WO2014077383A1 - Agent for ameliorating allergy - Google Patents

Agent for ameliorating allergy Download PDF

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Publication number
WO2014077383A1
WO2014077383A1 PCT/JP2013/081016 JP2013081016W WO2014077383A1 WO 2014077383 A1 WO2014077383 A1 WO 2014077383A1 JP 2013081016 W JP2013081016 W JP 2013081016W WO 2014077383 A1 WO2014077383 A1 WO 2014077383A1
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WO
WIPO (PCT)
Prior art keywords
allergy
galactopyranosyl
galactooligosaccharide
ameliorating
agent
Prior art date
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PCT/JP2013/081016
Other languages
French (fr)
Japanese (ja)
Inventor
松山　博昭
聡志 日暮
加藤　健
誠一郎 青江
Original Assignee
雪印メグミルク株式会社
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Application filed by 雪印メグミルク株式会社 filed Critical 雪印メグミルク株式会社
Priority to CN201380060243.8A priority Critical patent/CN104797260A/en
Publication of WO2014077383A1 publication Critical patent/WO2014077383A1/en
Priority to HK15108025.5A priority patent/HK1207311A1/en

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
    • A23L2/52—Adding ingredients
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/14—Pretreatment of feeding-stuffs with enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/163—Sugars; Polysaccharides
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/40—Feeding-stuffs specially adapted for particular animals for carnivorous animals, e.g. cats or dogs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/30—Foods or foodstuffs containing additives; Preparation or treatment thereof containing carbohydrate syrups; containing sugars; containing sugar alcohols, e.g. xylitol; containing starch hydrolysates, e.g. dextrin
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/20—Reducing nutritive value; Dietetic products with reduced nutritive value
    • A23L33/21—Addition of substantially indigestible substances, e.g. dietary fibres
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/702—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/20—Milk; Whey; Colostrum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients

Definitions

  • the present invention relates to an allergy ameliorating agent. More specifically, the present invention relates to an allergy ameliorating agent that is excellent in allergy improving action, suppresses metabolic inflammation, and suppresses secretion of inflammatory cytokines, thereby improving disturbance of the immune regulatory system in the living body.
  • the present invention further relates to an allergy ameliorating food / beverage, an allergy ameliorating nutritional composition, an allergy ameliorating feed or an allergy ameliorating pharmaceutical, which further contains an allergy ameliorating agent.
  • helper T cells There are two types of helper T cells: helper T1 (hereinafter referred to as “Th1”) cells and helper T2 (hereinafter referred to as “Th2”) cells.
  • Th1 helper T1
  • Th2 helper T2
  • cytokines cytokines produced by each and maintain a balance
  • an increase in inflammatory markers such as F4 / 80
  • NADPH oxidase subunits such as P40phox
  • secretion is promoted and allergic symptoms are thought to be induced. Therefore, it is possible to improve allergic diseases by suppressing the increase of inflammatory markers such as NADPH oxidase and macrophages and suppressing the secretion of inflammatory cytokines.
  • Antihistamines and steroids are known as drugs for allergic diseases.
  • these medicines may have side effects such as tinnitus, headache, and loss of appetite.
  • these substances cannot be added to food and drink at present from the viewpoints of safety and cost.
  • desensitization therapies that gradually weaken the response by administering a small amount of allergen for a long time, diets to avoid contact with allergens, etc. These include the need for long-term treatment and life Since there is a problem such as a decrease in quality (QOL), as an easy method, development of food and drink and feed that can be expected to prevent or treat allergic diseases by ingestion is desired.
  • QOL decrease in quality
  • Indigestible oligosaccharides flavonoids, polyunsaturated fatty acids, etc. are known as food ingredients that improve allergic symptoms. Indigestible oligosaccharides are said to be effective in maintaining the normal state of intestinal flora mainly composed of bifidobacteria, and the Th1 environment is induced by ingestion of raffinose, an indigestible oligosaccharide. It is known that the Th2 response of the systemic immune system is suppressed. In addition, acidic xylo-oligosaccharides have been shown to suppress inflammatory cytokines, and ingestion of oligosaccharides is considered to have an effect on allergy suppression from various aspects.
  • An object of the present invention is to provide an allergy ameliorating agent that improves the disturbance of the immune regulatory system in the living body by suppressing the secretion of inflammatory cytokines.
  • the inventors of the present invention have made extensive studies to solve the above problems, O- ⁇ -D-galactopyranosyl- (1 ⁇ 6)-[O- ⁇ -D-galactopyranosyl- (1 ⁇ 4)]-D-glucose or O- ⁇ -D-galactopyranosyl- (1 ⁇ 4)-[O
  • the present inventors have found that galactooligosaccharides having the structural formula represented by - ⁇ -D-galactopyranosyl- (1 ⁇ 6)]-D-glucose have an allergy ameliorating effect through suppression of inflammatory cytokine secretion. That is, the present invention includes the following modes.
  • an allergy ameliorating agent comprising a galactooligosaccharide having the structural formula represented by (2)
  • An allergy-improving food or drink, an allergy-improving nutritional composition, an allergy-improving feed, or an allergy-improving pharmaceutical comprising the allergy-improving agent according to any one of (1) to (3).
  • the allergy ameliorating agent of the present invention suppresses metabolic inflammation and suppresses secretion of inflammatory cytokines, thereby improving disturbance of the immune regulatory system in the living body.
  • the allergy ameliorating agent of the present application is O- ⁇ -D-galactopyranosyl- (1 ⁇ 6)-[O- ⁇ -D-galactopyranosyl- (1 ⁇ 4)]-D-glucose or O- ⁇ -D-galactopyranosyl-
  • the active ingredient is a galactooligosaccharide having a structural formula represented by (1 ⁇ 4)-[O- ⁇ -D-galactopyranosyl- (1 ⁇ 6)]-D-glucose.
  • the allergy ameliorating agent containing the galactooligosaccharide as an active ingredient suppresses the increase of inflammatory markers such as NADPH oxidase and macrophages and suppresses the secretion of inflammatory cytokines. Thereby, it is useful for prevention and / or treatment of various allergic diseases such as atopic dermatitis, allergic rhinitis, urticaria and hay fever. It is also useful for the prevention and / or treatment of rheumatism.
  • the galactooligosaccharide used in the improving agent described in the present application can be easily and economically produced because it uses lactose as a raw material, and because the sugar composition of the active ingredient is specified, it is safe.
  • O- ⁇ -D-galactopyranosyl- (1 ⁇ 6)-[O- ⁇ -D-galactopyranosyl- (1 ⁇ 4)]-D-glucose or O- ⁇ -D-galactopyranosyl- (1 ⁇ 4) )-[O- ⁇ -D-galactopyranosyl- (1 ⁇ 6)]-D-glucose is a galactooligosaccharide having a structural formula represented by, for example, Sumilacto LL in a solution containing 20% to 40% lactose.
  • the lactose-assimilating bacterium is main-cultured in a main culture medium having a lactose content of about 13% and a pH of 4 to 9, and then concentrated under reduced pressure to remove unreacted lactose from the culture solution, and further ⁇ -galactosidase acts.
  • the solution obtained by decomposing unreacted lactose remaining in the culture solution is passed through an activated carbon column, and the adsorbed galactooligosaccharide is eluted with 20% ethanol.
  • the main culture is cultured at 20 to 40 ° C., preferably around 30 ° C. for 5 to 15 days, the yield can be further increased.
  • the galactooligosaccharide prepared as described above can be concentrated by a reverse osmosis membrane (RO) membrane or the like, or dried by freeze drying or the like.
  • RO reverse osmosis membrane
  • Lactose used as a raw material for galactooligosaccharides is lactose prepared from milk from mammals such as cattle, goats, sheep, and humans, and aggregates, powders, or purified lactose, which are used to react with ⁇ -galactosidase. In the case of the main culture, it can be used in the state of an aqueous solution.
  • the above-described galactooligosaccharide may be used as it is as an allergy ameliorating agent, but if necessary, it is formulated into powders, granules, tablets, capsules, drinks, etc. according to conventional methods. It can also be used. Furthermore, galactooligosaccharides obtained by reverse osmosis membrane (RO) membrane treatment or the like can also be used as an allergy ameliorating agent as they are, and can be used as they are after drying. Moreover, it can also be formulated and used according to a conventional method. Furthermore, after formulating these, it is also possible to mix this with foods and beverages such as nutrients, yogurt, beverages, wafers, nutritional compositions, feeds and pharmaceuticals.
  • foods and beverages such as nutrients, yogurt, beverages, wafers, nutritional compositions, feeds and pharmaceuticals.
  • allergy improving food and beverage, allergic improving nutrition composition, allergy improving feed and allergy improving pharmaceutical include not only the active ingredient galactooligosaccharide, but also stabilizers, saccharides, lipids, Flavors, vitamins, minerals, flavonoids, polyphenols, and other raw materials usually contained in other foods, feeds and medicines can be contained. Further, in addition to the active ingredient galactooligosaccharide, it can also be used together with other components showing an allergy improving action, such as raffinose and acidic xylo-oligosaccharide. In addition, it may be prepared by blending raw materials usually contained in other foods and drinks, etc., using such allergy improving food and drink, allergy improving nutritional composition, allergy improving feed or allergy improving pharmaceuticals as raw materials. Is possible.
  • the compounding amount of galactooligosaccharides in allergic ameliorating foods, allergic ameliorating compositions, allergy ameliorating feeds, and allergy ameliorating pharmaceuticals is not particularly limited. Depending on the form of food, drink, feed and medicine, it generally contains 0.01 to 10% (mass / mass), preferably 0.1 to 5% (mass / mass) with respect to the total mass. Preferably it is.
  • the allergy ameliorating agent described in the present application can be formulated into an arbitrary form by adding an appropriate auxiliary agent to the above active ingredient, and can be made into an allergy ameliorating composition that can be administered orally.
  • diluents or excipients such as fillers, extenders, binders, disintegrants, surfactants, lubricants and the like that are usually used can be used.
  • excipient include sucrose, lactose, starch, crystalline cellulose, mannitol, light anhydrous silicic acid, magnesium aluminate, synthetic aluminum silicate, magnesium magnesium metasilicate, calcium carbonate, sodium hydrogen carbonate, calcium hydrogen phosphate
  • carboxymethylcellulose calcium and the like can be added in combination.
  • the present invention also relates to an allergy ameliorating method in which the allergy ameliorating agent of the above-described embodiment is orally ingested.
  • the allergy ameliorating method is useful for the treatment and / or prevention of various allergic diseases.
  • the allergy ameliorating method is effective for mammals such as humans as well as domestic animals such as dogs and cats.
  • Example 1 Commercially available ⁇ -galactosidase (trade name: Sumilac LL, Shin Nippon Chemical Industry Co., Ltd.) was added to 5000 ml of 20% lactose aqueous solution at 3 units / ml, and reacted at 40 ° C. (pH 5.0) for 3 hours. I let you. This solution is concentrated under reduced pressure to precipitate and remove lactose, and after passing through an activated carbon column (15 cm ⁇ 30 cm) to adsorb galactooligosaccharide, 3000 ml of 5% ethanol is passed through and remains. Lactose was removed.
  • galactooligosaccharide powder includes O- ⁇ -D-galactopyranosyl- (1 ⁇ 6)-[O- ⁇ -D-galactopyranosyl- (1 ⁇ 4)]-D-glucose or O- ⁇ -D-galactopyranosyl- ( 12% by mass of galactooligosaccharide having a structural formula represented by 1 ⁇ 4)-[O- ⁇ -D-galactopyranosyl- (1 ⁇ 6)]-D-glucose was contained.
  • the galactooligosaccharide thus obtained can be used as it is as an allergy ameliorating agent.
  • Penicillium roqueforti IFO 4135 strain was precultured (lactose 2.9%, dipotassium hydrogen phosphate 0.1%, magnesium sulfate heptahydrate 0.05%, sodium nitrate 0.2%, Potassium chloride 0.05%, iron sulfate 0.001%) was inoculated into 100 ml, and cultured at 30 ° C. for 5 days. 100 ml of the obtained preculture solution was added to the main culture medium (lactose 13%, dipotassium hydrogen phosphate 0.09%, magnesium sulfate heptahydrate 0.04%, sodium nitrate 0.17%, potassium chloride 0.04%).
  • This galactooligosaccharide powder includes O- ⁇ -D-galactopyranosyl- (1 ⁇ 6)-[O- ⁇ -D-galactopyranosyl- (1 ⁇ 4)]-D-glucose or O- ⁇ -D-galactopyranosyl- ( 1 ⁇ 4)-[O- ⁇ -D-galactopyranosyl- (1 ⁇ 6)]-D-glucose contained 9% by mass of galactooligosaccharide having the structural formula.
  • the galactooligosaccharide thus obtained can be used as it is as an allergy ameliorating agent.
  • TNCB 2,4,6-trinitro-1-chlorobenzene
  • mice in Group A control
  • Group B mice in each group
  • mice in Group C mice in each group
  • Saccharide was orally administered at 50 mg / kg of mouse body weight
  • galactooligosaccharide of Example Product 2 was orally administered daily for 2 weeks over 2 weeks.
  • Example Products 1 and 2 suspended in physiological saline were orally administered.
  • the right ear of the mouse was removed and its mass was measured, and the expression level of the inflammatory marker (F4 / 80, P40phox) mRNA was measured by a real-time PCR method.
  • the results are shown in Table 1.
  • the right ear mass and the expression level of inflammatory marker (F4 / 80, P40phox) mRNA after 2 weeks of administration were 50 mg or more of the galactooligosaccharides of Examples 1 and 2 per 1 kg of mouse body weight.
  • the B and C groups were significantly lower than the A group (control). From these results, it was found that the galactooligosaccharides of Examples 1 and 2 had an allergy improving action. Moreover, it became clear that this effect
  • Example 3 Preparation of tablets for allergy improvement
  • Each raw material was mixed so that the total amount was 100.0 parts by mass with the formulation shown in Table 2, and then molded into 1 g by a conventional method and compressed to produce tablets for allergy improvement.
  • Example 4 (Preparation of liquid nutritional composition for allergy improvement) 50 g of the galactooligosaccharide of Example product 2 was dissolved in 4,950 g of deionized water, heated to 50 ° C., and then stirred at 6,000 rpm for 30 minutes with a TK homomixer (TK ROBO MICS; manufactured by Tokushu Kika Kogyo Co., Ltd.). By mixing, a galactooligosaccharide solution of Example Product 2 having a galactooligosaccharide content of 50 g / 5 kg was obtained.
  • TK ROBO MICS manufactured by Tokushu Kika Kogyo Co., Ltd.
  • Example 5 Preparation of beverage for allergy improvement
  • 300 g of skim milk powder in 409 g of deionized water 1 g of the galactooligosaccharide of Example product 1 was dissolved, heated to 50 ° C., and then with an ultradisperser (ULTRA-TURRAX-25; manufactured by IKA Japan). The mixture was stirred and mixed at 9,500 rpm for 30 minutes.
  • maltitol 100g, acidulant 2g, reduced starch syrup 20g, fragrance 2g, deionized water 166g it is filled into a 100ml glass bottle, sterilized at 95 ° C for 15 seconds, hermetically sealed, and 10 drinks for allergy improvement (100ml) Prepared).
  • Example 6 Preparation of allergy-improving feed for dogs 2 kg of the galactooligosaccharide of Example product 2 was dissolved in 98 kg of deionized water, heated to 50 ° C., and then stirred and mixed for 40 minutes at 3,600 rpm with a TK homomixer (MARK II 160 type; manufactured by Tokushu Kika Kogyo Co., Ltd.). Thus, a galactooligosaccharide solution of Example Product 2 having a galactooligosaccharide content of 2 g / 100 g was obtained.
  • TK homomixer MARK II 160 type

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Abstract

The present invention addresses the problem of providing an agent for ameliorating allergy that has an effect of inhibiting the secretion of an inflammatory cytokine and thus ameliorating disorders in the immunoregulatory system in vivo, and products for ameliorating allergy such as foods, drinks, feeds and medicines, said products containing the agent for ameliorating allergy. An agent for ameliorating allergy which comprises, as an active ingredient, a galactooligosaccharide having a structural formula: O-β-D-galactopyranosyl-(1→6)-[O-β-D-galactopyranosyl-(1→4)]-D-glucose; or O-β-D-galactopyranosyl-(1→4)-[O-β-D-galactopyranosyl-(1→6)]-D-glucose.

Description

アレルギー改善剤Allergy improving agent
　本発明はアレルギー改善剤に関する。より詳しくは本発明はアレルギー改善作用に優れ、代謝性の炎症を抑制し、炎症性のサイトカインの分泌を抑制することにより、生体内の免疫調節系の乱れを改善するアレルギー改善剤に関する。本発明は、さらにアレルギー改善剤を含有する、アレルギー改善用飲食品、アレルギー改善用栄養組成物、アレルギー改善用飼料又はアレルギー改善用医薬品に関する。 The present invention relates to an allergy ameliorating agent. More specifically, the present invention relates to an allergy ameliorating agent that is excellent in allergy improving action, suppresses metabolic inflammation, and suppresses secretion of inflammatory cytokines, thereby improving disturbance of the immune regulatory system in the living body. The present invention further relates to an allergy ameliorating food / beverage, an allergy ameliorating nutritional composition, an allergy ameliorating feed or an allergy ameliorating pharmaceutical, which further contains an allergy ameliorating agent.
　近年、我が国を含む先進諸国において、アトピー性皮膚炎や花粉症などの種々のアレルギー疾患が増加しており、大きな社会問題となっている。アトピー性皮膚炎をはじめとするアレルギー疾患の発症には、遺伝的要因と環境要因の両方が関与しており、近年のアレルギー疾患患者数の増加には、大気汚染やライフスタイルの変化、食生活の変化などの環境要因の影響が大きいと考えられている。また、アレルギー疾患の発生機序は複雑であり、未だ解明されていない部分も多いが、免疫調節系の乱れにその原因があることが徐々にわかってきている。免疫調節にはヘルパーＴ細胞が重要な働きをするが、このヘルパーＴ細胞にはヘルパーＴ１（以下「Ｔｈ１」という）細胞とヘルパーＴ２（以下「Ｔｈ２」という）細胞の２種類があり、それぞれがサイトカインの産生を通して細胞性免疫と液性免疫を制御している。Ｔｈ１細胞とＴｈ２細胞は、それぞれの生産するサイトカインによって互いの活性化を抑制し、バランスを保っているが（Ｔｈ１／Ｔｈ２バランス）、なんらかの原因でＴｈ２細胞の誘導が強くなるとアレルギー疾患を発症する。
　この場合、活性酸素を産生するＮＡＤＰＨオキシダーゼのサブユニット（P40phoxなど）や炎症性サイトカインを分泌するマクロファージなどの炎症性マーカー（F4/80など）が上昇することにより、生体内での炎症性のサイトカインの分泌が促進し、アレルギー症状を誘導すると考えられている。したがって、ＮＡＤＰＨオキシダーゼやマクロファージなどの炎症性マーカーの上昇を抑制して、炎症性のサイトカインの分泌を抑制することにより、アレルギー疾患を改善することが可能である。 In recent years, various allergic diseases such as atopic dermatitis and hay fever have been increasing in developed countries including Japan, which has become a major social problem. Both genetic and environmental factors are involved in the development of allergic diseases such as atopic dermatitis. In recent years, the increase in the number of allergic disease patients has been caused by air pollution, lifestyle changes, and dietary habits. The influence of environmental factors such as changes in In addition, the mechanism of allergic diseases is complex, and there are many unexplained parts, but it is gradually becoming clear that the cause of the disorder in the immune regulatory system is. Helper T cells play an important role in immune regulation. There are two types of helper T cells: helper T1 (hereinafter referred to as “Th1”) cells and helper T2 (hereinafter referred to as “Th2”) cells. Cellular and humoral immunity is controlled through the production of cytokines. Although Th1 cells and Th2 cells suppress each other's activation by cytokines produced by each and maintain a balance (Th1 / Th2 balance), allergic diseases develop when the induction of Th2 cells becomes strong for some reason.
In this case, an increase in inflammatory markers (such as F4 / 80) such as NADPH oxidase subunits (such as P40phox) that produce active oxygen and macrophages that secrete inflammatory cytokines causes inflammatory cytokines in vivo. Secretion is promoted and allergic symptoms are thought to be induced. Therefore, it is possible to improve allergic diseases by suppressing the increase of inflammatory markers such as NADPH oxidase and macrophages and suppressing the secretion of inflammatory cytokines.
　アレルギー疾患に対する医薬として、抗ヒスタミン薬、ステロイド剤などが知られている。しかし、これらの医薬品を用いた場合、耳鳴り、頭痛、食欲不振などの副作用を伴うことがある。また、これらの物質は安全性及びコストなどの面から、現在のところ飲食品に添加することができない状況にある。さらに、少量のアレルゲンを長期間投与して徐々に反応を弱める減感作療法、アレルゲンとの接触を避けるための食事療法などがあるが、これらには、長期に渡る治療の必要性や生活の質（ＱＯＬ）が低下するなどの問題があるため、手軽な方法として、経口摂取することで、アレルギー疾患の予防または治療効果が期待できるような飲食品や飼料の開発が望まれている。 抗 Antihistamines and steroids are known as drugs for allergic diseases. However, these medicines may have side effects such as tinnitus, headache, and loss of appetite. In addition, these substances cannot be added to food and drink at present from the viewpoints of safety and cost. Furthermore, there are desensitization therapies that gradually weaken the response by administering a small amount of allergen for a long time, diets to avoid contact with allergens, etc. These include the need for long-term treatment and life Since there is a problem such as a decrease in quality (QOL), as an easy method, development of food and drink and feed that can be expected to prevent or treat allergic diseases by ingestion is desired.
　アレルギー症状を改善する食品成分として、難消化性オリゴ糖やフラボノイド類、多価不飽和脂肪酸などが知られている。難消化性オリゴ糖は、ビフィズス菌を主体とする腸内フローラの状態を正常に保つ上で有効であるとされており、難消化性オリゴ糖であるラフィノースの摂取によりＴｈ１環境が誘導されると共に全身免疫系のＴｈ２応答が抑制されることが知られている。また、酸性キシロオリゴ糖による炎症性サイトカインの抑制効果も認められており、オリゴ糖の摂取は様々な側面からアレルギー抑制に効果を示すと考えられている。さらに、Gal-(Gal)n-Glc(n=1～3、β-1,4結合)の一般式で表されるガラクトオリゴ糖（４’-ガラクトシルラクトース）には、ＩｇＥの産生抑制を介したアレルギー改善作用があることが報告されている。 難 Indigestible oligosaccharides, flavonoids, polyunsaturated fatty acids, etc. are known as food ingredients that improve allergic symptoms. Indigestible oligosaccharides are said to be effective in maintaining the normal state of intestinal flora mainly composed of bifidobacteria, and the Th1 environment is induced by ingestion of raffinose, an indigestible oligosaccharide. It is known that the Th2 response of the systemic immune system is suppressed. In addition, acidic xylo-oligosaccharides have been shown to suppress inflammatory cytokines, and ingestion of oligosaccharides is considered to have an effect on allergy suppression from various aspects. Furthermore, galactooligosaccharides (4′-galactosyl lactose) represented by the general formula of Gal- (Gal) n-Glc (n = 1-3, β-1,4 bond) are mediated through suppression of IgE production. It has been reported to have an allergy ameliorating effect.
特開２００７－１９７３３１号公報JP 2007-197331 A 特開２００１－２８８０９３号公報JP 2001-288093 A 特開２００７－２３０１８号公報JP 2007-23018 A 特開昭６２－７９７９１号公報JP 62-77991 A
　本発明は、炎症性のサイトカインの分泌を抑制することにより、生体内の免疫調節系の乱れを改善するアレルギー改善剤を提供することを課題とする。 An object of the present invention is to provide an allergy ameliorating agent that improves the disturbance of the immune regulatory system in the living body by suppressing the secretion of inflammatory cytokines.
　本発明者らは、上記の課題を解決するため鋭意検討を進めたところ、
　O-β-D-galactopyranosyl-(1→6)-[O-β-D-galactopyranosyl-(1→4)]-D-glucoseまたは
　O-β-D-galactopyranosyl-(1→4)-[O-β-D-galactopyranosyl-(1→6)]-D-glucoseで表される構造式を有するガラクトオリゴ糖に炎症性サイトカインの分泌抑制を介したアレルギー改善作用があることを見出した。
　すなわち本発明は、以下の様態を含むものである。
　（１）O-β-D-galactopyranosyl-(1→6)-[O-β-D-galactopyranosyl-(1→4)]-D-glucoseまたはO-β-D-galactopyranosyl-(1→4)-[O-β-D-galactopyranosyl-(1→6)]-D-glucoseで表される構造式を有するガラクトオリゴ糖を有効成分とするアレルギー改善剤。
　（２）ガラクトオリゴ糖が哺乳類の乳由来である（１）記載のアレルギー改善剤。
　（３）ガラクトオリゴ糖が牛乳由来である（１）または（２）に記載のアレルギー改善剤。
　（４）（１）乃至（３）のいずれか１つに記載のアレルギー改善剤を含むアレルギー改善用飲食品、アレルギー改善用栄養組成物、アレルギー改善用飼料又はアレルギー改善用医薬品。
　（５）（１）乃至（３）のいずれか１つに記載のアレルギー改善剤を経口摂取することによるアレルギーの改善方法。 The inventors of the present invention have made extensive studies to solve the above problems,
O-β-D-galactopyranosyl- (1 → 6)-[O-β-D-galactopyranosyl- (1 → 4)]-D-glucose or O-β-D-galactopyranosyl- (1 → 4)-[O The present inventors have found that galactooligosaccharides having the structural formula represented by -β-D-galactopyranosyl- (1 → 6)]-D-glucose have an allergy ameliorating effect through suppression of inflammatory cytokine secretion.
That is, the present invention includes the following modes.
(1) O-β-D-galactopyranosyl- (1 → 6)-[O-β-D-galactopyranosyl- (1 → 4)]-D-glucose or O-β-D-galactopyranosyl- (1 → 4) -[O-β-D-galactopyranosyl- (1 → 6)]-D-glucose, an allergy ameliorating agent comprising a galactooligosaccharide having the structural formula represented by
(2) The allergy ameliorating agent according to (1), wherein the galactooligosaccharide is derived from mammalian milk.
(3) The allergy ameliorating agent according to (1) or (2), wherein the galactooligosaccharide is derived from milk.
(4) An allergy-improving food or drink, an allergy-improving nutritional composition, an allergy-improving feed, or an allergy-improving pharmaceutical comprising the allergy-improving agent according to any one of (1) to (3).
(5) A method for improving allergy by orally ingesting the allergy ameliorating agent according to any one of (1) to (3).
　本発明のアレルギー改善剤は、代謝性の炎症を抑制し、炎症性のサイトカインの分泌を抑制することにより、生体内の免疫調節系の乱れを改善する。 The allergy ameliorating agent of the present invention suppresses metabolic inflammation and suppresses secretion of inflammatory cytokines, thereby improving disturbance of the immune regulatory system in the living body.
　以下に、実施形態を挙げて本発明の説明を行うが、本発明は以下の実施形態に限定されるものではない。
　［アレルギー改善剤］
　本件出願のアレルギー改善剤は、O-β-D-galactopyranosyl-(1→6)-[O-β-D-galactopyranosyl-(1→4)]-D-glucoseまたはO-β-D-galactopyranosyl-(1→4)-[O-β-D-galactopyranosyl-(1→6)]-D-glucoseで表される構造式を有するガラクトオリゴ糖を有効成分とする。
　上記ガラクトオリゴ糖を有効成分とするアレルギー改善剤は、ＮＡＤＰＨオキシダーゼやマクロファージなどの炎症性マーカーの上昇を抑制して、炎症性のサイトカインの分泌を抑制する。これにより、アトピー性皮膚炎、アレルギー性鼻炎、蕁麻疹、花粉症などの種々のアレルギー疾患の予防及び／又は治療に有用である。また、リュウマチの予防及び／又は治療にも有用である。
　なお、本件出願に記載の改善剤で使用するガラクトオリゴ糖は、乳糖を原料としているため、簡便且つ経済的に製造することができ、また、有効成分の糖構成が特定されているため、安全性や効力の面で十分な試験や検討が可能となることで、安定性及び安全性に優れている、さらに、優れたアレルギー改善効果を示すとともに、安定性及び安全性に優れているため、食品や医薬への応用が迅速かつ容易になる、といった効果も有するものである。
　有効成分として用いるO-β-D-galactopyranosyl-(1→6)-[O-β-D-galactopyranosyl-(1→4)]-D-glucoseまたはO-β-D-galactopyranosyl-(1→4)-[O-β-D-galactopyranosyl-(1→6)]-D-glucoseで表される構造式を有するガラクトオリゴ糖は、例えば、乳糖を２０％～４０％含有する溶液に、スミラクトＬＬなどの市販のβ－ガラクトシダーゼを数時間反応させた後、減圧濃縮して未反応の乳糖を除去し、得られた溶液を活性炭カラムに通して、吸着したガラクトオリゴ糖を２０％エタノールで溶出することによって得ることができる。また、特許文献４に記載されているように、アスペルギルス属やペニシリウム属、トリコデルマ属に属する乳糖資化性菌を乳糖含量が約３％でｐＨ６の培地で前培養することにより増殖させ、増殖させた該乳糖資化性菌を乳糖含量が約１３％でｐＨ４から９の本培養培地で本培養した後、減圧濃縮して該培養液から未反応の乳糖を除去し、さらにβ－ガラクトシダーゼを作用させて培養液中に残存する未反応の乳糖を分解して得られた溶液を活性炭カラムに通して、吸着したガラクトオリゴ糖を２０％エタノールで溶出することによって得ることができる。なお、この場合、本培養は、２０から４０℃、好ましくは３０℃前後で５から１５日間振とう培養すると収率を一層高めることができる。
　また、上記のように調製したガラクトオリゴ糖は、逆浸透膜（ＲＯ）膜等で濃縮することや、凍結乾燥等により乾燥することも可能である。 Hereinafter, the present invention will be described with reference to embodiments, but the present invention is not limited to the following embodiments.
[Allergy remedy]
The allergy ameliorating agent of the present application is O-β-D-galactopyranosyl- (1 → 6)-[O-β-D-galactopyranosyl- (1 → 4)]-D-glucose or O-β-D-galactopyranosyl- The active ingredient is a galactooligosaccharide having a structural formula represented by (1 → 4)-[O-β-D-galactopyranosyl- (1 → 6)]-D-glucose.
The allergy ameliorating agent containing the galactooligosaccharide as an active ingredient suppresses the increase of inflammatory markers such as NADPH oxidase and macrophages and suppresses the secretion of inflammatory cytokines. Thereby, it is useful for prevention and / or treatment of various allergic diseases such as atopic dermatitis, allergic rhinitis, urticaria and hay fever. It is also useful for the prevention and / or treatment of rheumatism.
The galactooligosaccharide used in the improving agent described in the present application can be easily and economically produced because it uses lactose as a raw material, and because the sugar composition of the active ingredient is specified, it is safe. As a result of being able to conduct sufficient tests and examinations in terms of efficacy and efficacy, it has excellent stability and safety, and also exhibits excellent allergy-improving effects and excellent stability and safety. It also has the effect that application to medicine and medicine becomes quick and easy.
O-β-D-galactopyranosyl- (1 → 6)-[O-β-D-galactopyranosyl- (1 → 4)]-D-glucose or O-β-D-galactopyranosyl- (1 → 4) )-[O-β-D-galactopyranosyl- (1 → 6)]-D-glucose is a galactooligosaccharide having a structural formula represented by, for example, Sumilacto LL in a solution containing 20% to 40% lactose. After reacting commercially available β-galactosidase for several hours, it was concentrated under reduced pressure to remove unreacted lactose, and the resulting solution was passed through an activated carbon column to elute the adsorbed galactooligosaccharide with 20% ethanol. Obtainable. Further, as described in Patent Document 4, a lactose-assimilating bacterium belonging to the genus Aspergillus, Penicillium, or Trichoderma is proliferated by pre-culturing in a medium having a lactose content of about 3% and pH 6. The lactose-assimilating bacterium is main-cultured in a main culture medium having a lactose content of about 13% and a pH of 4 to 9, and then concentrated under reduced pressure to remove unreacted lactose from the culture solution, and further β-galactosidase acts. The solution obtained by decomposing unreacted lactose remaining in the culture solution is passed through an activated carbon column, and the adsorbed galactooligosaccharide is eluted with 20% ethanol. In this case, if the main culture is cultured at 20 to 40 ° C., preferably around 30 ° C. for 5 to 15 days, the yield can be further increased.
In addition, the galactooligosaccharide prepared as described above can be concentrated by a reverse osmosis membrane (RO) membrane or the like, or dried by freeze drying or the like.
　ガラクトオリゴ糖の原料とする乳糖は、ウシやヤギ、ヒツジ、ヒト等の哺乳類の乳から調製した乳糖や、その凝集物、粉末、あるいは精製した乳糖をいい、これを用いてβ－ガラクトシダーゼと反応させる場合や、本培養する場合は水溶液の状態で使用することができる。 Lactose used as a raw material for galactooligosaccharides is lactose prepared from milk from mammals such as cattle, goats, sheep, and humans, and aggregates, powders, or purified lactose, which are used to react with β-galactosidase. In the case of the main culture, it can be used in the state of an aqueous solution.
　［アレルギー改善剤を含有する飲食品、飼料、医薬等のアレルギー改善用製品］
　本願発明の実施態様としては、前述のガラクトオリゴ糖をそのままアレルギー改善剤として使用してもよいが、必要に応じて、常法に従い、粉末剤、顆粒剤、錠剤、カプセル剤、ドリンク剤等に製剤化して用いることも出来る。また、さらに逆浸透膜（ＲＯ）膜処理等により得られたガラクトオリゴ糖についても、そのままアレルギー改善剤として使用することも可能であり、そのまま乾燥しても使用できる。また、常法に従い、製剤化して用いることもできる。
　さらに、これらを製剤化した後に、これを栄養剤やヨーグルト、飲料、ウエハース等の飲食品、栄養組成物、飼料及び医薬品に配合することも可能である。 [Allergy-improving products such as foods, drinks, feeds, and medicines that contain allergic agents]
As an embodiment of the present invention, the above-described galactooligosaccharide may be used as it is as an allergy ameliorating agent, but if necessary, it is formulated into powders, granules, tablets, capsules, drinks, etc. according to conventional methods. It can also be used. Furthermore, galactooligosaccharides obtained by reverse osmosis membrane (RO) membrane treatment or the like can also be used as an allergy ameliorating agent as they are, and can be used as they are after drying. Moreover, it can also be formulated and used according to a conventional method.
Furthermore, after formulating these, it is also possible to mix this with foods and beverages such as nutrients, yogurt, beverages, wafers, nutritional compositions, feeds and pharmaceuticals.
　本明細書における「アレルギー改善用飲食品、アレルギー改善用栄養組成物、アレルギー改善用飼料及びアレルギー改善用医薬品」は、有効成分のガラクトオリゴ糖のみを含む場合の他に、安定剤や糖類、脂質、フレーバー、ビタミン、ミネラル、フラボノイド、ポリフェノール等、他の飲食品、飼料及び医薬に通常含まれる原材料等を含有することができる。また、有効成分のガラクトオリゴ糖に加えて、他のアレルギー改善作用を示す成分、例えば、ラフィノースや酸性キシロオリゴ糖等とともに使用することも可能である。
　また、そのようなアレルギー改善用飲食品、アレルギー改善用栄養組成物、アレルギー改善用飼料又はアレルギー改善用医薬品を原材料として、他の飲食品等に通常含まれる原材料等を配合して調製することも可能である。 In the present specification, "allergy improving food and beverage, allergic improving nutrition composition, allergy improving feed and allergy improving pharmaceutical" include not only the active ingredient galactooligosaccharide, but also stabilizers, saccharides, lipids, Flavors, vitamins, minerals, flavonoids, polyphenols, and other raw materials usually contained in other foods, feeds and medicines can be contained. Further, in addition to the active ingredient galactooligosaccharide, it can also be used together with other components showing an allergy improving action, such as raffinose and acidic xylo-oligosaccharide.
In addition, it may be prepared by blending raw materials usually contained in other foods and drinks, etc., using such allergy improving food and drink, allergy improving nutritional composition, allergy improving feed or allergy improving pharmaceuticals as raw materials. Is possible.
　アレルギー改善用飲食品、アレルギー改善用栄養組成物、アレルギー改善用飼料及びアレルギー改善用医薬品におけるガラクトオリゴ糖の配合量は、特に制限はないが、成人一人一日あたりガラクトオリゴ糖を５０ｍｇ以上経口摂取させるためには、飲食品、飼料及び医薬の形態にもよるが、全質量に対して一般に０．０１～１０％（質量／質量）、好ましくは０．１～５％（質量／質量）含有していることが好ましい。 The compounding amount of galactooligosaccharides in allergic ameliorating foods, allergic ameliorating compositions, allergy ameliorating feeds, and allergy ameliorating pharmaceuticals is not particularly limited. Depending on the form of food, drink, feed and medicine, it generally contains 0.01 to 10% (mass / mass), preferably 0.1 to 5% (mass / mass) with respect to the total mass. Preferably it is.
　本件出願に記載のアレルギー改善剤は、上記の有効成分に適当な助剤を添加して任意の形態に製剤化して、経口投与が可能なアレルギー改善用組成物とすることができる。製剤化に際して、通常使用される充填剤、増量剤、結合剤、崩壊剤、界面活性剤、滑沢剤等の希釈剤又は賦形剤を用いることができる。賦形剤としては、例えばショ糖、乳糖、デンプン、結晶性セルロース、マンニット、軽質無水珪酸、アルミン酸マグネシウム、合成珪酸アルミニウム、メタ珪酸アルミン酸マグネシウム、炭酸カルシウム、炭酸水素ナトリウム、リン酸水素カルシウム、カルボキシルメチルセルロースカルシウム等の１種又は２種以上を組み合わせて加えることができる。 The allergy ameliorating agent described in the present application can be formulated into an arbitrary form by adding an appropriate auxiliary agent to the above active ingredient, and can be made into an allergy ameliorating composition that can be administered orally. In the formulation, diluents or excipients such as fillers, extenders, binders, disintegrants, surfactants, lubricants and the like that are usually used can be used. Examples of the excipient include sucrose, lactose, starch, crystalline cellulose, mannitol, light anhydrous silicic acid, magnesium aluminate, synthetic aluminum silicate, magnesium magnesium metasilicate, calcium carbonate, sodium hydrogen carbonate, calcium hydrogen phosphate One or two or more of carboxymethylcellulose calcium and the like can be added in combination.
　［アレルギーの改善方法］
　本発明は、上述の実施形態のアレルギー改善剤を経口摂取するアレルギーの改善方法にも関する。アレルギーの改善方法は、種々のアレルギー疾患の治療及び／又は予防に有用である。アレルギーの改善方法は、哺乳類、例えば人の他に、イヌ、ネコ等の家畜に効果がある。 [Allergy improvement methods]
The present invention also relates to an allergy ameliorating method in which the allergy ameliorating agent of the above-described embodiment is orally ingested. The allergy ameliorating method is useful for the treatment and / or prevention of various allergic diseases. The allergy ameliorating method is effective for mammals such as humans as well as domestic animals such as dogs and cats.
　以下に実施例、試験例を示し、本発明について詳細に説明するが、これらは単に例示するのみであり、本発明はこれらによって何ら限定されるものではない。 Hereinafter, the present invention will be described in detail with reference to examples and test examples, but these are merely illustrative, and the present invention is not limited by these.
　［実施例１］
　２０％乳糖水溶液５０００ｍｌに市販のβ－ガラクトシダーゼ（商品名：スミラクトＬＬ、新日本化学工業（株））を３単位／ｍｌになるように添加して、４０℃（ｐＨ５．０）で３時間反応させた。この溶液を減圧濃縮して乳糖を析出させて除去して、活性炭カラム（１５ｃｍ×３０ｃｍ）に通液してガラクトオリゴ糖を吸着させた後、５％エタノール３０００ｍｌを通液して、残存している乳糖を除去した。次に、２０％エタノール３０００ｍｌを通液して、得られた溶出画分を減圧濃縮した後、凍結乾燥して有効成分であるガラクトオリゴ糖の粉末（実施例品１）２０５ｇを得た。このガラクトオリゴ糖粉末には、O-β-D-galactopyranosyl-(1→6)-[O-β-D-galactopyranosyl-(1→4)]-D-glucoseまたはO-β-D-galactopyranosyl-(1→4)-[O-β-D-galactopyranosyl-(1→6)]-D-glucoseで表される構造式を有するガラクトオリゴ糖が１２質量％含まれていた。
　このようにして得られたガラクトオリゴ糖は、そのままアレルギー改善剤として使用可能である。 [Example 1]
Commercially available β-galactosidase (trade name: Sumilac LL, Shin Nippon Chemical Industry Co., Ltd.) was added to 5000 ml of 20% lactose aqueous solution at 3 units / ml, and reacted at 40 ° C. (pH 5.0) for 3 hours. I let you. This solution is concentrated under reduced pressure to precipitate and remove lactose, and after passing through an activated carbon column (15 cm × 30 cm) to adsorb galactooligosaccharide, 3000 ml of 5% ethanol is passed through and remains. Lactose was removed. Next, 3000 ml of 20% ethanol was passed through, and the obtained eluted fraction was concentrated under reduced pressure, and then lyophilized to obtain 205 g of an active ingredient galactooligosaccharide powder (Example product 1). This galactooligosaccharide powder includes O-β-D-galactopyranosyl- (1 → 6)-[O-β-D-galactopyranosyl- (1 → 4)]-D-glucose or O-β-D-galactopyranosyl- ( 12% by mass of galactooligosaccharide having a structural formula represented by 1 → 4)-[O-β-D-galactopyranosyl- (1 → 6)]-D-glucose was contained.
The galactooligosaccharide thus obtained can be used as it is as an allergy ameliorating agent.
　［実施例２］
　ペニシリウム・ロックフォルティ（Penicillium roqueforti）IFO 4135株を前培養培地（乳糖２．９％、リン酸水素二カリウム０．１％、硫酸マグネシウム七水和物０．０５％、硝酸ナトリウム０．２％、塩化カリウム０．０５％、硫酸鉄０．００１％）１００ｍｌに接種し、３０℃で５日間培養した。得られた前培養液１００ｍｌを、本培養培地（乳糖１３％、リン酸水素二カリウム０．０９％、硫酸マグネシウム七水和物０．０４％、硝酸ナトリウム０．１７％、塩化カリウム０．０４％、硫酸鉄０．００１％）５０００ｍｌに接種し、３０℃で１０日間振とう培養した。この培養液を遠心分離して菌体を除去した後、減圧濃縮して培養液中の乳糖を析出させて除去した。その溶液を活性炭カラム（１５ｃｍ×３０ｃｍ）に通液してガラクトオリゴ糖を吸着させた後、５％エタノール３０００ｍｌを通液して、残存している乳糖を除去した。次に、２０％エタノール３０００ｍｌを通液して、得られた溶出画分を減圧濃縮した後、凍結乾燥してガラクトオリゴ糖の粉末（実施例品２）５０ｇを得た。このガラクトオリゴ糖粉末には、O-β-D-galactopyranosyl-(1→6)-[O-β-D-galactopyranosyl-(1→4)]-D-glucoseまたはO-β-D-galactopyranosyl-(1→4)-[O-β-D-galactopyranosyl-(1→6)]-D-glucoseで表される構造式を有するガラクトオリゴ糖が９質量％含まれていた。
　このようにして得られたガラクトオリゴ糖は、そのままアレルギー改善剤として使用可能である。 [Example 2]
Penicillium roqueforti IFO 4135 strain was precultured (lactose 2.9%, dipotassium hydrogen phosphate 0.1%, magnesium sulfate heptahydrate 0.05%, sodium nitrate 0.2%, Potassium chloride 0.05%, iron sulfate 0.001%) was inoculated into 100 ml, and cultured at 30 ° C. for 5 days. 100 ml of the obtained preculture solution was added to the main culture medium (lactose 13%, dipotassium hydrogen phosphate 0.09%, magnesium sulfate heptahydrate 0.04%, sodium nitrate 0.17%, potassium chloride 0.04%). %, Iron sulfate 0.001%) was inoculated into 5000 ml and cultured with shaking at 30 ° C. for 10 days. The culture broth was centrifuged to remove the bacterial cells, and then concentrated under reduced pressure to precipitate and remove lactose in the culture broth. The solution was passed through an activated carbon column (15 cm × 30 cm) to adsorb galactooligosaccharide, and then 3000 ml of 5% ethanol was passed through to remove remaining lactose. Next, 3000 ml of 20% ethanol was passed through, and the obtained elution fraction was concentrated under reduced pressure, and then freeze-dried to obtain 50 g of a galactooligosaccharide powder (Example product 2). This galactooligosaccharide powder includes O-β-D-galactopyranosyl- (1 → 6)-[O-β-D-galactopyranosyl- (1 → 4)]-D-glucose or O-β-D-galactopyranosyl- ( 1 → 4)-[O-β-D-galactopyranosyl- (1 → 6)]-D-glucose contained 9% by mass of galactooligosaccharide having the structural formula.
The galactooligosaccharide thus obtained can be used as it is as an allergy ameliorating agent.
　［試験例１］
　(動物実験)
　実施例品１、２のガラクトオリゴ糖を使用して、アレルギー改善作用について評価した。実験には４週齢のＢＡＬＢ／ｃマウスを使用した。マウスの右耳の内側に０．３％の２，４，６－トリニトロ－１－クロロベンゼン（以下「ＴＮＣＢ」という）溶液を１０μｌ塗布して感作させた。１週間後、２から３日おきに０．３％ＴＮＣＢ溶液１０μｌをマウスの右耳の内側に塗布した。ＴＮＣＢ溶液の反復塗布と並行して、Ａ群（対照）、Ｂ群、Ｃ群（各群８匹ずつ）のマウスに、Ａ群には生理食塩水、Ｂ群には実施例品１のガラクトオリゴ糖をマウス体重１ｋｇあたり５０ｍｇ、Ｃ群には実施例品２のガラクトオリゴ糖をマウス体重１ｋｇあたり５０ｍｇを２週間に渡り連日経口投与した。Ｂ群、Ｃ群には実施例品１、２を生理食塩水に懸濁させたものを経口投与した。試験終了時に、マウスの右耳を摘出して質量を測定するとともに、炎症性マーカー（F4/80、P40phox）のmRNAの発現量をリアルタイムＰＣＲ法で測定した。その結果を表１に示す。 [Test Example 1]
(Animal experimentation)
Using the galactooligosaccharides of Example products 1 and 2, the allergy improving action was evaluated. For the experiment, 4-week-old BALB / c mice were used. 10 μl of 0.3% 2,4,6-trinitro-1-chlorobenzene (hereinafter referred to as “TNCB”) solution was applied to the inside of the right ear of the mouse for sensitization. One week later, 10 μl of 0.3% TNCB solution was applied to the inside of the right ear of the mouse every 2 to 3 days. In parallel with repeated application of the TNCB solution, mice in Group A (control), Group B, and Group C (8 mice in each group) were treated with saline in Group A, and the galacto-oligo of Example Product 1 in Group B. Saccharide was orally administered at 50 mg / kg of mouse body weight, and in Group C, galactooligosaccharide of Example Product 2 was orally administered daily for 2 weeks over 2 weeks. In Group B and Group C, Example Products 1 and 2 suspended in physiological saline were orally administered. At the end of the test, the right ear of the mouse was removed and its mass was measured, and the expression level of the inflammatory marker (F4 / 80, P40phox) mRNA was measured by a real-time PCR method. The results are shown in Table 1.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　この結果、２週間投与後の右耳の質量や炎症性マーカー（F4/80、P40phox）のmRNAの発現量は、実施例品１、２のガラクトオリゴ糖をマウス体重１ｋgあたり、それぞれ５０ｍｇ以上経口投与したＢ、Ｃ群では、Ａ群（対照）に比べ、有意に低下していた。この結果から、実施例品１、２のガラクトオリゴ糖にはアレルギー改善作用があることがわかった。また、この作用は実施例品１、２のガラクトオリゴ糖をラット体重１ｋｇ当たり５０ｍｇ以上投与した場合に認められることが明らかとなった。 As a result, the right ear mass and the expression level of inflammatory marker (F4 / 80, P40phox) mRNA after 2 weeks of administration were 50 mg or more of the galactooligosaccharides of Examples 1 and 2 per 1 kg of mouse body weight. The B and C groups were significantly lower than the A group (control). From these results, it was found that the galactooligosaccharides of Examples 1 and 2 had an allergy improving action. Moreover, it became clear that this effect | action is recognized when 50 mg or more of galactooligosaccharides of Example goods 1 and 2 are administered per 1 kg of rat body weight.
　［実施例３］
　（アレルギー改善用錠剤の調製）
　表２に示す配合で、合計が１００．０質量部となるように各原材料を混合後、常法により１ｇに成型、打錠してアレルギー改善用錠剤を製造した。 [Example 3]
(Preparation of tablets for allergy improvement)
Each raw material was mixed so that the total amount was 100.0 parts by mass with the formulation shown in Table 2, and then molded into 1 g by a conventional method and compressed to produce tablets for allergy improvement.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　［実施例４］
　（アレルギー改善用液状栄養組成物の調製）
　実施例品２のガラクトオリゴ糖５０ｇを４，９５０ｇの脱イオン水に溶解し、５０℃まで加熱後、ＴＫホモミクサー（ＴＫ　ＲＯＢＯ　ＭＩＣＳ；特殊機化工業社製）にて、６，０００ｒｐｍで３０分間撹拌混合して実施例品２のガラクトオリゴ糖含量５０ｇ／５ｋｇのガラクトオリゴ糖溶液を得た。このガラクトオリゴ糖溶液５．０ｋｇに、カゼイン５．０ｋｇ、大豆タンパク質５．０ｋｇ、魚油１．０ｋｇ、シソ油３．０ｋｇ、デキストリン１７．０ｋｇ、ミネラル混合物６．０ｋｇ、ビタミン混合物１．９５ｋｇ、乳化剤２．０ｋｇ、安定剤４．０ｋｇ、香料０．０５ｋｇを配合し、２００ｍｌのレトルトパウチに充填し、レトルト殺菌機(第１種圧力容器、ＴＹＰＥ:ＲＣＳ－４ＣＲＴＧＮ、日阪製作所製)で１２１℃、２０分間殺菌して、アレルギー改善用液状栄養組成物５０ｋｇを製造した。 [Example 4]
(Preparation of liquid nutritional composition for allergy improvement)
50 g of the galactooligosaccharide of Example product 2 was dissolved in 4,950 g of deionized water, heated to 50 ° C., and then stirred at 6,000 rpm for 30 minutes with a TK homomixer (TK ROBO MICS; manufactured by Tokushu Kika Kogyo Co., Ltd.). By mixing, a galactooligosaccharide solution of Example Product 2 having a galactooligosaccharide content of 50 g / 5 kg was obtained. 5.0 kg of this galactooligosaccharide solution, 5.0 kg of casein, 5.0 kg of soy protein, 1.0 kg of fish oil, 3.0 kg of perilla oil, 17.0 kg of dextrin, 6.0 kg of mineral mixture, 1.95 kg of vitamin mixture, emulsifier 2 1.0 kg, 4.0 kg stabilizer, 0.05 kg fragrance, and filled in a 200 ml retort pouch, 121 ° C. with a retort sterilizer (type 1 pressure vessel, TYPE: RCS-4CRTGN, manufactured by Nisaka Seisakusho) Sterilized for 20 minutes to produce 50 kg of a liquid nutritional composition for allergy improvement.
　　［実施例５］
　（アレルギー改善用飲料の調製）
　脱脂粉乳３００ｇを４０９ｇの脱イオン水に溶解した後、実施例品１のガラクトオリゴ糖１ｇを溶解し、５０℃まで加熱後、ウルトラディスパーサー（ＵＬＴＲＡ－ＴＵＲＲＡＸＴ－２５；ＩＫＡジャパン社製）にて、９，５００ｒｐｍで３０分間撹拌混合した。マルチトール１００ｇ、酸味料２ｇ、還元水飴２０ｇ、香料２ｇ、脱イオン水１６６ｇを添加した後、１００ｍｌのガラス瓶に充填し、９５℃、１５秒間殺菌後、密栓し、アレルギー改善用飲料１０本（１００ｍｌ入り）を調製した。 [Example 5]
(Preparation of beverage for allergy improvement)
After dissolving 300 g of skim milk powder in 409 g of deionized water, 1 g of the galactooligosaccharide of Example product 1 was dissolved, heated to 50 ° C., and then with an ultradisperser (ULTRA-TURRAX-25; manufactured by IKA Japan). The mixture was stirred and mixed at 9,500 rpm for 30 minutes. After adding maltitol 100g, acidulant 2g, reduced starch syrup 20g, fragrance 2g, deionized water 166g, it is filled into a 100ml glass bottle, sterilized at 95 ° C for 15 seconds, hermetically sealed, and 10 drinks for allergy improvement (100ml) Prepared).
　　［実施例６］
　（イヌ用アレルギー改善飼料の調製）
　実施例品２のガラクトオリゴ糖２ｋｇを９８ｋｇの脱イオン水に溶解し、５０℃まで加熱後、ＴＫホモミクサー（ＭＡＲＫ　ＩＩ　１６０型；特殊機化工業社製）にて、３，６００ｒｐｍで４０分間撹拌混合して実施例品２のガラクトオリゴ糖含量２ｇ／１００ｇのガラクトオリゴ糖溶液を得た。このガラクトオリゴ糖溶液１０ｋｇに大豆粕１２ｋｇ、脱脂粉乳１４ｋｇ、大豆油４ｋｇ、コーン油２ｋｇ、パーム油２３．２ｋｇ、トウモロコシ澱粉１４ｋｇ、小麦粉９ｋｇ、ふすま２ｋｇ、ビタミン混合物５ｋｇ、セルロース２．８ｋｇ、ミネラル混合物２ｋｇを配合し、１２０℃、４分間殺菌して、イヌ用アレルギー改善飼料１００ｋｇを製造した。 [Example 6]
(Preparation of allergy-improving feed for dogs)
2 kg of the galactooligosaccharide of Example product 2 was dissolved in 98 kg of deionized water, heated to 50 ° C., and then stirred and mixed for 40 minutes at 3,600 rpm with a TK homomixer (MARK II 160 type; manufactured by Tokushu Kika Kogyo Co., Ltd.). Thus, a galactooligosaccharide solution of Example Product 2 having a galactooligosaccharide content of 2 g / 100 g was obtained. 10 kg of this galactooligosaccharide solution, 12 kg of soybean meal, 14 kg of skim milk powder, 4 kg of soybean oil, 2 kg of corn oil, 23.2 kg of palm oil, 14 kg of corn starch, 9 kg of wheat flour, 2 kg of bran, 5 kg of vitamin mixture, 2.8 kg of cellulose, 2 kg of mineral mixture Was sterilized at 120 ° C. for 4 minutes to produce 100 kg of an allergy-improving feed for dogs.

Claims (5)

  1. 　O-β-D-galactopyranosyl-(1→6)-[O-β-D-galactopyranosyl-(1→4)]-D-glucoseまたは
    　O-β-D-galactopyranosyl-(1→4)-[O-β-D-galactopyranosyl-(1→6)]-D-glucoseで表される構造式を有するガラクトオリゴ糖を有効成分とするアレルギー改善剤。     O-β-D-galactopyranosyl- (1 → 6)-[O-β-D-galactopyranosyl- (1 → 4)]-D-glucose or O-β-D-galactopyranosyl- (1 → 4)-[O -β-D-galactopyranosyl- (1 → 6)]-D-glucose, an allergy ameliorating agent comprising a galactooligosaccharide having the structural formula represented by
  2. 　前記ガラクトオリゴ糖が、哺乳類の乳由来であることを特徴とする請求項１に記載のアレルギー改善剤。 2. The allergy ameliorating agent according to claim 1, wherein the galactooligosaccharide is derived from mammalian milk.
  3. 　前記ガラクトオリゴ糖が牛乳由来であることを特徴とする請求項１または２に記載のアレルギー改善剤。 The allergy ameliorating agent according to claim 1 or 2, wherein the galactooligosaccharide is derived from milk.
  4. 　請求項１乃至３のいずれか１項に記載の前記アレルギー改善剤を含むことを特徴とするアレルギー改善用飲食品、アレルギー改善用栄養組成物、アレルギー改善用飼料又はアレルギー改善用医薬品。 A food or drink for allergy improvement, a nutrition composition for allergy improvement, a feed for allergy improvement, or a pharmaceutical for allergy improvement, comprising the allergy ameliorating agent according to any one of claims 1 to 3.
  5. 　請求項１乃至３のいずれか１項に記載の前記アレルギー改善剤を経口摂取することを特徴とするアレルギーの改善方法。 An allergy ameliorating method comprising orally ingesting the allergy ameliorating agent according to any one of claims 1 to 3.
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JP2006136240A (en) * 2004-11-12 2006-06-01 Kumamoto Prefecture Bifidobacterium proliferation-promoting composition
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