WO2017145415A1

WO2017145415A1 - Immune development accelerator

Info

Publication number: WO2017145415A1
Application number: PCT/JP2016/075071
Authority: WO
Inventors: 達弥江原; 和泉　裕久; 毅松原
Original assignee: 森永乳業株式会社
Priority date: 2016-02-26
Filing date: 2016-08-26
Publication date: 2017-08-31
Also published as: PH12018501822A1; JPWO2017145415A1; JP6773368B2

Abstract

An immune development accelerator that comprises, as active ingredients, a bacterium belonging to Bifidobacterium breve such as Bifidobacterium breve M-16V (LMG23729), lactulose, raffinose and a galactooligosaccharide.

Description

Immune development promoter

The present invention relates to an immune development promoter.

In recent years, attention has been paid to the relationship between intestinal bacteria and the immune system, and research has been conducted on improvement of intestinal flora and immunoregulation by probiotics and prebiotics.
Regarding probiotics, Bifidobacterium breve and Lactobacillus rhamnosus have been reported to be effective for inflammation in chronic asthmatic model mice (Non-patent Document 1). . In addition, it is 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 when a mixture of galactooligosaccharide and inulin is administered to the mother of a mouse, food allergy in the fetus is prevented and immune tolerance is promoted (Non-patent Document 2). Indigestible oligosaccharides have been reported to have an immunomodulatory action on human monocyte-derived dendritic cells (Non-patent Document 3). Furthermore, a bifidobacteria growth promoting composition containing lactulose, fructooligosaccharide and / or galactooligosaccharide, and raffinose as active ingredients is known (Patent Document 2).

For the combination of probiotics and prebiotics, Bifidobacterium breve and scFOS (short chain fructooligosaccharides), lcFOS (long chain fructooligosaccharides), and ADS (pectin-derived acidic-oligosaccharides: pectin-derived acidic oligosaccharides) ) Have been reported to suppress airway inflammation in chronic asthma model mice (Non-patent Document 4). Bifidobacterium breve M-16V and galactooligosaccharides or fructooligosaccharides have been reported to increase Foxp3 mRNA in mesenteric lymph nodes (Non-patent Document 5).

However, it is not known that immune development is promoted by a combination of Bifidobacterium breve with lactulose, raffinose, and galactooligosaccharide.

JP 2014-065669 A Japanese Patent Laid-Open No. 10-175867

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

The present inventors have found that when a mammal is ingested with Bifidobacterium breve, lactulose, raffinose, and galactooligosaccharide, immune development can be promoted, and the present invention has been completed.

That is, the present invention provides an immune development promoter comprising bacteria belonging to Bifidobacterium breve, lactulose, raffinose, and galactooligosaccharide.
The above-mentioned immunodevelopment promoting agent is preferably characterized in that the promotion of immune development is the promotion of an increase in the number of T cells.
In addition, the above-mentioned immunity development promoter has a preferred embodiment in which the bacterium is Bifidobacterium breve M-16V (LMG23729).
In addition, the immune development promoter preferably has a weight ratio of lactulose, raffinose, and galactooligosaccharide of 1 to 9: 1 to 9: 1 to 9.
In addition, the immune development promoting agent is preferably configured such that the amount of the bacteria is 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 above-mentioned immune development promoter.
The present invention also provides a food and drink composition for promoting immune development, comprising as an active ingredient a bacterium belonging to Bifidobacterium breve, lactulose, raffinose, and galactooligosaccharide.
Moreover, the said food-drinks composition makes it a preferable aspect that a food-drinks composition is a health functional food.

Hereinafter, the present invention will be described in detail.
The immune development promoter of the present invention contains 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 together with lactulose, raffinose, and galactooligosaccharides by mammals. Brave M-16V (LMG23729) and the like. The bacteria are Belgian Coordinated Collections of microorganisms (BCCM) (http://bccm.belspo.be/, Universiteit Gent, Laboratorium voor Microbiologie, KL Ledeganckstraat, Belgian Coordinated Collections of Microorganisms 35 9000 Gent Belgium) is stored as a public deposit and can be obtained from the same organization under the strain number of LMG23729. Bifidobacterium breve M-16V is not limited to the deposited bacteria, and may be a bacterium substantially equivalent to the deposited bacteria. Bacteria substantially equivalent to the above-mentioned deposited bacteria are bacteria belonging to Bifidobacterium breve and can promote immune development when ingested together with lactulose, raffinose, and galactooligosaccharides. Furthermore, the base sequence of the 16S rRNA gene preferably has 99.86% or more, more preferably 99.93% or more, more preferably 100% homology to the base sequence of the 16S rRNA gene of the deposited bacterium. And preferably a bacterium having the same bacteriological properties as the above-mentioned deposited bacterium. In addition, Bifidobacterium breve M-16V includes mutants and genetically modified strains having the same bacterium as a parent strain.

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

Bacteria belonging to Bifidobacterium breve can be easily propagated by culturing the bacteria. The method of culturing is not particularly limited as long as the bacterium can grow, and the method usually used for culturing Bifidobacterium (Bifidobacteria) can be appropriately modified as necessary. For example, the culture temperature may be 25 to 50 ° C., preferably 30 to 40 ° C. The culture is preferably performed under anaerobic conditions, and for example, the culture can be performed while anaerobic gas such as carbon dioxide gas is passed. Moreover, you may culture | cultivate on microaerobic conditions, such as liquid stationary culture.

The medium used for the culture is not particularly limited, and a medium usually used for culture of Bifidobacterium can be appropriately modified as necessary. That is, as the carbon source, for example, saccharides such as galactose, glucose, fructose, mannose, cellobiose, maltose, lactose, sucrose, trehalose, starch, starch hydrolyzate, and molasses can be used according to the utilization. As the nitrogen source, for example, ammonium salts such as ammonia, ammonium sulfate, ammonium chloride, and ammonium nitrate, and nitrates can be used. Examples of inorganic salts that can be used include sodium chloride, potassium chloride, potassium phosphate, magnesium sulfate, calcium chloride, calcium nitrate, manganese chloride, and ferrous sulfate. Organic components such as peptone, soybean powder, defatted soybean meal, meat extract, yeast extract and the like may also be used. Moreover, as the prepared medium, for example, an MRS medium can be preferably used.

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

Raffinose is a trisaccharide (β-D-fructofuranosyl-α-D-galactopyranosyl- (1-6) -α-D-glucopyranoside, Gal α1 bound to fructose, galactose and glucose one by one. -6 Glc α1-2β Fru) and described in known methods such as “Food New Material Effective Utilization Technology Series No. 6,“ Raffinose ”, page 2, Confectionery Technology Center, 1996”. It can be manufactured by the method. For raffinose, a commercially available product (for example, manufactured by Nippon Sugar Sugar Co., Ltd.) can also be used.

Galacto-oligosaccharide (GOS) is an oligosaccharide having a structure represented by Gal- (Gal) n-Glc (n is 1 to 3, β-1,4 bond or β-1,6 bond) or a mixture thereof. . Galactooligosaccharides are industrially produced by transfer reaction with β-galactosidase using lactose as a raw material, and the main component is 4′-galactosyl lactose (4′-GL), which is a trisaccharide in which one galactose is bonded 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.

When a mammal is ingested with bacteria belonging to Bifidobacterium breve, lactulose, raffinose, and galactooligosaccharide, immune development can be promoted.

Bacteria belonging to Bifidobacterium breve and compositions containing lactulose, raffinose, and galactooligosaccharide can be widely used as medicines, foods and drinks, and feeds. For example, a drug for promoting immune development, a food and drink for promoting immune development, a food and drink composition for promoting immune development, a health functional food for promoting immune development, and a feed for promoting immune development can be provided.

The drug of the present invention is not particularly limited as long as it contains bacteria belonging to Bifidobacterium breve, and lactulose, raffinose, and galactooligosaccharide. The agent of the present invention may be a bacterium belonging to Bifidobacterium breve, as well as lactulose, raffinose, and galactooligosaccharide, and may be formulated by blending a physiologically acceptable liquid or solid pharmaceutical carrier. May be used. Bacteria belonging to Bifidobacterium breve, lactulose, raffinose, and galactooligosaccharide may be formulated as a single body, or may be separately formulated into two or three or more drugs.

The preparation form of the drug of the present invention is not particularly limited, and tablets (including sugar-coated tablets, enteric-coated tablets, buccal tablets), powders, capsules (including enteric capsules, soft capsules), granules (coated ones) ), Pills, troches, encapsulated liposomes, solutions, or pharmaceutically acceptable sustained-release preparations thereof. In formulating, carriers, excipients, binders, disintegrants, lubricants, stabilizers, flavoring agents, diluents, surfactants, solvents, etc. that are commonly used for ordinary oral drugs as pharmaceutical ingredients Additives can be used. In addition, as long as the effect of the present invention is not impaired, a bacterium belonging to Bifidobacterium breve, and lactulose, raffinose, and galactooligosaccharide, a known or future-found drug having an immune development promoting action, or You may use together 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 combined and commercialized as a separate drug without being contained in the drug of the present invention.

Carriers and excipients used in the above formulations include lactose, glucose, sucrose, mannitol, potato starch, corn starch, calcium carbonate, calcium phosphate, calcium sulfate, crystalline cellulose, licorice powder, gentian powder and the like as binders For example, starch, gelatin, syrup, polyvinyl alcohol, polyvinyl ether, polyvinyl pyrrolidone, 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., and as a colorant, red No. 2, yellow No. 4, and blue No. 1, etc. that are allowed to be added to pharmaceuticals, etc. It can be illustrated.

Tablets and granules include sucrose, hydroxypropylcellulose, purified shellac, gelatin, sorbitol, glycerin, ethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, cellulose phthalate acetate, hydroxypropylmethylcellulose phthalate, methyl methacrylate, 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, and lactulose, raffinose, and galactooligosaccharides in the manufacture of a medicament for promoting immune development. Another aspect of the present invention is a bacterium belonging to Bifidobacterium breve used for promoting immune development, and lactulose, raffinose, and galactooligosaccharide. Another aspect of the present invention is a method of promoting immune development, comprising administering a bacterium belonging to Bifidobacterium breve, lactulose, raffinose, and galactooligosaccharide, or a drug of the present invention to a mammal. Another aspect of the present invention is to prevent or treat a disease that can be prevented or treated by promoting immune development, wherein a bacterium belonging to Bifidobacterium breve and lactulose, raffinose, and galactooligosaccharide are administered to a mammal. Is the method. Mammals include humans, cows, sheep, goats, pigs, dogs, cats, horses and the like.

The amount of bacteria belonging to Bifidobacterium breve contained in the drug of the present invention is appropriately set according to the dosage form, usage, age of subject, sex, type of disease, degree of disease, and other conditions. Is preferably within the range of 1 × 10 ⁶ to 1 × 10 ¹² cfu / g or 1 × 10 ⁶ to 1 × 10 ¹² cfu / ml, preferably 1 × 10 ⁷ to 1 × 10 ¹¹ cfu / g. Or, 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, more preferably 3 to 7: 3 to 7. : It is preferably 3-7.

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

As the animal grows after birth, organs and tissues such as the large intestine and mesenteric lymph nodes increase, and the number of lymphocytes such as T cells also increases. Among T cells, Foxp3 positive cells, that is, regulatory T cells (regulatory T cells, Treg), which are one of helper T cells that suppress excessive immune responses, increase in the number of cells as they grow and occupy T cells. The ratio also tends to increase. The agent of the present invention can accelerate the increase in the number of regulatory T cells or promote the increase in the number of regulatory T cells. Therefore, in the present specification, “stimulation of immune development” can be replaced by “an increase in Foxp3 positive cells” or “an increase in regulatory T cells”.

In addition, R17 is a RORgt positive cell, ie, Th17 cell, which is one of inflammation-inducing helper T cells characterized by high production of IL-17. 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 Th17 cells or increase the number of Th17 cells. Therefore, in the present specification, “stimulation of immune development” can be replaced with “increased RORgt positive cells” or “increased Th17 cells”. “Immune development promotion” can be replaced with “an increase in at least one of Foxp3-positive cells, regulatory T cells, RORgt-positive cells, and Th17 cells”.

The present invention can increase regulatory T cells that suppress an excessive immune reaction during immune development, such as Foxp3-positive cells. Therefore, diseases for which the drug 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, infections, diseases derived from adipose tissue inflammation, etc. Diseases caused by such an excessive immune reaction can be mentioned.

Autoimmune diseases include scleroderma dermatitis, sarcoidosis, atherosclerosis, disseminated intravascular coagulation syndrome, Kawasaki disease, Graves disease (Graves' disease), nephrotic syndrome, chronic fatigue syndrome, Wegener's granulomatosis, Henoch Shaneline purpura, microscopic vasculitis in the kidney, chronic active hepatitis, uveitis, septic shock, toxic shock syndrome, septic syndrome, cachexia, AIDS (acquired immune deficiency syndrome), acute crossing Myelitis, Huntington's disease, Parkinson's disease, Alzheimer's disease, stroke, primary biliary cirrhosis, hemolytic anemia, multiglandular dysfunction syndrome type 1, multiglandular dysfunction syndrome type 2, Schmidt syndrome, adult ( Acute) respiratory distress syndrome, alopecia, alopecia areata, seronegative arthropathy, arthropathy, Reiter's disease, psoriatic arthropathy, Lamidia infection, arthropathy related to Yersinia salmonella infection, arteriosclerosis, autoimmune bullous disease, pemphigus vulgaris, decidual pemphigus, pemphigoid, linear IgA disease, autoimmune hemolytic anemia, Coombs Test positive hemolytic anemia, acquired pernicious anemia, juvenile pernicious anemia, myelomyelitis, Royal Free disease, chronic mucocutaneous candidiasis, giant cell arteritis, primary sclerosis hepatitis, autoimmune hepatitis of unknown cause Congenital immune deficiency-related diseases, hepatitis C, unclassifiable immunodeficiency (unclassifiable hypogammaglobulinemia), dilated cardiomyopathy, pulmonary fibrosis, unexplained fibrotic alveolitis, interstitial pneumonia , Interstitial lung disease associated with mixed connective tissue disease, interstitial lung disease due to systemic sclerosis, interstitial lung disease due to rheumatoid arthritis, lung disease due to systemic lupus erythematosus, dermatomyositis, multiple myositis Lung disease Lung disease due to Sjogren's disease, lung disease related to ankylosing spondylitis, lung disease due to panvasculitis, lung disease due to hemosiderinosis, drug-induced interstitial lung disease, radiation fibrosis, obstructive bronchiolitis, Chronic eosinophilic pneumonia, lymphocyte infiltrating lung disease, gouty arthritis, autoimmune hepatitis, type 1 autoimmune hepatitis (classical autoimmune or lupoid hepatitis), type 2 autoimmune hepatitis anti (anti LKM1 antibody-positive hepatitis), autoimmune hypoglycemia, insulin receptor abnormality B due to black epidermoma, hypoparathyroidism, osteoarthritis, primary sclerosing cholangitis, idiopathic leukopenia, autoimmunity Neutropenia, renal disease NOS, glomerulonephritis, microscopic vasculitis in the kidney, discoid lupus erythematosus, idiopathic male infertility NOS, sperm-related autoimmune disease, sympathetic ophthalmitis, pulmonary hypertension Yui Joint tissue disease, Goodpasture syndrome, pulmonary symptoms of polyarteritis nodosa, acute rheumatic fever, rheumatoid spondylitis, Still's disease, systemic sclerosis, Takayasu disease, arteritis, autoimmune thrombocytopenia, idiopathic platelets Reduction, autoimmune thyroid disease, hyperthyroidism, goiter / hypothyroidism (Hashimoto's disease), atrophic autoimmune hypothyroidism, primary myxedema, lens-induced uveitis, primary Examples include vasculitis, vitiligo and cutaneous lupus erythematosus. 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 , Eosinophilia syndrome, eosinophilic gastroenteritis and the like.

Examples of rejection include graft versus host rejection.

Infectious diseases include Salmonella, Shigella, Clostridium difficile, Mycobacterium (tuberculosis as disease), protozoa (malaria as disease), filamentous nematodes (filariasis as disease), schistosomiasis (as disease) Schistosomiasis), toxoplasma (toxoplasmosis as disease), leishmania (leishmaniasis as disease), HCV and HBV (hepatitis C and hepatitis B as disease), herpes simplex virus (herpes as disease) Infectious diseases due to such as.

Diseases derived from adipose tissue inflammation include metabolic syndrome, visceral obesity, insulin resistance, hyperglycemia, dyslipidemia, hypertriglycerideemia, low HDL cholesterolemia, increased blood pressure, arteriosclerotic disease, type 2 diabetes, Nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), etc. are mentioned.

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 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 to become a standard.

The administration time of the drug of the present invention is not particularly limited, and can be appropriately selected according to the state of the administration target. It may be administered prophylactically or used for maintenance therapy. Moreover, it is preferable that a dosage form is determined according to a formulation form, a patient's age, sex, other conditions, a patient's symptom, its grade, etc.

In any case, the drug of the present invention can be administered once or a plurality of times a day, or can be administered once every several days or weeks.

The drug of the present invention, or the bacteria belonging to Bifidobacterium breve, which is an active ingredient thereof, and lactulose, raffinose, and galactooligosaccharide can also be contained in food and drink (beverage or food).

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

As foods and beverages, the form and properties are not particularly limited as long as they can be taken orally without impairing the effects of promoting immune development, including bacteria belonging to Bifidobacterium breve, and lactulose, raffinose, and galactooligosaccharides Except making it, it can manufacture by a normal method using the raw material normally used for food-drinks.

The foods and drinks mentioned above are not limited to liquid, paste-like, gel-like solids, powders, etc. In addition to tablets, liquid foods, etc., for example, bread, macaroni, spaghetti, noodles, cake mix, fried Flour products such as flour and bread crumbs; instant noodles, cup noodles, retort / cooked food, cooking canned food, microwave food, instant soup / stew, instant miso soup / soup, canned soup, freeze-dried food, other instant foods, etc. Foods: Canned agricultural products, canned fruits, jams, marmalades, pickles, boiled beans, dried agricultural products, processed cereals (cereal products), canned fish, fish hams and sausages, marine products, marine delicacies, Processed marine products such as tsukudani; Livestock canned and pasted products, processed livestock products such as meat ham and sausage; processed milk, milk drinks, yogurts Milk and dairy products such as lactic acid bacteria beverages, cheese, ice cream, formula milk powder, cream, other dairy products; fats and oils such as butter, margarines, vegetable oils; soy sauce, miso, sauces, tomato processed seasonings, Basic seasonings such as mirin, vinegar, etc .; cooking mixes, curry ingredients, sauces, dressings, noodle soups, spices, and other complex seasonings and foods; frozen foods, semi Frozen foods such as cooked frozen foods and cooked frozen foods; caramel, candy, chewing gum, chocolate, cookies, biscuits, cakes, pie, snacks, crackers, Japanese confectionery, rice confectionery, bean confectionery, dessert confectionery, jelly, other confectionery Confectionery; carbonated drinks, natural fruit juices, fruit juice drinks, soft drinks with fruit juice, fruit drinks, fruit drinks with fruit granules, vegetable drinks, soy milk, Milk beverages, coffee beverages, tea beverages, powdered beverages, concentrated beverages, sports beverages, nutritional beverages, alcoholic beverages, other beverages such as other beverages, baby foods, sprinkles, and other commercial foods such as tea paste Formula milk for childcare; enteral nutrition; special-purpose foods, health functional foods (special health foods, nutritional functional foods, functional labeling foods); and dietary supplements.

The food / beverage products of the present invention can be sold as food / beverage products displaying the use for promoting immune development. In addition, such foods and drinks can be labeled as “for promoting immune development”. In addition, it is needless to say that words other than this can be used as long as they represent the effects that are secondarily produced by promoting immune development.

The “display” means all acts for informing the consumer of the use, and if it is a display that can recall and analogize the use, the purpose of the display, the content of the display, the display Regardless of the object and medium to be processed, all fall under the “display” of the present invention. However, it is preferable to display in such an expression that the consumer can directly recognize the application.
Specifically, the act of describing the above-mentioned use in the product or product packaging relating to the food or drink of the present invention, the product or product packaging describing the above-mentioned use is transferred, and displayed for delivery, transfer or delivery And importing, displaying advertisements on products, price lists or transaction documents for display or distribution, or describing the above uses in information containing these, electromagnetic (Internet, etc.) methods In particular, it is preferable to display on advertising materials at sales sites such as packaging, containers, catalogs, pamphlets, POPs, and other documents.

Also, the display is preferably a display permitted by the government or the like (for example, a display that is approved based on various systems determined by the government and is performed in a mode based on such approval). For example, health food, functional food, enteral nutrition food, special purpose food, nutritional functional food, quasi-drugs, etc. can be exemplified, other indications approved by the Consumer Affairs Agency, for example, Examples of health functional foods, more specifically, foods for specified health use, nutritional functional foods, functional labeling foods, and labels approved by a similar system. Examples of the latter include labeling as food for specified health use, labeling as conditionally specified food for specified health use, labeling that affects the structure and function of the body, labeling for reducing disease risk, and functionality based on scientific evidence The display etc. can be illustrated. In more detail, as food for specified health use stipulated in the Cabinet Office Ordinance (Cabinet Office Ordinance No. 57 of August 31, 2000) concerning the permission for special purpose labeling stipulated in the Health Promotion Act Display (especially the display of health use), a display similar to this, and the like.

Bacteria belonging to Bifidobacterium breve, as well as lactulose, raffinose, and galactooligosaccharides can be included in the feed as active ingredients and processed as a feed having an immune development promoting action as one aspect of the immune development promoting agent. It is.

The form of the feed is not particularly limited, for example, grains such as corn, wheat, barley, rye, milo; vegetable oils such as soybean oil meal, rapeseed oil meal, coconut oil meal, linseed oil meal; bran, wheat straw, rice bran, Dehydrated rice bran, etc .; Manufactured deer such as corn gluten meal, corn jam meal; Animal feed such as fish meal, non-fat dry milk, whey, yellow grease, tallow; Yeasts such as torula yeast, beer yeast; It can be produced by blending mineral feed such as calcium phosphate and calcium carbonate; fats and oils; simple amino acids; sugars and the like. Examples of the form of the feed include pet food, livestock feed, and fish feed.

The amount of bacteria belonging to Bifidobacterium breve, and lactulose, raffinose, and galactooligosaccharides contained in the food and drink (including feed) of the present invention is not particularly limited and may be appropriately selected. This is the same as the drug of the present invention described above.

Hereinafter, the present invention will be described more specifically 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 changes in the number of cells over time C57BL / 6J male mice were purchased from Japan SLC with their mothers. Infant mice were fed freely with maternal milk. Infant mice are divided into two groups so that weight does not become biased, one group is 14 days old and the other group is 21 days old, and each of them is dissected, and the large intestine and mesenteric lymph nodes (mesenteric lymph nodes) node (hereinafter sometimes referred to as “MLN”).

The obtained large intestine sample was cut vertically and transferred to DPBS (Dulbecco PBS) containing 5 mM EDTA, 1 mM mM DTT, and 2% fetal calf serum (FCS), and shaken in a 37 ° C. thermostat for 30 minutes. Thereafter, the sample was passed through a 70 μm mesh to remove the liquid containing epithelial cells. The remaining large intestine sample contains 0.5 mg / ml collagenase (Sigma), 25 μg / ml DNase® I (Roche Diagnostics), 50 μg / ml dispase (Gibco), 0.01 M HEPES, and 2% FCS Placed in RPMI 1640 medium and digested at 37 ° C for 30 minutes. The digested solution was filtered through a 70 μm mesh, and the obtained filtrate was centrifuged at 340 × g and 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 overlaid on the upper layer of the 90% Percoll solution. Centrifuge at 720 xg for 25 minutes at room temperature to recover lamina propria lymphocytes (lamina propria lymphocyte; hereinafter 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 treated with FITC-labeled anti-mouse CD4 antibody (clone number: RM4-5, manufactured by BD Biosciences), APC (allophycocyanin) -labeled anti-rat / mouse Foxp3 (clone number: FJK-16a, e -Bioscience), PE (phycoerythrin) labeled anti-human / mouse RORgt (clone number: AFKJS-9, e-Bioscience), Foxp3pStaining Buffer set (e-Bioscience) The cells were analyzed using a FACSCanto flow cytometer (BD Biosciences), and data analysis was performed using FlowJo software (TreeStar).

Tables 1 to 4 show the ratios (average value, unit:%) of Foxp3 positive cells and RORgt positive cells to CD4 positive cells. In large intestine LPL, the percentage of Foxp3 positive cells (Treg cells) in CD4 positive cells increased with increasing age, whereas in MLNL, the percentage of Foxp3 positive cells in CD4 positive cells changed with increasing age. (Tables 1 and 2). Note that CD4 positive cells in large intestine LPL and MLNL are almost T cells.

On the other hand, the ratio of RORgt positive cells (Th17 cells) in CD4 positive cells decreased with increasing age (Tables 3 and 4).
In addition, Tables 5 to 7 show the total number of cells in MLNL, the number of Foxp3-positive cells, and the number of RORgt-positive cells (average value, unit: × 10 ⁶ ). All cell numbers increased with increasing age.

[Example 2] Effect of Bifidobacterium breve and three oligosaccharides on the ratio of Foxp3 positive cells and RORgt positive cells in CD4 positive cells in mouse colon LPL and MLNL Bifidobacterium breve in starch M-16V cell powder (2.4 × 10 ¹¹ cfu / g, 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 Milk Industry Co., Ltd.), raffinose (product name: Nitten Raffinose, manufactured by Nippon Sesame Sugar Co., Ltd.), and galactooligosaccharide are mixed and dissolved in distilled water at a weight ratio of 1: 1: 1, for a total final concentration of 250 mg / ml. An oligosaccharide solution was prepared. Galactooligosaccharide is a product obtained by removing monosaccharide and disaccharide from a commercial product (product name Oligomate 55N, manufactured by Yakult Pharmaceutical Co., Ltd.), Galβ1-4Galβ1-4Glc (4′-galactosyl lactose) is about 65% by weight, About 15% by weight of Galβ1-6Galβ1-4Glc (6′-galactosyl lactose) is contained.

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

2 days old C57BL / 6J male mice were purchased from Japan SLC together with their mothers. Infant mice were fed freely with maternal milk. It was divided into the following three groups so that there was no bias in body weight at 5 days of age.

A: Vehicle group (administer physiological saline)
B: Bifidobacteria group (administered Bifidobacteria solution dilution)
C: Oligosaccharide + bifidobacteria group (administered oligosaccharide and bifidobacteria mixture)

Between 6 and 13 days of age after birth, each group of infant mice is given once a day with physiological saline, bifidobacterial solution dilution, or a mixture of bifidobacterial solution and oligosaccharide solution (1: 4). 100 μl was administered. That is, for each administration, 5 × 10 ⁷ cfu Bifidobacterium breve M-16V is used for the Bifidobacterium group, 20 mg oligosaccharide and 5 × 10 ⁷ cfu Bifidobacterium is used for the oligosaccharide + Bifidobacterium group. • Breve M-16V was administered.

At 14 days of age, each was dissected and the large intestine and MLN were collected, and in the same manner as in Example 1, the proportions of Foxp3 positive cells and RORgt positive cells in the CD4 positive cells in the large intestine LPL and MLNL were examined. The results are shown in Tables 8 and 9 (ratio of Foxp3 positive cells, average value, unit:%) and Tables 10 and 11 (ratio of RORgt positive cells, average 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, LPL and Bifidobacterium breve M-16V and oligosaccharide (lacturose + raffinose + galactooligosaccharide) were mixed and administered (M-16V + oligosaccharide). The percentage of Foxp3-positive cells in CD4-positive cells in MLNL increased compared to the Vehicle group (control) and the Bifidobacterium group (M-16V).
On the other hand, as shown in Tables 10 and 11, at the 14th day after birth, the mixed administration of M-16V and oligosaccharide did not affect the ratio 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 As in Example 2, milk administered with each administration The pups were dissected at 14 days of age, and the numbers of Foxp3-positive cells and RORgt-positive cells among CD4-positive cells in MLNL were examined. The results are shown in Tables 12 to 14 (average value, unit: × 10 ⁶ ). At the 14th day after birth, the total number of cells in MLNL, the number of CD4-positive Foxp3-positive cells, and the number of CD4-positive RORgt-positive cells increased by administration of Bifidobacterium breve M-16V and oligosaccharide.

The above results are summarized in Table 15 schematically.

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

Claims

An immune development promoter comprising bacteria belonging to Bifidobacterium breve and lactulose, raffinose, and galactooligosaccharide.
2. The immune development promoting agent according to claim 1, wherein the promotion of immune development is promotion of an increase in the number of T cells.
The immune development promoter according to claim 1 or 2, wherein the bacterium is Bifidobacterium breve M-16V (LMG23729).
The immune development promoter according to any one of claims 1 to 3, wherein the weight ratio of lactulose, raffinose, and galactooligosaccharide is 1 to 9: 1 to 9: 1 to 9.
The immune development promoting agent according to any one of claims 1 to 4, wherein the amount of the bacteria is 1 x 10 6 to 1 x 10 12 cfu with respect to 1 g of the total amount of lactulose, raffinose, and galactooligosaccharide. .
A feed comprising the immune development promoting agent according to any one of claims 1 to 5.
A food / beverage composition for promoting immune development, comprising bacteria belonging to Bifidobacterium breve and lactulose, raffinose, and galactooligosaccharide as active ingredients.
The food or beverage composition according to claim 7, wherein the food or beverage composition is a health functional food.