobacillus reuteri MG5346 ( L. reuteri MG5346, accession number: KCTC14507BP) and heat-killed Lactobacillus rhamnosus MG5200 ( L. rhamnosus MG5200, accession number: KCTC14424BP) comprising a mixed strain or a culture thereof, preventing or improving a feed additive composition for gastric cancer.
delete Mixing heat-killed cells; Including, the cell body is Bifidobacterium bifidum MG731 ( Bi. bifidum MG731, accession number: KCTC13452BP), Lactobacillus reuteri MG5346 ( L. reuteri MG5346, accession number: KCTC14507BP) and Lactobacillus rhamnosus MG5200 ( L. rhamnosus MG5200, accession number: KCTC14424BP), a method for producing a composition for preventing, improving or treating gastric cancer.
According to claim 9,
Freeze-drying the heat-killed cell mixture to powder; Further comprising a method for producing a composition for preventing, improving or treating gastric cancer.
The method of claim 9, wherein the Bifidobacterium bifiderm MG731, Lactobacillus reuteri MG5346 and Lactobacillus rhamnosus MG5200 are included in a cell number ratio of 1: 1 to 2: 1 to 2, preventing, improving or preventing gastric cancer A method for preparing a therapeutic composition.

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