WO2008023662A1 - Bacterial strain having immunostimulating activity, and beverage, food and immunostimulating agent comprising the bacterial strain - Google Patents

Bacterial strain having immunostimulating activity, and beverage, food and immunostimulating agent comprising the bacterial strain Download PDF

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Publication number
WO2008023662A1
WO2008023662A1 PCT/JP2007/066120 JP2007066120W WO2008023662A1 WO 2008023662 A1 WO2008023662 A1 WO 2008023662A1 JP 2007066120 W JP2007066120 W JP 2007066120W WO 2008023662 A1 WO2008023662 A1 WO 2008023662A1
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strain
brevis
bacterial strain
strains
belonging
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PCT/JP2007/066120
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French (fr)
Japanese (ja)
Inventor
Syuichi Segawa
Yasukazu Nakakita
Yoshihiro Takata
Hisako Yasui
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Sapporo Breweries Limited
Shinshu University
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Priority to JP2008530893A priority Critical patent/JPWO2008023662A1/en
Publication of WO2008023662A1 publication Critical patent/WO2008023662A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/005—Amino acids other than alpha- or beta amino acids, e.g. gamma amino acids
    • 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
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • 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
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • 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
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/52—Propionic acid; Butyric acids
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/225—Lactobacillus
    • C12R2001/24—Lactobacillus brevis

Definitions

  • Bacterial strain having immunostimulating action and beverage, food and immunostimulant containing the bacterial strain having immunostimulating action and beverage, food and immunostimulant containing the bacterial strain
  • the present invention relates to a strain having an immunostimulatory action, a beverage, a food and an immunostimulant containing the strain.
  • an immunostimulant is effective for enhancing immune function in the living body and maintaining health, and has been performed so far.
  • immunostimulants produced by microorganisms, mushrooms or plants are known as immunostimulants that have been confirmed to be effective with low toxicity.
  • cell wall polysaccharides of microorganisms, or polysaccharides derived from mushrooms such as Agarita sashitake (Patent Documents 1 and 2).
  • polysaccharides having a dalcoside bond have been disclosed as being particularly effective (Patent Documents 3, 4 and 5).
  • Patent Document 1 Japanese Patent Publication No.49-484
  • Patent Document 2 Japanese Patent Publication No. 56-46481
  • Patent Document 3 Japanese Patent Laid-Open No. 7-184595
  • Patent Document 4 Japanese Patent Laid-Open No. 10-194977
  • Patent Document 5 Japanese Patent Laid-Open No. 2005-220065
  • drugs such as immunomodulators and immunostimulants act directly on molecules involved in the immune response in the blood and affect the immune response necessary for maintaining homeostasis.
  • immunostimulants derived from lactic acid bacteria that have been reported so far have been used in the prevention and treatment of lifestyle-related diseases such as cancers, infectious diseases, and allergic diseases that weaken the effect of activating mucosal immunity. The effect was insufficient.
  • GABA ⁇ -aminobutyric acid
  • an object of the present invention is to provide a strain belonging to Lactobacillus brevis subspecies brevis, which has both an immunostimulatory action and the ability to produce ⁇ -aminobutyric acid (GABA).
  • Another object of the present invention is to provide beverages and foods containing the bacterial cells of the strain belonging to this ratatobatilla brevis subspecies brevis, and an immunostimulant containing these as active ingredients.
  • the present invention has been described in the following.
  • the present invention has been developed in a fizzy teno / nocolle beverage, in a difficult manner, such as Lactobacillus brevis subspecies brevis.
  • a strain capable of producing 7-aminobutyric acid (GABA) and having an immunostimulatory effect is provided.
  • Lactobacillus brevis lactic acid bacteria belonging to subspecies brevis, particularly effectively induces production of IgA from mouse Peyer's patches.
  • This IgA production promoting action activates mucosal immunity and is useful for maintaining and improving the body defense function.
  • Lactic acid bacteria are bacteria that have been used for fermented foods for a long time, and are much safer for the human body than chemically synthesized immunostimulants.
  • normal lactic acid bacteria cannot grow in effervescent alcoholic beverages, but the strains of the present invention can grow in foaming alcoholic beverages. Other lactic acid strains that do not have an immunostimulatory effect can be excluded.
  • the strain of the present invention produces 7-aminobutyric acid (GABA), it has an anti-stress action in addition to the above-mentioned immunostimulatory action, and it has cancer and correlates with physical stress and mental stress. Strong effects can be expected for the prevention and treatment of infectious diseases.
  • GABA 7-aminobutyric acid
  • Such strains include Ratatova SBC8044 (FERM BP—10631) force S of Chilas brevis subspecies brevis is particularly preferred.
  • the immunostimulatory action is preferably an IgA production promoting action.
  • IgA is one of immunoglobulins contained mainly in secretions such as intestinal mucus, saliva, tears, and breast milk, and plays an important role as a local defense mechanism of mucosa. Since the bacterial strain of the present invention can promote the production of IgA, it can enhance the local defense mechanism of mucosa and can be used for the prevention and treatment of lifestyle diseases such as infectious diseases and allergic diseases.
  • the present invention also provides beverages and foods containing the cells of the above strain.
  • the bacterial cells of the above strains have an immunostimulatory action and are safe for the human body, so that they can be used as beverages and foods as health food materials. Furthermore, since the above strain produces ⁇ -aminobutyric acid (GABA), it has anti-stress, blood pressure lowering and tranquilizing effects and is highly useful as a health food material.
  • GABA ⁇ -aminobutyric acid
  • the present invention also provides an immunostimulant containing the bacterial cells of the strain as an active ingredient.
  • the bacterial cells of the above strains have an IgA production promoting action, if an immunostimulant containing this bacterial cell as an active ingredient is produced, immunity superior in safety to chemically synthesized drugs is produced. It can be used as an activator.
  • the present invention it is possible to provide a strain belonging to Lactobacillus brevis subspecies brevis, which has a stronger immunostimulatory effect than lactic acid strains known so far. Moreover, since the strain of the present invention can grow in an effervescent alcoholic beverage, other lactic acid strains having no immunostimulatory action can be excluded from the strain of the present invention using this property. Furthermore, since the strain of the present invention produces ⁇ -aminobutyric acid (GABA), it has an anti-stress action in addition to an immunostimulatory action, and is associated with cancer and infection that are correlated with physical stress and mental stress. It is expected to have a strong effect on prevention and treatment of lifestyle-related diseases such as allergies and allergic diseases. In addition, according to the present invention, beverages, foods, and immunostimulants that contain the bacterial cells of the above strain, have excellent safety, and have an immunostimulatory action can be provided.
  • GABA ⁇ -aminobutyric acid
  • FIG.1 Ratatobatirus' brevis subsp. It shows the amount of IgA produced by mouse Peyer's patch cells.
  • the strain of the present invention is a strain belonging to Lactobacillus brevis subspecies brevis, is capable of growing in a sparkling alcoholic beverage, and produces ⁇ -aminobutyric acid (GABA). It has an immunostimulatory effect.
  • GABA ⁇ -aminobutyric acid
  • subspecies mosquitoes in Lactobacillus brevis which are brevis, gravesensis, otakiensis, and coagulans, respectively.
  • the strain belongs to the subspecies Brevis. Strains belonging to subspecies brevis can be separated by using the base sequence and sugar strength of 16S ribosomal DNA, differences in acid production, etc. as indicators, and can be classified as strains not belonging to subspecies grebsensis, subspecies otachysis and subspecies coagulance.
  • Immunostimulatory action refers to strengthening the immunity of a living body to provide resistance to diseases.
  • macrophages, neutrophils, NK cells and the like are activated to enhance the innate immune function and promote the production of immunoglobulins such as IgA.
  • IgA production promoting action can enhance mucosal immunity, and thus can be expected to have a strong effect on the prevention and treatment of lifestyle diseases such as infectious diseases and allergic diseases.
  • “Effervescent alcoholic beverages” include, for example, beer, miscellaneous sake, and happoshu. “Can grow in sparkling alcoholic beverages” means that lactic acid bacteria do not die in sparkling alcoholic beverages, and the number of cells increases due to cell division. The alcohol concentration of sparkling alcoholic beverages is 5 % Or more is preferable.
  • strains belonging to Ratatobacillus brevis subspecies brevis capable of growing in effervescent alcoholic beverages can be selected according to the method described in JP-A-2003-250557.
  • the strain of the strain belonging to Lactobacillus brevis subspecies Brevis Perform PCR with a specified primer set (primer set consisting of oligonucleotides consisting of the nucleic acid sequences described in SEQ ID NO: 1 and SEQ ID NO: 2 in JP-A-2003-250557) using Nom DNA as a saddle type!
  • the amplified DNA gyrase subunit B gene fragment may be cleaved with a restriction enzyme, the restriction enzyme cleave pattern after acrylamide gel electrophoresis may be analyzed, and a strain belonging to the group lib may be selected. Restriction enzyme cleavage patterns are classified into four groups, and the strains that can grow in sparkling alcoholic beverages have been found to belong to the group lib.
  • the strain can also be selected by examining the possibility of growth by transplanting and culturing a strain belonging to Lactobacillus brevis subspecies brevis in an effervescent alcoholic beverage.
  • the culture temperature is a suitable force at 30 ° C 15 ° C-45 ° C, preferably 20 ° C-37 ° C, especially
  • a strain producing ABA can be selected by analyzing the culture supernatant with an amino acid analyzer or the like and examining the GABA content.
  • a strain having an immunostimulatory effect can be selected by, for example, examining the IgA production promoting effect on mouse Peyer's patch cells.
  • Peyer's patch cells were isolated from the intestinal tract of the mouse and cultured, and a cell suspension prepared by sterilizing the cells of the test strain was added thereto and cultured for a certain period of time. It is sufficient to measure the production amount of IgA secreted from the plate cells by ELISA or the like and examine the presence or absence of an IgA production promoting action.
  • SBC8044 belonging to Ratatobacillus brevis subspecies brevis which can grow in sparkling alcoholic beverages, produce GABA, and has an immunostimulatory effect (date of contract: June 28, 2006; contract) No .: FERM BP—10631) is the International Depository Authority, National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (1st, 1st, Tsukuba, Higashi, Ibaraki, Japan, 1st 6th (zip code 305-8566)) And are available.
  • the beverage, food and immunostimulant of the present invention are strains belonging to Ratatobacillus' brevis subsp. Brevis, can be grown in an effervescent alcoholic beverage, produce GABA, It contains the above strain having an immunostimulatory effect.
  • the bacterial cells of the strain can be added to beverages and foods for the purpose of preventing and treating cancer and infectious diseases.
  • the said drink and food may consist only of a microbial cell of said strain.
  • area may be included.
  • the additives include apple fiber, soybean fiber, meat extract, black vinegar extract, gelatin, corn starch, honey, animal and vegetable oils and fats, monosaccharides such as glucose, disaccharides such as sucrose, fructose and mannitol, dextrose And polysaccharides such as starch, sugar alcohols such as erythritol, xylitol, and sorbitol, and vitamins such as vitamin C. These additives may be used alone or in combination.
  • the immunostimulatory agent of the present invention contains a microbial cell of the above-mentioned strain as an active ingredient, and if formulated by adding a carrier, an excipient and / or other additives, an immunostimulator having excellent safety It can be used as an agent.
  • Pharmaceutically acceptable additives include, for example, monosaccharides such as glucose, disaccharides such as sucrose, fructose and mannitol, polysaccharides such as dextrose and starch, sugar alcohols such as erythritol, xylitol and sorbitol, Vitamins such as vitamin C, gum acacia, calcium phosphate, alginate, calcium silicate, microcrystalline cellulose, polybulurpyrrolidone, cellulose derivatives, tragacanth, gelatin, syrup, methyl hydroxybenzoate, talc, magnesium stearate, water, Mineral oil is mentioned, and these additives may be single species or plural species.
  • monosaccharides such as glucose
  • disaccharides such as sucrose, fructose and mannitol
  • polysaccharides such as dextrose and starch
  • sugar alcohols such as erythritol, xylitol and sorbitol
  • Vitamins such as vitamin
  • the growth ability in beer was selected.
  • the selection method was performed according to the method described in Japanese Patent Application Laid-Open No. 2003-250557. That is, an oligo consisting of a nucleic acid sequence described in SEQ ID NO: 1 and SEQ ID NO: 2 in the sequence table of JP 2003-250557 A using the genomic DNA of a strain belonging to Lactobacillus brevis subsp.
  • restriction enzyme cleavage patterns can be broadly classified into four groups, and strains that can grow in beer have been found to belong to the group lib.
  • composition 1% proteose peptone, 1% beef extract, 0.5% yeast extract, 2% glucose, 0.1% Tween 80, 0.5% ammonium citrate, 0.01% (Magnesium sulfate, 0.005% manganese sulfate, 0.2% dipotassium phosphate) for 3 days. Centrifuge the culture at 1,500 rpm for 10 minutes to recover the cells of each strain. did. The obtained bacterial cells were washed with PBS, freeze-dried, and suspended in PBS so that the concentration was I mg / mL. The bacterial cell suspension thus obtained was autoclaved (121 ° C, 15 minutes) and used in the following experiments.
  • the intestinal tract was aseptically removed from a 6-week-old BALB mouse (female), and the Peyer's board was removed from it with scissors and immersed in RPMI1640 (GIBCO). Wash the Peyer plate thoroughly by repeatedly sucking the Peyer plate with a pipette, transfer the Peyer plate to an empty petri dish, and then add 10 mL of dispase solution (1.5 mL of 15 mg / mL dispase (DISPASE; GIBCO And 8.5 mL of RPMI1640 (GIBCO) was added and allowed to react for 40-45 minutes at 37 ° C with stirring. The cell suspension thus obtained was centrifuged at 1,500 rpm for 10 minutes.
  • Peyer's patch cells were separated and precipitated, and the supernatant was discarded, followed by washing with repeated addition of RPMI1640 medium containing 10% FBS, and then the number of Peyer's patch cells was 5 X 10 6 RPMI1640 medium containing 10% FBS was added and suspended at a volume of / mL, and used for the following experiments.
  • Mouse Peyer's plate cells are seeded on 96-well plates so that the cell density is 2.5 x 10 6 cells / mL, and then at 72 ° C in RPMI1640 medium containing 10% FBS under conditions of 37 ° C and 5% CO.
  • LPS Lipopolysaccharide
  • IgA ELISA kit IgA Mouse ELISA Quantitation Kit
  • FIG. 1 shows the amount of IgA produced by mouse Peyer's patch cells by the addition of a cell suspension of 21 strains belonging to the ratatobacillus brevis subsp. Brevis.
  • SBC8044 of Ratatobacillus brevis subspecies brevis induces the production of Ig A against mouse Peyer's patch cells, and the amount produced is the control and other strains belonging to latvis brevis subsp.
  • SBC8044 belonging to Ratatobacillus brevis subspecies brevis was tested for the ability to produce ⁇ -aminobutyric acid (GABA).
  • GABA ⁇ -aminobutyric acid
  • SBC8044 was inoculated into lOOmL liquid medium (3% malt extract (DIFCO), 2% yeast extract (DIFCO), 0.2% sodium glutamate, pH 6.0) and left to stand for 4 days. did. Thereafter, the culture solution of SBC8044 was centrifuged at 500 rpm for 10 minutes to recover the culture supernatant, and the amount of GABA contained in the culture supernatant was quantified by HPLC.
  • the HPLC conditions used are as follows.
  • Liquid B 45% by volume acetonitrile, 45% by volume ⁇ ⁇ ⁇ 1, 10% H 2 O
  • SBC8044 belonging to Ratatobacillus brevis subspecies brevis is a strain that can grow in an effervescent alcoholic beverage, produces GABA having an anti-stress action, and has an immune activation action. There was found.
  • a fruit juice lactic acid fermentation broth was produced using SBC8044 having an immunostimulatory action, and a sensory test was performed on the aroma and flavor of the obtained fruit juice lactic acid fermentation broth.
  • Table 2 shows the type, sugar content and pH of the fruit juice used for lactic acid fermentation.
  • Each fruit juice is prepared by diluting grapefruit concentrated juice, apple concentrated fruit juice, white grape concentrated fruit juice and lemon concentrated fruit juice with sterilized water to have a predetermined sugar content, and pH is adjusted by adding NaOH. It was adjusted. Lactic acid fermentation, the SB C8044 was inoculated in 1. 1 X 10 9 cells for each juice LOOmL, stirring is carried out for once a day, performed 72 hours, except when stirred under standing conditions 30 ° C It was. After completion of fermentation, the turbidity of each lactic acid fermentation broth was measured with a spectrophotometer (Tytec Corp.), and the amount of lactic acid was measured with F-kit D- / L-lactic acid (JK International Corp.). The turbidity of each lactic acid fermentation broth is used as an indicator of the growth of SBC8044. It was used as an index.
  • Table 3 shows the turbidity, lactic acid amount, and sensory test results of each lactic acid fermentation broth.
  • the strain of the present invention can grow in an effervescent alcoholic beverage, it is possible to exclude other lactic acid strains having no strain-stimulating immunostimulatory effect of the present invention by utilizing this property. Furthermore, the strain of the present invention, I Amino to produce butyric acid (GABA), anti-stress effect in addition to the immunopotentiating activity also has, Oh Ru cancers correlated with physical stress and mental stress, infections Furthermore, a strong effect can be expected for the prevention and treatment of lifestyle-related diseases such as allergic diseases.
  • GABA butyric acid

Abstract

Disclosed is a bacterial strain belonging to Lactobacillus brevis subspecies brevis which has both an immunostimulating activity and an ability of producing γ-amino butyric acid (GABA). Also disclosed is a beverage or food comprising a cell of the bacterial strain. Further disclosed is an immunostimulating agent comprising a cell of the bacterial strain as an active ingredient. The strain can proliferate in a sparkling alcoholic beverage, can produce γ-amino butyric acid (GABA) and has an immunostimulating activity.

Description

明 細 書  Specification
免疫賦活作用を有する菌株並びにその菌株を含有する飲料、食品及び 免疫賦活剤  Bacterial strain having immunostimulating action and beverage, food and immunostimulant containing the bacterial strain
技術分野  Technical field
[0001] 免疫賦活作用を有する菌株並びにその菌株を含有する飲料、食品及び免疫賦活 剤に関する。  [0001] The present invention relates to a strain having an immunostimulatory action, a beverage, a food and an immunostimulant containing the strain.
背景技術  Background art
[0002] 癌や感染症の治療では、抗癌剤や抗生物質等を用いた化学療法を中心に、外科 的療法及び放射線療法を組み合わせて治療方針が立てられてきた。しかし、近年で は、低下した免疫機能を活性化して、病原ウィルスや悪性腫瘍を自ら排除する免疫 療法が注目を集めている。例えば、 日常生活では、悪性腫瘍細胞や感染細胞体が 生体内のあらゆるところで発生する力 S、NK細胞がこれらと闘って癌や感染症から身 を守っている。免疫療法とは、こうした免疫システムの働きに目をつけて、これを高め て正常に保つことによってさまざまな疾患を治療していこうとするものである。  [0002] In the treatment of cancer and infectious diseases, a therapeutic policy has been established by combining surgical therapy and radiation therapy, mainly chemotherapy using anticancer drugs and antibiotics. However, in recent years, immunotherapy that activates reduced immune functions and eliminates pathogenic viruses and malignant tumors has attracted attention. For example, in everyday life, malignant tumor cells and infected cell bodies are generated everywhere in the body. S and NK cells fight these and protect themselves from cancer and infectious diseases. Immunotherapy aims to treat various diseases by paying attention to the function of the immune system and maintaining it normal.
[0003] 免疫賦活剤の探索は、生体における免疫機能を高め、健康を維持するために有効 であり、これまでも行われてきた。これらの中で、毒性が低ぐ有効性が確認されてい る免疫賦活剤としては、微生物ゃキノコ或!、は植物等が生産する免疫賦活物質が知 られる。例えば、微生物の細胞壁多糖類、又はァガリタスゃシィタケのようなキノコに 由来する多糖類が該当する（特許文献 1及び 2)。最近では、ダルコシド結合を有する 多糖類に特に効果があるとして開示されてレ、る（特許文献 3、 4及び 5)  [0003] The search for an immunostimulant is effective for enhancing immune function in the living body and maintaining health, and has been performed so far. Among these, immunostimulants produced by microorganisms, mushrooms or plants are known as immunostimulants that have been confirmed to be effective with low toxicity. For example, cell wall polysaccharides of microorganisms, or polysaccharides derived from mushrooms such as Agarita sashitake (Patent Documents 1 and 2). Recently, polysaccharides having a dalcoside bond have been disclosed as being particularly effective (Patent Documents 3, 4 and 5).
特許文献 1：特公昭 49— 484号公報  Patent Document 1: Japanese Patent Publication No.49-484
特許文献 2：特公昭 56— 46481号公報  Patent Document 2: Japanese Patent Publication No. 56-46481
特許文献 3：特開平 7— 184595号公報  Patent Document 3: Japanese Patent Laid-Open No. 7-184595
特許文献 4：特開平 10— 194977号公報  Patent Document 4: Japanese Patent Laid-Open No. 10-194977
特許文献 5：特開 2005— 220065号公報  Patent Document 5: Japanese Patent Laid-Open No. 2005-220065
発明の開示  Disclosure of the invention
発明が解決しょうとする課題 [0004] しかしながら、免疫調節物質や免疫刺激剤等の薬物は、血中で免疫反応に関与す る分子に直接作用し、生体の恒常性維持に必要な免疫反応にも影響を与えるため、 副作用が大きいという問題点がある。また、これまでに報告されている乳酸菌由来の 免疫賦活剤は、粘膜免疫を活性化する作用が弱ぐ癌、感染症、さらにはアレルギー 性疾患等の生活習慣病の予防及び治療にぉレ、て効果が不十分であった。さらに、 免疫賦活作用を有し、かつ、抗ストレス作用を有する γ—ァミノ酪酸 (GABA)を産生 する乳酸菌の菌株につレ、ては、これまで報告がなかった。 Problems to be solved by the invention [0004] However, drugs such as immunomodulators and immunostimulants act directly on molecules involved in the immune response in the blood and affect the immune response necessary for maintaining homeostasis. There is a problem that is large. In addition, immunostimulants derived from lactic acid bacteria that have been reported so far have been used in the prevention and treatment of lifestyle-related diseases such as cancers, infectious diseases, and allergic diseases that weaken the effect of activating mucosal immunity. The effect was insufficient. Furthermore, there has been no report so far on strains of lactic acid bacteria that produce γ-aminobutyric acid (GABA) having an immunostimulatory action and an anti-stress action.
[0005] そこで本発明の目的は、免疫賦活作用と γ—ァミノ酪酸 (GABA)の産生能を併せ 持つ、ラタトバチラス 'ブレビス亜種ブレビス（Lactobacillus brevis subspecies brevis)に属する菌株を提供することにある。また本発明の目的は、このラタトバチラ ス'ブレビス亜種ブレビスに属する菌株の菌体を含有する飲料及び食品並びにこれ らを有効成分として含有する免疫賦活剤を提供することにある。  [0005] Therefore, an object of the present invention is to provide a strain belonging to Lactobacillus brevis subspecies brevis, which has both an immunostimulatory action and the ability to produce γ-aminobutyric acid (GABA). Another object of the present invention is to provide beverages and foods containing the bacterial cells of the strain belonging to this ratatobatilla brevis subspecies brevis, and an immunostimulant containing these as active ingredients.
課題を解決するための手段  Means for solving the problem
[0006] 上記目的を達成するために、本発明は、ラタトバチラス.ブレビスの亜種ブレビス（L actobacillus brevis subspecies brevis)に j¾する困侏でめって、発泡十生/ノレコ ール飲料中で増殖可能であり、 7ーァミノ酪酸 (GABA)を産生し、免疫賦活作用を 有する菌株を提供する。  [0006] In order to achieve the above object, the present invention has been described in the following. The present invention has been developed in a fizzy teno / nocolle beverage, in a difficult manner, such as Lactobacillus brevis subspecies brevis. A strain capable of producing 7-aminobutyric acid (GABA) and having an immunostimulatory effect is provided.
[0007] 本発明者らは、ラタトバチラス'ブレビス種の乳酸菌のうち亜種ブレビスに属する菌 株力 特に効果的に、マウスパイエル板からの IgAの産生を誘導することを見出した 。この IgAの産生促進作用は粘膜免疫を活性化し、生体防御機能の維持と改善に有 用である。乳酸菌は、古くから発酵食品に利用されている細菌であり、化学合成され た免疫賦活剤と比較して、人体に対する安全性がはるかに優れている。また、通常の 乳酸菌は、発泡性アルコール飲料中で増殖不可能であるが、本発明の菌株は、発 泡性アルコール飲料中で増殖可能であるため、この性質を利用して本発明の菌株か ら免疫賦活作用を有しない他の乳酸菌株を除くことができる。さらに、本発明の菌株 は、 7ーァミノ酪酸 (GABA)を産生するため、上記の免疫賦活作用に加えて抗スト レス作用も有しており、身体的ストレス及び精神的ストレスと相関のある癌及び感染症 等の予防及び治療に対して強い効果が期待できる。このような菌株としては、ラタトバ チラス.ブレビス亜種ブレビスの SBC8044 (FERM BP— 10631)力 S特に好ましい。 [0007] The present inventors have found that among strains of Lactobacillus brevis lactic acid bacteria belonging to subspecies brevis, particularly effectively induces production of IgA from mouse Peyer's patches. This IgA production promoting action activates mucosal immunity and is useful for maintaining and improving the body defense function. Lactic acid bacteria are bacteria that have been used for fermented foods for a long time, and are much safer for the human body than chemically synthesized immunostimulants. In addition, normal lactic acid bacteria cannot grow in effervescent alcoholic beverages, but the strains of the present invention can grow in foaming alcoholic beverages. Other lactic acid strains that do not have an immunostimulatory effect can be excluded. Furthermore, since the strain of the present invention produces 7-aminobutyric acid (GABA), it has an anti-stress action in addition to the above-mentioned immunostimulatory action, and it has cancer and correlates with physical stress and mental stress. Strong effects can be expected for the prevention and treatment of infectious diseases. Such strains include Ratatova SBC8044 (FERM BP—10631) force S of Chilas brevis subspecies brevis is particularly preferred.
[0008] 上記の免疫賦活作用は、 IgAの産生促進作用であることが好ましい。 [0008] The immunostimulatory action is preferably an IgA production promoting action.
[0009] IgAは、主に腸管粘液、唾液、涙、母乳などの分泌液に含まれる免疫グロブリンの 1 つであり、粘膜局所の生体防御機構として重要な役割を果たしている。本発明の菌 株は、 IgAの産生を促進できるため、粘膜局所の生体防御機構を高めることができ、 感染症、さらにはアレルギー性疾患等の生活習慣病の予防及び治療に利用できる。 [0009] IgA is one of immunoglobulins contained mainly in secretions such as intestinal mucus, saliva, tears, and breast milk, and plays an important role as a local defense mechanism of mucosa. Since the bacterial strain of the present invention can promote the production of IgA, it can enhance the local defense mechanism of mucosa and can be used for the prevention and treatment of lifestyle diseases such as infectious diseases and allergic diseases.
[0010] また、本発明は、上記菌株の菌体を含有する飲料及び食品を提供する。 [0010] The present invention also provides beverages and foods containing the cells of the above strain.
[0011] 上記菌株の菌体は、免疫賦活作用を有し、人体に対して安全であることから、健康 食品素材として飲料及び食品に含有させて利用できる。さらに、上記菌株は γ —アミ ノ酪酸 (GABA)を産生するため、抗ストレス作用、血圧降下作用及び精神安定作用 を併せ持ち、健康食品素材として利用価値が高い。 [0011] The bacterial cells of the above strains have an immunostimulatory action and are safe for the human body, so that they can be used as beverages and foods as health food materials. Furthermore, since the above strain produces γ-aminobutyric acid (GABA), it has anti-stress, blood pressure lowering and tranquilizing effects and is highly useful as a health food material.
[0012] また、本発明は、上記菌株の菌体を有効成分として含有する免疫賦活剤を提供す [0012] The present invention also provides an immunostimulant containing the bacterial cells of the strain as an active ingredient.
[0013] 上記菌株の菌体は、 IgAの産生促進作用を有するため、この菌体を有効成分とし て含有する免疫賦活剤を製造すれば、化学合成された医薬品よりも安全性に優れた 免疫賦活剤として利用できる。 [0013] Since the bacterial cells of the above strains have an IgA production promoting action, if an immunostimulant containing this bacterial cell as an active ingredient is produced, immunity superior in safety to chemically synthesized drugs is produced. It can be used as an activator.
発明の効果  The invention's effect
[0014] 本発明によれば、これまでに知られる乳酸菌株よりも強い免疫賦活作用を有するラ クトバチラス'ブレビス亜種ブレビスに属する菌株を提供できる。また、本発明の菌株 は、発泡性アルコール飲料中で増殖可能であるため、この性質を利用して本発明の 菌株から免疫賦活作用を有しない他の乳酸菌株を除くことができる。さらに、本発明 の菌株は、 γ—ァミノ酪酸 (GABA)を産生するため、免疫賦活作用に加えて抗スト レス作用も有しており、身体的ストレス及び精神的ストレスと相関のある癌、感染症、さ らにはアレルギー性疾患等の生活習慣病の予防及び治療に対して強い効果が期待 できる。また本発明によれば、上記菌株の菌体を含有し、安全性に優れ、かつ、免疫 賦活作用を有する飲料、食品及び免疫賦活剤を提供できる。  [0014] According to the present invention, it is possible to provide a strain belonging to Lactobacillus brevis subspecies brevis, which has a stronger immunostimulatory effect than lactic acid strains known so far. Moreover, since the strain of the present invention can grow in an effervescent alcoholic beverage, other lactic acid strains having no immunostimulatory action can be excluded from the strain of the present invention using this property. Furthermore, since the strain of the present invention produces γ-aminobutyric acid (GABA), it has an anti-stress action in addition to an immunostimulatory action, and is associated with cancer and infection that are correlated with physical stress and mental stress. It is expected to have a strong effect on prevention and treatment of lifestyle-related diseases such as allergies and allergic diseases. In addition, according to the present invention, beverages, foods, and immunostimulants that contain the bacterial cells of the above strain, have excellent safety, and have an immunostimulatory action can be provided.
図面の簡単な説明  Brief Description of Drawings
[0015] [図 1]ラタトバチラス 'ブレビス亜種ブレビスに属する菌株の菌体縣濁液の添加により マウスパイエル板細胞が産生した IgAの量を示したものである。 [0015] [Fig.1] Ratatobatirus' brevis subsp. It shows the amount of IgA produced by mouse Peyer's patch cells.
発明を実施するための最良の形態  BEST MODE FOR CARRYING OUT THE INVENTION
[0016] 以下、本発明の好適な実施形態について詳細に説明する。 Hereinafter, preferred embodiments of the present invention will be described in detail.
[0017] 本発明の菌株は、ラタトバチラス.ブレビス亜種ブレビス（Lactobacillus brevis s ubspecies brevis)に属する菌株であって、発泡性アルコール飲料中で増殖可能 であり、 γーァミノ酪酸 (GABA)を産生し、免疫賦活作用を有することを特徴として いる。  [0017] The strain of the present invention is a strain belonging to Lactobacillus brevis subspecies brevis, is capable of growing in a sparkling alcoholic beverage, and produces γ-aminobutyric acid (GABA). It has an immunostimulatory effect.
[0018] ラタトバチラス 'ブレビス（Lactobacillus brevis)には、 4つの亜種（subspecies) カ存在し、それぞれ、ブレビス（brevis)、グレブセンシス（gravesensis)、オタキエン シス（otakiensis)、コアギュランス（coagulans)である力 上記菌株は、亜種ブレビ スに属する。亜種ブレビスに属する菌株は、 16Sリボゾーム DNAの塩基配列及び糖 力、らの酸生成の違い等を指標に分離でき、亜種グレブセンシス、亜種オタキエンシス 及び亜種コアギュランスに属さない菌株として分類できる。  [0018] There are four subspecies mosquitoes in Lactobacillus brevis, which are brevis, gravesensis, otakiensis, and coagulans, respectively. The strain belongs to the subspecies Brevis. Strains belonging to subspecies brevis can be separated by using the base sequence and sugar strength of 16S ribosomal DNA, differences in acid production, etc. as indicators, and can be classified as strains not belonging to subspecies grebsensis, subspecies otachysis and subspecies coagulance.
[0019] 「免疫賦活作用」とは、生体の免疫力を強化して病気に対する抵抗力をつけること をいう。例えば、マクロファージ、好中球及び NK細胞等を活性化して、自然免疫機 能を高めたり、 IgA等の免疫グロブリンの産生を促進したりすることが挙げられる。特 に、 IgA産生促進作用は、粘膜免疫を高めることができるため、感染症、さらにはァレ ルギー性疾患等の生活習慣病の予防及び治療に対して強い効果を期待できる。  [0019] "Immunostimulatory action" refers to strengthening the immunity of a living body to provide resistance to diseases. For example, macrophages, neutrophils, NK cells and the like are activated to enhance the innate immune function and promote the production of immunoglobulins such as IgA. In particular, IgA production promoting action can enhance mucosal immunity, and thus can be expected to have a strong effect on the prevention and treatment of lifestyle diseases such as infectious diseases and allergic diseases.
[0020] 「発泡性アルコール飲料」には、例えば、ビール、雑酒、発泡酒が含まれる。「発泡 性アルコール飲料中で増殖可能」とは、発泡性アルコール飲料中で乳酸菌が死滅せ ず、かつ、細胞分裂して細胞数が増えることをいい、発泡性アルコール飲料のアルコ ール濃度が 5%以上であることが好ましい。  [0020] "Effervescent alcoholic beverages" include, for example, beer, miscellaneous sake, and happoshu. “Can grow in sparkling alcoholic beverages” means that lactic acid bacteria do not die in sparkling alcoholic beverages, and the number of cells increases due to cell division. The alcohol concentration of sparkling alcoholic beverages is 5 % Or more is preferable.
[0021] ラタトバチラス'ブレビス亜種ブレビスに属する菌株は、自然界から容易に分離でき[0021] The strain belonging to Ratatobathiras brevis subsp. Brevis can be easily isolated from nature.
、 16Sリボゾーム DNAの塩基配列を調べることにより同定できる。また、 ATCC等の 細胞バンクから購入できる。 It can be identified by examining the base sequence of 16S ribosomal DNA. It can also be purchased from cell banks such as ATCC.
[0022] ラタトバチラス.ブレビス亜種ブレビスに属する菌株のうち、発泡性アルコール飲料 中で増殖可能である菌株は、特開 2003— 250557号公報に記載された方法に従つ て選抜できる。具体的には、ラクトバチルス 'ブレビス亜種ブレビスに属する菌株のゲ ノム DNAを铸型として所定のプライマーセット（特開 2003— 250557号公報の配列 表の配列番号 1及び配列番号 2に記載の核酸配列からなるオリゴヌクレオチドからな るプライマーセット）で PCRを行!/、、増幅された DNAジャィレースサブユニット B遺伝 子断片を制限酵素で切断し、アクリルアミドゲル電気泳動後の制限酵素切断パター ンを解析し、グループ libに属する菌株を選抜すればよい。制限酵素切断パターンは 、大きく 4つのグループに分類される力 S、発泡性アルコール飲料中で増殖可能な菌 株はグループ libに属することが判明している。 [0022] Among the strains belonging to Ratatobacillus brevis subspecies brevis, strains capable of growing in effervescent alcoholic beverages can be selected according to the method described in JP-A-2003-250557. Specifically, the strain of the strain belonging to Lactobacillus brevis subspecies Brevis Perform PCR with a specified primer set (primer set consisting of oligonucleotides consisting of the nucleic acid sequences described in SEQ ID NO: 1 and SEQ ID NO: 2 in JP-A-2003-250557) using Nom DNA as a saddle type! / The amplified DNA gyrase subunit B gene fragment may be cleaved with a restriction enzyme, the restriction enzyme cleave pattern after acrylamide gel electrophoresis may be analyzed, and a strain belonging to the group lib may be selected. Restriction enzyme cleavage patterns are classified into four groups, and the strains that can grow in sparkling alcoholic beverages have been found to belong to the group lib.
[0023] 上記の方法以外でも、ラクトバチルス ·ブレビス亜種ブレビスに属する菌株を、発泡 性アルコール飲料中に移植して培養し、増殖の可否を調べることによつても選抜でき る。培養温度は、 30°Cが適している力 15°C〜45°C、好ましくは 20°C〜37°C、特に[0023] In addition to the method described above, the strain can also be selected by examining the possibility of growth by transplanting and culturing a strain belonging to Lactobacillus brevis subspecies brevis in an effervescent alcoholic beverage. The culture temperature is a suitable force at 30 ° C 15 ° C-45 ° C, preferably 20 ° C-37 ° C, especially
30°C前後であれば培養可能である。 Cultivation is possible at around 30 ° C.
[0024] ラタトバチラス 'ブレビス亜種ブレビスに属する菌株のうち、 Ίーァミノ酪酸（以下、 G[0024] Among the strains belonging to Ratatobacillus' brevis subsp. Brevis, ァ aminobutyric acid (G)
ABA)を産生する菌株は、培養上清をアミノ酸分析装置等で分析し、 GABAの含有 量を調べることによって選抜できる。 A strain producing ABA) can be selected by analyzing the culture supernatant with an amino acid analyzer or the like and examining the GABA content.
[0025] ラタトバチラス'ブレビス亜種ブレビスに属する菌株のうち、免疫賦活作用を有する 菌株は、例えば、マウスパイエル板細胞に対する IgAの産生促進作用を試験すること によって選抜できる。 [0025] Among the strains belonging to Ratatobacillus' brevis subspecies brevis, a strain having an immunostimulatory effect can be selected by, for example, examining the IgA production promoting effect on mouse Peyer's patch cells.
[0026] 具体的には、マウスの腸管からパイエル板細胞を分離して培養し、そこに被検菌株 の菌体を滅菌して調製した菌体縣濁液を加えて一定時間培養し、パイエル板細胞か ら分泌される IgAの産生量を ELISA等で測定し、 IgAの産生促進作用の有無を調べ れば'よい。  [0026] Specifically, Peyer's patch cells were isolated from the intestinal tract of the mouse and cultured, and a cell suspension prepared by sterilizing the cells of the test strain was added thereto and cultured for a certain period of time. It is sufficient to measure the production amount of IgA secreted from the plate cells by ELISA or the like and examine the presence or absence of an IgA production promoting action.
[0027] 尚、発泡性アルコール飲料中で増殖可能であり、 GABAを産生し、免疫賦活作用 を有する、ラタトバチラス'ブレビス亜種ブレビスに属する SBC8044 (受託の日：200 6年 6月 28日；受託番号： FERM BP— 10631)は、国際寄託当局である独立行政 法人産業技術総合研究所特許生物寄託センター (日本国茨城県つくば巿東 1丁目 1番地 1 中央第 6 (郵便番号 305— 8566) )に寄託されており、入手可能である。  [0027] SBC8044 belonging to Ratatobacillus brevis subspecies brevis, which can grow in sparkling alcoholic beverages, produce GABA, and has an immunostimulatory effect (date of contract: June 28, 2006; contract) No .: FERM BP—10631) is the International Depository Authority, National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (1st, 1st, Tsukuba, Higashi, Ibaraki, Japan, 1st 6th (zip code 305-8566)) And are available.
[0028] 本発明の飲料、食品及び免疫賦活剤は、ラタトバチラス'ブレビス亜種ブレビスに属 する菌株であって、発泡性アルコール飲料中で増殖可能であり、 GABAを産生し、 免疫賦活作用を有する、上記の菌株を含有することを特徴としている。 [0028] The beverage, food and immunostimulant of the present invention are strains belonging to Ratatobacillus' brevis subsp. Brevis, can be grown in an effervescent alcoholic beverage, produce GABA, It contains the above strain having an immunostimulatory effect.
[0029] 上記菌株の菌体は、癌及び感染症等の予防及び治療を目的として飲料及び食品 に添加できる。上記飲料及び食品は、上記の菌株の菌体のみからなっていてもよぐ 当該分野で通常使用される添加物を含んでいてもよい。この添加物としては、例えば 、リンゴファイバー、大豆ファイバー、肉エキス、黒酢エキス、ゼラチン、コーンスター チ、蜂蜜、動植物油脂、グルコース等の単糖類、スクロース、フルクトース及びマンニ トール等の二糖類、デキストロース及びデンプン等の多糖類、エリスリトール、キシリト ール及びソルビトール等の糖アルコール類、ビタミン C等のビタミン類が挙げられ、こ れらの添加物は単独種又は複数種であつてもよい。 [0029] The bacterial cells of the strain can be added to beverages and foods for the purpose of preventing and treating cancer and infectious diseases. The said drink and food may consist only of a microbial cell of said strain. The additive normally used in the said field | area may be included. Examples of the additives include apple fiber, soybean fiber, meat extract, black vinegar extract, gelatin, corn starch, honey, animal and vegetable oils and fats, monosaccharides such as glucose, disaccharides such as sucrose, fructose and mannitol, dextrose And polysaccharides such as starch, sugar alcohols such as erythritol, xylitol, and sorbitol, and vitamins such as vitamin C. These additives may be used alone or in combination.
[0030] さらに、癌及び感染症等の予防の目的で、食品添加物として、特定保健用食品、 特殊栄養食品、栄養補助食品、健康食品、機能性食品や病者用食品等の飲食物に 酉己合することあでさる。 [0030] Furthermore, for the purpose of preventing cancer and infectious diseases, food additives such as foods for specified health use, special nutritional foods, nutritional supplements, health foods, functional foods and sick foods are used as food additives. I'm going to meet you.
[0031] 本発明の免疫賦活剤は、上記の菌株の菌体を有効成分として含み、担体、賦形剤 及び/又はその他の添加物を加えて製剤化すれば、安全性に優れた免疫賦活剤と して利用できる。薬学的に許容される添加物としては、例えば、グルコース等の単糖 類、スクロース、フルクトース及びマンニトール等の二糖類、デキストロース及びデン プン等の多糖類、エリスリトール、キシリトール及びソルビトール等の糖アルコール類 、ビタミン C等のビタミン類、アカシアゴム、リン酸カルシウム、アルギン酸塩、珪酸カル シゥム、微結晶性セルロース、ポリビュルピロリドン、セルロース誘導体、トラガカント、 ゼラチン、シロップ、ヒドロキシ安息香酸メチル、タルク、ステアリン酸マグネシウム、水 、鉱油が挙げられ、これらの添加物は単独種又は複数種であってもよい。  [0031] The immunostimulatory agent of the present invention contains a microbial cell of the above-mentioned strain as an active ingredient, and if formulated by adding a carrier, an excipient and / or other additives, an immunostimulator having excellent safety It can be used as an agent. Pharmaceutically acceptable additives include, for example, monosaccharides such as glucose, disaccharides such as sucrose, fructose and mannitol, polysaccharides such as dextrose and starch, sugar alcohols such as erythritol, xylitol and sorbitol, Vitamins such as vitamin C, gum acacia, calcium phosphate, alginate, calcium silicate, microcrystalline cellulose, polybulurpyrrolidone, cellulose derivatives, tragacanth, gelatin, syrup, methyl hydroxybenzoate, talc, magnesium stearate, water, Mineral oil is mentioned, and these additives may be single species or plural species.
実施例  Example
[0032] 以下、実施例を挙げて本発明を具体的に説明するが、本発明はこれらの実施例に 限定されるものではない。  Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited to these examples.
[0033] 1)実験に用いた各菌株について [0033] 1) About each strain used in the experiment
ラクトノくチラス 'ブレビス亜種ブレビス (Lactobacillus brevis subspecies brevi s)に属する 21菌株（SBC8044及び菌株 A〜T)は発明者が分離し、実験に用いる まで凍結乾燥菌体として 4°Cで保管した。 [0034] 2)ビール中での増殖能力の判定 21 strains (SBC8044 and strains A to T) belonging to Lactobacillus brevis subspecies brevi s were isolated by the inventor and stored at 4 ° C. as lyophilized cells until used in experiments. [0034] 2) Determination of growth ability in beer
上記のラタトバチラス'ブレビス亜種ブレビスに属する 21菌株について、ビール中で の増殖能を選抜した。選抜方法は、特開 2003— 250557号公報に記載された方法 に従って行なった。すなわち、ラクトバチルス'ブレビス亜種ブレビスに属する菌株の ゲノム DNAを铸型として所定のプライマーセット（特開 2003— 250557号公報の配 列表の配列番号 1及び配列番号 2に記載の核酸配列からなるオリゴヌクレオチドから なるプライマーセット）で PCRを行!/、、増幅された DNAジャィレースサブユニット B遺 伝子断片を制限酵素で切断し、アクリルアミドゲル電気泳動後の制限酵素切断バタ ーンを解析することにより判定した。この方法では、制限酵素切断パターンは、大きく 4つのグループに分類でき、ビール中で増殖可能である菌株は、グループ libに属す ることが判明している。  For 21 strains belonging to the above-mentioned Ratatobacillus brevis subspecies brevis, the growth ability in beer was selected. The selection method was performed according to the method described in Japanese Patent Application Laid-Open No. 2003-250557. That is, an oligo consisting of a nucleic acid sequence described in SEQ ID NO: 1 and SEQ ID NO: 2 in the sequence table of JP 2003-250557 A using the genomic DNA of a strain belonging to Lactobacillus brevis subsp. PCR with a primer set consisting of nucleotides) /, cleave the amplified DNA gyrase subunit B gene fragment with restriction enzymes, and analyze the restriction enzyme cleavage pattern after acrylamide gel electrophoresis Judged by. In this method, restriction enzyme cleavage patterns can be broadly classified into four groups, and strains that can grow in beer have been found to belong to the group lib.
[0035] その結果、調べたラタトバチラス'ブレビス亜種ブレビスに属する 21菌株は総てビー ル中で増殖する菌株であると判定された。尚、これらの菌株を実際にビール中に植 菌し増殖の可否を確認したが、上記方法の判定結果と同じであった。  As a result, it was determined that all the 21 strains belonging to the ratatobatillas brevis subspecies brevis examined were strains that grew in the beer. Although these strains were actually inoculated into beer and confirmed whether or not they could grow, the results were the same as the determination results of the above method.
[0036] 3)免疫賦活作用の評価  [0036] 3) Evaluation of immunostimulatory action
i)菌体懸濁液の調製  i) Preparation of cell suspension
上記の各菌株の免疫賦活作用を評価するために、各菌株の菌体懸濁液を調製し た。まず、各菌株を嫌気的条件下（N ： CO ： H = 90： 5： 5のガス組成）、 MRS液体  In order to evaluate the immunostimulatory action of each of the above strains, a cell suspension of each strain was prepared. First, each strain is subjected to anaerobic conditions (gas composition of N: CO: H = 90: 5: 5), MRS liquid
2 2 2  2 2 2
培地（ディフコ社製、組成： 1 %プロテオースペプトン、 1 %牛肉エキス、 0. 5%酵母ェ キス、 2%ブドウ糖、 0. l %Tween 80、 0. 5%クェン酸アンモユウム、 0. 01 %硫酸 マグネシウム、 0. 005%硫酸マンガン、 0. 2%リン酸二カリウム）で 3日間静置培養し 、その培養液を 1 , 500回転で 10分間遠心分離して、各菌株の菌体を回収した。得 られた菌体は、 PBSで洗浄した後に凍結乾燥し、 Img/mLとなるように PBSに懸濁 した。こうして得られた菌体懸濁液は、オートクレープ滅菌（121°C、 15分）して以下 の実験に使用した。  Medium (Difco), composition: 1% proteose peptone, 1% beef extract, 0.5% yeast extract, 2% glucose, 0.1% Tween 80, 0.5% ammonium citrate, 0.01% (Magnesium sulfate, 0.005% manganese sulfate, 0.2% dipotassium phosphate) for 3 days. Centrifuge the culture at 1,500 rpm for 10 minutes to recover the cells of each strain. did. The obtained bacterial cells were washed with PBS, freeze-dried, and suspended in PBS so that the concentration was I mg / mL. The bacterial cell suspension thus obtained was autoclaved (121 ° C, 15 minutes) and used in the following experiments.
[0037] ii)マウスパイエル板細胞に対するラタトバチラス 'ブレビス亜種ブレビスに属する菌株 の IgA産生促進作用（in vitro)：  [0037] ii) Stimulation of IgA production (in vitro) by strains belonging to the ratatobacillus brevis subspecies brevis on mouse Peyer's patch cells:
マウスパイエル板細胞にラタトバチラス 'ブレビス亜種ブレビスに属する 21菌株の菌 体縣濁液を加えて一定時間培養し、パイエル板細胞から分泌される IgAの量を調べ ることにより、各菌株の IgA産生促進作用を評価した。 21 strains belonging to ratatobacillus' brevis subsp.brevis on mouse Peyer's patch cells The body suspension was added and cultured for a certain period of time, and the IgA production promoting effect of each strain was evaluated by examining the amount of IgA secreted from Peyer's patch cells.
[0038] (マウスのパイエル板細胞の調製）  [0038] (Preparation of mouse Peyer's patch cells)
まず、 6週齢の BALBんマウス（雌)から腸管を無菌的に摘出し、そこからパイエル 板をハサミで外して、 RPMI1640 (GIBCO社）に浸漬した。ピペットでパイエル板を 吸い上げる操作を繰り返すことにより、パイエル板を十分に洗浄し、パイエル板を空 のシャーレに移した後に、 10mLのデイスパーゼ溶液（1 · 5mLの 15 mg/mL デ イスパーゼ（DISPASE； GIBCO社）と 8· 5mLの RPMI1640 (GIBCO社）を加えて 、 37°Cで 40〜45分間、撹拌しながら反応させた。こうして得られた細胞縣濁液は、 1 , 500回転で 10分間遠心分離してパイエル板細胞を沈殿させ、上清を捨てた後に 1 0%FBS含有 RPMI1640培地を加える操作を繰り返すことによりパイエル板細胞を 洗浄した。その後、パイエル板細胞の数が 5 X 106個/ mLになるように 10%FBS含 有 RPMI1640培地を加えて懸濁し、以下の実験に用いた。 First, the intestinal tract was aseptically removed from a 6-week-old BALB mouse (female), and the Peyer's board was removed from it with scissors and immersed in RPMI1640 (GIBCO). Wash the Peyer plate thoroughly by repeatedly sucking the Peyer plate with a pipette, transfer the Peyer plate to an empty petri dish, and then add 10 mL of dispase solution (1.5 mL of 15 mg / mL dispase (DISPASE; GIBCO And 8.5 mL of RPMI1640 (GIBCO) was added and allowed to react for 40-45 minutes at 37 ° C with stirring.The cell suspension thus obtained was centrifuged at 1,500 rpm for 10 minutes. Peyer's patch cells were separated and precipitated, and the supernatant was discarded, followed by washing with repeated addition of RPMI1640 medium containing 10% FBS, and then the number of Peyer's patch cells was 5 X 10 6 RPMI1640 medium containing 10% FBS was added and suspended at a volume of / mL, and used for the following experiments.
[0039] (IgAの ELISAによる測定）  [0039] (IgA ELISA measurement)
細胞密度が 2· 5 X 106個/ mLになるように 96ゥエルプレートにマウスパイエル板 細胞を播種し、 37°C、 5% COの条件下、 10%FBS含有 RPMI1640培地で 72時 Mouse Peyer's plate cells are seeded on 96-well plates so that the cell density is 2.5 x 10 6 cells / mL, and then at 72 ° C in RPMI1640 medium containing 10% FBS under conditions of 37 ° C and 5% CO.
2  2
間培養した。ノ ィエル板細胞を培養している各ゥエルに、各菌株の菌体縣濁液 (最終 濃度： 1 g/mL)を添加し、 7日間経過後に培養上清中に分泌された IgAの量を E LISAで定量した。ポジティブコントロールには、マウスの脾臓細胞に対して IgA産生 促進作用を有するリポポリサッカライド（以下、 LPS) (SIGMA社、最終濃度： 10 g /mUを、ネガティブコントロールには、 PBSを使用した。  Inter-cultured. To each well in which noel plate cells are cultured, a bacterial suspension of each strain (final concentration: 1 g / mL) is added, and the amount of IgA secreted into the culture supernatant after 7 days has passed. Quantified by E LISA. Lipopolysaccharide (hereinafter LPS) (SIGMA, final concentration: 10 g / mU) having an IgA production promoting action on mouse spleen cells was used as a positive control, and PBS was used as a negative control.
[0040] IgAの測定には、 IgA ELISAキット（IgA Mouse ELISA Quantitation Kit  [0040] IgA ELISA kit (IgA Mouse ELISA Quantitation Kit)
； BETHYL Laboratories)を用い、製造元のプロトコールに従って定量した。  Quantified using BETHYL Laboratories) according to the manufacturer's protocol.
[0041] 図 1は、ラタトバチラス 'ブレビス亜種ブレビスに属する 21菌株の菌体縣濁液の添加 によりマウスパイエル板細胞が産生した IgAの量を示したものである。その結果、ラタ トバチラス 'ブレビス亜種ブレビスの SBC8044は、マウスパイエル板細胞に対して Ig Aの産生を誘導し、その産生量はコントロール及び他のラタトバチラス 'ブレビス亜種 ブレビスに属する菌株（菌株 A〜T)によって誘導される量と比較して顕著に高いもの であり、 LPSの作用に匹敵するものであった。 IgA産生促進作用を有する SBC8044 (受託番号： FERM BP— 10631 ;受託の日： 2006年 6月 28日）は、国際寄託当局 である独立行政法人産業技術総合研究所特許生物寄託センター (日本国茨城県つ くば巿東 1丁目 1番地 1 中央第 6 (郵便番号 305— 8566) )に寄託した。 [0041] FIG. 1 shows the amount of IgA produced by mouse Peyer's patch cells by the addition of a cell suspension of 21 strains belonging to the ratatobacillus brevis subsp. Brevis. As a result, SBC8044 of Ratatobacillus brevis subspecies brevis induces the production of Ig A against mouse Peyer's patch cells, and the amount produced is the control and other strains belonging to latvis brevis subsp. Significantly higher than the amount induced by T) It was comparable to the action of LPS. SBC8044 (accession number: FERM BP—10631; date of accession: June 28, 2006), which has the effect of promoting IgA production, is the International Depository Agency, National Institute of Advanced Industrial Science and Technology, Patent Biology Center (Ibaraki, Japan) Prefectural Tsukuba Sagahigashi 1 Chome 1 1 Central 6 (Postal code 305-8566))
[0042] 4) γ -ァミノ酪酸（GABA)の産生能の測定 [0042] 4) Measurement of production ability of γ-aminobutyric acid (GABA)
ラタトバチラス.ブレビス亜種ブレビスに属する SBC8044の γ—ァミノ酪酸（以下、 GABA)の産生能について試験した。まず、 SBC8044を、 lOOmLの液体培地（3% 麦芽エキス（DIFCO社）、 2%酵母エキス（DIFCO社）、 0· 2%グルタミン酸ナトリウ ム、 pH6. 0)に植菌し、 4日間静置培養した。その後、 SBC8044の培養液を ， 500 回転で 10分間遠心分離して培養上清を回収し、その培養上清に含まれる GABAの 量を HPLCで定量した。使用した HPLCの条件は以下の通りである。  SBC8044 belonging to Ratatobacillus brevis subspecies brevis was tested for the ability to produce γ-aminobutyric acid (GABA). First, SBC8044 was inoculated into lOOmL liquid medium (3% malt extract (DIFCO), 2% yeast extract (DIFCO), 0.2% sodium glutamate, pH 6.0) and left to stand for 4 days. did. Thereafter, the culture solution of SBC8044 was centrifuged at 500 rpm for 10 minutes to recover the culture supernatant, and the amount of GABA contained in the culture supernatant was quantified by HPLC. The HPLC conditions used are as follows.
•HPLC装置： Agilent HPLC 1100  • HPLC system: Agilent HPLC 1100
'使用カラム： ZORBAX eclipse ΑΑΑ(4· 6 X 150mm、 3· 5 m) (島津ジーェ ルシ一社）  'Column used: ZORBAX eclipse ΑΑΑ (4.6 x 150mm, 3.5m) (Shimadzu GE)
•カラムオーブン： 40°C  • Column oven: 40 ° C
•流 丄. Oml min  • Fluent. Oml min
•蛍光検出器： Ex. 340nm、 Em. 450nm  • Fluorescence detector: Ex. 340nm, Em. 450nm
•HPLC試薬： lOmg/mL Agilent OPA試薬（0· 4M ホウ酸バッファー、 pHIO • HPLC reagent: lOmg / mL Agilent OPA reagent (0.4M borate buffer, pHIO
. 2) (島津ジーエルシー社） 2) (Shimadzu GL Corporation)
•溶離液： A液： 40mM NaH2P04 (pH7. 8)  • Eluent: Liquid A: 40 mM NaH2P04 (pH 7.8)
B液： 45体積％ァセトニトリル、 45体積％^^〇^1、 10%H O  Liquid B: 45% by volume acetonitrile, 45% by volume ^^ 〇 ^ 1, 10% H 2 O
2  2
• タイムテーブル (グラジェント）：表 1を参照  • Timetable (gradient): see Table 1
[0043] [表 1] 時間 （分） 溶媒 A 溶媒 B [0043] [Table 1] Time (min) Solvent A Solvent B
0 . 0 9 0 % 1 0 %  0. 0 9 0% 1 0%
4 . 0 9 0 % 1 0 %  4. 0 9 0% 1 0%
2 0 . 0 6 5 % 3 5 %  2 0. 0 6 5% 3 5%
2 5 . 0 1 0 % 9 0 %  2 5. 0 1 0% 9 0%
3 5 . 0 1 0 % 9 0 %  3 5. 0 1 0% 9 0%
3 6 . 0 9 0 % 1 0 %  3 6. 0 9 0% 1 0%
4 6 . 0 9 0 % 1 0 %  4 6. 0 9 0% 1 0%
[0044] その結果、 SBC8044は、培養上清中に GABAを 857. 7 mol/L産生した。 As a result, SBC8044 produced 857.7 mol / L of GABA in the culture supernatant.
[0045] 以上の結果より、ラタトバチラス 'ブレビス亜種ブレビスに属する SBC8044は、発泡 性アルコール飲料中で増殖可能であり、抗ストレス作用を有する GABAを産生し、免 疫賦活作用を有する菌株であることが判明した。 [0045] From the above results, SBC8044 belonging to Ratatobacillus brevis subspecies brevis is a strain that can grow in an effervescent alcoholic beverage, produces GABA having an anti-stress action, and has an immune activation action. There was found.
[0046] 5) SBC8044を用いて製造した果汁乳酸発酵液の官能検査 [0046] 5) Sensory test of the lactic acid fermentation broth manufactured using SBC8044
免疫賦活作用を有する SBC8044を用いて果汁乳酸発酵液を製造し、得られた果 汁乳酸発酵液の香りと風味について官能検査を行なった。  A fruit juice lactic acid fermentation broth was produced using SBC8044 having an immunostimulatory action, and a sensory test was performed on the aroma and flavor of the obtained fruit juice lactic acid fermentation broth.
[0047] 表 2は、乳酸発酵に使用した果汁について、果実の種類、糖度及び pHを示したも のである。 [0047] Table 2 shows the type, sugar content and pH of the fruit juice used for lactic acid fermentation.
[0048] [表 2] [0048] [Table 2]
Figure imgf000011_0001
Figure imgf000011_0001
[0049] 各果汁は、それぞれグレープフルーツ濃縮果汁、りんご濃縮果汁、白ぶどう濃縮果 汁及びレモン濃縮果汁を滅菌水で希釈して所定の糖度になるように調製し、 pHは N aOHを添加して調整した。乳酸発酵は、各果汁 lOOmLに対し 1. 1 X 109細胞の SB C8044を植菌し、 1日 1回の撹拌を行い、 30°Cで 72時間、攪拌時以外は静置条件 下で行なった。発酵終了後、各乳酸発酵液の濁度を分光光度計 (タイテック社)で測 定し、乳酸量を F— kit D— /L—乳酸 (J. K.インターナショナル社）で測定した。 各乳酸発酵液の濁度は、 SBC8044の増殖の指標とし、乳酸量は乳酸発酵の程度 の指標とした。 [0049] Each fruit juice is prepared by diluting grapefruit concentrated juice, apple concentrated fruit juice, white grape concentrated fruit juice and lemon concentrated fruit juice with sterilized water to have a predetermined sugar content, and pH is adjusted by adding NaOH. It was adjusted. Lactic acid fermentation, the SB C8044 was inoculated in 1. 1 X 10 9 cells for each juice LOOmL, stirring is carried out for once a day, performed 72 hours, except when stirred under standing conditions 30 ° C It was. After completion of fermentation, the turbidity of each lactic acid fermentation broth was measured with a spectrophotometer (Tytec Corp.), and the amount of lactic acid was measured with F-kit D- / L-lactic acid (JK International Corp.). The turbidity of each lactic acid fermentation broth is used as an indicator of the growth of SBC8044. It was used as an index.
[0050] 表 3は、各乳酸発酵液の濁度、乳酸量及び官能検査の結果を示したものである。  [0050] Table 3 shows the turbidity, lactic acid amount, and sensory test results of each lactic acid fermentation broth.
[0051] [表 3] [0051] [Table 3]
Figure imgf000012_0001
Figure imgf000012_0001
[0052] その結果、試験に用いた全ての果汁で SBC8044の乳酸発酵は進み、果汁乳酸 発酵液を製造するに至った。特に、りんご果汁及び白ぶどう果汁を用いた発酵では、 ヨーグルト風味はなぐベース果汁の風味が保持されており、乳酸発酵飲料として好 まし!/、性質を備えるものであった。  [0052] As a result, lactic acid fermentation of SBC8044 progressed in all the fruit juices used in the test, and the fruit juice lactic acid fermentation liquor was produced. In particular, in fermentation using apple juice and white grape juice, the yogurt flavor retains the flavor of the base juice, which is preferred as a lactic acid fermented beverage!
産業上の利用可能性  Industrial applicability
[0053] 本発明の菌株は、発泡性アルコール飲料中で増殖可能であるため、この性質を利 用して本発明の菌株力 免疫賦活作用を有しない他の乳酸菌株を除くことができる。 さらに、本発明の菌株は、 Ίーァミノ酪酸 (GABA)を産生するため、免疫賦活作用 に加えて抗ストレス作用も有しており、身体的ストレス及び精神的ストレスと相関のあ る癌、感染症、さらにはアレルギー性疾患等の生活習慣病の予防及び治療に対して 強い効果が期待できる。 [0053] Since the strain of the present invention can grow in an effervescent alcoholic beverage, it is possible to exclude other lactic acid strains having no strain-stimulating immunostimulatory effect of the present invention by utilizing this property. Furthermore, the strain of the present invention, I Amino to produce butyric acid (GABA), anti-stress effect in addition to the immunopotentiating activity also has, Oh Ru cancers correlated with physical stress and mental stress, infections Furthermore, a strong effect can be expected for the prevention and treatment of lifestyle-related diseases such as allergic diseases.

Claims

請求の範囲 The scope of the claims
[1] ラタトバチラス 'ブレビス亜種ブレビス（Lactobacillus brevis subspecies brevi s)に属する菌株であって、  [1] A strain belonging to Lactobacillus brevis subspecies brevi s,
発泡性アルコール飲料中で増殖可能であり、  Can grow in sparkling alcoholic beverages,
Ύーァミノ酪酸（GABA)を産生し、  Produces aminoaminobutyric acid (GABA),
免疫賦活作用を有する菌株。  A strain having an immunostimulatory effect.
[2] 前記免疫賦活作用は、 IgAの産生促進作用である、請求項 1記載の菌株。 [2] The strain according to claim 1, wherein the immunostimulatory action is an IgA production promoting action.
[3] クトバチラス *ブレビス亜種ブレビス（Lactobacillus brevis subspecies brevis[3] Kutobacillus * Lactobacillus brevis subspecies brevis
)の SBC8044 (FERM BP— 10631)である、請求項 1又は 2のいずれか一項記載 の菌株。 The strain according to any one of claims 1 and 2, which is SBC8044 (FERM BP-10631).
[4] 請求項 1〜3のいずれか一項記載の菌株の菌体を含有する飲料。  [4] A beverage containing the bacterial cell according to any one of claims 1 to 3.
[5] 請求項;!〜 3のいずれか一項記載の菌株の菌体を含有する食品。 [5] Claims; A food containing the bacterial cells of the strain according to any one of! To 3.
[6] 請求項;!〜 3のいずれか一項記載の菌株の菌体を有効成分として含有する免疫賦 活剤。 [6] An immunostimulant comprising the bacterial body of the strain according to any one of claims;! To 3 as an active ingredient.
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