Cell adhesion protectant

Abstract

To provide an intercellular adhesion protective agent.SOLUTION: Provided is an intercellular adhesion protective agent that contains at least one selected from the group consisting of euglena, paramylon, processed paramylon, and β-1,3-glucan.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an agent for protecting intercellular adhesion and the like. [Background technology]

[0002] Intercellular adhesion plays an important role in the body. For example, intestinal epithelial cells are connected by intestinal tight junctions, which play an important role in intestinal barrier function. Specifically, intestinal tight junctions normally physically prevent the entry of harmful foreign substances, pathogenic bacteria, allergens, etc. into the body, but when they are disrupted, they allow these substances to enter. In recent years, a state in which intestinal tight junctions are impaired has been called leaky gut, and it is associated with various diseases. Patent Document 1 reports that eucalyptus leaf extracts have the ability to promote tight junction formation. However, because the raw material is a cultivated tree, complex processes such as cultivation, harvesting, transportation, and extraction are required.

[0003] Euglena is a microalgae belonging to the genus Euglena, and is used as a food material. Euglena extracts are also applied to the skin. Paramylon is a β-1,3-glucan produced by Euglena, and has been reported to be useful for wound healing and allergy suppression. However, the relationship between Euglena and β-1,3-glucan and cell-cell adhesion is still unknown. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Publication No. 2018-197217 Summary of the Invention [Problem to be solved by the invention]

[0005] An objective of the present invention is to provide an agent for protecting intercellular adhesion. [Means for solving the problem]

[0006] In view of the above-mentioned problems, the present inventors conducted extensive research and found that the above-mentioned problems can be solved by an agent for protecting intercellular adhesion containing at least one selected from the group consisting of Euglena, paramylon, a paramylon processed product, and β-1,3-glucan. Further research based on this finding led to the completion of the present invention. Specifically, the present invention encompasses the following aspects.

[0007] Item 1. An agent for protecting intercellular adhesion, comprising at least one selected from the group consisting of Euglena, paramylon, a processed paramylon product, and β-1,3-glucan.

[0008] Item 2. The intercellular adhesion protecting agent according to Item 1, which contains at least one selected from the group consisting of Euglena, paramylon, and processed paramylon products.

[0009] Item 3. The intercellular adhesion protecting agent according to Item 1 or 2, which contains the Euglena, and the Euglena is Euglena gracilis.

[0010] Item 4. The intercellular adhesion protecting agent according to Item 1 or 2, which contains Euglena, and the Euglena is Euglena gracilis strain EOD-1 (accession number FERM BP-11530).

[0011] Item 5. The intercellular adhesion protecting agent according to Item 1, 2, or 4, which contains paramylon, and the paramylon is derived from Euglena gracilis.

[0012] Item 6. The intercellular adhesion protecting agent according to any one of Items 1 to 5, for use in protecting intestinal tight junctions.

[0013] Item 7. The intercellular adhesion protecting agent according to any one of Items 1 to 6, for use in suppressing leaky gut.

[0014] Item 8. The intercellular adhesion protecting agent according to any one of Items 1 to 7, for use in suppressing a decrease in intestinal barrier function caused by alcohol and / or inflammatory cytokines.

[0015] Item 9. The agent for protecting intercellular adhesion according to any one of Items 1 to 8, which is used to inhibit the invasion of at least one substance selected from the group consisting of allergens, viruses, and pathogenic bacteria into the body.

[0016] Item 10. The agent for protecting intercellular adhesion according to any one of Items 1 to 9, for use in suppressing diarrhea or loose stools.

[0017] Item 11. The intercellular adhesion protecting agent according to any one of Items 1 to 10, which is a food composition, a food additive, a cosmetic, a cosmetic additive, or a medicine.

[0018] Item 12. The agent for protecting intercellular adhesion according to any one of Items 1 to 11, which is an oral composition. [Effects of the Invention]

[0019] According to the present invention, an agent for protecting intercellular adhesion can be provided. [Brief explanation of the drawings]

[0020] [Figure 1] The figures show the results of the evaluation test for the protective effect on intercellular adhesion in Test Example 1. The vertical axis shows the relative transepithelial electrical resistance (TER) value, with the TER at the time of TNF-α addition (0 hours) set at 100. The horizontal axis shows the time elapsed since the addition of TNF-α. PM represents paramylon. For the paramylon-added groups (groups C and D), ** indicates a p-value of less than 0.01 compared to the control group (group B) (TNF-α added (5 ng / ml), PM not added (0 μg / ml)). [Figure 2]The results of the evaluation test for the protective effect on intercellular adhesion in Test Example 2 are shown. The vertical axis shows the relative TER value, with the TER at the time of ethanol addition (0 hours) set to 100. The horizontal axis shows the time elapsed since the addition of ethanol. EtOH represents ethanol, and PM represents paramylon. For the paramylon-added group (Group C), * indicates a p-value of less than 0.05 compared to the control group (Group B) (ethanol added (5%), PM not added (0 μg / ml)), and ** indicates a p-value of less than 0.01. DETAILED DESCRIPTION OF THE INVENTION

[0021] In this specification, the expressions "contain" and "comprise" include the concepts of "contain," "comprise," "consist essentially of," and "consist only of."

[0022] In this specification, the expression "and / or" includes both "and" and "or." That is, the expression "A and / or B" includes both "A or B" and "A and B."

[0023] In one aspect, the present invention relates to an agent for protecting intercellular adhesion (sometimes referred to herein as the "agent of the present invention") containing at least one selected from the group consisting of Euglena, paramylon, a paramylon processed product, and β-1,3-glucan. This is described below.

[0024] 1. Euglena Euglena is a microalgae belonging to the genus Euglena, and is not particularly limited thereto. Specific examples of Euglena include Euglena gracilis (Euglena gracilis), Euglena longa , Euglena caudata , Euglena oxyuris , Euglena tripteris , Euglena proxima , Euglena viridis , Euglena sociabilis , Euglena ehrenbergii , Euglena deses , Euglena pisciformis , Euglena spirogyra , Euglena acus , Euglena geniculata , Euglena intermedia , Euglena mutabilis , Euglena sanguinea , Euglena stellata , Euglena terricola , Euglena klebsi , Euglena rubra , Euglena cyclopicola Among these, from the viewpoint of ensuring the effects of the present invention, Euglena gracilis is preferred, and the Euglena gracilis EOD-1 strain (internationally deposited on June 28, 2013, with the International Patent Organism Depositary, National Institute of Technology and Evaluation (NITE-IPOD, Room 120, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan, Postal Code 292-0818) under the Budapest Treaty under Accession Number FERM BP-11530) is more preferred, from the viewpoint of ensuring the effects of the present invention.

[0025] The form of Euglena is not particularly limited as long as it contains the Euglena cell body or most of its components. Examples of the form of Euglena include a dry powder form of Euglena, a suspension of Euglena, and a Euglena extract. Among these, a dry powder form of Euglena is preferred.

[0026] The paramylon content of Euglena in a dry state is, for example, 50% or more, preferably 60% or more, and more preferably 70% or more.

[0027] Euglena may be of one type alone or a combination of two or more types.

[0028] 2. β-1,3-glucan, paramylon The β-1,3-glucan is not particularly limited as long as it has a single sugar chain (or sugar chain structure) as the main chain, in which glucose is linked only by β-1,3 bonds. β-1,3-glucans are not limited to linear ones, but also include those having branched chains.

[0029] The weight-average molecular weight of the β-1,3-glucan derivative is not particularly limited, but may be, for example, 1×10 4 ~2×10 6, preferably 5 x 10 4 ~1×10 6 , more preferably 1 × 10 5 ~1×10 6 The weight average molecular weight can be measured by the GPC method.

[0030] Although β-1,3-glucan may be obtained by chemical synthesis, natural β-1,3-glucans produced by various organisms are preferred from the viewpoint of availability, etc. Examples of natural β-1,3-glucans include paramylon, curdlan, laminaran, callose, lentinan, and schizophyllan. Among these, paramylon is preferred. Paramylon will be described below.

[0031] Paramylon is a β-1,3-glucan derived from Euglena, and is not particularly limited insofar as it is so.

[0032] The Euglena from which paramylon is derived is the same as that explained above in "1. Euglena."

[0033] The mass average molecular weight of paramylon is not particularly limited, but may be, for example, 1×10 4 ~5×10 6 , preferably 2 x 10 4 ~1×10 6 , more preferably 5 × 10 4 ~1×10 6 , and more preferably 1 × 10 5 ~5×10 5 is.

[0034] The mass average molecular weight can be measured by SEC-MALS analysis under the following conditions: Detector: Multi-angle scattering detector (DAWN HELEOS II manufactured by Wyatt Technology) Differential refractometer detector (Wyatt Technology Optilab T-rEX) Columns used: 2 TSKgel α-M (Tosoh) Mobile phase: DMSO with 0.05M potassium bromide Flow rate: 0.5 mL / min.

[0035] Paramylon exists within Euglena cells as paramylon particles, which are typically composed of triple helical structures formed by β-1,3-glucan chains highly aggregated in a regular pattern.

[0036] The shape of the paramylon particles is not particularly limited, but they are usually flat spheroids.

[0037] The particle size distribution of the paramylon particles is not particularly limited, but is, for example, 0.5 to 15 μm, preferably 1 to 6 μm. The average particle size of the paramylon particles is also not particularly limited, but is, for example, 1 to 10 μm, preferably 2 to 4 μm.

[0038] The form of paramylon is not particularly limited as long as it contains paramylon. Examples of the form of Euglena include a dry powder form of paramylon and a suspension of paramylon, and among these, a dry powder form of paramylon is preferred.

[0039] Paramylon may be used alone or in combination of two or more types.

[0040] 3. Method for producing Euglena and paramylon Euglena can be prepared in large quantities by a method including a step of culturing Euglena contained in a liquid (culturing step). The culturing step can be carried out, for example, according to a known method (e.g., the method described in Japanese Patent No. 5883532). In the culturing step, microalgae of the genus Euglena are typically cultured under aerobic conditions while stirring a liquid (culture solution) containing water, Euglena, and nutrients that can be utilized by Euglena.

[0041] Examples of nutrients include sugars (monosaccharides such as glucose (grape sugar) and fructose (fruit sugar)), minerals (e.g., sodium, potassium, magnesium, calcium, iron, zinc, molybdenum, copper, phosphorus, nitrogen, sulfur, or boron), and B vitamins (e.g., vitamin B1 (thiamine), vitamin B2 (riboflavin), niacin, pantothenic acid, vitamin B6 (pyridoxine, pyridoxal, or pyridoxamine), vitamin B12 (cyanocobalamin), folic acid, and biotin). The concentration of nutrients in the culture medium is not particularly limited as long as it allows Euglena to survive and grow.

[0042] The light conditions in the culture step are not particularly limited, and the culture step may be carried out under either light or dark conditions. When culturing heterotrophically, the culture is carried out under dark conditions. As light conditions, a normal light intensity for growing algae can be used. As dark conditions, for example, 10 μmol / m 2 / s, preferably in a completely dark place with no light at all.

[0043] The culture temperature in the culture step is not particularly limited as long as it is a temperature at which Euglena can grow, and the culture temperature (temperature of the culture solution) is, for example, 20°C to 35°C.

[0044] The pH of the liquid in the culture step is not particularly limited as long as it allows Euglena to grow, and examples of pH values ​​that allow Euglena to grow include a range of 3.0 to 5.5.

[0045] After the culturing step, it is preferable to concentrate the Euglena by centrifugation of the liquid, gravity separation, etc. The obtained Euglena can be subjected to additional treatment (e.g., suspension in a liquid, dispersion in water or oil, extraction, drying and powdering, etc.) depending on the desired form.

[0046] Paramylon particles can be produced by separating, isolating, or purifying Euglena according to or in accordance with known methods (e.g., the method described in Japanese Patent No. 5883532). Paramylon particles can be easily obtained, for example, by disrupting the Euglena cell membrane and recovering the intracellular components. Furthermore, paramylon particles may be purified as needed. Various methods for purifying paramylon particles are known (e.g., Japanese Patent No. 5883532), and these methods can be used. Examples of purification steps include surfactant treatment and washing. The obtained Euglena can be subjected to additional processing (e.g., suspension in liquid, dispersion in water or oil, dry powdering, etc.) depending on the desired form.

[0047] 4. Paramylon processed products Paramylon processed products are obtained by processing paramylon, such as physical or chemical treatment, and are not particularly limited insofar as such processing is carried out. Examples of paramylon processed products include fibrous paramylon and amorphous paramylon. Amorphous paramylon can be obtained by chemical treatment according to or in accordance with known methods, for example, the method described in JP 2011-184592 A.

[0048] As the processed paramylon product, fibrous paramylon is preferred. Fibrous paramylon will be described below.

[0049] Fibrillar paramylon is a β-1,3-glucan derived from Euglena, and is not particularly limited as long as it is in a fibrous form. Although amorphous paramylon obtained by chemically treating paramylon particles (such as by alkali treatment) has been reported, this is not recognized as fibrous when observed under an electron microscope, and is a mass with an irregular shape and size, so it is not included in the category of fibrous paramylon.

[0050] The weight-average molecular weight of the fibrous paramylon is not particularly limited, but may be, for example, 1×10 4 ~2×107 , preferably 1 x 10 5 ~5×10 5 is.

[0051] The weight average molecular weight can be measured by SEC-MALS analysis using the following method: Detector: Multi-angle scattering detector (DAWN HELEOS II manufactured by Wyatt Technology) Differential refractometer detector (Wyatt Technology Optilab T-rEX) Columns used: 2 TSKgel α-M (Tosoh) Mobile phase: DMSO with 0.05M potassium bromide Flow rate: 0.5 mL / min.

[0052] The diameter of the fibers of fibrous paramylon is not particularly limited, but is, for example, 10 to 500 nm, preferably 20 to 300 nm, and more preferably 50 to 200 nm. The diameter of the fibers of fibrous paramylon can usually be measured based on an electron microscope image of the fibrous paramylon.

[0053] The volume of fibrous paramylon settled in water is not particularly limited, but is, for example, 30 to 300 mL / g, preferably 50 to 250 mL / g, and more preferably 70 to 200 mL / g.

[0054] The submerged volume can be measured according to or by the following method: Measurements were performed according to the method described in "Dietary Fiber - Fundamentals and Applications - 3rd Edition, p. 111, Daiichi Publishing, Tokyo, edited by the Editorial Committee of the Japanese Society for Dietary Fiber Studies (2008)." Specifically, the procedure is as follows: 125 mg of the sample slurry test sample (equivalent to dry mass) is weighed into a 25 mL plastic tube, and the plastic tube is vigorously shaken by hand to agitate the contents. The contents are then transferred to a 25 mL graduated cylinder, and purified water is added to the tube until the total volume reaches 25 mL. The liquid in the graduated cylinder is stirred and then allowed to stand at 37°C for 24 hours. This allows the sample to settle, resulting in the formation of two layers separated by an interface: a layer (lower layer) containing primarily the settled sample, and a layer (upper layer) containing primarily water. The volume of the lower layer is determined from the graduated cylinder scale, and the resulting volume is divided by the sample mass (dry mass) to calculate the submerged volume (mL / g). The test is performed three or four times, and the average and standard deviation are calculated.

[0055] Fibrous paramylon has relatively high resistance to enzymatic degradation. For example, the amount of monomer (glucose) produced by degradation with β-glucanase is, for example, 0.1 to 50 mg, preferably 1 to 10 mg, per 1 g of fibrous paramylon.

[0056] This amount can be measured according to or by the following method: A reaction solution (30 mg test substance (dry weight), 5 mL buffer solution (Tokyo Chemical Industry Co., Ltd., B0156, potassium hydrogen phthalate-sodium hydroxide buffer (pH 4.0)), 0.1 mL enzyme solution (Japan Biocon Corporation, endo-1,3-β-Glucanase (enzyme content: 50 units / mL)), and purified water (10 mL reaction solution volume) was prepared and shaken horizontally at 45 rpm for 24 hours at 40°C. After shaking, the samples were immediately frozen and stored, and then lyophilized for concentration. After lyophilization, 0.5 mL of purified water was added to each sample and stirred (20-fold concentration). Centrifugation (10,000 G, 5 minutes, 4°C) and supernatant recovery were repeated twice. The glucose concentration in the recovered supernatant was measured using a measurement kit (Wako Pure Chemical Industries, Ltd., Glucose CII-Test Wako). Based on the measured values, the amount of glucose produced (mg) per 1 g of test substance was calculated.

[0057] Fibrous paramylon has relatively low solubility in alkaline solutions. For example, fibrous paramylon is insoluble in a 0.1 to 0.3 M aqueous solution of sodium hydroxide. Here, "insoluble" means, for example, that the absorbance (660 nm) of the solution after suspending fibrous paramylon in the aqueous solution (for example, immediately after suspension to after 1 hour) is, for example, 0.1 or more, preferably 1.0 or more.

[0058] Solubility can be measured according to or by the following method: 250 mg (dry weight) of the test substance was suspended in 10 mL of test solution (pure water, 0.1 M NaOH aqueous solution, 0.3 M NaOH aqueous solution) in a vial. After vigorously shaking the vial by hand for 20 seconds and then shaking it on a shaker at 80 rpm for 1 hour, the absorbance of the solution in the vial at 660 nm was measured. The absorbance was measured using a JASCO V-730 spectrophotometer.

[0059] The relative value of the crystallinity of fibrous paramylon to that of granular paramylon (crystallinity of fibrous paramylon / crystallinity of granular paramylon) is, for example, 0.60 to 0.90, and preferably 0.65 to 0.80.

[0060] Crystallinity can be measured according to or by the following method: XRD measurements were performed on the test substance under the following conditions: instrument: PANalytical X'Pert3 Powder, tube voltage: 45 kV, tube current: 40 mA, measurement range: 5.005 to 50.018°, measurement interval: 0.013°, analysis software: HighScore. Crystallinity was analyzed based on the ratio of the intensity of the amorphous portion to the intensity of the crystalline portion at 2θ = 5 to 80°. Analysis was performed after removing the background caused by the instrument from each measurement (background setting: Auto, bending factor: 0, granularity: 100), and the amorphous portion was determined by the tangent line passing through 2θ = 14, 29°. The conditions for the pending factor and granularity used to determine each amorphous portion were 0 / 20.

[0061] The fibrous paramylon may be in a form dispersed in a solvent such as water, or in a dried form. Even if the fibrous paramylon is in a dried form, it can be redispersed in water.

[0062] In this specification, the term "dry form" refers to a form having a water content of 15% by mass or less, preferably 10% by mass or less, and more preferably 5% by mass or less.

[0063] As the fibrous paramylon, preferably, defibrated paramylon particles obtained by physically defibrating the paramylon particles can be used. Alternatively, defibrated Euglena obtained by applying this defibration treatment to Euglena can also be used as the fibrous paramylon.

[0064] The defibration treatment is not particularly limited, as long as it is a treatment that can defibrate the β-1,3 glucan present in the paramylon particles without breaking the hydrogen bonds (for example, by breaking only 10% or less, 5% or less, 2% or less, or 1% or less of the hydrogen bonds of the β-1,3 glucan), or a treatment that can unravel some or all of the β-1,3-glucan chains present in the paramylon particles or the triple helix structures formed thereby. Preferably, the defibration treatment is carried out without breaking the hydrogen bonds of the β-1,3 glucan present in the paramylon particles, resulting in a fibrous form. Known treatments that can grind (shear) or pulverize (preferably grind (shear)) fine particles such as paramylon particles can be used as the defibration treatment.

[0065] The defibration treatment can be carried out using known equipment such as a grinder (shearer) or a pulverizer. Equipment used for the defibration treatment includes, for example, a stone mill, a jet mill, a twin-screw kneader, a high-pressure homogenizer, a high-pressure emulsifier, a twin-screw extruder, and a bead mill. Among these, preferred are stone mills and bead mills.

[0066] The defibration treatment can be carried out by either a wet method or a dry method. Wet defibration treatment is preferred because it allows for more efficient dispersion of fibrous paramylon in the solution. When wet defibration treatment is carried out, the solvent is not particularly limited as long as it is a solvent that can disperse fibrous paramylon, and water is preferably used.

[0067] The defibration treatment may be performed on one type of paramylon alone or in combination of two or more types. In addition, the paramylon may be partially defibrated, and as long as it contains defibrated paramylon, it is within the scope of the present invention.

[0068] 5.Applications Euglena, paramylon, paramylon processed products, and β-1,3-glucan have the effect of protecting intercellular adhesion, specifically, these components have the effect of protecting intercellular adhesion, preferably tight junctions, particularly intestinal tight junctions and tight junctions between intestinal epithelial cells, etc., i.e., the effect of promoting intercellular adhesion, the effect of strengthening intercellular adhesion, the effect of inhibiting dissociation of intercellular adhesion, etc. Therefore, at least one member selected from the group consisting of Euglena, paramylon, paramylon processed products, and β-1,3-glucan can be used as an active ingredient of an intercellular adhesion protective agent, etc.

[0069] Furthermore, at least one selected from the group consisting of Euglena, paramylon, a paramylon processed product, and β-1,3-glucan can be used as an active ingredient in compositions for various applications based on their protective effect on intercellular adhesion, the agent of the present invention, etc. Such applications include, for example, suppression of leaky gut, suppression of a decrease in intestinal barrier function (particularly a decrease in intestinal barrier function due to alcohol and / or inflammatory cytokines), suppression of diarrhea and loose stools, and suppression of the invasion of at least one selected from the group consisting of allergens, viruses, and pathogenic bacteria into the body.

[0070] Normal intestinal tight junctions can prevent viruses and pathogenic bacteria, as well as allergens, from entering the intestinal tract. However, damage to these junctions allows the entry of viruses and pathogenic bacteria. At least one selected from the group consisting of Euglena, paramylon, paramylon processed products, and β-1,3-glucan can restore damaged intercellular adhesion or maintain (protect) intercellular adhesion (repair, maintenance, or protection of intestinal tight junctions). This can inhibit the decline or inhibition of decline in intestinal barrier function, thereby inhibiting the entry of viruses and pathogenic bacteria into the body. Furthermore, maintaining intestinal function is expected to help maintain the health of healthy individuals.

[0071] Examples of viruses include enveloped viruses (viruses with an envelope) such as influenza viruses (e.g., types A and B), rubella viruses, Ebola viruses, coronaviruses, measles viruses, varicella-zoster viruses, mumps viruses, arboviruses, respiratory syncytial viruses, SARS viruses, hepatitis viruses (e.g., hepatitis B viruses, hepatitis C viruses), yellow fever viruses, AIDS viruses, rabies viruses, hantaviruses, dengue viruses, Nipah viruses, and lyssaviruses; and non-enveloped viruses (viruses without an envelope) such as adenoviruses, noroviruses, rotaviruses, human papillomaviruses, polioviruses, enteroviruses, coxsackieviruses, human parvoviruses, encephalomyocarditis viruses, polioviruses, and rhinoviruses.

[0072] Examples of pathogenic bacteria include Staphylococcus spp. (e.g., Staphylococcus aureus, Staphylococcus epidermidis), Enterococcus spp. (e.g., Enterococcus spp.), Streptococcus spp. (e.g., diplococcus, tetrastreptococcus, octastreptococcus, etc., Streptococcus pneumoniae, and hemolytic streptococcus), Bacillus spp. (e.g., Bacillus anthracis, Bacillus subtilis), Clostridium spp. (e.g., Clostridium tetani, Clostridium botulinum, and Clostridium perfringens), Corynebacterium spp. (e.g., Corynebacterium diphtheriae), Listeria spp., Lactobacillus spp., Bifidobacterium spp., Proteus spp. Examples of such bacteria include Pionibacterium (e.g., acnes, which causes acne), Actinomycetes, Escherichia (e.g., E. coli such as enterohemorrhagic E. coli (e.g., serotype O157, serotype O111, serotype O26, etc.), enteropathogenic E. coli, enteroinvasive E. coli, enterotoxigenic E. coli, enterodiffuse-adherent E. coli, and enteroaggregative E. coli), Salmonella, Pseudomonas (e.g., Pseudomonas aeruginosa), Helicobacter, Haemophilus influenzae, and Neisseria (e.g., Neisseria gonorrhoeae and Neisseria meningitidis).

[0073] Allergens include the above-mentioned viruses and pathogenic bacteria themselves, substances derived from the above-mentioned viruses and pathogenic bacteria (e.g., nucleic acids, sugar chains, peptides, proteins, etc.), and substances derived from various foods (e.g., eggs, milk, wheat, buckwheat, peanuts, shrimp, crab, soybeans, squid, salmon roe, salmon, mackerel, beef, chicken, pork, walnuts, yams, oranges, kiwi fruit, peaches, apples, bananas, gelatin, abalone, matsutake mushrooms, sesame, cashew nuts, etc.) (e.g., nucleic acids, sugar chains, peptides, proteins, etc.).

[0074] In addition to these, various uses have been reported in previous publications (e.g., Jpn. J. Food Microbiol., 36(1), 32-35, 2019, Curr Opin Gastroenterol. 2016 Mar;32(2):74-9.).

[0075] Preferably, it can be used for comprehensive applications including multiple applications (two or more, more preferably three or more, even more preferably four or more, still more preferably five or more, and even more preferably six or more) of the above applications.

[0076] The agent of the present invention can be used in various fields, for example, as a food composition (including health foods, health-promoting agents, and nutritional supplements (supplements, etc.)), food additives, cosmetics, cosmetic additives, medicines, reagents, feed, etc. The agent of the present invention is preferably an oral composition.

[0077] The form of the agent of the present invention is not particularly limited, and may take a form that is commonly used for each application depending on the application.

[0078] When the agent of the present invention is used as a food composition, the agent may be in the form of a liquid, gel, or solid food, for example, beverages such as juice, soft drinks, tea, soup, and soy milk, salad oil, dressing, yogurt, jelly, pudding, furikake, powdered milk for infants, cake mix, dairy products (for example, powder, liquid, gel, solid, etc.), bread, or confectionery (for example, cookies, etc.).

[0079] When the agent of the present invention is used as a cosmetic product, examples of the agent's form include emulsion, cosmetic liquid, face cream, hand cream, lotion, body soap, shampoo, rinse, cosmetic gel, pack, foundation, lip cream, and facial cleanser.

[0080] When the agent of the present invention is used for pharmaceutical purposes, examples of the form of the agent include formulations suitable for parenteral intake (particularly formulations for external use) such as ointments, liquid preparations for external use (liniments, lotions, etc.), sprays (aerosols for external use, pump sprays, etc.), creams, gels, patches (plasters, tapes such as plasters (reservoir type, matrix type, etc.), poultices, patches, microneedles, etc.), eye drops, eye ointments, nasal drops, suppositories, semi-solid rectal preparations, and enemas; and formulations suitable for oral intake (oral formulations) such as tablets (including orally disintegrating tablets, chewable tablets, effervescent tablets, lozenges, jelly drops, etc.), pills, granules, fine granules, powders, hard capsules, soft capsules, dry syrups, liquid preparations (including drinks, suspensions, and syrups), and jellies.

[0081] When the agent of the present invention is used as an additive, a health promoting agent, a nutritional supplement (such as a supplement), or the like, examples of the form of the agent include tablets (including orally disintegrating tablets, chewable tablets, effervescent tablets, lozenges, jelly drops, and the like), pills, granules, fine granules, powders, hard capsules, soft capsules, dry syrups, liquids (including suspensions and syrups), and jellies.

[0082] The agent of the present invention may further contain other ingredients as needed. The other ingredients are not particularly limited as long as they are ingredients that can be incorporated into food compositions (including health foods, health-promoting agents, and nutritional supplements (supplements, etc.)), food additives, cosmetics, cosmetic additives, medicines, reagents, feed, etc., and examples thereof include bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, colorants, flavorings, and chelating agents.

[0083] The content of the active ingredient in the agent of the present invention depends on the intended use, mode of use, condition of the subject to be applied, etc., and is not limited thereto, but can be, for example, 0.0001 to 100% by mass, preferably 0.001 to 50% by mass.

[0084] The amount of the agent of the present invention to be applied (e.g., administered, ingested, inoculated, etc.) is not particularly limited as long as it is an effective amount that exerts the effect of protecting intercellular adhesion, and is generally 0.1 to 10,000 mg / kg body weight per day in terms of the dry weight of the active ingredient. The above-mentioned application amount is preferably administered in divided doses once or more times a day (e.g., 1 to 3 times a day), and can be increased or decreased as appropriate depending on the age, pathological condition, and symptoms. [Example]

[0085] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0086] Reference example 1 As Euglena, dried powder (Kobe Eco-Solutions, paramylon content 70% or more) of Euglena gracilis EOD-1 strain (National Institute of Technology and Evaluation, Patent Organism Depositary Center (NITE-IPOD)) was prepared.

[0087] Reference example 2 Paramylon particles were prepared as follows.

[0088] The liquid from five flasks of the prepared Euglena gracilis EOD-1 strain (post-cultivation, before drying) was collected and centrifuged in a centrifuge tube (500 × g, 4 minutes, room temperature). The supernatant was then removed and collected. The collected supernatant was then placed in a centrifuge tube to disperse the precipitate, and the entire volume was transferred to a 100 mL measuring cylinder. The collected supernatant was then added to the measuring cylinder to bring the total volume to 90 mL.

[0089] [Enzyme treatment process] The liquid was diluted to 90 mL and transferred to a 200 mL beaker. Hydrochloric acid was added with stirring to adjust the pH to 3. A proteolytic enzyme (acid protease, product name "Protease YP-SS," manufactured by Yakult Pharmaceutical Co., Ltd., optimal pH 2.5-3.0) was added to the liquid to a concentration of 5 g / L. The liquid was subjected to enzyme treatment at 50°C for 2 hours while stirring.

[0090] [Surfactant treatment process] An aqueous solution of sodium dodecyl sulfate was added to the liquid that had undergone the enzyme treatment step so that the concentration of sodium dodecyl sulfate was 3.0% by mass / volume (w / v). While stirring the liquid containing sodium dodecyl sulfate, the pH of the liquid was adjusted to 3 by adding an aqueous solution of hydrochloric acid. The liquid was then stirred at 60°C for 30 minutes using a propeller stirrer (rotation speed: 200 rpm).

[0091] [Separation process] Paramylon was precipitated by centrifugation (1000 × g, 2 minutes, room temperature) and separated from the liquid that had undergone the surfactant treatment step. A surfactant treatment step was then carried out in the same manner, except that the concentration of sodium dodecyl sulfate was changed to 1.0% by mass / volume and the pH was not adjusted. The separation step was then carried out in the same manner as above. In this way, the surfactant treatment step and separation step were each carried out three times.

[0092] [Cleaning process] The paramylon precipitated by centrifugation in the separation step was suspended in pure water and allowed to stand at 40°C for 10 minutes. Next, paramylon was precipitated by centrifugation (1000 × g, 2 minutes, room temperature). This procedure was repeated three times.

[0093] [Drying process] The paramylon precipitated by centrifugation in the washing step was dried at 50°C to obtain paramylon particles. The obtained paramylon particles were used as paramylon in the following test examples.

[0094] Test Example 1. Evaluation test of intercellular adhesion protection effect 1 <Test Example 1-1. Preparation of an intestinal epithelial cell layer model> Caco-2 cells, derived from human colon cancer, were used as a model cell line for human intestinal epithelium. Caco-2 cells were subcultured in Dalbecco's Modified Eagle Medium (DMEM) (Sigma, D6046) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (complement inactivated by heating in a 56°C incubator for 40 minutes) and 1% antibiotic-antimycotic (Sigma, A5955) at 37°C in the presence of 5% CO2.

[0095] 750 μL of medium was placed in a 24-well plate for cell culture inserts (Corning, 353504), and an insert (apical (AP) side) (Cell Culture Insert, Transparent PET Membrane 24 Well 0.4 μm pore size, 353095) was placed therein to prepare a transwell. 2 × 10 5 250 μL of Caco-2 cell suspension (5 × 10 4 The cells were cultured for 14 days, with the medium changed every 3 days. This allowed Caco-2 cells to differentiate, resulting in an intestinal epithelial cell layer model with tight junctions.

[0096] <Test Example 1-2. Evaluation Test> The medium on the insert side was replaced with 250 μL of medium containing 0, 100, or 500 μg / mL of paramylon. After 30 minutes, the medium on the plate side was replaced with 750 μL of medium containing 0 or 5 ng / mL of TNF-α. The plates were then cultured for 48 hours. The combinations of paramylon (PM) concentration in the insert side medium and TNF-α concentration in the plate side medium are shown in the table below.

[0097] [Table 1]

[0098] 24 and 48 hours after the addition of TNF-α, electrical resistance was measured using a Merck Millicell ERS-2 electrical resistance measurement system, and TER (Transepithelial Electrical Resistance) was calculated. Measurement methods and TER calculation methods followed the attached instructions. TER at the time of TNF-α addition (0 hours) was set at 100, and the relative TER value was calculated. Williams' multiple comparison test was performed on the paramylon-added group against the control group, and the p-value was calculated.

[0099] The results are shown in Figure 1. The decrease in TER (a decrease in intercellular adhesion (mainly intercellular adhesion via tight junctions)) caused by inflammatory cytokines (TNF-α) was suppressed by the addition of paramylon. This demonstrated that paramylon has a protective effect on intercellular adhesion.

[0100] Test Example 2: Evaluation test of intercellular adhesion protection effect 2 An intestinal epithelial cell layer model prepared in the same manner as in Test Example 1-1 was used. The medium on the insert side was replaced with 225 μL of medium containing 0 or 550 μg / mL of paramylon. After 30 minutes, 25 μL of 50% ethanol (diluted with PBS) was added to the insert side and cultured for 2 hours. The combinations of paramylon (PM) concentration and ethanol concentration in the insert side medium are shown in the table below.

[0101] [Table 2]

[0102] Electrical resistance was measured using a Merck Millicel ERS-2 electrical resistance measurement system 20 minutes, 40 minutes, 60 minutes, and 2 hours after the addition of ethanol, and TER was calculated. The measurement method and TER calculation method followed the attached instructions. The TER at the time of ethanol addition (0 hours) was set at 100, and the relative TER value was calculated. A t-test was performed to determine whether there was a difference between the paramylon-added group and the control group, and the p-value was calculated.

[0103] The results are shown in Figure 2. The decrease in TER (a decrease in intercellular adhesion (mainly intercellular adhesion via tight junctions)) caused by ethanol was significantly suppressed by the addition of paramylon. This indicates that paramylon has a protective effect on intercellular adhesion.

Claims

1. An intercellular adhesion protecting agent containing Euglena and / or paramylon for use in suppressing alcohol-induced decline in intestinal barrier function.

2. The intercellular adhesion protecting agent according to claim 1, which contains Euglena, and the Euglena is Euglena gracilis.

3. The intercellular adhesion protecting agent according to claim 1, which contains Euglena, and the Euglena is Euglena gracilis strain EOD-1 (accession number FERM BP-11530).

4. The intercellular adhesion protecting agent according to claim 1, which contains paramylon, and the paramylon is derived from Euglena gracilis.

5. The intercellular adhesion protective agent according to claim 1, which contains paramylon and is derived from Euglena gracilis EOD-1 strain (accession number FERM BP-11530).

6. The intercellular adhesion protecting agent according to any one of claims 1 to 5, for use in protecting intestinal tight junctions.

7. The intercellular adhesion protecting agent according to any one of claims 1 to 6, for use in suppressing leaky gut.

8. An intercellular adhesion protecting agent described in any one of claims 1 to 7, wherein the alcohol is ethanol.

9. The intercellular adhesion protecting agent according to any one of claims 1 to 8, for use in inhibiting the invasion of at least one substance selected from the group consisting of allergens, viruses, and pathogenic bacteria into the body.

10. The agent for protecting intercellular adhesion according to any one of claims 1 to 9, for use in suppressing diarrhea or loose stools.

11. The agent for protecting intercellular adhesion according to any one of claims 1 to 10, which is a food composition, a food additive, a cosmetic, a cosmetic additive, or a medicine.

12. The agent for protecting intercellular adhesion according to any one of claims 1 to 11, which is an oral composition.

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