WO2024090415A1 - Lactic acid bacteria, innate immunity activator derived from said lactic acid bacteria, and food containing said lactic acid bacteria - Google Patents

Lactic acid bacteria, innate immunity activator derived from said lactic acid bacteria, and food containing said lactic acid bacteria Download PDF

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WO2024090415A1
WO2024090415A1 PCT/JP2023/038279 JP2023038279W WO2024090415A1 WO 2024090415 A1 WO2024090415 A1 WO 2024090415A1 JP 2023038279 W JP2023038279 W JP 2023038279W WO 2024090415 A1 WO2024090415 A1 WO 2024090415A1
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lactic acid
acid bacteria
products
innate immunity
food
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French (fr)
Japanese (ja)
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和久 関水
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株式会社ゲノム創薬研究所
株式会社アンテナ
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • 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
    • 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
    • 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
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology

Definitions

  • the present invention relates to lactic acid bacteria, natural immune activators derived from said lactic acid bacteria, and foods containing said lactic acid bacteria.
  • Lactic acid bacteria have long been used in fermented foods and in the production of foods, medicines, probiotics, etc. Lactic acid bacteria are characterized by being gram-positive, catalase-negative, not forming endospores, and not being motile.
  • lactic acid bacteria are most commonly used as probiotics. Establishing an efficient method for isolating highly functional lactic acid bacteria and culturing them in foods will be useful for developing foods that use functional lactic acid bacteria.
  • Lactic acid bacteria are found in a variety of conventionally known foods, and there have been reports of isolating lactic acid bacteria from foods and identifying the species. Examples of such reports are as follows:
  • Non-patent literature 1 Leuconostoc mesenteroides (isolation source: dairy products)
  • Non-patent literature 2 Pediococcus ethanolidurans (isolation source: apple wine)
  • Non-patent document 3 Lactobacillus versmoldensis (isolated from sausage)
  • Non-patent literature 4 Companilactobacillus versmoldensis (isolation source: sausage)
  • Non-patent literature 5 Streptococcus salivarius (isolation source: cheese)
  • Natural immunity is a defense mechanism that quickly eliminates foreign substances (bacteria, viruses, cancer cells) from the body without relying on antibodies, and is shared by all living organisms, from insects to mammals.
  • innate immunity is the first line of defense against the host, triggering subsequent immune responses, including antibody production.
  • various stimuli stimulate the secretion of cytokines by immune cells such as macrophages, which eliminate invading pathogens and transmit information to other immune cells.
  • invertebrates such as insects do not have adaptive immunity and rely solely on innate immunity to eliminate pathogens.
  • innate immune mechanisms of insects and mammals have many similarities.
  • insect blood cells called hemocytes phagocytose invading foreign substances in the same way as mammalian macrophages.
  • Toll-like receptor involved in the innate immune response in mammals is known to be highly homologous to the Toll receptor involved in the innate immune response in Drosophila.
  • the present inventors have developed an evaluation method that can easily measure innate immune activation responses in silkworms, which only have innate immune mechanisms (Patent Document 1, etc., Non-Patent Documents 6 and 7, etc.).
  • This evaluation method makes use of the fact that silkworms contract their muscles as a result of activation of innate immunity, and evaluates the innate immune activation ability based on the degree of muscle contraction.
  • the present inventors have shown that this evaluation method can be used to evaluate (screen) innate immune activators that have innate immune activation effects in vertebrates such as humans (Patent Document 1, etc.).
  • Patent Document 2 Non-Patent Document 8, Non-Patent Document 9, etc.
  • the present invention has been made in view of the above-mentioned background art, and aims to provide a lactic acid bacterium that has a high ability to activate natural immunity, and further to provide a natural immunity activator that contains the lactic acid bacterium, killed cells of the lactic acid bacterium, or a processed product of the lactic acid bacterium as an active ingredient, and a food product that contains the lactic acid bacterium or the natural immunity activator.
  • the present invention provides a lactic acid bacterium belonging to the genus Pediococcus, which has a deposit number of NITE BP-03720 at the National Patent Microorganisms Depositary (NPMD) of the National Institute of Technology and Evaluation (NITE).
  • NPMD National Patent Microorganisms Depositary
  • the present invention also provides an innate immune activator comprising the lactic acid bacterium, killed lactic acid bacterium, or a processed product of the lactic acid bacterium as an active ingredient
  • the present invention provides a natural immune activator, characterized in that the processed product of the lactic acid bacteria is at least one processed product selected from the group consisting of fermented products, cultured products, concentrates, pastes, dried products, liquefied products, diluted products, crushed products, sterilized products, and extracts from cultures of lactic acid bacteria.
  • the present invention also provides a food product containing the lactic acid bacteria.
  • the present invention also provides a food product containing the natural immune activator.
  • the present invention also provides a method for producing food, which includes a fermentation step using the lactic acid bacteria.
  • the present invention can provide lactic acid bacteria with high innate immune activation ability.
  • the present invention can also provide an innate immune activator that contains the lactic acid bacteria, killed cells of the lactic acid bacteria, or a processed product of the lactic acid bacteria as an active ingredient, and a food product that contains the lactic acid bacteria or the innate immune activator.
  • the strain of lactic acid bacteria of the present invention has been reported to have been isolated from food, i.e., it has been consumed in the past. For this reason, it can be said that the safety of the lactic acid bacteria and innate immune activators of the present invention is ensured to a certain extent, and the hurdle for adding them to food is low.
  • the lactic acid bacterium of the present invention is a lactic acid bacterium belonging to the genus Pediococcus, whose accession number at the National Patent Microorganisms Depositary (NPMD) of the National Institute of Technology and Evaluation (NITE) is NITE BP-03720.
  • NPMD National Patent Microorganisms Depositary
  • NITE National Institute of Technology and Evaluation
  • the lactic acid bacteria may be referred to as "lactic acid bacteria #4-3-1,”"lactic acid bacteria #4-3-1 strain,”"#4-3-1,” etc. Lactic acid bacteria #4-3-1 will be described in detail below.
  • Lactic acid bacteria #4-3-1 was first isolated from rice bran (bran bed).
  • Lactic Acid Bacteria #4-3-1 The morphology of Lactic Acid Bacteria #4-3-1 is as follows:
  • Lactobacillus #4-3-1 The physiological and chemical taxonomic properties of Lactobacillus #4-3-1 are as follows:
  • ⁇ Glycerol - ⁇ Erythritol: - ⁇ D-Arabinose: - ⁇ L-Arabinose: - D-Ribose: ⁇ D-Xylose: - ⁇ L-Xylose: - ⁇ D-Adonitol: - ⁇ -Methyl-D-xylopyranoside: - ⁇ D-Galactose: + ⁇ D-Glucose: + ⁇ D-Fructose: + ⁇ D-Mannose: + ⁇ L-Sorbose: - ⁇ L-Rhamnose: - ⁇ Dulcitol: - ⁇ Inositol: - ⁇ D-Mannitol: - ⁇ D-Sorbitol: - ⁇ -Methyl-D-mannopyranoside: - ⁇ -Methyl-D-glucopyranoside: - ⁇ N-Acet
  • Lactic Acid #4-3-1 which is used as an index for molecular biological systematic classification, are as shown in SEQ ID NO: 1 of the attached sequence listing. That is, the base sequence of the 16S rDNA region was amplified by PCR from the genomic DNA of lactic acid bacteria #4-3-1, and analyzed by a sequencer, resulting in the discovery of a base sequence corresponding to almost the entire length of the 16S rDNA.
  • SEQ ID NO:1 The base sequence of SEQ ID NO:1 is set forth below.
  • the base sequence of the 16S rDNA region of lactic acid bacteria #4-3-1 showed 99% homology with the base sequence of Pediococcus ethanolidurans strain Z-9 (accession number: NR_043291.2), which belongs to the Pediococcus genus, and therefore lactic acid bacteria #4-3-1 belongs to Pediococcus ethanolidurans.
  • lactic acid bacteria #4-3-1 of the present invention is a different lactic acid bacteria strain from the above strains.
  • Lactic Acid Bacteria #4-3-1 The physiological and chemical taxonomic properties of Lactic Acid Bacteria #4-3-1 were compared with the classification in Bergey's Manual of Systematic Bacteriology (vol. 3, 1989) and the contents of other literature, and further, taking into consideration the results of the 16S rDNA analysis, it was determined that the "Lactic Acid Bacteria #4-3-1" of the present invention is a novel microorganism belonging to the genus Pediococcus.
  • Lactic Acid Bacterium #4-3-1 is a novel isolated microbial strain.
  • Lactic acid bacteria #4-3-1 was deposited domestically at the National Institute of Technology and Evaluation (NPMD) of the National Institute of Technology and Evaluation (hereinafter abbreviated as "NITE"), Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan, and was deposited under the accession number: NITE P-03720 (deposit date: August 12, 2022).
  • NPMD National Institute of Technology and Evaluation
  • NITE P-03720 deposit date: August 12, 2022
  • Lactic acid bacteria #4-3-1 was then submitted to the National Institute of Technology and Evaluation (NITE) Patent Microorganisms Depository (NPMD), Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan, via an original deposit application, and an application was made to transfer the domestic deposit (original deposit date: August 12, 2022) to a deposit under the Budapest Treaty (transfer date (international deposit date): June 19, 2023).
  • NITE National Institute of Technology and Evaluation
  • the properties of the strain are easily mutated, so the lactic acid bacteria #4-3-1 of the present invention may not remain within the range of physiological properties shown above. Furthermore, such "mutations" include both natural and artificial mutations.
  • Lactic acid bacteria #4-3-1 may be cultured according to a general culture method used for microorganisms of the genus Pediococcus. The culture is preferably carried out under anaerobic conditions.
  • Carbon sources in the medium include organic carbon compounds such as D-ribose, D-galactose, D-glucose, D-fructose, D-mannose, D-mannitol, N-acetylglucosamine, amygdalin, arbutin, esculin, salicin, D-cellobiose, D-maltose, sucrose, D-trehalose, gentiobiose, molasses, starch syrup, and fats and oils
  • nitrogen sources include organic and inorganic nitrogen compounds such as meat extract, casein, peptone, yeast extract, dry yeast, germ, soybean flour, urea, amino acids, and ammonium salts.
  • inorganic salts such as sodium salts, potassium salts, calcium salts, magnesium salts, phosphates, iron salts, copper salts, zinc salts, cobalt salts, etc. may be added as necessary.
  • growth promoters such as biotin, vitamin B1, cystine, methyl oleate, lard oil, etc.
  • antifoaming agents such as silicone oil, surfactants, etc. may be added.
  • the prepared medium for example, MRS medium, GAM medium, etc. are preferably used.
  • the culture conditions may be the same as those generally used for the culture of Pediococcus genus microorganisms, as described above.
  • static culture is preferable.
  • static culture using a glass bottle with a lid may be used.
  • the culture temperature is preferably kept between 25° C. and 37° C., and more preferably between 30° C. and 37° C.
  • the culture pH is preferably around 7.
  • the culture period is a factor that varies depending on the medium composition used, the culture temperature, etc., but in the case of Lactic Acid Bacterium #4-3-1, a sufficient amount of the target product can be obtained by culture for preferably 12 to 72 hours, and more preferably 24 to 48 hours. It is also preferable to pick up colonies obtained by culture and perform single colony formation again on a medium.
  • Lactic acid bacteria #4-3-1 of the present invention has the ability to activate natural immunity both as the lactic acid bacteria themselves and as naturally or artificially mutated lactic acid bacteria of the lactic acid bacteria. That is, the present invention relates to a lactic acid bacterium belonging to the genus Pediococcus having the accession number NITE BP-03720, or a naturally or artificially mutated lactic acid bacterium thereof, which has the ability to activate innate immunity.
  • the "natural immunity activation ability" is measured by the method described in the Examples below.
  • the "processed product of lactic acid bacteria” includes at least one processed product selected from the group consisting of fermented products, cultured products, concentrates, pastes, dried products, liquefied products, diluted products, crushed products, sterilized products, and extracts from cultured products of lactic acid bacteria.
  • the "dried products” include spray-dried products, freeze-dried products, vacuum-dried products, drum-dried products, etc.
  • the present invention also relates to "a natural immune activator containing as an active ingredient the lactic acid bacteria of the present invention (Lactic acid bacteria #4-3-1 or naturally or artificially mutated lactic acid bacteria thereof), killed cells of the lactic acid bacteria, or a processed product of the lactic acid bacteria, characterized in that the processed product of the lactic acid bacteria is at least one processed product selected from the group consisting of fermented products, cultures, concentrates, pastes, dried products, liquefied products, diluted products, crushed products, sterilized products, and extracts from cultures of lactic acid bacteria.”
  • a natural immune activator containing as an active ingredient the lactic acid bacteria of the present invention (Lactic acid bacteria #4-3-1 or naturally or artificially mutated lactic acid bacteria thereof), killed cells of the lactic acid bacteria, or a processed product of the lactic acid bacteria, characterized in that the processed product of the lactic acid bacteria is at least one processed product selected from the group consisting of fermented products, cultures, concentrates, pastes, dried products, lique
  • the innate immunity activator of the present invention can contain the lactic acid bacteria of the present invention, killed cells of the lactic acid bacteria, or a processed product of the lactic acid bacteria in various states, such as a suspension, lactic acid bacteria bodies, culture supernatant, or medium components.
  • the lactic acid bacteria that are the active ingredient of the innate immunity activator of the present invention may be in the form of live bacteria, wet bacteria, dried bacteria, etc.
  • the active ingredient of the innate immunity activator of the present invention may be killed bacteria obtained by sterilizing the lactic acid bacteria, i.e., by subjecting the lactic acid bacteria to heat sterilization, radiation sterilization, crushing, etc.
  • the content of the lactic acid bacteria, killed lactic acid bacteria, and processed product of the lactic acid bacteria, which are the active ingredients in the innate immune activator of the present invention, relative to the entire innate immune activator is not particularly limited and can be selected appropriately depending on the purpose, but when the entire innate immune activator is taken as 100 parts by mass, the total amount of "lactic acid bacteria, killed lactic acid bacteria, and processed product of the lactic acid bacteria" is preferably 0.001 parts by mass or more, more preferably 0.01 parts by mass or more, and particularly preferably 0.1 parts by mass or more. It is also preferably 100 parts by mass or less, more preferably 99 parts by mass or less, and particularly preferably 95 parts by mass or less.
  • the active ingredients may be used alone or in combination of two or more kinds.
  • the content ratio of each active ingredient in the natural immune activator is not particularly limited and can be appropriately selected according to the purpose.
  • the innate immune activator of the present invention contains the lactic acid bacteria, killed lactic acid bacteria, or a processed product of the lactic acid bacteria as active ingredients, and can contain "other ingredients" in addition to these active ingredients.
  • the "other components" in the innate immune activator are not particularly limited and can be appropriately selected according to the purpose within a range that does not impair the effects of the present invention, and examples of such components include pharma- ceutically acceptable carriers.
  • the dosage form of the innate immune activator of the present invention is not particularly limited and can be appropriately selected depending on the desired administration method, for example, as described below.
  • Specific examples include oral solid preparations (tablets, coated tablets, granules, powders, hard capsules, soft capsules, etc.), oral liquid preparations (oral liquid preparations, syrups, elixirs, etc.), injections (solvents, suspensions, etc.), ointments, patches, gels, creams, external powders, sprays, and inhalation powders.
  • Oral solid preparations can be produced by adding, for example, excipients to the active ingredients, and, if necessary, additives such as binders, disintegrants, lubricants, colorants, and flavorings and odorants, in a conventional manner.
  • excipients include lactose, sucrose, sodium chloride, glucose, starch, calcium carbonate, kaolin, microcrystalline cellulose, and silicic acid.
  • binders include water, ethanol, propanol, simple syrup, glucose liquid, starch liquid, gelatin liquid, carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl starch, methyl cellulose, ethyl cellulose, shellac, calcium phosphate, and polyvinylpyrrolidone.
  • Disintegrants include, for example, dry starch, sodium alginate, agar powder, sodium hydrogen carbonate, calcium carbonate, sodium lauryl sulfate, monoglyceride stearate, lactose and the like.
  • Lubricants include, for example, purified talc, stearates, borax, polyethylene glycol, and the like.
  • colorants include titanium oxide and iron oxide.
  • flavoring and fragrance agents include sucrose, orange peel, citric acid, tartaric acid, and the like.
  • Oral liquid preparations can be produced by adding additives such as flavorings, buffers, stabilizers, edible (processed) oils, and animal and vegetable oils to the active ingredients in a conventional manner.
  • flavoring agents include sucrose, orange peel, citric acid, and tartaric acid.
  • buffering agent is sodium citrate.
  • Stabilizers include, for example, tragacanth, gum arabic, gelatin, and the like.
  • a pH adjusting agent for example, a buffering agent, a stabilizer, an isotonicity agent, a local anesthetic agent, etc.
  • injections for subcutaneous, intramuscular, intravenous, etc. can be produced by conventional methods.
  • pH adjusters and buffers include sodium citrate, sodium acetate, and sodium phosphate.
  • stabilizer include sodium pyrosulfite, EDTA, thioglycolic acid, and thiolactic acid.
  • isotonic agents include sodium chloride and glucose.
  • local anesthetics include procaine hydrochloride, lidocaine hydrochloride, and the like.
  • Ointments can be prepared, for example, by combining the active ingredients with known bases, stabilizers, humectants, preservatives, etc., and mixing them in a conventional manner.
  • bases include liquid paraffin, white petrolatum, white beeswax, octyldodecyl alcohol, paraffin, and the like.
  • preservatives include methyl parahydroxybenzoate, ethyl parahydroxybenzoate, and propyl parahydroxybenzoate.
  • Patches can be produced, for example, by applying the above-mentioned ointment in the form of a cream, gel, paste, etc., to a known support in a conventional manner.
  • the support examples include woven fabrics made of cotton, staple fiber, and chemical fibers, nonwoven fabrics, films such as soft polyvinyl chloride, polyethylene, polypropylene, and polyurethane, and foam sheets.
  • the innate immunity activator of the present invention can be suitably used for, for example, individuals, bacteria, etc. that require activation of the innate immune mechanism.
  • the composition can be administered to, for example, individuals who need to maintain their health or recover from fatigue; individuals who need to prevent or treat cancer or lifestyle-related diseases; individuals infected with bacteria, fungi, viruses, etc.; and the like.
  • animals to which the innate immune activator of the present invention may be administered include humans; laboratory animals such as mice and rats; monkeys; horses; livestock such as cows, pigs, goats, and chickens; and pets such as cats and dogs.
  • the method of administration of the natural immune activator is not particularly limited and can be appropriately selected depending on the dosage form described above, and examples of such methods include oral administration, intraperitoneal administration, injection into the blood, injection into the intestine, etc. Among these, oral administration is preferred because it is simple and exerts the above-mentioned effects.
  • the dosage of the natural immune activator is not particularly limited and can be appropriately selected depending on the age, body weight, and desired degree of effect of the individual to be administered, but for example, the daily dosage for an adult is preferably 1 mg or more, more preferably 10 mg or more, and particularly preferably 100 mg or more, as the amount of active ingredient. Also, it is preferably 30 g or less, more preferably 10 g or less, and particularly preferably 3 g or less.
  • the timing of administration is also not particularly limited and can be appropriately selected depending on the purpose. For example, the agent may be administered prophylactically or therapeutically.
  • the present invention also relates to a food product containing the above-mentioned lactic acid bacteria (Lactic Acid Bacteria #4-3-1) and a food product containing the above-mentioned innate immunity activator.
  • the present invention also relates to a method for producing a food product, the method comprising a step of fermenting the food product using the above-mentioned lactic acid bacteria (Lactic Acid Bacteria #4-3-1).
  • a food containing the above-mentioned lactic acid bacteria (Lactic acid bacteria #4-3-1) a food containing the above-mentioned innate immune activator", or "a food produced by a food production method having a fermentation step using the above-mentioned lactic acid bacteria (Lactic acid bacteria #4-3-1)” may be abbreviated as “the food of the present invention”.
  • the term “food” includes “drinks.”
  • lactic acid bacteria #4-3-1 is a lactic acid bacterium belonging to Pediococcus ethanolidurans.
  • Pediococcus ethanolidurans has been reported to have been isolated from food (apple wine) as described in Non-Patent Document 2.
  • Pediococcus ethanolidurans can be said to be a lactic acid bacterium that has been consumed in the past.
  • the content of lactic acid bacteria and natural immune activator in the food of the present invention is not particularly limited and can be selected appropriately depending on the purpose and the form (type) of the food, but when the entire food is taken as 100 parts by mass, the above total amount is preferably 0.001 parts by mass or more, more preferably 0.01 parts by mass or more, and particularly preferably 0.1 parts by mass or more. It is also preferably 100 parts by mass or less, more preferably 99 parts by mass or less, and particularly preferably 95 parts by mass or less.
  • any one of the above may be used alone, or two or more may be used in combination.
  • two or more are used in combination, there is no particular restriction on the content ratio of each substance in the above food, and it can be selected appropriately according to the purpose.
  • the food of the present invention has the ability to activate natural immunity.
  • the present invention is also a food for activating natural immunity.
  • the food of the present invention can further contain "other ingredients" in addition to the natural immunity activator of the present invention described above.
  • other ingredients are not particularly limited and can be selected appropriately according to the purpose within a range that does not impair the effects of the present invention, and examples include various food ingredients.
  • content of the “other ingredients” is not particularly limited and can be selected appropriately according to the purpose.
  • the food of the present invention may be a general food, or it may be a so-called health food (health functional food, food for specified health uses, food with nutrient functions, food with functional claims, etc.).
  • the type of general food is not particularly limited and can be selected appropriately depending on the purpose, and examples include confectioneries such as jelly, candy, chocolate, biscuits, gummies, etc.; beverages such as green tea, black tea, coffee, soft drinks, etc.; dairy products such as fermented milk, yogurt, ice cream, lacto ice cream, etc.; vegetable and fruit processed products such as vegetable drinks, fruit drinks, jams, etc.; liquid foods such as soups; grain processed products such as breads, noodles, etc.; various seasonings, etc. There are no particular limitations on the method for producing these foods, and they can be produced, for example, according to any of the usual methods for producing various foods.
  • the type of health food is not particularly limited and can be appropriately selected depending on the purpose.
  • examples include oral solid preparations such as tablets, granules, and capsules; oral liquid preparations such as oral liquid preparations and syrups; and the like.
  • the method for producing the above oral solid preparation and oral liquid preparation is not particularly limited and can be appropriately selected depending on the purpose.
  • the food of the present invention is particularly useful as a functional food, health food, etc., intended for activating the natural immune system and building resistance to infectious diseases.
  • the production method can be carried out by a method well known to those skilled in the art.
  • Those skilled in the art can produce the desired food by appropriately combining a step of mixing the (killed) cells or treated products of the lactic acid bacteria of the present invention with other ingredients, a molding step, a sterilization step, a fermentation step, a baking step, a drying step, a cooling step, a granulation step, a packaging step, etc.
  • 0.5 g of rice bran was collected with 10 ⁇ L of Aze and spread on MRS agar medium containing calcium carbonate (CaCO 3 -MRS agar medium).
  • MRS agar medium containing calcium carbonate CaCO 3 -MRS agar medium
  • 1 mL of physiological saline was added to 0.5 g of rice bran to suspend it, and after leaving it to stand for a while, the supernatant was diluted 10 -3 or 10 -5 with physiological saline and spread on CaCO 3 -MRS agar medium with 10 ⁇ L of Aze, and each plate was anaerobically cultured at 30°C for 3 to 6 days. Anaeropack Kenki was used for the culture.
  • colonies that had formed a clear zone around them were cultured again on CaCO 3 -MRS agar medium, and it was confirmed that they formed a clear zone. These were selected as candidate strains of lactic acid bacteria.
  • 16S rDNA was amplified from colonies of candidate lactic acid bacteria strains by colony PCR, and the sequence was subjected to a homology search with the 16S rDNA sequences of bacteria registered in a database using BLAST to identify the bacterial species with the highest sequence homology.
  • the above test samples were diluted to various concentrations, and 50 ⁇ L of each was injected into silkworm muscle specimens to measure muscle contraction values (C values).
  • Injection of 50 ⁇ L of saline and 0.2 mL of air served as negative and positive controls, respectively.
  • Dose-response curves were created using medical statistics software Prizm.
  • Table 1 shows the bacterial species and the measurement results of the natural immune activation ability for each candidate strain collected from rice bran (bran bed).
  • Table 2 shows the bacterial species and the values of innate immune activation ability of known lactic acid bacteria strains that the inventors have previously measured using the same method.
  • the values of the natural immune activation ability shown in Tables 1 and 2 are relative values when the activity (U/mg) per 1 mg of sample in Public Example 4 (Lactococcus lactis) is set to 1.
  • the lactic acid bacteria of the present invention have a high ability to activate natural immunity, and therefore are widely used in the production of general foods, health foods, and other foods.
  • SEQ ID NO:1 is the base sequence of almost the entire length of the 16S rDNA of an unknown strain belonging to the genus Pediococcus.

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Abstract

The present invention addresses the problem of providing lactic acid bacteria having high ability to activate innate immunity. The present invention also addresses the problem of providing: an innate immunity activator containing, as an active ingredient, said lactic acid bacteria, dead cells of the lactic acid bacteria, or a treated product of the lactic acid bacteria; and a food containing said lactic acid bacteria or said innate immunity activator. [Solution] The problem is solved by: lactic acid bacteria belonging to the genus Pediococcus the accession number of which in the Patent Microorganism Depository Center (NPMD) of the National Institute of Technology and Evaluation for Independent Administrative Agency (NITE) is NITE BP-03720; or an innate immunity activator comprising, as an active ingredient, said lactic acid bacteria, dead cells of said lactic acid bacteria or a treated product of said lactic acid bacteria, the innate immunity activator being characterized in that the treated product of said lactic acid bacteria is at least one treated product selected from the group consisting of fermented products, cultured products, concentrated products, paste products, dried products, liquefied products, diluted products, crushed products, sterilized products, and extracts from the cultured products.

Description

乳酸菌、該乳酸菌由来の自然免疫活性化剤及び該乳酸菌を含有する食品Lactic acid bacteria, natural immune activator derived from said lactic acid bacteria, and food containing said lactic acid bacteria
　本発明は、乳酸菌や、該乳酸菌由来の自然免疫活性化剤及び該乳酸菌を含有する食品に関する。 The present invention relates to lactic acid bacteria, natural immune activators derived from said lactic acid bacteria, and foods containing said lactic acid bacteria.
　乳酸菌は、古くから醗酵食品に利用され、食品、医薬品、プロバイオティクス等の生産に利用されている。乳酸菌は、グラム陽性である、カタラーゼ陰性である、内生胞子を形成しない、運動性がない、等の特徴がある。 Lactic acid bacteria have long been used in fermented foods and in the production of foods, medicines, probiotics, etc. Lactic acid bacteria are characterized by being gram-positive, catalase-negative, not forming endospores, and not being motile.
　また、乳酸菌はプロバイオティクスとして最も利用されている。機能性の高い乳酸菌を効率的に分離する方法の確立、及び、これらの食品中での培養方法を確立することは、機能性乳酸菌を利用した食品の開発に有用である。 Furthermore, lactic acid bacteria are most commonly used as probiotics. Establishing an efficient method for isolating highly functional lactic acid bacteria and culturing them in foods will be useful for developing foods that use functional lactic acid bacteria.
　乳酸菌は、従来知られている種々の食品の中に含まれており、食品から乳酸菌を分離し、菌種を同定したという報告がなされている。そのような報告の例を挙げる。 Lactic acid bacteria are found in a variety of conventionally known foods, and there have been reports of isolating lactic acid bacteria from foods and identifying the species. Examples of such reports are as follows:
　非特許文献１：Leuconostoc mesenteroides（分離源：乳製品）
　非特許文献２：Pediococcus ethanolidurans（分離源：リンゴ酒）
　非特許文献３：Lactobacillus versmoldensis（分離源：ソーセージ）
　非特許文献４：Companilactobacillus versmoldensis（分離源：ソーセージ）
　非特許文献５：Streptococcus salivarius（分離源：チーズ） Non-patent literature 1: Leuconostoc mesenteroides (isolation source: dairy products)
Non-patent literature 2: Pediococcus ethanolidurans (isolation source: apple wine)
Non-patent document 3: Lactobacillus versmoldensis (isolated from sausage)
Non-patent literature 4: Companilactobacillus versmoldensis (isolation source: sausage)
Non-patent literature 5: Streptococcus salivarius (isolation source: cheese)
　プロバイオティクスで注目されている機能性の一つに「自然免疫促進活性」がある。自然免疫とは、抗体によらずに生体内の異物（細菌・ウイルス・癌細胞）を速やかに排除する防御機構であり、昆虫から哺乳動物に至るまで生物が共通してもっている。 One of the functions of probiotics that has attracted attention is their "natural immunity-promoting activity." Natural immunity is a defense mechanism that quickly eliminates foreign substances (bacteria, viruses, cancer cells) from the body without relying on antibodies, and is shared by all living organisms, from insects to mammals.
　哺乳類において自然免疫は、生体防御の最前線であり、抗体産生を含むその後の免疫反応を引き起こす。哺乳動物における自然免疫では様々な刺激によりマクロファージ等の免疫担当細胞によるサイトカイン分泌が促され、侵入した病原体の排除や、他の免疫担当細胞への情報伝達が行われる。 In mammals, innate immunity is the first line of defense against the host, triggering subsequent immune responses, including antibody production. In mammals, various stimuli stimulate the secretion of cytokines by immune cells such as macrophages, which eliminate invading pathogens and transmit information to other immune cells.
　一方、昆虫等の無脊椎動物は獲得免疫をもっておらず、病原体の排除は自然免疫だけによっている。自然免疫機構は昆虫と哺乳動物の間で多くの共通点があることが知られている。例えばヘモサイトと呼ばれる昆虫の血液細胞は哺乳類におけるマクロファージと同様に侵入した異物を貪食する。また、哺乳動物での自然免疫応答に関与するToll-like Receptorは、ショウジョウバエで自然免疫応答に関与するToll Receptorと相同性が高いことが知られている。 On the other hand, invertebrates such as insects do not have adaptive immunity and rely solely on innate immunity to eliminate pathogens. It is known that the innate immune mechanisms of insects and mammals have many similarities. For example, insect blood cells called hemocytes phagocytose invading foreign substances in the same way as mammalian macrophages. In addition, the Toll-like receptor involved in the innate immune response in mammals is known to be highly homologous to the Toll receptor involved in the innate immune response in Drosophila.
　これまでに、本発明者らにより、自然免疫機構しかないカイコにおいて、自然免疫活性化反応を簡便に測定できる評価方法が開発されている（特許文献１等、非特許文献６、非特許文献７等）。この評価方法では、自然免疫の活性化に伴い、カイコの筋収縮が起こることを利用し、筋収縮の度合いにより、自然免疫活性化能を評価している。本発明者らは、該評価方法でヒト等の脊椎動物に対して自然免疫活性化作用を有する自然免疫活性化剤の評価（スクリーニング）ができることを示している（特許文献１等）。 　To date, the present inventors have developed an evaluation method that can easily measure innate immune activation responses in silkworms, which only have innate immune mechanisms (Patent Document 1, etc., Non-Patent Documents 6 and 7, etc.). This evaluation method makes use of the fact that silkworms contract their muscles as a result of activation of innate immunity, and evaluates the innate immune activation ability based on the degree of muscle contraction. The present inventors have shown that this evaluation method can be used to evaluate (screen) innate immune activators that have innate immune activation effects in vertebrates such as humans (Patent Document 1, etc.).
　また、本発明者らは、この評価方法（スクリーニング方法）を利用することにより、食品中の自然免疫活性物質を同定する方法を確立している（特許文献２、非特許文献８、非特許文献９等）。 The inventors have also established a method for identifying natural immune active substances in food by using this evaluation method (screening method) (Patent Document 2, Non-Patent Document 8, Non-Patent Document 9, etc.).
　免疫機構の異常は、様々な疾患を引き起こす原因となる。従って、このような免疫機構を所望に調節することが可能な、優れた自然免疫活性化剤や自然免疫を活性化させる食品の開発が望まれている。 Abnormalities in the immune system can lead to a variety of diseases. Therefore, there is a need to develop superior natural immune activators and foods that activate the natural immune system, which can regulate such immune systems as desired.
国際公開第２００８／１２６９０５号WO 2008/126905 特開２０１９－１８２７７６号公報JP 2019-182776 A
　本発明は上記背景技術に鑑みてなされたものであり、その課題は、高い自然免疫活性化能を有する乳酸菌を提供することであり、更に、該乳酸菌、該乳酸菌の死菌又は該乳酸菌の処理物を有効成分とする自然免疫活性化剤や、該乳酸菌や該自然免疫活性化剤を含有する食品を提供することにある。 The present invention has been made in view of the above-mentioned background art, and aims to provide a lactic acid bacterium that has a high ability to activate natural immunity, and further to provide a natural immunity activator that contains the lactic acid bacterium, killed cells of the lactic acid bacterium, or a processed product of the lactic acid bacterium as an active ingredient, and a food product that contains the lactic acid bacterium or the natural immunity activator.
　本発明者は、上記の課題を解決すべく鋭意検討を重ねた結果、乳酸菌の中でも、特定の菌株には、他の菌株と比較して高い自然免疫活性化能があることを見出し、かかる菌株を同定することにより、上記課題を解決できることを見出して、本発明を完成するに至った。 As a result of extensive research into solving the above problems, the inventors discovered that certain strains of lactic acid bacteria have a higher ability to activate natural immunity than other strains, and discovered that the above problems can be solved by identifying such strains, which led to the completion of the present invention.
　すなわち、本発明は、独立行政法人製品評価技術基盤機構（ＮＩＴＥ）の特許微生物寄託センター（ＮＰＭＤ）における受託番号がＮＩＴＥ　ＢＰ－０３７２０であるペディオコッカス属に属する乳酸菌を提供するものである。 In other words, the present invention provides a lactic acid bacterium belonging to the genus Pediococcus, which has a deposit number of NITE BP-03720 at the National Patent Microorganisms Depositary (NPMD) of the National Institute of Technology and Evaluation (NITE).
　また、本発明は、前記の乳酸菌、該乳酸菌の死菌又は該乳酸菌の処理物を有効成分とする自然免疫活性化剤であって、
　該乳酸菌の処理物が、乳酸菌の、醗酵物、培養物、濃縮物、ペースト化物、乾燥物、液状化物、希釈物、破砕物、殺菌加工物、及び、培養物からの抽出物よりなる群から選ばれる少なくとも１つの処理物であることを特徴とする自然免疫活性化剤を提供するものである。 The present invention also provides an innate immune activator comprising the lactic acid bacterium, killed lactic acid bacterium, or a processed product of the lactic acid bacterium as an active ingredient,
The present invention provides a natural immune activator, characterized in that the processed product of the lactic acid bacteria is at least one processed product selected from the group consisting of fermented products, cultured products, concentrates, pastes, dried products, liquefied products, diluted products, crushed products, sterilized products, and extracts from cultures of lactic acid bacteria.
　また、本発明は、前記の乳酸菌を含有する食品を提供するものである。 The present invention also provides a food product containing the lactic acid bacteria.
　また、本発明は、前記の自然免疫活性化剤を含有する食品を提供するものである。 The present invention also provides a food product containing the natural immune activator.
　また、本発明は、前記の乳酸菌を用いて醗酵する工程を有する、食品の製造方法を提供するものである。 The present invention also provides a method for producing food, which includes a fermentation step using the lactic acid bacteria.
　本発明によれば、高い自然免疫活性化能を有する乳酸菌を提供することができる。また、本発明によれば、該乳酸菌、該乳酸菌の死菌又は該乳酸菌の処理物を有効成分とする自然免疫活性化剤や、該乳酸菌や該自然免疫活性化剤を含有する食品を提供することができる。 The present invention can provide lactic acid bacteria with high innate immune activation ability. The present invention can also provide an innate immune activator that contains the lactic acid bacteria, killed cells of the lactic acid bacteria, or a processed product of the lactic acid bacteria as an active ingredient, and a food product that contains the lactic acid bacteria or the innate immune activator.
　本発明の乳酸菌の菌種は、食品から分離されたという報告がある。すなわち、過去に食経験がある。
　このため、本発明の乳酸菌や、自然免疫活性化剤は、安全性がある程度確保されているといえ、食品に添加するためのハードルが低い。 The strain of lactic acid bacteria of the present invention has been reported to have been isolated from food, i.e., it has been consumed in the past.
For this reason, it can be said that the safety of the lactic acid bacteria and innate immune activators of the present invention is ensured to a certain extent, and the hurdle for adding them to food is low.
本発明の乳酸菌＃４－３－１の１６Ｓ ｒＤＮＡと、Pediococcus ethanolidurans strain Z-9（登録番号：NR_043291.2）の１６Ｓ ｒＤＮＡのＢＬＡＳＴを用いた相同性解析の結果を示す図である。This figure shows the results of a homology analysis using BLAST between the 16S rDNA of lactic acid bacteria #4-3-1 of the present invention and the 16S rDNA of Pediococcus ethanolidurans strain Z-9 (registration number: NR_043291.2). 図１の続きの図である。This is a continuation of Figure 1.
　以下、本発明について説明するが、本発明は以下の実施の形態に限定されるものではなく、任意に変形して実施することができる。 The present invention will be described below, but it is not limited to the following embodiment and can be modified in any way.
　本発明の乳酸菌は、独立行政法人製品評価技術基盤機構（ＮＩＴＥ）の特許微生物寄託センター（ＮＰＭＤ）における受託番号がＮＩＴＥ　ＢＰ－０３７２０であるペディオコッカス属に属する乳酸菌である。
　なお、本明細書において、該乳酸菌を「乳酸菌＃４－３－１」、「乳酸菌＃４－３－１株」、「＃４－３－１」等と記述する場合がある。
　以下、乳酸菌＃４－３－１について詳述する。 The lactic acid bacterium of the present invention is a lactic acid bacterium belonging to the genus Pediococcus, whose accession number at the National Patent Microorganisms Depositary (NPMD) of the National Institute of Technology and Evaluation (NITE) is NITE BP-03720.
In this specification, the lactic acid bacteria may be referred to as "lactic acid bacteria #4-3-1,""lactic acid bacteria #4-3-1 strain,""#4-3-1," etc.
Lactic acid bacteria #4-3-1 will be described in detail below.
　乳酸菌＃４－３－１は、米糠（糠床）を分離源として初めて分離された。 Lactic acid bacteria #4-3-1 was first isolated from rice bran (bran bed).
　乳酸菌＃４－３－１の形態は、以下の通りである。 The morphology of Lactic Acid Bacteria #4-3-1 is as follows:
　グラム染色結果：陽性
　細胞形態：球形
　好気／嫌気：嫌気
　乳酸生成能：あり Gram staining result: positive Cell morphology: spherical Aerobic/anaerobic: anaerobic Lactic acid production: yes
　乳酸菌＃４－３－１の生理学的、化学分類学的性質は以下の通りである。 The physiological and chemical taxonomic properties of Lactobacillus #4-3-1 are as follows:
（１）カタラーゼ：－
（２）酸性フォスファターゼ：＋
（３）アルカリフォスファターゼ：－
（４）ナフトール－ＡＳ－ＢＩ－フォスフォヒドロラーゼ：＋
（５）エステラーゼ（Ｃ４）：＋
（６）α－ガラクトシダーゼ：－
（７）エステラーゼリパーゼ（Ｃ８）：＋
（８）β－ガラクトシダーゼ：－
（９）リパーゼ（Ｃ１４）：＋
（１０）β－グルクロニダーゼ：－
（１１）ロイシンアリルアミダーゼ：＋
（１２）α－グルコシダーゼ：＋
（１３）バリンアリルアミダーゼ：＋
（１４）β－グルコシダーゼ：＋
（１５）シスチンアリルアミダーゼ：＋
（１６）Ｎ－アセチル－β－グルコサミニダーゼ：＋
（１７）トリプシン：－
（１８）α－マンノシダーゼ：－
（１９）α－キモトリプシン：－
（２０）α－フコシダーゼ：－ (1) Catalase: -
(2) Acid phosphatase: +
(3) Alkaline phosphatase: -
(4) Naphthol-AS-BI-phosphohydrolase: +
(5) Esterase (C4): +
(6) α-galactosidase: -
(7) Esterase lipase (C8): +
(8) β-galactosidase: -
(9) Lipase (C14): +
(10) β-glucuronidase: -
(11) Leucine aryl amidase: +
(12) α-glucosidase: +
(13) valine aryl amidase: +
(14) β-glucosidase: +
(15) Cystine aryl amidase: +
(16) N-acetyl-β-glucosaminidase: +
(17) Trypsin: -
(18) α-mannosidase: -
(19) α-chymotrypsin: -
(20) α-fucosidase: -
（２１）下記の糖類等からの酸及びガスの生成能 (21) Ability to produce acids and gases from the following sugars, etc.
・グリセロール（Glycerol）：－
・エリトリトール（Erythritol）：－
・Ｄ－アラビノース（D-Arabinose）：－
・Ｌ－アラビノース（L-Arabinose）：－
・Ｄ－リボース（D-Ribose）：：－
・Ｄ－キシロース（D-Xylose）：－
・Ｌ－キシロース（L-Xylose）：－
・Ｄ－アドニトール（D-Adonitol）：－
・β－メチル－Ｄ－キシロピラノサイド（β-Methyl-D-xylopyranoside）：－
・Ｄ－ガラクトース（D-Galactose）：＋
・Ｄ－グルコース（D-Glucose）：＋
・Ｄ－フルクトース（D-Fructose）：＋
・Ｄ－マンノース（D-Mannose）：＋
・Ｌ－ソルボース（L-Sorbose）：－
・Ｌ－ラムノース（L-Rhamnose）：－
・ズルシトール（Dulcitol）：－
・イノシトール（Inositol）：－
・Ｄ－マンニトール（D-Mannitol）：－
・Ｄ－ソルビトール（D-Sorbitol）：－
・α－メチル－Ｄ－マンノピラノサイド（α-Methyl-D-mannopyranoside）：－
・α－メチル－Ｄ－グルコピラノサイド（α-Methyl-D-glucopyranoside）：－
・Ｎ－アセチルグルコサミン（N-Acetyl glucosamine）：＋
・アミグダリン（Amygdalin）：＋
・アルブチン（Arbutin）：－
・エスクリンクエン酸第二鉄（Esculin ferric citrate）：＋
・サリシン（Salicin）：＋
・Ｄ－セロビオース（D-Cellobiose）：＋
・Ｄ－マルトース（D-Maltose）：＋
・Ｄ－ラクトース（D-Lactose）：－
・Ｄ－メリビオース（D-Melibiose)：－
・Ｄ－スクロース（D-Sucrose）：＋
・Ｄ－トレハロース（D-Trehalose）：＋
・インスリン（Insulin）：－
・Ｄ－メレジトース（D-Melezitose）：＋
・Ｄ－ラフィノース（D-Raffinose）：＋
・スターチ（Starch）：＋
・グリコーゲン（Glycogen）：－
・キシリトール（Xylitol）：－
・ゲンチオビオース（Gentiobiose）：＋
・Ｄ－ツラノース（D-Turanose）：－
・Ｄ－リキソース（D-Lyxose）：－
・Ｄ－タガトース（D-Tagatose）：＋
・Ｄ－フコース（D-Fucose）：－
・Ｌ－フコース（L-Fucose）：－
・Ｄ－アラビトール（D-Arabitol）：－
・Ｌ－アラビトール（L-Arabitol）：－
・グルコネート（Gluconate）：－
・２－ケト－グルコネート（2-Keto-gluconate）：－
・５－ケト－グルコネート（5-Keto-gluconate）：－ ・Glycerol: -
・Erythritol: -
・D-Arabinose: -
・L-Arabinose: -
D-Ribose:
・D-Xylose: -
・L-Xylose: -
・D-Adonitol: -
β-Methyl-D-xylopyranoside: -
・D-Galactose: +
・D-Glucose: +
・D-Fructose: +
・D-Mannose: +
・L-Sorbose: -
・L-Rhamnose: -
・Dulcitol: -
・Inositol: -
・D-Mannitol: -
・D-Sorbitol: -
α-Methyl-D-mannopyranoside: -
α-Methyl-D-glucopyranoside: -
・N-Acetyl glucosamine: +
・Amygdalin: +
・Arbutin:-
・Esculin ferric citrate: +
・Salicin: +
・D-Cellobiose: +
・D-Maltose: +
・D-Lactose: -
・D-Melibiose: -
D-Sucrose: +
D-Trehalose: +
・Insulin:-
・D-Melezitose: +
・D-Raffinose: +
・Starch: +
・Glycogen:-
・Xylitol: -
・Gentiobiose: +
・D-Turanose: -
・D-Lyxose: -
・D-Tagatose: +
・D-Fucose: -
・L-Fucose: -
・D-Arabitol: -
・L-Arabitol: -
Gluconate:-
・2-Keto-gluconate:-
5-Keto-gluconate:
　分子生物学的な系統分類の指標として用いられている１６ＳｒＤＮＡに関する乳酸＃４－３－１の解析結果は、添付した配列表の配列番号１の通りである。
　すなわち、乳酸菌＃４－３－１のゲノムＤＮＡから、ＰＣＲにより、１６ＳｒＤＮＡ領域の塩基配列を増幅し、シーケンサーによる解析を行った結果、１６ＳｒＤＮＡのほぼ全長に当たる塩基配列が見出された。 The analysis results of Lactic Acid #4-3-1 regarding 16S rDNA, which is used as an index for molecular biological systematic classification, are as shown in SEQ ID NO: 1 of the attached sequence listing.
That is, the base sequence of the 16S rDNA region was amplified by PCR from the genomic DNA of lactic acid bacteria #4-3-1, and analyzed by a sequencer, resulting in the discovery of a base sequence corresponding to almost the entire length of the 16S rDNA.
　配列番号１の塩基配列を以下に記載する。 The base sequence of SEQ ID NO:1 is set forth below.
TATACTGCAGTCGAACGCACTTTCGTTAATTGATTTTGAGATGCTTGCATCGAAGATGATTTTAACTATAAAGTGAGTGGCGAACGGGTGAGTAACACGTGGGTAACCTGCCCAGAAGTGGGGGATAACACCTGGAAACAGATGCTAATACCGCATAACAAAATTAACCGCATGGTTGATTTTTAAAAGATGGCTTCGGCTATCACTTCTGGATGGACCCGCGGCGTATTAGCTAGTTGGTGAGATAAAGGCTCACCAAGGCAGTGATACGTAGCCGACCTGAGAGGGTAATCGGCCACATTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGCAAGTCTGATGGAGCAACGCCGCGTGAGTGATGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTGGAGAAGAACGTGTGTGAGAGTAACTGTTCATGCAGTGACGGTATCCAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTCTTTTAAGTCTAATGTGAAAGCCTTCGGCTTAACCGAAGAAGTGCATTGGAAACTGGGAGACTTGAGTGCAGAAGAGGAGAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTCTCTGGTCTGCAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAATGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAAGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCCAAGAACCTTACCAGGTCTTGACATCTTCTGCTAACCTAAGAGATTAGGCGTTCCCTTCGGGGACGGAATGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTATTAGTTGCCAGCATTAAGTTGGGCACTCTAGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGACGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGGTACAACGAGTTGCGAGACCGCGAGGTTTAGCTAATCTCTTAAAACCGTTCTCAGTTCGGACTGCAGGCTGCAACTCGCCTGCACGAAGTTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCAAAGCCGGTGGAGTAACCTTCGGGAGCTAGCCGTCTAAGGTGGGACAGATGATTGGGGTGAAGTCGTAACA TATACTGCAGTCGAACGCACTTTCGTTAATTGATTTTGAGATGCTTGCATCGAAGATGATTTTAACTATAAAGTGAGTGGCGAACGGGTGAGTAACACGTGGGTAACCTGCCCAGAAGTGGGGGATAACACCTGGAAACAGATGCTAATACCGCATAACAAAATTAACCGCATGGTTGATTTTTAAAAGATGGCTTCGGCTATCACTTCTGGATGGACCCGCGGCGTATTAGCTAGTTGGTGAGATAAAGGCTCACCAAGGCAGTGATACGTAGCCGACCTGAGAGGGTAATCGGCCACATTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGCAAGTC TGATGGAGCAACGCCGCGTGAGTGATGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTGGAGAAGAACGTGTGTGAGAGTAACTGTTCATGCAGTGACGGTATCCAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTCTTTTAAGTCTAATGTGAAAGCCTTCGGCTTAACCGAAGAAGTGCATTGGAAACTGGGAGACTTGAGTGCAGAAGAGGAGAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTCTCTGGTCTGCAACTGACGCTG AGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAATGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAAGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCCAAGAACCTTACCAGGTCTTGACATCTTCTGCTAACCTAAGAGATTAGGCGTTCCCTTCGGGGACGGAATGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTATTAGTTGCC AGCATTAAGTTGGGCACTCTAGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGACGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGGTACAACGAGTTGCGAGACCGCGAGGTTTAGCTAATCTCTTAAAACCGTTCTCAGTTCGGACTGCAGGCTGCAACTCGCCTGCACGAAGTTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCAAAGCCGGTGGAGTAACCTTCGGGAGCTAGCCGTCTAAGGTGGGACAGATGATTGGGGTGAAGTCGTAACA
　上記塩基配列をＮＣＢＩのＢＬＡＳＴ解析で相同性検索を行ったところ、乳酸菌＃４－３－１の１６ＳｒＤＮＡ領域の塩基配列は、ペディオコッカス属であるPediococcus ethanolidurans strain Z-9の塩基配列（登録番号：NR_043291.2）と相同性９９％を示したので、乳酸菌＃４－３－１は、ペディオコッカス・エタノリデュランス（Pediococcus ethanolidurans）に属するものである。
　しかしながら、１６ＳｒＤＮＡ領域だけを比較したときですら完全には一致していないので、本発明の乳酸菌＃４－３－１は、上記の株とは異なる乳酸菌株である。 When the above base sequence was subjected to a homology search using NCBI BLAST analysis, the base sequence of the 16S rDNA region of lactic acid bacteria #4-3-1 showed 99% homology with the base sequence of Pediococcus ethanolidurans strain Z-9 (accession number: NR_043291.2), which belongs to the Pediococcus genus, and therefore lactic acid bacteria #4-3-1 belongs to Pediococcus ethanolidurans.
However, even when only the 16S rDNA region was compared, there was no complete agreement, and therefore the lactic acid bacteria #4-3-1 of the present invention is a different lactic acid bacteria strain from the above strains.
　乳酸菌＃４－３－１の生理学的・化学分類学的性質を、バージース・マニュアル・オブ・システマティックバクテリオロジー（Bergey’s Manual of Systematic Bacteriology，vol.3 1989）による分類及びその他の文献の記載内容に照らし合わせ、更に、上記１６ＳｒＤＮＡ解析の結果を考慮して判断した結果、本発明の「乳酸菌＃４－３－１」は、ペディオコッカス（Pediococcus）属に属する新規の微生物である。
　また、乳酸菌＃４－３－１の１６ＳｒＤＮＡ領域の塩基配列に一致する１６ＳｒＤＮＡ領域の塩基配列を有する微生物が存在しないこと、ペディオコッカス属に属する既知の株等と比べて高い自然免疫活性作用を示すこと等を含め総合的に検討した結果、乳酸菌＃４－３－１は単離された新規な微生物株であると判断した。 The physiological and chemical taxonomic properties of Lactic Acid Bacteria #4-3-1 were compared with the classification in Bergey's Manual of Systematic Bacteriology (vol. 3, 1989) and the contents of other literature, and further, taking into consideration the results of the 16S rDNA analysis, it was determined that the "Lactic Acid Bacteria #4-3-1" of the present invention is a novel microorganism belonging to the genus Pediococcus.
In addition, after a comprehensive review, including the fact that there are no microorganisms having a base sequence in the 16S rDNA region that matches the base sequence in the 16S rDNA region of Lactic Acid Bacterium #4-3-1 and that it exhibits higher natural immune activation effects than known strains belonging to the Pediococcus genus, it was determined that Lactic Acid Bacterium #4-3-1 is a novel isolated microbial strain.
　乳酸菌＃４－３－１は、千葉県木更津市かずさ鎌足２－５－８　１２２号室、独立行政法人製品評価技術基盤機構（National Institute of Technology and Evaluation；以下、「ＮＩＴＥ」と略記する）の特許微生物寄託センター（ＮＰＭＤ）に国内寄託され、受託番号：ＮＩＴＥ　Ｐ－０３７２０（受託日：２０２２年８月１２日）として受託された微生物である。
　乳酸菌＃４－３－１は、その後、千葉県木更津市かずさ鎌足２－５－８　１２２号室、独立行政法人製品評価技術基盤機構（ＮＩＴＥ）の特許微生物寄託センター（ＮＰＭＤ）に、原寄託申請書を提出して、国内寄託（原寄託日：２０２２年８月１２日）から、ブダペスト条約に基づく寄託への移管申請を行い（移管日（国際寄託日）：２０２３年６月１９日）、生存が証明され、ブダペスト条約に基づく寄託（国際寄託）への移管申請が受領された結果、受託番号：ＮＩＴＥ　ＢＰ－０３７２０を付与された。 Lactic acid bacteria #4-3-1 was deposited domestically at the National Institute of Technology and Evaluation (NPMD) of the National Institute of Technology and Evaluation (hereinafter abbreviated as "NITE"), Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan, and was deposited under the accession number: NITE P-03720 (deposit date: August 12, 2022).
Lactic acid bacteria #4-3-1 was then submitted to the National Institute of Technology and Evaluation (NITE) Patent Microorganisms Depository (NPMD), Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan, via an original deposit application, and an application was made to transfer the domestic deposit (original deposit date: August 12, 2022) to a deposit under the Budapest Treaty (transfer date (international deposit date): June 19, 2023). As a result of the verification of viability and the acceptance of the application for transfer to a deposit (international deposit) under the Budapest Treaty, the deposit was assigned the deposit number: NITE BP-03720.
　細菌の一般的な性状として、その菌株としての性質は変異し易いため、本発明の乳酸菌＃４－３－１は、先に示した生理学的性状の範囲内に留まらない可能性も有している。また、かかる「変異」には、自然的な変異と人工的な変異の両方を含む。 As a general property of bacteria, the properties of the strain are easily mutated, so the lactic acid bacteria #4-3-1 of the present invention may not remain within the range of physiological properties shown above. Furthermore, such "mutations" include both natural and artificial mutations.
　以下に、乳酸菌＃４－３－１の培養方法について記載する。乳酸菌＃４－３－１の培養方法は、ペディオコッカス属の微生物に対して行われる一般的な培養方法に準じて行えばよい。
　培養は嫌気条件下で行うことが好ましい。培地中の炭素源としては、例えば、Ｄ－リボース、Ｄ－ガラクトース、Ｄ－グルコース、Ｄ－フルクトース、Ｄ－マンノース、Ｄ－マンニトール、Ｎ－アセチルグルコサミン、アミグダリン、アルブチン、エスクリン、サリシン、Ｄ－セロビオース、Ｄ－マルトース、シュクロース、Ｄ－トレハロース、ゲンチオビオース、糖蜜、水飴、油脂類等の有機炭素化合物が用いられ、窒素源としては、肉エキス、カゼイン、ペプトン、酵母エキス、乾燥酵母、胚芽、大豆粉、尿素、アミノ酸、アンモニウム塩等の有機・無機窒素化合物を用いることができる。
　また、塩類は、ナトリウム塩、カリウム塩、カルシウム塩、マグネシウム塩、リン酸塩、鉄塩、銅塩、亜鉛塩、コバルト塩等の無機塩類を必要に応じて適宜添加することができる。更に、ビオチン、ビタミンＢ１、シスチン、オレイン酸メチル、ラード油等の生育促進物質を添加することが、目的物の産生量を増加させる点で好ましい。
　また、シリコン油、界面活性剤等の消泡剤を添加してもよい。調製済みの培地としては、例えば、ＭＲＳ培地、ＧＡＭ培地等を用いることが好ましい。 The method for culturing Lactic Acid Bacteria #4-3-1 is described below. Lactic acid bacteria #4-3-1 may be cultured according to a general culture method used for microorganisms of the genus Pediococcus.
The culture is preferably carried out under anaerobic conditions. Carbon sources in the medium include organic carbon compounds such as D-ribose, D-galactose, D-glucose, D-fructose, D-mannose, D-mannitol, N-acetylglucosamine, amygdalin, arbutin, esculin, salicin, D-cellobiose, D-maltose, sucrose, D-trehalose, gentiobiose, molasses, starch syrup, and fats and oils, and nitrogen sources include organic and inorganic nitrogen compounds such as meat extract, casein, peptone, yeast extract, dry yeast, germ, soybean flour, urea, amino acids, and ammonium salts.
In addition, inorganic salts such as sodium salts, potassium salts, calcium salts, magnesium salts, phosphates, iron salts, copper salts, zinc salts, cobalt salts, etc. may be added as necessary. Furthermore, it is preferable to add growth promoters such as biotin, vitamin B1, cystine, methyl oleate, lard oil, etc., in order to increase the production amount of the target product.
In addition, antifoaming agents such as silicone oil, surfactants, etc. may be added. As the prepared medium, for example, MRS medium, GAM medium, etc. are preferably used.
　培養条件は、先に記したようにペディオコッカス属の微生物に対して行われる一般的な培養条件に準じて行えばよい。液体培養法であれば静置培養が望ましい。小規模であれば蓋付きガラス瓶による静置培養法を用いてもよい。
　培養温度は、２５℃～３７℃間に保つことが好ましく、３０℃～３７℃で行うことがより好ましい。培養ｐＨは７付近で行うことが好ましい。培養期間は、用いた培地組成、培養温度等により変動するファクターであるが、乳酸菌＃４－３－１の場合、好ましくは１２～７２時間、より好ましくは２４～４８時間で充分な量の目的物を確保することができる。
　培養して得られたコロニーをピックアップし、再度培地上でシングルコロニー形成を行うことも好ましい。 The culture conditions may be the same as those generally used for the culture of Pediococcus genus microorganisms, as described above. For liquid culture, static culture is preferable. For small-scale culture, static culture using a glass bottle with a lid may be used.
The culture temperature is preferably kept between 25° C. and 37° C., and more preferably between 30° C. and 37° C. The culture pH is preferably around 7. The culture period is a factor that varies depending on the medium composition used, the culture temperature, etc., but in the case of Lactic Acid Bacterium #4-3-1, a sufficient amount of the target product can be obtained by culture for preferably 12 to 72 hours, and more preferably 24 to 48 hours.
It is also preferable to pick up colonies obtained by culture and perform single colony formation again on a medium.
　本発明の乳酸菌＃４－３－１は、該乳酸菌自身として、また、該乳酸菌の自然的若しくは人工的に変異した乳酸菌として、自然免疫活性化能を有する。
　すなわち、本発明は、受託番号がＮＩＴＥ　ＢＰ－０３７２０であるペディオコッカス属に属する乳酸菌又はその自然的若しくは人工的に変異した乳酸菌であって、自然免疫活性化能を有する乳酸菌である。
　「自然免疫活性化能」は、後記する実施例に記載の方法により測定される。 Lactic acid bacteria #4-3-1 of the present invention has the ability to activate natural immunity both as the lactic acid bacteria themselves and as naturally or artificially mutated lactic acid bacteria of the lactic acid bacteria.
That is, the present invention relates to a lactic acid bacterium belonging to the genus Pediococcus having the accession number NITE BP-03720, or a naturally or artificially mutated lactic acid bacterium thereof, which has the ability to activate innate immunity.
The "natural immunity activation ability" is measured by the method described in the Examples below.
　「乳酸菌＃４－３－１又はその自然的若しくは人工的に変異した乳酸菌」、「該乳酸菌の死菌」、「該乳酸菌の処理物」は、何れも自然免疫活性化能を有する。
　ここで、「乳酸菌の処理物」としては、乳酸菌の、醗酵物、培養物、濃縮物、ペースト化物、乾燥物、液状化物、希釈物、破砕物、殺菌加工物、及び、培養物からの抽出物よりなる群から選ばれる少なくとも１つの処理物が挙げられる。「乾燥物」としては、噴霧乾燥物、凍結乾燥物、真空乾燥物、ドラム乾燥物等が挙げられる。 "Lactic acid bacteria #4-3-1 or naturally or artificially mutated lactic acid bacteria thereof,""killed cells of said lactic acid bacteria," and "processed products of said lactic acid bacteria" all have the ability to activate natural immunity.
Here, the "processed product of lactic acid bacteria" includes at least one processed product selected from the group consisting of fermented products, cultured products, concentrates, pastes, dried products, liquefied products, diluted products, crushed products, sterilized products, and extracts from cultured products of lactic acid bacteria. The "dried products" include spray-dried products, freeze-dried products, vacuum-dried products, drum-dried products, etc.
　本発明は、「前記した本発明の乳酸菌（乳酸菌＃４－３－１又はその自然的若しくは人工的に変異した乳酸菌）、該乳酸菌の死菌又は該乳酸菌の処理物を有効成分とする自然免疫活性化剤であって、該乳酸菌の処理物が、乳酸菌の、醗酵物、培養物、濃縮物、ペースト化物、乾燥物、液状化物、希釈物、破砕物、殺菌加工物、及び、培養物からの抽出物よりなる群から選ばれる少なくとも１つの処理物であることを特徴とする自然免疫活性化剤」にも関する。 The present invention also relates to "a natural immune activator containing as an active ingredient the lactic acid bacteria of the present invention (Lactic acid bacteria #4-3-1 or naturally or artificially mutated lactic acid bacteria thereof), killed cells of the lactic acid bacteria, or a processed product of the lactic acid bacteria, characterized in that the processed product of the lactic acid bacteria is at least one processed product selected from the group consisting of fermented products, cultures, concentrates, pastes, dried products, liquefied products, diluted products, crushed products, sterilized products, and extracts from cultures of lactic acid bacteria."
　本発明の自然免疫活性化剤は、前記の本発明の乳酸菌、該乳酸菌の死菌又は該乳酸菌の処理物を種々の状態で含むことができる。かかる状態としては、例えば、懸濁液、乳酸菌体、培養上清液、培地成分を含む状態が挙げられる。
　本発明の自然免疫活性化剤の有効成分である乳酸菌は、生菌体、湿潤菌、乾燥菌等が適宜使用可能である。また、本発明の自然免疫活性化剤の有効成分は、乳酸菌に、殺菌、すなわち、加熱殺菌処理、放射線殺菌処理、破砕処理等を施した死菌であってもよい。 The innate immunity activator of the present invention can contain the lactic acid bacteria of the present invention, killed cells of the lactic acid bacteria, or a processed product of the lactic acid bacteria in various states, such as a suspension, lactic acid bacteria bodies, culture supernatant, or medium components.
The lactic acid bacteria that are the active ingredient of the innate immunity activator of the present invention may be in the form of live bacteria, wet bacteria, dried bacteria, etc. The active ingredient of the innate immunity activator of the present invention may be killed bacteria obtained by sterilizing the lactic acid bacteria, i.e., by subjecting the lactic acid bacteria to heat sterilization, radiation sterilization, crushing, etc.
　本発明の自然免疫活性化剤中の有効成分である、乳酸菌、該乳酸菌の死菌、該乳酸菌の処理物の、自然免疫活性化剤全体に対する含有量は、特に制限がなく、目的に応じて適宜選択することができるが、自然免疫活性化剤全体を１００質量部としたときに、「乳酸菌、該乳酸菌の死菌、該乳酸菌の処理物」の合計量として、０．００１質量部以上であることが好ましく、０．０１質量部以上であることがより好ましく、０．１質量部以上であることが特に好ましい。また、１００質量部以下であることが好ましく、９９質量部以下であることがより好ましく、９５質量部以下であることが特に好ましい。 The content of the lactic acid bacteria, killed lactic acid bacteria, and processed product of the lactic acid bacteria, which are the active ingredients in the innate immune activator of the present invention, relative to the entire innate immune activator is not particularly limited and can be selected appropriately depending on the purpose, but when the entire innate immune activator is taken as 100 parts by mass, the total amount of "lactic acid bacteria, killed lactic acid bacteria, and processed product of the lactic acid bacteria" is preferably 0.001 parts by mass or more, more preferably 0.01 parts by mass or more, and particularly preferably 0.1 parts by mass or more. It is also preferably 100 parts by mass or less, more preferably 99 parts by mass or less, and particularly preferably 95 parts by mass or less.
　また、前記有効成分は、何れか１種を単独で使用してもよいし、２種以上を併用してもよい。２種以上を併用する場合の、前記自然免疫活性化剤中の各々の有効成分の含有比については、特に制限はなく目的に応じて適宜選択することができる。 Furthermore, the active ingredients may be used alone or in combination of two or more kinds. When two or more kinds are used in combination, the content ratio of each active ingredient in the natural immune activator is not particularly limited and can be appropriately selected according to the purpose.
　本発明の自然免疫活性化剤は、前記乳酸菌、前記乳酸菌の死菌又は前記乳酸菌の処理物を有効成分として含有するが、それら有効成分に加えて、「その他の成分」を含有することができる。 The innate immune activator of the present invention contains the lactic acid bacteria, killed lactic acid bacteria, or a processed product of the lactic acid bacteria as active ingredients, and can contain "other ingredients" in addition to these active ingredients.
　前記自然免疫活性化剤における、上記「その他の成分」としては、特に制限はなく、本発明の効果を損なわない範囲内で、目的に応じて適宜選択することができ、例えば、薬学的に許容され得る担体等が挙げられる。 The "other components" in the innate immune activator are not particularly limited and can be appropriately selected according to the purpose within a range that does not impair the effects of the present invention, and examples of such components include pharma- ceutically acceptable carriers.
　かかる担体としては、特に制限はなく、例えば、後述する剤型等に応じて適宜選択される。また、自然免疫活性化剤中の「その他の成分」の含有量としても、特に制限はなく、目的に応じて適宜選択することができる。 There are no particular limitations on such carriers, and they can be selected appropriately depending on, for example, the dosage form described below. In addition, there are no particular limitations on the amount of "other ingredients" contained in the natural immune activator, and they can be selected appropriately depending on the purpose.
　本発明の自然免疫活性化剤の剤型としては、特に制限はなく、例えば、後述するような所望の投与方法に応じて適宜選択することができる。
　具体的には、例えば、経口固形剤（錠剤、被覆錠剤、顆粒剤、散剤、ハードカプセル剤、ソフトカプセル剤等）、経口液剤（内服液剤、シロップ剤、エリキシル剤等）、注射剤（溶剤、懸濁剤等）、軟膏剤、貼付剤、ゲル剤、クリーム剤、外用散剤、スプレー剤、吸入散布剤等が挙げられる。 The dosage form of the innate immune activator of the present invention is not particularly limited and can be appropriately selected depending on the desired administration method, for example, as described below.
Specific examples include oral solid preparations (tablets, coated tablets, granules, powders, hard capsules, soft capsules, etc.), oral liquid preparations (oral liquid preparations, syrups, elixirs, etc.), injections (solvents, suspensions, etc.), ointments, patches, gels, creams, external powders, sprays, and inhalation powders.
　経口固形剤としては、例えば、上記有効成分に、賦形剤、更には必要に応じて結合剤、崩壊剤、滑沢剤、着色剤、矯味・矯臭剤等の添加剤を加え、常法により製造することができる。 Oral solid preparations can be produced by adding, for example, excipients to the active ingredients, and, if necessary, additives such as binders, disintegrants, lubricants, colorants, and flavorings and odorants, in a conventional manner.
　賦形剤としては、例えば、乳糖、白糖、塩化ナトリウム、ブドウ糖、デンプン、炭酸カルシウム、カオリン、微結晶セルロース、珪酸等が挙げられる。
　結合剤としては、例えば、水、エタノール、プロパノール、単シロップ、ブドウ糖液、デンプン液、ゼラチン液、カルボキシメチルセルロース、ヒドロキシプロピルセルロース、ヒドロキシプロピルスターチ、メチルセルロース、エチルセルロース、シェラック、リン酸カルシウム、ポリビニルピロリドン等が挙げられる。
　崩壊剤としては、例えば、乾燥デンプン、アルギン酸ナトリウム、カンテン末、炭酸水素ナトリウム、炭酸カルシウム、ラウリル硫酸ナトリウム、ステアリン酸モノグリセリド、乳糖等が挙げられる。
　滑沢剤としては、例えば、精製タルク、ステアリン酸塩、ホウ砂、ポリエチレングリコール等が挙げられる。
　着色剤としては、例えば、酸化チタン、酸化鉄等が挙げられる。
　記矯味・矯臭剤としては、例えば、白糖、橙皮、クエン酸、酒石酸等が挙げられる。 Examples of excipients include lactose, sucrose, sodium chloride, glucose, starch, calcium carbonate, kaolin, microcrystalline cellulose, and silicic acid.
Examples of binders include water, ethanol, propanol, simple syrup, glucose liquid, starch liquid, gelatin liquid, carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl starch, methyl cellulose, ethyl cellulose, shellac, calcium phosphate, and polyvinylpyrrolidone.
Disintegrants include, for example, dry starch, sodium alginate, agar powder, sodium hydrogen carbonate, calcium carbonate, sodium lauryl sulfate, monoglyceride stearate, lactose and the like.
Lubricants include, for example, purified talc, stearates, borax, polyethylene glycol, and the like.
Examples of colorants include titanium oxide and iron oxide.
Examples of flavoring and fragrance agents include sucrose, orange peel, citric acid, tartaric acid, and the like.
　経口液剤としては、例えば、上記有効成分に、矯味・矯臭剤、緩衝剤、安定化剤、食用（加工）油、動植物油等の添加剤を加え、常法により製造することができる。 Oral liquid preparations can be produced by adding additives such as flavorings, buffers, stabilizers, edible (processed) oils, and animal and vegetable oils to the active ingredients in a conventional manner.
　矯味・矯臭剤としては、例えば、白糖、橙皮、クエン酸、酒石酸等が挙げられる。
　緩衝剤としては、例えば、クエン酸ナトリウム等が挙げられる。
　安定化剤としては、例えば、トラガント、アラビアゴム、ゼラチン等が挙げられる。 Examples of flavoring agents include sucrose, orange peel, citric acid, and tartaric acid.
An example of the buffering agent is sodium citrate.
Stabilizers include, for example, tragacanth, gum arabic, gelatin, and the like.
　注射剤としては、例えば、上記有効成分に、ｐＨ調節剤、緩衝剤、安定化剤、等張化剤、局所麻酔剤等を添加し、常法により皮下用、筋肉内用、静脈内用等の注射剤を製造することができる。 As for injections, for example, a pH adjusting agent, a buffering agent, a stabilizer, an isotonicity agent, a local anesthetic agent, etc. can be added to the above-mentioned active ingredient, and injections for subcutaneous, intramuscular, intravenous, etc. can be produced by conventional methods.
　ｐＨ調節剤及び該緩衝剤としては、例えば、クエン酸ナトリウム、酢酸ナトリウム、リン酸ナトリウム等が挙げられる。
　安定化剤としては、例えば、ピロ亜硫酸ナトリウム、ＥＤＴＡ、チオグリコール酸、チオ乳酸等が挙げられる。
　等張化剤としては、例えば、塩化ナトリウム、ブドウ糖等が挙げられる。
　局所麻酔剤としては、例えば、塩酸プロカイン、塩酸リドカイン等が挙げられる。 Examples of pH adjusters and buffers include sodium citrate, sodium acetate, and sodium phosphate.
Examples of the stabilizer include sodium pyrosulfite, EDTA, thioglycolic acid, and thiolactic acid.
Examples of isotonic agents include sodium chloride and glucose.
Examples of local anesthetics include procaine hydrochloride, lidocaine hydrochloride, and the like.
　軟膏剤としては、例えば、上記有効成分に、公知の基剤、安定剤、湿潤剤、保存剤等を配合し、常法により混合し、製造することができる。 Ointments can be prepared, for example, by combining the active ingredients with known bases, stabilizers, humectants, preservatives, etc., and mixing them in a conventional manner.
　基剤としては、例えば、流動パラフィン、白色ワセリン、サラシミツロウ、オクチルドデシルアルコール、パラフィン等が挙げられる。
　保存剤としては、例えば、パラオキシ安息香酸メチル、パラオキシ安息香酸エチル、パラオキシ安息香酸プロピル等が挙げられる。 Examples of bases include liquid paraffin, white petrolatum, white beeswax, octyldodecyl alcohol, paraffin, and the like.
Examples of preservatives include methyl parahydroxybenzoate, ethyl parahydroxybenzoate, and propyl parahydroxybenzoate.
　貼付剤としては、例えば、公知の支持体に上記軟膏剤としてのクリーム剤、ゲル剤、ペースト剤等を、常法により塗布し、製造することができる。 Patches can be produced, for example, by applying the above-mentioned ointment in the form of a cream, gel, paste, etc., to a known support in a conventional manner.
　支持体としては、例えば、綿、スフ、化学繊維からなる織布、不織布、軟質塩化ビニル、ポリエチレン、ポリプロピレン、ポリウレタン等のフィルム、発泡体シート等が挙げられる。 Examples of the support include woven fabrics made of cotton, staple fiber, and chemical fibers, nonwoven fabrics, films such as soft polyvinyl chloride, polyethylene, polypropylene, and polyurethane, and foam sheets.
　本発明の自然免疫活性化剤は、例えば、自然免疫機構の活性化を必要とする個体、細菌等に対して好適に使用できる。
　具体的には、例えば、健康維持や疲労回復を必要とする個体；癌や生活習慣病の予防や治療を必要とする個体；細菌、真菌、ウイルス等に感染した個体；等に投与することにより使用することができる。 The innate immunity activator of the present invention can be suitably used for, for example, individuals, bacteria, etc. that require activation of the innate immune mechanism.
Specifically, the composition can be administered to, for example, individuals who need to maintain their health or recover from fatigue; individuals who need to prevent or treat cancer or lifestyle-related diseases; individuals infected with bacteria, fungi, viruses, etc.; and the like.
　本発明の自然免疫活性化剤の投与対象動物としては、特に制限はないが、例えば、ヒト；マウス、ラット等の実験動物；サル；ウマ；ウシ、ブタ、ヤギ、ニワトリ等の家畜；ネコ、イヌ等のペット；等が挙げられる。 　There are no particular limitations on the animals to which the innate immune activator of the present invention may be administered, but examples include humans; laboratory animals such as mice and rats; monkeys; horses; livestock such as cows, pigs, goats, and chickens; and pets such as cats and dogs.
　また、上記自然免疫活性化剤の投与方法としては、特に制限はなく、例えば、上記した剤型等に応じ、適宜選択することができ、経口投与、腹腔内投与、血液中への注射、腸内への注入等が挙げられる。中でも、経口投与が、簡便で上記効果を発揮する点から好ましい。 The method of administration of the natural immune activator is not particularly limited and can be appropriately selected depending on the dosage form described above, and examples of such methods include oral administration, intraperitoneal administration, injection into the blood, injection into the intestine, etc. Among these, oral administration is preferred because it is simple and exerts the above-mentioned effects.
　上記自然免疫活性化剤の投与量としては、特に制限・限定はなく、投与対象である個体の年齢、体重、所望の効果の程度等に応じて適宜選択することができるが、例えば、成人への１日の投与量は、有効成分の量として、１ｍｇ以上であることが好ましく、１０ｍｇ以上であることがより好ましく、１００ｍｇ以上であることが特に好ましい。また、３０ｇ以下であることが好ましく、１０ｇ以下であることがより好ましく、３ｇ以下であることが特に好ましい。
　また、投与時期としても、特に制限はなく、目的に応じて適宜選択することができ、例えば、予防的に投与されてもよいし、治療的に投与されてもよい。 The dosage of the natural immune activator is not particularly limited and can be appropriately selected depending on the age, body weight, and desired degree of effect of the individual to be administered, but for example, the daily dosage for an adult is preferably 1 mg or more, more preferably 10 mg or more, and particularly preferably 100 mg or more, as the amount of active ingredient. Also, it is preferably 30 g or less, more preferably 10 g or less, and particularly preferably 3 g or less.
The timing of administration is also not particularly limited and can be appropriately selected depending on the purpose. For example, the agent may be administered prophylactically or therapeutically.
　本発明は、前記した乳酸菌（乳酸菌＃４－３－１）を含有する食品や、前記した自然免疫活性化剤を含有する食品にも関する。また、本発明は、前記した乳酸菌（乳酸菌＃４－３－１）を用いて醗酵する工程を有する、食品の製造方法にも関する。
　以下、「前記した乳酸菌（乳酸菌＃４－３－１）を含有する食品」、「前記した自然免疫活性化剤を含有する食品」又は「前記した乳酸菌（乳酸菌＃４－３－１）を用いて醗酵する工程を有する、食品の製造方法により製造された食品」を「本発明の食品」と略記する場合がある。
　また、本明細書において、「食品」の中には「飲用品」が含まれる。 The present invention also relates to a food product containing the above-mentioned lactic acid bacteria (Lactic Acid Bacteria #4-3-1) and a food product containing the above-mentioned innate immunity activator. The present invention also relates to a method for producing a food product, the method comprising a step of fermenting the food product using the above-mentioned lactic acid bacteria (Lactic Acid Bacteria #4-3-1).
Hereinafter, "a food containing the above-mentioned lactic acid bacteria (Lactic acid bacteria #4-3-1)", "a food containing the above-mentioned innate immune activator", or "a food produced by a food production method having a fermentation step using the above-mentioned lactic acid bacteria (Lactic acid bacteria #4-3-1)" may be abbreviated as "the food of the present invention".
In this specification, the term "food" includes "drinks."
　前記のように、乳酸菌＃４－３－１は、ペディオコッカス・エタノリデュランス（Pediococcus ethanolidurans）に属する乳酸菌である。
　ペディオコッカス・エタノリデュランス（Pediococcus ethanolidurans）は、非特許文献２に記載のように、食品（リンゴ酒）から分離されたことが報告されている。すなわち、ペディオコッカス・エタノリデュランス（Pediococcus ethanolidurans）は、食経験がある乳酸菌といえる。 As described above, lactic acid bacteria #4-3-1 is a lactic acid bacterium belonging to Pediococcus ethanolidurans.
Pediococcus ethanolidurans has been reported to have been isolated from food (apple wine) as described in Non-Patent Document 2. In other words, Pediococcus ethanolidurans can be said to be a lactic acid bacterium that has been consumed in the past.
　本発明の食品中の、乳酸菌、自然免疫活性化剤の含有量は、特に制限がなく、目的や食品の態様（種類）に応じて、適宜選択することができるが、食品全体を１００質量部としたときに、上記の合計量で、０．００１質量部以上であることが好ましく、０．０１質量部以上であることがより好ましく、０．１質量部以上であることが特に好ましい。また、１００質量部以下であることが好ましく、９９質量部以下であることがより好ましく、９５質量部以下であることが特に好ましい。 The content of lactic acid bacteria and natural immune activator in the food of the present invention is not particularly limited and can be selected appropriately depending on the purpose and the form (type) of the food, but when the entire food is taken as 100 parts by mass, the above total amount is preferably 0.001 parts by mass or more, more preferably 0.01 parts by mass or more, and particularly preferably 0.1 parts by mass or more. It is also preferably 100 parts by mass or less, more preferably 99 parts by mass or less, and particularly preferably 95 parts by mass or less.
　また、上記の何れか１種を単独で使用してもよいし、２種以上を併用してもよい。２種以上を併用する場合の、上記食品中の各々の物質の含有量比には、特に制限はなく、目的に応じて適宜選択することができる。 Furthermore, any one of the above may be used alone, or two or more may be used in combination. When two or more are used in combination, there is no particular restriction on the content ratio of each substance in the above food, and it can be selected appropriately according to the purpose.
　本発明の食品は、自然免疫活性化能を有する。すなわち、本発明は、自然免疫活性化食品でもある。本発明の食品は、上記した本発明の自然免疫活性化剤に加えて、更に、「その他の成分」を含有することができる。 The food of the present invention has the ability to activate natural immunity. In other words, the present invention is also a food for activating natural immunity. The food of the present invention can further contain "other ingredients" in addition to the natural immunity activator of the present invention described above.
　上記「その他の成分」としては、特に制限はなく、本発明の効果を損なわない範囲内で目的に応じて適宜選択することができ、例えば、各種食品原料等が挙げられる。また、「その他の成分」の含有量は、特に制限はなく、目的に応じて適宜選択することができる。 The above-mentioned "other ingredients" are not particularly limited and can be selected appropriately according to the purpose within a range that does not impair the effects of the present invention, and examples include various food ingredients. In addition, the content of the "other ingredients" is not particularly limited and can be selected appropriately according to the purpose.
　本発明の食品は、一般食品であってもよいし、いわゆる健康食品（保健機能食品、特定保健用食品、栄養機能食品、機能性表示食品等）であってもよい。 The food of the present invention may be a general food, or it may be a so-called health food (health functional food, food for specified health uses, food with nutrient functions, food with functional claims, etc.).
　一般食品の種類としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、ゼリー、キャンディー、チョコレート、ビスケット、グミ等の菓子類；緑茶、紅茶、コーヒー、清涼飲料等の嗜好飲料；醗酵乳、ヨーグルト、アイスクリーム、ラクトアイス等の乳製品；野菜飲料、果実飲料、ジャム類等の野菜・果実加工品；スープ等の液体食品；パン類、麺類等の穀物加工品；各種調味料；等が挙げられる。
　これらの食品の製造方法としては、特に制限はなく、例えば、通常の各種食品の製造方法に応じて、適宜製造することができる。 The type of general food is not particularly limited and can be selected appropriately depending on the purpose, and examples include confectioneries such as jelly, candy, chocolate, biscuits, gummies, etc.; beverages such as green tea, black tea, coffee, soft drinks, etc.; dairy products such as fermented milk, yogurt, ice cream, lacto ice cream, etc.; vegetable and fruit processed products such as vegetable drinks, fruit drinks, jams, etc.; liquid foods such as soups; grain processed products such as breads, noodles, etc.; various seasonings, etc.
There are no particular limitations on the method for producing these foods, and they can be produced, for example, according to any of the usual methods for producing various foods.
　健康食品の種類としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、錠剤、顆粒剤、カプセル剤等の経口固形剤；内服液剤、シロップ剤等の経口液剤；等が挙げられる。
　上記経口固形剤、経口液剤の製造方法は、特に制限はなく、目的に応じて適宜選択することができる。 The type of health food is not particularly limited and can be appropriately selected depending on the purpose. Examples include oral solid preparations such as tablets, granules, and capsules; oral liquid preparations such as oral liquid preparations and syrups; and the like.
The method for producing the above oral solid preparation and oral liquid preparation is not particularly limited and can be appropriately selected depending on the purpose.
　本発明の食品は、自然免疫機構の活性化や感染症に対して抵抗力を付けること等を目的とした、機能性食品、健康食品等として、特に有用である。
　本発明の乳酸菌、該死菌若しくは処理物等を食品の製造に使用する場合、製造方法は当業者に周知の方法によって行うことができる。当業者であれば、本発明の乳酸菌の（死）菌体又は処理物を他の成分と混合する工程、成形工程、殺菌工程、醗酵工程、焼成工程、乾燥工程、冷却工程、造粒工程、包装工程等を適宜組み合わせ、目的の食品を作ることが可能である。 The food of the present invention is particularly useful as a functional food, health food, etc., intended for activating the natural immune system and building resistance to infectious diseases.
When the lactic acid bacteria of the present invention, killed cells or treated products thereof, etc. are used in the production of foods, the production method can be carried out by a method well known to those skilled in the art. Those skilled in the art can produce the desired food by appropriately combining a step of mixing the (killed) cells or treated products of the lactic acid bacteria of the present invention with other ingredients, a molding step, a sterilization step, a fermentation step, a baking step, a drying step, a cooling step, a granulation step, a packaging step, etc.
　以下に、実施例を挙げて本発明を更に具体的に説明するが、本発明は、その要旨を超えない限りこれらの実施例に限定されるものではない。 The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to these examples as long as they do not exceed the gist of the invention.
［乳酸菌の分離］
　分離源として、数種の米糠（糠床）を使用し、以下のようにして乳酸菌を分離した。 [Isolation of lactic acid bacteria]
Several types of rice bran (rice bran bed) were used as the isolation source, and lactic acid bacteria were isolated as follows.
　米糠０．５ｇを１０μＬエーゼで採取し、炭酸カルシウムを含むＭＲＳ寒天培地（ＣａＣＯ３－ＭＲＳ寒天培地）に塗布した。または、米糠０．５ｇに生理食塩水１ｍＬを加えて懸濁し、しばらく静置した後、上清を生理食塩水で１０－３又は１０－５希釈し、１０μＬエーゼでＣａＣＯ３－ＭＲＳ寒天培地に塗布し、各プレートを３０℃にて３～６日間嫌気培養した。培養にはアネロパック・ケンキを使用した。 0.5 g of rice bran was collected with 10 μL of Aze and spread on MRS agar medium containing calcium carbonate (CaCO 3 -MRS agar medium). Alternatively, 1 mL of physiological saline was added to 0.5 g of rice bran to suspend it, and after leaving it to stand for a while, the supernatant was diluted 10 -3 or 10 -5 with physiological saline and spread on CaCO 3 -MRS agar medium with 10 μL of Aze, and each plate was anaerobically cultured at 30°C for 3 to 6 days. Anaeropack Kenki was used for the culture.
　生成したコロニーのうち、周囲に透明帯を形成したコロニーを再度ＣａＣＯ３－ＭＲＳ寒天培地上で培養し、透明帯を形成することを確認した。これを乳酸菌の候補株とした。 Among the colonies that were produced, colonies that had formed a clear zone around them were cultured again on CaCO 3 -MRS agar medium, and it was confirmed that they formed a clear zone. These were selected as candidate strains of lactic acid bacteria.
［１６ｓ　ｒＤＮＡシークエンスによる乳酸菌種の同定］
　乳酸菌の候補株のコロニーを、コロニーＰＣＲにより１６Ｓ　ｒＤＮＡを増幅し、その配列についてＢＬＡＳＴを用いてデータベース上に登録されている菌の１６Ｓ　ｒＤＮＡ配列との相同性検索を行い、最も配列相同性の高い菌種を同定した。 [Identification of lactic acid bacteria species by 16s rDNA sequencing]
16S rDNA was amplified from colonies of candidate lactic acid bacteria strains by colony PCR, and the sequence was subjected to a homology search with the 16S rDNA sequences of bacteria registered in a database using BLAST to identify the bacterial species with the highest sequence homology.
［乳酸菌のオートクレーブ死菌体の調製］
　ＭＲＳ液体培地１４ｍＬで２日間培養した乳酸菌のフルグロース（full growth）１０ｍＬを、ＭＲＳ液体培地１００ｍＬに加え１日間培養し、約１００ｍＬの乳酸菌のフルグロースを得た。 [Preparation of autoclaved lactic acid bacteria cells]
10 mL of full growth of lactic acid bacteria cultured in 14 mL of MRS liquid medium for 2 days was added to 100 mL of MRS liquid medium and cultured for 1 day to obtain about 100 mL of full growth of lactic acid bacteria.
　これをオートクレーブ１２１℃、２０分で処理し、８０００ｒｐｍ、５分の遠心で菌体を回収した後、生理食塩水で１回洗浄し、生理食塩水１ｍＬに再懸濁し、自然免疫活性化能の測定のための試験サンプルとした。
　また、サンプルの一部を遠心エバポレーターで蒸発乾固し、乾燥重量を測定して濃度を算出した。 This was autoclaved at 121°C for 20 minutes, and the bacterial cells were collected by centrifugation at 8,000 rpm for 5 minutes. The cells were then washed once with saline and resuspended in 1 mL of saline to prepare a test sample for measuring innate immune activation ability.
In addition, a portion of the sample was evaporated to dryness using a centrifugal evaporator, and the dry weight was measured to calculate the concentration.
［自然免疫活性化能の測定（カイコ筋収縮試験）］
　カイコの筋収縮試験（非特許文献６に記載の試験方法）により、各候補株の自然免疫活性化能を測定した。 [Measurement of innate immune activation (silkworm muscle contraction test)]
The natural immune activation ability of each candidate strain was measured using a silkworm muscle contraction test (the test method described in Non-Patent Document 6).
　上記の試験サンプルを種々の濃度に希釈し、５０μＬをカイコ筋肉標本に注射して、筋収縮値（contraction value；Ｃ値）を測定した。
　筋収縮値（contraction value；Ｃ値）は、サンプル注射前のカイコ筋肉標本の体長（ｘ）、カイコ筋肉標本が最も収縮した時（約１０分後）の体長（ｙ）の測定値から、Ｃ＝（ｘ－ｙ）／ｘにより算出した。 The above test samples were diluted to various concentrations, and 50 μL of each was injected into silkworm muscle specimens to measure muscle contraction values (C values).
The muscle contraction value (C value) was calculated from the measured values of the body length (x) of the silkworm muscle specimen before sample injection and the body length (y) when the silkworm muscle specimen was most contracted (approximately 10 minutes later) by the formula C = (xy)/x.
　Ｃ値を縦軸に、サンプルの投与量を横軸にしたグラフを作成し、得られた用量応答曲線からＣ＝０．１５を与えるサンプル量を求めた。Ｃ＝０．１５を与えるサンプル量を、１Ｕ（１ユニット）と定義し、サンプル１ｍｇ当たりの活性（Ｕ／ｍｇ）を算出した。
　なお、生理食塩水５０μＬ、空気０．２ｍＬを注射したときをそれぞれネガティブ及びポジティブコントロールとした。用量応答曲線は、医療統計ソフトＰｒｉｚｍを使用して作成した。 A graph was prepared with the C value on the vertical axis and the dose of the sample on the horizontal axis, and the amount of sample giving C = 0.15 was determined from the resulting dose-response curve. The amount of sample giving C = 0.15 was defined as 1 U (1 unit), and the activity per mg of sample (U/mg) was calculated.
Injection of 50 μL of saline and 0.2 mL of air served as negative and positive controls, respectively. Dose-response curves were created using medical statistics software Prizm.
　米糠（糠床）から採取された各候補株について、菌種と、自然免疫活性化能の測定結果を表１に示す。 Table 1 shows the bacterial species and the measurement results of the natural immune activation ability for each candidate strain collected from rice bran (bran bed).
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　また、本発明者が過去に同じ方法で自然免疫活性化能を測定した公知の乳酸菌株について、菌種と、自然免疫活性化能の値を表２に示す。 　Table 2 shows the bacterial species and the values of innate immune activation ability of known lactic acid bacteria strains that the inventors have previously measured using the same method.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　なお、表１及び表２に示した自然免疫活性化能の値は、公知例４（ラクトコッカス・ラクティス）におけるサンプル１ｍｇ当たりの活性（Ｕ／ｍｇ）を１とした場合の相対値である。 The values of the natural immune activation ability shown in Tables 1 and 2 are relative values when the activity (U/mg) per 1 mg of sample in Public Example 4 (Lactococcus lactis) is set to 1.
　本発明の乳酸菌は、高い自然免疫活性化能を有するので、一般食品、健康食等の食品等の製造に広く利用されるものである。 The lactic acid bacteria of the present invention have a high ability to activate natural immunity, and therefore are widely used in the production of general foods, health foods, and other foods.
　ＮＩＴＥ　ＢＰ－０３７２０ NITE BP-03720
　配列番号１は、ペディオコッカス（Pediococcus）属に属する未知の菌株の、１６Ｓ ｒＤＮＡのほぼ全長にあたる塩基配列である。 SEQ ID NO:1 is the base sequence of almost the entire length of the 16S rDNA of an unknown strain belonging to the genus Pediococcus.

Claims (6)

  1. 　独立行政法人製品評価技術基盤機構（ＮＩＴＥ）の特許微生物寄託センター（ＮＰＭＤ）における受託番号がＮＩＴＥ　ＢＰ－０３７２０であるペディオコッカス属に属する乳酸菌。 A lactic acid bacterium belonging to the genus Pediococcus, whose accession number at the National Institute of Technology and Evaluation (NITE)'s Patent Microorganism Depositary (NPMD) is NITE BP-03720.
  2. 　請求項１に記載の乳酸菌又はその自然的若しくは人工的に変異した乳酸菌であって、自然免疫活性化能を有する乳酸菌。 The lactic acid bacterium according to claim 1 or a naturally or artificially mutated lactic acid bacterium thereof, which has the ability to activate natural immunity.
  3. 　請求項１若しくは請求項２に記載の乳酸菌、該乳酸菌の死菌又は該乳酸菌の処理物を有効成分とする自然免疫活性化剤であって、
    　該乳酸菌の処理物が、乳酸菌の、醗酵物、培養物、濃縮物、ペースト化物、乾燥物、液状化物、希釈物、破砕物、殺菌加工物、及び、培養物からの抽出物よりなる群から選ばれる少なくとも１つの処理物であることを特徴とする自然免疫活性化剤。     A natural immunity activator comprising the lactic acid bacterium according to claim 1 or 2, a killed lactic acid bacterium, or a processed product of the lactic acid bacterium as an active ingredient,
    The natural immune activator is characterized in that the processed product of the lactic acid bacteria is at least one processed product selected from the group consisting of fermented products, cultured products, concentrates, pastes, dried products, liquefied products, diluted products, crushed products, sterilized products, and extracts from cultures of lactic acid bacteria.
  4. 　請求項１又は請求項２に記載の乳酸菌を含有する食品。 A food product containing the lactic acid bacteria according to claim 1 or 2.
  5. 　請求項３に記載の自然免疫活性化剤を含有する食品。 A food product containing the natural immune activator described in claim 3.
  6. 　請求項１又は請求項２に記載の乳酸菌を用いて醗酵する工程を有する、食品の製造方法。 A method for producing a food product, comprising a step of fermenting the food product using the lactic acid bacteria according to claim 1 or 2.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008126905A1 (en) * 2007-04-10 2008-10-23 Genome Pharmaceuticals Institute Co., Ltd. Evaluation method and screening method for substance having action of activating/suppressing innate immunity, agent and food product for activating/suppressing innate immune mechanism and method for producing the same
JP2013193996A (en) * 2012-03-21 2013-09-30 Ishikawa Prefectural Public Univ Corp Lactobacillus isolated from traditional fermented food in ishikawa prefecture, and functionality and utilization of cultured material of the same
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WO2008126905A1 (en) * 2007-04-10 2008-10-23 Genome Pharmaceuticals Institute Co., Ltd. Evaluation method and screening method for substance having action of activating/suppressing innate immunity, agent and food product for activating/suppressing innate immune mechanism and method for producing the same
JP2013193996A (en) * 2012-03-21 2013-09-30 Ishikawa Prefectural Public Univ Corp Lactobacillus isolated from traditional fermented food in ishikawa prefecture, and functionality and utilization of cultured material of the same
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