Composition for enhancing intestinal colonization of bacteria

Abstract

To provide a technique for enhancing intestinal colonization of bacteria.SOLUTION: A composition for enhancing intestinal colonization of bacteria, comprises at least one selected from the group consisting of a sugar represented by Galβ(1→3)Xxx1 [wherein Xxx1 represents an N-acetylamino sugar or a 6-sulfo-N-acetylamino sugar], and a sugar represented by Galβ(1→3)Xxx1-Xxx2 [wherein Xxx1 represents an N-acetylamino sugar or a 6-sulfo-N-acetylamino sugar, and Xxx2 represents any sugar].SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a composition for enhancing bacterial colonization of the intestinal tract. [Background technology]

[0002] Live microorganisms that have a beneficial effect on the health of the host by improving the intestinal environment are called probiotics, and representative examples of these are lactobacilli and bifidobacteria. Beneficial functions of probiotics include suppressing diarrhea and constipation, increasing good bacteria in the intestines and reducing bad bacteria, improving the intestinal environment, preventing intestinal infections, regulating immune function and the nervous system, suppressing allergies, and lowering cholesterol levels.

[0003] The global food probiotics market is expected to reach US$60.5 billion in 2022 and US$100.1 billion in 2030, with a CAGR of 6.5% from 2023 to 2030. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] J Nutr. 1997 Jan;127(1):89-94. doi: 10.1093 / jn / 127.1.89. Summary of the Invention [Problem to be solved by the invention]

[0005] Exogenous probiotics taken orally in the form of yogurt or fermented milk are hindered by the bacterial flora already established in the intestine, making it difficult for them to establish themselves in the human intestine (Non-Patent Document 1). To date, sugars such as inulin have been reported to have the effect of increasing the proliferation of beneficial bacteria in the intestine, but no sugars have been reported to enhance bacterial colonization of the intestinal tract.

[0006] An objective of the present invention is to provide a technique for enhancing bacterial colonization of the intestinal tract. [Means for solving the problem]

[0007] In view of the above problems, the present inventors have conducted extensive research and have discovered Galβ(1→3)Xxx 1 [Xxx 1 represents N-acetylamino sugar or 6-sulfo-N-acetylamino sugar.] and Galβ(1→3)Xxx 1 -Xxx 2 [Xxx 1 represents N-acetylamino sugar or 6-sulfo-N-acetylamino sugar, and Xxx 2 represents any sugar.] has been found to enhance bacterial colonization in the intestinal tract. Based on this finding, the present inventors have conducted further research and have completed the present invention. That is, the present invention encompasses the following aspects.

[0008] Term 1. Galβ(1→3)Xxx 1 [Xxx 1 represents N-acetylamino sugar or 6-sulfo-N-acetylamino sugar.] and Galβ(1→3)Xxx 1 -Xxx 2 [Xxx 1 represents N-acetylamino sugar or 6-sulfo-N-acetylamino sugar, and Xxx 2 represents any sugar.]. A composition for enhancing bacterial colonization of the intestinal tract, comprising at least one sugar selected from the group consisting of sugars represented by the following formulas:

[0009] Section 2. Xxx 1 Item 2. The composition for enhancing the activity of a protein according to Item 1, wherein is an N-acetylamino sugar.

[0010] Section 3. Xxx 1 Item 2. The composition for enhancing the activity of α-glucosamine according to Item 1, wherein is N-acetylgalactosamine or N-acetylglucosamine.

[0011] Term 4. Galβ(1→3)Xxx 1Item 1. The composition for enhancing the activity of a sugar represented by the formula:

[0012] Item 5. The composition for enhancement according to Item 1, wherein the bacterium is an enterobacterium.

[0013] Item 6. The composition for enhancement according to Item 1, wherein the bacteria are lactic acid bacteria or bifidobacteria.

[0014] Item 7. The composition for enhancement according to Item 1, wherein the bacterium is a bacterium of the genus Lacticaseibacillus.

[0015] Item 8. The enhancing composition according to any one of Items 1 to 7, which is in an orally ingestible form.

[0016] Item 9. The enhancing composition according to any one of Items 1 to 7, which is used to be taken in combination with the bacterium or contains the bacterium.

[0017] Item 10. The enhancing composition according to any one of Items 1 to 7, which is a food composition, a food additive, a health-promoting agent, a nutritional supplement, or a medicine.

[0018] Section 11. Bacteria and Galβ(1→3)Xxx 1 [Xxx 1 represents N-acetylamino sugar or 6-sulfo-N-acetylamino sugar.] and Galβ(1→3)Xxx 1 -Xxx 2 [Xxx 1 represents N-acetylamino sugar or 6-sulfo-N-acetylamino sugar, and Xxx 2 represents any sugar.].

[0019] Item 12. A bacterium with enhanced intestinal colonization ability, obtained by the production method described in Item 11.

[0020] Section 13. Bacteria and Galβ(1→3)Xxx 1 [Xxx 1represents N-acetylamino sugar or 6-sulfo-N-acetylamino sugar.] and Galβ(1→3)Xxx 1 -Xxx 2 [Xxx 1 represents N-acetylamino sugar or 6-sulfo-N-acetylamino sugar, and Xxx 2 represents any sugar.]. A method for enhancing colonization ability of bacteria in the intestinal tract, comprising contacting a bacterial cell with at least one selected from the group consisting of sugars represented by the following formula: [Effects of the Invention]

[0021] The present invention can provide a composition for enhancing intestinal colonization of bacteria. The present invention can also provide a method for producing bacteria with enhanced intestinal colonization ability, bacteria with enhanced intestinal colonization ability, a method for enhancing intestinal colonization of bacteria, etc. [Brief explanation of the drawings]

[0022] [Figure 1] The method of Test Example 2 is outlined below. [Figure 2] 1 shows the results of Test Example 2. The vertical axis shows the number of L. rhamnosus bacteria in the feces. The horizontal axis shows the group names. The control group was administered with physiological saline. [Figure 3] 1 shows the results of Test Example 3. The vertical axis shows fluorescence intensity (representing the amount of attached L. rhamnosus bacteria), and the horizontal axis shows the test sugar. DETAILED DESCRIPTION OF THE INVENTION

[0023] In this specification, the expressions "contain" and "comprise" include the concepts of "contain," "comprise," "consist essentially of," and "consist only of."

[0024] 1. Composition for enhancing intestinal colonization In one aspect, the present invention provides a method for producing Galβ(1→3)Xxx 1 and Galβ(1→3)Xxx 1 -Xxx 2The present invention relates to a composition for enhancing bacterial colonization of the intestinal tract (hereinafter sometimes referred to as the "composition of the present invention"), which contains at least one kind selected from the group consisting of saccharides represented by the following formula (hereinafter sometimes referred to as the "oligosaccharide of the present invention"). This will be explained below.

[0025] Xxx 1 indicates N-acetylamino sugar or 6-sulfo-N-acetylamino sugar.

[0026] In one embodiment of the present invention, Xxx 1 is preferably an N-acetylamino sugar.

[0027] N-acetylamino sugars are sugars in which the amino group of a sugar (amino sugar) obtained by replacing the hydroxy group (typically the hydroxy group at the 2-position) of a hexose with an amino group is substituted with an acetyl group, and are not particularly limited as long as this is the case. Specific examples of N-acetylamino sugars include N-acetylgalactosamine, N-acetylglucosamine, and N-acetylmannosamine. In one embodiment of the present invention, among these, N-acetylgalactosamine and N-acetylglucosamine are preferred, and N-acetylgalactosamine is more preferred.

[0028] 6-Sulfo-N-acetylaminosugars are sugars (amino sugars) obtained by replacing the hydroxy group (-OH) at the 6th position of a hexose with -OS(=O)2-OH and replacing hydroxy groups other than those at the 6th and 3rd positions (typically the hydroxy group at the 2nd position) with amino groups, in which the amino group is substituted with an acetyl group, and are not particularly limited as long as such sugars are used. Specific examples of 6-sulfo-N-acetylaminosugars include 6-sulfo-N-acetylgalactosamine, 6-sulfo-N-acetylglucosamine, and 6-sulfo-N-acetylmannosamine. In one embodiment of the present invention, among these, 6-sulfo-N-acetylgalactosamine and 6-sulfo-N-acetylglucosamine are preferred, and 6-sulfo-N-acetylgalactosamine is more preferred.

[0029] Xxx 2 represents any sugar. Examples of the arbitrary sugar include monosaccharides and oligosaccharides.

[0030] The monosaccharide is not particularly limited, and any known monosaccharide can be used, such as heptose, hexose, pentose, tetraose, or triose, with hexose being preferred.

[0031] Examples of hexoses include galactose, glucose, N-acetylglucosamine, N-acetylgalactosamine, mannose, fructose, allose, talose, gulose, altrose, idose, psicose, sorbose, and tagatose, in which some of the hydroxy groups may be reduced and substituted with hydrogen atoms, or some of the hydroxy groups may be protected with known protecting groups or substituted with functional groups such as sulfate groups.

[0032] Oligosaccharides are sugars in which two or more monosaccharide molecules are linked to one molecule via glycosidic bonds. The number of monosaccharide molecules constituting an oligosaccharide is, for example, 2 to 20, preferably 2 to 10, more preferably 2 to 5, and even more preferably 2 to 3. The type of monosaccharide constituting an oligosaccharide is not particularly limited, and the monosaccharides listed above can be used. Furthermore, the combination of monosaccharides constituting an oligosaccharide is also not particularly limited. Specific examples of oligosaccharides include oligosaccharides containing two constituent monosaccharide molecules (e.g., lactose, Galβ(1→3)GalNAc, Galβ(1→4)GlcNAc, Galβ(1→6)GlcNAc, sucrose, maltose, trehalose, turanose, or cellobiose), oligosaccharides containing three constituent monosaccharide molecules (e.g., raffinose, melezitose, or maltotriose), oligosaccharides containing four constituent monosaccharide molecules (e.g., acarbose or stachyose), and oligosaccharides containing five or more constituent monosaccharide molecules, with oligosaccharides containing two constituent monosaccharide molecules being preferred.

[0033] Galβ(1→3)Xxx 1 is a sugar formed by combining the hydroxyl group at position 1 of galactose (Gal) with an N-acetylamino sugar or a 6-sulfo-N-acetylamino sugar (Xxx 1 ) is a sugar formed by linking the hydroxyl group at the 3-position of Xxx via a β-glycosidic bond. 1 is N-acetylgalactosamine (GalNAc), structural formula (A) (galacto-N-biose), Xxx 1 is N-acetylglucosamine (GlcNAc), structural formula (B) (lacto-N-biose), Xxx 1 is 6-sulfo-N-acetylgalactosamine (6sulfo GalNAc), and structural formula (C) (6-sulfo-galacto-N-biose), and Xxx 1 When is 6-sulfo-N-acetylglucosamine (6sulfo GlcNAc), the structural formula (D) (6-sulfo-lacto-N-biose) is shown below.

[0034] [ka]

[0035] [ka]

[0036] [ka]

[0037] [ka]

[0038] Galβ(1→3)Xxx 1 -Xxx 2 Galβ(1→3)Xxx 1 Xxx 1 Hydroxy group of Xxx 2It is a sugar in which the hydroxyl group of the α- and β-glycosides is linked by a glycosidic bond. The glycosidic bond is in the form of Galβ(1→3)Xxx. 1 -Xxx 2 There are no particular limitations on the bond, as long as it can be assimilated by bacteria (particularly enterobacteria). The bond can be, for example, a bond that can be cleaved in vivo (particularly in the digestive tract), and specific examples include an α-1,4-glycosidic bond and a β-1,4-glycosidic bond.

[0039] The composition of the present invention is particularly preferably Galβ(1→3)Xxx 1 In this embodiment, the composition of the present invention particularly preferably contains at least one sugar selected from the group consisting of galacto-N-biose and lacto-N-biose. In one embodiment of the present invention, the composition of the present invention contains galacto-N-biose.

[0040] The sugar also includes salt forms. The salt is not particularly limited and can be, for example, a pharmaceutically acceptable salt. Both acid salts and basic salts can be used. Examples of acid salts include inorganic acid salts such as hydrochloride, hydrobromide, sulfate, nitrate, and phosphate; organic acid salts such as acetate, propionate, tartrate, fumarate, maleate, malate, citrate, methanesulfonate, and paratoluenesulfonate. Examples of basic salts include alkali metal salts such as sodium salt and potassium salt; and alkaline earth metal salts such as calcium salt and magnesium salt; salts with ammonia; and salts with organic amines such as morpholine, piperidine, pyrrolidine, monoalkylamine, dialkylamine, trialkylamine, mono(hydroxyalkyl)amine, di(hydroxyalkyl)amine, and tri(hydroxyalkyl)amine.

[0041] As an example, the structural formula of the sodium salt of structural formula (C) and structural formula (D) is shown below.

[0042] [ka]

[0043] [ka]

[0044] The sugar also includes a solvate. The solvate is not particularly limited and may be, for example, a solvate with a pharmaceutically acceptable solvent. Examples of the solvent include water and pharmaceutically acceptable organic solvents (e.g., ethanol, glycerol, acetic acid, etc.).

[0045] The oligosaccharides of the present invention may be used singly or in combination of two or more.

[0046] As the oligosaccharide of the present invention, commercially available products can be used as they are, or those synthesized according to known methods can be used. For example, they can be synthesized according to the method described in JP 2017-195793 A.

[0047] Specifically, the oligosaccharide of the present invention may be, for example, a galactose donor and Xxx 1 or Xxx 1 -Xxx 2 and a saccharide represented by the formula (I) in the presence of β-galactosidase.

[0048] Xxx 1 or Xxx 1 -Xxx 2 The sugar represented by the formula (I) is as described above.

[0049] Xxx 1 is a 6-sulfo-N-acetylamino sugar, Xxx 1 and Xxx 1 -Xxx 2 The sugar represented by is an N-acetylamino sugar, or an N-acetylamino sugar with a hydroxy group and Xxx 2and the hydroxy group of the above linked by a glycosidic bond with a sulfation reagent (for example, sulfur trioxide pyridine complex, sulfur trioxide trimethylamine complex, etc.).

[0050] The galactose donor is not particularly limited, and a wide variety of galactose donors can be used, including those described in JP 2017-195793 A. Specific examples include p-nitrophenyl-β-D-galactopyranoside, o-nitrophenyl-β-D-galactopyranoside, 4-methoxyphenyl-β-D-galactopyranoside, phenyl-β-D-galactopyranoside, lacto-N-biose, and galactobiose.

[0051] The β-galactosidase is not particularly limited, but from the viewpoint of reaction efficiency, the β-galactosidase described in JP 2017-195793 A is preferred. Particularly preferred is the β-galactosidase derived from the Bacillus GS-MAIU strain (NITE International Patent Organism Depositary Center accession number: NITE P-02240).

[0052] The reaction can be carried out according to or in accordance with the method described in JP 2017-195793 A.

[0053] After the reaction, the solution containing the oligosaccharide of the present invention obtained by the reaction may be further subjected to a purification step. Purification can be performed by known methods (liquid separation, distillation, chromatography, recrystallization, etc.). Furthermore, the excess glycosyl acceptor can be recovered and reused after the reaction is completed.

[0054] The composition of the present invention preferably contains the sugar as an active ingredient in a purified form. The content of the sugar as an active ingredient in the composition of the present invention (e.g., Galβ(1→3)Xxx 1 and Galβ(1→3)Xxx 1 -Xxx 2 The total content of Galβ(1→3)Xxx 1The sugar content (the total content of galacto-N-biose and lacto-N-biose, the galacto-N-biose content, or the lacto-N-biose content) is preferably 30% by mass or more, more preferably 50% by mass or more, even more preferably 70% by mass or more, still more preferably 80% by mass or more, particularly preferably 90% by mass or more, and particularly preferably 95% by mass or more, relative to 100% by mass of the sugars contained in the composition of the present invention.

[0055] The composition of the present invention is used to enhance intestinal colonization by bacteria, which means, for example, that bacteria colonize the intestinal tract for a longer period and / or to a greater extent.

[0056] Enhancement of bacterial colonization of the intestinal tract includes, and can be substituted by, increased expression of intestinal colonization factors and / or improved adhesion of bacteria to mucin.

[0057] The bacterium is not particularly limited as long as it can colonize the intestinal tract. The bacterium is preferably an enterobacterium. More preferably, the bacterium includes lactic acid bacteria and bifidobacteria, and even more preferably, bacteria of the genus Lacticaseibacillus, and particularly preferably, Lacticaseibacillus rhamnosus.

[0058] The bacteria can be exogenously ingested. Exogenously ingested bacteria may have difficulty colonizing in the intestine due to interference from bacterial flora already established in the intestine. For this reason, the composition of the present invention is preferably used to be ingested in combination with the bacteria, or contains the bacteria. The timing of their combination is not particularly limited; for example, the composition of the present invention and the Lacticaseibacillus bacteria may be ingested simultaneously or on the same day, or one may be ingested one or more days (e.g., 2 to 14 days) before or after the other is ingested.

[0059] The composition of the present invention can be used in various fields, for example, as a food additive, a food composition (including health-promoting agents and nutritional supplements (supplements, etc.)), a medicine, and the like.

[0060] The composition of the present invention is not particularly limited and can be applied to various mammals (for example, humans, mice, rats, rabbits, dogs, cats, cows, horses, pigs, monkeys, etc.).

[0061] The composition of the present invention is usually taken orally, but is not limited to this.

[0062] The form of the composition of the present invention is not particularly limited, and may take any form commonly used for each application depending on the application.

[0063] When the composition of the present invention is used as a food additive, a medicine, a health-promoting agent, a nutritional supplement (such as a supplement), or the like, the composition may be in the form of, for example, a tablet (including orally disintegrating tablets, chewable tablets, effervescent tablets, lozenges, jelly drops, and the like), a pill, a granule, fine granules, a powder, a hard capsule, a soft capsule, a dry syrup, a liquid (including drinks, suspensions, and syrups), or a jelly.

[0064] The composition of the present invention, when used as a food composition, may take the form of liquid, gel, or solid foods, for example: (1) beverages such as soft drinks, carbonated drinks, fruit drinks, vegetable juices, lactic acid bacteria drinks, dairy drinks, soy milk, mineral water, tea drinks, coffee drinks, sports drinks, alcoholic drinks, and jelly drinks; (2) processed vegetables such as tomato puree, canned mushrooms, dried vegetables, and pickles; and (3) processed fruit products such as dried fruit, jam, fruit puree, and canned fruit. (4) Spices such as curry powder, wasabi, ginger, spice blends, seasoning powder, etc.; (5) Noodles (including fresh and dried noodles) such as pasta, udon, soba, ramen, macaroni, etc.; (6) Breads such as bread, sweet bread, cooked bread, donuts, etc.; (7) Pregelatinized rice, oatmeal, wheat gluten, batter, etc.; (8) Baked goods, biscuits, rice snacks, candy, chocolate, chewing gum, snacks, frozen desserts, candied sweets, Japanese sweets, Western sweets, semi-fresh sweets (9) Sweets, pudding, ice cream, and other confectioneries; (10) bean products, such as red beans, tofu, natto, soybean flour, yuba (tofu skin), boiled beans, and peanuts; (11) honey and royal jelly processed foods; (12) meat products, such as ham, sausage, and bacon; (13) dairy products, such as yogurt, pudding, condensed milk, cheese, fermented milk, butter, and ice cream; (14) processed fish, such as dried fish, kamaboko (fish paste), chikuwa (fish sausage), and fish sausage; processed seaweed, such as dried wakame seaweed, kelp, and tsukudani (simmered food in soy sauce); and cod roe. (14) processed fish eggs such as herring roe, salmon roe, and dried mullet roe; (15) seasonings such as dashi stock, soy sauce, vinegar, mirin, consommé base, Chinese base, concentrated dashi, dressing, mayonnaise, ketchup, and miso; edible oils and fats such as salad oil, sesame oil, linoleic oil, diacylglycerol, and safflower oil; (16) soups (including powders and liquids), cooked and semi-cooked foods, side dishes, retort foods, chilled foods, and semi-cooked foods (for example, seasonings for seasoned rice and seasonings for crab and egg omelets).

[0065] The composition of the present invention may further contain other ingredients as needed. These other ingredients are not particularly limited as long as they are suitable for incorporation into food additives, food compositions, pharmaceuticals, health-promoting agents, nutritional supplements (e.g., supplements), etc., and examples thereof include amino acids, alcohols, polyhydric alcohols, polymeric compounds such as sugars, gums, and polysaccharides, surfactants, antiseptics, antibacterial agents, and disinfectants, pH adjusters, chelating agents, antioxidants, enzyme components, binders, disintegrants, lubricants, fluidizers, and coolants, as well as minerals, cell activators, tonics, excipients, thickeners, stabilizers, preservatives, isotonicity agents, dispersants, adsorbents, disintegration aids, wetting agents or moisture regulators, moisture-proofing agents, colorants, flavorings or fragrances, fragrances, reducing agents, solubilizers, solubilizers, foaming agents, thickeners or viscosifiers, solvents, bases, emulsifiers, plasticizers, buffers, and gloss enhancers.

[0066] The content of the active ingredient in the composition of the present invention depends on the intended use, mode of use, condition of the subject to be applied, etc., and is not limited, but can be, for example, 0.0001 to 100% by mass, preferably 0.001 to 50% by mass. The content, calculated on a dry weight basis, can be, for example, 50% by mass or more, 60% by mass or more, 70% by mass or more, 80% by mass or more, 90% by mass or more, 95% by mass or more, or 99% by mass or more.

[0067] The amount of the composition 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 its effect, and is generally 0.1 to 10,000 mg / kg body weight per day in terms of dry weight of the oligosaccharide of the present invention. The above-mentioned application amount is preferably applied 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.

[0068] 2. Method for enhancing intestinal adhesion In one aspect, the present invention relates to a method for producing a bacterium and a Galβ(1→3)Xxx 1 [Xxx 1 represents N-acetylamino sugar or 6-sulfo-N-acetylamino sugar.] and Galβ(1→3)Xxx 1 -Xxx 2 [Xxx1 represents N-acetylamino sugar or 6-sulfo-N-acetylamino sugar, and Xxx 2 represents any sugar.] with at least one selected from the group consisting of sugars represented by the following formula (I):

[0023] . The present invention also relates to a bacterium with enhanced intestinal colonization ability obtained by the production method.

[0069] The contacting method is not particularly limited, and typically includes contacting the bacteria with the oligosaccharide of the present invention in an aqueous solution. The aqueous solution is not particularly limited as long as it does not inhibit the survival of the bacteria, and may be, for example, a culture medium.

[0070] The medium (e.g., culture solution) preferably contains a carbon source. Examples of carbon sources include carbohydrates such as monosaccharides, disaccharides, oligosaccharides, and polysaccharides; invert sugar obtained by hydrolyzing sucrose; glycerol; carbon-1 compounds (hereinafter referred to as C1 compounds) such as methanol, formaldehyde, formate, carbon monoxide, and carbon dioxide; oils such as corn oil, palm oil, and soybean oil; acetate; animal fats and oils; animal oils; fatty acids such as saturated fatty acids and unsaturated fatty acids; lipids; phospholipids; glycerolipids; glycerol fatty acid esters such as monoglycerides, diglycerides, and triglycerides; polypeptides such as microbial proteins and plant proteins; renewable carbon sources such as hydrolyzed biomass carbon sources; extracts of organisms or tissues such as yeast extract, meat extract, and liver extract; or combinations thereof. Examples of nitrogen sources that can be used include inorganic ammonium salts such as ammonium sulfate, ammonium chloride, and ammonium phosphate; organic nitrogen sources such as soybean hydrolysates; ammonia gas; and aqueous ammonia. As organic trace nutrient sources, it is desirable to include appropriate amounts of required substances such as vitamin B1 and L-homoserine, or yeast extract, etc. In addition to these, small amounts of potassium phosphate, magnesium sulfate, iron ions, manganese ions, etc. may be added as necessary. The medium used in the present invention may be either a natural medium or a synthetic medium.

[0071] The contact time is, for example, 30 minutes or more, 2 hours or more, 4 hours or more, 8 hours or more, or 16 hours or more, and, for example, 360 hours or less, 240 hours or less, 120 hours or less, 60 hours or less, or 48 hours or less.

[0072] The temperature during contact can be appropriately set depending on the optimum temperature for bacterial survival, for example, 20 to 45°C, preferably 30 to 40°C.

[0073] The concentration of the oligosaccharide of the present invention in the aqueous solution is, for example, 0.01 to 30% by mass, preferably 0.02 to 10% by mass, more preferably 0.05 to 5% by mass, even more preferably 0.1 to 2% by mass, and particularly preferably 0.3 to 1.2% by mass.

[0074] The content of the oligosaccharides of the present invention relative to 100% by mass of sugars in the aqueous solution is preferably 1% by mass or more, 5% by mass or more, 10% by mass or more, 20% by mass or more, 30% by mass or more, 40% by mass or more, 50% by mass or more, 60% by mass or more, 70% by mass or more, 80% by mass or more, 90% by mass or more, or 95% by mass or more.

[0075] The culture can be anaerobic depending on the properties of the bacteria.

[0076] After the cultivation is completed, the cells and viable cells can be collected by solid-liquid separation such as filtration or centrifugation. Alternatively, the culture liquid can be used as is.

[0077] The intestinal colonization can be evaluated, for example, according to or in accordance with any of the methods of Test Examples 1 to 3 described below.

[0078] Enhanced intestinal colonization means, for example, that the expression level of at least one intestinal colonization factor is, for example, 1.2-fold or more, 1.3-fold or more, 1.4-fold or more, 1.5-fold or more, 1.6-fold or more, 1.7-fold or more, 1.8-fold or more, 1.9-fold or more, 2.0-fold or more, 2.1-fold or more, 2.2-fold or more, 2.3-fold or more, 2.4-fold or more, 2.5-fold or more, 2.6-fold or more, 2.7-fold or more, 2.8-fold or more, or 2.9-fold or more compared to a control (a case where glucose is used instead of the oligosaccharide of the present invention). The expression level can be evaluated according to or in accordance with the method of Test Example 1.

[0079] Enhanced intestinal adhesion means, for example, that the amount of adhesion to mucin is 1.1 times or more, 1.2 times or more, 1.3 times or more, or 1.4 times or more compared to a control (when glucose is used instead of the oligosaccharide of the present invention). The expression level can be evaluated according to or in accordance with the method of Test Example 3. [Example]

[0080] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0081] The β-galactosidase used in the following Synthesis Examples 1 and 2 was obtained according to the method described in Example 2 of JP 2017-195793 A.

[0082] Synthesis Example 1: Synthesis of Galβ(1→3) GalNAc (galacto-N-biose) According to the method described in JP 2017-195793 A, a sugar (Galβ(1→3) GalNAc) was synthesized in which the hydroxyl group at position 1 of galactose (Gal) and the hydroxyl group at position 3 of N-acetylgalactosamine (GalNAc) are linked via a β-glycosidic bond. Specifically, the synthesis was carried out as follows.

[0083] β-PNP-galactose (5 mg, 16.6 μmol) was dissolved in 495 μL of distilled water. Subsequently, 100 μL of β-galactosidase solution and 2.5 mL of N-acetylgalactosamine solution (50 mM Tris-HCl buffer (pH 8.0)) (N-acetylgalactosamine: 200 mg, 0.91 mmol) were added, and the mixture was incubated at 37°C for 48 hours. After the reaction was completed, the enzyme was inactivated by heating at 100°C for 3 minutes to terminate the reaction. The reaction mixture was desalted and filtered to remove insoluble components, and then applied to a 5 μm Inertsil Amide (14 × 250 mm) column (GL Sciences HPLC System PLC761) (column temperature: 40°C, eluent: acetonitrile / water = 7 / 3, flow rate: 9.5 mL / min). Detection was performed using UV at 215 nm to obtain Galβ(1→3) GalNAc (4.93 mg, 12.7 μmol, 77.5%). 1 H-NMR (D2O) δ5.01 (d, 1H, J = 3.6 Hz), 4.50 (d, 1 H, J = 8.0 Hz), 4.30 (d, 1 H, J = 7.6 Hz), 4.08 (dd), 4.05 (d, 1 H), 3.98-3.66 (m), 3.56-3.40 (m), 3.32 (t, 1H), 1.82 (s, 3H).

[0084] Synthesis Example 2: Synthesis of Galβ(1→3)GlcNAc (lacto-N-biose) According to the method described in JP 2017-195793 A, a sugar (Galβ(1→3)GlcNAc) was synthesized in which the hydroxyl group at position 1 of galactose (Gal) and the hydroxyl group at position 3 of N-acetylglucosamine (GlcNAc) are linked via a β-glycosidic bond. Specifically, the synthesis was carried out as follows.

[0085] β-PNP-galactose (5 mg, 16.6 μmol) was dissolved in 495 μL of distilled water. Subsequently, 100 μL of β-galactosidase solution and 2.5 mL of N-acetylglucosamine saturated solution (50 mM Tris-HCl buffer (pH 8.0)) (N-acetylglucosamine: 625 mg, 2.83 mmol) were added, and the mixture was incubated at 37°C for 24 hours. After the reaction was completed, the enzyme was inactivated by heating at 100°C for 3 minutes to terminate the reaction. The reaction mixture was desalted, filtered to remove insoluble components, and then applied to a 5 μm Inertsil Amide (14 × 250 mm) column (GL Sciences HPLC System PLC761) (column temperature: 40°C, eluent: acetonitrile / water = 7 / 3, flow rate: 9.5 mL / min). Detection was performed with UV at 215 nm to obtain lacto-N-biose (Galβ(1→3)GlcNAc) (6.17 mg, 16.1 μmol, 97%). 1 H-NMR (D2O) 4.96 (d, 1 H, J = 3.6 Hz), 4.54 (d, 1 H, J = 8.4 Hz), 4.26 (m), 3.87 (dd, 1H), 3.74- 3.25 (m), 1.82 (s, 3 H).

[0086] Synthesis Example 3. Synthesis of 6-sulfo-N-acetylgalactosamine (6sulfo GalNAc) N-acetylgalactosamine (72 mg, 0.30 mmol) and sulfur trioxide pyridine complex (48 mg, 0.30 mmol) were dissolved in DMF (3.6 mL) and stirred at room temperature for 24 hours. After the reaction was completed, the mixture was subjected to gel filtration (Sephadex G10) to obtain the desired 6-sulfo-N-acetylgalactosamine (13.5 mg).

[0087] Synthesis Example 4: Synthesis of Galβ(1→3)6sulfo GalNAc (6-sulfo-galacto-N-biose) PNP-galactose (27.3 mg, 90.6 μmol) and 6-sulfo-N-acetylgalactosamine (292.8 mg, 0.90 mmol) were dissolved in 600 μL of 50 mM phosphate buffer (pH 6.0). β-galactosidase (155 μL, 30 mU, derived from Bacillus sp. GS-MAIU strain (NITE P-02240)) was added and incubated at 37°C for 48 hours. After the reaction was completed, the mixture was heated at 100°C for 3 minutes and centrifuged at 15,000 × g for 5 minutes. The supernatant was applied to a gel filtration column (Sephadex G10) to obtain the desired 6-sulfo-galacto-N-biose (12.5 mg, 42.8%). 1 H-NMR (D2O) δ5.11 (d, 1H, J = 4.0 Hz), 4.61 (d, 1 H, J = 8.0 Hz), 4.39 (d, 1 H, J = 7.6 Hz), 4.33 (d, 1 H, J = 7.3 Hz), 4.27-4.24 (m), 4.20-4.17 (m), 4.12-4.03 (m), 3.96-3.74 (m), 3.68-3.59 (m), 3.56-3.49 (m), 3.41 (dd, 1 H, J = 8.0 Hz, J = 10.4 Hz), 1.92 (s, 3 H).

[0088] Test Example 1. Analysis of bacterial colonization factors in the intestinal tract Bacteria were cultured in the presence of test sugars, and the expression levels of intestinal colonization factors in the resulting bacteria were analyzed. Specifically, this was done as follows.

[0089] 50 mL of GAM glycolysis medium (1.0% peptone, 0.3% soybean peptone, 1.0% protease peptone, 1.35% digested serum powder, 0.5% yeast extract, 0.22% meat extract, 0.12% liver extract, 0.25% potassium dihydrogen phosphate, 0.30% sodium chloride, 0.03% L-cysteine hydrochloride, 0.03% sodium thioglycolate, pH 7.1) supplemented with 0.6% galacto-N-biose, a sugar in which an acetylated amino sugar is capped with β(1→3)-linked galactose, glucose, or galactose was added. Lactic acid bacteria (Lacticaseibacillus rhamnosus JCM1136) were added to the medium. T ) was inoculated into 500 μl of the culture medium and cultured anaerobically at 37°C. 600 Bacteria were harvested when the β-solenoid structure (β-solenoid structure) reached 0.3. RNA was extracted from these bacteria using the RiboPure-Bacteria Kit (Invitrogen Corp., Carlsbad, CA, USA) and the MICROBExpress Bacterial mRNA Purification Kit (Invitrogen Corp.), and gene expression analysis (RNA-seq) was performed at Seibu Giken Co., Ltd. Analysis of gene expression changes in response to glucose (log2 fold change) revealed that the expression of two genes (RS13130 and RS13565) predicted to function as colonization factors for this bacterium increased significantly by 3.161-fold and 2.955-fold, respectively, when galacto-N-biose was added to the medium (Table 1). These factors have a β-solenoid structure typical of colonization factors.

[0090] [Table 1]

[0091] Test Example 2: Analysis of bacterial colonization in the intestinal tract After mice were fed the bacteria and test sugars for a certain period of time, the bacteria were detected in the feces (which is thought to reflect the bacterial flora in the intestinal tract), and the colonization of the bacteria in the intestinal tract was analyzed based on the amount of the bacteria.

[0092] Six-week-old male BALB / cA mice (CLEA Japan, Inc.) were administered antibiotics (vancomycin 0.5 g / L, doripenem 0.25 g / L) in their drinking water for 10 days, and then divided into three groups of eight mice each. On days 0, 2, and 4, the L. rhamnosus group was challenged with L. rhamnosus JCM1136. T (1×10 9 cells), and L. rhamnosus JCM1136 in the L. rhamnosus + galacto-N-biose group. T (1×10 9 Mice were administered 50 mg of galacto-N-biose (50 cells) and saline (50 mg), while the control group received saline (Fig. 1). Two days after the end of administration (6 days after administration), fecal DNA was extracted using a QIAamp Fast DNA Stool Mini kit (Qiagen), and L. rhamnosus bacterial counts were compared using real-time PCR (StepOne; Applied Biosystems) with L. rhamnosus-specific primers. The results showed that the bacterial counts were significantly higher in mice administered galacto-N-biose (Fig. 2).

[0093] Test Example 3. Analysis of bacterial adhesion to mucin Bacteria were cultured in the presence of the test sugars, and the adhesion of the resulting bacteria to mucin was analyzed as follows.

[0094] Mucin was exfoliated from the small and large intestinal mucosa of four 4-week-old male Tsl:ICR Germfree mice (Sankyo Labo Services Co., Ltd.), diluted four-fold with PBS buffer containing EDTA, PMSF, and IAA (phosphate-buffered saline containing 1 mM EDTA, 25 μg / ml phenylmethylsulfonyl fluoride, and 5 mM iodoacetamide), and then precipitated with ethanol. The precipitated fraction was suspended in MilliQ water and lyophilized. The resulting solution was then stored at a specific gravity of 1.39 g / cm. 3The cesium chloride solution was dissolved in PBS buffer containing EDTA, PMSF, and IAA, and subjected to cesium chloride equilibrium density gradient centrifugation (Beckman Coulter, centrifuge: Optima MAX-XP, rotor: MLS-50, 160,000 g, 15°C, 72 h). Polysaccharides were detected by PAS staining and by UV method. 280 The protein content was detected by 1.3-1.5 g / cm3. 3 The fractions were collected and dialyzed using MilliQ, followed by lyophilization to obtain mucin. Mucin was dissolved in HHBS buffer (HEPES-buffered Hank's salt solution) at a concentration of 1 mg / ml, and 100 μl of the solution was dispensed into each well of a 96-well black cell culture plate for fluorescence measurement (Sumitomo Bakelite MS-8096K) and left overnight at 4°C. The next day, each well was washed three times with 200 μl of HHBS buffer to prepare a mucin-coated plate.

[0095] Lactic acid bacteria (Lacticaseibacillus rhamnosus JCM1136 T ) was cultured anaerobically overnight at 37°C in GAM glycolysis medium supplemented with 0.6% galacto-N-biose or glucose. The following day, the bacteria were harvested (10,000 g, 4°C, 5 min), washed three times with anaerobic PBS buffer, and then suspended in anaerobic PBS buffer containing 10 μM CEDA-SE [5-(and-6)-Carboxyfluorescein Diacetate, Succinimidyl Ester]. The bacteria were then cultured anaerobically at 37°C for 30 min and fluorescently stained. The bacteria were then washed three times with anaerobic PBS buffer and subjected to OD analysis in anaerobic PBS buffer. 600The pH was adjusted to 2.0. 100 μl of the resulting lactic acid bacteria suspension was applied to each well of the mucin-coated plate and anaerobically cultured at 37°C for 1 h. After washing each well three times with 200 μl of HHBS buffer, fluorescence at an excitation wavelength of 490 nm was measured (multimode microplate reader Varioskan LUX, excitation / emission, 490 / 517 nm). The results demonstrated that bacteria cultured in galacto-N-biose-containing medium (GNB-cultured bacteria) adhered to mucin in significantly greater numbers than bacteria cultured in glucose-containing medium (glucose-cultured bacteria) (Figure 3).

Claims

1. Galβ(1→3)Xxx 1 [Xxx 1 represents N-acetylamino sugar or 6-sulfo-N-acetylamino sugar.] and Galβ(1→3)Xxx 1 -Xxx 2 [Xxx 1 represents N-acetylamino sugar or 6-sulfo-N-acetylamino sugar, and Xxx 2 represents any sugar.]. A composition for enhancing bacterial colonization of the intestinal tract, comprising at least one sugar selected from the group consisting of sugars represented by the following formulas:

2. Xxx 1 The enhancing composition of claim 1, wherein is an N-acetylamino sugar.

3. Xxx 1 The enhancing composition of claim 1, wherein is N-acetylgalactosamine or N-acetylglucosamine.

4. Galβ(1→3)Xxx 1 The enhancing composition according to claim 1, comprising a sugar represented by the formula:

5. The enhancing composition of claim 1 , wherein the bacteria is an enterobacterium.

6. The enhancing composition according to claim 1 , wherein the bacteria are lactic acid bacteria or bifidobacteria.

7. The enhancing composition according to claim 1 , wherein the bacterium is a bacterium of the genus Lacticaseibacillus.

8. The enhancing composition according to any one of claims 1 to 7, which is in an orally ingestible form.

9. The enhancing composition according to any one of claims 1 to 7, which is used to be taken in combination with the bacterium or contains the bacterium.

10. The enhancing composition according to any one of claims 1 to 7, which is a food composition, a food additive, a health-promoting agent, a nutritional supplement, or a medicine.

11. Bacteria and Galβ(1→3)Xxx 1 [Xxx 1 represents N-acetylamino sugar or 6-sulfo-N-acetylamino sugar.] and Galβ(1→3)Xxx 1 -Xxx 2 [Xxx 1 represents N-acetylamino sugar or 6-sulfo-N-acetylamino sugar, and Xxx 2 represents any sugar.].

12. A bacterium with enhanced intestinal colonization ability, obtainable by the production method according to claim 11.

13. Bacteria and Galβ(1→3)Xxx 1 [Xxx 1 represents N-acetylamino sugar or 6-sulfo-N-acetylamino sugar.] and Galβ(1→3)Xxx 1 -Xxx 2 [Xxx 1 represents N-acetylamino sugar or 6-sulfo-N-acetylamino sugar, and Xxx 2 represents any sugar.]. A method for enhancing colonization ability of bacteria in the intestinal tract, comprising contacting a bacterial cell with at least one selected from the group consisting of sugars represented by the following formula:

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