JP6629021B2 - Immunoglobulin A secretagogue - Google Patents

Description

  The present invention relates to an immunoglobulin A secretion promoter and a method for efficiently producing a galactosyl oligosaccharide contained in the agent.

  In the living body, the mucous membrane is exposed to microorganisms such as bacteria and viruses, various antigens, toxins, and the like, and thus has a defense mechanism against them. As one of them, a mucosal immune mechanism such as intestinal immunity is known. For example, in the intestinal mucosa, immunoglobulin A is secreted by the immune cells of Peyer's patches, thereby preventing the invasion of microorganisms from the intestinal mucosa, neutralizing toxins, preventing allergen invasion, and controlling intestinal symbiotic bacteria. Has been done. Therefore, it is thought that if secretion of immunoglobulin A is promoted, mucosal immunity is activated and resistance to pathogen infection and poisoning by its toxin is increased.

  On the other hand, galactosyl oligosaccharides such as galactosyl lactose are components contained in human breast milk, and their safety is said to be high. Certain galactosyl oligosaccharides are known to have bifidobacterial growth promoting activity (Patent Document 1), and it is also said that bifidobacteria promote the secretion of immunoglobulin A. However, since galactosyl oligosaccharides have also been reported to suppress immunity (Patent Documents 2 and 3), it is impossible to predict what effects galactosyl oligosaccharides have on the immune system. It is the current situation. Even if the galactosyl oligosaccharide can promote the secretion of immunoglobulin A, if it is mediated by the growth of bifidobacteria, the amount of bifidobacteria is small (or not at all) on the mucous membrane or the amount of bifidobacteria retained is small. For elderly people (or none at all) and antibiotics, the effect cannot be expected.

  The galactosyl oligosaccharide is desirably produced by an enzymatic reaction from the viewpoint of efficiency and the like. However, in the enzymatic reaction, a by-product having a molecular weight similar to that of the target sugar is easily generated. In this case, it is difficult to purify the target sugar with high purity by column chromatography or the like.

JP-A-5-140178 JP-A-2003-040779 JP 2000-516591 A

  An object of the present invention is to provide an agent containing galactosyl oligosaccharide, which is said to be highly safe, as an active ingredient, and capable of promoting secretion of immunoglobulin A without the intervention of bacteria such as bifidobacteria. Furthermore, it is another object of the present invention to provide a method for producing the galactosyl oligosaccharide efficiently and by suppressing the generation of by-products (especially, sugars having a molecular weight close to the target sugar).

  The present inventors have made intensive studies and as a result, even if a specific galactosyl oligosaccharide (α-galactosyl lactulose, β-galactosyl lactulose, α-galactosyl melibiose, or β-galactosyl melibiose) is present in the absence of bifidobacteria, It has been found that the secretion of immunoglobulin A (in this specification, sometimes referred to as “IgA”) can be promoted by directly acting on immune cells. Furthermore, they have found that by reacting a specific sugar acceptor with a galactose donor in the presence of galactosidase, these galactosyl oligosaccharides can be produced efficiently and with reduced by-product formation. The present invention has been completed as a result of further studies based on these findings. That is, the present invention includes the following embodiments.

  That is, the present invention includes the following embodiments.

Item 1. An immunoglobulin A secretion promoter comprising at least one galactosyl oligosaccharide selected from the group consisting of α-galactosyl lactulose, β-galactosyl lactulose, α-galactosyl melibiose, and β-galactosyl melibiose.
Item 2. Item 2. The agent according to Item 1, which is a Bifidobacterium non-mediated immunoglobulin A secretion promoter.
Item 3. Item 3. The agent according to Item 1 or 2, wherein the content of the galactosyl oligosaccharide is 80% by mass or more based on 100% by mass of the oligosaccharide contained in the immunoglobulin A production promoter.
Item 4. Item 4. The agent according to any one of Items 1 to 3, wherein the galactosyl oligosaccharide is at least one selected from the group consisting of α-galactosyl lactulose, α-galactosyl melibiose, and β-galactosyl melibiose.
Item 5. Item 5. The agent according to any one of Items 1 to 4, which is an oral preparation.
Item 6. A mucosal immunostimulator comprising at least one galactosyl oligosaccharide selected from the group consisting of α-galactosyl lactulose, β-galactosyl lactulose, α-galactosyl melibiose, and β-galactosyl melibiose.
Item 7. Item 7. The agent according to Item 6, wherein the mucosa is intestinal mucosa.
Item 8. A sugar receptor selected from the group consisting of melibiose and lactulose,
Reacting with a galactose donor in the presence of galactosidase,
A method for producing a galactosyl oligosaccharide selected from the group consisting of α-galactosyl lactulose, β-galactosyl lactulose, α-galactosyl melibiose, and β-galactosyl melibiose.
Item 9. Item 9. The production method according to Item 8, wherein the temperature during the reaction is 20 ° C or higher and lower than 40 ° C.
Item 10. Item 10. The method according to Item 8 or 9, wherein the total content of the sugar acceptor and the galactose donor in the reaction solution is 20 to 80% by mass relative to 100% by mass of the reaction solvent.
Item 11. The production according to any one of Items 8 to 10, wherein the galactosyl oligosaccharide to be produced is a galactosyl oligosaccharide selected from the group consisting of α-galactosyl lactulose, α-galactosyl melibiose, and β-galactosyl melibiose. Method.
Item 12. Item 12. The method according to any one of Items 8 to 11, wherein the galactosidase is derived from an organism belonging to the genus Aspergillus.

  ADVANTAGE OF THE INVENTION According to this invention, the immunoglobulin A secretion promoter which can promote the secretion of immunoglobulin A without the intervention of bacteria, such as bifidobacteria, can be provided. Since the immunoglobulin A secretion enhancer of the present invention contains galactosyl oligosaccharide as an active ingredient, it is considered to be highly safe. Moreover, since the active ingredient galactosyl oligosaccharide hardly causes a decrease in pH in the oral cavity, the immunoglobulin A secretion enhancer of the present invention is hardly caused by oral ingestion. Furthermore, since the active ingredient galactosyl oligosaccharide also has a bifidobacterial growth activity, the immunoglobulin A secretagogue of the present invention not only has an effect by mucosal immunostimulation (improvement of resistance, etc.) but also an improvement of intestinal flora. It can also exert effects (promoting digestion and absorption, improving bowel movements, preventing aging, etc.).

  ADVANTAGE OF THE INVENTION According to this invention, it becomes possible to manufacture the galactosyl oligosaccharide which is an active ingredient efficiently and by suppressing generation | occurrence | production of a by-product (especially, sugar whose molecular weight is close to the target sugar). This makes it possible to provide an immunoglobulin A secretagogue containing galactosyl oligosaccharide, which is an active ingredient, having high activity of promoting secretion of immunoglobulin A and containing the same in high purity.

The measurement result of the IgA level of Peyer's patch cell culture supernatant is shown (Example 5). The vertical axis indicates the measured IgA concentration (ng / mL). "**" indicates that the P value for the negative control is 0.01 or less. The measurement result of the turbidity of the culture solution of Bifidobacterium is shown (Example 6). The vertical axis indicates turbidity (OD ₆₀₀ ). The horizontal axis shows the elapsed time (unit: time) from the start of culture. The measurement result of the turbidity of the culture solution of Bifidobacterium is shown (Example 6). The vertical axis indicates turbidity (OD ₆₀₀ ). The horizontal axis shows the elapsed time (unit: time) from the start of culture. The measurement result of the turbidity of the culture solution of Bifidobacterium is shown (Example 6). The vertical axis indicates turbidity (OD ₆₀₀ ). The horizontal axis shows the elapsed time (unit: time) from the start of culture. The result of pH measurement of a mixture of saliva and sugar is shown (Example 7). The vertical axis indicates the measured pH.

1. The present invention provides an immunoglobulin comprising at least one galactosyl oligosaccharide selected from the group consisting of α-galactosyl lactulose, β-galactosyl lactulose, α-galactosyl melibiose, and β-galactosyl melibiose. A secretion promoting agent (also referred to herein as the “agent of the present invention”). Hereinafter, this will be described.

  α-Galactosyl lactulose is a galactosyl oligosaccharide in which a hydroxyl group on the anomeric carbon of galactose and a hydroxyl group at the non-reducing end of lactulose are α-glycosidically bonded, and is not particularly limited.

  β-galactosyl lactulose is a galactosyl oligosaccharide in which a hydroxyl group on the anomeric carbon of galactose and a hydroxyl group at the non-reducing end of lactulose are β-glycosidically bound, and are not particularly limited.

  α-Galactosyl melibiose is a galactosyl oligosaccharide in which a hydroxyl group on the anomeric carbon of galactose and a hydroxyl group at the non-reducing terminal of melibiose are α-glycosidically linked, and is not particularly limited.

  β-galactosyl melibiose is a galactosyl oligosaccharide in which a hydroxyl group on the anomeric carbon of galactose and a hydroxyl group at the non-reducing terminal of melibiose are β-glycosidically bound, and are not particularly limited.

  The galactosyl oligosaccharide as an active ingredient is preferably at least one selected from the group consisting of α-galactosyl lactulose, α-galactosyl melibiose, and β-galactosyl melibiose. The galactosyl oligosaccharide is preferably at least one selected from the group consisting of α-galactosyl lactulose, β-galactosyl lactulose, and β-galactosyl melibiose, from the viewpoint of having a higher immunoglobulin A secretion action, and more preferably Is at least one member selected from the group consisting of α-galactosyl lactulose and β-galactosyl melibiose, and more preferably α-galactosyl lactulose. The galactosyl oligosaccharide is preferably at least one selected from the group consisting of α-galactosyl lactulose and β-galactosyl lactulose from the viewpoint of low cariogenicity.

  Galactosyl oligosaccharides may be used alone or in combination of two or more.

  The use of the agent of the present invention is not particularly limited as long as the effects of the present invention are exhibited. The agent of the present invention can be used, for example, as pharmaceuticals, health enhancers, nutritional supplements (supplements and the like), food additives and the like. Further, the agent of the present invention can be suitably used for intestinal immunotherapy, supplement therapy and the like.

  The formulation of the agent of the present invention is not particularly limited, and may be a formulation generally used in each application depending on the use of the agent of the present invention. When the application is a pharmaceutical, a health enhancer, a nutritional supplement (supplement, etc.) and the like, for example, a tablet (intra-oral disintegrating tablet, chewable tablet, effervescent tablet, troche, jelly-drop, etc.) Suitable for oral ingestion of pills, pills, granules, fine granules, powders, hard capsules, soft capsules, dry syrups, liquids (including drinks, suspensions and syrups), and jellies Formulation forms (oral preparation forms), preparation forms suitable for parenteral ingestion (parenteral preparation forms) such as injections, patches, lotions, and creams, and oral preparation forms are preferred. When the application is a food additive or the like, examples of the formulation include liquids, granules, and powders.

  The agent of the present invention may consist solely of the active ingredient galactosyl oligosaccharide, or may optionally contain other components according to the use or the form of the preparation. Other components are not particularly limited, but include, for example, high molecular compounds such as amino acids, alcohols, polyhydric alcohols, saccharides, gums, and polysaccharides, surfactants, preservatives, antibacterial agents, bactericides, and pH adjustment. Agents, chelating agents, antioxidants, enzyme components, binders, disintegrants, lubricants, fluidizers, fresheners, minerals, cell activators, nutritional tonics, excipients, thickeners , Stabilizing agents, preservatives, isotonic agents, dispersants, adsorbents, disintegration aids, wetting agents or wetting regulators, moisturizers, colorants, flavors or fragrances, fragrances, reducing agents, solubilization Agents, dissolution aids, foaming agents, thickeners or thickeners, solvents, bases, emulsifiers, plasticizers, buffers, brighteners and the like.

  When the agent of the present invention contains other components other than galactosyl oligosaccharide which is an active ingredient, the content of galactosyl oligosaccharide is determined according to the use or the formulation of the agent of the present invention, and the content capable of exerting its effect. There is no particular limitation as long as The content is, for example, 0.1 to 90% by mass relative to 100% by mass of the agent of the present invention.

  By the production method of the present invention described later, galactosyl oligosaccharide as an active ingredient can be produced while suppressing generation of by-products (particularly, sugars having a molecular weight close to that of a target saccharide). Therefore, by utilizing this method, the agent of the present invention can contain galactosyl oligosaccharide, which is an active ingredient having a high immunoglobulin A secretion promoting action, and other oligosaccharides which are considered to have relatively low action. It is possible to mix more while reducing the amount. In this respect, the content of the active ingredient galactosyl oligosaccharide is, for example, 80% by mass or more, preferably 90% by mass or more, more preferably 95% by mass, based on 100% by mass of the oligosaccharide contained in the agent of the present invention. % Or more, more preferably 97% by mass or more, even more preferably 99% by mass or more, and particularly preferably 100% by mass.

  When the agent of the present invention is taken as a medicine, a health enhancer, a nutritional supplement (supplement, etc.), the amount of intake should be appropriately set according to the symptoms, age, body weight, dosage form, etc. of the recipient. Can be. The intake amount is, for example, such that the daily intake of galactosyl oligosaccharide as an active ingredient is 0.01 g to 100 g per adult. The ingestion may be performed once a day, or may be performed a plurality of times (for example, 2 to 4 times).

  When the agent of the present invention is used as a food additive or the like, the amount of the additive can be appropriately set according to the type of food or the like. The amount of addition is, for example, an amount such that the amount of galactosyl oligosaccharide, which is an active ingredient in an amount of food consumed by an adult per day, is 0.01 g to 100 g. The method of incorporating the agent of the present invention into food can be appropriately set according to the type of food and the like. For example, the agent of the present invention may be mixed in a food, the food may be immersed in an aqueous solution containing the agent of the present invention, or the agent of the present invention may be sprayed on the food. When spraying, the agent of the present invention can be mixed with a volatile solvent such as ethanol and sprayed. Further, for example, when processing the food by heating, the agent of the present invention may be applied to any food material before and after heating.

  The agent of the present invention can promote the secretion of immunoglobulin A onto mucous membranes (eg, buccal mucosa, gastric mucosa, intestinal mucosa, olfactory epithelium, oral mucosa, endometrium, and preferably intestinal mucosa). . As a result, mucosal immunity is activated, and resistance to pathogen infection and poisoning by its toxin is further increased. From this viewpoint, the active ingredient of the agent of the present invention is used as an active ingredient of mucosal (for example, buccal mucosa, gastric mucosa, intestinal mucosa, olfactory epithelium, oral mucosa, endometrium, and preferably intestinal mucosa) immunostimulant Is also useful. Further, since the active ingredient of the agent of the present invention also has a bifidobacteria growth activity, it can also be used as an active ingredient such as a bifidobacterium growth promoter, an intestinal flora improver, a digestion absorption enhancer, a bowel movement improver, an antiaging agent, and the like. Useful.

  Galactosyl oligosaccharide as an active ingredient of the agent of the present invention can exert an immunoglobulin A secretion promoting action even in the absence of bacteria such as bifidobacteria. Therefore, the agent of the present invention can more suitably be used as a bacterial (preferably bifidobacterium) -independent immunoglobulin A secretion promoter. When used as a bifidobacterial non-mediated immunoglobulin A secretion enhancer, immunoglobulin A can be effectively used even on mucous membranes or specimens (elderly people or antibiotic users) with little (or no) bifidobacterium retention. Secretion can be promoted.

2. A method for producing a galactosyl oligosaccharide The present invention comprises a step of reacting a sugar acceptor selected from the group consisting of melibiose and lactulose with a galactose donor in the presence of galactosidase, α-galactosyl lactulose, β-galactosyl lactulose, α The present invention relates to a method for producing a galactosyl oligosaccharide selected from the group consisting of galactosyl melibiose and β-galactosyl melibiose (in this specification, sometimes referred to as “the production method of the present invention”). Hereinafter, this will be described.

  The sugar acceptor is appropriately selected depending on the galactosyl oligosaccharide to be produced. If the production target is α-galactosyl lactulose or β-galactosyl lactulose, lactulose is selected as the sugar acceptor.If the production target is α-galactosyl melibiose or β-galactosyl melibiose, melibiose is selected as the sugar acceptor. Is done.

  Galactosidase is appropriately selected depending on the galactosyl oligosaccharide to be produced. When the production target is α-galactosyl lactulose or α-galactosyl melibiose, α-galactosidase is selected as galactosidase, and when the production target is β-galactosyl lactulose or β-galactosyl melibiose, β-galactosidase is selected as galactosidase. .

  α-Galactosidase is an enzyme classified under EC No. EC 3.2.1.22, and is not particularly limited as long as it is used. As α-galactosidase, those derived from various organisms can be used. For example, α-galactosidase derived from Aspergillus organisms (preferably Aspergillus niger), coffee beans, Xanthomonas organisms (preferably Xanthomonas manihotis) and the like can be mentioned. Among them, from the viewpoint that the galactosyl oligosaccharide to be produced can be produced efficiently and by suppressing the production of by-products (particularly, sugars having a molecular weight close to the target sugar), it is preferably derived from an Aspergillus organism. Preferably, α-galactosidase derived from Aspergillus niger is used. As α-galactosidase, one type may be used alone, or two or more types may be used in combination.

  β-galactosidase is an enzyme classified under EC No. EC 3.2.1.23, and is not particularly limited as long as it is so. As β-galactosidase, those derived from various organisms can be used. For example, β-galactosidase derived from Aspergillus organisms (preferably Aspergillus oryzae), cattle (testis, liver, etc.), Escherichia coli, Xanthomonas manihotis, Bacteroides fragilis, Jack bean, Streptococcus pneumonia, Kluyveromyces lactis, Xanthomonas sp and the like. Among them, from the viewpoint that the galactosyl oligosaccharide to be produced can be produced efficiently and by suppressing the production of by-products (particularly, sugars having a molecular weight close to the target sugar), it is preferably derived from an Aspergillus organism. Preferably, β-galactosidase derived from Aspergillus oryzae is used. As β-galactosidase, one type may be used alone, or two or more types may be used in combination.

  The galactosidase may be a galactosidase immobilized on a carrier. A known method can be employed for immobilizing galactosidase on a carrier. The carrier is not particularly limited as long as galactosidase can be immobilized, and examples thereof include a carrier having a carboxyl group capable of amide bonding with an amino group contained in galactosidase. As a specific example, immobilizing galactosidase on a carrier by forming an amide bond between an activated ester group of sepharose (carrier) in which a carboxyl group is esterified with N-hydroxysuccinimide (NHS) and an amino group of galactosidase. Can be. Another specific example is an immobilization method in which a carrier is activated with cyanogen bromide to form an amide bond with an amino group of galactosidase.

  The galactose donor is not particularly limited as long as it is a sugar capable of donating galactose, and examples thereof include galactose and oligosaccharides (preferably disaccharides) containing galactose as a constituent monosaccharide. Such an oligosaccharide is not particularly limited as long as it is an oligosaccharide that can be decomposed by the galactosidase used in the production method of the present invention to produce galactose.For example, the hydroxyl group on the anomeric carbon of galactose and the hydroxyl group of a monosaccharide or oligosaccharide Is a glycoside-linked oligosaccharide.

  When the galactosidase used in the production method of the present invention is α-galactosidase, oligosaccharides that can be used as a galactose donor include, for example, melibiose, melibiulose, galactosucrose, α (1-2) galaviose, α (1-3) galaviose, α (1-6) galabiose, α (1-4) galabiose, melibiosamin, N-acetyl melibiosamin, α-PNP galactose, and the like.If the galactosidase used in the production method of the present invention is β-galactosidase, galactose Oligosaccharides that can be used as donors include, for example, lactulose, lactose, neolactose, β-PNP galactose, allolactose, N-acetyllactosamine, N-acetylallolactosamine and the like.

  Among these, preferred galactose donors are galactosyl donors, which are preferable from the viewpoint that galactosyl oligosaccharides to be produced can be produced efficiently and by suppressing the production of by-products (particularly, sugars having a molecular weight close to the target sugar). In the case of α-galactosyl lactulose, galactose can be mentioned, and in the case of β-galactosyl lactulose, lactulose can be mentioned (in this case, lactulose functions as both a galactose donor and a sugar acceptor). In the case of ribobiose, melibiose is used (in this case, melibiose functions as both a galactose donor and a sugar acceptor), and in the case of β-galactosyl melibiose, lactose is used.

  As the galactose donor, one kind may be used alone, or two or more kinds may be used in combination.

  The reaction is performed in a suitable solvent. The solvent is not particularly limited as long as it can dissolve the sugar acceptor and the galactose donor and does not deactivate galactosidase. Examples of such a solvent include water, acetate buffer, phosphate buffer, citrate buffer, borate buffer, tartrate buffer, Tris buffer, phosphate buffered saline, and the like, and are preferable. Include water, acetate buffer and the like. Further, a small amount of an organic solvent may be mixed in the solvent.

  The reaction temperature is not particularly limited as long as galactosidase is not deactivated, but the galactosyl oligosaccharide to be produced is produced efficiently and by suppressing the generation of by-products (particularly, sugars having a molecular weight close to the target sugar). From the viewpoint that it is possible, the temperature is preferably lower than the optimum temperature of galactosidase. More specifically, the reaction temperature is, for example, 10 to 40 ° C., preferably 15 to 35 ° C., more preferably 20 to 35 ° C., and still more preferably 23 to 32 ° C. lower than the optimal temperature of galactosidase. No. From the same viewpoint, when the optimal temperature of galactosidase is 55 to 60 ° C, the reaction temperature is, for example, 15 to 50 ° C, preferably 20 to 45 ° C, more preferably 20 to 40 ° C, and further more preferably 25 to 40 ° C, more preferably 25 to 35 ° C.

  The reaction time is not particularly limited as long as the enzyme activity of galactosidase can be sufficiently exhibited. The reaction time is, for example, 1 to 200 hours, preferably 5 to 200 hours, more preferably 10 to 170 hours. From the viewpoint that the galactosyl oligosaccharide to be produced can be produced efficiently and by suppressing the generation of by-products (especially, sugars having a molecular weight similar to the target sugar), a preferable reaction time is as follows: When the production target is α-galactosyl lactulose, 100 to 200 hours (more preferably 130 to 200 hours, even more preferably 150 to 190 hours), and when the object to be produced is β-galactosyl lactulose, 5 to 50 hours (more preferably 12 to 36 hours, even more preferably 18 to 30 hours). Hours), 40 to 150 hours (more preferably 60 to 130 hours, more preferably 80 to 120 hours) when the object to be produced is α-galactosyl melibiose, and 1 to 24 when the object to be produced is β-galactosyl melibiose. Time (more preferably 4 to 18 hours, even more preferably 8 to 16 hours).

  The reaction pH is not particularly limited as long as the enzyme activity of galactosidase is sufficiently exhibited. The reaction pH is, for example, pH 3 to 10, preferably pH 4 to 6.

  When the galactose donor and the sugar acceptor are different from each other, the molar ratio of the galactose donor and the sugar acceptor in the reaction is not particularly limited as long as an enzymatic reaction by galactosidase occurs. The molar ratio is, for example, 1 mol of the galactose donor from the viewpoint that the galactosyl oligosaccharide to be produced can be produced efficiently and while suppressing generation of by-products (particularly, sugars having a molecular weight close to the target sugar). The sugar acceptor is, for example, 0.5 to 15 mol, preferably 1 to 10 mol, more preferably 2 to 8 mol, and still more preferably 3 to 7 mol.

  From the viewpoint that the galactosyl oligosaccharide to be produced can be produced efficiently and by suppressing the generation of by-products (particularly, sugars having a molecular weight close to the target sugar), the concentration of the sugar receptor and the galactose receptor in the reaction solution Is desirable. More specifically, the total content of the sugar acceptor and the galactose donor in the reaction solution is, for example, 20 to 80% by mass, preferably 25 to 75% by mass, more preferably 100% by mass of the reaction solvent. It is 30 to 70% by mass. The content is preferably 30 to 50% by mass (more preferably 35 to 45% by mass) with respect to 100% by mass of the reaction solvent when α-galactosyl lactulose is produced, and β-galactosyl lactulose when produced. Is preferably 50 to 70% by mass (more preferably 55 to 65% by mass) with respect to 100% by mass of the reaction solvent, and preferably 25 to 70% by mass when the object to be produced is α-galactosyl melibiose. 45% by mass (more preferably 30 to 40% by mass), preferably 30 to 50% by mass (more preferably 35 to 45% by mass) based on 100% by mass of the reaction solvent when β-galactosyl melibiose is produced. It is.

  After the above reaction, the solution containing the galactosyl oligosaccharide to be produced obtained by the reaction may be further subjected to a purification step. As a purification means, a known method (liquid separation, distillation, chromatography, recrystallization, etc.) can be adopted. According to the production method of the present invention, a galactosyl oligosaccharide to be produced can be produced by suppressing the production of by-products (particularly, sugars having a molecular weight close to that of the target sugar). Can be obtained with high purity.

  Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited to these examples.

Example 1 : Synthesis of α-galactosyl lactulose Galactose (350 mg, 1.94 mmol) and lactulose (3.4 g, 9.9 mmol) were dissolved in a sodium acetate buffer (pH = 4.0, 50 mM, 8.6 mL). Subsequently, 0.6 mg (18 U) of α-galactosidase (Sumiteam AGS: optimal temperature 60 ° C., manufactured by Nippon Chemical Co., Ltd.) was added, and the mixture was incubated at 30 ° C. for 168 hours. After completion of the reaction, the reaction was stopped by heating at 100 ° C. for 3 minutes to inactivate the enzyme. The reaction mixture was desalted and filtered to remove insoluble components, and then applied to an Inertsil Amide 5 μm (14 × 250 mm) column manufactured by GL Sciences (HPLC system PLC761) (column temperature: 40 ° C., eluent: acetonitrile / (Water = 7/3), flow rate: 9.5 mL / min), and detection yielded the desired α-galactosyl lactulose (60.9 mg) by RI (yield: 6.2%). In the column purification, there was no by-product other than the desired α-galactosyl lactulose, and the purification was easy.

Example 2 : Synthesis of β-galactosyl lactulose Lactulose (2.6 g, 7.6 mmol) was treated with sodium acetate buffer (pH = 5.0, 50 mM, 4.2 mL), β-galactosidase (Smilact L: optimal temperature 55 ° C, Shin Nippon Chemical Co., Ltd.) 10 mg (100 U) was added and incubated at 30 ° C. for 24 hours. After completion of the reaction, the reaction was stopped by heating at 100 ° C. for 3 minutes to inactivate the enzyme. The reaction mixture was desalted and filtered to remove insoluble components, and then applied to an Inertsil Amide 5 μm (14 × 250 mm) column manufactured by GL Sciences (HPLC system PLC761) (column temperature: 40 ° C., eluent: acetonitrile / Water = 7/3, flow rate: 9.5 mL / min), and detection was performed by RI to obtain the desired β-galactosyl lactulose (47.8 mg) (yield: 1.2%). In the column purification, there was no by-product other than the desired β-galactosyl lactulose, and the column could be easily purified.

Example 3 Synthesis of α-Galactosyl Melibiose Melibiose (280 mg, 0.82 mmol) was dissolved in distilled water (700 μL). Next, sodium acetate buffer (pH = 5.0, 50 mM, 100 μL) and 0.104 mg (3 U) of α-galactosidase (Sumiteam AGS: optimum temperature 60 ° C., manufactured by Nippon Chemical Co., Ltd.) were added, and the mixture was added at 30 ° C. For 96 hours. After completion of the reaction, the reaction was stopped by heating at 100 ° C. for 3 minutes to inactivate the enzyme. The reaction mixture was desalted and filtered to remove insoluble components, and then applied to an Inertsil Amide 5 μm (14 × 250 mm) column manufactured by GL Sciences (HPLC system PLC761) (column temperature: 40 ° C., eluent: acetonitrile / Water = 7/3, flow rate: 9.5 mL / min), and detection yielded the desired α-galactosyl melibiose (75.9 mg) by RI (yield: 1.8%). In the column purification, there was no by-product other than the desired α-galactosyl melibiose, and the purification was easy.

Example 4 : Synthesis of β-galactosyl melibiose Lactose (90 mg, 0.26 mmol) and melibiose (450 mg, 1.32 mmol) were dissolved in distilled water (1.2 mL). Next, a sodium acetate buffer (pH = 5.0, 50 mM, 100 μL) and 10 mg (100 U) of β-galactosidase (Sumilac L: optimal temperature of 55 ° C., manufactured by Nippon Chemical Co., Ltd.) were added, and the mixture was added at 30 ° C. For 12 hours. After completion of the reaction, the reaction was stopped by heating at 100 ° C. for 3 minutes to inactivate the enzyme. The reaction mixture was desalted and filtered to remove insoluble components, and then applied to an Inertsil Amide 5 μm (14 × 250 mm) column manufactured by GL Sciences (HPLC system PLC761) (column temperature: 40 ° C., eluent: acetonitrile / Water = 7/3, flow rate: 9.5 mL / min), and detection yielded the desired β-galactosyl melibiose (19.7 mg) by RI (yield: 15%). In the column purification, there was no by-product other than the desired β-galactosyl melibiose, and the purification was easy.

Example 5 : IgA-inducing activity test Each of the galactosyl oligosaccharides synthesized in Examples 1 to 4 was cultured (5% CO _{2 in} RPMI medium at 37 ° C.) in a mouse Peyer's patch cell (Journal of Applied Microbiology 116, 980-989, 2013) (1.25 × 10 ⁶ cells / mL) in a medium (100 μL) to a final concentration of 100 μg / mL, and cultured for 5 days. After the culture, the IgA level in the culture supernatant was measured using a mouse IgA ELISA kit (Bethyl Laboratories, Montgomery, TX, USA). The case where no galactosyl oligosaccharide was added (negative control) and the case where lipopolysaccharide (LPS) derived from Escherichia coli was added in place of galactosyl oligosaccharide to a final concentration of 5 μg / mL (positive control for IgA induction) were also used. Similarly, the IgA level of the culture supernatant was measured. The average value of n = 3 for the negative control and the positive control, and n = 6 for the others, the standard error and the P value were determined. FIG. 1 shows the measurement results. In FIG. 1, “saline” indicates a negative control, “LPS” indicates a positive control, “3” indicates β-galactosyl lactulose, “4” indicates α-galactosyl lactulose, and “8” indicates α-galactosyl melvio. “9” indicates β-galactosyl melibiose, and “13” indicates melibiose.

  As shown in FIG. 1, when the galactosyl oligosaccharide synthesized in Examples 1 to 4 was added, the IgA level in the medium was higher than that of the negative control. This suggests that these galactosyl oligosaccharides have an IgA secretion promoting effect. In addition, since this test system was performed in the absence of other bacteria such as bifidobacteria, it was suggested that this IgA secretion promoting action was a non-bacterial (such as bifidobacterium) -mediated action. Furthermore, melibiose classified into the same galactosyl oligosaccharide did not show any IgA secretion promoting effect. This suggests that the galactosyl oligosaccharides synthesized in Examples 1 to 4 have IgA secretion-promoting properties not properties of galactosyl oligosaccharides in general but properties specific to these sugars.

Example 6 : Bifidobacterium growth activity test
In a 96-well plate, 172 μL of Basal Media (0.2% yeast extract, 1.0% peptone, 0.5% sodium acetate, 0.2% diammonium citrate, 0.02% magnesium sulfate, 0.2% dipotassium hydrogen phosphate, 0.1% cysteine hydrochloride) and Example 1 20 μL of a 10% aqueous solution of each of the galactosyl oligosaccharides synthesized in Steps 4 to 4 was added (final concentration: 1%), and the mixture was allowed to stand overnight in an anaerobic state. On the other hand, bifidobacteria (B. longum JCM1222, B. Animalis sbsp. Lactis JCM10602, or B. breve JCM1192) Cells of, OD ₆₀₀ = 0.1 and comprising as Reducing reagent (2% cysteine hydrochloride and 11% sodium carbonate ) To obtain a cell suspension. 8 μL of this cell suspension was added to a 96-well plate after standing overnight, and culture was started under anaerobic conditions at 37 ° C. Turbidity (OD ₆₀₀ ) was measured over time (up to 48 hours). The turbidity was measured in the same manner when no galactosyl oligosaccharide was added (negative control) and when glucose was added instead of galactosyl oligosaccharide (positive control for bifidobacterial growth). The measurement results are shown in FIGS. 2 to 4, “3” indicates β-galactosyl lactulose, “4” indicates α-galactosyl lactulose, “7” indicates β-galactosyl melibiose, and “8” indicates α-galactosyl melibiose.

  As shown in FIGS. 2 to 4, when the galactosyl oligosaccharide synthesized in Examples 1 to 4 was added, the turbidity after a certain period of time was higher than that of the negative control. This suggested that these galactosyl oligosaccharides have bifidobacterial growth activity.

Example 7 : Cariogenicity test The oral cavity was rinsed with tap water and gargled with distilled water, and then saliva was collected. To 0.2 mL of a 1% aqueous solution of α-galactosyl lactulose, β-galactosyl lactulose, sucrose, lactulose, or xylitol, 0.6 mL of fresh saliva and 0.2 mL of Brainheart Infusion Medium (Nissui Pharmaceutical Co., Ltd.) were added. The mixture was heated at 37 ° C., and the pH change was measured over time with a pH meter (Compact pH Meter B-71X, manufactured by HORIBA). When the organic matter is metabolized by the oral bacteria in saliva to produce an organic acid, the pH of the mixed solution is lowered. In this case, the added sugar can be evaluated as having a palatogenicity. FIG. 5 shows the measurement results.

  When sucrose is added, the pH is greatly reduced after 5 hours, whereas when α-galactosyl lactulose or β-galactosyl lactulose is added, like xylitol, which is known as a sugar that is less likely to cause tooth decay, The decrease in pH was small. From this result, it was found that α-galactosyl lactulose or β-galactosyl lactulose is a sugar chain that is hardly metabolized by oral bacteria, that is, hardly forms caries.

Claims (12)

An immunoglobulin A secretion promoter comprising at least one galactosyl oligosaccharide selected from the group consisting of α-galactosyl lactulose, β-galactosyl lactulose, α-galactosyl melibiose, and β-galactosyl melibiose. 2. The agent according to claim 1, which is a bifidobacterium non-mediated immunoglobulin A secretion promoter. The agent according to claim 1 or 2, wherein the content of the galactosyl oligosaccharide is 80% by mass or more with respect to 100% by mass of the oligosaccharide contained in the immunoglobulin A production promoter. The agent according to any one of claims 1 to 3, wherein the galactosyl oligosaccharide is at least one selected from the group consisting of α-galactosyl lactulose, α-galactosyl melibiose, and β-galactosyl melibiose. The agent according to any one of claims 1 to 4, which is in an oral preparation form. A mucosal immunostimulator comprising at least one galactosyl oligosaccharide selected from the group consisting of α-galactosyl lactulose, β-galactosyl lactulose, α-galactosyl melibiose, and β-galactosyl melibiose. The agent according to claim 6, wherein the mucous membrane is an intestinal mucosa. α-galactosyl lactulose, β-galactosyl lactulose, α-galactosyl melibiose, and a method for producing a galactosyl oligosaccharide selected from the group consisting of β-galactosyl melibiose,
A sugar receptor selected from the group consisting of melibiose and lactulose,
The step of reacting a galactose donor in the presence of galactosidase seen including,
When the galactosyl oligosaccharide to be produced is β-galactosyl lactulose, the galactose donor is lactulose, and the total content of the sugar acceptor and the galactose donor in a reaction solution is 100 mass of the reaction solvent. % To 50 to 70% by mass,
When the galactosyl oligosaccharide to be produced is α-galactosylmelibiose, the galactosidase is a galactosidase derived from an organism belonging to the genus Aspergillus.
Production method. The method according to claim 8, wherein the reaction temperature is 20 ° C. or more and less than 40 ° C. 10. The galactosyl oligosaccharide to be produced is α-galactosyl lactulose, α-galactosyl melibiose, or β-galactosyl melibiose, and the total content of the sugar acceptor and the galactose donor in the reaction solution is 100 The production method according to claim 8 or 9, wherein the content is 20 to 80% by mass relative to the mass%. The galactosyl oligosaccharide to be produced is a galactosyl oligosaccharide selected from the group consisting of α-galactosyl lactulose, α-galactosyl melibiose, and β-galactosyl melibiose, according to any one of claims 8 to 10, wherein Production method. The method according to any one of claims 8 to 11, wherein the galactosidase is derived from an organism belonging to the genus Aspergillus.