Intestinal barrier protective agent and prophylactic agent against intestinal barrier disruption-related disease

Abstract

Provided is an intestinal bacterium for protecting intestinal barrier function by including Bifidobacterium adolescentis as an active ingredient, which exhibits higher efficacy than conventional strains, wherein as an active component of the intestinal barrier protective agent, there is a cell wall polysaccharide fraction and / or cytoplasmic fraction of Bifidobacterium adolescentis, comprising a polysaccharide having a repeating structure of rhamnose, glucose, and galactose at a ratio of 4-3:1:2-1, fractionated using an ultrafiltration membrane to obtain molecules with a molecular weight of 10,000-30,000, or uridine diphosphate-N-acetylmuramic acid-L-alanine-D-glutamic acid-L-lysine.

Description

Title of Invention: INTESTINAL BARRIER PROTECTIVE AGENT AND PROPHYLACTIC AGENT AGAINST INTESTINAL BARRIER DISRUPTION-RELATED DISEASE Technical Field

[0001] The present invention relates to an intestinal barrier protective agent and a prophylactic agent against an intestinal barrier disruption-related disease, each containing, as an active ingredient, Bifidobacterium adolescentis or a component derived from Bifidobacterium adolescentis. Background Art

[0002] In recent years, attention has been focused on the influence of intestinal bacteria on the pathology of metabolic disorders in the host, particularly the pathogenesis of obesity and diabetes. Currently, intestinal bacteria are considered to be a cause of obesity, along with dietary habits and lack of exercise. Therefore, novel treatment methods for obesity are being explored, including altering intestinal bacterial flora of obese patients and improving the intestinal environment.

[0003] Impairment of the intestinal bacterial flora (dysbiosis) is caused by various factors (stress, aging, alcohol, smoking, poor diet, antibiotics, lack of physical activity, etc.), and as a result, the intestinal barrier function is disrupted by three stimulations: stimulation by lipopolysaccharides (LPS), which are a toxin derived from intestinal bacteria; stimulation by inflammatory cytokines in the intestinal tract; and stimulation by oxidative stress. It is known that this disruption allows LPS and inflammatory cytokines to enter and circulate in the bloodstream and to reach adipose tissue and the liver, which determine insulin sensitivity, causing chronic inflammation and insulin resistance (NPL 1 and 2).

[0004] Therefore, protecting the intestinal barrier function is considered to allow prevention of diseases related to disruption of the intestinal barrier, such as obesity and diabetes.

[0005] Although intestinal bacteria for protecting the intestinal barrier function have been explored to date, those reported so far include the intestinal barrier protection effect of Bifidobacterium bifidum and Bifidobacterium animalis against cytokine stimulations (NPL 3 to 6), the intestinal barrier protection effect of Bifidobacterium longum and Bifidobacterium animalis against LPS stimulations (NPL 7 and 8), and the like. However, no intestinal bacteria having intestinal barrier protection effects against a plurality of stimulations have been reported. Citation List Non Patent Literature

[0006] NPL 1: Coppe. J-P, et al., PLoS Biol. 6.2853.2008 NPL 2: Winer. D. A, et al., Cell Metab. 23.413. 2016 NPL 3: Al-Sadi. R, et al., Int. J. Mol. Sci. 22.8070.2021 NPL 4: Hsieh. C. Y, et al., Physiol Rep. 3.3. e12327.2015 NPL 5: Wang. X, et al., Biomedicines. 9.641. 2011 NPL 6: Castro-Herrera. V. M, et al., Nutrients. 12.1719.2020 NPL 7: Zhao. L, et al., J. Funct. Foods. 92.105030.2022 NPL 8: Kim. J.Y, et al., Front Microbiol. 13.817591.2022 Summary of Invention Technical Problem

[0007] The present invention is to address problems relating to identifying intestinal bacteria for protecting the intestinal barrier function with higher efficacy than conventional ones, and further to identifying their active substances. Solution to Problem

[0008] The present inventors conducted extensive studies in order to address the above problems, and as a result, found that specific Bifidobacteria exhibit excellent intestinal barrier function protection effects, and further identified their active substances, and completed the present invention.

[0009] Specifically, the present invention is as follows. [1] An intestinal barrier protective agent including, as an active ingredient, Bifidobacterium adolescentis. [2] The intestinal barrier protective agent according to [1], wherein the intestinal barrier protection is protection of the intestinal barrier from one or more stimulations selected from among LPS stimulation, inflammatory cytokine stimulation, and oxidative stress stimulation. [3] A prophylactic agent against an intestinal barrier disruption-related disease, including the intestinal barrier protective agent according to [1] or [2]. [4] The prophylactic agent against an intestinal barrier disruption-related disease according to [3], wherein the intestinal barrier disruption-related disease is selected from among diabetes, obesity, inflammatory bowel disease, fatty liver, Parkinson’s disease, cancer, irritable bowel syndrome, dyslipidemia, atopic dermatitis, asthma, dementia, insomnia, depression, and rheumatoid arthritis. [5] An intestinal barrier protective agent including, as an active ingredient, a cell wall polysaccharide fraction and / or cytoplasmic fraction of Bifidobacterium adolescentis. [6] An intestinal barrier protective agent including, as an active ingredient, a polysaccharide having a repeating structure of rhamnose, glucose, and galactose in a ratio of (4 to 3):1:(2 to 1), wherein the polysaccharide is recovered from a 10,000- to 30,000-molecular-weight-cutoff ultrafiltration fraction. [7] An intestinal barrier protective agent, including, as an active ingredient, uridine diphosphate-N-acetylmuramic acid-L-alanine-D-glutamic acid-L-lysine. [8] A polysaccharide having a repeating structure of rhamnose, glucose, and galactose in a ratio of (4 to 3):1:(2 to 1), wherein the polysaccharide is recovered from a 10,000- to 30,000-molecular-weight-cutoff ultrafiltration fraction. [9] A method of producing a polysaccharide having a repeating structure of rhamnose, glucose, and galactose in a ratio of (4 to 3):1:(2 to 1) , wherein the polysaccharide is recovered from a 10,000- to 30,000-molecular-weight-cutoff ultrafiltration fraction, the method including: subjecting Bifidobacterium adolescentis to a lysis treatment, then obtaining a fraction corresponding to a molecular weight range defined by ultrafiltration membranes having molecular weight cutoffs of 10,000 to 30,000, and additionally collecting these fractions through hydrophilic interaction liquid chromatography performed under the following conditions or equivalent conditions: [Chromatography conditions] Flow rate: 1.0 mL / min Mobile phase A: MeCN, B: H2O Gradient: 0 to 3.3 min: A=95, B=5, 3.3 to 83.3 min: A=95^0, B=5^100 Column: YMC-triart Diol HILIC (^4.6 x 250 mm, 5 pm) Temperature: 40°C Injection volume: 2 to 10 pL (5 mg / mL in H2O) Elution time: 22 to 35 min.

[10] The method of producing a polysaccharide according to [9], wherein the lysis treatment includes a treatment with a muramidase, followed by a treatment with a nuclease and a protease.

[11] A prophylactic agent against an intestinal barrier disruption-related disease, including the intestinal barrier protective agent according to any one of [5] to [7].

[12] The prophylactic agent against an intestinal barrier disruption-related disease according to

[11] , wherein the intestinal barrier disruption-related disease is selected from among diabetes, obesity, inflammatory bowel disease, fatty liver, Parkinson’s disease, cancer, irritable bowel syndrome, dyslipidemia, atopic dermatitis, asthma, dementia, insomnia, depression, and rheumatoid arthritis. Advantageous Effects of Invention

[0010] The intestinal barrier protective agent of the present invention contains, as an active ingredient, Bifidobacterium adolescentis present in the intestines, a cell wall polysaccharide fraction and / or cytoplasmic fraction of Bifidobacterium adolescentis, a polysaccharide derived from the fraction having a repeating structure of rhamnose, glucose, and galactose in a ratio of (4 to 3):1:(2 to 1) , wherein the polysaccharide is recovered from a 10,000- to 30,000-molecular-weight-cutoff ultrafiltration fraction, or uridine diphosphate-N-acetylmuramic acid-L-alanine-D-glutamic acid-L-lysine. Therefore, it can safely protect the intestinal barrier for a long period of time.

[0011] In addition, since the intestinal barrier protective agent of the present invention can protect the intestinal barrier, it is also useful as a prophylactic agent for diseases related to the disruption of the intestinal barrier.

[0012] In addition, the polysaccharide of the present invention has a repeating structure of rhamnose, glucose, and galactose in a ratio of (4 to 3):1:(2 to 1), and a molecular weight of 10,000 to 30,000 as determined by an ultrafiltration membrane, and this polysaccharide can be used in various applications. Brief Description of Drawings

[0013] [Fig. 1] Fig. 1 is a diagram showing the relationship between the time and the TEER in the LPS-stimulated monolayer membrane of Example 1. [Fig. 2] Fig. 2 is a diagram showing the TEER at 48 hours in the LPS-stimulated monolayer membrane of Example 1. [Fig. 3] Fig. 3 is a diagram showing the relationship between the time and the TEER in the monolayer membrane stimulated with inflammatory cytokines of Example 1. TI indicates TNF-a+IFN-Y-[Fig. 4] Fig. 4 is a diagram showing the TEER at 48 hours in the monolayer membrane stimulated with inflammatory cytokines of Example 1. TI indicates TNF-a+IFN-Y-[Fig. 5] Fig. 5 is a diagram showing the relationship between the time and the TEER in the oxidative stress-stimulated monolayer membrane of Example 1. [Fig. 6] Fig. 6 is a diagram showing the TEER at 6 hours in the oxidative stress-stimulated monolayer membrane of Example 1. [Fig. 7] Fig. 7 shows diagrams showing the influence on localization of tight junction proteins in Example 1 (+: with YIT 13021 added, -: without YIT 13021). [Fig. 8] Fig. 8 is a diagram showing the relationship between the time and the TEER in the unstimulated monolayer membrane of Example 2. [Fig. 9] Fig. 9 is a diagram showing the TEER at 6 hours in the unstimulated monolayer membrane of Example 2. [Fig. 10] Fig. 10 is a diagram showing the TEER at 48 hours in the LPS-stimulated monolayer membrane of Example 3. [Fig. 11] Fig. 11 is a diagram showing the TEER at 48 hours in the monolayer membrane stimulated with inflammatory cytokines of Example 3. TI indicates TNF-a+IFN-Y. [Fig. 12] Fig. 12 is a diagram showing the TEER at 6 hours in the oxidative stress-stimulated monolayer membrane of Example 3. [Fig. 13] Fig. 13 is a diagram showing the TEER at 24 hours in the unstimulated monolayer membrane of Example 4. [Fig. 14] Fig. 14 is a diagram showing the TEER at 24 hours in the unstimulated monolayer membrane of Example 5 (heat-killed bacterial cells). [Fig. 15] Fig. 15 is a diagram showing the TEER at 24 hours in the unstimulated monolayer membrane of Example 5 (viable bacterial cells). [Fig. 16] Fig. 16 is a diagram showing the TEER at 24 hours in the unstimulated monolayer membrane of Example 6 (viable bacterial cells cultured in combination with starch granules). [Fig. 17] Fig. 17 is a diagram showing the results of TEER values for cell wall polysaccharide fractions (including cytoplasmic fractions) under unstimulated conditions. [Fig. 18] Fig. 18 is a diagram showing the results of size exclusion chromatography (SEC) analysis of cell wall polysaccharide fractions (including cytoplasmic fractions). [Fig. 19] Fig. 19 is a diagram showing the results of TEER values under unstimulated conditions when cell wall polysaccharide fractions (including cytoplasmic fractions) were fractionated in ultrafiltration membranes and these fractions were then added at a concentration of 1 pg / mL. [Fig. 20] Fig. 20 is a diagram showing the results of hydrophilic interaction liquid chromatography (HILIC) analysis of fractions with a molecular weight of 10,000 or less and isolated fractions. [Fig. 21] Fig. 21 is a diagram showing the results of TEER values under unstimulated conditions when collected fractions with a molecular weight of 10,000 or less were added at a concentration of 1 pg / mL. [Fig. 22] Fig. 22 is a diagram showing 1H-NMR spectra of collected fractions 2 with a molecular weight of 10,000 or less. [Fig. 23] Fig. 23 is a diagram showing the structure of uridine diphosphate-N-acetylmuramic acid-L-alanine-D-glutamic acid-L-lysine. [Fig. 24] Fig. 24 is a diagram showing the results of HILIC analysis of fractions with a molecular weight of 10,000 to 30,000 and isolated fractions. [Fig. 25] Fig. 25 is a diagram showing the results of TEER values under unstimulated conditions when collected fractions with a molecular weight of 10,000 to 30,000 were added at a concentration of 1 pg / mL. [Fig. 26] Fig. 26 is a diagram showing the 1H-NMR spectra of the collected fractions 1 and 2 with a molecular weight of 10,000 to 30,000. [Fig. 27] Fig. 27 is a diagram showing the results of constituent sugar analysis of the fraction 2. [Fig. 28] Fig. 28 is a diagram showing the structure of a polysaccharide. Description of Embodiments

[0014] Among intestinal barrier protective agents of the present invention (hereinafter referred to as “protective agents of the present invention”), one containing, as an active ingredient, Bifidobacterium adolescentis is referred to as a protective agent 1 of the present invention. In addition, among protective agents of the present invention, one containing, as an active ingredient, a cell wall polysaccharide fraction and / or cytoplasmic fraction of Bifidobacterium adolescentis, a polysaccharide having a repeating structure of rhamnose, glucose, and galactose in a ratio of (4 to 3):1:(2 to 1) , wherein the polysaccharide is recovered from a 10,000- to 30,000-molecular-weight-cutoff ultrafiltration fraction, or uridine diphosphate-N-acetylmuramic acid-L-alanine-D-glutamic acid-L-lysine is referred to as a protective agent 2 of the present invention.

[0015] First, among protective agents of the present invention, a protective agent 1 of the present invention, which contains, as an active ingredient, Bifidobacterium adolescentis, will be described.

[0016] Bifidobacterium adolescentis strains used in the protective agent 1 of the present invention may be any strain known as Bifidobacterium adolescentis or any strain isolated by conventional methods (for example, (1) Research on Bifidobacteria, edited by Tomotari Mitsuoka, Japan Bifidus Foundation, (2) Comparison of intestinal bacteria composition identified by various analytical methods, Tomohiko Fujisawa, Japanese Journal of Bacteriology 2014, and (3) PMID: 28394924 (where Bifidobacterium adolescentis is isolated from mouse feces, but is applicable to humans)), and examples thereof include Bifidobacterium adolescentis YIT 13021, Bifidobacterium adolescentis YIT 13609, Bifidobacterium adolescentis YIT 12828, Bifidobacterium adolescentis type strain (ATCC15703), Bifidobacterium adolescentis YIT 13022, Bifidobacterium adolescentis YIT 11033, Bifidobacterium adolescentis YIT 11034, and Bifidobacterium adolescentis YIT 11040. These Bifidobacterium adolescentis strains can be used alone or two or more thereof can be used in combination.

[0017] Among these Bifidobacterium adolescentis strains, Bifidobacterium adolescentis YIT 13021, Bifidobacterium adolescentis YIT 13609 and Bifidobacterium adolescentis YIT 12828 are preferable, and Bifidobacterium adolescentis YIT 13021 is more preferable.

[0018] Bifidobacterium adolescentis strains used in the protective agent 1 of the present invention include not only the above bacterial strains, but also strains that have undergone artificial, genetic modification or the like, while maintaining the properties of Bifidobacterium adolescentis. In addition, Bifidobacterium adolescentis exhibits efficacy in the form of either viable bacteria or non-viable bacteria, but viable bacteria are preferable.

[0019] Among the Bifidobacterium adolescentis strains, Bifidobacterium adolescentis YIT 13021 is internationally deposited as Bifidobacterium adolescentis YIT 13021 (NITE BP-03808, date of deposit: January 24, 2023) at the NITE Patent Microorganisms Depositary (NPMD), National Institute of Technology and Evaluation (#122, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba 292-0818, Japan), which is an international depositary authority under the Budapest Treaty.

[0020] Bifidobacterium adolescentis YIT 13609, which was originally deposited domestically as YIT 13609 (NITE P-04064, date of deposit: January 15, 2024) at the NITE Patent Microorganisms Depositary (NPMD), National Institute of Technology and Evaluation (#122, 2-58 Kazusakamatari, Kisarazu-shi, Chiba 292-0818, Japan), was requested for transfer to an international deposit on December 9, 2024 and is internationally deposited as YIT 13609 (NITE BP-04064, date of deposit: January 15, 2024) at the above Patent Microorganisms Depositary, which is an international depositary authority under the Budapest Treaty.

[0021] Bifidobacterium adolescentis YIT 12828, which was originally deposited domestically as YIT 12828 (NITE P-04065, date of deposit: January 15, 2024) at the above Patent Microorganisms Depositary, was requested for transfer to an international deposit on December 9, 2024, and is internationally deposited as YIT 12828 (NITE BP-04065, date of deposit: January 15, 2024) at the above Patent Microorganisms Depositary, which is an international depositary authority under the Budapest Treaty.

[0022] Bifidobacterium adolescentis YIT 13022 is internationally deposited as Bifidobacterium adolescentis YIT 13022 (NITE BP-03809, date of deposit: January 24, 2023) at the above international depositary authority.

[0023] Bifidobacterium adolescentis YIT 11033 is internationally deposited as Bifidobacterium adolescentis YIT 11033 (NITE BP-03810, date of deposit: January 24, 2023) at the above international depositary authority.

[0024] Bifidobacterium adolescentis YIT 11034 is internationally deposited as Bifidobacterium adolescentis YIT 11034 (NITE BP-03811, date of deposit: January 24, 2023) at the above international depositary authority.

[0025] Bifidobacterium adolescentis YIT 11040 is internationally deposited as Bifidobacterium adolescentis YIT 11040 (NITE BP-03812, date of deposit: January 24, 2023) at the above international depositary authority.

[0026] When the protective agent 1 of the present invention contains, as an active ingredient, Bifidobacterium adolescentis, it can protect the intestinal barrier from one or more stimulations selected from among LPS stimulation, inflammatory cytokine stimulation and oxidative stress stimulation, preferably, it can protect the intestinal barrier from two or more of the above stimulations, and more preferably, it can protect the intestinal barrier from three of the above stimulations.

[0027] Here, LPS stimulation refers to the action of lipopolysaccharides (LPS), which are a toxin derived from intestinal bacteria, on intestinal epithelial cells.

[0028] Inflammatory cytokine stimulation refers to the action of inflammatory cytokines such as TNF-a, IFN-y, and IL-6 on intestinal epithelial cells.

[0029] Oxidative stress stimulation refers to the action of reactive oxygen species such as hydrogen peroxide generated by oxidative stress on intestinal epithelial cells.

[0030] Here, as shown in examples described below, protection of the intestinal barrier means that, even with one or more stimulations selected from among LPS stimulation, inflammatory cytokine stimulation, and oxidative stress stimulation, preferably two or more of the above stimulations, and more preferably three of the above stimulations, the expression or localization of tight junction proteins present in the intestinal tract is not disturbed or disrupted, or a decrease in the index value of the intestinal barrier strength (transepithelial electrical resistance value; TEER) is suppressed.

[0031] The form of the protective agent 1 of the present invention is not particularly limited, and like conventionally known Bifidobacteria, examples thereof include food and beverages such as fermented milk, yogurt, and bacterial powder supplements, as well as pharmaceutical products such as lactic acid bacteria and Bifidobacteria preparations. Among these, fermented foods such as fermented milk and pharmaceutical products such as lactic acid bacteria and Bifidobacteria preparations are preferable.

[0032] The protective agent 1 of the present invention can protect the intestinal barrier when it is administered to mammals, including humans. In this case, the protective agent 1 of the present invention may contain Bifidobacterium adolescentis in an amount such that the intake amount is about 105 to 1010 cfu per day.

[0033] As described above, since the protective agent 1 of the present invention can protect the intestinal barrier, it can be used as a prophylactic agent for diseases related to the disruption of the intestinal barrier (hereinafter referred to as a “prophylactic agent 1 of the present invention”). Here, since the prophylactic agent 1 of the present invention can also protect against disturbance of the intestinal barrier, diseases related to the disruption of the intestinal barrier also include diseases related to the disturbance of the intestinal barrier. Examples of intestinal barrier disruption-related diseases include diabetes, obesity, inflammatory bowel disease (Crohn’s disease, ulcerative colitis, etc.), fatty liver, Parkinson’s disease, cancer, irritable bowel syndrome, dyslipidemia, atopic dermatitis, asthma, dementia, insomnia, depression, and rheumatoid arthritis. Among these intestinal barrier disruption-related diseases, obesity and diabetes are preferable.

[0034] The prophylactic agent 1 of the present invention can prevent intestinal barrier disruption-related diseases when it is administered to mammals, including humans. In this case, the prophylactic agent 1 of the present invention may contain Bifidobacterium adolescentis in an amount such that the intake amount is about 105 to 1010 cfu per day.

[0035] The fact that the above diseases are related to the disruption of the intestinal barrier is described in the following literature. <Inflammatory bowel disease, obesity, and fatty liver> Lee. B, et al., J Immunol Res. 2018.2645465.2018 <Parkinson’s disease> Van Ijzendoorn. S.C.D, et al., J Parkinsons Dis. 9. S323.2019 <Cancer and irritable bowel syndrome> Oshima. T, et al., J Gastroenterol. 51.768. 2016 <Dyslipidemia> Flaig. B, et al., Nutrients. 15.228. 2023 <Atopic dermatitis and asthma> Niewiem. M, et al., Nutrients. 14.1893.2022 <Dementia> Stadlbauer. V, et al., BMC Geriatr. 20.248. 2020 <Insomnia and depression> Li. Y, et al., Front Psychiatry. 9.669. 2018 <Rheumatoid arthritis> Matei. D. E, et al., Med. 2.864. e9.2021

[0036] Among the Bifidobacterium adolescentis strains used in the protective agent 1 of the present invention and the prophylactic agent 1 of the present invention, Bifidobacterium adolescentis YIT 13021, Bifidobacterium adolescentis YIT 13022, Bifidobacterium adolescentis YIT 11033, Bifidobacterium adolescentis YIT 11034, Bifidobacterium adolescentis YIT 11040 and the like have a property of adhering to and assimilating starch granules. Such a property is not found in the Bifidobacterium adolescentis type strain (ATCC15703). This fact and the test methods and the like for confirming the above property are described in the international application filed on January 31, 2023 (PCT / JP2023 / 3018).

[0037] Therefore, when using Bifidobacterium adolescentis having the above property in the protective agent 1 of the present invention and the prophylactic agent 1 of the present invention, it is preferable to incorporate starch granules together with these. This inhibits adhesion of intestinal bacteria such as Eubacterium rectale, which compete in the intestine, to the starch granules, thereby allowing the strain itself to adhere and colonize, thereby improving efficacy. Here, in order to more easily obtain the above property, it is preferable to culture Bifidobacterium adolescentis in a medium supplemented with soluble starches, starch granules, dextrin or the like.

[0038] Here, the term “starch granules” refers to starches derived from tubers such as potatoes and sweet potatoes, cereals such as wheat, rice, and corn, legumes such as soybeans and adzuki beans, or tapioca, banana and the like, which have not undergone a heating step involving water (such as boiling or steaming) except for a drying step during production, or which have not been gelatinized even if subjected to a heating step involving water, and which retain a natural granular state and are insoluble in water at room temperature. More specifically, the starch granules are uncooked starches derived from uncooked tubers such as potatoes and sweet potatoes, cereals such as wheat, rice, and corn, legumes such as soybeans and adzuki beans, or tapioca, banana and the like, have granular shapes such as spherical, oval, or polyhedral shapes, and have a diameter of typically 1 to 120 pm, although it depends on the plant species. The diameter can be measured using a microscope or a particle size measuring instrument.

[0039] When the starch granules are contained in the protective agent 1 of the present invention and the prophylactic agent 1 of the present invention, the starch granules may be contained in an amount such that the intake amount is about 0.5 to 100 g per day.

[0040] The protective agent 1 of the present invention and the prophylactic agent 1 of the present invention described above can safely protect the intestinal barrier and prevent intestinal barrier disruption-related diseases for a long period of time.

[0041] In addition, the protective agent 1 of the present invention protects the intestinal barrier, but can also strengthen the intestinal barrier when there is no stimulation. Therefore, the protective agent 1 of the present invention can be used as an intestinal barrier strengthening agent. Here, strengthening of the intestinal barrier refers to increasing the index value of the intestinal barrier strength (transepithelial electrical resistance value; TEER).

[0042] Next, among the protective agents of the present invention, a protective agent 2 of the present invention which contains, as an active ingredient, any of the following components derived from Bifidobacterium adolescentis will be described. Here, in the protective agent 2 of the present invention, protection of the intestinal barrier means increasing the index value of the intestinal barrier strength (transepithelial electrical resistance value; TEER) compared to a case where nothing is added. (1) a cell wall polysaccharide fraction and / or cytoplasmic fraction of Bifidobacterium adolescentis (2) a polysaccharide having a repeating structure of rhamnose, glucose, and galactose in a ratio of (4 to 3):1:(2 to 1), wherein the polysaccharide is recovered from a 10,000- to 30,000-molecular-weight-cutoff ultrafiltration fraction (3) uridine diphosphate-N-acetylmuramic acid-L-alanine-D-glutamic acid-L-lysine

[0043] The active ingredient of the protective agent 2 of the present invention, (1) the cell wall polysaccharide fraction and / or cytoplasmic fraction of Bifidobacterium adolescentis, will be described.

[0044] The cell wall polysaccharide fraction and / or cytoplasmic fraction of Bifidobacterium adolescentis are obtained by subjecting Bifidobacterium adolescentis to a lysis treatment.

[0045] As the Bifidobacterium adolescentis strain used above, the Bifidobacterium adolescentis strain used in the protective agent 1 of the present invention containing, as an active ingredient, the above Bifidobacterium adolescentis can be used.

[0046] The above Bifidobacterium adolescentis is preferably cultured before being subjected to a lysis treatment. Culturing may be performed under conventionally known conditions. In addition, after culturing, bacterial collection by centrifugation or the like, or washing with PBS or the like may be performed according to conventional methods.

[0047] The lysis treatment of Bifidobacterium adolescentis is not particularly limited as long as a cell wall polysaccharide fraction and / or cytoplasmic fraction can be obtained from Bifidobacterium adolescentis, and for example, a known method using an appropriate combination of a plurality of enzymes such as muramidase, nuclease, and protease can be used.

[0048] Among these methods using a plurality of enzymes, a preferable method is a method in which the sample is treated with a muramidase and then treated with a nuclease and a protease. Examples of muramidases include Mutanolysin, Lysozyme, and Labiase. Examples of nucleases include Benzonase. Examples of proteases include Pronase and Trypsin.

[0049] The preferable method is performed as follows. First, Bifidobacterium adolescentis is sufficiently cultured in advance according to a conventional method, and then collected by centrifugation and washed with PBS. To 1 mg of viable bacteria (Bifidobacterium adolescentis), 1 to 100 U, preferably 4 U, of muramidase is added, and the mixture is reacted at 37 to 60°C, preferably 37°C, for 12 hours or longer, preferably 16 to 24 hours. Next, as necessary, insoluble substances are removed by centrifugation or the like to obtain a supernatant, 1 to 100 U, preferably 1.4 U, of nuclease is added thereto with respect to 1 mg of viable bacteria, and the mixture is reacted at 37 to 60°C, preferably 37°C, for 12 hours or longer, preferably 16 to 24 hours. Next, 0.0001 to 0.5 mg, preferably 0.006 mg, of protease is added thereto with respect to 1 mg of viable bacteria, and the mixture is reacted at 37 to 60°C, preferably 37°C, for 12 hours or longer, preferably 16 to 24 hours. After the reaction, purification may be performed using a dialysis membrane or the like. The molecular weight cutoff of the dialysis membrane is 3,500 to 14,000, and preferably 3,500 to 8,000.

[0050] As described above, the cell wall polysaccharide fraction and / or cytoplasmic fraction of Bifidobacterium adolescentis of (1) are obtained. The physical properties of the cell wall polysaccharide fraction and / or cytoplasmic fraction of Bifidobacterium adolescentis are as follows. Appearance: white to light grayish-brown solid

[0051] The cell wall polysaccharide fraction and / or cytoplasmic fraction of Bifidobacterium adolescentis have the intestinal barrier protection effect.

[0052] Next, the active ingredient of the protective agent 2 of the present invention, (2) the polysaccharide having a repeating structure of rhamnose, glucose, and galactose in a ratio of (4 to 3):1:(2 to 1), and preferably a repeating structure of rhamnose, glucose, and galactose in a ratio of 3:1:1 or 4:1:2, wherein the polysaccharide is recovered from a 10,000- to 30,000-molecular-weight-cutoff ultrafiltration fraction, will be described. Here, the polysaccharide has a structure represented by the following formula and is a novel substance.

[0053] [C1] (4 to 3):1:(2 to 1)

[0054] The physical properties of the polysaccharide are as follows. Appearance: white to light yellow solid

[0055] The polysaccharide can be produced by subjecting Bifidobacterium adolescentis to a lysis treatment, then obtaining a fraction corresponding to a molecular weight range defined by ultrafiltration membranes having molecular weight cutoffs of 10,000 to 30,000, and additionally collecting these fractions through hydrophilic interaction liquid chromatography performed under the following conditions or equivalent conditions: [Chromatography conditions] Flow rate: 1.0 mL / min Mobile phase A: MeCN, B: H2O Gradient: 0 to 3.3 min: A=95, B=5, 3.3 to 83.3 min: A 95A, B=5^100 Column: YMC-triart Diol HILIC (^4.6 x 250 mm, 5 pm) Temperature: 40°C Injection volume: 2 to 10 pL (5 mg / mL in H2O) Detection: charged aerosol detector (CAD) Elution time: 22 to 35 min.

[0056] The Bifidobacterium adolescentis and lysis treatment used in the above are the same as described above.

[0057] After the lysis treatment, the cell wall polysaccharide fraction and / or cytoplasmic fraction of Bifidobacterium adolescentis are obtained. These are purified using an ultrafiltration membrane to obtain fractions with a molecular weight cutoff of 10,000 to 30,000. The ultrafiltration membranes used in this case are not particularly limited as long as fractions having the above molecular weight are obtained, and for example, it is preferable to use a combination of ultrafiltration membranes with a molecular weight cutoff of 100,000, a molecular weight cutoff of 30,000, and a molecular weight cutoff of 10,000, and it is more preferable to use a combination of ultrafiltration membranes with a molecular weight cutoff of 30,000 and a molecular weight cutoff of 10,000.

[0058] After purification, fractions recovered from a 10,000- to 30,000-molecular-weight-cutoff ultrafiltration are obtained. The fractions are subjected to hydrophilic interaction liquid chromatography under the above conditions, and polysaccharides having a repeating structure of rhamnose, glucose, and galactose in a ratio of (4 to 3):1:(2 to 1) and being contained in an ultrafiltration fraction corresponding to a molecular weight range of 10,000 to 30,000 are collected.

[0059] In the hydrophilic interaction liquid chromatography, in addition to the above conditions, the conditions can be appropriately changed as long as the chromatography is performed under equivalent conditions to obtain polysaccharides having a repeating structure of rhamnose, glucose, and galactose in a ratio of (4 to 3):1:(2 to 1) and being contained in an ultrafiltration fraction corresponding to a molecular weight range of 10,000 to 30,000 are collected. Such equivalent conditions can be set by those skilled in the art by appropriately changing the flow rate, the mobile phase composition, the type of the column carrier, the particle size of the column carrier, the length of the column, the temperature, the injection volume, and the concentration of the injection solution, with reference to the above conditions, and specifically, the following conditions are exemplified.

[0060] [Chromatography conditions] Flow rate: 0.5 to 1.0 mL / min Mobile phase A: MeCN, B: H2O Gradient:  (any one of a) to c)) a) 0 to 2 min: A=95, B=5, 2 to 25 min: A 95-0, B=5-100, b) 0 to 2 min: A=95, B=5, 2 to 50 min: A 95-0, B=5-100 c) 0 to 2 min: A=95, B=5, 2 to 100 min: A=95-0, B=5-100 Column: YMC-triart Diol HILIC (^4.6 x 150 mm, 5 pm) Temperature: 40°C Injection volume: 2 to 10 pL (5 mg / mL in H2O) Detection: charged aerosol detector (CAD) Elution time: a flow rate of 1.0 mL / min; a) 8 to 11 min, b) 13 to 19 min, c) 25 to 35 min a flow rate of 0.5 mL / min; a) 10 to 13 min, b) 15 to 21 min, c) 27 to 37 min

[0061] As described above, the polysaccharide of (2), having a repeating structure of rhamnose, glucose, and galactose in a ratio of (4 to 3):1:(2 to 1), wherein the polysaccharide is recovered from a 10,000- to 30,000-molecular-weight-cutoff ultrafiltration fraction, is obtained. Whether this polysaccharide has been obtained can be confirmed, for example, by structural analysis or constituent sugar analysis using NMR, HPLC or the like. Specifically, in SEC analysis using a Shodex SUGAR KS-804 column (F6378035, commercially available from Showa Denko K.K.), a peak can be confirmed around an elution time of 8 to 9 minutes. More specifically, confirmation can be performed using the method described in examples described below.

[0062] The polysaccharide having a repeating structure of rhamnose, glucose, and galactose in a ratio of (4 to 3):1:(2 to 1), wherein the polysaccharide is recovered from a 10,000- to 30,000-molecular-weight-cutoff ultrafiltration fraction has an intestinal barrier protection effect.

[0063] Finally, the active ingredient of the protective agent 2 of the present invention, (3) uridine diphosphate-N-acetylmuramic acid-L-alanine-D-glutamic acid-L-lysine, will be described.

[0064] This uridine diphosphate-N-acetylmuramic acid-L-alanine-D-glutamic acid-L-lysine (CAS No. 143436-84-4, etc.) is a known compound, is a constituent component in the cytoplasm, and is represented by the following chemical formula (Nicolas Gisch, Birte Buske, Holger Heine, Buko Lindner, Ulrich Zahringer, Synthesis of biotinylated muramyl tripeptides with NOD2-stimulating activity, Bioorganic & Medicinal Chemistry Letters 2011(21) 3362.).

[0065] [C2]

[0066] This uridine diphosphate-N-acetylmuramic acid-L-alanine-D-glutamic acid-L-lysine may be obtained using a commercially available product, obtained based on the above literature, or obtained from the cell wall polysaccharide fraction and / or cytoplasmic fraction of Bifidobacterium adolescentis of the (1) above, based on examples described below.

[0067] This uridine diphosphate-N-acetylmuramic acid-L-alanine-D-glutamic acid-L-lysine has an intestinal barrier protection effect.

[0068] The protective agent 2 of the present invention can protect the intestinal barrier when it contains, as an active ingredient, any of the above (1) to (3).

[0069] The form of the protective agent 2 of the present invention is not particularly limited as long as it contains any of the above (1) to (3) and is suitable for administration, and can be, for example, a pharmaceutical product for humans, a food or beverage, a supplement or the like.

[0070] As a pharmaceutical product, any of the above (1) to (3) can be formulated without change or as necessary, in combination with a pharmaceutically acceptable carrier. Examples of pharmaceutically acceptable carriers include glucose, lactose, starch, mannitol, dextrin, fatty acid glycerides, polyethylene glycol, hydroxyethyl starch, ethylene glycol, polyoxyethylene sorbitan fatty acid esters, amino acids, gelatin, albumin, water, and normal saline. In addition, as necessary, conventional additives such as stabilizers, wetting agents, emulsifiers, binders, isotonic agents, and excipients can be appropriately added.

[0071] In addition, the dosage form is not particularly limited, and examples thereof include liquids, powders, granules, capsules, and tablets, which can be produced according to conventional methods. The content of the active ingredient in the protective agent 2 of the present invention is not particularly limited, and for example, it is preferably 1 to 100 mass%, more preferably 5 to 70 mass%, and particularly preferably 10 to 60 mass%.

[0072] When the protective agent 2 of the present invention is in the form of a food or beverage, it can be prepared using any of the above (1) to (3) without change or by mixing, as necessary, known additives, materials and the like used in food and beverages according to conventional methods. Examples of food and beverages include starch-based foods such as bread, biscuits, pancakes, noodles, and tablet candies, confectioneries such as gum, candy, and Japanese sweets, meat foods such as ham and sausages, fish meat foods such as chikuwa and kamaboko, seafood products, seasonings such as dressings, soy sauce, jam, and furikake, and beverages such as tea, juice, soft drinks, and alcoholic beverages.

[0073] When the protective agent 2 of the present invention is in the form of a supplement, it can be prepared using any of the above (1) to (3) without change or by mixing, as necessary, known additives, materials and the like used in supplements according to conventional methods.

[0074] The protective agent 2 of the present invention can protect the intestinal barrier when it is administered to mammals, including humans. In this case, the protective agent 2 of the present invention may be administered in the following amounts per day: (1) an amount of 0.000060 to 270 mg in the case of the cell wall polysaccharide fraction and / or cytoplasmic fraction of Bifidobacterium adolescentis, (2) an amount of 0.00000324 to 15 mg in the case of the polysaccharide having a repeating structure of rhamnose, glucose, and galactose in a ratio of (4 to 3):1:(2 to 1), wherein the polysaccharide is recovered from a 10,000- to 30,000- molecular-weight-cutoff ultrafiltration fraction, and (3) an amount of 0.00000420 to 19 mg in the case of the uridine diphosphate-N-acetylmuramic acid-L-alanine-D-glutamic acid-L-lysine.

[0075] As described above, since the protective agent 2 of the present invention described above can protect the intestinal barrier, it can be used as a prophylactic agent for diseases related to the disruption of the intestinal barrier (hereinafter referred to as a “prophylactic agent 2 of the present invention”). Here, since the prophylactic agent 2 of the present invention can also protect against disturbance of the intestinal barrier, diseases related to the disruption of the intestinal barrier also include diseases related to the disturbance of the intestinal barrier. Examples of intestinal barrier disruption-related diseases include diabetes, obesity, inflammatory bowel disease (Crohn’s disease, ulcerative colitis, etc.), fatty liver, Parkinson’s disease, cancer, irritable bowel syndrome, dyslipidemia, atopic dermatitis, asthma, dementia, insomnia, depression, and rheumatoid arthritis. Among these intestinal barrier disruption-related diseases, obesity and diabetes are preferable.

[0076] The prophylactic agent 2 of the present invention can prevent intestinal barrier disruption-related diseases when it is administered to mammals, including humans. In this case, the prophylactic agent 2 of the present invention may be administered in the following amounts per day: (1) an amount of 0.000060 to 270 mg in the case of the cell wall polysaccharide fraction and / or cytoplasmic fraction of Bifidobacterium adolescentis, (2) an amount of 0.00000324 to 15 mg in the case of the polysaccharide having a repeating structure of rhamnose, glucose, and galactose in a ratio of (4 to 3):1:(2 to 1), wherein the polysaccharide is recovered from a 10,000- to 30,000-molecular-weight-cutoff ultrafiltration fraction, and (3) an amount of 0.00000420 to 19 mg in the case of the uridine diphosphate-N-acetylmuramic acid-L-alanine-D-glutamic acid-L-lysine.

[0077] The fact that the above diseases are related to the disruption of the intestinal barrier is described in the same literature used in the description of the prophylactic agent 1 of the present invention.

[0078] The protective agent 2 of the present invention and the prophylactic agent 2 of the present invention can safely protect the intestinal barrier and prevent intestinal barrier disruption-related diseases for a long period of time.

[0079] In addition, the protective agent 2 of the present invention protects the intestinal barrier, but can also strengthen the intestinal barrier when there is no stimulation. Therefore, the protective agent 2 of the present invention can also be used as an intestinal barrier strengthening agent. Here, strengthening of the intestinal barrier refers to increasing the index value of the intestinal barrier strength (transepithelial electrical resistance value; TEER) compared to a case where nothing is added. Examples

[0080] Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited to these examples. Here, in all of the following examples, a significance test was performed using a t-test, where * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001.

[0081] Example 1 Intestinal barrier protection effect: The test bacterial strain used was Bifidobacterium adolescentis YIT 13021 (NITE BP-03808) (hereinafter referred to as “YIT 13021”). The control bacterial strain used was the Bifidobacterium breve type strain (ATCC15700) (hereinafter referred to as “YIT 4014”). These bacterial strains were cultured in an mGAM medium supplemented with 1% glucose, and their intestinal barrier protection effects against LPS stimulation, inflammatory cytokine stimulation or oxidative stress stimulation were examined using unstimulated strains as controls. In addition, the influence of YIT 13021 on the localization of tight junction proteins was also examined.

[0082] (1) LPS stimulation 1.1 Cells Cryopreserved human colon epithelial cell line T84 was used. T84 cells were subcultured in a DMEM / F-12 medium (10% FBS / F-12) containing 10% FBS and 100 pg / mL of streptomycin at 5% CO2 and 37°C.

[0083] 1.2 Preparation of monolayer membrane A T84 monolayer membrane was prepared using a 24-well cell culture insert plate (Millipore). T84 cells at 80% confluence were collected from a culture dish and seeded at 60,000 cells / well in 400 pL of 10% FBS / F-12 on the Apical side of the wells, 800 pL of 10% FBS / F-12 was added to the Basolateral side, and the cells were cultured at 5% CO2 and 37°C. The medium was replaced every 2 or 3 days with 400 pL of the medium on the Apical side and 800 pL of the medium on the Basolateral side. The T84 cells were cultured for 10 days, and thus a monolayer membrane was prepared.

[0084] 1.3 Evaluation of protection effect on LPS-stimulated monolayer membrane For LPS, LPS derived from E. coli O55 (WAKO, #128-05171) was used and dissolved in 10% FBS / F-12 to achieve a test concentration of 10 ng / mL. LPS stimulation was performed by adding an LPS-containing medium to both Apical and Basolateral sides of the prepared monolayer membrane. Heat-killed bacterial cells of YIT 13021 or YIT 4014 were prepared by a known method, and added only to the Apical side of the monolayer membrane at a concentration of 100 pg / mL (about 2.4 x 108 cells / mL). The protection effect against damage to the monolayer membrane due to stimulation was evaluated by the transepithelial electrical resistance (TEER) value (Q / cm2), which is a value obtained by dividing the measured resistance value (Q) measured using Millicell MRS-2 (Millipore) by the culture area (cm2) of the cell culture insert plate. The TEER value of the monolayer membrane at each elapsed time after the start of stimulation was divided by the TEER value at 0 hours (immediately after the start of stimulation) to calculate the barrier disruption as a rate of change (%) from the start of stimulation.

[0085] 1.4 Results In order to examine the influence of YIT 13021 or YIT 4014 on the intestinal barrier function, T84 cells were seeded on the cell culture insert plate and cultured for 10 days to prepare a monolayer membrane. LPS and heat-killed bacterial cells of the strains were added to the monolayer membrane, and the TEER values were measured every 24 hours until 48 hours after addition (Fig. 1). As a result, 48 hours after addition, no decrease in the TEER value was observed in the monolayer membrane to which YIT 13021 was added, and the decrease in the TEER value due to LPS stimulation was significantly reduced, whereas the results for YIT 4014 were the same as those for LPS stimulation alone (Fig. 2).

[0086] (2) Inflammatory cytokine stimulation 2.1 Cells The same cells as in 1.1 of (1) LPS stimulation above were used.

[0087] 2.2 Preparation of monolayer membrane A monolayer membrane was prepared in the same manner as in 1.2 of (1) LPS stimulation above.

[0088] 2.3 Evaluation of protection effect on monolayer membrane stimulated with inflammatory cytokines The inflammatory cytokines used were TNF-a (R&D Systems, #210-TA-005) and IFN-y (R&D Systems, #285-IF-100) . Both cytokines were dissolved in equal amounts in 10% FBS / F-12 to achieve a test concentration of 1 ng / mL. Stimulation with TNF-a and IFN-y was performed by adding a cytokine-containing medium only to the Basolateral side of the prepared monolayer membrane. The measurement of the TEER value of the monolayer membrane at each elapsed time from the start of stimulation and the calculation of the rate of change (%) were performed in the same manner as in 1.3 of the (1) LPS stimulation above.

[0089] 2.4 Results Inflammatory cytokines TNF-a and IFN-y were added to the T84 monolayer membrane, and either YIT 13021 or YIT 4014 was added simultaneously, to evaluate the protection effects against barrier disruption caused by the inflammatory cytokines. The TEER values at 24 and 48 hours were evaluated (Fig. 3). TI indicates TNF-a+IFN-y. As a result, in the monolayer membrane with inflammatory cytokine stimulation alone or with YIT 4014 added, no significant change was observed at 24 hours, but the TEER value decreased to about 50% at 48 hours. On the other hand, in the monolayer membrane to which YIT 13021 was added, the TEER value increased by about 20% compared to Ctrl at 24 hours. The decrease due to stimulation at 48 hours was also limited to about 70%, and the decrease in the TEER value due to inflammatory cytokine stimulation was significantly reduced (Fig. 4).

[0090] (3) Oxidative stress stimulation 3.1 Cells The same cells as in 1.1 of (1) LPS stimulation above were used.

[0091] 3.2 Preparation of monolayer membrane A monolayer membrane was prepared in the same manner as in 1.2 of (1) LPS stimulation above.

[0092] 3.3 Evaluation of protection effect on oxidative stress-stimulated monolayer membrane For oxidative stress, H2O2 (Mitsubishi, #K-8230) was used and diluted in 10% FBS / F-12 to achieve a test concentration of 1,500 pM. Stimulation with H2O2 was performed by adding a H2O2-containing medium to both Apical and Basolateral sides of the prepared monolayer membrane. The measurement of the TEER value of the monolayer membrane at each elapsed time from the start of stimulation and the calculation of the rate of change (%) were performed in the same manner as in 1.3 of the (1) LPS stimulation above.

[0093] 3.4 Results H2O2 was added to the T84 monolayer membrane, and either YIT 13021 or YIT 4014 was respectively added simultaneously, to evaluate the protection effects against barrier disruption caused by the oxidative stress. The TEER values were measured every hour until 6 hours after addition (Fig. 5). As a result, no protection effect against H2O2 stimulation was observed for YIT 4014, but in the monolayer membrane with YIT 13021 added, the decrease in the TEER value was significantly reduced compared to the case of H2O2 stimulation alone, and the protection effect against H2O2 stimulation was observed (Fig. 6).

[0094] (4) Influence on localization of tight junction proteins 4.1 Cells The same cells as in 1.1 of (1) LPS stimulation above were used.

[0095] 4.2 Preparation of monolayer membrane A monolayer membrane was prepared in the same manner as in 1.2 of (1) LPS stimulation above.

[0096] 4.3 Immunostaining of monolayer membrane The monolayer membrane was washed twice with PBS, a 10% neutral buffered formalin solution was then added to a 24-well cell culture insert plate, and fixation was performed for 15 minutes. After the washing operation was performed twice, permeabilization with 0.25% Triton X-100 / PBS was performed for 5 minutes. The washing operation was additionally performed twice and blocking with 10% BSA / PBS at 37°C was performed for 60 minutes. After removing the blocking solution, Anti-ZO-1 antibodies (Abcam, #ab216880) diluted 100-fold in PBS were added and incubated overnight at 4°C. The washing operation was performed twice, and anti-rabbit Alexa 546 antibodies (Invitrogen, #A11010) diluted 500fold in PBS were added, and incubated at 37°C for 60 minutes in the dark. Finally, the washing operation was performed three times, the membrane was cut out from the cell culture insert plate and placed on a glass slide, a drop of VECTASHIELD with DAPI (VECTOR LABORATORIES) as a mounting agent was added, and a coverslip was placed to prepare a glass slide for observation. The cells were observed using an all-in-one fluorescence microscope BZ-X700 (Keyence).

[0097] 4.4 Results The monolayer membrane was subjected to each stimulation in the same manner as in the above (1) to (3), 48 hours after stimulation with inflammatory cytokines and LPS and 6 hours after stimulation with oxidative stress, proteins ZO-1 constituting tight junctions were immunostained, and each monolayer membrane was observed (the - panels in Fig. 7). Alternatively, after YIT 13021 was added simultaneously with each stimulation in the same manner as in the above (1) to (3), the monolayer membrane was observed after immunostaining ZO-1. Based on these observations, the protection effect of YIT 13021 on the barrier function was evaluated (Fig. 7). The protection effects of YIT 13021 against LPS stimulation, inflammatory cytokine stimulation, and oxidative stress stimulation were evaluated. As a result, it was confirmed that, upon stimulation alone, a part of the network-like localization of ZO-1 was lost, and the presence of ZO-1 was observed not only at the cell membrane but also in the cytoplasm, indicating that the tight junctions were disturbed. On the other hand, it was found that, in the monolayer membrane to which YIT 13021 was added simultaneously with stimulation, the network-like localization of ZO-1 was lost and its abundance in the cytoplasm was reduced, indicating that YIT 13021 suppressed the disturbance of the tight junctions. Based on these results, the intestinal barrier protection effect was confirmed from the perspective of the localization of tight junction proteins.

[0098] Example 2 Intestinal barrier strengthening effect: The test bacterial strains used were YIT 13021 and YIT 4014. In order to examine the influence of these strains on the intestinal barrier function, T84 cells prepared in the same manner as in Example 1 were seeded on the cell culture insert plate and cultured for 10 days to prepare a monolayer membrane. Heat-killed bacterial cells of YIT 13021 or YIT 4014 were respectively added to the Apical side of the monolayer membrane (without stimulation), and TEER values were measured every hour until 6 hours after addition (Fig. 8). As a result, a significant increase in the TEER value in the monolayer membrane 6 hours after YIT 13021 was added was observed, compared to the untreated Ctrl (Fig. 9). On the other hand, in the monolayer membrane with YIT 4014 added, the results were comparable to those of the untreated Ctrl.

[0099] Based on these results, it was found that YIT 13021 had an intestinal barrier strengthening effect.

[0100] Example 3 Intestinal barrier protection effect: For Bifidobacterium adolescentis strains other than YIT 13021, the same intestinal barrier protection effect of YIT 13021 was examined. Bifidobacterium adolescentis strains used were YIT 13609 (NITE BP-04064) and YIT 12828 (NITE BP-04065).

[0101] (1) LPS stimulation In the same manner as in (1) LPS stimulation in Example 1, heat-killed bacterial cells of the strains were added to the Apical side of the monolayer membrane, and the TEER value was measured after 48 hours. As a result, it was found that 48 hours after addition, YIT 13609 and YIT 12828 exhibited the intestinal barrier protection effect against LPS stimulation comparable to that of YIT 13021 (Fig. 10).

[0102] (2) Inflammatory cytokine stimulation In the same manner as in (2) inflammatory cytokine stimulation in Example 1, heat-killed bacterial cells of the strains were added to the Apical side of the monolayer membrane, and the TEER value was measured after 48 hours. As a result, it was found that 48 hours after addition, YIT 13609 and YIT 12828 exhibited the intestinal barrier protection effect against inflammatory cytokine stimulation comparable to that of YIT 13021 (Fig. 11).

[0103] (3) Oxidative stress stimulation In the same manner as in (3) oxidative stress stimulation in Example 1, heat-killed bacterial cells of the strains were added to the Apical side of the monolayer membrane, and the TEER value was measured after 6 hours. As a result, it was found that 6 hours after addition, YIT 13609 and YIT 12828 exhibited the intestinal barrier protection effect against oxidative stress stimulation comparable to that of YIT 13021 (Fig. 12).

[0104] Example 4 Intestinal barrier strengthening effect: The test bacterial strains used were YIT 13021, YIT 13609 and YIT 12828. In order to examine the influence of these strains on the intestinal barrier function, T84 cells prepared in the same manner as in Example 1 were seeded on the cell culture insert plate and cultured for 10 days to prepare a monolayer membrane. Heat-killed bacterial cells of YIT 13021, YIT 13609 or YIT 12828 were respectively added to the Apical side of the monolayer membrane, and the TEER value was measured after 24 hours. As a result, it was found that YIT 13609 and YIT 12828 exhibited the intestinal barrier strengthening effect comparable to that of YIT 13021 (Fig. 13).

[0105] Example 5 Intestinal barrier strengthening effect: In order to examine the influence of viable bacteria (YIT 13021) on the intestinal barrier function, T84 cells prepared in the same manner as in Example 1 were seeded on the cell culture insert plate and cultured for 10 days to prepare a monolayer membrane. Viable bacteria or heat-killed bacterial cells (YIT 13021) were added at each concentration to the Apical side of the monolayer membrane, and the TEER values were measured and compared after 24 hours. As a result, it was found that the heat-killed bacterial cells exhibited a significant increase in the TEER value from an addition concentration of 10 pg / mL (Fig. 14), and the viable bacteria exhibited a significant increase in the TEER value from an addition concentration of 1 pg / mL, indicating that the viable bacteria exhibited the barrier strengthening effect in a lower concentration range (Fig. 15).

[0106] Example 6 Intestinal barrier strengthening effect: YIT 13021 has a property of adhering to and assimilating starch granules, and the presence of starch granules during culturing may influence the properties of bacterial cells. Therefore, the influence of viable bacteria (YIT 13021), cultured by changing the carbon source in the mGAM medium from glucose to starch granules, on the intestinal barrier function was examined. T84 cells prepared in the same manner as in Example 1 were seeded on the cell culture insert plate and cultured for 10 days to prepare a monolayer membrane. Viable bacteria (YIT 13021) cultured in the presence of starch granules were added at each concentration to the Apical side of the monolayer membrane, and the TEER values were measured and compared after 24 hours. As a result, it was found that, in the case of viable bacteria (YIT 13021) cultured in the presence of starch granules, a significant increase in the TEER value was confirmed from an addition concentration of 0.1 pg / mL, and the viable bacteria cultured in combination with starch granules exhibited the barrier strengthening effect from a lower concentration range (Fig. 16) compared to the viable bacteria (YIT 13021) cultured using glucose as a carbon source (Fig. 15).

[0107] Example 7 Intestinal barrier protection effect of cell wall polysaccharide fraction of Bifidobacterium adolescentis: Bacterial strains belonging to Bifidobacterium adolescentis were cultured and then subjected to a lysis treatment to obtain cell wall polysaccharide fractions, which were then analyzed.

[0108] (1) Bacterial strain The bacterial strain used was Bifidobacterium adolescentis YIT 13021 (NITE BP-03808) (hereinafter referred to as “YIT 13021”).

[0109] (2) Culture The YIT 13021 bacterial strain was cultured at 37°C in a modified GAM medium supplemented with 1% soluble starch (191-03985, commercially available from FUJIFILM Wako Pure Chemical Corporation) (hereinafter referred to as “1% Starch mGAM”) under anaerobic conditions.

[0110] (3) Lysis treatment Preparation of 5 mM Tris-maleate buffer (pH 6.8) containing 2 mM MgCl2 1.91 g (8.05 mmol) of Tris-maleate (Sigma 93328-25G) was dissolved in 800 mL of ultrapure water, and the pH was adjusted to 6.8 with a 0.1 mol / L sodium hydroxide aqueous solution (37851-08, commercially available from Kanto Chemical Co., Inc.). Ultrapure water was added to this solution to make a volume of 1.6 L, and the solution was filtered and sterilized using a 0.2 pm filter (Thermo 567-0020).  109.78 mg (0.540 mmol) of magnesium chloride hexahydrate (25009-30, commercially available from Kanto Chemical Co., Inc.) was added to 270 mL of the filtrate to prepare a 5 mM Tris-maleate buffer (pH 6.8) containing 2 mM MgCl2.

[0111] Viable bacteria (400 mg, dry bacterial cell weight) cultured in a 1% Starch mGAM medium were suspended in a 5 mM Tris-maleate buffer (pH 6.8) containing 2 mM MgCl2 at a concentration of 13.9 mg / mL, Mutanolysin was added as a muramidase at 4 U per 1 mg of the bacterial cells, and the mixture was reacted at 37°C for 24 hours while shaking.

[0112] The remaining reaction solution was centrifuged at 24°C and 12,000 *g for 45 minutes, the insoluble substance was precipitated, and the supernatant was collected. Benzonase was added as a nuclease to the supernatant at 1.4 U per 1 mg of the bacterial cells, and the mixture was reacted at 37°C for 24 hours. Subsequently, Pronase was added as a protease at 0.006 mg per 1 mg of the bacterial cells, and the mixture was reacted at 37°C for 24 hours while shaking. After dialysis was performed in pure water at room temperature for 72 hours using a dialysis membrane with a molecular weight cutoff of 3,500, a cell wall polysaccharide fraction (42.5 mg) was obtained by freezing-drying.

[0113] The TEER value of the cell wall polysaccharide fraction under unstimulated conditions was measured based on the following method. The results are shown in Fig. 17. Based on these results, it was found that the cell wall polysaccharide fraction had the intestinal barrier protection effect.

[0114] <TEER value measurement under unstimulated conditions> A T84 monolayer membrane was prepared using a 24-well cell culture insert plate (Millipore). T84 cells at 80% confluence were collected from a culture dish and seeded at 60,000 cells / well in 400 pL of 10% FBS / F-12 on the Apical side of the wells, 800 pL of 10% FBS / F-12 was added to the Basolateral side, and the cells were cultured at 5% CO2 and 37°C. The medium was replaced every 2 or 3 days with 400 pL of the medium on the Apical side and 800 pL of the medium on the Basolateral side. The T84 cells were cultured for 10 days, and thus a monolayer membrane was prepared.

[0115] The barrier strengthening effect was evaluated based on the transepithelial electrical resistance (TEER) value (Q / cm2) which was a value obtained by adding a test substance at each concentration to the Apical side of the prepared monolayer membrane, measuring the measured resistance value (Q) using Millicell MRS-2 (Millipore) after 24 hours, and dividing the measured value by the culture area (cm2) of the cell culture insert plate. The TEER value of the monolayer membrane at each elapsed time after the start of stimulation was divided by the TEER value at 0 hours (immediately after the start of stimulation) to calculate the barrier disruption as a rate of change (%) from the start of stimulation.

[0116] Example 8 Intestinal barrier protection effect of fractions with a molecular weight cutoff of 10,000 or less: The cell wall polysaccharide fraction having the intestinal barrier protection effect obtained in Example 7 was dissolved in ultrapure water at a concentration of 5 mg / mL, and SEC measurement was performed under the following conditions. The results are shown in Fig. 18.

[0117] <Size exclusion chromatography (SEC) measurement> Column: Shodex SUGAR KS-804 (^8.0 mm x 300 mm) Mobile phase: 50 mM NaCl aq. Flow rate: 1.0 mL / min Temperature: 80°C Detection: refractive index detector (RI)

[0118] Based on the SEC, it was found that the cell wall polysaccharide fraction contained substances with molecular weights of 10,000, 30,000, 100,000, and 400,000 or more. Next, this was fractionated into those with molecular weights of 100,000 or more, 100,000 to 30,000, 30,000 to 10,000, and 10,000 or less using an ultrafiltration membrane with a molecular weight cutoff of 100,000 (Millipore, UFC910008), an ultrafiltration membrane with a molecular weight cutoff of 30,000 (Millipore, UFC903008), and an ultrafiltration membrane with a molecular weight cutoff of 10,000 (Millipore, UFC901008). The TEER values for these fractions under unstimulated conditions were measured based on the following method. The results are shown in Fig. 19.

[0119] <TEER value measurement under unstimulated conditions> The same measurement as the TEER value measurement under unstimulated conditions in Example 7 was performed.

[0120] Based on these results, it was found that fractions with a molecular weight cutoff of 30,000 or less (fractions with a molecular weight cutoff of 10,000 to 30,000 and fractions with a molecular weight cutoff of 10,000 or less) were active.  Next, the fractions with a molecular weight of 10,000 or less were subjected to hydrophilic interaction liquid chromatography (HILIC) analysis (Fig. 20). In addition, liquid passage was performed 24 times (a total amount of 1.2 mg), and fractions 1 to 4 in Fig. 20 were isolated each time (1 (16 to 20.5 minutes): 0.45 mg, 2 (20.5 to 22 minutes): 0.25 mg, 3 (22 to 25 minutes): 0.40 mg, and 4 (51 to 56 minutes): 0.29 mg).

[0121] <HILIC analysis> Flow rate: 1.0 mL / min Mobile phase A: MeCN, B: H2O Gradient: 0 to 3.3 min: A=95, B=5, 3.3 to 83.3 min: A=95^0, B=5^100 Column: YMC-triart Diol HILIC (¢4.6 x 250 mm, 5 pm) Temperature: 40°C Injection volume: 10 pL (5 mg / mL in H2O)

[0122] For the fractions 1 to 4 obtained above, TEER value measurement was performed in the same manner as described above. The results are shown in Fig. 21. Only in the fraction 2, the strengthening of the intestinal barrier protection effect was observed. The fraction 2 was subjected to structural analysis using 1H-NMR (700 MHz in D2O at 25°C). The results are shown in Fig. 22.

[0123] <1H-NMR structural analysis> The fraction 2 was dissolved in deuterium oxide (32070-1A, commercially available from Kanto Chemical Co., Inc), and 1H-NMR (700 MHz) measurement was performed at 25°C.

[0124] Based on the 1H-NMR results, the fraction 2 had a signal at ■, and it was found to be the cell wall precursor uridine diphosphate-N-acetylmuramic acid-L-alanine-D-glutamic acid-L-lysine (literature value (Table 1): Nicolas Gisch, et al., Bioorganic Med. Chem. Lett. 21.3362.2011). The structure is shown in Fig. 23. In addition, the molecular weight of uridine diphosphate-N-acetylmuramic acid-L-alanine-D-glutamic acid-L-lysine was 1,008, the fraction 2 was subjected to mass spectrometry, and the [M+1] was 1008.8 (not shown). Since the fraction 2 contained the cell wall precursor, it was found that the cell wall polysaccharide fraction measured so far also contained cytoplasmic fractions.

[0125] (Signal of uridine diphosphate-N-acetylmuramic acid-L-alanine-D-glutamic acid-L-lysine)

[0126] [Table 1] Position UDP-MurNAc-L-Ala-D-Glu-L-Lys literature value> UDP-MurNAc-L-Ala derivative H-6u 7.96 (d, / = 8.3 Hz, 1H) 7.93 (d, / = 7.8 Hz, 1H) H-lRib 5.98 (d, / = 4.4 Hz, 1H) 5.98-5.98 (m, 1H) H-5u 5.97 (d, / =8.2 Hz, 1H) 5.95 (d, / = 7.8 Hz, 1H) H-lMurNA 5.47 (dd, / = 7.0, 3.0 Hz, 1H) 5.46-5.45 (m, 1H) H-2Rib 4.39-4.35 (m, 1H) 4.35-4.12 (m, 10H) H-3Rib 4.38-4.34 (m, 1H) H-2igiu 4.32-4.28 (m, 1H) H-2Lys 4.31-4.26 (m, 1H) CHAla 4.29 (q, / = 7.3 Hz, 1H) H-4Rib 4.30-4.26 (m, 1H) C / ZCHjmufNAc 4.27-4.21 (m, 1H) H-5aRib 4.26-4.21 (m, 1H) H-5bRlb 4.21-4.16 (m, 1H) H-2mufNAc 4.12 (ddd, / = 10.3,2.8, 2.7 Hz, 1H) H-5mufNAc 3.97-3.93 (m, 1H) 3.97-3.63 (m, 5H) H-6aMurNAc 3.88 (dd, / = 12.4,1.9 Hz, 1H) H-6bMurNAc 3.83 (dd, / = 12.3, 4.0 Hz, 1H) H-3mufNAc 3.79 (dd, / =9.7, 9.3 Hz, 1H) H-4mufNAc 3.64 (dd, / = 9.7, 9.7 Hz, 1H) H-6tys 3.00 (t, / = 7.4 Hz, 2H) 2.99-2.98 (m, 2H) H-4,g1u 2.36 (t, / =7.5 Hz, 2H) 2.30-2.28 (m, 2H) H-3aiGiu 2.22-2.15 (m, 1H) 2.15-2.14 (m, 1H) COCHjMurNAc 2.01 (s, 3H) 2.00 (s, 3H) H-3biGiu 2.04-1.95 (m, 1H) 1.90-1.89 (m, 1H) H-3aLys 1.92-1.84 (tn, 1H) 1.82-1.77 (m, 2H) H-3bLy3 1.79-1.72 (m, 1H) H-5Lys 1.73-1.64 (m, 2H) 1.69-1.65 (m, 2H) H-4Lys 1.47-1.41 (m, 2H) 1.44-1.41 (m, 2H) CHjAla 1.43 (d, / = 7.2 Hz, 3H) 1.43 (d, / = 6.5 Hz, 3H) CHC / fsMurNAc 1.39 (d, / = 6.7 Hz, 3H) 1.39 (d, / = 6.5 Hz, 3H)

[0127] Example 9 Intestinal barrier protection effect of fractions corresponding to a molecular weight range of 10,000 to 30,000: The fractions corresponding to a molecular weight range of 10,000 to 30,000 obtained in Example 8 were subjected to HILIC analysis (Fig. 24). In addition, liquid passage was performed 48 times (a total amount of 2.4 mg), fractions 1 to 4 in Fig. 24 were isolated each time (1 (22 to 30 minutes): 1.19 mg, 2 (30 to 35 minutes): 1.26 mg, 3 (35 to 42.5 minutes): 1.01 mg, and 4 (51.5 to 57.5 minutes): 0.79 mg).

[0128] <HILIC analysis> Flow rate: 1.0 mL / min Mobile phase A: MeCN, B: H2O, 0 to 3.3 min: A=95, B=5, 3.3 to 83.3 min: A 95-0, B=5-100 Column: YMC-triart Diol HILIC (^4.6 x 250 mm, 5 pm) Temperature: 40°C Injection volume: 10 pL (5 mg / mL in H2O)

[0129] For the fractions 1 to 4 obtained above, TEER value measurement was performed in the same manner as described above. The results are shown in Fig. 25. The strengthening of the intestinal barrier protection effect was observed in the fractions 1 and 2. The fractions 1 and 2 were subjected to structural analysis using 1H-NMR (700 MHz in D2O at 25°C) in the same manner as above. The results are shown in Fig. 26.

[0130] According to these results, it was suggested that the fraction 1 and the fraction 2 were the same substance because their NMR spectra were similar. In addition, the • near 5 ppm was a signal derived from an octasaccharide, and the • near 1 ppm was a signal derived from rhamnose. Next, the fraction 2 was subjected to constituent sugar analysis.

[0131] <Constituent sugar analysis> The constituent sugars were analyzed and quantified by derivation with 1-phenyl-3-methyl-5-pyrazolone (PMP). For calibration curve creation, the standard products L-Arabinose (Ara), 6-deoxy talose (6dT), L-Fucose (Fuc), D-Galactosamine (GalN), D-Galactose (Gal), D-Galacturonic acid (GalA), D- Glucosamine (GlcN), D-Glucose (Glc), D-Glucuronic acid (GlcA), D-Mannose (Man), Muramic acid (MurA), L-Rhamnose (Rha), D-Ribose (Rib), and D-Xylose (Xyl) were used. Aqueous solutions of the sugars GlcN, Man, Rib, 6-Deoxy Tal, GlcA, Glc and Xyl were mixed to prepare a standard product mixed solution 1 with concentrations of 0.0625, 0.25, 1 and 4 mM. Similarly, aqueous solutions of the sugars MurA, GalN, Rha, GalA, Gal, Ara and Fuc were mixed to prepare a standard product mixed solution 2 with concentrations of 0.0625, 0.25, 1 and 4 mM. The internal standard used was 0.0625 mM D-Talose (Tal). The fraction 2 was dissolved in ultrapure water at a concentration of 5 mg / mL to prepare an analysis sample.

[0132] 400 pL of ultrapure water and 500 pL of an 8 M trifluoroacetic acid aqueous solution were added to each screw-cap test tube, 100 pL of the analysis sample or the standard product mixed solutions 1 and 2 at each concentration were added, the tube was capped, and the mixture was stirred and then subjected to acid decomposition at 100°C for 2 hours. After cooling, the mixture was evaporated to dryness using a centrifugal evaporator. 400 pL of ultrapure water was added, the mixture was stirred, 100 pL of the mixture was then transferred to a new screw-cap test tube, 100 pL of the internal standard, 100 pL of a 0.6 M sodium hydroxide aqueous solution, and 200 pL of a 0.5 M PMP methanol solution were added, the tube was capped, and the mixture was stirred and then reacted at 70°C for 30 minutes. After cooling, 1 mL of a 0.1 M hydrochloric acid aqueous solution was added to make the mixture acidic. The mixture was washed three times with about 1 mL of chloroform.

[0133] The aqueous layer after washing with chloroform was filtered through a 0.45 pm filter (Millipore UFC30HVNB), and HPLC analysis was performed under the following conditions. The number of moles of each sugar was calculated from the peak area using a calibration curve of the standard product of each sugar. Column washing was performed every 4 to 6 analyses.

[0134] (HPLC analysis) Column temperature: 40°C Column: Waters CORTECS UPLC T3 (^2.1 mm x 100 mm, 1.6 pm) Flow rate: 0.3 mL / min Mobile phase: 10 mM triethylamine (TEA)-formic acid (FA) (pH 4.7) / MeCN=75 / 25 Measurement time: 8 min Detection: UV 245 nm

[0135] (Column washing) Column temperature: 40°C Flow rate: 0.3 mL / min Mobile phase: A H2O, B MeCN Gradient: 0 to 3 min: A=75, B=25, 3 to 6 min: A=75^5, B=25^95, 6 to 9 min: A=5, B=95, 9 to 12 min: A=5^75, B=95^25%, 12 to 15 min: A=75, B=25 Detection: UV 245 nm

[0136] Based on the constituent sugar analysis results, it was found that the fraction 2 contained rhamnose (Rha), glucose (Glc) and galactose (Gal) in a ratio of Rha:Glc:Gal=3.7:1.0:1.5 (Fig. 27). Based on the 1H-NMR results, the number of sugars in the repeating structure was initially thought to be 8, but since the sugar ratio appeared to be 4:1:2 or 3:1:1, it was thought to be 5 to 7 sugars. From the above, it was found that the chemical structure of the active substance isolated from the fractions recovered from a 10,000- to 30,000-molecular-weight-cutoff ultrafiltration was a repeating structure of 5 to 7 sugars containing rhamnose as the main sugar and additionally containing glucose and galactose, and the repeating structure had rhamnose, glucose, and galactose in a ratio of (4 to 3):1:(2 to 1) (Fig. 28). Industrial Applicability

[0137] The intestinal barrier protective agent of the present invention can be used to protect the intestinal barrier or as a prophylactic agent for diseases related to the disruption of the intestinal barrier.

[138] Rule 26, 02.04.2025 0-1 0-1-1 Form PCT / RO / 134 Indications relating to the deposited microorganism or other biological material (PCT Rule 13bis) were prepared by the following. JPO-PAS i530 0-2 International Application No . 0-3 Applicant's or agent's file reference PF-240021-WO 1 1-1 The following indication relates to the microorganism or biological material described in the Detailed Description of the Invention . Paragraph number 0019 1-3 1-3-1 1-3-2 1-3-3 1-3-4 Indication of deposit Name of depositary institution Address of depositary institution Date of deposit Accession number NPMD NITE Patent Microorganisms Depositary (NPMD), National Institute of Technology and Evaluation #122, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba 292-0818, Japan January 24, 2023 (24.01.2023) NPMD NITE BP-03808 1-5 Designated states for which indications are made All designated States 2 2-1 The following indication relates to the microorganism or biological material described in the Detailed Description of the Invention . Paragraph number 0020 2-3 2-3-1 2-3-2 2-3-3 2-3-4 Indication of deposit Name of depositary institution Address of depositary institution Date of deposit Accession number NPMD NITE Patent Microorganisms Depositary (NPMD), National Institute of Technology and Evaluation #122, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba 292-0818, Japan January 15, 2024 (15.01.2024) NPMD NITE BP-04064 2-5 Designated states for which indications are made All designated States 3 3-1 The following indication relates to the microorganism or biological material described in the Detailed Description of the Invention . Paragraph number 0021 3-3 3-3-1 3-3-2 3-3-3 3-3-4 Indication of deposit Name of depositary institution Address of depositary institution Date of deposit Accession number NPMD NITE Patent Microorganisms Depositary (NPMD), National Institute of Technology and Evaluation #122, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba 292-0818, Japan January 15, 2024 (15.01.2024) NPMD NITE BP-04065 3-5 Designated states for which indications are made All designated States 4 4-1 The following indication relates to the microorganism or biological material described in the Detailed Description of the Invention . Paragraph number 0022 4-3 4-3-1 4-3-2 4-3-3 4-3-4 Indication of deposit Name of depositary institution Address of depositary institution Date of deposit Accession number NPMD NITE Patent Microorganisms Depositary (NPMD), National Institute of Technology and Evaluation #122, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba 292-0818, Japan January 24, 2023 (24.01.2023) NPMD NITE BP-03809 4-5 Designated states for which indications are made All designated States 5 5-1 The following indication relates to the microorganism or biological material described in the Detailed Description of the Invention . Paragraph number 0023 5-3 5-3-1 5-3-2 5-3-3 5-3-4 Indication of deposit Name of depositary institution Address of depositary institution Date of deposit Accession number NPMD NITE Patent Microorganisms Depositary (NPMD), National Institute of Technology and Evaluation #122, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba 292-0818, Japan January 24, 2023 (24.01.2023) NPMD NITE BP-03810 5-5 Designated states for which indications are made All designated States 6 6-1 The following indication relates to the microorganism or biological material described in the Detailed Description of the Invention . Paragraph number 0024 6-3 6-3-1 6-3-2 6-3-3 6-3-4 Indication of deposit Name of depositary institution Address of depositary institution Date of deposit Accession number NPMD NITE Patent Microorganisms Depositary (NPMD), National Institute of Technology and Evaluation #122, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba 292-0818, Japan January 24, 2023 (24.01.2023) NPMD NITE BP-03811 6-5 Designated states for which indications are made All designated States 7 7-1 The following indication relates to the microorganism or biological material described in the Detailed Description of the Invention . Paragraph number 0025 7-3 7-3-1 7-3-2 7-3-3 7-3-4 Indication of deposit Name of depositary institution Address of depositary institution Date of deposit Accession number NPMD NITE Patent Microorganisms Depositary (NPMD), National Institute of Technology and Evaluation #122, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba 292-0818, Japan January 24, 2023 (24.01.2023) NPMD NITE BP-03812 7-5 Designated states for which indications are made All designated States For receiving Office use only 0-4 This sheet was received with the international application (Yes / No) (Checked) 0-4-1 Authorized officer Tsuneo Nagai For International Bureau use only 0-5 This sheet was received by the International Bureau on : 0-5-1 Authorized officer

Claims

1. An intestinal barrier protective agent comprising, as an active ingredient, Bifidobacterium adolescentis.

2. The intestinal barrier protective agent according to claim 1, wherein the intestinal barrier protection is protection of the intestinal barrier from one or more stimulations selected from among LPS stimulation, inflammatory cytokine stimulation, and oxidative stress stimulation.

3. A prophylactic agent against an intestinal barrier disruption-related disease, comprising the intestinal barrier protective agent according to claim 1 or 2.

4. The prophylactic agent against an intestinal barrier disruption-related disease according to claim 3, wherein the intestinal barrier disruption-related disease is selected from among diabetes, obesity, inflammatory bowel disease, fatty liver, Parkinson’s disease, cancer, irritable bowel syndrome, dyslipidemia, atopic dermatitis, asthma, dementia, insomnia, depression, and rheumatoid arthritis.

5. An intestinal barrier protective agent comprising, as an active ingredient, a cell wall polysaccharide fraction and / or a cytoplasmic fraction of Bifidobacterium adolescentis.

6. An intestinal barrier protective agent comprising, as an active ingredient, a polysaccharide having a repeating structure of rhamnose, glucose, and galactose in a ratio of (4 to 3):1:(2 to 1), wherein the polysaccharide is recovered from a 10,000- to 30,000-molecular-weight-cutoff ultrafiltration fraction.

7. An intestinal barrier protective agent, comprising, as an active ingredient, uridine diphosphate-N-acetylmuramic acid-L-alanine-D-glutamic acid-L-lysine.

8. A polysaccharide having a repeating structure of rhamnose, glucose, and galactose in a ratio of (4 to 3):1:(2 to 1), wherein the polysaccharide is recovered from a 10,000- to 30,000-molecular-weight-cutoff ultrafiltration fraction.

9. A method of producing a polysaccharide having a repeating structure of rhamnose, glucose, and galactose in a ratio of (4 to 3):1:(2 to 1), wherein the polysaccharide is recovered from a 10,000- to 30,000-molecular-weight-cutoff ultrafiltration fraction, the method comprising: subjecting Bifidobacterium adolescentis to a lysis treatment, then obtaining a fraction corresponding to a molecular weight range defined by ultrafiltration membranes having molecular weight cutoffs of 10,000 to 30,000, and additionally collecting these fractions through hydrophilic interaction liquid chromatography performed under the following conditions or equivalent conditions: [Chromatography conditions] Flow rate: 1.0 mL / min Mobile phase A: MeCN, B: H2O Gradient: 0 to 3.3 min: A=95, B=5, 3.3 to 83.3 min: A=95^0, B=5^100 Column: YMC-triart Diol HILIC (^4.6 x 250 mm, 5 pm) Temperature: 40°C Injection volume: 2 to 10 pL (5 mg / mL in H2O) Elution time: 22 to 35 min.

10. The method of producing a polysaccharide according to claim 9, wherein the lysis treatment includes a treatment with a muramidase, followed by a treatment with a nuclease and a protease.

11. A prophylactic agent against an intestinal barrier disruption-related disease, comprising the intestinal barrier protective agent according to any one of claims 5 to 7.

12. The prophylactic agent against an intestinal barrier disruption-related disease according to claim 11, wherein the intestinal barrier disruption-related disease is selected from among diabetes, obesity, inflammatory bowel disease, fatty liver, Parkinson’s disease, cancer, irritable bowel syndrome, dyslipidemia, atopic dermatitis, asthma, dementia, insomnia, depression, and rheumatoid arthritis.

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