Intestinal barrier protectant

Abstract

This study aims to elucidate the active mechanism responsible for the intestinal barrier protection effect of Bifidobacterium adolescentis. [Solution] An intestinal barrier protectant comprising a polysaccharide having a repeating structure of the cell wall polysaccharide fraction and / or intracytoplasmic fraction of Bifidobacterium adolescentis, rhamnose, glucose, and galactose in a ratio of 4-3:1:2-1, with a fractional molecular weight of 1-30,000 determined by ultrafiltration membrane, or uridine diphosphate-N-acetylmuramic acid-L-alanine-D-glutamic acid-L-lysine as an active ingredient.

Description

Technical Field

[0001] The present invention relates to an intestinal barrier protective agent.

Background Art

[0002] In recent years, attention has been focused on the fact that intestinal bacteria affect the pathogenesis of host metabolic disorders, particularly the pathogenesis of obesity and diabetes. Currently, intestinal bacteria are considered to be a cause of obesity, along with eating habits and lack of exercise, and new treatment methods for obesity by changing the intestinal microbiota of obese patients and further improving the intestinal environment are being explored.

[0003] Dysbiosis of the intestinal microbiota is caused by various factors (stress, aging, alcohol, smoking, diet, antibiotics, lack of physical activity, etc.). As a result, the intestinal barrier function is disrupted by three stimuli: stimulation by lipopolysaccharide (LPS), a toxin derived from intestinal bacteria, stimulation by inflammatory cytokines in the intestine, and stimulation by oxidative stress. It is known that due to this disruption, LPS and inflammatory cytokines flow into the blood and circulate, reaching adipose tissue and the liver that determine insulin sensitivity, causing chronic inflammation and insulin resistance (Non-Patent Documents 1 and 2).

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

[0005] Although exploration of intestinal bacteria for protecting the intestinal barrier function has been carried out so far, what has been reported so far are the intestinal barrier protective effects against cytokine stimulation of Bifidobacterium bifidum and Bifidobacterium animalis (Non-Patent Documents 3 to 6), and the intestinal barrier protective effects against LPS stimulation of Bifidobacterium longum and Bifidobacterium animalis (Non-Patent Documents 7 to 8), etc.

[0006] Furthermore, the applicant has also reported Bifidobacterium adolescentis as an intestinal bacterium that has previously not been reported to have an intestinal barrier protective effect against multiple stimuli (Patent Document 1).

[0007] However, the active component responsible for the intestinal barrier protection effect of Bifidobacterium adolescentis remained completely unknown. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] Japanese Patent Application No. 2024-44857 [Non-patent literature]

[0009] [Non-Patent Document 1] Coppe. JP, et al., PLoS Biol. 6. 2853. 2008 [Non-Patent Document 2] Winer. D. A, et al., Cell Metab. 23. 413. 2016 [Non-Patent Document 3] Al-Sadi. R, et al., Int. J. Mol. Sci. 22. 8070. 2021 [Non-Patent Document 4] Hsieh. C. Y, et al., Physiol Rep. 3. 3. e12327. 2015 [Non-Patent Document 5] Wang. X, et al., Biomedicines. 9. 641. 2011 [Non-Patent Document 6] Castro-Herrera. V. M, et al., Nutrients. 12. 1719. 2020 [Non-Patent Document 7] Zhao. L, et al., J. Funct. Foods. 92. 105030. 2022 [Non-Patent Document 8] Kim. JY, et al., Front Microbiol. 13. 817591. 2022 [Overview of the project] [Problems that the invention aims to solve]

[0010] Therefore, the object of the present invention is to clarify the active component of the intestinal barrier protective effect possessed by Bifidobacterium adolescentis. [Means for solving the problem]

[0011] As a result of diligent research to solve the above problems, the present inventors have discovered that the active component of the intestinal barrier protective effect of Bifidobacterium adolescentis is a polysaccharide having a repeating structure of 4-3:1:2-1 in the cell wall polysaccharide fraction and / or cytoplasmic fraction of Bifidobacterium adolescentis, rhamnose, glucose, and galactose, with a molecular weight cutoff of 10,000-30,000 by ultrafiltration membrane, or uridine diphosphate-N-acetylmuramic acid-L-alanine-D-glutamic acid-L-lysine, and have completed the present invention.

[0012] Furthermore, we discovered that among the above active ingredients, rhamnose, glucose, and galactose have a repeating structure of 4-3:1:2-1, and that polysaccharides with a molecular weight cutoff of 10,000-30,000 by ultrafiltration membrane are novel polysaccharides, thus completing the present invention.

[0013] In other words, the present invention is as follows: [1] An intestinal barrier protectant containing the cell wall polysaccharide fraction and / or cytoplasmic fraction of Bifidobacterium adolescentis as the active ingredient. [2] An intestinal barrier protectant containing a polysaccharide as an active ingredient, which has a repeating structure of rhamnose, glucose, and galactose in a ratio of 4-3:1:2-1 and a fractional molecular weight of 10,000-30,000 determined by ultrafiltration membrane. [3]An intestinal barrier protecting agent containing uridine diphosphate-N-acetylmuramic acid-L-alanine-D-glutamic acid-L-lysine as an active ingredient. [4]A polysaccharide characterized in that rhamnose, glucose, and galactose have a repeating structure of 4 - 3:1:2 - 1 and a molecular weight cut-off by an ultrafiltration membrane of 10,000 - 30,000. [5]Bifidobacterium adlescentis is lysed, and then, by an ultrafiltration membrane, a fraction with a molecular weight cut-off of 10,000 - 30,000 is obtained, and further, hydrophilic interaction chromatography carried out under the following conditions or equivalent conditions thereto, [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 and collecting under the above conditions, a method for producing a polysaccharide characterized in that rhamnose, glucose, and galactose have a repeating structure of 4 - 3:1:2 - 1 and a molecular weight cut-off by an ultrafiltration membrane of 10,000 - 30,000. [6]The method for producing a polysaccharide according to [5], wherein the lysis treatment is carried out by treating with muramidase and then with nuclease and protease.

Advantages of the Invention

[0016] [Figure 1] This figure shows the TEER values ​​of the cell wall polysaccharide fraction (including the intracytoplasmic fraction) under unstimulated conditions. [Figure 2] This figure shows the results of size exclusion chromatography (SEC) analysis of the cell wall polysaccharide fraction (including the intracytoplasmic fraction). [Figure 3] This figure shows the TEER values ​​under unstimulated conditions when the cell wall polysaccharide fraction (including the intracytoplasmic fraction) was fractionated using an ultrafiltration membrane and then added to a concentration of 1 μg / mL. [Figure 4] This figure shows the results of hydrophilic interaction chromatography (HILIC) analysis of fractions with a molecular weight of 10,000 or less, as well as the fractions that were separated. [Figure 5] This figure shows the TEER values ​​under non-stimulant conditions when a fraction extracted from molecules with a molecular weight of 10,000 or less was added at a concentration of 1 μg / mL. [Figure 6] This figure shows the 1H-NMR spectrum of fraction 2, which was collected from molecules with a molecular weight of 10,000 or less. [Figure 7] This is a diagram showing the structure of uridine diphosphate-N-acetylmuramic acid-L-alanine-D-glutamic acid-L-lysine. [Figure 8] This figure shows the results of HILIC analysis of fractions with molecular weights of 1 to 30,000, and the separated fractions. [Figure 9] This figure shows the TEER values ​​under no-stimulation conditions when fractions collected from molecules with molecular weights of 10,000 to 30,000 were added at a concentration of 1 μg / mL. [Figure 10]This figure shows the 1H-NMR spectra of fractions 1 and 2, collected from molecules with molecular weights of 10,000 to 30,000. [Figure 11] This figure shows the results of the constituent sugar analysis of fraction 2. [Figure 12] This is a diagram showing the structure of a polysaccharide. [Modes for carrying out the invention]

[0017] The intestinal barrier protective agent of the present invention (hereinafter referred to as "the protective agent of the present invention") contains any of the following components as an active ingredient. Here, protection of the intestinal barrier means increasing the intestinal barrier strength index value (transepithelial electrical resistance; TEER) compared to the case where nothing is added. (1) Cell wall polysaccharide fraction and / or cytoplasmic fraction of Bifidobacterium adolescentis (2) Polysaccharides having a repeating structure of rhamnose, glucose, and galactose in a ratio of 4-3:1:2-1, and a molecular weight cutoff of 10,000-30,000 by ultrafiltration membrane. (3) Uridine diphosphate-N-acetylmuramic acid-L-alanine-D-glutamic acid-L-lysine

[0018] The active ingredient of the protective agent of the present invention, (1) the cell wall polysaccharide fraction and / or intracytoplasmic fraction of Bifidobacterium adolescentis, will be described below.

[0019] The cell wall polysaccharide fraction and / or intracytoplasmic fraction of Bifidobacterium adolescentis are obtained by lysing Bifidobacterium adolescentis.

[0020] The Bifidobacterium adolescentis used above may be any known strain of Bifidobacterium adolescentis or any strain isolated by conventional methods (for example, (1) Research on Bifidobacteria, edited by Tomotari Mitsuoka, Japan Bifidobacteria Center; (2) Intestinal Microbiota Detection Methods and Intestinal Constituent Bacteria, Norihiko Fujisawa, Japanese Journal of Bacteriology 2014; (3) PMID: 28394924 (Bifidobacterium adolescentis is isolated from mouse feces, but applicable to humans)). For example, Bifidobacterium adolescentis YIT 13021, Bifidobacterium adolescentis YIT 13609, Bifidobacterium adolescentis YIT 12828, Bifidobacterium adolescentis reference strain (ATCC15703), Bifidobacterium adolescentis YIT Examples include 13022, Bifidobacterium adolescentis YIT 11033, Bifidobacterium adolescentis YIT 11034, and Bifidobacterium adolescentis YIT 11040.

[0021] Among these Bifidobacterium adolescentis species, Bifidobacterium adolescentis YIT 13021, Bifidobacterium adolescentis YIT 13609, and Bifidobacterium adolescentis YIT 12828 are preferred, with Bifidobacterium adolescentis YIT 13021 being more preferred.

[0022] The Bifidobacterium adolescentis used in the protective agent of the present invention includes not only the aforementioned strains, but also strains that have been artificially genetically modified while maintaining the properties of Bifidobacterium adolescentis.

[0023] Of the above Bifidobacterium adolescentis species, Bifidobacterium adolescentis YIT 13021 has been internationally deposited as Bifidobacterium adolescentis YIT 13021 (NITE BP-03808, deposit date: January 24, 2023) at the Patent Microorganism Depository Center of the National Institute of Technology and Evaluation (NITE), an independent administrative agency that is the international depositary authority under the Budapest Convention (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan 292-0818).

[0024] Bifidobacterium adolescentis YIT 13609 is deposited in Japan as YIT 13609 (NITE P-04064, deposit date: January 15, 2024) at the Patent Microorganism Depository Center of the National Institute of Technology and Evaluation (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture 292-0818, Japan).

[0025] Bifidobacterium adolescentis YIT 12828 is deposited domestically with the above-mentioned Patent Microorganism Depository Center as YIT 12828 (NITE P-04065, deposit date: January 15, 2024).

[0026] 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).

[0027] 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).

[0028] 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).

[0029] 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).

[0030] It is preferable to culture the Bifidobacterium adolescentis mentioned above before lysis. Culturing can be carried out under conventionally known conditions. After culturing, the cells may be collected by centrifugation or other methods and washed with PBS or similar, according to standard procedures.

[0031] The lysis treatment of Bifidobacterium adolescentis described above is not particularly limited as long as it yields a cell wall polysaccharide fraction and / or an intracytoplasmic fraction from Bifidobacterium adolescentis. For example, known methods using a combination of multiple enzymes such as muramidase, nuclease, and protease can be used.

[0032] Among these methods using multiple enzymes, a preferred method involves treating the sample with muramidase, followed by treatment with a nuclease and a protease. Examples of muramidases include mutanolysin, lysozyme, and labiases. Examples of nucleases include benzonase. Examples of proteases include pronase and trypsin.

[0033] The preferred method described above is carried out as follows: First, Bifidobacterium adolescentis is thoroughly cultured beforehand according to a conventional method, then collected by centrifugation and washed with PBS. 1 to 100 U, preferably 4 U, of muramidase is added to 1 mg of live Bifidobacterium adolescentis, and the mixture is reacted at 37 to 60°C, preferably 37°C, for 12 hours or more, preferably 16 to 24 hours. Then, if necessary, insoluble matter is removed by centrifugation, etc., to obtain the supernatant, and 1 to 100 U, preferably 1.4 U, of nuclease is added to 1 mg of live bacteria, and the mixture is reacted at 37 to 60°C, preferably 37°C, for 12 hours or more, preferably 16 to 24 hours. Next, 0.0001 to 0.5 mg, preferably 0.006 mg, of protease is added to 1 mg of live bacteria, and the mixture is reacted at 37 to 60°C, preferably 37°C, for 12 hours or more, 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, preferably 3,500 to 8,000.

[0034] As described above, the cell wall polysaccharide fraction and / or intracytoplasmic fraction of Bifidobacterium adolescentis (1) are obtained. The physical properties of this cell wall polysaccharide fraction and / or intracytoplasmic fraction of Bifidobacterium adolescentis are as follows. Characteristics: White to light grayish-brown solid

[0035] The cell wall polysaccharide fraction and / or cytoplasmic fraction of Bifidobacterium adolescentis have a protective effect on the intestinal barrier.

[0036] Next, we will describe a polysaccharide in which the active ingredient of the protective agent of the present invention, (2) rhamnose, glucose, and galactose, has a repeating structure of 4-3:1:2-1, preferably a repeating structure of 3:1:1 or 4:1:2, and whose fractional molecular weight by ultrafiltration membrane is 1 to 30,000. This polysaccharide has a structure represented by the following formula and is a novel substance.

[0037] [ka]

[0038] The physical properties of this polysaccharide are as follows: Characteristics: White to pale yellow solid

[0039] This polysaccharide is obtained by lysing Bifidobacterium adolescentis, then by ultrafiltration to obtain fractions with a molecular weight cutoff of 1 to 30,000, and further by hydrophilic interaction chromatography performed under the following conditions or equivalent conditions. [Chromatography conditions] Flow rate: 1.0 mL / min Mobile phase A: MeCN, B: H2O Gradient: 0-3.3 min: A=95, B=5; 3.3-83.3 min: A=95→0, B=5→100 Column: YMC-triart Diol HILIC (φ4.6 x 250 mm, 5 μm) Temperature: 40℃ Injection volume: 2~10 uL (5 mg / mL in H2O) Detection: Charged aerosol detector (CAD) Dissolution time: 22-35 min It can be manufactured by extracting it.

[0040] The Bifidobacterium adolescentis and lysis treatment used in the above are the same as described above.

[0041] After lysis, the cell wall polysaccharide fraction and / or intracytoplasmic 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 membrane used is not particularly limited as long as it can obtain the above molecular weights, but it is preferable to use a combination of ultrafiltration membranes with molecular weight cutoffs of 100,000, 30,000, and 10,000, and more preferably a combination of ultrafiltration membranes with molecular weight cutoffs of 30,000 and 10,000.

[0042] After purification, fractions with a molecular weight cutoff of 10,000 to 30,000 are obtained by ultrafiltration. Hydrophilic interaction chromatography is performed on these fractions under the conditions described above to collect polysaccharides in which rhamnose, glucose, and galactose form a repeating structure of 4-3:1:2-1, and which have a molecular weight cutoff of 10,000 to 30,000 by ultrafiltration.

[0043] In the hydrophilic interaction chromatography described above, in addition to the above conditions, these conditions can be appropriately modified to obtain polysaccharides having a repeating structure of rhamnose, glucose, and galactose in a ratio of 4-3:1:2-1, and a fractional molecular weight of 10,000-30,000 obtained by ultrafiltration. Such equivalent conditions can be set by those skilled in the art by appropriately changing the flow rate, mobile phase composition, column support type, column support particle size, column length, temperature, injection volume, and injection solution concentration, with reference to the above conditions. Specifically, these include the following:

[0044] [Chromatography conditions] Flow rate: 0.5~1.0 mL / min Mobile phase A: MeCN, B: H2O Gradient: (a)~c) a) 0-2min: 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 (φ4.6 x 150 mm, 5 μm) Temperature: 40℃ Injection volume: 2~10 uL (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

[0045] As described above, a polysaccharide is obtained in which rhamnose, glucose, and galactose in (2) form a repeating structure of 4-3:1:2-1, and the molecular weight cutoff by ultrafiltration is 10,000-30,000. Whether this polysaccharide has been obtained can be confirmed, for example, by structural analysis or constituent sugar analysis using NMR, HPLC, etc. Specifically, in SEC analysis using a Shodex SUGAR KS-804 column (Showa Denko F6378035), a peak can be observed around the elution time of 8-9 minutes. More specifically, it can be confirmed by the method described in the examples below.

[0046] This polysaccharide, in which rhamnose, glucose, and galactose form a repeating structure of 4-3:1:2-1, and whose molecular weight cutoff by ultrafiltration is 10,000-30,000, has an intestinal barrier protective effect.

[0047] Finally, we will explain (3) uridine diphosphate-N-acetylmuramic acid-L-alanine-D-glutamic acid-L-lysine, which is the active ingredient of the protective agent of the present invention.

[0048] This uridine diphosphate-N-acetylmuramic acid-L-alanine-D-glutamic acid-L-lysine (CAS No. 143436-84-4) is a known compound, a component of 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).

[0049] [ka]

[0050] This uridine diphosphate-N-acetylmuramic acid-L-alanine-D-glutamic acid-L-lysine may be obtained using commercially available products, obtained based on the above-mentioned literature, or obtained from the cell wall polysaccharide fraction and / or intracytoplasmic fraction of (1) Bifidobacterium adolescentis as described in the examples below.

[0051] This uridine diphosphate-N-acetylmuramic acid-L-alanine-D-glutamic acid-L-lysine has an intestinal barrier protective effect.

[0052] The protective agent of the present invention can protect the intestinal barrier by using any of the above (1) to (3) as an active ingredient.

[0053] The form of the protective agent 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 human use, a food or beverage, a supplement, etc.

[0054] As pharmaceuticals, any of the above (1) to (3) can be formulated as is or, as needed, 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, physiological saline, etc. Furthermore, conventional additives such as stabilizers, wetting agents, emulsifiers, binders, isotonic agents, and excipients may be added as needed.

[0055] Furthermore, the dosage form is not particularly limited, and examples include liquids, powders, granules, capsules, tablets, etc., which can be manufactured according to conventional methods. The content of the active ingredient in the protective agent of the present invention is not particularly limited, for example, 1 to 100% by mass is preferred, more preferably 5 to 70% by mass, and particularly preferably 10 to 60% by mass.

[0056] When the protective agent of the present invention is made into a food or beverage, any of the above (1) to (3) can be used as is or, as necessary, combined with additives, materials, etc., commonly used in food and beverages, and prepared according to conventional methods. Examples of food and beverages include starch-based foods such as bread, biscuits, pancakes, noodles, and candy tablets; confectionery such as gum, candy, and Japanese sweets; meat products such as ham and sausages; fish products 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.

[0057] When the protective agent of the present invention is in the form of a supplement, any of the above (1) to (3) can be prepared as is or, as necessary, by combining it with additives, materials, etc., commonly used in supplements, and following conventional methods.

[0058] The protective agent of the present invention can protect the intestinal barrier when administered to mammals, including humans. In this case, the protective agent of the present invention should be administered in an amount of (1) 0.000060 to 270 mg per day if it is the cell wall polysaccharide fraction and / or intracellular fraction of Bifidobacterium adolescentis, (2) 0.00000324 to 15 mg per day if it is a polysaccharide having a repeating structure of rhamnose, glucose, and galactose in a ratio of 4 to 3:1:2 to 1, with a fractional molecular weight of 1 to 30,000 by ultrafiltration membrane, or (3) 0.00000420 to 19 mg per day if it is uridine diphosphate-N-acetylmuramic acid-L-alanine-D-glutamic acid-L-lysine.

[0059] As described above, the protective agent of the present invention can protect the intestinal barrier and can therefore be used as a preventive agent for diseases related to the breakdown of the intestinal barrier (hereinafter referred to as "the preventive agent of the present invention"). Furthermore, since the preventive agent of the present invention can also protect against the disruption of the intestinal barrier, diseases related to the breakdown of the intestinal barrier also include diseases related to the disruption of the intestinal barrier. Examples of diseases related to the breakdown of the intestinal barrier include diabetes, obesity, inflammatory bowel disease (Crohn's disease, ulcerative colitis, etc.), fatty liver, Parkinson's disease, cancer, irritable bowel disease, dyslipidemia, atopic dermatitis, asthma, dementia, insomnia, depression, and rheumatoid arthritis. Among these diseases related to the breakdown of the intestinal barrier, obesity and diabetes are preferred.

[0060] The preventive agent of the present invention can prevent intestinal barrier disruption-related diseases when administered to mammals, including humans. In this case, the preventive agent of the present invention should be administered in an amount of (1) 0.000060 to 270 mg per day if it is the cell wall polysaccharide fraction and / or intracytoplasmic fraction of Bifidobacterium adolescentis, (2) 0.00000324 to 15 mg per day if it is a polysaccharide having a repeating structure of rhamnose, glucose, and galactose in a ratio of 4 to 3:1:2 to 1 and a molecular weight cutoff of 1 to 30,000 by ultrafiltration membrane, or (3) 0.00000420 to 19 mg per day if it is uridine diphosphate-N-acetylmuramic acid-L-alanine-D-glutamic acid-L-lysine.

[0061] The fact that the above-mentioned diseases are associated with a 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 <Atopic dermatitis, 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

[0062] The protective agent and preventive agent of the present invention can safely protect the intestinal barrier and prevent intestinal barrier breakdown-related diseases for a long period of time.

[0063] Furthermore, while the protective agent of the present invention protects the intestinal barrier, it can also enhance the intestinal barrier in the absence of any irritants. Therefore, the protective agent of the present invention can also be used as an intestinal barrier enhancer. Here, enhancing the intestinal barrier refers to increasing the intestinal barrier strength indicator value (transepithelial electrical resistance; TEER) compared to the case where nothing is added. [Examples]

[0064] The present invention will be described in detail below with reference to examples of the present invention, but the present invention is not limited in any way to these examples.

[0065] Example 1 Intestinal barrier protection effect of the cell wall polysaccharide fraction of Bifidobacterium adolescentis: After culturing strains belonging to Bifidobacterium adolescentis, the cell wall polysaccharide fraction was obtained by lysis and analyzed.

[0066] (1) Strain The strain used was Bifidobacterium adolescentis YIT 13021 (hereinafter referred to as "YIT 13021").

[0067] (2)Culture The YIT 13021 strain was cultured under anaerobic conditions at 37°C in a modified GAM medium (hereinafter referred to as "1% Starch mGAM") supplemented with 1% soluble starch (Fujifilm Wako Pure Chemical Industries, Ltd. 191-03985).

[0068] (3) Lysolysis 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 aqueous solution (Kanto Chemical 37851-08). Ultrapure water was added to this solution to make 1.6 L, and the solution was filtered and sterilized using a 0.2 μm filter (Thermo 567-0020). 109.78 mg (0.540 mmol) of magnesium chloride hexahydrate (Kanto Chemical 25009-30) was added to 270 mL of the filtrate to prepare a 5 mM Tris-maleate buffer (pH 6.8) containing 2 mM MgCl2.

[0069] 400 mg (dry cell weight) of viable bacteria cultured in 1% Starch mGAM medium was suspended in 5 mM Tris-maleate buffer (pH 6.8) containing 2 mM MgCl2 to a concentration of 13.9 mg / mL. 4 U of Mutanolysin was added as muramidase per 1 mg of bacterial cells, and the mixture was reacted at 37°C for 24 hours with shaking.

[0070] The remaining reaction mixture was centrifuged at 24°C and 12,000 × g for 45 minutes to precipitate insoluble matter, and the supernatant was collected. Benzonase was added to the supernatant as a nuclease at a dose of 1.4 U per 1 mg of bacterial cells, and the mixture was reacted at 37°C for 24 hours. Subsequently, pronase was added as a protease at a dose of 0.006 mg per 1 mg of bacterial cells, and the mixture was reacted at 37°C with shaking for 24 hours. After dialysis in pure water at room temperature for 72 hours using a dialysis membrane with a molecular weight cutoff of 3,500, the cell wall polysaccharide fraction (42.5 mg) was obtained by freeze-drying.

[0071] The TEER value of this cell wall polysaccharide fraction was measured under no-stimulation conditions using the following method. The results are shown in Figure 1. From these results, it was found that the cell wall polysaccharide fraction has a protective effect on the intestinal barrier.

[0072] <Measuring TEER values ​​under no stimulation> T84 monolayers were prepared using a 24-well cell culture insert plate (Millipore). 80% confluent T84 cells were harvested from a culture dish 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 5% CO2 and 37°C. The culture medium was changed every 2-3 days, with 400 μL of medium on the apical side and 800 μL on the basal side. T84 monolayers were formed after 10 days of culture.

[0073] The test substance was added to the prepared monolayer apical membrane at various concentrations, and the measured resistance value (Ω) was measured after 24 hours using Millicell MRS-2 (Millipore). The measured resistance value was then measured in the cell culture insert plate over a specific culture area (cm²). 2 The transepithelial electrical resistance (TEER) value (Ω / cm²) is the value obtained by dividing by ). 2 The barrier-enhancing effect was evaluated using the following method. The TEER value of the monolayer membrane at each time interval from the start of stimulation was divided by the TEER value at 0 hours (immediately after the start of stimulation) to calculate the rate of change (%) from the start of stimulation.

[0074] Example 2 Intestinal barrier protection effect of fractions with a molecular weight of 10,000 or less: The cell wall polysaccharide fraction with intestinal barrier protective activity obtained in Example 1 was dissolved in ultrapure water to a concentration of 5 mg / mL, and the SEC was measured under the following conditions. The results are shown in Figure 2.

[0075] <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)

[0076] SEC revealed that the cell wall polysaccharide fraction contained molecules with molecular weights of 10,000, 30,000, 100,000, and 400,000 or more. Next, this was fractionated into molecules with molecular weights of 100,000 or more, 100,000 to 30,000, 30,000 to 10,000, and 10,000 or less using ultrafiltration membranes with molecular weight cutoffs of 100,000 (Millipore, UFC910008), 30,000 (Millipore, UFC903008), and 10,000 (Millipore, UFC901008). The TEER values ​​for these molecules under unstimulated conditions were measured according to the following method. The results are shown in Figure 3.

[0077] <Measuring TEER value under no stimulation conditions> The same measurement as the TEER value measurement under non-stimulation conditions in Example 1 above was performed.

[0078] From these results, it was found that the fractions with a molecular weight of 30,000 or less (fractions of 10,000 - 30,000 and fractions of 10,000 or less) were active. Next, hydrophilic interaction chromatography (HILIC) analysis was performed on the fraction with a molecular weight of 10,000 or less (Figure 4). Also, the elution was carried out 24 times (total 1.2 mg), and in each run, fractions 1 - 4 in Figure 4 were collected (1 (16 - 20.5 minutes): 0.45 mg, 2 (20.5 - 22 minutes): 0.25 mg, 3 (22 - 25 minutes): 0.40 mg, 4 (51 - 56 minutes): 0.29 mg).

[0079] [[ID=No. 4]]<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 (Φ4.6x250mm, 5μm) Temperature: 40°C Injection volume: 10 μL (5 mg / mL in H2O)

[0080] For the fractions 1 - 4 obtained above, TEER values were measured in the same manner as above. The results are shown in Figure 5. An enhancement of the protective effect on the intestinal barrier was observed only in fraction 2. Regarding this fraction 2 1 1H - NMR (700 MHz in D2O at 25°C) was used for structural analysis. The results are shown in Figure 6.

[0081] < 1 <1H - NMR Structural Analysis> Fraction 2 was dissolved in heavy water (Kanto Chemical 32070 - 1A), and 1 1H - NMR (700 MHz) was measured at 25°C.

[0082] 1From the results of 1H-NMR, fraction 2 had signals at ■, indicating that it was uridine diphosphate-N-acetylmuramic acid-L-alanine-D-glutamic acid-L-lysine, a cell wall precursor (literature value (Table 1): Nicolas Gisch, et al., Bioorganic Med. Chem. Lett. 21, 3362, 2011). The structure is shown in Figure 7. The molecular weight of uridine diphosphate-N-acetylmuramic acid-L-alanine-D-glutamic acid-L-lysine is 1008, but when fraction 2 was analyzed by mass spectrometry, [M+1] was 1008.8 (not shown). Since fraction 2 contains cell wall precursors, it was found that the previously measured cell wall polysaccharide fraction also contains intracellular fractions.

[0083] (Signals of uridine diphosphate-N-acetylmuramic acid-L-alanine-D-glutamic acid-L-lysine)

Table 1

[0084] Example 3 Intestinal barrier protective effect of fractions with a fraction molecular weight of 10,000 to 30,000: HILIC analysis was performed on the fraction with a fraction molecular weight of 10,000 to 30,000 obtained in Example 2 (Figure 8). Also, the liquid was passed through 48 times (total 2.4 mg), and each time, fractions 1 to 4 in Figure 8 were collected (1 (22 - 30 minutes): 1.19 mg, 2 (30 - 35 minutes): 1.26 mg, 3 (35 - 42.5 minutes): 1.01 mg, 4 (51.5 - 57.5 minutes): 0.79 mg).

[0085] <HILIC analysis> Flow rate: 1.0 mL / min Mobile phase A: MeCN, D: H2O, 0 - 3.3 min: A = 95, D = 5, 3.3 - 83.3 min: A = 95 → 0, D = 5 → 100 Column: YMC-triart Diol HILIC (φ4.6x250mm, 5μm) Temperature: 40°C Injection volume: 10 μL (5 mg / mL in H2O)

[0086] The TEER values ​​were measured for fractions 1-4 obtained above in the same manner as described above. The results are shown in Figure 9. Enhanced protective effect of the intestinal barrier was observed in fractions 1 and 2. For fractions 1 and 2, the same method as described above was used. 1 Structural analysis was performed using 1H-NMR (700 MHz in D2O at 25°C). The results are shown in Figure 10.

[0087] These results suggest that fractions 1 and 2 are the same substance, given the similarity of their NMR spectra. Furthermore, the ● around 5 ppm indicates signals from octasaccharides, and the ● around 1 ppm indicates signals from rhamnose. Next, the constituent sugars of fraction 2 were analyzed.

[0088] <Constituent sugar analysis> The constituent sugars were analyzed and quantified by derivatization with 1-phenyl-3-methyl-5-pyrazolone (PMP). The following standards were used for creating the 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). Aqueous solutions of the sugars GlcN, Man, Rib, 6-DeoxyTal, GlcA, Glc, and Xyl were mixed to prepare standard mixture 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 standard mixture 2, with 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 to prepare the analytical sample.

[0089] 400 μL of ultrapure water and 500 μL of 8M trifluoroacetic acid aqueous solution were added to each screw-cap test tube. 100 μL of the analytical sample or standard mixture 1 and 2 of each concentration were added, the tubes were capped and stirred, and then acid-decomposed at 100°C for 2 hours. After cooling, the mixture was dried using a centrifugal evaporator. 400 μL of ultrapure water was added and stirred. 100 μL of this mixture was transferred to a new screw-cap test tube, and 100 μL of internal standard, 100 μL of 0.6 M sodium hydroxide aqueous solution, and 200 μL of 0.5 M PMP methanol solution were added. The tubes were capped and stirred, and then reacted at 70°C for 30 minutes. After cooling, 1 mL of 0.1 M hydrochloric acid aqueous solution was added to make the mixture acidic. The tubes were washed three times with approximately 1 mL of chloroform.

[0090] The aqueous layer after chloroform washing was filtered through a 0.45 μm 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 the calibration curve of the standard for each sugar. Column washing was performed every 4-6 analyses.

[0091] (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

[0092] (Column washing) Column temperature: 40℃ 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

[0093] Analysis of the constituent sugars revealed that fraction 2 contains rhamnose (Rha), glucose (Glc), and galactose (Gal), in a ratio of Rha:Glc:Gal = 3.7:1.0:1.5 (Figure 11). The number of sugars in the repeating structure is: 1Based on the 1H-NMR results, we initially thought it was an octasaccharide, but since the sugar ratio appears to be 4:1:2 or 3:1:1, we considered it to be a 5-7saccharide. From the above, we found that the chemical structure of the active substance isolated from the fraction with a molecular weight cutoff of 10,000-30,000 by ultrafiltration membrane is a repeating structure of 5-7saccharides with rhamnose as the main sugar, and glucose and galactose also included, with rhamnose, glucose, and galactose in a repeating ratio of 4-3:1:2-1 (Figure 12). [Industrial applicability]

[0094] The intestinal barrier protective agent of the present invention can be used for protecting the intestinal barrier and preventing diseases related to intestinal barrier breakdown.

Claims

1. An intestinal barrier protectant containing the cell wall polysaccharide fraction and / or cytoplasmic fraction of Bifidobacterium adolescentis as the active ingredient.

2. An intestinal barrier protectant containing a polysaccharide as its active ingredient, which has a repeating structure of rhamnose, glucose, and galactose in a ratio of 4-3:1:2-1, and whose fractional molecular weight by ultrafiltration membrane is 10,000-30,000.

3. An intestinal barrier protectant containing uridine diphosphate-N-acetylmuramic acid-L-alanine-D-glutamic acid-L-lysine as its active ingredient.

4. 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 of 10,000-30,000 by ultrafiltration membrane.

5. Bifidobacterium adolescentis is lysed, and then fractions with a molecular weight cutoff of 10,000 to 30,000 are obtained by ultrafiltration. These fractions are then subjected to hydrophilic interaction chromatography under the following conditions or equivalent conditions. [chromatographic conditions] Flow rate: 1.0 mL / min Moving phase A: MeCN, D: H 2 O Gradient: 0-3.3 min: A=95, D=5; 3.3-83.3 min: A=95→0, D=5→100 Column: YMC-triart Diol HILIC (φ4.6 x 250 mm, 5 μm) Temperature: 40℃ Injection volume: 2–10 μL (5 mg / mL in H) 2 O) Dissolution time: 22-35 min A method for producing polysaccharides characterized by extraction using a rhamnose, glucose, and galactose repeating structure in a ratio of 4-3:1:2-1, and having a fractional molecular weight of 10,000-30,000 by ultrafiltration membrane.

6. A method for producing polysaccharides according to claim 5, wherein the lysis treatment involves treating with muramidase followed by treatment with nuclease and protease.

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