Immunoglobulin a secretion promoter

Abstract

To provide a substance which has an effect of promoting secretion of immunoglobulin A for a long time on relatively aged animals.SOLUTION: An immunoglobulin A secretion promoter contains LACTO-N-BIOSE I as an active ingredient. Foods and drinks and drugs having a mucous membranes immune-stimulation action contain the promoter.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an immunoglobulin A secretagogue, and a food or drink product and a pharmaceutical product for stimulating mucosal immunity that utilize the same. [Background technology]

[0002] Immunoglobulin A (IgA) is a type of immunoglobulin and consists of two heavy chains and two light chains. IgA forms a dimer in exocrine fluids such as the intestinal tract and respiratory tract, and plays an important role in mucosal immunity. Therefore, substances that promote IgA secretion are expected to enhance mucosal immune function and have preventive effects against infectious diseases and allergic diseases. It has long been known that various oligosaccharides, such as galactosyl oligosaccharides (Patent Document 1), fructooligosaccharides (Patent Documents 2 and 3), and galactooligosaccharides (Non-Patent Document 1), promote IgA secretion.

[0003] On the other hand, lacto-N-biose I (LNB) is a milk oligosaccharide unique to human breast milk and has been known to have the effect of selectively promoting the growth of bifidobacteria (Patent Document 4), but it was not known to have the effect of promoting IgA secretion. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Laid-Open No. 2017-057174 [Patent Document 2] Japanese Patent Application Laid-Open No. 2008-115169 [Patent Document 3] Japanese Patent Application Laid-Open No. 2003-201239 [Patent Document 4] Japanese Patent Application Laid-Open No. 2008-290972 [Non-patent literature]

[0005] [Non-Patent Document 1] Takeharu Sato et al., Journal of the Japanese Society of Nutrition and Food Science, Vol. 61, No. 2, 79-88 (2008) Summary of the Invention [Problem to be solved by the invention]

[0006] As mentioned above, the IgA secretion-promoting effect of several oligosaccharides has already been reported, but these reports only confirmed short-term effects in young animals. The present invention was made against this background, and aims to provide a substance that has a long-term IgA secretion-promoting effect in relatively elderly animals. [Means for solving the problem]

[0007] As a result of extensive research to solve the above problems, the inventors discovered that LNB has the effect of promoting IgA secretion even in relatively elderly animals, and that this effect persists for a long period of time. Based on these findings, the present invention was completed.

[0008] That is, the present invention provides the following (1) to (3). (1) An IgA secretion promoter characterized by containing LNB as an active ingredient.

[0009] (2) A food or drink for stimulating mucosal immunity, comprising the IgA secretion promoter according to (1).

[0010] (3) A mucosal immunity-stimulating drug comprising the IgA secretion promoter described in (1). [Effects of the Invention]

[0011] The present invention provides a novel IgA secretion enhancer, which is expected to have preventive effects against infectious diseases, allergic diseases, and the like by promoting IgA secretion and enhancing mucosal immune function. [Brief explanation of the drawings]

[0012] [Figure 1] A graph showing the change in fecal IgA levels in mice fed a crude diet (5 mice in the LNB-administered group, 6 mice in the control group). [Figure 2] A graph showing the change in fecal IgA levels in mice fed a purified diet (7 mice in the LNB-administered group, 7 mice in the control group). [Figure 3] FIG. 1 shows the results of comparing antigen-specific spleen cell proliferation (LNB-administered group: 4 mice, control group: 7 mice). [Figure 4] Figure showing the comparison of antigen-specific cytokine (IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p40, IFN-gamma) production (5 mice in the LNB-treated group, 6 mice in the control group). [Figure 5] FIG. 1 shows the effect of adding LNB to spleen cell culture on enhancing antigen-specific IgA antibody production. DETAILED DESCRIPTION OF THE INVENTION

[0013] The present invention will be described in detail below. The IgA secretion promoter of the present invention is characterized by containing LNB as an active ingredient.

[0014] The method for producing LNB is not particularly limited, and for example, the method described in Japanese Patent Application Laid-Open No. 2008-290972 can be used. Specifically, a method in which a substrate containing lactose and N-acetylglucosamine is used as a starting material and sequentially reacted with β-galactosidase derived from pig testis and β-galactosidase produced by Bacillus circulans (Japanese Patent Application Laid-Open No. 6-253878), or a method using microorganisms, animal cells, or insect cells capable of producing glycoconjugates from sugar nucleotides and glycoconjugate precursors (Japanese Patent Application Laid-Open No. 2003-189891) can also be used. Furthermore, enzymatic methods for producing LNB can also be used.

[0015] The enzymatic method for producing LNB involves the use of (i) a carbohydrate raw material in combination with an enzyme that phosphorylates the carbohydrate raw material to produce α-glucose-1-phosphate, and (ii) enzymes that convert α-glucose-1-phosphate to UDP-glucose and UDP-galactose to α-galactose-1-phosphate in combination with their cofactors in the presence of N-acetylglucosamine, phosphate, lacto-N-biose phosphorylase (EC 2.4.1.211), and UDP-glucose-4-epimerase (EC 5.1.3.2). This method involves three enzymatic reactions in the reaction system: (1) phosphorolysis of the carbohydrate raw material, (2) conversion of glucose-1-phosphate to galactose-1-phosphate, and (3) synthesis of LNB from N-acetylglucosamine. In reaction (1), glucose-1-phosphate is produced by a reaction between phosphoric acid, a carbohydrate raw material (e.g., sucrose), and an enzyme (e.g., sucrose phosphorylase) that phosphorolyzes the carbohydrate raw material to produce α-glucose-1-phosphate. In reaction (2), glucose-1-phosphate produced in reaction (1) above is converted to UDP-glucose and UDP-galactose is converted to galactose-1-phosphate by a reaction between glucose-1-phosphate produced in the system and an enzyme that converts α-glucose-1-phosphate to UDP-glucose, an enzyme that converts UDP-galactose to α-galactose-1-phosphate (e.g., UDP-glucose-hexose-1-phosphate uridylyltransferase), and UDP-glucose-4-epimerase. In the reaction (3), the galactose-1-phosphate produced in the reaction (2) above reacts with lacto-N-biose phosphorylase and N-acetylglucosamine in the system to produce the desired LNB.

[0016] The intake or administration amount of the IgA secretion promoter of the present invention can be determined appropriately depending on the age, sex, weight, severity of symptoms, and administration method of the subject.

[0017] LNB, the active ingredient of the IgA secretion promoter of the present invention, can exert its IgA secretion-promoting effect even in the absence of bacteria such as bifidobacteria. Therefore, the IgA secretion promoter of the present invention can be used as a bacteria- (preferably bifidobacteria-) non-mediated IgA secretion promoter. When used as a bifidobacteria-non-mediated IgA secretion promoter, it can effectively promote IgA secretion even in mucosa or specimens (such as the elderly or those taking antibiotics) that retain little (or no) bifidobacteria.

[0018] The IgA secretion promoter of the present invention can be incorporated into compositions such as food and drink products and pharmaceuticals together with appropriate additives, and provided as food and drink products or pharmaceuticals for stimulating mucosal immunity.

[0019] In the present invention, the term "food and drink for stimulating mucosal immunity" refers to a food and drink used for the purpose of stimulating mucosal immunity. The "mucosa" in the term "food and drink for stimulating mucosal immunity" refers to the mucosa of the digestive tract, such as the buccal mucosa, gastric mucosa, intestinal mucosa, and oral mucosa.

[0020] The food and drink products for stimulating mucosal immunity of the present invention include health foods, functional foods, foods for specified health uses, nutritional supplements, and foods for special dietary uses (such as foods for formula-fed infants or foods for the sick). Furthermore, when the food and drink products for stimulating mucosal immunity of the present invention are used for mammals other than humans, the food and drink products for stimulating mucosal immunity of the present invention also include pet food and feed. Furthermore, the food and drink products for stimulating mucosal immunity of the present invention may be described or labeled as activating mucosal immunity in accordance with the functional claims (nutritional component functional claims or health use claims) approved under legal provisions for health functional foods such as foods for specified health uses and foods with nutrient functions.

[0021] The form of the food or drink may be any form suitable for consumption, such as solid, liquid, granular, particulate, powder, capsule, cream, or paste.

[0022] Specific types of food and beverages include dairy drinks (raw milk, regular milk, concentrated milk, processed milk such as low-fat milk, skim milk, coffee milk drinks, fruit milk drinks), soft drinks, carbonated drinks, nutritional drinks, fruit drinks, and other beverages (including concentrated liquids and powders for adjusting these drinks); powdered milk such as whole milk powder, skim milk powder, and adjusted milk powder; dairy products such as yogurt (soft yogurt, hard yogurt, plain yogurt, frozen yogurt, yogurt with fruit pulp, etc.), butter, cheese, and condensed milk; frozen desserts such as ice cream, ice sorbet, and shaved ice; noodles such as soba, udon, vermicelli, gyoza wrappers, Chinese noodles, and instant noodles; candy, chocolate, and other confectionery products. Examples include, but are not limited to, baked goods such as gum, candy, gummy candy, chewing gum, caramel, chocolate, tablet candy, snacks, biscuits, and other confectioneries, jelly, jam, cream, and other confectioneries; processed seafood and livestock foods such as kamaboko, chikuwa, hamburger steak, ham, and sausage; oils and fats and oil-processed foods such as margarine, mayonnaise, shortening, whipped cream, and dressing; condiments such as soy sauce, sauce, vinegar, and mirin; and breads such as white bread and sweet buns.

[0023] The mucosal immunity-stimulating food and drink of the present invention may be appropriately blended with commonly used additives depending on the type of food and drink, including, for example, sweeteners such as sugar, fructose, isomerized liquid sugar, glucose, aspartame, stevia, etc., acidulants such as citric acid, malic acid, tartaric acid, etc., excipients such as dextrin and starch, binders, diluents, flavorings, colorants, buffers, thickeners, gelling agents, stabilizers, preservatives, emulsifiers, dispersants, suspending agents, antiseptics, etc.

[0024] The amount of LNB to be incorporated in the mucosal immunity-stimulating food and beverage of the present invention may be any amount that can exert mucosal immunity-stimulating effects by promoting IgA secretion, and may be appropriately determined taking into consideration the general intake amount of the target food and beverage, the form of the food and beverage, efficacy and effects, taste, palatability, cost, etc.

[0025] In the present invention, a mucosal immunity-stimulating drug refers to a drug used for the purpose of activating mucosal immunity. The "mucosa" in a mucosal immunity-stimulating drug refers to mucosa of the digestive tract, such as the buccal mucosa, gastric mucosa, intestinal mucosa, and oral mucosa, as well as olfactory epithelium, endometrium, etc.

[0026] The mucosal immunity-stimulating pharmaceutical of the present invention can be administered orally or parenterally to act on the mucosa. Formulations suitable for oral administration include tablets (including orally disintegrating tablets, chewable tablets, effervescent tablets, lozenges, and jelly drops), pills, granules, fine granules, powders, hard capsules, soft capsules, dry syrups, liquids (including drinks, suspensions, and syrups), and jellies. They can also be formulated for parenteral administration, such as injections, patches, lotions, and creams. The above-mentioned formulations can be prepared according to known methods using LNB with pharmaceutically acceptable bases and carriers, as well as additives (e.g., excipients, diluents, binders, lubricants, disintegrants or disintegration aids, solubilizers, stabilizers, preservatives, antiseptics, bulking agents, dispersants, lubricants, humectants, buffers, flavors, etc.) appropriately selected depending on the dosage form.

[0027] The mucosal immunity-stimulating pharmaceutical of the present invention can be administered orally or parenterally to mammals such as humans, mice, rats, rabbits, dogs, cats, etc. The amount of LNB contained in the mucosal immunity-stimulating pharmaceutical of the present invention, i.e., the daily dose, can be appropriately determined depending on the age and sex of the subject, the form of the pharmaceutical, the expected effect, etc. [Example]

[0028] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0029] The effects of LNB intake on intestinal and systemic immune responses were evaluated by fecal IgA levels and antigen-specific immune responses in primary cultures of spleen cells.

[0030] Example 1: LNB administration test while rearing on crudely purified diet Feces were collected from DO11.10 female mice (16-17 weeks old) fed an NMF diet (Oriental Yeast Co., Ltd.). Fecal IgA levels were measured using a commercially available mouse IgA assay kit (Betchil). Individuals with IgA levels of 800 μg / g or higher were selected. The LNB-treated group received a 2.5% (w / w) solution of LNB as drinking water, while the control group received water for 12-13 weeks. The control group consisted of six mice, and the LNB-treated group consisted of five mice. Feces were collected every two weeks, and IgA levels were measured. The results are shown in Figure 1. As shown in Figure 1, IgA levels remained higher than those of the control group from 2 to 13 weeks after LNB administration.

[0031] Example 2: LNB administration test under purified diet conditions Feces were collected from DO11.10 female mice (14–19 weeks old) fed an NMF diet (Oriental Yeast Co., Ltd.). Fecal IgA levels were measured using a commercially available mouse IgA assay kit (Betchil). Individuals with IgA levels of 800 μg / g or higher were selected and administered AIN-93M purified diet (Oriental Yeast Co., Ltd.) for 3 weeks. After this, the LNB-treated group received a 2.5% (w / w) aqueous solution, while the control group received water for 12–13 weeks. Each group consisted of seven mice. Feces were collected every two weeks, and IgA levels were measured. The results are shown in Figure 2. As shown in Figure 2, IgA levels remained higher than those of the control group from 2–10 weeks after LNB administration. However, IgA levels began to decline in both the LNB-treated and control groups from the fourth week of administration.

[0032] Immediately after the administration period, spleen cells were collected from the mice and cultured. Antigen-specific immune responses were induced by the addition of ovalbumin (1.5 and 7.5 μM), and cell proliferation and cytokine production were compared. Cell proliferation was assessed using a BrdU incorporation assay kit (Roche), and cytokines were assessed using the culture supernatant with a commercially available cytokine assay kit (eBioscience). Cell proliferation was measured in wells without antigen stimulation, divided by the measured value in wells stimulated with antigen, and the results are shown as the stimulation index (SI). The results of comparing cell proliferation and cytokine production are shown in Figures 3 and 4, respectively. As shown in these figures, no clear differences were observed in spleen cell proliferation or cytokine production between the LNB-treated and control groups.

[0033] It is known that ingestion of bifidobacteria increases IFN-γ and suppresses IL-4, which are antigen-specifically produced by the spleen (E. Neau et al., Appl Environ Microbiol Vol.82(6) Page. 1722 (2016)). However, as mentioned above, no clear difference in cytokine production was observed between the LNB-treated and control groups. Therefore, these results suggest that the increase in IgA secretion due to LNB administration is not mediated by bifidobacteria.

[0034] Example 3: Test of adding LNB to in vitro spleen cell culture Spleen cells were collected from DO11.10 female mice (7 weeks old) fed an NMF diet (Oriental Yeast Co., Ltd.) using standard methods, and an antigen-specific immune response was induced by adding the antigen ovalbumin (1.0 μM). Simultaneously, LNB (12.5 and 25 μM) was added to the culture medium for one week, and IgA antibodies in the culture supernatant were measured using a commercially available mouse IgA assay kit (Betchil). The results are shown in Figure 5. As shown in Figure 5, the addition of LNB significantly increased the amount of IgA in the culture supernatant. Because spleen cells do not come into direct contact with intestinal bacteria, this result suggests that the increase in IgA levels caused by LNB is not mediated by intestinal bacteria. [Industrial Applicability]

[0035] The present invention can be used in industries related to food and beverages and pharmaceuticals.

Claims

1. A non-bacterial immunoglobulin A secretion promoter characterized by containing lacto-N-biose I as an active ingredient, and characterized by being used for individuals who have little or no bifidobacteria.

2. The immunoglobulin A secretion enhancer according to claim 1, characterized in that the person who has little or no bifidobacteria is elderly or taking antibiotics.

3. A food or drink for promoting non-bacterial-mediated immunoglobulin A secretion, comprising the non-bacterial-mediated immunoglobulin A secretion promoter according to claim 1 or 2.

4. A pharmaceutical for promoting non-bacterial-mediated immunoglobulin A secretion, comprising the non-bacterial-mediated immunoglobulin A secretion promoter according to claim 1 or 2.

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