Composition for activating gastrointestinal stem cells

Abstract

To provide a composition having the function of maintaining or promoting (activating) gastrointestinal epithelial stem cells.SOLUTION: Provided are a composition for activating gastrointestinal stem cells, comprising Lactobacillus lactic acid bacteria or a processed product of bacterial cells thereof, and a method for activating gastrointestinal stem cells, comprising contacting the composition with gastrointestinal stem cells.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a composition for activating gastrointestinal stem cells and a method for activating gastrointestinal stem cells. [Background technology]

[0002] The gastrointestinal epithelium is a single cell layer that covers the outermost layer of the gastrointestinal mucosa, and not only digests food and absorbs nutrients and water, but also functions as the first barrier to prevent the invasion of enteric bacteria and pathogens into the body. The gastrointestinal epithelium undergoes active turnover, and maintaining this homeostasis is an important factor supporting the health of the tissue.

[0003] Intestinal epithelial tissue is composed of "villi" protruding to the luminal side and "crypts" that invaginate into the mucosa side and contain proliferating epithelial cells. In order to keep this intestinal epithelial tissue, which is important for maintaining life, healthy at all times, all epithelial cells are replaced with new ones in just 3 to 5 days (see, for example, Non-Patent Document 1). This active turnover is made possible by a cell supply system carried by a very small number of intestinal epithelial stem cells localized at the very bottom of the intestinal crypts. The maintenance of the properties of intestinal epithelial stem cells is not cell-autonomous, but is maintained by the support of various cells in a unique microenvironment called the stem cell niche (see, for example, Non-Patent Document 2). These functions decrease with age, leading to a decrease in intestinal function and are thought to be involved in age-related diseases such as glucose metabolism disorders. It is also known that DNA damage and carcinogenic stress caused by oxidative stress and cell division stress can induce cellular senescence in intestinal epithelial tissue (see, for example, Non-Patent Document 3).

[0004] It has been previously known that calorie restriction and the NAD+ precursor nicotinamide riboside activate the SIRT1 / mTORC1 pathway and improve stem cell proliferation capacity, which declines with age (see, for example, non-patent literature 4 and 5).

[0005] On the other hand, methods of utilizing lactic acid bacteria against the aging of digestive tract cells are also being studied. As an example, a method of recovering intestinal barrier function using a specific strain of lactic acid bacteria Lactococcus lactis that has oxidative stress resistance (IL-8 suppression ability) focusing on the increase in interleukin 8 (IL-8) associated with increased oxidative stress in the intestine is known (see, for example, Patent Document 1). In addition, a method of utilizing a specific strain of Lactobacillus fermentum that has oxidative stress resistance (IL-8 suppression ability) and the function of regulating gene expression in small intestinal stem cells has been disclosed (see, for example, Patent Document 2). [Prior art documents] [Patent documents]

[0006] [Patent Document 1] JP 2020-191831 A [Patent Document 2] JP 2022-117889 A [Non-patent literature]

[0007] [Non-Patent Document 1] Cell Biol., 15: 19-33 (2014) [Non-Patent Document 2] Biochemistry 93(4): 503-511 (2021) [Non-Patent Document 3] Japanese Journal of Anti-Aging 9(1): 47-052 (2013) [Non-Patent Document 4] Aging Cell. 18: e12935 (2019) [Non-Patent Document 5] Cell. 166: 436-450 (2016) Summary of the Invention [Problem to be solved by the invention]

[0008] The methods disclosed in Patent Documents 1 and 2 aim to reduce oxidative stress in gastrointestinal epithelial cells, and do not directly maintain or promote (activate) gastrointestinal epithelial stem cells. Toward the realization of a society in which people live long and healthy lives, there is a demand for methods and compositions that can activate gastrointestinal stem cells using additional, hitherto unknown components or novel pathways. [Means for solving the problem]

[0009] As a result of extensive investigations, the present inventors have found that gastrointestinal stem cells can be activated by a specific lactic acid bacterium or a processed product of its cells, and have thus completed the present invention.

[0010] According to one embodiment, the present invention relates to a composition for activating gastrointestinal stem cells, comprising Lactobacillus lactic acid bacteria or a treated cell product thereof.

[0011] In the composition for activating gastrointestinal stem cells, the Lactobacillus lactic acid bacteria is preferably Lacticase Bacillus paracasei strain R0701 (NITE P-03952) or Lacticase Bacillus paracasei strain JCM1111.

[0012] In the composition for activating gastrointestinal stem cells, the treated cell product of the Lactobacillus lactic acid bacteria is a culture solution of the lactic acid bacteria, and the substrate of the culture solution preferably contains soybeans.

[0013] In the composition for activating gastrointestinal stem cells, the treated cell product of the Lactobacillus lactic acid bacteria is preferably a fermentation product or an extract product.

[0014] In the composition for activating gastrointestinal stem cells, the gastrointestinal tract is preferably the oral cavity, pharynx, esophagus, stomach, small intestine, large intestine, or anus.

[0015] The composition for activating gastrointestinal stem cells is preferably a food or drink composition.

[0016] The composition for activating gastrointestinal stem cells is preferably a cosmetic composition, a quasi-drug composition, or a pharmaceutical composition.

[0017] According to another embodiment, the present invention relates to a method for activating gastrointestinal stem cells, comprising the step of contacting the gastrointestinal stem cells with the above-mentioned composition for activating gastrointestinal stem cells, except for the method for treating humans.

[0018] According to another embodiment, the present invention relates to a method for activating gastrointestinal stem cells, comprising the steps of contacting the above-mentioned composition for activating gastrointestinal stem cells with stem cell niche cells, and co-culturing the stem cell niche cells with gastrointestinal stem cells, except for the method for treating humans.

[0019] In the method for activating gastrointestinal stem cells, the Lactobacillus lactic acid bacteria is preferably Lacticase Bacillus paracasei strain R0701 (NITE P-03952) or Lacticase Bacillus paracasei strain JCM1111.

[0020] In the method for activating gastrointestinal stem cells, it is preferable that the treated cell product of the Lactobacillus lactic acid bacteria is a culture solution of the lactic acid bacteria, the substrate of the culture solution contains soybeans, and the activation is performed without passing through the SIRT1 / mTORC1 pathway. Effect of the Invention

[0021] The composition for activating gastrointestinal stem cells and the method for activating gastrointestinal stem cells according to the present invention make it possible to maintain or promote gastrointestinal epithelial stem cells, leading to the improvement of organ function decline due to aging and the suppression of diseases that increase with aging. In addition, this leads to repair and regeneration promotion therapy for epithelial disorders such as epithelial regeneration failure in inflammatory bowel disease and radiation enteritis, or extensive mucosal damage after mucosal resection. [Brief description of the drawings]

[0022] [Figure 1]FIG. 1 is a graph showing the results of in vitro evaluation of the maintenance / promotion function of small intestinal stem cells. [Diagram 2] FIG. 2 is a graph showing the results of in vitro evaluation of the maintenance / promotion function of gastric epithelial stem cells. [Diagram 3] FIG. 3 is a graph showing the results of in vitro evaluation of the maintenance / promotion function of small intestinal stem cells in aged mice. [Figure 4] FIG. 4 is a graph showing the results of in vitro evaluation of the maintenance / promotion function of taste bud stem cells. [Diagram 5] FIG. 5 is a graph showing the results of in vivo evaluation of the maintenance / promotion function of small intestinal stem cells, depicting the influence of Paneth cells. [Figure 6] FIG. 6 is a graph showing the results of in vivo evaluation of the maintenance / promotion function of small intestinal stem cells, comparing the effects of Paneth cells in multiple subjects. [Figure 7] FIG. 7 is a graph showing the effect of a selective SIRT1 inhibitor as an in vivo evaluation result of the maintenance / promotion function of small intestinal stem cells. [Figure 8] FIG. 8 is a graph showing the results of in vivo evaluation of the maintenance / promotion function of small intestinal stem cells, comparing the effects of Paneth cells and a SIRT1-selective inhibitor in multiple subjects. [Figure 9] FIG. 9 is a graph showing the results of in vivo evaluation of the maintenance / promotion function of small intestinal stem cells, comparing the effects of Paneth cells and a SIRT1-selective inhibitor for subject 7. [Figure 10] FIG. 10 is a graph showing the results of in vivo evaluation of the maintenance / promotion function of small intestinal stem cells, comparing the effects of Paneth cells and a SIRT1-selective inhibitor for subject 8. [Figure 11] FIG. 11 is a graph showing the number of stem cell marker-positive cells in the crypts of the small intestine of mice administered with test sample 1, which is an in vivo evaluation result of the maintenance / promotion function of small intestinal stem cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] Hereinafter, an embodiment of the present invention will be described. However, the present invention is not limited to the embodiment described below.

[0024] [First embodiment: Composition for activating gastrointestinal stem cells] According to a first embodiment, the present invention relates to a composition for activating gastrointestinal stem cells, which comprises Lactobacillus lactic acid bacteria or a treated component of the bacteria.

[0025] The digestive tract refers to the path that food takes from the mouth to the anus, and specifically includes the mouth, pharynx, esophagus, stomach, small intestine (duodenum, jejunum, ileum), large intestine (cecum, colon, rectum), and anus. Specific examples of digestive tract stem cells that are activated by the composition of the present invention include, but are not limited to, tongue epithelial stem cells, taste bud stem cells, esophageal epithelial stem cells, gastric epithelial stem cells, and intestinal epithelial stem cells.

[0026] The gastrointestinal stem cells to be activated may be cells present in the gastrointestinal tract of a living organism. Alternatively, the gastrointestinal stem cells may be cells derived from a living body and existing outside the body, isolated cells, or cells forming tissues or organs together with other cells.Furthermore, the gastrointestinal stem cells may be cells induced to differentiate from pluripotent stem cells.

[0027] The living organism is not particularly limited as long as it has a digestive tract, but includes birds, mammals, fish, amphibians, reptiles, insects, crustaceans, arachnids, myriapods, mollusks, echinoderms, annelids, rotifers, nematodes, flatworms, and cnidarians. Mammals include primates such as humans, rodents such as mice and rats, carnivores such as dogs and cats, even-toed ungulates such as cows and sheep, odd-toed ungulates such as horses, lagomorphs such as rabbits, proboscideans such as elephants, marsupials such as kangaroos, and cetaceans such as dolphins and whales. Preferably, the digestive tract stem cells are cells in the human body or cells derived from humans.

[0028] Activation of gastrointestinal stem cells refers to maintaining or enhancing the self-renewal ability of the stem cells. In vitro, the maintenance or enhancement of the self-renewal ability of the stem cells can be evaluated by the number of organoids formed in organoid culture. Since organoid colonies are obtained from a single stem cell, the number of organoids in organoid culture can be used to evaluate the functionality of stem cells. In vivo, it can be evaluated by the number of stem cell marker-positive cells in immunohistochemical staining of pathological specimens. Stem cells can be identified by positive expression of leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5), olfactomedin 4 (Olfm4), and B cell-specific Moloney-MLV integration site 1 (Bmi-1).

[0029] The composition for activating gastrointestinal stem cells contains, as an active ingredient, either or both of the following (a) or (b): (a) Lactobacillus lactic acid bacteria (b) Treated Lactobacillus lactic acid bacteria cells Each component will be described below.

[0030] (a) Lactobacillus lactic acid bacteria Lactobacillus lactic acid bacteria refers to lactic acid bacteria belonging to the genus Lactobacillus. Examples of Lactobacillus lactic acid bacteria include Lactobacillus delbrueckii, Lactobacillus acidophilus, Lactobacillus gasseri, Lacticaceae bacillus ... Lactobacillus casei, Lactobacillus johnsonii, Lactobacillus crispatus, Lactobacillus helveticus, Lacticaseibacillus rhamnosus, Latilactobacillus sakei, Latilactobacillus curvatus, Lactiplantibacillus plantarum, Limosilactobacillus reuteri, Limosilactobacillus fermentum, Levilactobacillus brevis Examples of the Lactobacillus lactic acid bacteria include, but are not limited to, Lactobacillus brevis. The Lactobacillus lactic acid bacteria are preferably those belonging to the species Lacticaseibacillus paracasei.

[0031] The lactic acid bacteria of the genus Lactobacillus are most preferably Lacticaceae Bacillus paracasei R0701 strain, Lacticaceae Bacillus paracasei JCM1111 strain, or bacteria substantially equivalent thereto, and mutant strains having substantially equivalent functions. Lacticaceae Bacillus paracasei R0701 strain was deposited on July 27, 2023 at the Patent Microorganism Deposit Center of the National Institute of Technology and Evaluation (NITE) Biotechnology Center (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) under the accession number P-03952. The symbol R0701 relating to the strain is a number uniquely assigned to the strain by B&S Corporation, and the function of Lacticaceae Bacillus paracasei R0701 strain was revealed for the first time by the present invention. Hereinafter, in this specification, the Lacticacei Bacillus paracasei R0701 strain may be abbreviated to simply "R0701 strain". The Lacticacei Bacillus paracasei JCM1111 strain may be provided by the Microorganism Materials Development Laboratory, BioResource Research Center, RIKEN (3-1-1 Takanodai, Tsukuba, Ibaraki, Japan). Hereinafter, in this specification, the Lacticacei Bacillus paracasei JCM1111 strain may be abbreviated to simply "JCM1111 strain".

[0032] A bacterium substantially equivalent to the R0701 or JCM1111 strain is a bacterium having a 16S rRNA gene with, for example, 95% or more, preferably 98% or more, more preferably 99% or more, and even more preferably 100% sequence homology with the base sequence of the 16S rRNA gene of the R0701 or JCM1111 strain, and may be a bacterium having the same bacteriological properties as the R0701 or JCM1111 strain. In addition, a mutant strain of the R0701 or JCM1111 strain is any strain obtained from the parent strain R0701 or JCM1111, and may include a strain obtained by natural mutation or artificial gene mutation. Among such mutant strains, a mutant strain having a function substantially equivalent to that of the parent strain R0701 or JCM1111 may also be included in the composition of the present invention. The substantially equivalent function may be a function of activating gastrointestinal stem cells.

[0033] The bacterial cells may be in a dry or wet state, and may be live or dead. The composition containing live bacteria preferably further contains a soybean component. The soybean component is intended to contain soybean-derived components, including soybeans, soybean extracts, soybean milk, soybean meal, ground soybeans, and other soybean processed products such as tofu. The inclusion of the soybean component in addition to the live bacteria has the advantage that the soybean component is metabolized by the live bacteria to produce a metabolic product of the soybean component. In a composition containing live bacteria and a soybean component, the ratio of live bacteria to soybean component is 10:1. 5 ~10 10 It is preferable to use 0.001 to 1000 mg of soybean components per 10 6 ~10 9 It is more preferable that the amount of soybean component is 0.1 to 10 mg per piece.

[0034] (b) Treated Lactobacillus lactic acid bacteria cells The active ingredient contained in the composition of the present invention may be the bacterial cell itself (a), or may be a product obtained by subjecting the bacterial cell to some kind of treatment, and such a product is called a bacterial cell-treated product. The treatment of the bacterial cell may be any treatment, including, for example, crushing, disintegration, grinding, sterilization, solid-liquid separation, drying, separation, drug treatment, enzyme treatment, extraction treatment, etc., and the products of these multiple treatments, as well as their suspensions, dilutions, extracts, and supernatants, may also be included in the bacterial cell-treated product. Furthermore, the treated bacterial cell product may include a culture solution obtained by adding bacterial cells to a medium serving as a substrate, and a fermentation product obtained by adding bacterial cells to a substrate. In these treated bacterial cell products, the bacterial cells may be live or dead. The treated bacterial cell product may be a liquid, a solid, or a mixture thereof.

[0035] In a preferred embodiment, the treated bacterial cell product may be, for example, a component derived from a culture medium of the bacterial cells. The culture solution-derived component may be a culture solution or a component thereof obtained by adding the bacteria to a substrate and culturing the bacteria under a predetermined condition. The component may be, for example, a culture solution supernatant, a filtrate of the culture solution, or an extract of the culture solution, or a mixture thereof. More preferably, the culture solution-derived component of the bacteria is a culture solution or a component thereof obtained by adding the bacteria to a medium using soybean as a substrate and culturing the bacteria at 25 to 40°C for about 1 to 5 days. The medium using soybean as a substrate may be in the form of a boiled soybean pulp containing soybean pulp components, in the form of soybean milk from which soybean pulp components have been removed, or in the form of soybean milk from which acid and solid components have been removed. The culture solution using a medium using soybean as a substrate may also be called a soybean lactic acid bacteria fermentation product. In this case, the treated bacteria may be an extract consisting of a fraction extracted from the soybean lactic acid bacteria fermentation product with an alcohol such as ethanol. In this case, water can be removed from the extract by a conventional method, such as freeze-drying or spray-drying. The culture broth using soy milk as a substrate is advantageous in that the soybean lactic acid bacteria fermentation product is easily handled as a solid content, the clarified product obtained by filtering the culture broth using soy milk as a substrate is advantageous in that the soybean lactic acid bacteria fermentation product is easily handled as a liquid content, and the alcohol extracts thereof are advantageous in that they can be handled in a state in which proteins and the like have been removed and can be used after being concentrated.

[0036] In another preferred embodiment, the treated bacterial cell product may be, for example, a fermentation product of the bacterial cell. The fermentation product is a product obtained as a result of fermentation by the lactic acid bacteria using sugars or the like as a nutrient source. Specifically, fermentation products obtained by fermenting milk or the like as a substrate include, but are not limited to, yogurt and cheese.

[0037] In another preferred embodiment, the treated bacterial cell may be, for example, an enzyme-treated bacterial cell. The product obtained by enzymatically treating the bacterial cell is in a state in which the cell wall of the lactic acid bacteria is decomposed by the enzyme and the contents are dissolved. The enzyme is not particularly limited as long as it can decompose the cell wall of the lactic acid bacteria, and for example, lysozyme, β-N-acetyl-D-glucosaminidase, muramidase, achromopeptidase, etc. can be used.

[0038] The composition for activating gastrointestinal stem cells contains either (a) or (b) or both of them as an active ingredient, and may contain any additional ingredient suitable for the use and purpose of the composition. In this case, (a) may be a single type of bacteria or a mixture of multiple types of bacteria. Similarly, (b) may be a processed product of a single type of bacterial cell, a mixture of multiple types of bacteria that have been treated, or a mixture of multiple types of processed products obtained by treating a single type of bacterial cell.

[0039] A method for preparing a composition for activating gastrointestinal stem cells according to the present embodiment will be described. First, the active ingredient, Lactobacillus lactic acid bacteria, can be cultured and grown by a conventionally known method. Specifically, as a medium for growth, MRS (DE MAN, ROGOSA and SHARPE) agar medium and MRS broth can be mentioned. These MRS media contain glucose, protein hydrolysate, yeast extract, fatty acid, and minerals. Culture conditions can be, for example, 20 to 40°C and 1 to 5 days, but are not limited to specific conditions. The grown Lactobacillus lactic acid bacteria can be used as is or optionally after the above treatment and mixed with any additional components to prepare a composition for activating gastrointestinal stem cells.

[0040] The composition for activating gastrointestinal stem cells according to the present embodiment may vary in product form and additional components depending on its use and purpose. The uses of the composition and its product form are described below. The composition for activating gastrointestinal stem cells may be a composition used for the following purposes, for example. (i) Food and drink composition (ii) Cosmetic Composition (iii) Quasi-drug composition or pharmaceutical composition (iv) Compositions for experimental or biological tissue production

[0041] (i) Food and drink composition In some embodiments, the composition for activating gastrointestinal stem cells may be a food and drink composition. The food and drink composition may also include functional food compositions or specific health food compositions. The food and drink composition may be a food and drink composition for humans or animals, and may include, but is not limited to, bread, confectionery, noodles, meat products, seafood products, cereal products, processed vegetables, processed fruits, processed eggs, dairy products, flour, instant confectionery base, edible oils and fats, soup base, powdered drinks, seasonings, food additives, beverages, feed, etc. More specifically, general foods such as jelly foods, ice cream, cakes, candies, caramel, chewing gum, Japanese sweets, snacks, chocolate, Ramune candy, gummy candies, puddings, yogurt, soup, miso soup, rice, rice balls, meat, bread, udon, soba, ramen, pasta, konjac, pickles, natto, fried soybeans, flour, potato starch, gelatin, bread crumbs, pastes, retort foods, frozen foods, chilled foods, and instant foods; Examples of such beverages include drinking water, green tea beverages, black tea beverages, coffee beverages, fermented tea beverages (such as oolong tea), vegetable juice, milk, dairy beverages, fermented milk beverages, energy drinks, sports beverages, jelly beverages, and alcoholic beverages; seasonings such as furikake, sauce, soy sauce, fish sauce, miso, cooking sake, vinegar, mirin, oyster sauce, sauce, mayonnaise, ketchup, salt, spices, herbs, curry powder, edible oil, noodle soup, umami seasoning, spices, and flavor seasonings; and processed foods such as capsules, tablets, sugar-coated agents, granules, powders, liquid agents, edible films, and jellies, but are not limited to these.

[0042] In the food and drink composition, the Lactobacillus lactic acid bacteria or a processed product thereof may be contained as an active ingredient in an amount of, for example, about 0.001 to 1000 mg, preferably about 0.1 to 10 mg per 10 g of the food and drink composition. However, the content of the Lactobacillus lactic acid bacteria or a processed product thereof in the cosmetic composition is not limited to a specific numerical range.

[0043] The food and beverage composition may contain optional ingredients in addition to the active ingredient and the raw ingredients of the food, such as, but not limited to, excipients, binders, disintegrants, disintegration inhibitors, absorption promoters, adsorbents, lubricants, colorants, preservatives, fragrances, flavorings, sweeteners, etc. These optional ingredients may be blended within the range that achieves the purpose of the cosmetic composition according to the present embodiment. These food and beverage compositions may be produced by conventional methods commonly known in the art.

[0044] (ii) Cosmetic Composition In another embodiment, the composition for activating gastrointestinal stem cells may be a cosmetic composition, which may include oral cosmetics such as toothpaste, liquid toothpaste, and oral rinse, anti-aging cosmetics, cleansing cosmetics, hair cosmetics, bath cosmetics, medical cosmetics, and makeup cosmetics.

[0045] In the cosmetic composition, the Lactobacillus lactic acid bacteria or a processed product thereof may be contained as an active ingredient in an amount of, for example, about 0.001 to 1000 mg, preferably about 0.1 to 100 mg per 10 g of the cosmetic composition. However, the content of the Lactobacillus lactic acid bacteria or a processed product thereof in the cosmetic composition is not limited to a specific numerical range.

[0046] The cosmetic composition may contain optional ingredients in addition to the active ingredient, such as water, alcohol, surfactants, moisturizers, thickeners, waxes, petrolatum, hydrocarbon saturated fatty acids, unsaturated fatty acids, silicon oils and derivatives thereof, triglycerides such as tri(caprylic / capric)glyceryl and trioctanoic acid glyceryl, ester oils such as isopropyl stearate, natural fats and oils, preservatives, bactericides, UV absorbers, anti-inflammatory agents, antioxidants, chelating agents, animal and plant extracts, pH adjusters, vitamins, titanium oxide, talc, mica, silica, zinc oxide, iron oxide, silicon, and powders obtained by processing these, but are not limited thereto. These optional ingredients may be blended within the scope of achieving the objective of the cosmetic composition according to the present embodiment. These cosmetic compositions may be manufactured by conventional methods commonly known in the cosmetic field. The cosmetic composition may be in any form, such as a liquid, solid, powder, or emulsion composition.

[0047] (iii) Quasi-drug composition or pharmaceutical composition In another embodiment, the composition for activating gastrointestinal stem cells may be a quasi-drug composition or a pharmaceutical composition. Quasi-drug compositions include intestinal regulators, vitamins, throat fresheners, stomach fresheners, candies, gums, toothpastes and liquid toothpastes, mouthwashes, beverages, etc. Pharmaceutical compositions include, in particular, compositions for preventing, alleviating, and treating gastrointestinal symptoms.

[0048] In the quasi-drug composition, the Lactobacillus lactic acid bacteria or the processed product thereof may be contained as an active ingredient in an amount of, for example, about 0.001 to 1000 mg, preferably about 0.1 to 100 mg per 10 g of the quasi-drug composition. However, the content of the Lactobacillus lactic acid bacteria or the processed product thereof in the quasi-drug composition is not limited to a specific numerical range. In addition, in the pharmaceutical composition, the Lactobacillus lactic acid bacteria or the processed product thereof may be contained as an active ingredient in an amount of, for example, about 0.001 to 1000 mg, preferably about 0.1 to 100 mg per 10 g of the pharmaceutical composition. However, the content of the Lactobacillus lactic acid bacteria or the processed product thereof in the pharmaceutical composition is not limited to a specific numerical range.

[0049] In the quasi-drug composition or pharmaceutical composition, optional ingredients may be included in addition to the active ingredient, for example, pharma- ceutically acceptable bases, carriers, and additives (e.g., excipients, binders, disintegrants, lubricants, solvents, sweeteners, colorants, odorants, surfactants, moisturizers, preservatives, pH adjusters, thickeners, etc.) may be included as necessary, but are not limited to these. These optional ingredients can be blended within the scope of achieving the purpose of the quasi-drug composition or pharmaceutical composition according to the present embodiment. Such bases, carriers, additives, etc. are specifically disclosed, for example, in the Pharmaceutical Additives Dictionary 2021 (Yakuji Nippo Co., Ltd.), and a person skilled in the art can appropriately select the substances described in the document according to the purpose and include them in the composition.

[0050] The quasi-drug composition or pharmaceutical composition can be prepared by a conventional method generally known in the art.The formulation form is not particularly limited, and can be prepared into a formulation such as a tablet, a coated tablet, a powder, a pill, a granule, a fine granule, a chewable agent, a capsule, a liquid, an emulsion, a chewable agent, a soft tablet, a drink, etc.The administration form of the pharmaceutical composition can be, but is not limited to, oral, sublingual, nasal, enteral, or transdermal administration; intravenous, intramuscular, or subcutaneous injection; spray, or inhalation.

[0051] (iv) Compositions for experimental or biological tissue production In some embodiments, the composition for activating gastrointestinal stem cells may be a composition for experimental use or for the production of biological tissue. The composition for activating gastrointestinal stem cells can activate the proliferation function of stem cells, and therefore can be used, for example, as an experimental reagent for gastrointestinal stem cells, or as a composition for the production of biological tissue for experimental use or transplantation.

[0052] [Second embodiment: Method for activating gastrointestinal stem cells] According to a second embodiment, the present invention relates to a method for activating gastrointestinal stem cells, comprising the step of contacting gastrointestinal stem cells with the gastrointestinal stem cell activating composition according to the first embodiment.

[0053] The method for activating gastrointestinal stem cells may be, for example, any of in vitro, ex vivo, and in vivo methods. Therefore, the gastrointestinal stem cells to be contacted with the gastrointestinal stem cell activation composition according to the first embodiment may be isolated cells or cells in living tissues or organs in vivo or ex vivo.

[0054] The step of contacting the gastrointestinal stem cell activation composition with gastrointestinal stem cells can be carried out, in an in vitro or ex vivo method, by, for example, adding the gastrointestinal stem cell activation composition described in the first embodiment to a culture solution for isolated cells, or tissues, organs, etc. that contain gastrointestinal stem cells. Alternatively, in an in vivo method, for example, the gastrointestinal stem cell activation composition can be ingested by a living body as one of the various compositions described in the first embodiment.

[0055] According to the method for activating gastrointestinal stem cells according to the present embodiment, gastrointestinal stem cells can be activated without going through the SIRT1 / mTORC1 pathway. The SIRT1 / mTORC1 pathway is disclosed in detail in Non-Patent Documents 4 and 5, and is one of the pathways that restores stem cell proliferation ability that decreases with age by improving the activity of mTORC1, a protein complex containing SIRT1, an NAD+-dependent protein deacetylase, and mTOR, a type of protein kinase involved in intracellular signal transduction. The fact that the SIRT1 / mTORC1 pathway is not passed through can be confirmed by the fact that the proliferation ability of gastrointestinal stem cells is not reduced even when the method is carried out in the presence of a substance that can selectively suppress the activity of these proteins, for example, a SIRT1 inhibitor. Activating gastrointestinal stem cells without going through the SIRT1 / mTORC1 pathway is advantageous in that gastrointestinal stem cells can be activated additively or synergistically, particularly by combining with a component that activates a known pathway.

[0056] According to a variant of the second embodiment, the present invention relates to a method for activating gastrointestinal stem cells, comprising the steps of contacting a gastrointestinal stem cell activation composition according to the first embodiment with stem cell niche cells, and allowing the stem cell niche cells to coexist with gastrointestinal stem cells.

[0057] The stem cell niche refers to a microenvironment required for stem cells to maintain their properties in the body, and the cells that constitute the niche are called stem cell niche cells. Stem cell niche cells vary depending on the type of stem cell. For example, when the gastrointestinal stem cells are small intestinal epithelial stem cells, the stem cell niche cells are Paneth cells. Paneth cells are a lineage of terminally differentiated cells of the small intestinal epithelium, and are cells that are adjacent to small intestinal epithelial stem cells in the body and located at the base of the small intestinal crypts. Other combinations of gastrointestinal stem cells and stem cell niche cells include, but are not limited to, intestinal epithelial stem cells and intestinal neurons or tuft cells, enteroendocrine cells, interstitial cells, colonic stem cells and their surrounding non-stem cells, gastric epithelial stem cells and their niche cells, taste bud stem cells and their niche cells, and esophageal stem cells and their niche cells.

[0058] Even in the modified form, the in vivo method can be carried out, for example, by having a living body ingest the gastrointestinal stem cell activation composition as the various compositions described in the first embodiment. By having a living body ingest the composition, the composition can be brought into contact with stem cell niche cells, and the stem cell niche cells coexist with gastrointestinal stem cells in the living body.

[0059] In the in vitro or ex vivo method, the step of contacting the gastrointestinal stem cell activation composition according to the first embodiment with Paneth cells can be carried out by including the gastrointestinal stem cell activation composition described in the first embodiment in a culture solution for isolated stem cell niche cells or tissues or organs containing stem cell niche cells. The isolated stem cell niche cells are preferably cells derived from the same species or the same individual as the target gastrointestinal stem cells to be activated, or Paneth cells derived from pluripotent stem cells. The step of allowing the stem cell niche cells to coexist with gastrointestinal stem cells can be carried out by co-culturing the stem cell niche cells and the target gastrointestinal stem cells when the stem cell niche cells are isolated cells, and can be carried out by leaving the stem cell niche cells to stand without any special operation when the stem cell niche cells are in a tissue or organ and already coexist with the gastrointestinal stem cells.

[0060] According to the method of this modified embodiment, the gastrointestinal stem cell activation composition can be brought into contact with stem cell niche cells, without directly contacting the gastrointestinal stem cells, and the gastrointestinal stem cells can be activated via the activated stem cell niche cells. The method of this modified embodiment can provide a new option for activating gastrointestinal stem cells, particularly in vitro or ex vivo, and is particularly advantageous in the preparation of tissues for transplantation. EXAMPLES

[0061] The present invention will be described in more detail below with reference to examples, which are not intended to limit the present invention.

[0062] <Lacticase Bacillus paracasei strain R0701> The bacteriological properties of Lacticaceae Bacillus paracasei strain R0701 (NITE P-03952, hereinafter referred to as R0701 strain) were confirmed according to the method described in Bergey's manual of systematic bacteriology Vol. 2 (1986).

[0063] Table 1 shows the shape and other information of the Lacticaceae Bacillus paracasei R0701 strain. In Table 1, "+" indicates positive and "-" indicates negative. [Table 1]

[0064] Table 2 shows the results of sugar assimilation tests using API 50CHL (manufactured by bioMérieux) and the test items. In Table 2, "+" indicates fermentation and "-" indicates no fermentation. [Table 2]

[0065] <Preparation of lactobacillus-fermented soymilk extract> A test sample which was a gastrointestinal stem cell activating composition according to the present invention, and a comparative test sample which was a gastrointestinal stem cell activating composition not corresponding to the present invention, were prepared by the following method.

[0066] Subject 1 Soy milk was prepared from soybeans in a conventional manner, and then sterilized in an autoclave (121°C, 15 minutes) to obtain a soy milk medium. The R0701 strain was inoculated into 10 mL of soy milk medium and cultured at 30°C for 48 hours to obtain a preculture solution. Next, 0.001-fold volume of the preculture solution (approximately 10 6cfu) was added and cultured at 30°C for 96 hours. After sterilization at 90°C for 10 minutes, an equal amount of ethanol was added and stirred. The ethanol-fermented liquid was naturally filtered through filter paper with a retention particle size of 5 μm to obtain a filtrate. 10 mL of the obtained filtrate was used in a rotary evaporator to remove the ethanol, and then freeze-dried to obtain about 100 mg of a dried product, which was designated as sample 1. Sample 1 did not contain any live R0701 strain, but contained some dead bacteria and metabolites.

[0067] Subjects 2-5 Soy milk prepared in the same manner as in Test Sample 1 was adjusted to pH 4.0 with hydrochloric acid, the soy milk was coagulated, and then filtered through filter paper with a particle retention size of 5 μm to obtain a filtrate. The obtained filtrate was adjusted to pH 6.7-6.8 with sodium hydroxide, and again filtered through filter paper with a particle size of 5 μm. Thereafter, it was sterilized in an autoclave (121°C, 15 minutes) and used as a medium (clarified soy milk medium). Lactic acid bacteria (R0701 strain) were inoculated into 10 mL of the clarified soy milk medium and cultured at 30°C for 48 hours to obtain a preculture solution. Next, 0.001-fold volume of the preculture solution (approximately 10 6 cfu) was added and cultured at 30°C for 96 hours. After sterilization at 65°C for 10 minutes, the culture solution was divided into four equal parts, and ethanol was added to each part to make 0%, 25%, 50%, and 75%. The culture solution was stirred. Each sample was naturally filtered through filter paper with a retention particle size of 1 μm to obtain filtrate. The obtained filtrate was dried to an equivalent amount of clarified soy milk medium. That is, 5 mL (0%), 6.7 mL (25%), 10 mL (50%), and 20 mL (75%) were each subjected to rotary evaporator to remove ethanol, and then freeze-dried to obtain about 100 mg of dried material, which were named specimen 2 (0%), specimen 3 (25%), specimen 4 (50%), and specimen 5 (75%), respectively. None of specimens 2 to 5 contained live R0701 strain, but contained some dead bacteria and metabolites.

[0068] Subject 6 Lactic acid bacteria (R0701 strain) were inoculated into 10 mL of de Man-Rogosa-Sharpe (MRS) medium (OXOID) and cultured statically at 30° C. for 48 hours to prepare a preculture solution. Next, 0.001 volume of preculture solution (approximately 10 7 cfu) was added and cultured at 30°C for 96 hours. After sterilization at 65°C for 10 minutes, an equal amount of ethanol was added and stirred. The ethanol-added culture solution was naturally filtered through filter paper with a retention particle size of 1 μm to obtain a filtrate. 10 mL of the obtained filtrate was used in a rotary evaporator to remove the ethanol, and then freeze-dried to obtain about 200 mg of a dried product, which was designated as sample 6. Sample 6 did not contain any live R0701 strain, but contained some dead bacteria and metabolites.

[0069] Subject 7 Lacticaseibacillus paracasei subsp. paracasei JCM1111 strain was inoculated into 10 mL of the clarified soy milk medium prepared in the same manner as in samples 2 to 5, and the mixture was subjected to static culture at 30°C for 48 hours to prepare a preculture solution. Next, 0.001-fold volume of the preculture solution (approximately 10 6 cfu) was added and cultured at 30°C for 96 hours. After sterilization at 65°C for 10 minutes, an equal volume of ethanol was added and stirred. The ethanol-fermented liquid was naturally filtered through filter paper with a retention particle size of 1 μm to obtain a filtrate. 10 mL of the obtained filtrate was used in a rotary evaporator to remove the ethanol, and then freeze-dried to obtain about 100 mg of a dried product, which was designated as sample 7. The JCM1111 strain was obtained from the Microorganism Materials Development Laboratory, BioResource Research Center, RIKEN.

[0070] Subject 8 As in the case of the specimen 6, the R0701 strain was inoculated into 10 mL of MRS medium and cultured at 30° C. for 24 hours to prepare a preculture solution. 7cfu) was added and cultured at 30°C for 30 hours. The culture solution was centrifuged at 4,000×g for 5 minutes to obtain lactic acid bacteria cells as a precipitate. The obtained lactic acid bacteria cells were washed three times with phosphate-buffered saline (PBS) and then suspended in 10 mL of a protective solution (10% sucrose, 1% sodium glutamate). 1 mL of the cell suspension was dispensed into vials and freeze-dried to prepare specimen 8. After freeze-drying, the viable cell count when resuspended in 2 mL of sterile water per vial was approximately 1×10 10 cfu / mL.

[0071] Comparison sample 1 Soy milk was prepared from soybeans in a conventional manner, and then an equal amount of ethanol was added and stirred. The ethanol-added soy milk was naturally filtered through a filter paper with a particle size of 5 μm to obtain a filtrate. 10 mL of the obtained filtrate was used in a rotary evaporator to remove the ethanol, and then freeze-dried to obtain about 100 mg of a dried product, which was used as Comparative Sample 1. Comparative Sample 1 did not contain lactic acid bacteria or processed products thereof.

[0072] Comparison sample 2 The soy milk prepared in the same manner as in Test Sample 1 was adjusted to pH 4.0 with hydrochloric acid, the soy milk was coagulated, and then filtered through filter paper with a retention particle size of 5 μm to obtain a filtrate. The obtained filtrate was adjusted to pH 6.7-6.8 with sodium hydroxide, filtered again through filter paper with a particle size of 5 μm, and sterilized in an autoclave (121°C, 15 minutes). An equal amount of ethanol was added to 100 mL of clarified soy milk medium and stirred, and then naturally filtered through filter paper with a retention particle size of 1 μm to obtain a filtrate. The ethanol was removed from 10 mL of the obtained filtrate using a rotary evaporator, and then the filtrate was freeze-dried to obtain about 100 mg of a dried product, which was used as Comparative Test Sample 2. Comparative Test Sample 2 did not contain lactic acid bacteria or processed products thereof.

[0073] Comparative sample 3 An equal amount of ethanol was added to 100 mL of de Man-Rogosa-Sharpe (MRS) medium (OXOID) and stirred. The ethanol-added medium was naturally filtered through a filter paper with a retention particle size of 1 μm to obtain a filtrate. 10 mL of the obtained filtrate was evaporated in a rotary evaporator to remove the ethanol, and then freeze-dried to obtain about 270 mg of a dried product, which was used as Comparative Sample 3. Comparative Sample 3 did not contain lactic acid bacteria or processed products thereof.

[0074] The bacterial species, culture medium, and extraction conditions for test specimens 1 to 6 and comparative specimens 1 to 3 are shown in Table 3 below. [Table 3]

[0075] Test method 1: In vitro evaluation of the maintenance / promotion function of gastrointestinal stem cells The in vitro maintenance / promotion function of gastrointestinal stem cells in the subjects obtained above was evaluated by the number of organoids formed from small intestinal epithelial cells, gastric epithelial cells, or tongue epithelial cells. Since organoid colonies are obtained from a single stem cell, the number of organoids in organoid culture can be used to evaluate the functionality of stem cells. Organoid cultures from intestinal crypts, gastric epithelium, and circumvallate papillae isolated from the upper part of the mouse small intestine were performed using the following method. The isolated cells were embedded in Matrigel (Corning) at 10 cells / μL. A total of 300 isolated crypt cells, gastric epithelial cells, or circumvallate papilla cells were seeded per well of a 48-well plate and cultured in DMEM / F12 medium (Thermo Scientific) supplemented with 1xN2 (Thermo Scientific), 1xB27 (Thermo Scientific), 1mM N-acetyl-L-cysteine ​​(Sigma), 50ng / ml EGF (Thermo Scientific), 100ng / ml Noggin (Peprotech), and 1000ng / ml R-spondin (R&D) + CHIR99021 (or Wnt3a). For the evaluation of the subject, at the start of organoid culture, the subject solution or the control solution dissolved in 5mL of ethanol was added to the culture medium at a concentration of 0.05% or 0.15% by volume. In addition, EX527 (1μM), a selective inhibitor of SIRT1, was also added to the medium as necessary. The medium was replaced every other day. The number of organoids was counted under a microscope 5 days after the start of culture.

[0076] Test method 2: In vivo evaluation of the maintenance / promotion function of gastrointestinal stem cells - co-culture of stem cells and Paneth cells The in vivo maintenance / promotion function of the test specimens obtained above in gastrointestinal stem cell was evaluated by the number of organoids formed from isolated small intestinal stem cells and isolated Paneth cells obtained from mice administered the test specimen or comparison specimen. LgR5-GFP mice, aged 3.5 months or older, in which the intestinal epithelial stem cell marker LgR5 was labeled with GFP, were orally administered the test specimen or comparison specimen every day using a feeding needle. Each specimen was dissolved in 5 mL of ethanol, diluted 1 / 100 with 25% ethanol, and 300 μL was administered to mice weighing 30 g (10 μL / g BW). After administration for at least one month, small intestinal stem cells and Paneth cells were obtained using the following method.

[0077] Mouse small intestinal crypt suspension was centrifuged at 250×g for 5 min, and the resulting pellet was gently resuspended in 1 mL of TrypLE Express reagent (Life Technologies) + 120 μL of DNase I (10 U / μL, Roche). The mixture was heated in a 32°C water bath for 1.5 min, then placed on ice. 12 mL of chilled MEM was added, and the crypts were gently crushed. After centrifugation, the pellet was resuspended in MEM containing CD24-APC (1 / 500 Biolegend) and reacted on ice for 15 min. After centrifugation, the pellet was resuspended in MEM containing 1.5 μM propidium iodide (PI) (Thermo Scientific). The suspension was filtered through a 40 μm mesh (Corning), and the cells were separated using a FACS Aria (Becton Dickinson) as follows. Intestinal stem cells were isolated as Lgr5-EGFPhi CD24low / - PI- and Paneth cells were CD24hi SideScatterhi LgR5 EGFP- PI-.

[0078] Isolated intestinal stem cells or Paneth cells were suspended in medium containing 1x N2 supplement, 1x B27 supplement, and 10 μM Y-27632 (WAKO). Intestinal stem cells (2,000 cells) and Paneth cells (2,000 cells, with or without) were seeded in 30 μL Matrigel containing 1 μM Jagged-1 (AnaSpec) and 10 μM Y-27632. Matrigel drops containing intestinal stem cells with or without Paneth cells were allowed to solidify on a 48-well plate for 15 min in a 37°C incubator. Medium containing 1x N2 supplement, 1x B27 supplement, 1 mM N-acetyl-cysteine, 50 ng / ml EGF, 200 ng / ml Noggin, and 1 μg / ml R-Spondin was layered on top of the Matrigel drops and cultured at 37°C. When inhibitors were used, EX527 (Santa Cruz) was added to the medium at 1 μM. The medium was replaced every two days. The number of organoids was measured by counting the number of colonies with a lumen on the fifth day of culture.

[0079] Test method 3: In vivo evaluation of the maintenance / promotion function of gastrointestinal stem cells - organoid culture from intestinal crypts The in vivo maintenance / promotion function of gastrointestinal stem cells of the test specimens obtained above was evaluated by the number of organoids formed from the small intestinal crypts obtained from mice administered the test specimens. Specifically, the test specimens or control specimens were orally administered daily to mice aged 3.5 months or older using a feeding needle. Each specimen was dissolved in 5 mL of ethanol, diluted 1 / 100 with 25% ethanol, and 300 μL was administered to mice weighing 30 g (10 μL / g BW). After at least one month of administration, organoids were cultured using intestinal crypts isolated from the upper part of the small intestine. The isolated intestinal crypts were embedded in Matrigel (Corning) at 10 cells / μL. A total of 300 isolated crypts were seeded per well of a 48-well plate and cultured in DMEM / F12 medium (Thermo Scientific) supplemented with 1x N2 supplement (Thermo Scientific), 1x B27 supplement (Thermo Scientific), 1mM N-acetyl-L-cysteine ​​(Sigma), 50ng / ml EGF (Thermo Scientific), 200ng / ml Noggin (Peprotech), and 1000ng / ml R-spondin 1 recombinant protein (R&D) + CHIR99021 (WAKO). The medium was changed every other day. The number of organoids was counted under a microscope 5 days after the start of culture.

[0080] Test method 4: In vivo evaluation of the maintenance / promotion function of gastrointestinal stem cells - intestinal crypt Olfm4 positive cell count The maintenance / promotion function of gastrointestinal stem cells in vivo in the test specimens obtained above was evaluated by quantifying stem cell marker-positive cells in small intestinal pathological specimens in mice administered the test specimen or comparative specimen. Specifically, the test specimen or comparative specimen was orally administered to mice aged 3.5 months or older every day using a feeding needle. Each specimen was dissolved in 5 mL of ethanol, diluted 1 / 100 with 25% ethanol, and 300 μL was administered to mice weighing 30 g (10 μL / g BW). After administration for at least one month, a fragment of the jejunum (1-4 cm from the pylorus) was collected and the number of Olfm4-positive stem cells was counted by immunostaining with an antibody specific to the Olfm4 gene, a marker gene for intestinal epithelial stem cells. The jejunum fragment was fixed overnight in 10% formalin at room temperature, embedded in paraffin, and sectioned. The quantification of Olfm4-positive cells was performed by counting Olfm4-positive cells from intact and highly oriented crypts under a microscope.

[0081] Test Example 1: In vitro evaluation of the maintenance / promotion function of small intestinal stem cells The function of the extract from soy milk fermented with the R0701 strain (subject 1) in maintaining and promoting small intestinal stem cells was evaluated. The method was the same as Test Method 1 above, and the evaluation was based on the number of organoids formed from mouse-derived small intestinal crypt cells. The control was cultured in a medium that did not contain the extract.

[0082] The results are shown in Figure 1. As is clear from Figure 1, it was confirmed that the number of organoids obtained increased by culturing in a composition for activating the digestive tract, to which an extract of soy milk fermented with the R0701 strain was added at a concentration of 0.05% as a test solution. Since an organoid colony is obtained from a single stem cell, the number of organoids in an organoid culture depends on the functionality of the stem cell. Therefore, it was confirmed that the extract of soy milk fermented with the R0701 strain has the ability to activate small intestinal stem cells.

[0083] Test Example 2: In vitro evaluation of the maintenance / promotion function of gastric epithelial stem cells The maintenance / promotion function of gastric epithelial stem cells of subject 1 was evaluated. The method was according to the above-mentioned test method 1, and the evaluation was performed based on the number of organoids formed from mouse-derived gastric fundus gland cells. The control was cultured in a medium containing no extract. The results are shown in Figure 2. As is clear from Figure 2, the number of organoids obtained was increased by culturing the subject liquid in a composition for activating the digestive tract, to which an extract of soy milk fermented with the R0701 strain was added at a concentration of 0.05% or 0.15%. Since organoid colonies are obtained from a single stem cell, the number of organoids in organoid culture depends on the functionality of the stem cell. Therefore, it was confirmed that the extract of soy milk fermented with the R0701 strain has the ability to activate gastric epithelial stem cells.

[0084] Test Example 3: In vitro evaluation of the maintenance / promotion function of small intestinal stem cells in aged mice The maintenance / promotion function of small intestinal stem cells in old mice (subject 1) was evaluated. The method was according to the above-mentioned test method 1, and the evaluation was performed based on the number of organoids formed from small intestinal crypt cells derived from old mice (24 months old). Note that the one cultured in a medium containing no extract was used as the control. The results are shown in Figure 3. As is clear from Figure 3, it was observed that the number of organoids obtained was significantly increased by culturing in a composition for activating the digestive tract to which an extract of soy milk fermented with the R0701 strain was added at a concentration of 0.15% as the subject liquid. Since organoid colonies are obtained from a single stem cell, the number of organoids in organoid culture depends on the functionality of the stem cell. Therefore, it was confirmed that the extract of soy milk fermented with the R0701 strain has the ability to activate small intestinal stem cells weakened by aging.

[0085] Test Example 4: In vitro evaluation of the maintenance / promotion function of taste bud stem cells The maintenance / enhancement function of taste bud stem cells in subject 1 was evaluated. The method was according to the above-mentioned test method 1, and the evaluation was performed based on the number of organoids formed from mouse-derived circumvallate papilla cells. The control was cultured in a medium containing no extract. The results are shown in Figure 4. As is clear from Figure 4, it was found that the number of organoids obtained was significantly increased by culturing in a composition for activating the digestive tract to which an extract of soy milk fermented with the R0701 strain was added at a concentration of 0.15% as the subject liquid. Since organoid colonies are obtained from a single stem cell, the number of organoids in organoid culture depends on the functionality of the stem cell. Therefore, it was confirmed that the extract of soy milk fermented with the R0701 strain has the ability to activate taste bud stem cells.

[0086] Test Example 5: In vivo evaluation of the maintenance / promotion function of small intestinal stem cells (1) The maintenance / promotion function of small intestinal stem cells in subject 1 was evaluated in a mouse administration test. The method was according to the above-mentioned test method 2, and the number of organoids formed by co-culture of small intestinal stem cells and Paneth cells obtained from mice administered subject 1 was evaluated. Note that mice raised without soy milk extract were used as controls. The results are shown in Figure 5. As is clear from Figure 5, the number of organoids formed was significantly increased in the co-culture of stem cells and Paneth cells from mice raised by administering an extract of soy milk fermented with the R0701 strain, compared to the co-culture of stem cells and Paneth cells from control mice. Since organoid colonies are obtained from a single stem cell, the number of organoids in organoid culture depends on the functionality of the stem cells. Therefore, it was confirmed that the composition for activating the digestive tract containing an extract of soy milk fermented with the R0701 strain has the ability to activate small intestinal stem cells. In addition, the number of organoids formed was also increased when stem cells from control mice were co-cultured with Paneth cells from mice administered subject 1. In other words, it was confirmed that a composition containing a soymilk extract fermented with the R0701 strain has the function of enhancing stem cell function by activating Paneth cells.

[0087] Test Example 6: In vivo evaluation of the maintenance / promotion function of small intestinal stem cells (2) The maintenance / promotion function of small intestinal stem cells of clarified soymilk fermented with the R0701 strain of the present invention (specimen 2) and its extracts (specimens 3-5) and comparative specimen 2 were evaluated in a mouse administration test. The method was according to the above test method 2, and the number of organoids formed by co-culture of Paneth cells obtained from mice administered with specimens 2-5 and comparative specimen 2 with stem cells from control mice was evaluated. Note that mice kept without receiving the soymilk extract served as controls.

[0088] The results are shown in FIG. 6. As is clear from FIG. 6, the co-culture of stem cells with Paneth cells from mice fed with clarified soy milk fermented with the R0701 strain (specimen 2) and its extracts (specimens 3 to 5) significantly increased the number of organoids formed compared to the co-culture of Paneth cells from control-fed mice and comparative specimen 2-fed mice. Since organoid colonies are obtained from a single stem cell, the number of organoids in organoid culture depends on the functionality of the stem cell. Therefore, it was confirmed that the composition for activating the digestive tract, which contains clarified soy milk fermented with the R0701 strain and its extract, has the ability to activate small intestinal stem cells. It was also confirmed that the composition has the ability to activate small intestinal stem cells under various extraction conditions.

[0089] Test Example 7: In vivo evaluation of the maintenance / promotion function of small intestinal stem cells (3) The function of the extract from soy milk fermented with the R0701 strain of the present invention (subject 1) in maintaining / promoting small intestinal stem cells was compared with that of comparative specimen 1 in a mouse administration test. The method was according to the above-mentioned test method 2, and the number of organoids formed by co-culture of Paneth cells obtained from mice administered with subject 1 and comparative specimen 1 with stem cells from control mice was evaluated. Note that mice kept without the extract were used as controls.

[0090] The results are shown in FIG. 7. As is clear from FIG. 7, the number of organoids formed in the co-culture of stem cells with Paneth cells from mice fed with an extract of soy milk fermented with the R0701 strain (subject 1) was significantly increased compared to the co-culture of Paneth cells from control mice. Since organoid colonies are obtained from a single stem cell, the number of organoids in the organoid culture depends on the functionality of the stem cell. Therefore, it was confirmed that the composition for activating the digestive tract containing an extract of soy milk fermented with the R0701 strain has the ability to activate small intestinal stem cells.

[0091] In addition, the number of organoids formed also increased when Paneth cells from mice administered comparative sample 1 were co-cultured. However, the activity was suppressed by the addition of EX527, a SIRT1 inhibitor. On the other hand, when Paneth cells from mice administered sample 1 were co-cultured, no suppression by EX527 was observed. In other words, it was confirmed that the composition for activating the digestive tract containing the soy milk extract fermented with the R0701 strain has a function of activating stem cell functions different from the existing SIRT1 / mTORC1 pathway.

[0092] Test Example 8: In vivo evaluation of the maintenance / promotion function of small intestinal stem cells (4) The maintenance / promotion function of small intestinal stem cells of an extract from clarified soy milk (sample 4), an extract from soy milk cultured in MRS medium (sample 6), and extracts from each medium only without lactic acid bacteria (comparison samples 2 and 3) were evaluated in a mouse administration test. Mice raised without the extract served as controls, and the method was the same as above-mentioned test method 2, and the number of organoids formed by co-culture of Paneth cells obtained from mice administered sample 4 and sample 6 with stem cells from control mice was evaluated.

[0093] The results are shown in FIG. 8. As is clear from FIG. 8, the co-culture of stem cells with Paneth cells from mice fed with soy milk extract (subject 4) and MRS medium culture extract (subject 6) significantly increased the number of organoids formed compared to the co-culture of Paneth cells from control-fed mice, comparative specimens 2, and comparative specimens 3-fed mice. Since organoid colonies are obtained from a single stem cell, the number of organoids in organoid culture depends on the functionality of the stem cells. Therefore, it was confirmed that the composition for activating the digestive tract containing the extract cultured with the R0701 strain has the ability to activate Paneth cells, thereby activating small intestinal stem cells.

[0094] Furthermore, the results showed that activation by sample 6 was inhibited by EX527, whereas activation by sample 4 was not inhibited. In other words, it was predicted that the mechanism of small intestinal stem cell activation differs depending on the culture substrate, and that substrates containing soybeans in particular are useful for stem cell activation not mediated by the SIRT1 / mTORC1 pathway. In any case, there is no doubt that the composition for gastrointestinal activation containing an extract cultured with the R0701 strain has the ability to activate small intestinal stem cells.

[0095] Test Example 9 In vivo evaluation of the maintenance / promotion function of small intestinal stem cells (5) The function of an extract of clarified soy milk fermented with JCM1111 strain (subject 7) in maintaining / promoting small intestinal stem cells was evaluated in an administration test in mice. The method was the same as Test Method 2 above, and the number of organoids formed by co-culturing Paneth cells obtained from subject 7 or control mice with stem cells from control mice was evaluated. Mice kept without the extract served as controls.

[0096] The results are shown in FIG. 9. As is clear from FIG. 9, the number of organoids formed in the co-culture of stem cells with Paneth cells from mice fed with an extract of clarified soy milk fermented with the JCM1111 strain (subject 7) was significantly increased compared to the co-culture of Paneth cells from control-fed mice. Since organoid colonies are obtained from a single stem cell, the number of organoids in the organoid culture depends on the functionality of the stem cell. Therefore, it was confirmed that the composition for activating the digestive tract containing an extract of clarified soy milk fermented with the JCM1111 strain has the ability to activate small intestinal stem cells. In addition, this activity was not suppressed by the addition of EX527, a SIRT1 inhibitor, and therefore it was shown to have the same activity as the composition for activating the digestive tract containing a soy milk extract fermented with the R0701 strain.

[0097] Test Example 10 In vivo evaluation of the maintenance / promotion function of small intestinal stem cells (6) The maintenance / promotion function of the R0701 strain live bacteria (subject 8) in small intestinal stem cells was evaluated in an administration test in mice. The method was according to the above-mentioned test method 2, and the number of organoids formed by co-culturing Paneth cells obtained from mice administered with subject 8 and soybean extract (equivalent to comparative sample 2) with stem cells from control mice was evaluated. The administration of the subject was performed in the following manner. Subject 8 was administered 10 8 The mice were suspended in sterile water to obtain cfu / individual, and soybean extract (equivalent to comparative sample 2) was suspended in PBS to obtain 1 mg / 30 g BW and administered daily. Mice were similarly administered PBS and bred as controls. The administration period was 2 months.

[0098] The results are shown in FIG. 10. As is clear from FIG. 10, the number of organoids formed in the co-culture of stem cells with Paneth cells from mice fed with the R0701 strain live bacteria (specimen 8) and soybean components was significantly increased compared to the co-culture of Paneth cells from control mice and mice fed only with specimen 8. Since organoid colonies are obtained from a single stem cell, the number of organoids in the organoid culture depends on the functionality of the stem cell. Therefore, it was confirmed that the R0701 strain live bacteria has the ability to activate small intestinal stem cells in the presence of soybean components. In addition, this activity was not suppressed by the addition of EX527, a SIRT1 inhibitor, and therefore it was shown to have the same activity as the composition for activating the digestive tract containing a soybean milk extract fermented with the R0701 strain.

[0099] Test Example 11: In vivo evaluation of the maintenance / promotion function of small intestinal stem cells (7) The maintenance / promotion function of small intestinal stem cells in test subject 1 was evaluated in a mouse administration test. Mice kept without the extract were used as controls, and the number of stem cell marker-positive cells in the small intestinal crypts of test subject 1-administered mice and control mice was measured according to the above test method 3.

[0100] The results are shown in Figure 11. As is clear from Figure 11, the number of stem cell marker-positive cells in the small intestinal crypts of the mice that were administered sample 1 was found to be greater than that of the control mice. Therefore, it was confirmed that the composition for activating the digestive tract containing an extract of soymilk fermented with the R0701 strain has the ability to activate small intestinal stem cells.

Claims

1. A composition for activating gastrointestinal stem cells, comprising Lactobacillus lactic acid bacteria or a processed product of said bacteria.

2. The composition for activating gastrointestinal stem cells according to claim 1, wherein the Lactobacillus lactic acid bacteria is Lacticaceobacillus paracasei R0701 strain (NITE P-03952) or Lacticaceobacillus paracasei JCM1111 strain.

3. The composition for activating gastrointestinal stem cells according to claim 1 , wherein the treated cell product of the Lactobacillus lactic acid bacteria is a culture solution of the lactic acid bacteria, and a substrate of the culture solution contains soybeans.

4. The composition for activating gastrointestinal stem cells according to claim 1 , wherein the treated cell product of the Lactobacillus lactic acid bacteria is a fermentation product or an extract product.

5. The composition for activating gastrointestinal stem cells according to claim 1 , wherein the gastrointestinal tract is the oral cavity, pharynx, esophagus, stomach, small intestine, large intestine, or anus.

6. The composition for activating gastrointestinal stem cells according to claim 1, which is a food or drink composition.

7. The composition for activating gastrointestinal stem cells according to claim 1 , which is a cosmetic composition, a quasi-drug composition, or a pharmaceutical composition.

8. A method for activating gastrointestinal stem cells (excluding methods for treating humans), comprising the step of contacting the composition according to claim 1 with gastrointestinal stem cells.

9. A method for activating gastrointestinal stem cells (excluding methods for treating humans), comprising the steps of contacting the composition described in claim 1 with stem cell niche cells and co-culturing the stem cell niche cells with gastrointestinal stem cells.

10. The method according to claim 8 or 9, wherein the Lactobacillus lactic acid bacterium is Lacticaceobacillus paracasei R0701 strain (NITE P-03952) or Lacticaceobacillus paracasei JCM1111 strain.

11. The method according to claim 8 or 9, wherein the treated cell product of the Lactobacillus lactic acid bacterium is a culture solution of the lactic acid bacterium, the substrate of the culture solution contains soybean, and the activation is performed without passing through the SIRT1 / mTORC1 pathway.

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