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

Intestinal barrier protector and preventive agent for diseases associated with intestinal barrier breakdown

[0001] The present invention relates to an agent for protecting an intestinal barrier and an agent for preventing diseases associated with intestinal barrier breakdown, which contain, as an active ingredient, Bifidobacterium adolescentis or a component derived from Bifidobacterium adolescentis.

[0002] In recent years, attention has been drawn to the influence that intestinal bacteria have on the pathology of metabolic disorders in the host, particularly the pathogenesis of obesity and diabetes. Currently, intestinal bacteria are thought to be a cause of obesity, along with dietary habits and lack of exercise, and novel treatments for obesity are being sought that involve changing the intestinal flora of obese patients and further improving the intestinal environment.

[0003] Dysbiosis is caused by various factors (stress, aging, alcohol, smoking, diet, antibiotics, lack of physical activity, etc.), and as a result, the intestinal barrier function is disrupted by three stimuli: lipopolysaccharide (LPS), a toxin derived from enterobacteria, inflammatory cytokines in the intestinal tract, and oxidative stress. This disruption allows LPS and inflammatory cytokines to enter the bloodstream and circulate, reaching the adipose tissue and liver, which determine insulin sensitivity, and causing chronic inflammation and insulin resistance (Non-Patent Documents 1 and 2).

[0004] Therefore, it is believed that protecting the intestinal barrier function can also prevent diseases associated with the breakdown of the intestinal barrier, such as obesity and diabetes.

[0005] Although intestinal bacteria that protect the intestinal barrier function have been searched for, reports have been made so far of the intestinal barrier protective effects of Bifidobacterium bifidum and Bifidobacterium animalis against cytokine stimulation (Non-Patent Documents 3 to 6), and the intestinal barrier protective effects of Bifidobacterium longum and Bifidobacterium animalis against LPS stimulation (Non-Patent Documents 7 to 8), but no intestinal bacteria have been reported that have the ability to protect the intestinal barrier against multiple stimuli.

[0006] Coppe. JP, et al., PLoS Biol. 6. 2853. 2008Winer. D. A, et al., Cell Metab. 23. 413. 2016Al-Sadi. R, et al., Int. J. Mol. Sci. 22. 8070. 2021Hsieh. C. Y, et al., Physiol Rep. 3. 3. e12327. 2015Wang. et al., Front Microbiol. 13. 817591. 2022

[0007] The present invention aims to find an intestinal bacterium that is more effective than conventional ones in protecting the intestinal barrier function, and further to find its active substance.

[0008] As a result of intensive research to solve the above problems, the present inventors discovered that a specific bifidobacterium has an excellent protective effect on the intestinal barrier function, and further discovered its active ingredient, thereby completing the present invention.

[0009] That is, the present invention provides the following: [1] An intestinal barrier protection agent characterized by containing Bifidobacterium adolescentis as an active ingredient. [2] The intestinal barrier protection agent according to [1], wherein the intestinal barrier protection is protection of the intestinal barrier from one or more stimuli selected from LPS stimulation, inflammatory cytokine stimulation, and oxidative stress stimulation. [3] An agent for preventing diseases associated with intestinal barrier breakdown, comprising the intestinal barrier protection agent according to [1] or [2]. [4] The agent for preventing diseases associated with intestinal barrier breakdown according to [3], wherein the disease associated with intestinal barrier breakdown is selected from diabetes, obesity, inflammatory bowel disease, fatty liver, Parkinson's disease, cancer, irritable bowel disease, dyslipidemia, atopy, asthma, dementia, insomnia, depression, and rheumatoid arthritis. [5] An intestinal barrier protection agent containing a cell wall polysaccharide fraction and / or a cytoplasmic fraction of Bifidobacterium adolescentis as an active ingredient. [6] An intestinal barrier protector comprising, as an active ingredient, a polysaccharide having a repeating structure of rhamnose, glucose, and galactose in a 4-3:1:2-1 ratio, and having a molecular weight cutoff of 10,000 to 30,000 using an ultrafiltration membrane. [7] An intestinal barrier protector 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 4-3:1:2-1 ratio, and having a molecular weight cutoff of 10,000 to 30,000 using an ultrafiltration membrane. [9] Bifidobacterium adolescentis is lysed, and then a fraction with a molecular weight cutoff of 10,000 to 30,000 is obtained by ultrafiltration. This fraction is then subjected to hydrophilic interaction chromatography under the following conditions or equivalent conditions: [Chromatography conditions] Flow rate: 1.0 mL / min Mobile phase A: MeCN, B: H2O Gradient: 0-3.3 min: A=95, B=5, 3.3-83.3 min: A=95→0, B=5→100 Column: YMC-triart Diol HILIC (φ4.6x250mm, 5μm) Temperature: 40°C Injection volume: 2-10 μL (5 mg / mL in H2O) Elution time: 22-35 min 2. A method for producing a polysaccharide having a repeating structure of rhamnose, glucose and galactose in a ratio of 4-3:1:2-1, and a molecular weight cutoff of 10,000 to 30,000 by ultrafiltration membrane, comprising:

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

[11] A preventive agent for intestinal barrier breakdown-associated diseases, comprising the intestinal barrier protection agent according to any one of [5] to [7].

[12] The preventive agent for intestinal barrier breakdown-associated diseases according to

[11] , wherein the intestinal barrier breakdown-associated disease is selected from diabetes, obesity, inflammatory bowel disease, fatty liver, Parkinson's disease, cancer, irritable bowel disease, dyslipidemia, atopy, asthma, dementia, insomnia, depression, and rheumatoid arthritis.

[0010] The intestinal barrier protection agent of the present invention contains as its active ingredient Bifidobacterium adolescentis present in the intestine, a cell wall polysaccharide fraction and / or cytoplasmic fraction of Bifidobacterium adolescentis, a polysaccharide derived from said fraction and having a repeating structure of rhamnose, glucose and galactose in a ratio of 4-3:1:2-1 and having a molecular weight cutoff of 10,000 to 30,000 when filtered through an ultrafiltration membrane, or uridine diphosphate-N-acetylmuramic acid-L-alanine-D-glutamic acid-L-lysine, and is therefore capable of protecting the intestinal barrier safely for a long period of time.

[0011] Furthermore, since the intestinal barrier protecting agent of the present invention can protect the intestinal barrier, it is also useful as a preventive agent for diseases associated with breakdown of the intestinal barrier.

[0012] Furthermore, the polysaccharide of the present invention has a repeating structure of rhamnose, glucose, and galactose in a ratio of 4-3:1:2-1, and has a molecular weight cutoff of 10,000 to 30,000 using an ultrafiltration membrane, and can be used for a variety of purposes.

[0013] 1 shows the relationship between TEER and time in monolayers stimulated with LPS in Example 1. 2 shows the relationship between TEER after 48 hours in monolayers stimulated with LPS in Example 1. 3 shows the relationship between TEER and time in monolayers stimulated with inflammatory cytokines in Example 1. TI represents TNF-α+IFN-γ. 4 shows the relationship between TEER after 48 hours in monolayers stimulated with inflammatory cytokines in Example 1. TI represents TNF-α+IFN-γ. 5 shows the relationship between TEER and time in monolayers stimulated with oxidative stress in Example 1. 6 shows the relationship between TEER and time in monolayers stimulated with oxidative stress in Example 1. 6 shows the relationship between TEER and time in monolayers stimulated with oxidative stress in Example 1. 7 shows the effect on the localization of tight junction proteins in Example 1 (+: YIT 13021 added, -: no YIT 13021 added). 8 shows the relationship between TEER and time in monolayers unstimulated in Example 2. 9 shows the relationship between TEER and time in monolayers unstimulated in Example 2. 1 shows the TEER after 48 hours for LPS-stimulated monolayers of Example 3. 2 shows the TEER after 48 hours for inflammatory cytokine-stimulated monolayers of Example 3. TI represents TNF-α+IFN-γ. 3 shows the TEER after 6 hours for oxidative stress-stimulated monolayers of Example 3. 4 shows the TEER after 24 hours for unstimulated monolayers of Example 4. 5 shows the TEER after 24 hours for unstimulated monolayers of Example 5 (heat-killed cells). 6 shows the TEER after 24 hours for unstimulated monolayers of Example 5 (viable cells). 7 shows the TEER after 24 hours for unstimulated monolayers of Example 6 (viable cells cultured in combination with starch granules). 8 shows the TEER values ​​of cell wall polysaccharide fractions (including cytoplasmic fractions) under unstimulated conditions. 1 shows the results of size exclusion chromatography (SEC) analysis of cell wall polysaccharide fractions (including cytoplasmic fractions). 2 shows the results of TEER values ​​under unstimulated conditions when the cell wall polysaccharide fractions (including cytoplasmic fractions) were fractionated using an ultrafiltration membrane and then added to a concentration of 1 μg / mL.This figure shows the results of hydrophilic interaction chromatography (HILIC) analysis of fractions with a molecular weight of 10,000 or less, and the fractions that were separated. This figure shows the results of TEER values ​​under no stimulation when fractions collected from a molecular weight of 10,000 or less were added at 1 μg / mL. Fraction 2 collected from a molecular weight of 10,000 or less. １ 1 shows the H-NMR spectrum. 2 shows the structure of uridine diphosphate-N-acetylmuramic acid-L-alanine-D-glutamic acid-L-lysine. 3 shows the results of HILIC analysis of fractions with molecular weights of 10,000 to 30,000 and the fractions collected. 4 shows the results of TEER values ​​under non-stimulation when fractions collected from molecular weights of 10,000 to 30,000 are added to give a concentration of 1 μg / mL. 5 shows the results of TEER values ​​under non-stimulation when fractions 1 and 2 collected from molecular weights of 10,000 to 30,000 are added to give a concentration of 1 μg / mL. １ 1 shows a H-NMR spectrum. FIG. 2 shows the results of the analysis of the constituent sugars of fraction 2. FIG. 3 shows the structure of a polysaccharide.

[0014] Among the intestinal barrier protecting agents of the present invention (hereinafter referred to as "protective agents of the present invention"), those containing Bifidobacterium adolescentis as an active ingredient are referred to as Protective Agent of the Present Invention 1. Furthermore, among the protective agents of the present invention, those containing as an active ingredient a cell wall polysaccharide fraction and / or a cytoplasmic fraction of Bifidobacterium adolescentis, a polysaccharide having a repeating structure of rhamnose, glucose, and galactose in a ratio of 4-3:1:2-1 and having a molecular weight cutoff of 10,000 to 30,000 using an ultrafiltration membrane, or uridine diphosphate-N-acetylmuramic acid-L-alanine-D-glutamic acid-L-lysine are referred to as Protective Agent of the Present Invention 2.

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

[0016] The Bifidobacterium adolescentis used in the protective agent 1 of the present invention may be any of the strains known as Bifidobacterium adolescentis or those isolated by conventional methods (for example, (1) Research on Bifidobacteria, edited by Mitsuoka Tomotari, Japan Bifidobacteria Center; (2) Methods for Identifying Intestinal Bacteria and Intestinal Constituent Bacteria, Fujisawa Michihiko, Japanese Journal of Bacteriology 2014; (3) PMID: 28394924 (Bifidobacterium adolescentis is isolated from mouse feces, but is applicable to humans)). Examples of such strains 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, Bifidobacterium adolescentis YIT 11040, etc. These Bifidobacterium adolescentis can be used alone or in combination of two or more types.

[0017] Among these Bifidobacterium adolescentis, Bifidobacterium adolescentis YIT 13021, Bifidobacterium adolescentis YIT 13609, and Bifidobacterium adolescentis YIT 12828 are preferred, with Bifidobacterium adolescentis YIT 13021 being more preferred.

[0018] In addition to the above-mentioned strains, the Bifidobacterium adolescentis used in the protective agent 1 of the present invention also includes strains that have been artificially genetically manipulated while maintaining the properties of Bifidobacterium adolescentis. Furthermore, although both live and dead Bifidobacterium adolescentis are effective, live bacteria are preferred.

[0019] Of the above Bifidobacterium adolescentis, Bifidobacterium adolescentis YIT 13021 has been deposited as Bifidobacterium adolescentis YIT 13021 (NITE BP-03808, deposit date: January 24, 2023) at the National Institute of Technology and Evaluation, Japan Patent Microorganisms Depositary Center (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818, Japan), which is the international depositary authority of the Budapest Treaty.

[0020] Bifidobacterium adolescentis YIT 13609 was domestically deposited as YIT 13609 (NITE P-04064, date of deposit: January 15, 2024) at the Patent Microorganisms Depositary of the National Institute of Technology and Evaluation (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818), and a request for transfer to international deposit was made on December 9, 2024, and the strain has been internationally deposited as YIT 13609 (NITE BP-04064, date of deposit: January 15, 2024) at the Patent Microorganisms Depositary, the international depositary authority of the Budapest Treaty.

[0021] Bifidobacterium adolescentis YIT 12828 was domestically deposited at the above-mentioned Patent Microorganisms Depositary as YIT 12828 (NITE P-04065, date of deposit: January 15, 2024), and a request for transfer to international deposit was made on December 9, 2024, and the strain has now been internationally deposited at the above-mentioned Patent Microorganisms Depositary, the international depositary authority under the Budapest Treaty, as YIT 12828 (NITE BP-04065, date of deposit: January 15, 2024).

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

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

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

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

[0026] The protective agent 1 of the present invention contains the above-mentioned Bifidobacterium adolescentis as an active ingredient, and is therefore capable of protecting the intestinal barrier from one or more stimuli selected from LPS stimulation, inflammatory cytokine stimulation, and oxidative stress stimulation, preferably from two or more of the above stimuli, and more preferably from the three above stimuli.

[0027] Here, LPS stimulation refers to the action of lipopolysaccharide (LPS), a toxin derived from enterobacteria, on intestinal epithelial cells.

[0028] Inflammatory cytokine stimulation refers to the action of inflammatory cytokines such as TNF-α, IFN-γ, 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, protection of the intestinal barrier means that the expression or localization of tight junction proteins present in the intestinal tract is not disrupted or disrupted even when subjected to one or more stimuli selected from LPS stimulation, inflammatory cytokine stimulation, and oxidative stress stimulation, preferably two or more of the above stimuli, and more preferably three of the above stimuli, or that a decrease in the index value of intestinal barrier strength (transepithelial electrical resistance; TEER) is suppressed, as will be shown in the Examples described below.

[0031] The form of the protective agent 1 of the present invention is not particularly limited, and, like conventionally known bifidobacteria, examples include foods and beverages such as fermented milk, yogurt, and bacterial powder supplements, as well as pharmaceuticals such as lactic acid bacteria and bifidobacterial preparations. Of these, fermented foods such as fermented milk, and pharmaceuticals such as lactic acid bacteria and bifidobacterial preparations are preferred.

[0032] The intestinal barrier can be protected by administering the protective agent 1 of the present invention to mammals, including humans. In this case, the protective agent 1 of the present invention contains 100 mg of Bifidobacterium adolescentis per day. ５ ~10 １０ It is sufficient to add it in an amount that will result in an intake of about cfu.

[0033] As described above, the protective agent 1 of the present invention can protect the intestinal barrier and therefore can be used as a preventive agent for diseases associated with intestinal barrier disruption (hereinafter referred to as "preventive agent 1 of the present invention"). Since the preventive agent 1 of the present invention can also protect against intestinal barrier disruption, diseases associated with intestinal barrier disruption also include diseases associated with intestinal barrier disruption. Examples of diseases associated with intestinal barrier disruption include diabetes, obesity, inflammatory bowel disease (Crohn's disease, ulcerative colitis, etc.), fatty liver, Parkinson's disease, cancer, irritable bowel disease, dyslipidemia, atopy, asthma, dementia, insomnia, depression, rheumatoid arthritis, etc. Among these diseases associated with intestinal barrier disruption, obesity and diabetes are preferred.

[0034] The preventive agent 1 of the present invention can prevent diseases associated with intestinal barrier disruption by administering it to mammals, including humans. In this case, the preventive agent 1 of the present invention contains 100 mg of Bifidobacterium adolescentis per day. ５ ~10 １０It is sufficient to add it in an amount that will result in an intake of about cfu.

[0035] The fact that the above-mentioned diseases are associated with breakdown of the intestinal barrier is described in the following literature. <Inflammatory bowel disease, obesity, fatty liver> Lee. B, et al., J Immunol Res. 2018. 2645465. 2018 <Parkinson's disease> Van Ijzendoorn. SC D, et al., J Parkinsons Dis. 9. S323. 2019 <Cancer, irritable bowel disease> Oshima. T, et al., J Gastroenterol. 51. 768. 2016 <Dyslipidemia> Flaig. B, et al., Nutrients. 15. 228. 2023 <Atopy, asthma> Niewiem. M, et al., Nutrients. 14. 1893. 2022 <Dementia> Stadlbauer. V, et al., BMC Geriatr. 20. 248. 2020 <Insomnia, 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 and the preventive agent 1 of the present invention, Bifidobacterium adolescentis YIT 13021, Bifidobacterium adolescentis YIT 13022, Bifidobacterium adolescentis YIT 11033, Bifidobacterium adolescentis YIT 11034, and Bifidobacterium adolescentis YIT 11040 have the property of adhering to and assimilating starch granules. This property is not found in the Bifidobacterium adolescentis type strain (ATCC15703). This property, along with test methods for confirming this property, is described in the international application (PCT / JP2023 / 3018) filed January 31, 2023.

[0037] Therefore, when Bifidobacterium adolescentis having the above-mentioned properties is used in the protective agent 1 of the present invention and the preventive agent 1 of the present invention, it is preferable to contain starch granules together with these. This suppresses the adhesion of enterobacteria such as Eubacterium lectare, which compete with them in the intestine, to the starch granules, allowing the bacteria to adhere and colonize, thereby enhancing the effect. Here, in order to more easily obtain the above-mentioned properties, it is preferable to culture Bifidobacterium adolescentis in a medium supplemented with soluble starch, starch granules, dextrin, etc.

[0038] Here, starch granules refer to starches derived from potatoes, sweet potatoes, and other potatoes; grains, such as wheat, rice, and corn; beans, such as soybeans and adzuki beans; or tapioca, bananas, and the like, which have not undergone a heating process involving water (e.g., boiling or steaming) except for the drying process during production, or which have undergone a heating process involving water but have not gelatinized, retaining their natural granular state and being insoluble in water at room temperature. More specifically, starch granules refer to uncooked starches derived from potatoes, such as sweet potatoes, grains, such as wheat, rice, and corn; beans, such as soybeans and adzuki beans; or tapioca, bananas, and the like, which have granular shapes, such as spherical, ovoid, or polyhedral, and typically have a diameter of 1 to 120 μm, depending on the plant species. This diameter can be measured using a microscope or a particle size measuring device.

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

[0040] The protective agent 1 of the present invention and the preventive agent 1 of the present invention described above can safely protect the intestinal barrier and prevent diseases associated with intestinal barrier breakdown for a long period of time.

[0041] Furthermore, the intestinal barrier protector 1 of the present invention not only protects the intestinal barrier but can also enhance the intestinal barrier in the absence of any irritation. Therefore, the intestinal barrier protector 1 of the present invention can also be used as an intestinal barrier enhancer. Here, enhancing the intestinal barrier means increasing the index value of intestinal barrier strength (transepithelial electrical resistance; TEER).

[0042] Next, we will explain the protective agent of the present invention, which contains one of the following components derived from Bifidobacterium adolescentis as an active ingredient. In the protective agent of the present invention, "protection of the intestinal barrier" refers to increasing the index value of intestinal barrier strength (transepithelial electrical resistance; TEER) compared to when 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 4-3:1:2-1, with a molecular weight cutoff of 10,000 to 30,000 using an ultrafiltration membrane. (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 the cytoplasmic fraction of Bifidobacterium adolescentis, will now be described.

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

[0045] The Bifidobacterium adolescentis used above can be the Bifidobacterium adolescentis used in the protective agent 1 of the present invention, which contains Bifidobacterium adolescentis as an active ingredient.

[0046] It is preferable to culture the above-mentioned Bifidobacterium adolescentis before lysis treatment. The culture may be carried out under conventionally known conditions. After the culture, the cells may be collected by centrifugation or washed with PBS or the like according to a conventional method.

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

[0048] Among these methods using multiple enzymes, a preferred method is to treat the tissue with muramidase, followed by treatment 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 above-mentioned preferred method is carried out as follows. First, Bifidobacterium adolescentis is sufficiently cultured in advance according to conventional methods, then collected by centrifugation and washed with PBS. Muramidase is added at 1 to 100 U, preferably 4 U, per 1 mg of viable Bifidobacterium adolescentis cells, and the mixture is incubated at 37 to 60°C, preferably 37°C, for 12 hours or more, preferably 16 to 24 hours. Next, centrifugation or other methods are performed to remove insoluble matter, and the supernatant is obtained. Nuclease is added at 1 to 100 U, preferably 1.4 U, per 1 mg of viable cells, and the mixture is incubated at 37 to 60°C, preferably 37°C, for 12 hours or more, preferably 16 to 24 hours. Next, protease is added at 0.0001 to 0.5 mg, preferably 0.006 mg, per 1 mg of viable cells, and the mixture is incubated at 37 to 60°C, preferably 37°C, for 12 hours or more, preferably 16 to 24 hours. After the reaction, purification may be carried out using a dialysis membrane, etc. The molecular weight cutoff of the dialysis membrane is 3,500 to 14,000, preferably 3,500 to 8,000.

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

[0051] The cell wall polysaccharide fraction and / or cytoplasmic fraction of Bifidobacterium adolescentis has an intestinal barrier protecting effect.

[0052] Next, we will explain (2) the polysaccharide, which is the active ingredient of the protective agent 2 of the present invention, and which has a repeating structure of rhamnose, glucose, and galactose in a ratio of 4-3:1:2-1, preferably 3:1:1 or 4:1:2, and has a molecular weight cutoff of 10,000 to 30,000 using an ultrafiltration membrane. This polysaccharide has a structure represented by the following formula and is a novel substance.

[0053]

[0054] The physical properties of this polysaccharide are as follows: Appearance: White to pale yellow solid

[0055] This polysaccharide can be produced by lysing Bifidobacterium adolescentis, filtering the lysed product through an ultrafiltration membrane to obtain a fraction with a molecular weight cutoff of 10,000 to 30,000, and then subjecting the fraction to hydrophilic interaction chromatography under the following conditions or equivalent: [Chromatography conditions] Flow rate: 1.0 mL / min Mobile phase A: MeCN, B: HO Gradient: 0-3.3 min: A=95, B=5, 3.3-83.3 min: A=95→0, B=5→100 Column: YMC-triart Diol HILIC (φ4.6×250 mm, 5 μm) Temperature: 40°C Injection volume: 2-10 μL (5 mg / mL in HO) Detection: Charged aerosol detector (CAD) Elution time: 22-35 min

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

[0057] After the lysis treatment, a cell wall polysaccharide fraction and / or a cytoplasmic fraction of Bifidobacterium adolescentis is obtained. This is then purified using an ultrafiltration membrane to obtain a fraction with a molecular weight cutoff of 10,000 to 30,000. The ultrafiltration membrane used in this process is not particularly limited as long as it can obtain the above molecular weights, but 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, a fraction with a molecular weight cutoff of 10,000 to 30,000 is obtained using an ultrafiltration membrane. This fraction is then subjected to hydrophilic interaction chromatography under the conditions described above to collect a polysaccharide with a repeating structure of rhamnose, glucose, and galactose in a ratio of 4-3:1:2-1 and a molecular weight cutoff of 10,000 to 30,000 using an ultrafiltration membrane.

[0059] In addition to the above conditions, the hydrophilic interaction chromatography can be performed under equivalent conditions that can yield a polysaccharide having a repeating structure of rhamnose, glucose, and galactose in a ratio of 4-3:1:2-1 and a molecular weight cutoff of 10,000 to 30,000 through an ultrafiltration membrane, by appropriately modifying these conditions. Such equivalent conditions can be set by a person skilled in the art by appropriately modifying the flow rate, mobile phase composition, type of column carrier, particle size of the column carrier, column length, temperature, injection volume, and injection solution concentration, with reference to the above conditions. Specific examples include the following:

[0060] [Chromatographic conditions] Flow rate: 0.5-1.0 mL / min Mobile phase: A: MeCN, B: H2O Gradient: (a)-c) a) 0-2 min: A = 95, B = 5, 2-25 min: A = 95→0, B = 5→100, b) 0-2 min: A = 95, B = 5, 2-50 min: A = 95→0, B = 5→100 c) 0-2 min: A = 95, B = 5, 2-100 min: A = 95→0, B = 5→100 Column: YMC-triart Diol HILIC (φ4.6x150 mm, 5 μm) Temperature: 40 ℃ Injection volume: 2-10 μL (5 mg / mL in H2O) Detection: Charged aerosol detector (CAD) Elution time: flow rate 1.0 mL / min; a) 8-11 min, b) 13-19 min, c) 25-35 min flow rate 0.5 mL / min; a) 10-13 min, b) 15-21 min, c) 27-37 min

[0061] As described above, a polysaccharide (2) having a repeating structure of rhamnose, glucose, and galactose in a ratio of 4-3:1:2-1 and a molecular weight cutoff of 10,000 to 30,000 through an ultrafiltration membrane is obtained. Whether or not this polysaccharide has been obtained can be confirmed by, for example, structural analysis using NMR, HPLC, or the like, or by analysis of the constituent sugars. Specifically, in SEC analysis using a Shodex SUGER KS-804 column (Showa Denko F6378035), a peak can be confirmed at an elution time of approximately 8 to 9 minutes. More specifically, it can be confirmed by the method described in the Examples below.

[0062] This polysaccharide has a repeating structure of rhamnose, glucose, and galactose in a ratio of 4-3:1:2-1, and has a molecular weight cutoff of 10,000-30,000 through an ultrafiltration membrane, and has an intestinal barrier protecting 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 explained.

[0064] Uridine diphosphate-N-acetylmuramic acid-L-alanine-D-glutamic acid-L-lysine (e.g., CAS No. 143436-84-4) is a known compound that is a cytoplasmic component 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]

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

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

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

[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 human medicine, food or drink, supplement, etc.

[0070] Pharmaceuticals can be formulated using any of the above (1) to (3) as is or, if 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 physiological saline. Furthermore, conventional additives such as stabilizers, wetting agents, emulsifiers, binders, isotonicity agents, and excipients can also be added as needed.

[0071] The dosage form is not particularly limited, and examples thereof include liquids, powders, granules, capsules, tablets, etc., 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 is, for example, preferably 1 to 100% by mass, more preferably 5 to 70% by mass, and particularly preferably 10 to 60% by mass.

[0072] When the protective agent 2 of the present invention is in the form of a food or beverage, any of the above (1) to (3) can be used as is or, if necessary, can be blended with additives, ingredients, etc. used in known food and beverage products, and prepared according to a conventional method. Examples of food and beverage products include starch-based foods such as bread, biscuits, pancakes, noodles, and tablet candy, confectioneries such as gum, candy, and Japanese sweets, meat foods such as ham and sausage, fish foods such as chikuwa and kamaboko, seafood foods, seasonings such as dressing, 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, any of the above (1) to (3) can be used as is or, if necessary, can be blended with additives, materials, etc. used in known supplements and prepared according to conventional methods.

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

[0075] As described above, the protective agent 2 of the present invention can protect the intestinal barrier, and therefore can be used as a preventive agent for diseases associated with intestinal barrier disruption (hereinafter referred to as "preventive agent 2 of the present invention"). Since the preventive agent 2 of the present invention can also protect against intestinal barrier disruption, diseases associated with intestinal barrier disruption also include diseases associated with intestinal barrier disruption. Examples of diseases associated with intestinal barrier disruption include diabetes, obesity, inflammatory bowel disease (Crohn's disease, ulcerative colitis, etc.), fatty liver, Parkinson's disease, cancer, irritable bowel disease, dyslipidemia, atopy, asthma, dementia, insomnia, depression, rheumatoid arthritis, etc. Among these diseases associated with intestinal barrier disruption, obesity and diabetes are preferred.

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

[0077] The fact that the above-mentioned diseases are associated with the breakdown of the intestinal barrier is described in the same literature as that used in the description of the preventive agent 1 of the present invention.

[0078] The protective agent 2 of the present invention and the preventive agent 2 of the present invention can safely protect the intestinal barrier and prevent diseases associated with intestinal barrier breakdown for a long period of time.

[0079] Furthermore, the intestinal barrier protector 2 of the present invention not only protects the intestinal barrier but can also enhance the intestinal barrier in the absence of any irritation. Therefore, the intestinal barrier protector 2 of the present invention can also be used as an intestinal barrier enhancer. Here, "enhancing the intestinal barrier" means increasing the index value of intestinal barrier strength (transepithelial electrical resistance; TEER) compared to when nothing is added.

[0080] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. In all of the following examples, significant differences were tested by t-test, with * indicating p<0.05, ** indicating p<0.01, and *** indicating p<0.001.

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

[0082] (1) LPS stimulation 1.1 Cells Cryopreserved human colon epithelial cell line T84 was used. T84 cells were cultured in DMEM / F-12 medium (10% FBS / F-12) containing 10% FBS and 100 μg / mL streptomycin under 5% CO ２ Subculture was carried out at 37°C.

[0083] 1.2 Preparation of Monolayer Membranes T84 monolayer membranes were prepared using a 24-well cell culture insert plate (Millipore). T84 cells were harvested from a culture dish at 80% confluence and seeded on the apical side of the wells with 400 μL of 10% FBS / F-12 at 60,000 cells / well. 800 μL of 10% FBS / F-12 was added to the basal side. The monolayer membranes were incubated in 5% CO. ２ The cells were cultured at 37°C for 10 days. The medium was replaced every 2 to 3 days with 400 μL of medium on the apical side and 800 μL on the basal side. T84 cells were cultured for 10 days to produce a monolayer membrane.

[0084] 1.3 Evaluation of protective effect on LPS-stimulated monolayer membranes LPS derived from Escherichia coli O55 (WAKO, #128-05171) was used and dissolved in 10% FBS / F-12 to prepare a test concentration of 10 ng / mL. Stimulation with LPS was performed by adding an LPS-containing medium to both the apical and basal sides of the prepared monolayer membrane. Heat-killed cells of YIT 13021 or YIT 4014 were prepared by a known method and diluted to 100 μg / mL (approximately 2.4 × 10 ８ The protective effect against damage to the monolayer membrane due to stimuli was evaluated by multiplying the actual resistance (Ω) measured using a Millicell MRS-2 (Millipore) by the culture area (cm) of the cell culture insert plate. 2 ) divided by the transepithelial electrical resistance (TEER) value (Ω / cm 2 The TEER value of the monolayer membrane at each time point after the start of stimulation was divided by the TEER value at time 0, which was immediately after the start of stimulation, to calculate the percentage change in barrier breakdown from the start of stimulation.

[0085] 1.4 Results To examine the effects of YIT 13021 or YIT 4014 on intestinal barrier function, T84 cells were seeded onto cell culture insert plates and cultured for 10 days to prepare monolayers. LPS and heat-killed cells of the respective strains were added to the monolayers, and TEER values ​​were measured every 24 hours for up to 48 hours (Fig. 1). 48 hours after addition, no decrease in TEER was observed in monolayers treated with YIT 13021, and the decrease in TEER due to LPS stimulation was significantly suppressed. For YIT 4014, the decrease was the same as that observed with LPS stimulation alone (Fig. 2).

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

[0087] 2.2 Preparation of Monolayer Membrane Monolayer membranes were prepared in the same manner as in 1.2 of (1) LPS stimulation above.

[0088] 2.3 Evaluation of protective effect on monolayer membranes stimulated with inflammatory cytokines. The inflammatory cytokines used were TNF-α (R&D Systems, #210-TA-005) and IFN-γ (R&D Systems, #285-IF-100). Equal amounts of both cytokines were dissolved in 10% FBS / F-12 to prepare a test concentration of 1 ng / mL. Stimulation with TNF-α and IFN-γ was performed by adding cytokine-containing medium only to the basal side of the prepared monolayer membrane. Measurement of the TEER value of the monolayer membrane after each elapsed time from the start of stimulation and calculation of the percentage change (%) were performed as described in 1.3 of (1) LPS stimulation above.

[0089] 2.4 Results We evaluated the protective effects of the inflammatory cytokines TNF-α and IFN-γ on barrier disruption induced by inflammatory cytokines by adding YIT 13021 or YIT 4014 to T84 monolayers. TEER values ​​were assessed after 24 and 48 hours (Figure 3). TI indicates TNF-α + IFN-γ. Monolayers stimulated with inflammatory cytokines alone or with YIT 4014 showed little change after 24 hours, but TEER values ​​decreased to approximately 50% after 48 hours. In contrast, monolayers stimulated with YIT 13021 showed an increase of approximately 20% compared to Ctrl after 24 hours. The decrease after 48 hours was limited to approximately 70%, demonstrating significant suppression of the decrease in TEER values ​​due to inflammatory cytokine stimulation (Figure 4).

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

[0091] 3.2 Preparation of Monolayer Membrane Monolayer membranes were prepared in the same manner as in 1.2 of (1) LPS stimulation above.

[0092] 3.3 Evaluation of protective effect against oxidative stress-stimulated monolayer membranes. ２ O ２ (Mitsubishi, #K-8230) was used and diluted with 10% FBS / F-12 to prepare a test concentration of 1500 μM. ２ O ２ The stimulation with H was performed on both the apical and basal sides of the prepared monolayer membrane. ２ O ２ Measurement of the TEER value of the monolayer membrane at each time point after the start of stimulation and calculation of the rate of change (%) were performed in the same manner as in 1.3 of (1) LPS stimulation above.

[0093] 3.4 Results H on the monolayer of T84 ２ O ２ The protective effect of YIT 13021 or YIT 4014 on barrier breakdown due to oxidative stress was evaluated by adding YIT 13021 or YIT 4014 simultaneously. TEER values ​​were measured every hour for up to 6 hours after addition (Fig. 5).２ O ２ Although no protective effect against irritation was observed in monolayers containing YIT 13021, the H ２ O ２ The decrease in TEER value was significantly suppressed compared to the case of stimulation alone, and H ２ O ２ A protective effect against irritation was observed (Fig. 6).

[0094] (4) Effect 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 Monolayer membranes were prepared in the same manner as in 1.2 of (1) LPS stimulation above.

[0096] 4.3 Immunostaining of Monolayer Membranes After washing the monolayer membranes twice with PBS, they were placed in a 24-well cell culture insert plate and fixed in 10% neutral buffered formalin for 15 minutes. After two washes, they were permeabilized with 0.25% Triton X-100 / PBS for 5 minutes. After two more washes, they were blocked with 10% BSA / PBS at 37°C for 60 minutes. After removing the blocker, anti-ZO-1 antibody (Abcam, #ab216880) diluted 1:100 in PBS was added and incubated overnight at 4°C. After two washes, anti-rabbit Alexa 546 antibody (Invitrogen, #A11010) diluted 5:500 in PBS was added and incubated at 37°C in the dark for 60 minutes. Finally, after washing three times, the membrane from the cell culture insert plate was cut out and placed on a glass slide. A drop of the mounting medium VECTASHIELD with DAPI (VECTOR LABORATORIES) was placed on the slide, and a cover glass was placed on top to prepare the slide for observation. The cells were observed using an all-in-one fluorescence microscope BZ-X700 (Keyence).

[0097] 4.4 Results Monolayers were subjected to the same stimuli as described above (1) to (3). After 48 hours of inflammatory cytokine and LPS stimulation and 6 hours of oxidative stress stimulation, the monolayers were immunostained for ZO-1, a protein that constitutes tight junctions, and observed (Fig. 7 ). Alternatively, ZO-1 immunostained monolayers were simultaneously stimulated as described above (1) to (3) and YIT 13021 was added, and the monolayers were then observed (Fig. 7 ). The protective effect of YIT 13021 on barrier function was evaluated (Fig. 7). The results of evaluating the protective effect of YIT 13021 against LPS, inflammatory cytokine, and oxidative stress stimulation showed that stimulation alone disrupted the ZO-1 network, revealing the presence of ZO-1 not only in the cell membrane but also in the cytoplasm, confirming disruption of tight junctions. On the other hand, in monolayers to which YIT 13021 was added simultaneously with stimulation, the meshwork localization of ZO-1 was disrupted and its presence in the cytoplasm was reduced, indicating that YIT 13021 suppressed the disruption of tight junctions. These results confirmed the protective effect of YIT 13021 on the intestinal barrier from the perspective of the localization of tight junction proteins.

[0098] Example 2: Intestinal Barrier Enhancement: YIT 13021 and YIT 4014 were used as test strains. To examine their effects on intestinal barrier function, T84 cells prepared as in Example 1 were seeded onto cell culture insert plates and cultured for 10 days to prepare monolayer membranes. Heat-killed YIT 13021 or YIT 4014 cells were added to the apical side of the monolayer membrane (no stimulation), and TEER values ​​were measured every hour for up to 6 hours (Figure 8). As a result, a significant increase in TEER value was observed in the monolayer membrane 6 hours after addition of YIT 13021 compared with the untreated Ctrl (Figure 9). On the other hand, the TEER value in the monolayer membrane added with YIT 4014 was similar to that in the untreated Ctrl.

[0099] These results demonstrate that YIT 13021 has the effect of strengthening the intestinal barrier.

[0100] Example 3 Intestinal Barrier Protective Effect: Bifidobacterium adolescentis other than YIT 13021 was also tested for its intestinal barrier protective effect similar to that of YIT 13021. The Bifidobacterium adolescentis strains used were YIT 13609 (NITE BP-04064) and YIT 12828 (NITE BP-04065).

[0101] (1) LPS stimulation Heat-killed cells of each strain were added to the apical side of the monolayer membrane in the same manner as in (1) LPS stimulation in Example 1, and the TEER value was measured 48 hours later. The results showed that 48 hours after addition, YIT 13609 and YIT 12828 had the same intestinal barrier protective effect against LPS stimulation as YIT 13021 (Figure 10).

[0102] (2) Inflammatory cytokine stimulation Heat-killed cells of each strain were added to the apical side of the monolayer membrane in the same manner as in (2) Inflammatory cytokine stimulation in Example 1, and the TEER value was measured 48 hours later. As a result, it was found that 48 hours after addition, YIT 13609 and YIT 12828 had the same intestinal barrier protective effect against inflammatory cytokine stimulation as YIT 13021 ( FIG. 11 ).

[0103] (3) Oxidative Stress Stimulation Heat-killed cells of each strain were added to the apical side of the monolayer membrane in the same manner as in (3) Oxidative Stress Stimulation in Example 1, and the TEER value was measured 6 hours later. As a result, it was found that YIT 13609 and YIT 12828 had the same intestinal barrier protective effect against oxidative stress stimulation as YIT 13021 6 hours after addition ( FIG. 12 ).

[0104] Example 4: Intestinal Barrier Enhancement: YIT 13021, YIT 13609, and YIT 12828 were used as test strains. To examine their effects on intestinal barrier function, T84 cells prepared as in Example 1 were seeded onto cell culture insert plates and cultured for 10 days to produce monolayer membranes. Heat-killed cells of YIT 13021, YIT 13609, or YIT 12828 were added to the apical side of the monolayer membrane, and the TEER value was measured 24 hours later. The results showed that YIT 13609 and YIT 12828 had the same intestinal barrier enhancing effect as YIT 13021 ( FIG. 13 ).

[0105] Example 5: Intestinal Barrier Enhancement: To examine the effect of live YIT 13021 bacteria on intestinal barrier function, T84 cells prepared as in Example 1 were seeded onto cell culture insert plates and cultured for 10 days to produce monolayer membranes. Live or heat-killed YIT 13021 bacteria were added to the apical side of the monolayer membrane at various concentrations, and TEER values ​​were measured and compared 24 hours later. As a result, a significant increase in TEER value was observed with the heat-killed bacteria starting at an added concentration of 10 μg / mL ( FIG. 14 ), while a significant increase in TEER value was observed with the live bacteria starting at an added concentration of 1 μg / mL, demonstrating that the live bacteria exert a barrier-enhancing effect even at low concentrations ( FIG. 15 ).

[0106] Example 6: Intestinal Barrier Enhancement: YIT 13021 has the characteristic of adhering to and assimilating starch granules, and the presence or absence of starch granules during culture may affect the properties of the bacterial cell. Therefore, the carbon source in mGAM medium was changed from glucose to starch granules, and the effect of live YIT 13021 cultured on intestinal barrier function was examined. T84 cells prepared as in Example 1 were seeded on cell culture insert plates and cultured for 10 days to produce monolayer membranes. Live YIT 13021 cells cultured in the presence of starch granules were added to the apical side of the monolayer membrane at various concentrations, and TEER values ​​were measured and compared after 24 hours. As a result, a significant increase in TEER value was confirmed in live YIT 13021 cells cultured in the presence of starch granules, starting from an added concentration of 0.1 μg / mL, indicating that live YIT 13021 cells cultured in combination with starch granules exhibited a barrier-enhancing effect even at low concentrations ( FIG. 16 ) compared to live YIT 13021 cells cultured using glucose as a carbon source ( FIG. 15 ).

[0107] Example 7 Protective effect of cell wall polysaccharide fraction of Bifidobacterium adolescentis on intestinal barrier: After culturing a strain belonging to Bifidobacterium adolescentis, the cell wall polysaccharide fraction was obtained by lysis treatment and analyzed.

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

[0109] (2) Cultivation The YIT 13021 strain was cultured in a modified GAM medium supplemented with 1% soluble starch (Fujifilm Wako Pure Chemical Industries, Ltd. 191-03985) (hereinafter referred to as "1% Starch mGAM") under anaerobic conditions at 37°C.

[0110] (3) Lysis 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 adjusted to pH 6.8 with 0.1 mol / L sodium hydroxide solution (Kanto Chemical 37851-08). Ultrapure water was added to this solution to make a 1.6 L volume, and the solution was sterilized by filtration using a 0.2 μm filter (Thermo 567-0020). To 270 mL of the filtrate, 109.78 mg (0.540 mmol) of magnesium chloride hexahydrate (Kanto Chemical 25009-30) was added to prepare 5 mM Tris-maleate buffer (pH 6.8) containing 2 mM MgCl2.

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

[0112] The remaining reaction mixture was centrifuged at 12,000 × g at 24°C for 45 minutes to precipitate insoluble material, and the supernatant was collected. Benzonase (nuclease) was added to the supernatant at 1.4 U per 1 mg of bacterial cells, and the mixture was incubated at 37°C for 24 hours. Pronase (protease) was then added at 0.006 mg per 1 mg of bacterial cells, and the mixture was incubated at 37°C for 24 hours with shaking. The mixture was dialyzed in pure water at room temperature using a dialysis membrane with a molecular weight cutoff of 3,500 for 72 hours, followed by lyophilization to obtain a cell wall polysaccharide fraction (42.5 mg).

[0113] The TEER value of this cell wall polysaccharide fraction under non-stimulatory conditions was measured using the following method, and the results are shown in Figure 17. These results demonstrate that the cell wall polysaccharide fraction has a protective effect on the intestinal barrier.

[0114] <TEER Measurements Without Stimulation> T84 monolayers were prepared using 24-well cell culture insert plates (Millipore). T84 cells were harvested from 80% confluent culture dishes and seeded at 60,000 cells / well in 400 μL of 10% FBS / F-12 on the apical side of the wells. 800 μL of 10% FBS / F-12 was added to the basal side, and the cells were cultured at 37°C in 5% CO2. Medium was replaced every 2–3 days, with 400 μL of medium on the apical side and 800 μL on the basal side. T84 monolayers were cultured for 10 days to prepare monolayers.

[0115] The test substance was added at each concentration to the apical portion of the prepared monolayer membrane, and the actual resistance (Ω) measured after 24 hours using a Millicell MRS-2 (Millipore) was calculated based on the culture area (cm) of the cell culture insert plate. 2 ) divided by the transepithelial electrical resistance (TEER) value (Ω / cm 2 The barrier enhancement effect was evaluated by the TEER measurement. The TEER value of the monolayer membrane at each time point after the start of stimulation was divided by the TEER value at time 0, immediately after the start of stimulation, to calculate the percentage change in barrier breakdown from the start of stimulation.

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

[0117] <Size Exclusion Chromatography (SEC) Measurement> Column: Shodex SUGER KS-804 (φ8.0 mm × 300 mm) Mobile phase: 50 mM NaCl aq. Flow rate: 1.0 mL / min Temperature: 80°C Detection: Refractive index detector (RI)

[0118] SEC revealed that the cell wall polysaccharide fraction contained fractions with molecular weights of 10,000, 30,000, 100,000, and 400,000 or greater. This fraction was then fractionated 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) into fractions with molecular weights of 100,000 or greater, 100,000 to 30,000, 30,000 to 10,000, and less than 10,000. The TEER values ​​of these fractions under unstimulated conditions were measured using the following method. The results are shown in Figure 19.

[0119] <TEER Value Measurement Without Stimulation> Measurement was carried out in the same manner as in the TEER value measurement without stimulation in Example 7 above.

[0120] These results indicated that the fractions with a molecular weight cutoff of 30,000 or less (the fractions between 10,000 and 30,000 and the fraction below 10,000) were active. Next, the fractions with a molecular weight of 10,000 or less were analyzed by hydrophilic liquid interaction chromatography (HILIC) (Figure 20). The liquid was passed through the column 24 times (a total of 1.2 mg), and fractions 1 to 4 in Figure 20 were collected each time (1 (16-20.5 min): 0.45 mg, 2 (20.5-22 min): 0.25 mg, 3 (22-25 min): 0.40 mg, 4 (51-56 min): 0.29 mg).

[0121] <HILIC analysis> Flow rate: 1.0 mL / min Mobile phase A: MeCN, B: H2O Gradient: 0-3.3 min: A=95, B=5, 3.3-83.3 min: A=95→0, B=5→100 Column: YMC-triart Diol HILIC (Φ4.6x250 mm, 5 μm) Temperature: 40 ℃ Injection volume: 10 μL (5 mg / mL in H2O)

[0122] The TEER values ​​of the fractions 1 to 4 obtained above were measured in the same manner as above. The results are shown in Figure 21. Only fraction 2 was found to have an enhanced protective effect on the intestinal barrier. １ Structural analysis was carried out using H-NMR (700 MHz in DO at 25°C), and the results are shown in Figure 22.

[0123] < １H-NMR structural analysis> Fraction 2 was dissolved in heavy water (Kanto Chemical 32070-1A) and heated at 25°C. 1 H-NMR (700 MHz) was measured.

[0124] １ H-NMR results showed that fraction 2 had a signal at ■, indicating that it was 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 Figure 23. Furthermore, the molecular weight of uridine diphosphate-N-acetylmuramic acid-L-alanine-D-glutamic acid-L-lysine is 1008, but mass spectrometry analysis of fraction 2 revealed an [M+1] of 1008.8 (not shown). Since fraction 2 contains cell wall precursors, it was determined that the cell wall polysaccharide fractions measured up to that point also contained cytoplasmic fractions.

[0125] (Uridine diphosphate-N-acetylmuramic acid-L-alanine-D-glutamic acid-L-lysine signal)

[0126]

[0127] Example 9: Intestinal barrier protection effect of the fraction with a molecular weight cutoff of 10,000 to 30,000: HILIC analysis was performed on the fraction with a molecular weight cutoff of 10,000 to 30,000 obtained in Example 8 (Figure 24). The liquid was passed through the column 48 times (total of 2.4 mg), and fractions 1 to 4 in Figure 24 were collected each time (1 (22-30 min): 1.19 mg, 2 (30-35 min): 1.26 mg, 3 (35-42.5 min): 1.01 mg, 4 (51.5-57.5 min): 0.79 mg).

[0128] <HILIC analysis> Flow rate: 1.0 mL / min Mobile phase A: MeCN, B: H2O, 0-3.3 min: A=95, B=5, 3.3-83.3 min: A=95→0, B=5→100 Column: YMC-triart Diol HILIC (φ4.6x250 mm, 5 μm) Temperature: 40 ℃ Injection volume: 10 μL (5 mg / mL in H2O)​

[0129] The TEER values ​​of the fractions 1 to 4 obtained above were measured in the same manner as above. The results are shown in Figure 25. Fractions 1 and 2 were found to have enhanced protective effects on the intestinal barrier. Fractions 1 and 2 were measured in the same manner as above. １ Structural analysis was carried out using H-NMR (700 MHz in DO at 25°C), and the results are shown in Figure 26.

[0130] These results suggest that Fraction 1 and Fraction 2 are the same substance, as their NMR spectra were similar. The ● at around 5 ppm was a signal derived from an octasaccharide, and the ● at around 1 ppm was a signal derived from rhamnose. Fraction 2 was then analyzed for its constituent sugars.

[0131] <Analysis of sugar components> Sugar components were quantified by derivatization with 1-phenyl-3-methyl-5-pyrazolone (PMP). The following standards were used to prepare a calibration curve: L-arabinose (Ara), 6-deoxtalose (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). Standard mixture 1 was prepared by mixing aqueous solutions of GlcN, Man, Rib, 6-Deoxy Tal, GlcA, Glc, and Xyl sugars at concentrations of 0.0625, 0.25, 1, and 4 mM. Similarly, standard mixture 2 was prepared by mixing aqueous solutions of MurA, GalN, Rha, GalA, Gal, Ara, and Fuc sugars at concentrations of 0.0625, 0.25, 1, and 4 mM. 0.0625 mM D-Talose (Tal) was used as the internal standard. Fraction 2 was dissolved in ultrapure water to a concentration of 5 mg / mL and used as the analytical sample.

[0132] 400 μL of ultrapure water and 500 μL of 8M trifluoroacetic acid solution were added to each screw-cap test tube, and 100 μL of the analytical sample or standard mixture 1 or 2 at each concentration was added. The tubes were capped and stirred, followed by acid hydrolysis at 100°C for 2 hours. After cooling, the tubes were dried in a centrifugal evaporator. 400 μL of ultrapure water was added and stirred, and 100 μL was transferred to a new screw-cap test tube. 100 μL of internal standard, 100 μL of 0.6 M sodium hydroxide solution, and 200 μL of 0.5 M PMP methanol solution were added. The tubes were capped and stirred, followed by incubation at 70°C for 30 minutes. After cooling, 1 mL of 0.1 M hydrochloric acid solution was added to acidify the tube. The tubes were washed three times with approximately 1 mL of chloroform.

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

[0134] (HPLC analysis) Column temperature: 40°C Column: Waters CORTECS UPLC T3 (φ2.1 mm × 100 mm, 1.6 μm) 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: 0-3 min: A = 75, B = 25, 3-6 min: A = 75 → 5, B = 25 → 95, 6-9 min: A = 5, B = 95, 9-12 min: A = 5 → 75, B = 95 → 25%, 12-15 min: A = 75, B = 25 Detection: UV 245 nm

[0136] The results of the sugar component analysis showed that fraction 2 contained rhamnose (Rha), glucose (Glc), and galactose (Gal), with a ratio of Rha:Glc:Gal = 3.7:1.0:1.5 (Figure 27).１ Based on the H-NMR results, we had initially suspected it to be an octasaccharide, but because the sugar ratio was thought to be 4:1:2 or 3:1:1, we concluded it was a pentose- or heptasaccharide. Based on the above, we determined that the chemical structure of the active substance isolated from the fraction with a molecular weight cutoff of 10,000 to 30,000 using an ultrafiltration membrane was a repeating structure of pentose- to heptasaccharides, with rhamnose as the main sugar and glucose and galactose as the other sugars, with the repeating structure of rhamnose, glucose, and galactose in a 4-3:1:2-1 ratio (Figure 28).

[0137] The intestinal barrier protecting agent of the present invention can be used to protect the intestinal barrier and as a preventive agent for diseases associated with the breakdown of the intestinal barrier.

[0138] [Rule 26 amendment 02.04.2025]

Claims

1. An intestinal barrier protection agent characterized by containing Bifidobacterium adolescentis as an active ingredient.

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

3. A preventive agent for diseases associated with intestinal barrier breakdown, comprising the intestinal barrier protecting agent according to claim 1 or 2.

4. The preventive agent for intestinal barrier breakdown-associated diseases according to claim 3, wherein the intestinal barrier breakdown-associated diseases are selected from diabetes, obesity, inflammatory bowel disease, fatty liver, Parkinson's disease, cancer, irritable bowel disease, dyslipidemia, atopy, asthma, dementia, insomnia, depression, and rheumatoid arthritis.

5. An intestinal barrier protector containing as an active ingredient a cell wall polysaccharide fraction and / or a cytoplasmic fraction of Bifidobacterium adolescentis.

6. An intestinal barrier protector whose active ingredient is a polysaccharide with a repeating structure of rhamnose, glucose, and galactose in a ratio of 4-3:1:2-1, and a molecular weight cutoff by an ultrafiltration membrane of 10,000 to 30,000.

7. An intestinal barrier protector containing uridine diphosphate-N-acetylmuramic acid-L-alanine-D-glutamic acid-L-lysine as the active ingredient.

8. A polysaccharide characterized by a repeating structure of rhamnose, glucose, and galactose in a ratio of 4-3:1:2-1, and a molecular weight cutoff by ultrafiltration membrane of 10,000 to 30,000.

9. Bifidobacterium adolescentis is lysed, and then subjected to ultrafiltration to obtain a fraction with a molecular weight cutoff of 10,000 to 30,000. This fraction is then subjected to hydrophilic interaction chromatography under the following conditions or equivalent conditions: [Chromatography conditions] Flow rate: 1.0 mL / min Mobile phase A: MeCN, B: H2O Gradient: 0-3.3 min: A=95, B=5, 3.3-83.3 min: A=95→0, B=5→100 Column: YMC-triart Diol HILIC (φ4.6x250 mm, 5 μm) Temperature: 40°C Injection volume: 2-10 μL (5 mg / mL in H2O) Elution time: 22-35 min 2. A method for producing a polysaccharide having a repeating structure of rhamnose, glucose and galactose in a ratio of 4-3:1:2-1, and a molecular weight cutoff of 10,000 to 30,000 by ultrafiltration membrane, comprising:

10. The method for producing a polysaccharide according to claim 9, wherein the lysis treatment is carried out by treating with muramidase, followed by treatment with a nuclease and a protease.

11. A preventive agent for diseases associated with intestinal barrier breakdown, comprising the intestinal barrier protecting agent according to any one of claims 5 to 7.

12. The preventive agent for intestinal barrier breakdown-associated diseases according to claim 11, wherein the intestinal barrier breakdown-associated diseases are selected from diabetes, obesity, inflammatory bowel disease, fatty liver, Parkinson's disease, cancer, irritable bowel disease, dyslipidemia, atopy, asthma, dementia, insomnia, depression, and rheumatoid arthritis.

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