WO2004071520A1 - Bifidobacterium preparation for improving immune functions - Google Patents

Bifidobacterium preparation for improving immune functions Download PDF

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Publication number
WO2004071520A1
WO2004071520A1 PCT/JP2004/001683 JP2004001683W WO2004071520A1 WO 2004071520 A1 WO2004071520 A1 WO 2004071520A1 JP 2004001683 W JP2004001683 W JP 2004001683W WO 2004071520 A1 WO2004071520 A1 WO 2004071520A1
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stress
fraction
bifidobacteria
preparation
bifidobacterial
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PCT/JP2004/001683
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French (fr)
Japanese (ja)
Inventor
Akira Hosono
Hiroki Kobo
Ryo Nakamura
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The Food Science Institute Foundation
Nihon University
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Publication of WO2004071520A1 publication Critical patent/WO2004071520A1/en

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    • 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
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/745—Bifidobacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • 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
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates to a bifidobacterial preparation that improves the deterioration of the intestinal immune system of animals (including humans) induced by stress.
  • the bifidobacterial preparation of the present invention comprises administering a nutritional product containing a bifidobacterial preparation to an animal (including a human) before, during and / or after a stress event.
  • the present invention also relates to a nutritional product comprising an antioxidant system and a bifidobacterial preparation.
  • stress or stress response a common group of biological reactions to various external harmful factors (stressors), that is, medically physiologically described as general adaptation syndrome, is called stress or stress response. is there.
  • stress a common group of biological reactions to various external harmful factors (stressors), that is, medically physiologically described as general adaptation syndrome, is called stress or stress response.
  • stress Today, however, it is common to see changes in the body due to external factors, including social psychological factors, as “stress”.
  • stress one of the three symptoms of Se lye who advocated the concept of stress was the thymus' lymphatic atrophy. Causes quantitative and qualitative changes to the immune system.
  • the mechanism by which stress causes the quantitative and qualitative changes described above in an individual's immune system has been analyzed in a field called “psycho-neuroimmunology” or “neuroendocrine immunology”.
  • the immune system as a body defense system interacts with the nervous system and the endocrine system, and works to maintain homeostasis through the cross of the so-called “nerve-endocrine-one immune system”. Is becoming clear.
  • the intestinal tract immune system is characterized by IgA secretion to the mucosal surface and plays an important role in infection protection, but the effects of various stresses on this IgA secretion have not been clarified so far.
  • enterobacteria-derived components expected as probiotics have an intestinal immunity-modulating action (eg, Non-Patent Document 3).
  • Bifidobacteriun ⁇ Lactobacillus Some Gram-positive bacteria have functions that increase lymphocyte proliferation activity and IgA production, and are expected to have immunomodulatory effects of probiotics. Anti-tumor effects and adjuvant activities have been reported for Gram-positive bacterial cell wall components such as peptide daricans, but water-soluble high-molecular-weight polysaccharides among ⁇ /// cer / OT cell-derived components It has been reported that the ingredients also have high immunostimulatory action.
  • This active ingredient contains high-molecular polysaccharides composed mainly of glucose and galactose, and the constituent sugars are -4Galp- (or -5Gal i l-), _6Glcp ⁇ , Gal f l_,- Does not contain galactofuranoside such as 6Gal f l-
  • -4Galp- or -5Gal i l-
  • _6Glcp ⁇ Gal f l_
  • Gal f l_ Does not contain galactofuranoside such as 6Gal f l-
  • Non-Patent Document 1 Keller, SE et al: Science. 221: 1301-1303, 1983; Ke Her, SE et al: Proc. Natl. Acad. Sci. USA. 85: 9297-9301, 1988
  • Non-Patent Document 2 Hof iman-Goeiz, L. et al .: I ⁇ unol. Today. 15: 382-387, 1994
  • Non-Patent Document 3 Hosono m: FOOD Style 21 2002. 9 (Vol. 6 No. 9) Disclosure of Invention
  • the present invention provides a bifidobacterial preparation having an action of improving immunity resulting from stress in animals including humans, in particular, a decrease in function of the intestinal tract immune system.
  • the present invention also provides a bifidobacterial preparation having an action of imparting stress resistance to animals including humans. Stress includes excessive physical activity, extreme mental fatigue, and disease state.
  • the present invention was made on the basis of the finding that // ingestion of bacterial components of A3 ⁇ 4 bacteria can improve the functional deterioration of intestinal immunity induced by stress. is there.
  • a bifidobacteria preparation that improves stress resistance and Z or stress-induced dysfunction caused by microbial cells of genus / 7'ffl7,
  • fraction is a cell wall fraction, a cytoplasmic membrane fraction, a protoplast, or a water-soluble fraction
  • BifidobacteriuEM (hereinafter also referred to as “Bifidobacteria”) was studied by Reutei :, Mitsuoka, Scardovi and others, and was classified into 11 species in the 8th edition of Bergey's Manual. Later, classification by genotype using DNA homology was introduced by Scardovi, and many new bacterial species were recognized based on DNA homology. Berg According to the ey's Manual of Systematic Bacter iology (1986), it is classified into 24 species, and 26 species are currently recognized as Bifidobacteriun as shown in Table 1. 1992. Classification and ecology of intestinal flora: p. 55-72.
  • B. bifidum 58 Human infant and adult feces; human vagina; lactating calf feces
  • B. gallicum 61 Human feces also, ⁇ . Adolescent is, Manni] ⁇ 1 and sorbi! ⁇ 1 was divided into four biovars a, b, c, and d depending on the assimilation property. While B. catenulatim, B. angulatunk B. dentium, and Reported as a new strain isolated from human feces, vagina and oral cavity, sewage and calf. As shown in Table 2, these four strains are distinctly different from those of ⁇ zo c / in terms of DNA homology, but their glycolytic properties are considered to be similar (Tomookatsu Mitsuoka) Edit. 199 2. Classification and ecology of intestinal flora: p. 55-72.
  • candidate genus of 77 genera includes all of the bacterial species (live and dead) shown in Table 1 and Table 2. Bifidobacteria culture methods are known
  • bifidobacteria cultures also included are bifidobacteria cultures, purified bifidobacteria cells, freeze-dried cells, or commercially available bifidobacteria preparations. As for bifidobacteria preparations, there are many products that claim 1 X 10 1D Zg or more.
  • the present invention provides a crushed cell body fraction, a cell wall fraction, a pro-plus cocoon, a cytoplasmic membrane fraction, or a water-soluble fraction, and when subjected to various stresses, IgA antibodies in the intestinal mucosal system Including all those having the effect of reducing IgA production and / or mitigation of mitogenic activity on lymphocytes in / or cytokine production.
  • the cell-insoluble fraction containing the soluble cytoplasmic fraction of bifidobacteria and other membrane fractions can be easily separated by ultracentrifugation after sonication of the cells.
  • Preparation of cell wall fractions, protoplasts, cytoplasmic membrane fractions, or water-soluble fractions is well known [for example, Hiroshi Matsuzawa, Suiharu Lee, “Microbial Experiments (Neurochemistry Laboratory)”, edited by Japan Biochemical Society, ( Tokyo Chemical Co., Ltd., p. 179; Japanese Patent No. 3174611; Japanese Unexamined Patent Publication No. 9-241179], and the present invention includes these known techniques.
  • Bifidobacteria are distributed in the digestive tract and faeces of various human animals, the vagina and oral cavity of bees, the digestive tract of bees, and sewage, and it is "sumi" between humans and animal species and between infants and adults. Is recognized. At present, 10 species of bifidobacteria derived from humans have been reported, but in Japan, bifidum, B. infant is, B. breve, B. longum, B. adolescent is 5 species, and other species have not been reported (edited by Tomohiro Mitsuoka. 1992. Classification and ecology of intestinal flora: p. 55-72. Academic Publishing Center).
  • the candidate bifidobacteria are the above-mentioned 10 species resident in chicks, namely B. bifidu m, B. infant is, B. breve, B. long ⁇ B. adolescent is, B. angulatum, B. ca teniilati B. pseudoca tenula turn, B. dentiun ⁇ B. zozo.
  • the target is a domestic cattle
  • the bifidobacteria derived from cattle in Table 1, i.e. B. bifiduE, B. adolescent is, B. pseudoca tenula turn, B. globosum, B.
  • B. animal is ⁇ B. thermophilum and X B. ffi ⁇ If the subject is a chicken, the same idea is applied to B. pseudolong belly, B. animal is, B. thermop. hilum, B. pullorm ⁇ XM ⁇ zo / ⁇ ⁇ are candidates. In addition, bifidobacteria isolated from nature will be candidates.
  • Bifidobacteria of microorganism preservation organizations include: ⁇ . Zo ATCC 15707 T (Intes tin of adult), B. (Intes tine of infant), B. Man ti ski CC 15697 T strain (Intes tine of infant), B. (Intestine of adult), caienuJanAmi (Int. J. Syst. Bact. 24: 6-20, 1974) and the like.
  • the present invention includes such a combination.
  • the preparation containing the disrupted bifidobacteria, cell wall fraction, protoplast, cytoplasmic membrane fraction, or water-soluble fraction of the present invention is administered to animals containing baboons whose immune function has been reduced by stress for the purpose of improving immune function.
  • animals containing baboons whose immune function has been reduced by stress can also be used as a health functional food for humans whose immune function has been reduced by stress.
  • an effective amount thereof can be added to the feed for the purpose of improving immune function.
  • Antibiotics that exert an advantageous effect on immune function such as vitamin A, selenium, vitamin C or] 3-carotene can be added to the bifidobacteria preparation.
  • the effective amount of a bifidobacteria preparation for humans whose immune function has been reduced due to stress is to improve immune function or improve immune function is empirically more than 10 9 viable bacteria per day Since the intestinal regulating effect is recognized in this study, this number is considered to be a standard (Ogata T. et al .: Bioscience Microflora, 16 (2): 5 3-58, 1997).
  • An effective amount of improvement can be determined from known immunological parameters (eg, activation of leukocyte function).
  • the form of the bifidobacteria preparation of the present invention can be, for example, a lump, liquid, syrup, or powder.
  • various additives such as a bulking agent, a sweetener, and other sugars It can contain quality, vitamins, fragrances, coloring agents, etc.
  • Forms include liquid or powder food forms, and pharmaceutical forms such as powders, powders, liquids, suspensions, tablets, and foaming agents.
  • FIG. 1 is a diagram showing the measurement results of blood corticosterone during exercise stress, restraint stress, water invasion restraint stress, and non-stress loading period in BALB / c mice.
  • exercise stress image
  • restraint stress ⁇
  • water invasion restraint stress ⁇
  • non-stress X.
  • Fig. 2 shows the total measurement results in the intestinal contents after 5 days of stress applied to BALB / c mice.
  • Fig. 3 is a diagram showing the results of measuring Peyer's patch cells in the presence of Con A or BP and measuring IgA in the culture supernatant after loading the above-mentioned strains on BALB / c mice for 5 days.
  • Fig. 4 shows that Bifidobacterium pseudocatenulatum l ⁇ W ⁇ (BP) was orally administered at 10 mg / day for 5 days, after restraint stress (5 hours per day) was loaded for 5 consecutive days, and then in the intestinal contents It is a figure which shows the result of having measured the total IgA amount.
  • FIG. 5 is a diagram showing the results of measuring the IFN-a in the culture supernatant of the same Peyer's patch cells cultured in the presence of Con A or BP.
  • Bifidobacterium pseudocatenulatum 7041 (Japan Bifidobacterium Center 1) 3 Anaerobic culture was performed at 7 ° C for 48 hours.
  • the cells are collected by centrifugation (6,000 X g, 5 ° C, 10 minutes), washed 3 times with ultrapure water (6,000 X g, 5 ° C, 10 minutes), and then frozen. Dried and stored at 20 ° C. Freeze-dried cells are suspended in ultrapure water at 4 mg / mL, then sonicated under ice-cooling (Otake, 15 minutes), and further centrifuged at 800X g, 5 ° C for 5 minutes. Metal scraps generated by the treated cells and sonication were removed. This was freeze-dried to obtain cell debris (BP) (BP was stored at -20 ° C until Atsey).
  • BP cell debris
  • mice Female BALB / c mice were purchased at 6 weeks of age (Claire Japan), acclimated for 3 days, and then divided into 4 groups, 8 per group. Next, the exercise stress group only was trained for 5 days from the 4th to the 8th day, with running water swimming exercise (water temperature 32 ° C, 7 L / min for 5 minutes x 3 sets). Each group was stressed under the following conditions for 5 days from 11 to 15 days. During the experimental period (15 days), each mouse was given free access to solid food MF (oriental yeast) for mice and water.
  • solid food MF orientation yeast
  • the stress conditions are exercise stress, water temperature 32 ° C, 7L / miri running water swimming exercise load (10 minX3 set per day), restraint stress (in a 50 mL polypropylene tube with many small holes) Mouse is fixed) Load group (3 hours a day), water invasion restraint stress (Mouse is fixed in a 50 mL polypropylene tube with many small holes, and this is 1 cm in depth, approx. 32 ° C) It was placed in a cage containing a large amount of water). It was classified as a load group (3 hours a day) and a non-stress load group.
  • Blood was collected from the orbital venous plexus for 5 days before stress was applied (Day 10) and during the stress period (Days 11, 12, 13, 14, and 15), and the amount of corticosterone in the plasma was measured using the RIA method. More measured.
  • Goat anti-mouse IgA antibody (ICN Pharmaceuticals) was added to a 96-well microphone mouthplate (MaxSorp, Nunc) and immobilized at 4 ° C overnight. After blocking with urine serum albumin (BSA), a sample diluted with 1% BSA-PBS-Tween solution (sample buffer) to the appropriate magnification was added to each well, and incubated at 4 ° C. . Piotinylated anti-mouse IgA antibody (Sigma) diluted 1,000-fold with sample buffer was added to each well and incubated at room temperature for 2 hours.
  • BSA-PBS-Tween solution sample buffer
  • Streptavidin-alkaline phosphatase complex (Zymed) diluted 1,000-fold with 1% BSA-PBS-Tween solution was added to each well and incubated at room temperature for 1 hour. Then, 0.1% phenyltriphosphate ninatrium-containing diethanolamine buffer was added to each well, incubated at 37 ° C, and the absorbance was measured at 405 nm (BioRad). The total IgA content in the intestinal contents was also measured by the same method.
  • the total IgA content in the intestinal contents was significantly lower in the exercise stress group than in the non-stress group (control group) by 0.05, one-way analysis of variance and Tukey's test.
  • the restraint stress and water invasion restraint stress load groups tended to show lower values than the unstressed group (Fig. 2).
  • mice Female BALB / c mice were purchased at 6 weeks of age (CLEA Japan) and acclimated for 3 days, then 8 animals per group: 1) restraint stress load + BP administration, 2) restraint stress load + BP not applied 3) Restraint stress unloaded + BP administration, and 4) Restraint stress unloaded + BP non-administered.
  • the total amount of IgA in the intestinal contents tended to be higher in the BP administration group than in the BP non-administration group in the restraint stress load group (Fig. 4).
  • the total IgA level in the BP non-administered group in the restraint stress load group was almost the same as the total IgA level in the BP-administered group and the non-BP-administered group in the non-stressed group.
  • the amount of IFN- ⁇ produced by Peyer's patch cells was remarkably increased by administering BP to the restraint stress and non-restraint stress groups.
  • a bifidobacteria preparation useful for improving the functional deterioration of the intestinal tract immune system resulting from stress is provided.
  • the preparation is useful as a health functional food. Stress includes excessive physical exercise, mental extreme fatigue, and disease states.
  • Stress includes excessive physical exercise, mental extreme fatigue, and disease states.
  • a bifidobacterial preparation having the formula:
  • a bifidobacteria preparation having an effect of imparting stress resistance to animals including humans has been provided. Stress includes excessive physical exercise, extreme mental fatigue, and disease state.

Abstract

It is found out that oral administration of Bifidobacterium cell components contributes to the improvement in stress-induced hypofunction of immunity in the intestinal tract.

Description

免疫機能改善を目的としたビフィズス菌製剤 技術分野  Bifidobacteria preparation for improving immune function
本発明は、 ストレスにより誘発された動物 （ヒトを含む） の腸管免疫系の機能 低下を改善するビフィズス菌製剤に関する。 本発明のビフィズス菌製剤は、 ス卜 レス事象の前、 ストレス事象中および/またはストレス事象後に、 ビフィズス菌 製剤を含む栄養製品を動物 （ヒトを含む） に投与することを含む。 本発明はまた、 抗酸化剤系およびビフィズス菌製剤を含む栄養製品にも関する。 背景技術  The present invention relates to a bifidobacterial preparation that improves the deterioration of the intestinal immune system of animals (including humans) induced by stress. The bifidobacterial preparation of the present invention comprises administering a nutritional product containing a bifidobacterial preparation to an animal (including a human) before, during and / or after a stress event. The present invention also relates to a nutritional product comprising an antioxidant system and a bifidobacterial preparation. Background art
元来、 種々の外的有害因子 （ストレッサー） に対する共通した一群の生体反応、 すなわち一般適応症候群 （general adaptat ion syndrome ) として医学的生理学 的に記載されたのがストレスまたはストレス反応とよばれるものである。 しかし、 今日においては社会的心理学的因子を含め広く外的要因による生体の変化を "ス トレス" としてとらえることが一般的である。 1930年代、 ストレスの概念を提唱 した Se lyeのいう 3症候のひとつに、 胸腺 ' リンパ線の萎縮があげられていること からも明らかなように、 心理的倩動因子を含む "ス卜レス" は免疫系に量的質的 変化をもたらす。  Originally, a common group of biological reactions to various external harmful factors (stressors), that is, medically physiologically described as general adaptation syndrome, is called stress or stress response. is there. Today, however, it is common to see changes in the body due to external factors, including social psychological factors, as “stress”. In the 1930s, one of the three symptoms of Se lye who advocated the concept of stress was the thymus' lymphatic atrophy. Causes quantitative and qualitative changes to the immune system.
ストレスによる末梢血白血球数、 リンパ球数、 リンパ球分画の変動については、 これまでの報告は必ずしも一定していない。 古典的にはストレスにより末梢血顆 粒球の増加に伴い総白血球数は増加するが、 リンパ球数は減少すると報告されて いる （例えば非特許文献 1 ) 。 しかし、 運動負荷を物理的ストレスのモデルとし た近年の報告では、 運動負荷時には一過性の末梢血リンパ球 （NK細胞数、 T細胞 数、 B細胞数） 数の増加がみられ、 運動終了後にリンパ球数の減少がみられる （例 えば非特許文献 2 )。 T細胞サブセットの変化についても報告により異なり、 スト レスにより CD4/CD8比は低下するという報告が多いが、 逆に増加するという報告も ある。 Previous reports of changes in peripheral blood leukocyte counts, lymphocyte counts, and lymphocyte fractions due to stress are not necessarily constant. Classically, it has been reported that the total number of white blood cells increases with the increase in peripheral blood condyles due to stress, but the number of lymphocytes decreases (for example, Non-Patent Document 1). However, a recent report using physical stress as a model of physical stress showed that the number of transient peripheral blood lymphocytes (number of NK cells, number of T cells, number of B cells) increased during exercise, and exercise ended A later decrease in lymphocyte counts (eg Non-patent document 2). Changes in T cell subsets also differ depending on the report, and many reports indicate that the CD4 / CD8 ratio decreases with stress, but there are reports that it increases conversely.
一方、 ストレスによる免疫系の機能的変化については、 NK細胞活性、 phytohema gglut inや concanaval in Aなどのマイトジェン刺激によりリンパ球増殖反応、 抗体 産生能などは、 種々のストレスによりいずれも抑制される。  On the other hand, regarding the functional change of the immune system due to stress, NK cell activity, lymphocyte proliferation reaction, antibody production ability, etc. are all suppressed by various stresses by mitogen stimulation such as phytohemagglutin and concanaval in A.
ストレスが個体の免疫系に上記のような量的質的変化を与える機構については、 "精神神経免疫学" あるいは "神経内分泌免疫学" とよばれる分野において解析 が行われている。 それによると、 生体防御系としての免疫系は、 神経系や内分泌 系と相互に影響し合い、 いわゆる 「神経—内分泌一免疫系」 のクロス 1 ^一クを通 して恒常性維持に働くことが明らかになりつつある。 しかしながら、 全身系，末 梢系免疫システムとは異なった誘導 ·制御機構をもつ腸管免疫系に対するストレ スの作用についてはほとんど解析が行われていないのが現状である。 すなわち、 腸管免疫系は粘膜面への IgA分泌が特徴的であり、 感染防御において重要な役割を 果たしているが、 この IgA分泌に対する各種ストレスの影響はこれまで明らかにさ れてない。  The mechanism by which stress causes the quantitative and qualitative changes described above in an individual's immune system has been analyzed in a field called “psycho-neuroimmunology” or “neuroendocrine immunology”. According to it, the immune system as a body defense system interacts with the nervous system and the endocrine system, and works to maintain homeostasis through the cross of the so-called “nerve-endocrine-one immune system”. Is becoming clear. However, at present, little has been analyzed on the effects of stress on the intestinal tract immune system, which has a different induction and control mechanism than the systemic and terminal immune systems. In other words, the intestinal tract immune system is characterized by IgA secretion to the mucosal surface and plays an important role in infection protection, but the effects of various stresses on this IgA secretion have not been clarified so far.
一方、 プロバイオティクスとして期待される腸内細菌由来成分は腸管免疫調節 作用 （例えば非特許文献 3 ) がある。  On the other hand, enterobacteria-derived components expected as probiotics have an intestinal immunity-modulating action (eg, Non-Patent Document 3).
Bifidobacteriun ^Lactobacillus どのグラム陽性菌の中には、 リンパ球の増 殖活性を高めたり、 IgA産生を亢進させるはたらきのあるものが存在し、 プロバイ ォテイクスの免疫調節作用が期待されている。 ペプチドダリカンなどのグラム陽 性菌の菌体壁成分には抗腫瘍効果やアジュバント活性などが報告されているが、 β /// c er/OTの菌体由来成分のうち水溶性高分子多糖成分にも高い免疫賦活作 用があることが報告されている。 この活性成分はグルコースとガラク 1 スを主 成分とする高分子多糖を含み、 その構成糖の結合型は、 - 4Galp- (または- 5Gal i l- ) 、 _6Glcp卜が多く、 Gal f l_、 -6Gal f l-といったガラクトフラノシドも含んでい る。 さらに |S -ダルコシド結合をとる構造と活性との相関も示唆されている。 Bifidobacteriun ^ Lactobacillus Some Gram-positive bacteria have functions that increase lymphocyte proliferation activity and IgA production, and are expected to have immunomodulatory effects of probiotics. Anti-tumor effects and adjuvant activities have been reported for Gram-positive bacterial cell wall components such as peptide daricans, but water-soluble high-molecular-weight polysaccharides among β /// cer / OT cell-derived components It has been reported that the ingredients also have high immunostimulatory action. This active ingredient contains high-molecular polysaccharides composed mainly of glucose and galactose, and the constituent sugars are -4Galp- (or -5Gal i l-), _6Glcp 卜, Gal f l_,- Does not contain galactofuranoside such as 6Gal f l- The Furthermore, there is a suggestion of a correlation between the structure taking the S-darcoside bond and the activity.
上記のマイトジェン活性の高い seudocatenulatum 7041由来菌体成分のマウ スへの 6日間の経口投与によって、 腸管免疫系の誘導部位であるパイエル板の CD4 陽性細胞はインタ一ロイキン- 6 (IL-6) やインタ一フエロン _ γ (IFN- r ) などの サイトカインの産生量が亢進し、 腸管粘膜に外分泌される総 IgA量は増加すること が明らかになつている。 このことから、 菌体成分によって粘膜免疫系への感作が 起こると、 IL- 6によって IgA形質細胞への最終分化の誘導が促進され、 さらに上皮 細胞 （IEC) 側では IFN -ァによってポリ Igレセプター （plgR) の誘導促進がおこり. 結果として管腔側への SIgAの分泌が促進されていることが示唆される。  As a result of oral administration of the above-mentioned seudocatenulatum 7041-derived bacterial component with high mitogenic activity to mice for 6 days, CD4 positive cells on Peyer's patches, which are the induction site of the intestinal tract immune system, become interleukin-6 (IL-6) and It has become clear that the production of cytokines such as interferon_γ (IFN-r) increases, and the total amount of IgA secreted into the intestinal mucosa increases. From this, when sensitization to the mucosal immune system occurs by bacterial components, IL-6 promotes the induction of terminal differentiation into IgA plasma cells, and on the epithelial cell (IEC) side, poly-Ig-a is induced by IFN-a. Induction of receptor (plgR) induction occurred. As a result, secretion of SIgA toward the luminal side was promoted.
さらに、 プロバイオテイクスとして期待される菌体によって腸管内で産生され る代謝産物 （短鎖脂肪酸など） が腸管上皮細胞間リンパ球 （IEC) などに作用して いる可能性も考えられ、 SIgA産生に影響を与えていることが推察される。  Furthermore, there is a possibility that metabolites (such as short-chain fatty acids) produced in the intestine by the cells expected as probiotics may act on intestinal epithelial cell lymphocytes (IEC), etc. It is inferred that it affects
〔非特許文献 1〕 Ke l l er, S. E. et al ： Science. 221 : 1301-1303, 1983 ; Ke Her, S. E. et al. ： Proc. Nat l. Acad. Sci. USA. 85 : 9297-9301, 1988 〔非特許文献 2〕 Hof iman-Goeiz, L. et al. ： I膽 unol. Today. 15 : 382-387, 1994  [Non-Patent Document 1] Keller, SE et al: Science. 221: 1301-1303, 1983; Ke Her, SE et al: Proc. Natl. Acad. Sci. USA. 85: 9297-9301, 1988 [Non-Patent Document 2] Hof iman-Goeiz, L. et al .: I 膽 unol. Today. 15: 382-387, 1994
〔非特許文献 3〕 細野 m ： FOOD Style 21 2002. 9 (Vol. 6 No. 9) 発明の開示  [Non-Patent Document 3] Hosono m: FOOD Style 21 2002. 9 (Vol. 6 No. 9) Disclosure of Invention
本発明は、 ヒトを含む動物における、 ストレスの結果として生じる免疫能、 と りわけ腸管免疫系の機能低下を改善する作用を有するビフィズス菌製剤を提供す る。 また、 本発明は、 ヒトを含む動物に対するストレス抵抗性を付与する作用を 有するビフィズス菌製剤を提供する。 ストレスは、 身体的な過度の運動、 精神的 な極度の疲労、 疾患状態を含む。  The present invention provides a bifidobacterial preparation having an action of improving immunity resulting from stress in animals including humans, in particular, a decrease in function of the intestinal tract immune system. The present invention also provides a bifidobacterial preparation having an action of imparting stress resistance to animals including humans. Stress includes excessive physical activity, extreme mental fatigue, and disease state.
本発明は、 // A¾ 菌の菌体成分を経口摂取すると、 ストレスにより誘 発される腸管免疫の機能低下を改善し得るという知見に基づいてなされたもので ある。 The present invention was made on the basis of the finding that // ingestion of bacterial components of A¾ bacteria can improve the functional deterioration of intestinal immunity induced by stress. is there.
すなわち、 本発明は  That is, the present invention
( 1 ) /7'ffl7属の菌の菌体成分を有効成分とする、 ストレス抵抗性お よび Zまたはストレスにより誘発された免疫能の機能低下を改善するビフ ィズス菌製剤、  (1) A bifidobacteria preparation that improves stress resistance and Z or stress-induced dysfunction caused by microbial cells of genus / 7'ffl7,
(2) 腸管免疫の機能低下である （1) のビフィズス菌製剤、  (2) The bifidobacteria preparation of (1), which is a decrease in intestinal immunity function,
( 3 ) Bifidobac teriun WfiBifidobac terium pseudoca tenula 7041であ る （2) のビフィズス菌製剤、  (3) Bifidobac teriun Wfi Bifidobacterium pseudoca tenula 7041 (2) Bifidobacterium preparation,
(4) 菌体成分が菌体の超音波破砕物および Zまたはその分画物である （1) のビフィズス菌製剤、  (4) The bifidobacteria preparation of (1), wherein the bacterial cell component is an ultrasonic disruption of the bacterial cell and Z or a fraction thereof,
(5) 分画物が細胞壁画分、 細胞質膜画分、 プロトプラスト、 または水溶性画 分である （4) のビフィズス菌製剤、  (5) The bifidobacteria preparation of (4), wherein the fraction is a cell wall fraction, a cytoplasmic membrane fraction, a protoplast, or a water-soluble fraction,
(6) ストレス抵抗性および Zまたはストレスにより誘発された免疫能の機能 低下を改善するビフィズス菌製剤を製造するための、 BUidobacteriimW 菌の菌体成分を有効成分の使用、  (6) Use of a bacterial component of BUidobacteriimW as an active ingredient to produce a bifidobacterial preparation that improves stress resistance and functional decline in immune function induced by Z or stress,
( 7 ) Bifidobacteriui の WfiBifidobacterium pseudocatenulatum 7041であ る (6) の使用、  (7) Use of (6) which is WifBifidobacterium pseudocatenulatum 7041 of Bifidobacteriui,
(8) 菌体成分が菌体の超音波破碎物および Zまたはその分画物である （7) の使用、  (8) Use of (7), wherein the bacterial cell component is an ultrasonic debris of the bacterial cell and Z or a fraction thereof,
(9) 分画物が細胞壁画分、 細胞質膜画分、 プロトプラスト、 または水溶性画 分である （8) の使用、  (9) Use of (8), wherein the fraction is a cell wall fraction, a cytoplasmic membrane fraction, a protoplast, or a water-soluble fraction,
からなる。 Consists of.
BifidobacteriuEM (以下 「ビフィズス菌」 ともいう） 内の菌種については、 Re utei：、 Mitsuoka, Scardoviらによって研究が行われ、 Bergey' s Manualの第 8版で は 11菌種に分類された。 その後、 DNAホモロジ一を用いた遺伝子型による分類が Sc ardoviにより導入され、 DNAホモロジ一を基準に多くの新菌種が認められた。 Berg ey' s Manual of Sys temat ic Bacter iology (1986年） では 24菌種に分類され、 さ らに現在、 表 1に示したように、 Bifidobacteriun として 26菌種が認められてい る （光岡知足編集. 1992. 腸内フローラの分類と生態： p. 55-72 . 学会出版セン 夕一） 。 BifidobacteriuEM (hereinafter also referred to as “Bifidobacteria”) was studied by Reutei :, Mitsuoka, Scardovi and others, and was classified into 11 species in the 8th edition of Bergey's Manual. Later, classification by genotype using DNA homology was introduced by Scardovi, and many new bacterial species were recognized based on DNA homology. Berg According to the ey's Manual of Systematic Bacter iology (1986), it is classified into 24 species, and 26 species are currently recognized as Bifidobacteriun as shown in Table 1. 1992. Classification and ecology of intestinal flora: p. 55-72.
種 DNAの GC% 由来 Origin of GC% of species DNA
B. bifidum 58 ヒト乳児および成人の糞便； ヒト膣；哺乳期の子 牛の糞便  B. bifidum 58 Human infant and adult feces; human vagina; lactating calf feces
B. longum 58 ヒト成人および乳児の类便；（ヒト clinical ) B. longum 58 Human adult and infant stool; (human clinical)
B. infantis 58 ヒト乳児の类便；（ヒト膣 ） B. infantis 58 Human infants; (human vagina)
B. bieve 58 ヒ ト乳児および成人の糞便； （ヒ ト膣および clinical)  B. bieve 58 feces of infants and adults; (human vagina and clinical)
B. adolescentis 58 ヒト成人の I便；下水；（牛のル一メン；サルぉ よびィヌの II便）  B. adolescentis 58 Human adult stool I; sewage; (cow rumen; monkey and inu stool II)
B. angulatum 59 ヒト成人の糞便；下水  B. angulatum 59 Human adult feces; sewage
B. catenulatum 55 ヒ卜成人および乳児の 便； (下水） B. catenulatum 55 stool for adults and infants; (sewage)
B. seudocatenul atum 57.5 ヒト乳児および哺乳期の子牛の类便；下水B. seudocatenul atum 57.5 Human feces and suckling calves; sewage
B. dentium 61 ヒトむしばおよび clinical (ヒト成人および乳児の 糞便；ヒトロ腔および膣） B. dentium 61 human bark and clinical (human adult and infant feces; human lobe and vagina)
B. globosum 64 豚、 哺乳期の子牛、 ラット、 ゥサギおよび子羊の 便；牛のル一メン；（下水）  B. globosum 64 pig, suckling calf, rat, rabbit and lamb stool; cattle rumen; (sewage)
B. pseudolongum 60 牛、 フットおひマウスの炎便  B. pseudolongum 60 cows
B. cumcuh 64 ゥサ の獎便  B. cumcuh 64
B. choerinum 66 子豚の糞便；（下水）  B. choerinum 66 Piglet stool; (sewage)
B. animalis 60 ラット、 鶏、 ゥサギ、 子牛の 便；下水 B. animalis 60 rat, chicken, rabbit, calf stool; sewage
B.thennophilum 60 豚、 子豚、 鶏、 子牛の ¾便、 牛のルーメン；下水B.thennophilum 60 Pig, Piglet, Chicken, Calf ¾ stool, Cattle lumen; Sewage
B. bourn 60 牛のルーメン；子豚の棄便 B. bourn 60 cattle lumen; abandonment of piglets
B. magnum 60 ゥサギの糞便  B. magnum 60 Usagi's feces
B. pullorum 67 鶏の糞便  B. pullorum 67 chicken feces
B. suis 62 子豚の糞便  B. suis 62 Piglet stool
B. minimum 61.5 下水  B. minimum 61.5 sewage
B. subtile 61.5 下水  B. subtile 61.5 Sewage
B. coryneforme Apis mellifera L. supsp. melliieraの腸 Intestine of B. coryneforme Apis mellifera L. supsp. Melliiera
B. asteroides 59 A. mellifera L. supsp. mellifera ligusticaifeよび caucasicaの腸； (A. cerana F. ) B. asteroides 59 A. mellifera L. supsp. Mellifera ligusticaife and caucasica intestines; (A. cerana F.)
B. indicum 60 A. cerana F.および A. dorsata F.の腸 B. indicum 60 A. cerana F. and A. dorsata F. intestines
B. gallinaium 65.7 鶏の盲腸 B. gallinaium 65.7 Chicken cecum
B. gallicum 61 ヒトの糞便 また、 β. adolescent is 、 マンニ] ^一ルとソルビ! ^一ルの資化性により 4つの biovar a、 b、 c、 および dに分けられた。 一方、 B. catenulatim, B. angulatunk B. dentium、 および
Figure imgf000007_0001
らによりヒト糞便、 膣およ び口腔、 下水や子牛から分離される新菌種として報告された。 これらの 4菌種は 表 2に示すように DNAの相同性において^ ゾ c / とは明らかに異なった菌 種であるが、 糖分解性状は類似していることがしられている （光岡知足編集. 199 2. 腸内フローラの分類と生態： p. 55-72 . 学会出版センター） 。 B. gallicum 61 Human feces Also, β. Adolescent is, Manni] ^ 1 and sorbi! ^ 1 was divided into four biovars a, b, c, and d depending on the assimilation property. While B. catenulatim, B. angulatunk B. dentium, and
Figure imgf000007_0001
Reported as a new strain isolated from human feces, vagina and oral cavity, sewage and calf. As shown in Table 2, these four strains are distinctly different from those of ^ zo c / in terms of DNA homology, but their glycolytic properties are considered to be similar (Tomookatsu Mitsuoka) Edit. 199 2. Classification and ecology of intestinal flora: p. 55-72.
表 2  Table 2
DNAホモロジ一 (%) DNA homology (%)
B. adoles- 6. angu6. catenu - B. pseudoca B. dencentis latum latim -tenulatum tium B. adoles- 6. angu6. Catenu-B. pseudoca B. dencentis latum latim -tenulatum tium
DSM ATCC DSM DSM DSM DSM ATCC DSM DSM DSM
20083T 27535T 20103， 「 20438T 20436T 20083 T 27535 T 20103, “20438 T 20436 T
B. adolescentts B. adolescentts
DSM 20083τ 100 14 19 20 17DSM 20083 τ 100 14 19 20 17
B. angulatum B. angulatum
ATCC 27535T 15 100 9 10 8ATCC 27535 T 15 100 9 10 8
B. catenulatim B. catenulatim
DSM 20103T 30 14 100 51 16DSM 20103 T 30 14 100 51 16
B. pseudocatenulatum B. pseudocatenulatum
DSM 20438T 27 11 51 100 14DSM 20438 T 27 11 51 100 14
B. dentium B. dentium
DSM 20436T 20 13 13 16 100 DSM 20436 T 20 13 13 16 100
本発明においては、 候補となる 77 属の菌種は表 1および表 2に示 す菌種 （生菌および死菌） すべてを含む。 ビフィズス菌の培養方法は公知であるIn the present invention, candidate genus of 77 genera includes all of the bacterial species (live and dead) shown in Table 1 and Table 2. Bifidobacteria culture methods are known
〔光岡知足著、 "腸内菌の世界" （叢文社、 1980) 〕 。 また、 ビフィズス菌培養 物、 精製ビフィズス菌体、 凍結乾燥菌体、 あるいは市販のビフィズス菌製剤等も 含む。 ビフィズス菌製剤については、 1 X 101D個 Zg以上を訴求した商品も多くみ られるようになっている。 本発明は、 上記菌体の破砕物、 細胞壁画分、 プロ卜プラス卜、 細胞質膜画分、 または水溶性画分であって、 各種ストレス負荷したとき、 腸管粘膜系における IgA 抗体おょぴ総 IgA産生低下、 ならびに / におけるリンパ球に対するマイトジ ェン作用低下、 あるいはサイトカイン産生低下を抑制する作用を有するものすベ てを含む。 [Tomochika Mitsuoka, “The World of Enteric Bacteria” (Monobunsha, 1980)]. Also included are bifidobacteria cultures, purified bifidobacteria cells, freeze-dried cells, or commercially available bifidobacteria preparations. As for bifidobacteria preparations, there are many products that claim 1 X 10 1D Zg or more. The present invention provides a crushed cell body fraction, a cell wall fraction, a pro-plus cocoon, a cytoplasmic membrane fraction, or a water-soluble fraction, and when subjected to various stresses, IgA antibodies in the intestinal mucosal system Including all those having the effect of reducing IgA production and / or mitigation of mitogenic activity on lymphocytes in / or cytokine production.
ビフィズス菌の可溶性の細胞質画分とそれ以外の膜画分を含む菌体不溶性画分 は、 菌体を超音波処理の後、 超遠心分離により簡便に分けることができる。 細胞 壁画分、 プロトプラスト、 細胞質膜画分、 または水溶性画分の調製は公知 〔例え ば、 松沢 洋， 李泳春， "微生物実験法 （新生化学実験講座） "， （社）日本生化 学会編， （株)東京化学同人， p. 179；特許 3174611号；特開平 9- 241179公開公報〕 であり、 本発明はこれらの公知技術を含む。  The cell-insoluble fraction containing the soluble cytoplasmic fraction of bifidobacteria and other membrane fractions can be easily separated by ultracentrifugation after sonication of the cells. Preparation of cell wall fractions, protoplasts, cytoplasmic membrane fractions, or water-soluble fractions is well known [for example, Hiroshi Matsuzawa, Suiharu Lee, “Microbial Experiments (Neurochemistry Laboratory)”, edited by Japan Biochemical Society, ( Tokyo Chemical Co., Ltd., p. 179; Japanese Patent No. 3174611; Japanese Unexamined Patent Publication No. 9-241179], and the present invention includes these known techniques.
ビフィズス菌は、 ヒトゃ各種動物の消化管や糞便、 ヒ卜の膣や口腔、 ミツバチ の消化管、 下水などに分布し、 ヒトと動物種間や乳児と成人の間には "すみわ け" が認められる。 現在、 ヒト由来ビフィズス菌として 10菌種が報告されている が、 わが国においては、 ヒトから分離されるビフィズス菌種として報告されてい るの bifidum、 B. infant is, B. breve, B. longum, B. adolescent is 5 種であり、 その他の菌種については報告されていない （光岡知足編集. 1992. 腸 内フローラの分類と生態： p. 55-72 . 学会出版センター） 。  Bifidobacteria are distributed in the digestive tract and faeces of various human animals, the vagina and oral cavity of bees, the digestive tract of bees, and sewage, and it is "sumi" between humans and animal species and between infants and adults. Is recognized. At present, 10 species of bifidobacteria derived from humans have been reported, but in Japan, bifidum, B. infant is, B. breve, B. longum, B. adolescent is 5 species, and other species have not been reported (edited by Tomohiro Mitsuoka. 1992. Classification and ecology of intestinal flora: p. 55-72. Academic Publishing Center).
そこで、 ストレスにより誘発される免疫抑制を改善する適用対象がヒ卜の場合 は、 候補となるビフィズス菌はヒ卜に常在する上記 10菌種、 すなわち、 B. bifidu m、 B. infant is, B. breve, B. long鼠 B. adolescent is, B. angulatum, B. ca teniilati B. pseudoca tenula turn, B. dentiun^^ B. ゾゾ あげられる。 また、 対象が家畜の牛を想定する場合は、 表 1の牛由来のビフィズス菌、 すなわ ち、 B. bifiduE, B. adolescent is、 B. pseudoca tenula turn, B. globosum, B. ps eudolongu , B. animal isヽ B. thermophilumお X B. ffi^候補としてあげられ る。 対象が鶏の場合は同様の考えで B. pseudolong腹、 B. animal is、 B. thermop hilum、 B. pullorm^XM ^ゾ / ω^、候補としてあげられる。 さらに今後自然 界から分離されるビフィズス菌も候補となる。 Therefore, if the target of application to improve stress-induced immunosuppression is baboon, the candidate bifidobacteria are the above-mentioned 10 species resident in chicks, namely B. bifidu m, B. infant is, B. breve, B. long 鼠 B. adolescent is, B. angulatum, B. ca teniilati B. pseudoca tenula turn, B. dentiun ^^ B. zozo. If the target is a domestic cattle, the bifidobacteria derived from cattle in Table 1, i.e. B. bifiduE, B. adolescent is, B. pseudoca tenula turn, B. globosum, B. ps eudolongu, B. animal is ヽ B. thermophilum and X B. ffi ^ If the subject is a chicken, the same idea is applied to B. pseudolong belly, B. animal is, B. thermop. hilum, B. pullorm ^ XM ^ zo / ω ^ are candidates. In addition, bifidobacteria isolated from nature will be candidates.
微生物保存機関のビフィズス菌株としては、 Β. ゾ ATCC 15707T株 （Intes t i ne of adul t) 、 B.
Figure imgf000009_0001
(Intes t ine of infant) 、 B. Man ti ski CC 15697T株 (Intes t ine of inf ant) 、 B.
Figure imgf000009_0002
(Intest ine of adul t) 、 および caienuJa nAmi (Int. J. Sys t. Bact. 24 : 6-20, 1974) 等を例示することができる。 Bifidobacteria of microorganism preservation organizations include: Β. Zo ATCC 15707 T (Intes tin of adult), B.
Figure imgf000009_0001
(Intes tine of infant), B. Man ti ski CC 15697 T strain (Intes tine of infant), B.
Figure imgf000009_0002
(Intestine of adult), caienuJanAmi (Int. J. Syst. Bact. 24: 6-20, 1974) and the like.
近年、 ビフィズス菌のなかでも、 さらに菌種により、 あるいは菌株により作用 がすこしずつ異なることが明らかにされてきており、 最近の利用傾向は特徴の異 なる数種の菌を組み合わせて、 より機能性を高めた商品や、 腸内ビフィズス菌の 増殖因子となるオリゴ糖などと組合わせた商品が多くみられるようになつている。 そこで、 本発明はこのような組み合わせを含む。  In recent years, it has been clarified that among Bifidobacteria, the action varies slightly depending on the species or strain, and the recent usage trend is more functional by combining several types of bacteria with different characteristics. There are many products that have been improved in combination with oligosaccharides that are growth factors for intestinal bifidobacteria. Therefore, the present invention includes such a combination.
本発明のビフィズス菌破砕物、 細胞壁画分、 プロトプラスト、 細胞質膜画分、 または水溶性画分を含む製剤は、 ストレスにより免疫機能の低下したヒ卜を含む 動物に免疫機能改善を目的として投与することができる。 また、 ストレスにより 免疫機能の低下したヒトに対して、 保健機能食品として利用することができる。 また、 ストレスにより免疫機能の低下した動物に対して、 免疫機能改善を目的と して、 その有効量を飼料に添加することができる。  The preparation containing the disrupted bifidobacteria, cell wall fraction, protoplast, cytoplasmic membrane fraction, or water-soluble fraction of the present invention is administered to animals containing baboons whose immune function has been reduced by stress for the purpose of improving immune function. be able to. It can also be used as a health functional food for humans whose immune function has been reduced by stress. In addition, for animals whose immune function has been reduced by stress, an effective amount thereof can be added to the feed for the purpose of improving immune function.
ビフィズス菌製剤には、 免疫機能に有利な効果を発揮する抗酸化剤、 例えば、 ビタミン A、 セレン、 ビタミン Cあるいは ]3 -カロチンなどを添加することができる。 ストレスにより免疫機能の低下したヒトが、 免疫機能改善、 あるいは免疫機能 低下の改善を目的して、 ビフィズス菌製剤を摂取する場合の有効量は、 経験的に 1日あたり生菌数が 109以上で整腸効果が認められているので、 この数字は一つの 目安となると考えられる （Ogata T. e t al.： Biosc ience Microf lora, 16 (2)： 5 3-58, 1997 ) が、 免疫機能改善の有効量は公知の免疫学的パラメーター （例えば、 白血球機能の活性化） から決定可能である。 本発明のビフィズス菌製剤の形態は、 例えば、 塊状、 液状、 シロップ状、 粉末 状とすることができ、 これらの形態に応じて、 種々の添加剤、 例えば増量剤、 甘 味剤、 他の糖質、 ビタミン類、 香料、 着色剤等を含有させることがでる。 形態は 液状または粉末の食品形態や、 粉剤、 散剤、 液剤、 懸濁剤、 錠剤、 発泡剤等の医 薬品形態等である。 図面の簡単な説明 Antibiotics that exert an advantageous effect on immune function, such as vitamin A, selenium, vitamin C or] 3-carotene can be added to the bifidobacteria preparation. The effective amount of a bifidobacteria preparation for humans whose immune function has been reduced due to stress is to improve immune function or improve immune function is empirically more than 10 9 viable bacteria per day Since the intestinal regulating effect is recognized in this study, this number is considered to be a standard (Ogata T. et al .: Bioscience Microflora, 16 (2): 5 3-58, 1997). An effective amount of improvement can be determined from known immunological parameters (eg, activation of leukocyte function). The form of the bifidobacteria preparation of the present invention can be, for example, a lump, liquid, syrup, or powder. Depending on these forms, various additives such as a bulking agent, a sweetener, and other sugars It can contain quality, vitamins, fragrances, coloring agents, etc. Forms include liquid or powder food forms, and pharmaceutical forms such as powders, powders, liquids, suspensions, tablets, and foaming agents. Brief Description of Drawings
図 1は、 BALB/cマウスへの運動ストレス、 拘束ストレス、 水侵拘束ストレスお よび非ストレス負荷期間中の血中コルチコステロンの測定結果を示す図である。 ここで、 運動ス卜レス：像、 拘束ストレス：騸、 水侵拘束ストレス▲：非ス卜 レス： Xである。  FIG. 1 is a diagram showing the measurement results of blood corticosterone during exercise stress, restraint stress, water invasion restraint stress, and non-stress loading period in BALB / c mice. Here, exercise stress: image, restraint stress: 騸, water invasion restraint stress ▲: non-stress: X.
図 2は、 BALB/cマウスへの同上ストレスを 5日間負荷後の腸内容物中の総 の 測定結果を示す図である。  Fig. 2 shows the total measurement results in the intestinal contents after 5 days of stress applied to BALB / c mice.
図 3は、 BALB/cマウスへの同上ス卜レスを 5日間負荷後のパイエル板細胞を Con Aあるいは BP存在下で培養し、 その培養上清中の IgAを測定した結果を示す図であ る。  Fig. 3 is a diagram showing the results of measuring Peyer's patch cells in the presence of Con A or BP and measuring IgA in the culture supernatant after loading the above-mentioned strains on BALB / c mice for 5 days. The
図 4は、 Bifidobacterium pseudocatenulatum l ^ W^^ (BP) を 10 mg/ 日、 5日間経口投与したマウスに対し、 拘束ストレス （1日 5時間） を連続 5日間負 荷した後、 腸内容物中総 IgA量を測定した結果を示す図である。  Fig. 4 shows that Bifidobacterium pseudocatenulatum l ^ W ^^ (BP) was orally administered at 10 mg / day for 5 days, after restraint stress (5 hours per day) was loaded for 5 consecutive days, and then in the intestinal contents It is a figure which shows the result of having measured the total IgA amount.
図 5は、 同上におけるパイエル板細胞を Con Aあるいは BP存在下で培養し、 その 培養上清中の IFN-ァを測定した結果を示す図である。 発明を実施するための最良の形態  FIG. 5 is a diagram showing the results of measuring the IFN-a in the culture supernatant of the same Peyer's patch cells cultured in the presence of Con A or BP. BEST MODE FOR CARRYING OUT THE INVENTION
次に、 実施例により本発明を説明する。  Next, an example explains the present invention.
[実施例 1 ] ビフィズス菌の菌体破砕物の調製  [Example 1] Preparation of Bifidobacterium crushed material
Bifidobacterium pseudocatenulatum 7041 〔日本ビフィズス菌センタ一〕 を 3 7°C、 48 時間嫌気培養した。 Bifidobacterium pseudocatenulatum 7041 (Japan Bifidobacterium Center 1) 3 Anaerobic culture was performed at 7 ° C for 48 hours.
培養後菌体を遠心 （6, 000 X g、 5°C、 10分間 ） して回収し、 超純水で 3回洗浄 (6, 000 X g、 5°C、 10分間 ） した後、 凍結乾燥して一 20°Cで保存した。 凍結乾燥 菌体は超純水で 4 mg/mLに懸濁した後、 氷冷却下超音波処理 （大岳製作所製、 15分 間 ） し、 さらに 800X g、 5°C、 5分間の遠心によって未処理の細胞や超音波処理に よって生じた金属くずを除去した。 これを凍結乾燥し、 細胞破碎物 （BP) を得た (BPはアツセィまで- 20°Cで保存） 。  After incubation, the cells are collected by centrifugation (6,000 X g, 5 ° C, 10 minutes), washed 3 times with ultrapure water (6,000 X g, 5 ° C, 10 minutes), and then frozen. Dried and stored at 20 ° C. Freeze-dried cells are suspended in ultrapure water at 4 mg / mL, then sonicated under ice-cooling (Otake, 15 minutes), and further centrifuged at 800X g, 5 ° C for 5 minutes. Metal scraps generated by the treated cells and sonication were removed. This was freeze-dried to obtain cell debris (BP) (BP was stored at -20 ° C until Atsey).
[実施例 2 ] 各種ストレス負荷の腸管免疫に及ぼす影響 [Example 2] Effects of various stress loads on intestinal immunity
雌性 BALB/cマウスを 6週齢で購入 （日本クレア） し、 3日間順化飼育した後、 1 群 8匹として 4群に分けた。 次に運動ストレス群のみ 4〜8日目までの 5日間、 流 水遊泳運動のトレ一ニング （水温 32°C、 7 L/minの流水中にて 5分間 X 3セット ） を行い、 さらに 2日間おいて 11〜15日目の 5日間は以下の条件で各群にそれぞれの ストレスを負荷した。 実験期間中 （15日間） は、 それぞれのマウスにはマウス用 固型飼料 MF (オリエンタル酵母） および水を自由摂取させた。  Female BALB / c mice were purchased at 6 weeks of age (Claire Japan), acclimated for 3 days, and then divided into 4 groups, 8 per group. Next, the exercise stress group only was trained for 5 days from the 4th to the 8th day, with running water swimming exercise (water temperature 32 ° C, 7 L / min for 5 minutes x 3 sets). Each group was stressed under the following conditions for 5 days from 11 to 15 days. During the experimental period (15 days), each mouse was given free access to solid food MF (oriental yeast) for mice and water.
ストレス条件は、 運動ストレスとして水温 32°C、 7L/miriの流水遊泳運動負荷 （1 日あたり 10 minX3セッ卜） 群、 拘束ストレス （小さい穴を多数開けた 50 mL容ポ リプロピレン製チューブ中にマウスを固定） 負荷群 （1日 3時間） 、 さらに、 水侵 拘束ストレス （小さい穴を多数開けた 50 mL容ポリプロピレン製チューブ中にマウ スを固定し、 これを水深 1 cm、 約 32°Cの水をたくわえたケ一ジ内に静置） 負荷群 (1日 3時間） 、 および非ストレス負荷群とした。  The stress conditions are exercise stress, water temperature 32 ° C, 7L / miri running water swimming exercise load (10 minX3 set per day), restraint stress (in a 50 mL polypropylene tube with many small holes) Mouse is fixed) Load group (3 hours a day), water invasion restraint stress (Mouse is fixed in a 50 mL polypropylene tube with many small holes, and this is 1 cm in depth, approx. 32 ° C) It was placed in a cage containing a large amount of water). It was classified as a load group (3 hours a day) and a non-stress load group.
ストレスを負荷する前 （10日目） 、 ストレス負荷期間 （11、 12、 13、 14および 1 5日目） の 5日間、 眼窩静脈叢から採血し、 血漿中のコルチコステロン量を RIA法に より測定した。  Blood was collected from the orbital venous plexus for 5 days before stress was applied (Day 10) and during the stress period (Days 11, 12, 13, 14, and 15), and the amount of corticosterone in the plasma was measured using the RIA method. More measured.
ストレス負荷終了後 （15日目） 、 マウスからパイエル板細胞を採取し、 各種サ ィトカイン刺激による培養条件での培養上清中の総 IgA量を ELISA法により測定し た。 ELISA法の概略は以下のとおりである。 After the end of stress loading (15th day), Peyer's patch cells were collected from mice, and the total IgA content in the culture supernatant under culture conditions stimulated with various cytokines was measured by ELISA. It was. The outline of the ELISA method is as follows.
ャギ抗マウス IgA抗体 （ICN Pharmaceut icals ) を 96ウェルマイク口プレート (MaxSorp, Nunc社製） に添加し、 4°Cで一晩固相化した。 ゥシ血清アルブミン （B SA) でブロッキング後、 1 % BSA- PBS-Tween溶液 （サンプルバッファ一） で適当な 倍率に希釈した測定サンプルを各ゥエルに添加し、 4 °Cでー晚ィンキュベーショ ンした。 サンプルバッファ一で 1, 000倍に希釈したピオチン化抗マウス IgA抗体 (S igma) を各ゥエルに添加し、 室温で 2時間インキュベーションした。 1 % BSA- PBS - Tween溶液で 1， 000倍に希釈したストレプトアビジン-アルカリホスファタ一ゼ複合 体 （Zymed) を各ゥエルに添加し、 室温で 1時間インキュベーションした。 そして 0. 1 %フエ二ルリン酸ニナトリゥム含ジエタノールァミン緩衝液を各ゥエルに添加し、 37°Cでインキュベーション後、 405 nmにて吸光値を測定した （BioRad) 。 腸内容 物中の総 IgA量についても同様の方法で測定した。  Goat anti-mouse IgA antibody (ICN Pharmaceuticals) was added to a 96-well microphone mouthplate (MaxSorp, Nunc) and immobilized at 4 ° C overnight. After blocking with urine serum albumin (BSA), a sample diluted with 1% BSA-PBS-Tween solution (sample buffer) to the appropriate magnification was added to each well, and incubated at 4 ° C. . Piotinylated anti-mouse IgA antibody (Sigma) diluted 1,000-fold with sample buffer was added to each well and incubated at room temperature for 2 hours. Streptavidin-alkaline phosphatase complex (Zymed) diluted 1,000-fold with 1% BSA-PBS-Tween solution was added to each well and incubated at room temperature for 1 hour. Then, 0.1% phenyltriphosphate ninatrium-containing diethanolamine buffer was added to each well, incubated at 37 ° C, and the absorbance was measured at 405 nm (BioRad). The total IgA content in the intestinal contents was also measured by the same method.
血中コルチコステロン量は、 運動ストレス、 拘束ストレス、 および水侵拘束ス トレス負荷群において、 いずれも非ストレス群 （対照群） に比べて高いレベルを 維持したが、 ストレスの種類による大きな差異は認められなかった （図 1 ) 。  Blood corticosterone levels remained high in the exercise stress, restraint stress, and water stress restraint stress groups compared to the non-stress group (control group), but there were significant differences depending on the type of stress. It was not recognized (Fig. 1).
腸内容物中の総 IgA量は、 運動ストレス負荷群において、 非ストレス群 （対照 群） に対して有意 ぐ 0. 05、 一元配置分散分析および Tukey s test ) に低下し た。 拘束ストレスおよび水侵拘束ストレス負荷群においては、 非ストレス群に対 して低値を示す傾向がみられた （図 2 ) 。  The total IgA content in the intestinal contents was significantly lower in the exercise stress group than in the non-stress group (control group) by 0.05, one-way analysis of variance and Tukey's test. The restraint stress and water invasion restraint stress load groups tended to show lower values than the unstressed group (Fig. 2).
パイエル板細胞の IgA産生量は、 運動ス卜レス、 拘束ストレス群が非ストレス群 に対して顕著に低下する傾向が認められた （図 3 ) 。  The IgA production of Peyer's patch cells tended to decrease markedly in the motor stress and restraint stress group compared to the non-stress group (Fig. 3).
以上の結果から、 マウスに運動ストレスあるいは拘束ストレスを負荷すると、 血中コルチコステロンレベルが上昇し、 腸管免疫系における総 IgA分泌が抑制され ることが明らかとなった。  From the above results, it was clarified that when exercise stress or restraint stress is applied to mice, the blood corticosterone level increases and total IgA secretion in the intestinal tract immune system is suppressed.
[実施例 3 ] ストレス負荷による腸管免疫機能低下に対するビフィズス菌体破 砕物の効果 [Example 3] Bifidobacteria destruction for intestinal immune function decline due to stress load Crushed effect
雌性 BALB/cマウスを 6週齢で購入 （日本クレア） し、 3日間順化飼育した後、 1 群 8匹として、 1 ) 拘束ストレス負荷 +BP投与、 2 ) 拘束ストレス負荷 +BP非投 与、 3 ) 拘束ストレス非負荷 +BP投与、 および 4 ) 拘束ストレス非負荷 +BP非投 与の 4群に分けた。  Female BALB / c mice were purchased at 6 weeks of age (CLEA Japan) and acclimated for 3 days, then 8 animals per group: 1) restraint stress load + BP administration, 2) restraint stress load + BP not applied 3) Restraint stress unloaded + BP administration, and 4) Restraint stress unloaded + BP non-administered.
実施例 1で調製した BPを 10 mg/日、 9日間あらかじめ経口投与したマウスに対し、 さらに BPの経口投与とともに拘束ストレス （1日 5時間） を連続 5日間負荷した （こ の場合、 BPを 14日間投与し、 投与期間中の最後の 5日間にストレスを負荷した） 。 実験終了後、 実施例 2と同様に、 腸内容物中総 IgA量、 およびパイエル板細胞の 培養上清中の IFN-ァ量を ELISA法にて測定した。 測定法は実施例 2と同様に行い、 このときの捕捉用抗体はラット抗マウス IFN-ァ抗体、 検出用抗体をピオチン標識 ラット抗マウス用 IFN-ァ抗体 （いずれも PharMingen) を用いた。  In addition to oral administration of BP, restraint stress (5 hours per day) was further applied to mice that had been orally administered with BP prepared in Example 1 at 10 mg / day for 9 days in advance. For 14 days and stressed during the last 5 days of administration). After the experiment was completed, in the same manner as in Example 2, the total amount of IgA in the intestinal contents and the amount of IFN-a in the culture supernatant of Peyer's patch cells were measured by ELISA. The measurement was carried out in the same manner as in Example 2. At this time, the capture antibody was rat anti-mouse IFN-a antibody, and the detection antibody was piotin-labeled rat anti-mouse IFN-a antibody (both PharMingen).
腸内容物中の総 IgA量は、 拘束ストレス負荷群において、 BP投与群が BP非投与群 に対して高値を示す傾向が認められた （図 4 ) 。 また、 拘束ストレス負荷群にお ける BP非投与群の総 IgA量は、 非ス卜レス負荷群における BP投与群および BP非投与 群の総 IgA量とほぼ同じレベルであった。  The total amount of IgA in the intestinal contents tended to be higher in the BP administration group than in the BP non-administration group in the restraint stress load group (Fig. 4). The total IgA level in the BP non-administered group in the restraint stress load group was almost the same as the total IgA level in the BP-administered group and the non-BP-administered group in the non-stressed group.
一方、 パイエル板細胞の IFN- τ産生量は、 拘束ストレス負荷群および非拘束ス トレス負荷群に対して BPを投与することにより、 顕著に亢進した。  On the other hand, the amount of IFN-τ produced by Peyer's patch cells was remarkably increased by administering BP to the restraint stress and non-restraint stress groups.
以上の結果から、 BPの経口投与は、 ストレス負荷による腸管粘膜における IgA産 生の低下を抑制することが明らかとなった。 これは、 BP投与によって IFN-ァ産生 応答が亢進し、 分泌型 I gA産生が高まることによるものと考えられる。  From the above results, it was clarified that oral administration of BP suppresses the decrease in IgA production in the intestinal mucosa caused by stress. This is thought to be because IFN-a production response is enhanced by BP administration and secretory IgA production is increased.
すなわち、 Biiidobacteriwn pseudocatenulatum 7041の菌体破碎物の経口摂取 は、 ストレスによる腸管免疫の低下を改善することが明らかとなった。  In other words, it was clarified that oral intake of Biiidobacteriwn pseudocatenulatum 7041 cell disruption improves the decrease in intestinal immunity caused by stress.
このことは、 プロバイオティクスとして期待される / // 菌体成分が、 ストレスによる腸管免疫系の機能低下を改善化できることを示すものである。 産業上の利用の可能性 This indicates that the probiotics / // mycelium component can improve the functional deterioration of the intestinal tract immune system due to stress. Industrial applicability
本発明により、 ストレスの結果として生じる腸管免疫系の機能低下を改善する のに有用なビフィズス菌製剤が提供された。 該製剤は保健機能食品として有用で ある。 ストレスは、 身体的な過度の運動、 精神的な極度の疲労、 疾患状態を含む 本発明により、 ヒトを含む動物における、 ストレスの結果として生じる免疫能、 とりわけ腸管免疫系の機能低下を改善する作用を有するビフィズス菌製剤が提供 された。 また、 ヒトを含む動物に対するストレス抵抗性を付与する作用を有する ビフィズス菌製剤が提供された。 ストレスは、 身体的な過度の運動、 精神的な極 度の疲労、 疾患状態を含む。  According to the present invention, a bifidobacteria preparation useful for improving the functional deterioration of the intestinal tract immune system resulting from stress is provided. The preparation is useful as a health functional food. Stress includes excessive physical exercise, mental extreme fatigue, and disease states.According to the present invention, the ability to improve immunity resulting from stress in animals including humans, in particular, the functional deterioration of the intestinal tract immune system is improved. A bifidobacterial preparation having the formula: In addition, a bifidobacteria preparation having an effect of imparting stress resistance to animals including humans has been provided. Stress includes excessive physical exercise, extreme mental fatigue, and disease state.

Claims

請求の範囲 The scope of the claims
1 . /// / ffi!癘の菌の菌体成分を有効成分とする、 ス卜レス抵抗性およ び/またはス卜レスにより誘発された免疫能の機能低下を改善するビフィズ ス菌製剤。 1. /// / ffi! Bifidobacteria preparations that improve the resistance to stress and / or the decline in immune function induced by stress, using as an active ingredient the fungus body component .
2 . 腸管免疫の機能低下である請求項 1記載のビフィズス菌製剤。  2. The bifidobacterial preparation according to claim 1, which is a decrease in intestinal immunity function.
3 . Bifidobacteriun o ifidobacieriwn pseudoca tenula turn 7041である 請求項 2記載のビフィズス菌製剤。  3. The bifidobacteria preparation according to claim 2, which is Bifidobacteriun o ifidobacieriwn pseudoca tenula turn 7041.
4. 菌体成分が菌体の超音波破砕物および Zまたはその分画物である請求項 1 記載のビフィズス菌製剤。  4. The bifidobacterial preparation according to claim 1, wherein the bacterial cell component is an ultrasonic disruption product and Z or a fraction thereof.
5 . 分画物が細胞壁画分、 細胞質膜画分、 プロトプラスト、 または水溶性画分 である請求項 4記載のビフィズス菌製剤。  5. The bifidobacterial preparation according to claim 4, wherein the fraction is a cell wall fraction, a cytoplasmic membrane fraction, a protoplast, or a water-soluble fraction.
6 . ストレス抵抗性および Zまたはストレスにより誘発された免疫能の機能低 下を改善するビフィズス菌製剤を製造するための、 Bifidobacteriu の の 菌体成分を有効成分の使用。  6. Use of Bifidobacteriu's bacterial component as an active ingredient to produce a bifidobacteria preparation that improves stress resistance and reduced immune function induced by Z or stress.
7 . BifidobacteriumWiyWfiBifidobacteriwn pseudoca tenula tuin 7041である 請求項 6記載の使用。  7. Use according to claim 6, which is Bifidobacterium WiyWfi Bifidobacteriwn pseudoca tenula tuin 7041.
8 . 菌体成分が菌体の超音波破碎物および Zまたはその分画物である請求項 7 記載の使用。  8. The use according to claim 7, wherein the bacterial cell component is an ultrasonic lysate of the bacterial cell and Z or a fraction thereof.
9 . 分画物が細胞壁画分、 細胞質膜画分、 プロトプラスト、 または水溶性画分 である請求項 8記載の使用。 9. The use according to claim 8, wherein the fraction is a cell wall fraction, a cytoplasmic membrane fraction, a protoplast, or a water-soluble fraction.
PCT/JP2004/001683 2003-02-17 2004-02-17 Bifidobacterium preparation for improving immune functions WO2004071520A1 (en)

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