JP6773368B2 - Immune development promoter - Google Patents

Description

The present invention relates to an immunodevelopmental promoter.

In recent years, attention has been paid to the relationship between gut flora and the immune system, and research is being conducted on the improvement of intestinal flora and immune regulation by probiotics and prebiotics.
Regarding probiotics, it has been reported that Bifidobacterium breve and Lactobacillus rhamnosus are effective for inflammation in model mice of chronic asthma (Non-Patent Document 1). .. In addition, it has been reported that Bifidobacterium longum ATCC BAA-999 and / or Bifidobacterium Breve LMG23729 can prevent allergic diseases in infants (Patent Document 1).

Regarding prebiotics, it has been reported that administration of a mixture of galactooligosaccharide and inulin to the mother of a mouse prevents food allergies in the fetus and promotes immune tolerance (Non-Patent Document 2). In addition, it has been reported that indigestible oligosaccharides have an immunomodulatory effect on human monocyte-derived dendritic cells (Non-Patent Document 3). Further, a bifidobacteria growth promoting composition (Patent Document 2) containing lactulose, fructooligosaccharide and / or galactooligosaccharide, and raffinose as active ingredients is known.

The combination of probiotics and prebiotics, Bifidobacterium breve and ScFOS (short chain fructooligosaccharides), LcFOS (long chain fructooligosaccharides), and A O S (pectin-derived acidic -oligosaccharides: Pectin derived acid It has been reported that indigestible oligosaccharides such as oligosaccharides suppress airway inflammation in model mice of chronic asthma (Non-Patent Document 4). It has also been reported that Bifidobacterium breve M-16V and galactooligosaccharides or fructooligosaccharides increase the mRNA of Foxp3 in the mesenteric lymph nodes (Non-Patent Document 5).

However, it is not known that the combination of Bifidobacterium breve with lactulose, raffinose, and galactooligosaccharide promotes immune development.

Japanese Unexamined Patent Publication No. 2014-0565669 Japanese Unexamined Patent Publication No. 10-175867

Sager, S. et al., Respiratory Research, 15:46, 2014 Bouchaud, G. et al., Allergy, 71 (1): 68-76, 2016 Lehmann, S. et al., PLOS ONE, DOI: 10.1371 / journal.pone.0132304, July 6, 2015 Sager, S. et al., Biochim. Biophys. Acta, 1842: 573-583, 2014 de Kivit, S. et al., Allergy, 67: 343-352, 2012

An object of the present invention is to provide a technique for promoting immune development.

The present inventors have found that ingestion of bifidobacteria breve and lactulose, raffinose, and galactooligosaccharide in mammals can promote immune development, and have completed the present invention.

That is, the present invention provides a bacterium belonging to Bifidobacterium breve and an immunodevelopment promoter containing lactulose, raffinose, and galactooligosaccharide.
The immunodevelopment-promoting agent preferably has an immunodevelopment promotion of promoting an increase in the number of T cells.
Further, the immunodevelopment promoter preferably has the bacterium Bifidobacterium Breve M-16V (LMG23729) as a preferred embodiment.
Further, the immunodevelopment promoter preferably has a weight ratio of lactulose, raffinose, and galactooligosaccharide of 1 to 9: 1 to 9: 1-9.
Further, the immunodevelopment promoter preferably has an amount of the bacteria of 1 × 10 ⁶ to 1 × 10 ¹² cfu with respect to 1 g of the total amount of lactulose, raffinose, and galactooligosaccharide.

The present invention also provides a feed containing the immunodevelopmental promoter.
The present invention also provides a food or drink composition for promoting immune development, which comprises a bacterium belonging to Bifidobacterium breve and lactulose, raffinose, and galactooligosaccharide as active ingredients.
Further, the food or drink composition preferably has a food or drink composition as a food with health claims.

Hereinafter, the present invention will be described in detail.
The immunodevelopmental promoter of the present invention includes bacteria belonging to Bifidobacterium breve and three types of oligosaccharides, lactulose, raffinose, and galactooligosaccharide.

Bacteria belonging to Bifidobacterium breve are not particularly limited as long as they can promote immune development when ingested by mammals together with lactulose, raffinose, and galactooligosaccharide. For example, Bifidobacterium. Breve M-16V (LMG23729) and the like can be mentioned. The bacterium is a Belgian conservation agency, the Belgian Coordinated Collections of Microorganisims (BCCM) (http://bccm.belspo.be/, Universiteit Gent, Laboratorium voor Microbiologie, KL Ledeganckstraat, It is stored as a public deposit in 35 9000 Gent Belgium) and can be obtained from the same institution under the strain number of LMG23729. Bifidobacterium breve M-16V is not limited to the above-mentioned deposit bacterium, and may be a bacterium substantially equivalent to the above-mentioned deposit bacterium. Bacteria substantially equivalent to the above-mentioned deposited bacteria are bacteria belonging to Bifidobacterium breve, and can promote immune development when ingested by mammals together with lactulose, raffinose, and galactooligosaccharide. Further, the base sequence of the 16SrRNA gene has a homology of preferably 99.86% or more, more preferably 99.93% or more, and more preferably 100% with respect to the base sequence of the 16SrRNA gene of the depositary bacterium. However, it is preferably a bacterium having the same bacteriological properties as the above-mentioned deposited bacterium. In addition, Bifidobacterium Breve M-16V also includes mutant strains and transgenic strains having the same bacterium as a parent strain.

The bacterium belonging to Bifidobacterium breve may be a bacterial cell or a culture containing the bacterial cell. The bacterium may be a live bacterium or a dead bacterium, and may be both a live bacterium and a dead bacterium, but a live bacterium is preferable. In addition, various additional operations such as freeze-drying can be performed after culturing as long as the effects of the present invention are not impaired. The additional operation preferably has a high viability of viable bacteria.

Bacteria belonging to Bifidobacterium breve can be easily grown by culturing the bacterium. The method for culturing is not particularly limited as long as the bacterium can grow, and a method usually used for culturing a bacterium belonging to the genus Bifidobacterium (Bifidobacterium) can be appropriately modified and used as necessary. For example, the culture temperature may be 25 to 50 ° C, preferably 30 to 40 ° C. In addition, the culture is preferably carried out under anaerobic conditions, and for example, the culture can be performed while aerating an anaerobic gas such as carbon dioxide. Further, the cells may be cultured under microaerobic conditions such as liquid static culture.

The medium used for culturing is not particularly limited, and a medium usually used for culturing Bifidobacterium spp. Can be appropriately modified and used as necessary. That is, as the carbon source, for example, sugars such as galactose, glucose, fructose, mannose, cellobiose, maltose, lactose, sucrose, trehalose, starch, starch hydrolysate, and waste sugar honey can be used depending on the assimilation property. As the nitrogen source, for example, ammonium salts such as ammonia, ammonium sulfate, ammonium chloride, and ammonium nitrate, and nitrates can be used. Further, as the inorganic salts, for example, sodium chloride, potassium chloride, potassium phosphate, magnesium sulfate, calcium chloride, calcium nitrate, manganese chloride, ferrous sulfate and the like can be used. In addition, organic components such as peptone, soybean flour, defatted soybean meal, meat extract, and yeast extract may be used. Further, as the prepared medium, for example, MRS medium can be preferably used.

Lactulose is a disaccharide consisting of fructose and galactose (4-O-β-D-galactopyranosyl-D-fructose, Gal β1-4 Fru), and is a known method, for example, JP-A-3-169888. And, it can be produced by the method described in JP-A-6-228179. Further, as the lactulose, a commercially available product (for example, manufactured by Morinaga Milk Industry Co., Ltd.) can be used.

Raffinose is a trisaccharide (β-D-fructose furanosyl-α-D-galactopyranosyl- (1-6) -α-D-glucopyranoside, Gal α1) in which fructose, galactose, and glucose are bound one by one. -6 Glc α1-2β Fru), which is described in a known method, for example, "Food New Material Effective Utilization Technology Series No. 6," Raffinose ", Page 2, Japan Confectionery Research Center, 1996". It can be manufactured by the method. As raffinose, a commercially available product (for example, manufactured by Nippon Beet Sugar Mfg. Co., Ltd.) can also be used.

Galactooligosaccharide (GOS) is an oligosaccharide having a structure represented by Gal- (Gal) n-Glc (n is 1-3, β-1,4 bond or β-1,6 bond) or a mixture thereof. .. Galactooligosaccharides are industrially produced from lactose by a rearrangement reaction with β-galactosidase, and the main component is 4'-galactosyl lactose (4'-GL), which is a trisaccharide in which one galactose is bound to the non-reducing end of lactose. ). As the galactooligosaccharide, a commercially available product (for example, manufactured by Yakult Pharmaceutical Co., Ltd.) can also be used. The galactooligosaccharide may be one kind or a mixture of two or more kinds.

Ingestion of bacteria belonging to Bifidobacterium breve and lactulose, raffinose, and galactooligosaccharides in mammals can promote immune development.

Bacteria belonging to Bifidobacterium breve, and compositions containing lactulose, raffinose, and galactooligosaccharides can be widely used as pharmaceuticals, foods and drinks, and feeds. For example, a medicine for promoting immune development, a food or drink for promoting immune development, a food or drink composition for promoting immune development, a food for health function for promoting immune development, or a feed for promoting immune development can be provided.

The agent of the present invention is not particularly limited as long as it contains a bacterium belonging to Bifidobacterium breve, and lactulose, raffinose, and galactooligosaccharide. As the agent of the present invention, bacteria belonging to Bifidobacterium breve, as well as lactulose, raffinose, and galactooligosaccharide may be used as they are, and a physiologically acceptable liquid or solid preparation carrier is blended and formulated. You may use it. Bacteria belonging to Bifidobacterium breve and lactulose, raffinose, and galactooligosaccharide may be formulated as a single agent, or may be separately formulated as two or three or more agents.

The formulation form of the agent of the present invention is not particularly limited, and tablets (including sugar-coated tablets, enteric coated tablets, and buccal tablets), powders, capsules (including enteric capsules and soft capsules), and granules (coated ones). ), Rounds, troches, encapsulated liposomes, liquids, or pharmaceutically acceptable sustained-release preparations thereof and the like can be exemplified. In formulation, carriers, excipients, binders, disintegrants, lubricants, stabilizers, flavoring agents, diluents, surfactants, solvents, etc., which are commonly used as formulation ingredients in ordinary oral drugs, are used. Additives can be used. In addition, as long as the effect of the present invention is not impaired, bacteria belonging to Bifidobacterium breve, as well as lactulose, raffinose, and galactooligosaccharide, and known or future drugs having an immunodevelopmental promoting action, or It may be used in combination with a pharmaceutical composition. The pharmaceutical composition to be used in combination may be contained as one of the active ingredients in the drug of the present invention, or may be commercialized by combining them as separate drugs without being contained in the drug of the present invention.

As the carrier and excipient used in the above preparation, lactose, glucose, sucrose, mannitol, horse bell starch, corn starch, calcium carbonate, calcium phosphate, calcium sulfate, crystalline cellulose, kanzo powder, gentiana powder and the like are used as binders. For example, starch, gelatin, syrup, polyvinyl alcohol, polyvinyl ether, polyvinylpyrrolidone, hydroxypropyl cellulose, ethyl cellulose, methyl cellulose, carboxymethyl cellulose and the like can be exemplified.

Examples of the disintegrant include starch, agar, gelatin powder, sodium carboxymethyl cellulose, calcium carboxymethyl cellulose, crystalline cellulose, calcium carbonate, sodium hydrogen carbonate, sodium alginate and the like.

Further, as a lubricant, magnesium stearate, hydrogenated vegetable oil, polyethylene glycol, etc. are used, and as a colorant, Red No. 2, Yellow No. 4, Blue No. 1, etc., which are allowed to be added to pharmaceutical products, are used. It can be exemplified.

Tablets and granules include sucrose, hydroxypropyl cellulose, purified cellac, gelatin, sorbitol, glycerin, ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, polyvinylpyrrolidone, phthalate cellulose acetate, hydroxypropyl methyl cellulose phthalate, methyl methacrylate, as required. It can also be coated with a methacrylic acid polymer or the like.

Another aspect of the present invention is the use of bacteria belonging to Bifidobacterium breve, as well as lactulose, raffinose, and galactooligosaccharides in the manufacture of a medicament for promoting immune development. In addition, another aspect of the present invention is a bacterium belonging to Bifidobacterium breve used for promoting immune development, and lactulose, raffinose, and galactooligosaccharide. In addition, another aspect of the present invention is a method for promoting immune development, in which a bacterium belonging to Bifidobacterium breve and lactulose, raffinose, and galactooligosaccharide, or a drug of the present invention are administered to a mammal. In addition, another aspect of the present invention is the prevention or treatment of a disease that can be prevented or treated by promoting immune development, in which a bacterium belonging to Bifidobacterium breve and lactulose, raffinose, and galactooligosaccharide are administered to a mammal. The method. Examples of mammals include humans, cows, sheep, goats, pigs, dogs, cats, horses and the like.

The amount of bacteria belonging to Bifidobacterium breve contained in the agent of the present invention is appropriately set according to the dosage form, usage, age, sex, type of disease, degree of disease, and other conditions. However, it is usually preferably in the range of 1 × 10 ⁶ to 1 × 10 ¹² cfu / g or 1 × 10 ⁶ to 1 × 10 ¹² cfu / ml, and 1 × 10 ⁷ to 1 × 10 ¹¹ cfu / g. Alternatively, it is more preferably in the range of 1 × 10 ⁷ to 1 × 10 ¹¹ cfu / ml. If the bacterium is dead, cfu / g or cfu / ml can be replaced with individual cells / g or individual cells / ml. “Cfu” represents a colony forming unit.

The amount of bacteria belonging to Bifidobacterium breve is 1 × 10 ⁶ to 1 × 10 ¹² cfu, preferably 1 × 10 ⁷ to 1 × 10 per 1 g of the total amount of lactulose, raffinose, and galactooligosaccharide. ^It is preferably ¹² cfu, more preferably 1 × 10 ⁸ to 1 × 10 ¹² cfu.
The weight ratio of lactulose, raffinose, and galactooligosaccharide is 1 to 9: 1 to 9: 1 to 9, preferably 2 to 8: 2 to 8: 2 to 8, and more preferably 3 to 7: 3 to 7. : It is preferably 3 to 7.

The agents of the present invention are useful in promoting immune development in mammals. Immunity develops as the animal grows after birth. Promoting immune development includes both accelerating the development of such immunity and increasing the degree of immune development. In addition, promotion of immune development includes promotion of increase in the number of T cells.

After birth and growth of an animal, organs and tissues such as the large intestine and mesenteric lymph nodes become larger, and the number of lymphocytes such as T cells also increases. Among T cells, Foxp3-positive cells, that is, regulatory T cells (Treg), which is one of the helper T cells that suppress an excessive immune response, increase in number with growth and occupy T cells. The proportion also tends to increase. The agent of the present invention can accelerate the increase in the number of such regulatory T cells, or promote the increase in the number of regulatory T cells. Therefore, in the present specification, "promotion of immune development" can be replaced with "increase in Foxp3-positive cells" or "increase in regulatory T cells".

In addition, RORgt-positive cells, that is, Th17 cells, which are one of the inflammation-inducing helper T cells characterized by high IL-17 production, decrease in proportion in T cells as they grow after birth, but T As the number of cells increases, the number of Th17 cells tends to increase. The agent of the present invention can accelerate the increase in the number of such Th17 cells, or promote the increase in the number of Th17 cells. Therefore, in the present specification, "immunity development promotion" can be replaced with "RORgt-positive cell increase" or "Th17 cell increase". Also, "promoting immune development" can be replaced with "increase in at least one of Foxp3-positive cells, regulatory T cells, RORgt-positive cells, and Th17 cells."

The present invention is capable of increasing regulatory T cells, such as Foxp3-positive cells, that suppress an excessive immune response during immune development. Therefore, diseases for which the agent of the present invention is effective, or diseases or symptoms that can be prevented or treated by promoting immune development include autoimmune diseases, allergic diseases, rejection reactions, infectious diseases, diseases derived from adipose tissue inflammation, and the like. Diseases caused by such excessive immune response can be mentioned.

Autoimmune diseases include dermatitis due to strong skin disease, sarcoidosis, atherosclerosis, disseminated intravascular coagulation syndrome, Kawasaki disease, Graves disease (Basedou disease), nephrosis syndrome, chronic fatigue syndrome, Wegener's granulomatosis, and Henoch.・ Shaneline purpura, microangiitis in the kidney, chronic active hepatitis, vasculitis, septic shock, toxin shock syndrome, blood loss syndrome, malaise, AIDS (acquired immunodeficiency syndrome), acute crossing Sexual myelitis, Huntington butoh disease, Parkinson's disease, Alzheimer's disease, stroke, primary biliary liver cirrhosis, hemolytic anemia, polyglandular dysfunction syndrome type 1, polyglandular dysfunction syndrome type 2, Schmidt syndrome, adults ( Acute) respiratory distress syndrome, alopecia, circular alopecia, serum reaction negative arthropathy, arthritis, Reiter's disease, psoriatic arthritis, chlamydia infection, arthritis related to Elsina salmonella infection, arteriosclerosis, autoimmune blisters Sexual disorders, vulgaris vulgaris, foliate vesicles, varicella, linear IgA disease, autoimmune hemolytic anemia, Coombs test positive hemolytic anemia, acquired malignant anemia, juvenile malignant anemia, myelitis Royal Free disease, chronic mucosal candidiasis, giant cell arteritis, primary sclerosing hepatitis, unexplained autoimmune hepatitis, acquired immunodeficiency-related diseases, hepatitis C, unclassifiable immunodeficiency (unclassifiable type) Due to hypogamma globulinemia), dilated cardiomyopathy, pulmonary fibrosis, fibrotic alveolar inflammation of unknown cause, interstitial pneumonia, interstitial lung disease associated with mixed connective tissue disease, systemic sclerosis Interstitial lung disease, rheumatoid arthritis interstitial lung disease, systemic erythematosus lung disease, dermatitis, lung disease associated with polymyositis, Schegren's disease lung disease, tonic spondylitis-related lung disease, pandemic Pulmonary disease due to vasculitis, lung disease due to hemodiderin deposition, drug-induced interstitial lung disease, radiation fibrosis, obstructive bronchitis, chronic eosinophilic pneumonia, lymphocyte-invasive lung disease, gouty Artimmune, autoimmune hepatitis, type 1 autoimmune hepatitis (classical autoimmune or rupoid hepatitis), type 2 autoimmune hepatitis anti (anti-LKM1 antibody positive hepatitis), autoimmune hypoglycemia, insulin due to melanoma In receptor dysfunction type B, hypothyroidism, osteoarthritis, primary sclerosing cholangitis, idiopathic leukopenia, autoimmune neutrophilia, renal disease NOS, glomerulonephritis, kidney Microangiitis, discoid erythematosus, idiopathic male infertility NOS, sperm-related autoimmune disease, sympathetic ophthalmitis, binding due to pulmonary hypertension Tissue disease, Good Pasture syndrome, Pulmonary symptoms of nodular polyarteritis, Acute rheumatic fever, rheumatic spondylitis, Still's disease, systemic sclerosis, high anxiety, arteritis, autoimmune thrombocytopenia, idiopathic thrombocytopenia Disease, autoimmune thyroid disease, hyperthyroidism, thyroidoma / hypothyroidism (Hashimoto disease), atrophic autoimmune hypothyroidism, primary mucinous edema, crystalline dysplasia, primary blood vessels Examples include flames, white spots, and skin erythroides. NOS indicates "not classified elsewhere".

Allergic diseases include atopic dermatitis, atopic allergy, food allergy, pollen allergy, allergic rhinitis (pollen allergy), anaphylaxis, pet allergy, latex allergy, drug allergy, allergic rhinitis conjunctivitis, eosinophilic esophagitis. , Eosinophilic syndrome, eosinophilic gastroenteritis and the like.

Examples of the rejection include graft-versus-host rejection and the like.

Infectious diseases include salmonella, erythema, crustridium difficile, mycobacteria (tuberculosis as a disease), protozoa (malaria as a disease), filamentous nematodes (filariasis as a disease), and blood-sucking insects (as a disease). Hematopoietic disease), Toxoplasma (Toxoplasmosis as a disease), Leishmania (Leashmania as a disease), HCV and HBV (Hepatitis C and Hepatitis B as a disease), Simple herpesvirus (herpes as a disease) Infectious diseases caused by such as.

Diseases derived from adipose tissue inflammation include metabolic syndrome, visceral obesity, insulin resistance, hyperglycemia, dyslipidemia, hypertriglycerideemia, low HDL cholesterolemia, elevated blood pressure, arteriosclerotic disease, type 2 diabetes, Examples thereof include non-alcoholic steatohepatitis (NAFLD) and non-alcoholic steatohepatitis (NASH).

The dose of the drug of the present invention can be appropriately selected depending on the age, sex, condition, other conditions, etc. of the administration subject. The amount of bacteria belonging to Bifidobacterium breve is preferably in the range of 2 × 10 ⁴ to 2 × 10 ⁹ cfu / kg / day, more preferably 2 × 10 ⁶ to 2 × 10 ⁹ cfu / kg / day. It is good to use the amount that becomes as a guide.

The administration time of the drug of the present invention is not particularly limited, and it can be appropriately selected according to the condition of the administration target. In addition, it may be administered prophylactically or may be used for maintenance therapy. In addition, it is preferable that the administration form is determined according to the formulation form, the age, sex, other conditions of the patient, the symptom of the patient and the degree thereof.

In any case, the drug of the present invention can be administered once a day or in multiple divided doses, or may be administered once every few days or weeks.

The agent of the present invention, or a bacterium belonging to Bifidobacterium breve, which is an active ingredient thereof, and lactulose, raffinose, and galactooligosaccharide can also be contained in foods and drinks (beverages or foods).

In addition, bacteria belonging to Bifidobacterium breve, lactulose, raffinose, and galactooligosaccharide, or the drug of the present invention are contained in foods and drinks as active ingredients, and an immunodevelopment-promoting action is exhibited as one aspect of the immunodevelopment-promoting agent. It is also possible to process it as a food or drink to have. That is, the present invention provides a food or drink composition for promoting immune development, which comprises a bacterium belonging to Bifidobacterium breve, and lactulose, raffinose, and galactooligosaccharide as active ingredients.

Foods and drinks are not particularly limited in morphology and properties as long as they do not impair the effect of promoting immune development and can be ingested orally, and contain bacteria belonging to Bifidobacterium breve, lactulose, raffinose, and galactooligosaccharides. Other than the above, it can be produced by a usual method using raw materials usually used for foods and drinks.

Foods and drinks such as those described above may be liquid, pasty, gel-like solids, powders, etc., in addition to tablet confectionery, liquid foods, etc., for example, bread, macaroni, spaghetti, noodles, cake mix, and fried foods. Wheat flour products such as flour and bread flour; instant noodles, cup noodles, retort / cooked foods, canned foods, microwave foods, instant soups / stews, instant miso soup / soups, canned soups, freeze / dry foods, and other instant foods. Foods: Canned agricultural products, canned fruits, jams and marmalades, pickles, boiled beans, dried agricultural products, processed agricultural products such as cereals (processed grain products); canned fishery products, fish hams and sausages, fish paste products, fishery delicacies, Processed marine products such as boiled tsukuda; processed livestock products such as canned livestock / pastes, livestock ham / sausage; processed milk, milk drinks, yogurts, lactic acid bacteria drinks, cheese, ice creams, prepared milk powder, creams, etc. Milk and dairy products such as dairy products; oils and fats such as butter, margarines, vegetable oils; basic seasonings such as soy sauce, miso, sauces, tomato processed seasonings, mirins, vinegars, etc .; cooking mix, curry ingredients Complex seasonings / foods such as kind, sauces, dressings, noodle soups, spices, and other complex seasonings; frozen foods such as raw material frozen foods, semi-cooked frozen foods, and cooked frozen foods; caramel, candy, Confectionery such as chewing gum, chocolate, cookies, biscuits, cakes, pies, snacks, crackers, Japanese sweets, rice sweets, bean sweets, dessert sweets, jelly, and other sweets; carbonated drinks, natural fruit juices, fruit juice drinks, soft drinks with fruit juice , Fruit meat drinks, fruit drinks with fruits, vegetable drinks, soy milk, soy milk drinks, coffee drinks, tea drinks, powder drinks, concentrated drinks, sports drinks, nutritional drinks, alcoholic drinks, and other favorite drinks, Baby foods, sprinkles, other commercial foods such as tea mashing paste, etc .; Prepared milk powder for childcare; Enteric nutritional foods; Special purpose foods, health functional foods (specified health foods, nutritional functional foods, functional foods); Examples include nutritional supplements.

The food and drink of the present invention can be sold as a food and drink labeled for use in promoting immune development. In addition, such foods and drinks can be labeled as "for promoting immune development". In addition to this, it goes without saying that any wording that expresses the secondary effect of promoting immune development can be used.

The "display" means all actions for informing the consumer of the above-mentioned use, and if the display can remind or infer the above-mentioned use, the purpose of the display, the content of the display, and the display. Regardless of the object, medium, etc., all fall under the "indication" of the present invention. However, it is preferable to display it in an expression that allows the consumer to directly recognize the above application.
Specifically, the act of describing the above-mentioned use on the product or product packaging related to the food or drink of the present invention, or transferring the product or product packaging containing the above-mentioned use, and displaying it for delivery, transfer or delivery. However, the act of importing, advertisements related to products, price lists or transaction documents, the above-mentioned uses are described and displayed or distributed, or the above-mentioned uses are described in the information containing these, and the electromagnetic (Internet, etc.) method The act of providing the product can be illustrated, and it is particularly preferable to display it on advertising materials such as packaging, containers, catalogs, pamphlets, POPs, and other documents.

In addition, as the display, it is preferable that the display is permitted by the government or the like (for example, a display obtained based on various systems established by the government and performed in a manner based on such approval). For example, labeling as health foods, functional foods, enteric nutritional foods, special-purpose foods, nutritionally functional foods, pharmaceutical non-standard products, etc. can be exemplified, and other labeling approved by the Consumer Affairs Agency, for example, Examples of foods with functional health, more specifically foods for specified health use, foods with nutritional function, foods with functional claims, and labeling approved by a similar system can be exemplified. Examples of the latter include labeling as a food for specified health use, labeling as a food for specified health use as a condition, labeling to the effect that it affects the structure and function of the body, disease risk reduction labeling, and functionality based on scientific evidence. Can be illustrated. More specifically, as a food for specified health use specified in the Cabinet Office Ordinance (Cabinet Office Ordinance No. 57, August 31, 2001) regarding permission for special use labeling prescribed in the Health Promotion Law. (In particular, indications for health uses) and similar indications can be exemplified.

Bacteria belonging to Bifidobacterium breve, as well as lactulose, raffinose, and galactooligosaccharide may be contained in the feed as active ingredients and processed as a feed having an immunodevelopment promoting action as one aspect of an immunodevelopment promoting agent. Is.

The form of the feed is not particularly limited, and for example, cereals such as corn, wheat, barley, rye, and mylo; vegetable oil meals such as soybean oil meal, rapeseed oil cake, palm oil meal, and flaxseed oil meal; bran, wheat bran, rice bran, etc. Bran such as defatted rice bran; production cereals such as corn gluten meal and corn jam meal; animal feeds such as fish meal, defatted milk powder, whey, yellow grease and tallow; yeasts such as tolla yeast and beer yeast; It can be produced by blending mineral feeds such as calcium phosphate and calcium carbonate; fats and oils; simple amino acids; sugars and the like. Examples of the form of feed include pet food, livestock feed, fish feed and the like.

The amounts of the bacteria belonging to Bifidobacterium breve and lactulose, raffinose, and galactooligosaccharide contained in the food and drink (including feed) of the present invention are not particularly limited and may be appropriately selected, but preferably. It is the same as the above-mentioned agent of the present invention.

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

[Example 1] Ratio of Foxp3-positive cells and RORgt-positive cells among CD4-positive cells in mouse colonic lamina propria lymphocytes and mesenteric lymph node lymphocytes, and age-related changes in the number of cells C57BL / 2 days old I purchased a 6J male mouse with a mother beast from Japan SLC. Infant mice were fed the mother's milk freely. Infant mice were divided into two groups so that the weight was not biased. One group was 14 days old and the other group was 21 days old. The large intestine and mesenteric lymph nodes were dissected. node. It may be referred to as "MLN" below.) Was collected.

The resulting colon sample was cut vertically, transferred to DPBS (Dulbecco PBS) containing 5 mM EDTA, 1 mM DTT, and 2% fetal bovine serum (FCS), and shaken in a constant temperature bath at 37 ° C. for 30 minutes. The sample was then passed through a 70 μm mesh to remove fluid containing epithelial cells. Remaining colon samples containing 0.5 mg / ml collagenase (Sigma), 25 μg / ml DNase I (Roche Diagnostics), 50 μg / ml digest (Gibco), 0.01 M HEPES, and 2% FCS. Placed in RPMI 1640 medium and digested at 37 ° C. for 30 minutes. The solution after digestion was filtered through a 70 μm mesh, and the obtained filtrate was centrifuged at 340 × g at 4 ° C. for 5 minutes to obtain a cell suspension. The obtained cell suspension and Percoll solution were mixed to obtain a 40% Percoll cell suspension, which was layered on top of the 90% Percoll solution. Centrifuge at 720 x g at room temperature for 25 minutes to collect lamina propria lymphocytes (hereinafter sometimes referred to as "LPL") present at the boundary between 40% Percoll solution and 90% Percoll solution. did.

Mesenteric lymph node lymphocytes (MLN lymphocytes, hereinafter sometimes referred to as "MLNL") were ground on a 70 μm mesh to obtain a cell suspension that passed through the mesh.

The obtained LPL and MLNL were used as FITC-labeled anti-mouse CD4 antibody (clone number: RM4-5, manufactured by BD Biosciences), APC (alophycocyanin) -labeled anti-rat / mouse Foxp3 (clone number: FJK-16a, e). -Staining with PE (Phycoerythrin) labeled anti-human / mouse RORgt (clone number: AFKJS-9, e-Bioscience), Foxp3 Staining Buffer set (e-Bioscience) , FACSCanto flow cytometer (manufactured by BD Biosciences) was used to analyze cells, and FlowJo software (manufactured by TreeStar) was used to analyze the data.

The ratio (mean value, unit:%) of Foxp3-positive cells and RORgt-positive cells among CD4-positive cells is shown in Tables 1 to 4. In colon LPL, the proportion of Foxp3-positive cells (Treg cells) in CD4-positive cells increased with increasing age, whereas in MLNL, the proportion of Foxp3-positive cells in CD4-positive cells changed with increasing age. There was no (Tables 1 and 2). The CD4 positive cells in the large intestine LPL and MLNL are almost T cells.

On the other hand, the proportion of RORgt-positive cells (Th17 cells) among CD4-positive cells decreased with increasing age (Tables 3 and 4).
The total number of cells in MLNL, Foxp3-positive cells, and RORgt-positive cells (mean value, unit: × 10 ⁶ ) are shown in Tables 5 to 7. The number of all cells increased with increasing age.

[Example 2] Effect of bifidobacteria brave and tri-oligosaccharides on the ratio of Foxp3-positive cells and RORgt-positive cells in CD4-positive cells in mouse colon LPL and MLNL Bifidobacterium brave dispersed in starch M-16V cell powder (2.4 × 10 ¹¹ cfu / g, manufactured by Morinaga Milk Industry Co., Ltd.) was suspended in physiological saline to prepare a 2.5 × 10 ⁹ cfu / ml bifidobacteria solution.

Lactulose (manufactured by Morinaga Dairy Co., Ltd.), raffinose (product name: Nitten Raffinose, manufactured by Nippon Beet Sugar Mfg. Co., Ltd.), and galactooligosaccharide were mixed and dissolved in distilled water at a weight ratio of 1: 1: 1 to a total final concentration of 250 mg / ml. An oligosaccharide solution was prepared. Galactooligosaccharide is a commercially available product (product name: Oligomate 55N, manufactured by Yakult Pharmaceutical Co., Ltd.) from which monosaccharides and disaccharides have been removed, and contains approximately 65% by weight of Galβ1-4Galβ1-4Glc (4'-galactosyllactose). It contains about 15% by weight of Galβ1-6Galβ1-4Glc (6'-galactosyllactose).

Physiological saline solution, diluted solution of the above Bifizus bacterium solution diluted 5 times with physiological saline (final concentration 5 × 10 ⁸ cfu / ml Bifizus bacterium), and Bifizus bacterium solution and oligosaccharide solution in a volume ratio of 1: 4. The mixture (final concentration 5 × 10 ⁸ cfu / ml Bifizus bacterium, 200 mg / ml oligosaccharide) was used as the administration product.

Two-day-old C57BL / 6J male mice were purchased from Japan SLC along with their mothers. Infant mice were fed the mother's milk freely. They were divided into the following three groups so that their weights would not be biased at 5 days of age.

A: Vehicle group (administered saline)
B: Bifidobacterium group (administered diluted bifidobacteria solution)
C: Oligosaccharide + bifidobacteria group (administer a mixed solution of oligosaccharide and bifidobacteria)

From 6 to 13 days of age, each group of infant mice was given saline, bifidobacteria diluted solution, or a mixture of bifidobacteria solution and oligosaccharide solution (1: 4) once daily. 100 μl was administered. That is, 5 × 10 ⁷ cfu bifidobacteria Breve M-16V for the bifidobacteria group and 20 mg oligosaccharide and 5 × 10 ⁷ cfu bifidobacteria for the oligosaccharide + bifidobacteria group per administration. -Breve M-16V was administered.

Dissection was performed at 14 days of age, and the large intestine and MLN were collected, and the ratio of Foxp3-positive cells and RORgt-positive cells in the CD4-positive cells in the large intestine LPL and MLNL was examined in the same manner as in Example 1. The results are shown in Tables 8 and 9 (ratio of Foxp3-positive cells, mean value, unit:%) and Tables 10 and 11 (ratio of RORgt-positive cells, mean value, unit:%). In the table, "M-16V" means Bifidobacterium breve M-16V, and "M-16V + oligosaccharide" means a mixture of Bifidobacterium breve M-16V and lactulose, raffinose and galactooligosaccharide. ..

As shown in Tables 8 and 9, at 14 days after birth, the mixed administration (M-16V + oligosaccharide) of bifidobacteria breve M-16V and oligosaccharide (lacturose + raffinose + galactooligosaccharide) resulted in LPL and The proportion of Foxp3-positive cells in CD4-positive cells in MLNL was increased compared to the Vehicle group (control) and the bifidobacteria group (M-16V).
On the other hand, as shown in Tables 10 and 11, at 14 days after birth, the mixed administration of M-16V and oligosaccharide did not affect the proportion of RORgt-positive cells in CD4-positive cells in LPL and MLNL.

[Example 3] Effect of Bifidobacterium breve and three oligosaccharides on the number of CD4-positive Foxp3-positive cells and RORgt-positive cells in mouse MLNL Milk to which each administration was administered in the same manner as in Example 2. The pups were dissected at 14 days of age to determine the number of CD4 + Foxp3-positive and RORgt-positive cells in MLNL. The results are shown in Tables 12 to 14 (mean value, unit: × 10 ⁶ ). At 14 days of age, mixed administration of Bifidobacterium Breve M-16V and oligosaccharides increased the total number of cells in MLNL, the number of CD4-positive Foxp3-positive cells, and the number of CD4-positive RORgt-positive cells.

The above results are schematically shown in Table 15.

The immunodevelopmental promoter of the present invention is effective in promoting the immune development of mammals. Immune development promoters are useful for the prevention and treatment of various diseases for which it is effective to promote immune development.

Claims (11)

An immune development promoter for babies containing bacteria belonging to Bifidobacterium breve, lactulose, raffinose, and galactooligosaccharides.
An immunodevelopmental promoter whose immunodevelopment promotion is at least one of Foxp3-positive cells , R ORgt-positive cells, Th17 cells, and mesenteric lymph node lymphocytes. The immunodevelopmental promoter according to claim 1, wherein the Foxp3-positive cells are Foxp3-positive regulatory T cells. The immunodevelopmental promoter according to claim 1 or 2 , wherein the baby is healthy. The immunodevelopmental promoter according to any one of claims 1 to 3, wherein the bacterium is Bifidobacterium Breve M-16V. The immunodevelopmental promoter according to any one of claims 1 to 4 , wherein the weight ratio of lactulose, raffinose, and galactooligosaccharide is 1 to 9: 1 to 9: 1-9. The immunodevelopmental promoter according to any one of claims 1 to 5 , wherein the amount of the bacterium is 1 × 10 ⁶ to 1 × 10 ¹² cfu with respect to 1 g of the total amount of lactulose, raffinose, and galactooligosaccharide. .. An immune development-promoting feed for infants containing bacteria belonging to Bifidobacterium breve, lactulose, raffinose, and galactooligosaccharides.
A feed, wherein the immune development promotion is the promotion of at least one of Foxp3-positive cells, RORgt-positive cells, Th17 cells, and mesenteric lymph node lymphocytes . The feed according to claim 7, wherein the Foxp3 positive cells are Foxp3 positive regulatory T cells. A food and drink composition for promoting immune development for infants, which comprises a bacterium belonging to Bifidobacterium breve and lactulose, raffinose, and galactooligosaccharide as active ingredients.
A food or drink composition in which the promotion of immune development promotes the increase of at least one of Foxp3-positive cells , R ORgt-positive cells, Th17 cells, and mesenteric lymph node lymphocytes. The food or drink composition according to claim 9, wherein the Foxp3 positive cells are Foxp3 positive regulatory T cells. The food or drink composition according to claim 9 or 10 , wherein the food or drink composition is a food with health claims.