WO2023054625A1 - Threonic-acid-containing composition and method for producing same - Google Patents

Threonic-acid-containing composition and method for producing same Download PDF

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Publication number
WO2023054625A1
WO2023054625A1 PCT/JP2022/036544 JP2022036544W WO2023054625A1 WO 2023054625 A1 WO2023054625 A1 WO 2023054625A1 JP 2022036544 W JP2022036544 W JP 2022036544W WO 2023054625 A1 WO2023054625 A1 WO 2023054625A1
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bacteria
threonic acid
acid
genus
bifidobacterium
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PCT/JP2022/036544
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French (fr)
Japanese (ja)
Inventor
一弥 戸田
真 吉本
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森永乳業株式会社
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Priority to JP2023551868A priority Critical patent/JPWO2023054625A1/ja
Publication of WO2023054625A1 publication Critical patent/WO2023054625A1/en

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
    • A23L2/52—Adding ingredients
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • 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
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/191—Carboxylic acids, e.g. valproic acid having two or more hydroxy groups, e.g. gluconic acid
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/745—Bifidobacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A61P21/06—Anabolic agents
    • 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

Definitions

  • the present invention relates to a threonic acid-containing composition and a method for producing the same.
  • Muscles are the foundation of daily activities such as standing, walking, and maintaining posture. It exists regardless of gender. In addition, people who like to exercise, athletes, and the like desire to increase their muscle mass in order to improve their performance. Furthermore, as people become more health-conscious, how to increase muscle mass is becoming a social issue in improving quality of life (QOL), improving frailty, and preventing locomotive syndrome. It has become a challenge. In addition, “frailty” is a weakened state between a healthy state and a state in which life functions are impaired or a state requiring nursing care due to increased vulnerability to stress due to a decrease in physiological reserve in old age. It is a word indicating a state (Non-Patent Document 1).
  • Non-Patent Document 2 Muscle is mainly composed of protein and free amino acids. Also, branched-chain amino acids are known to act as stimuli to promote protein synthesis. Therefore, conventionally, foods and drinks containing various proteins and amino acids have been used to increase muscle mass (Non-Patent Document 2). In recent years, it has also been reported that ingestion of Bifidobacterium bacteria can thicken myotubes and increase muscle mass, and is expected to be a new active ingredient for increasing muscle mass ( Patent document 1).
  • an object of the present invention is to provide useful means for increasing muscle mass.
  • threonic acid activates muscle protein synthesis-related genes and promotes muscle hypertrophy. They also found that many Bifidobacterium bacteria and lactic acid bacteria produce threonic acid, and that culturing these bacteria in the presence of ascorbic acid promotes threonic acid production. Based on these findings, the inventors have conceived that threonic acid and threonic acid-producing bacteria can be used as an active ingredient in a composition for increasing muscle mass, and have completed the present invention.
  • the first aspect of the present invention provides bacteria of the genus Bifidobacterium, bacteria of the genus Lactobacillus, bacteria of the genus Lacticaseibacillus, bacteria of the genus Lactiplantibacillus, rimosi One or two or more bacteria selected from bacteria of the genus Limosilactobacillus, bacteria of the genus Levilactobacillus, bacteria of the genus Ligilactobacillus, and bacteria of the genus Latilactobacillus, ascorbic acids
  • a method for producing a threonic acid-containing composition or a composition for building muscle comprising the steps of culturing in a medium containing threonic acid and recovering a threonic acid-containing fraction from the culture after culturing.
  • the bacterium is preferably Bifidobacterium breve, more preferably Bifidobacterium breve FERM BP-11175.
  • a second aspect of the present invention is a combination of threonic acid and bacteria of the genus Bifidobacterium, Lactobacillus, Lacticaseibacillus, Lactipplantibacillus, Remocilactobacillus, and Levilactobacillus. , Rezilactobacillus, and Lactylactobacillus, and one or more selected from cultures of said bacteria. is.
  • a third aspect of the present invention relates to bacteria belonging to the genus Bifidobacterium, bacteria belonging to the genus Lactobacillus, bacteria belonging to the genus Lacticaseibacillus, bacteria belonging to the genus Lactiplantibacillus, bacteria belonging to the genus Remosilactobacillus, bacteria belonging to the genus Levilactobacillus, and bacteria belonging to the genus Lysilactobacillus.
  • compositions according to the third aspect are preferably used for increasing muscle mass.
  • compositions according to the second and third aspects of the present invention are hereinafter also referred to as "compositions of the present invention".
  • the bacterium is preferably Bifidobacterium breve, more preferably Bifidobacterium breve FERM BP-11175.
  • the composition of the present invention is preferably food or drink.
  • the compositions of the invention are preferably pharmaceuticals.
  • the present invention it is possible to provide a composition that can be taken continuously, is highly safe, and can effectively increase muscle mass. Therefore, by increasing muscle mass, daily life and exercise can be improved, and frailty and locomotive syndrome can be improved, so it is possible to support a healthy life in a wide range of age groups.
  • the present invention is a method for producing a composition for muscle building, comprising the steps of culturing a threonic acid-producing bacterium in an ascorbic acid-containing medium and recovering a threonic acid-containing fraction from the culture after culturing.
  • the first aspect of the present invention provides bacteria belonging to the genus Bifidobacterium, bacteria belonging to the genus Lactobacillus, bacteria belonging to the genus Lacticaseibacillus, bacteria belonging to the genus Lactiplantibacillus, bacteria belonging to the genus Remocilactobacillus, bacteria belonging to the genus Levilactobacillus, bacterium A step of culturing one or more bacteria selected from bacteria belonging to the genus Lactobacillus and bacteria belonging to the genus Lactobacillus in an ascorbic acid-containing medium, and recovering a threonic acid-containing fraction from the culture after culturing.
  • threonic acid-containing composition is not limited as long as it contains threonic acid, but contains any of bacterial cells, culture supernatant, medium, and purified threonic acid. Well, it may be a mixture of these. Further, the "threonic acid-containing composition” as used herein may contain a threonic acid-containing fraction described later.
  • NPMD Patent Microorganism Depositary Center
  • NITE Kazusa Kamatari, Kisarazu City, Chiba Prefecture 292-0818
  • IPTD is an abbreviation for the National Institute of Advanced Industrial Science and Technology Patent Organism Depositary Center. In 2012, the National Institute of Advanced Industrial Science and Technology inherited the international deposit status from the National Institute of Advanced Industrial Science and Technology to the National Institute of Technology and Evaluation, and the patent microorganism deposit business has been integrated into NPMD.
  • DSMZ is an abbreviation for Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH. The address is Inhoffenstr. 7B 38124 Braunschweig Germany. Strains with accession numbers beginning with “DSM” herein have been deposited with the DSMZ.
  • the following bacteria can be used when producing a threonic acid-containing composition containing bacterial cells.
  • the following bacteria can also be used when producing a threonic acid-containing composition containing any of culture supernatant, medium, and purified threonic acid.
  • Bifidobacterium bacteria include Bifidobacterium longum subspecies longum, Bifidobacterium longum subspecies infantis, Bifidobacterium breve, Bifidobacterium animalis sub Species animalis, Bifidobacterium animalis subspecies lactis, Bifidobacterium pseudolongum, Bifidobacterium pseudocatenulatum, Bifidobacterium adrecentis, and Bifidobacterium bifidum etc.
  • Bifidobacterium longum subspecies longum for example, Bifidobacterium longum subspecies longum NITE BP-02621 (alias: BB536 or Bifidobacterium longum subsp. longum ATCC BAA-999, for example, JP-A-2012- 223134, etc.), Bifidobacterium longum subspecies longum ATCC 15707, Bifidobacterium longum subspecies longum JCM1217 and the like can be used.
  • Bifidobacterium longum subspecies infantis for example, Bifidobacterium longum subspecies infantis M-63 (accession number NITE BP-02623), Bifidobacterium longum subspecies Species Infantis ATCC 15697 can be used.
  • Bifidobacterium breve for example, Bifidobacterium breve MCC1274 (accession number FERM BP-11175,) Bifidobacterium breve M-16V (accession number NITE BP-02622, commercially available, For example, "Bifidobacterium breve M-16V" manufactured by Morinaga Milk Industry Co., Ltd. may be used.), Bifidobacterium breve ATCC 15700, etc. may be used.
  • Bifidobacterium animalis subspecies animalis for example, Bifidobacterium animalis subspecies animalis ATCC25527 can be used.
  • Bifidobacterium pseudolongum for example, Bifidobacterium pseudolongum Subspecies pseudolongum JCM1205T can be used.
  • Bifidobacterium pseudocatenulatum for example, Bifidobacterium pseudocatenulatum DSM20438 can be used.
  • Bifidobacterium animalis subspecies lactis for example, Bifidobacterium animalis subspecies lactis DSM10140 can be used.
  • Bifidobacterium adolescentis JCM1275 can be used as Bifidobacterium adolescentis.
  • Bifidobacterium bifidum includes, for example, Bifidobacterium bifidum MCC1092 (accession number NITE BP-02429), Bifidobacterium bifidum MCC1319 (accession number NITE BP-02431), Bifidobacterium bifidum MCC1868 (Accession number NITE BP-02432), Bifidobacterium bifidum MCC1870 (accession number NITE BP-02433), Bifidobacterium bifidum JCM1255, etc. can be used.
  • Lactobacillus acidophillus Lactobacillus acidophillus, Lactobacillus acidophillus, Lactobacillus delbrueckii subsp. (Lactobacillus delbrueckii subsp. bulgaricus), Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus johnsonii, Lactobacillus helveticus, Lactobacillus amylovorus amylovorus).
  • Lactobacillus acidophilus Lactobacillus acidophilus MCC1847 (NITE BP-01695) can be used.
  • Lactobacillus gasseri Lactobacillus gasseri MCC1846 (NITE BP-01669), Lactobacillus gasseri JCM1131, and the like can be used.
  • Lactobacillus helveticus Lactobacillus helveticus JCM1120, Lactobacillus helveticus MCC1848 (NITE BP-01671), Lactobacillus helveticus MCC1844 (NITE BP-02185) and the like can be used.
  • the new classification of the genus Lactobacillus in the production method of the present invention (classified in the genus Lactobacillus in the old classification) includes Lacticaseibacillus casei (old classification name is Lactobacillus casei), Lacticaseibacillus paracasei (formerly classified as Lactobacillus paracasei), Lacticaseibacillus rhamnosus (formerly classified as Lactobacillus rhamnosus), and the like.
  • Lactobacillus casei for example, Lactobacillus casei JCM1134, Lactobacillus casei ATCC393, etc. can be used.
  • Lactobacillus paraceasi JCM8130 Lactobacillus paraceasi MCC1849 (NITE BP-01633), Lactobacillus paraceasi MCC1375 (NITE BP-11313) and the like can be used as Lactobacillus paraceasi.
  • Lactobacillus rhamnosus Lactobacillus rhamnosus JCM1136, Lactobacillus rhamnosus MCC1855 (LCS742, commercially available, for example, "LCS-742" manufactured by Morinaga Milk Industry Co., Ltd. can be used), lacto Bacillus rhamnosus ATCC53103, Lactobacillus rhamnosus ATCC53103, etc. can be used.
  • Lactiplantibacillus plantarum (old classification name is Lactiplantibacillus plantarum). Lactobacillus plantarum), Lactiplantibacillus pentosus (formerly known as Lactiplantibacillus pentosus). Lactiprantivibacillus plantarum ATCC14917 can be used as Lactiprantivibacillus plantarum.
  • Limosilactobacillus fermentum (old classification name) is Lactobacillus fermentum), and Limosilactobacillus reuteri (formerly classified as Lactobacillus reuteri).
  • Remosilactobacillus fermentum Remosilactobacillus fermentum SBS-1 can be used.
  • Remocilactobacillus reuteri JCM1112 Remocilactobacillus reuteri DSM17938, Remocilactobacillus reuteri ATCC PTA 6475, Remocilactobacillus reuteri ATCC PTA 5289, etc. can be used. .
  • Ligilactobacillus salivarius (formerly classified as lacto Bacillus salivarius).
  • Lactobacillus curvatus (formerly classified as Lactobacillus curvatus).
  • any one or a combination of two or more of the above bacteria can be used.
  • Bifidobacterium breve Bifidobacterium longum subspecies longum, Bifidobacterium longum subspecies infantis, Bifidobacterium animalis subspecies animalis, Bifidobacterium pseudolongum, Bifidobacterium pseudocatenulatum, Bifidobacterium adrecentis, Bifidobacterium animalis subspecies lactis, Lactobacillus gasseri, Lactobacillus helveticus, Lacti It is one or two or more selected from Bacillus casei, Lacticaseibacillus paracasei, Lacticaseibacillus rhamnosus, and Remocilactobacillus reuteri.
  • Bifidobacterium breve Bifidobacterium longum subspecies longum, Bifidobacterium longum subspecies infantis, Lactobacillus gasseri, Lactobacillus helveticus, Lacticaseibacillus It is one or two or more selected from Paracasei and Lacticaceae Bacillus rhamnosus. Even more preferred is Bifidobacterium breve or Bifidobacterium longum subspecies longum. Most preferred is Bifidobacterium breve FERM BP-11175 or Bifidobacterium longum NITE BP-02621.
  • the bacterium specified by the exemplified bacterial name is not limited to the strain itself (hereinafter also referred to as "deposited strain” for convenience of explanation) that has been deposited or registered with a predetermined institution under the bacterial name, Strains substantially equivalent thereto (also referred to as “derivative strains” or “derived strains”) are also included. That is, it is not limited to the strain itself deposited with the depositary institution under the above accession number, but also includes substantially equivalent strains.
  • a "strain substantially equivalent to the deposited strain” means that it belongs to the same species as the deposited strain, and the nucleotide sequence of the 16S rRNA gene is different from the nucleotide sequence of the 16S rRNA gene of the deposited strain. , preferably 97% or more, more preferably 98% or more, even more preferably 99% or more, even more preferably 100% identity, and preferably the same mycological properties as the deposited strain. refers to the shares held.
  • a strain substantially equivalent to the deposited strain may be, for example, a derivative of the deposited strain as a parent strain. Derivative strains include strains bred from the deposited strain and strains that arise naturally from the deposited strain.
  • Breeding methods include modification by genetic engineering techniques and modification by mutation treatment. Mutagenesis treatments include X-ray irradiation, ultraviolet irradiation, and treatment with mutating agents such as N-methyl-N'-nitro-N-nitrosoguanidine, ethyl methanesulfonate, and methyl methanesulfonate. be done. Strains naturally occurring from the deposited strain include strains naturally occurring during use of the deposited strain. Such strains include mutants naturally occurring by culturing (eg, subculturing) the deposited strain. Derivative strains may be constructed with one modification, or may be constructed with two or more modifications.
  • ascorbic acids are added to the medium when culturing the bacteria.
  • “ascorbic acids” means one or more selected from ascorbic acid, ascorbic acid derivatives, and salts thereof. L-isomers of ascorbic acids can usually be used. L-ascorbic acid can be used as ascorbic acid, and erythorbic acid, which is a stereoisomer of L-ascorbic acid, is also included.
  • ascorbic acid derivatives include salts of inorganic acid esters of L-ascorbic acid such as L-ascorbic acid phosphate and L-ascorbic acid sulfate, and L-ascorbic acid derivatives such as L-ascorbic acid-2-glucoside. Preferred examples include saccharides and the like. Salts of ascorbic acid or derivatives thereof are not particularly limited, but examples include alkali metal salts such as sodium salts and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, ammonium salts, triethanolamine salts, triethylamine salts, and the like.
  • ascorbic acids in the present invention include sodium L-ascorbate, potassium L-ascorbate, calcium L-ascorbate, magnesium L-ascorbate, sodium erythorbate, potassium erythorbate, calcium erythorbate, and magnesium erythorbate. , L-ascorbic acid-2-glucoside, and the like.
  • the amount of ascorbic acid added is not particularly limited, but is preferably 1 mg/L to 100 g/L, more preferably 10 mg/L to 10 g/L, and still more preferably 100 mg/L to 1 g/L, relative to the medium. be able to. Also, the timing of adding ascorbic acids is not particularly limited, but it is usually added from the start of culture.
  • the culture step in the production method of the present invention is not particularly limited as long as the bacteria can grow.
  • the culturing method for example, the method normally used for culturing the bacteria can be used as it is or after being modified as appropriate.
  • the culture temperature may be, for example, 25-50°C, preferably 35-42°C.
  • Culturing is preferably carried out under anaerobic conditions, for example, while passing anaerobic gas such as carbon dioxide.
  • Cultivation can also be performed under microaerobic conditions such as liquid stationary culture. Cultivation can be carried out, for example, until threonic acid accumulates within and/or is secreted outside the cells to a desired extent, or until the bacteria proliferate to a desired extent. For example, it may be 12-72 hours.
  • the medium used for culture is not particularly limited as long as it contains ascorbic acids and the bacteria can grow.
  • a medium commonly used for culturing the bacteria can be used as it is or after being appropriately modified. That is, as carbon sources, for example, sugars such as galactose, glucose, fructose, mannose, cellobiose, maltose, lactose, sucrose, trehalose, starch, starch hydrolysate, blackstrap molasses, etc. can be used depending on the assimilation. .
  • ammonia ammonium salts such as ammonium sulfate, ammonium chloride and ammonium nitrate, and nitrates
  • inorganic salts include sodium chloride, potassium chloride, potassium phosphate, magnesium sulfate, calcium chloride, calcium nitrate, manganese chloride, and ferrous sulfate.
  • Organic ingredients such as peptone, soybean flour, defatted soybean meal, meat extract and yeast extract may also be used.
  • reinforced Clostridial medium Reinforced Clostridial medium
  • MRS medium de Man, Rogosa, and Sharpe medium
  • mMRS medium modified MRS medium
  • TOSP medium TOS propionate medium
  • TOSP Mup medium TOS propionate mupirocin medium
  • the production method of the present invention includes a step of collecting a threonic acid-containing fraction from the culture after the culturing step.
  • threonic acid-containing fraction may be any of bacterial cells, culture supernatant, medium, or a mixture thereof, as long as it contains threonic acid.
  • threonic acid is an active ingredient that exerts a muscle-increasing effect.
  • ascorbic acids to the medium and culturing threonic acid-producing bacteria, the presence of threonic acid was confirmed in the culture supernatant and bacterial cell crushing supernatant. It was clarified that it was metabolized, accumulated inside the cells, and also secreted outside the cells.
  • a step of treating the collected threonic acid-containing fraction by centrifugation, crushing, washing, freeze drying, spray drying, or the like is performed. may contain.
  • a crushed composition, washed composition, freeze-dried composition, or spray-dried composition is obtained. A process may be included.
  • the recovered threonic acid-containing fraction when producing a threonic acid-containing composition containing a culture supernatant, is treated by centrifugation, supernatant recovery, concentration, freeze drying, spray drying, or the like. may include a step of In the production method of the present invention, when producing a threonic acid-containing composition containing a culture supernatant, it includes a step of obtaining a concentrated composition, a freeze-dried composition, or a spray-dried composition. good too.
  • a step of treating the recovered threonic acid-containing fraction by centrifugation, medium recovery, concentration, freeze drying, spray drying, or the like is included. may contain.
  • a step of obtaining a concentrated composition, a freeze-dried composition, or a spray-dried composition when producing a threonic acid-containing composition containing a medium, it may include a step of obtaining a concentrated composition, a freeze-dried composition, or a spray-dried composition. .
  • a step of purifying threonic acid contained in the threonic acid-containing fraction may be included.
  • the step of purifying threonic acid is not particularly limited as long as it can purify threonic acid, but it is possible to use a step of crushing cells, centrifugation, membrane separation, solvent extraction, or the like.
  • the production method of the present invention can be a method for producing threonic acid.
  • the production method of the present invention When used as a method for producing threonic acid, it may contain a collected threonic acid-containing fraction.
  • the threonic acid-containing fraction recovered in this manner usually contains threonic acid of preferably 0.01 ⁇ g/g or more, more preferably 0.1 ⁇ g/g or more, and still more preferably 1 ⁇ g/g of threonic acid per total dry weight. g or more, still more preferably 5 ⁇ g/g or more, even more preferably 10 ⁇ g/g or more.
  • threonic acid usually means L-form.
  • threonic acid may be contained in the form of threonate, and examples of such salts include, but are not limited to, sodium salts, potassium salts, calcium salts, and the like.
  • the identification and quantification of threonic acid can be appropriately carried out using known analysis methods. For example, as shown in Examples below, it can be performed by liquid chromatography mass spectrometry (LC/MS). For example, Prominence (manufactured by Shimadzu Corporation) is used for HPLC, and TSQ Quantum Discovery MAX (Thermo company) can be used.
  • the production method of the present invention may include a step of treating the recovered threonic acid-containing fraction after the step of recovering the threonic acid-containing fraction.
  • the method of treating the threonic acid-containing fraction is not particularly limited as long as it does not impair the muscle-increasing action, and includes dilution, concentration, heating, freeze-drying, spray-drying, crushing, fractionation, and the like. That is, in the production method of the present invention, the steps of treating the recovered threonic acid-containing fraction include a step of separating and recovering bacterial cells, a step of separating and recovering the culture supernatant, a step of separating and recovering the medium, and a step of separating and recovering the medium containing threonic acid.
  • the production method of the present invention may be a step of purifying threonic acid from the fraction.
  • the production method of the present invention can be a method for producing threonic acid.
  • the production method of the present invention may include a step of mixing the collected threonic acid-containing fraction with the food raw material.
  • the production method of the present invention may include a step of converting the recovered threonic acid-containing fraction into a form such as powder, granules, paste, emulsion, wetting agent, capsule, tablet, or the like.
  • the production method of the present invention may include a step of sterilizing the recovered threonic acid-containing fraction after the step of recovering the threonic acid-containing fraction.
  • the step of sterilizing the threonic acid-containing fraction is not particularly limited as long as it does not impair the muscle volume-increasing action, and includes heat treatment, crushing treatment, pressure treatment, treatment with chemicals, and the like.
  • a step of sterilizing the cells may be included, and the sterilization may be heat treatment, crushing treatment, or pressure treatment. That is, when the resulting composition for increasing muscle mass contains bacterial cells as the threonic acid-containing fraction, the bacterial cells may be viable cells or dead cells. It may be a mixture of bodies.
  • the process of treating the recovered threonic acid-containing fraction, the process of mixing it with food ingredients, the process of making it into a liquid or the like, and the process of sterilizing are optional steps, and the order of the steps can be changed as appropriate. It is possible.
  • composition of the second aspect of the present invention comprises threonic acid, bacteria of the genus Bifidobacterium, bacteria of the genus Lactobacillus, bacteria of the genus Lacticaseibacillus, bacteria of the genus Lactipplantibacillus, bacteria of the genus Remocilactobacillus, and Reviract. containing one or more bacteria selected from bacteria belonging to the genus Bacillus, bacteria belonging to the genus Redilactobacillus, and bacteria belonging to the genus Rachilactobacillus, and one or more bacteria selected from cultures of the above bacteria to increase muscle mass provided for use.
  • a composition according to a second alternative aspect of the present invention contains purified threonic acid, or a threonic acid-containing fraction, for use in muscle building.
  • composition of the present invention can be preferably produced by the production method of the present invention described above.
  • threonic acid is usually included in the bacteria and/or culture thereof. Therefore, from another aspect, the composition of the present invention, as a third aspect, includes bacteria belonging to the genus Bifidobacterium, bacteria belonging to the genus Lactobacillus, bacteria belonging to the genus Lacticaseibacillus, bacteria belonging to the genus Lactipplantibacillus, and bacteria belonging to the genus Remocilactobacillus.
  • compositions can preferably serve for muscle building applications.
  • the type of bacteria in the composition of the present invention conforms to the description of the production method of the present invention described above.
  • the bacterium and/or its culture to be contained in the composition of the present invention may refer to the threonic acid-containing fraction recovered in the production method.
  • the content of the bacterium and/or its culture in the composition of the present invention is not particularly limited, but is, for example, 1.0 ⁇ 10 7 cfu or more per 1 g of the composition in terms of the amount of the bacterium. preferably 1.0 ⁇ 10 8 cfu or more, and still more preferably 1.0 ⁇ 10 9 cfu or more.
  • cfu refers to a colony forming unit. In this specification, for example, the value obtained when cultured at 38° C. in a solid medium containing 10% by mass of reconstituted skim milk can be used. Also, when the cells are dead cells, the cfu can be replaced with cells.
  • the content of the bacterial cells is preferably 0.1 mg or more, more preferably 1 mg or more, and still more preferably 10 mg or more per 1 g of the composition in terms of the amount of solids in the culture. .
  • the content of threonic acid in the composition of the present invention is not particularly limited, it is preferably 0.01 ⁇ g or more, more preferably 0.1 ⁇ g or more, and still more preferably 1 ⁇ g or more per 1 g of the composition. Preferably, it may contain 5 ⁇ g or more, and still more preferably 10 ⁇ g or more. These may be in the range of content when they are usually distributed as an oral composition.
  • the form of the composition of the present invention is not particularly limited. It may be a spray dried composition, a crushed composition, a fractionated composition, or a sterilized composition.
  • the form of the composition of the present invention may be liquid, powder, granules, paste, emulsion, wetting agent, capsule, tablet and the like.
  • the sterilization method is not limited, and it may be a composition sterilized by heat, a composition sterilized with a drug, or a composition sterilized by crushing. .
  • the composition of the present invention has the effect of increasing muscle mass in animals, including humans.
  • “Muscle mass” includes increasing muscle volume and/or weight and inhibiting muscle volume and/or weight loss. Increase in muscle volume and/or weight and inhibition of decrease in muscle volume and/or weight may be due to muscle hypertrophy (thickening of myotubes or muscle fibers), It may be due to an increase in the number of fibers.
  • the action of the composition of the present invention to increase muscle mass is believed to be due to the action of threonic acid activating protein synthesis-related genes such as p70S6K and promoting muscle protein synthesis.
  • composition of the present invention When the composition of the present invention is to be orally ingested (administered), it is preferably in the form of a food or drink. That is, another aspect of the present invention is the combination of threonic acid and Bifidobacterium, Lactobacillus, Lacticaseibacillus, Lactipplantibacillus, Remocilactobacillus, A composition containing one or more bacteria selected from bacteria belonging to the genus Levilactobacillus, bacteria belonging to the genus Rydilactobacillus, and bacteria belonging to the genus Lachilactobacillus, and one or more bacteria selected from cultures of the bacteria It is the use of things.
  • Another aspect of the present invention is the use of Bifidobacterium, Lactobacillus, Lacticaseibacillus, Lactipplantibacillus, Remocilactobacillus, Levilactobacillus, in muscle building.
  • Another aspect of the present invention is to provide bacteria of the genus Bifidobacterium, bacteria of the genus Lactobacillus, bacteria of the genus Lacticaseibacillus, bacteria of the genus Lactiplantibacillus, bacteria of the genus Remosilactobacillus, bacteria of the genus Levilactobacillus, and genus Lyzilactobacillus.
  • bacteria of the genus Bifidobacterium including administering to a subject one or more selected from bacteria and bacteria of the genus Lachilactobacillus, and one or more selected from cultures of said bacteria, said bacteria and/or said A method for increasing muscle bulk wherein the culture contains 1 ⁇ g/g or more of threonic acid per total dry weight thereof.
  • This specification also discloses, as a fourth aspect, a muscle building composition containing threonic acid as an active ingredient.
  • threonic acid activates protein synthesis-related genes in skeletal muscle cells, promotes muscle hypertrophy, and exerts a muscle-increasing effect.
  • Such invention can also be translated into the use of threonic acid in muscle mass building.
  • This invention can also be translated into a method for increasing muscle mass, including administering threonic acid to a subject.
  • the food and drink composition of the present invention can increase muscle mass, it prevents and/or improves symptoms such as muscle atrophy and diseases caused by decreased muscle mass, and prevents decreased muscle mass due to lack of exercise and aging. Prevention and/or amelioration is expected.
  • Diseases accompanied by symptoms such as muscle atrophy include, for example, hypotonia, muscle atrophy, muscle dystrophy, muscle degeneration, inflammatory muscle disease, myasthenia and sarcopenia.
  • Diseases caused by decreased muscle mass include osteoporosis, bone fracture, diabetes, chronic obstructive pulmonary disease, chronic kidney disease, dementia, and the like.
  • the food and drink composition of the present invention is also useful for healthy people and people who play sports because they can maintain and improve their daily lives and improve exercise efficiency.
  • "amelioration" of symptoms or diseases includes curing of diseases, alleviation of symptoms, reduction in severity of diseases or symptoms, and delay of progression of diseases or symptoms.
  • "prevention" of a symptom or disease includes preventing the occurrence of the symptom or disease, delaying the occurrence, and reducing the risk of the occurrence.
  • the subject (administrator) to whom the food and drink compositions of the second to fourth aspects of the present invention are administered (administrator) and the subject (consumer) to be ingested are not particularly limited as long as they are animals, but are usually humans. Moreover, any of adults, children, infants, newborns (including low-weight infants), and the like may be used. Moreover, sex is not specifically limited.
  • administering to a subject may be synonymous with “making a subject ingest”.
  • the intake is usually voluntary (free intake), but may be forced (forced intake).
  • the intake (administration) timing of the food and drink composition of the present invention is not particularly limited, and can be appropriately selected according to the condition of the intake (administration) subject.
  • the intake (administration) amount of the food and drink composition of the present invention is appropriately selected depending on the age, sex, condition, other conditions, etc. of the intake (administration) subject.
  • the intake (administration) amount of the food and drink composition of the present invention is preferably in the range of 100 ⁇ g/day to 10 g/day, for example, 1 mg/day in terms of the solid content of the bacteria and/or culture thereof for adults.
  • a range of -1 g/day is more preferred, and 10 mg/day to 500 mg/day is even more preferred.
  • the range for adults is preferably 1 ng/day to 100 ⁇ g/day, more preferably 1 ng/day to 1 ⁇ g/day, and even more preferably 10 ng/day to 500 ng/day.
  • the food and drink composition of the present invention can be ingested (administered) once a day or divided into multiple times a day, regardless of the amount and period of ingestion (administration).
  • the intake (administration) period of the food and drink composition of the present invention is not particularly limited. In addition, there is no particular upper limit for the intake (administration) period, and continuous long-term intake (administration) is possible.
  • the form and properties are not particularly limited as long as they can be orally ingested (administered) without impairing the effects of the present invention. It can be produced by a normal method using raw materials that are usually used for food and drink.
  • Food and drink regardless of the form such as liquid, paste, gel-like solid, powder, etc., for example, nutritional supplements (supplements), tablet confectionery; liquid diet (nutrition for ingestion); bread, macaroni, spaghetti Flour products such as , noodles, cake mixes, fried chicken powder, bread crumbs; instant noodles, cup noodles, retort/prepared foods, cooked canned foods, microwave oven foods, instant soups/stews, instant miso soups/soups, canned soups, freeze-dried foods , and other instant foods; canned agricultural products, canned fruits, jams and marmalades, pickled vegetables, boiled beans, dried agricultural products, processed agricultural products such as cereals (processed grains); canned marine products, fish meat hams and sausages , fish paste products, marine delicacies, processed fish products such as tsukudani; processed livestock products such as canned livestock products, pastes, meat hams and sausages; processed milk, milk drinks, yogurts, lactic acid bacteria drinks, cheese, ice creams Milk and dairy products
  • confectionery such as caramels, candies, chewing gums, chocolates, cookies, biscuits, cakes, pies, snacks, crackers, Japanese confectionery, rice confectionery, bean confectionery, dessert confectionery, jelly, and other confectionery; carbonated drinks, natural fruit juices , fruit juice drinks, soft drinks containing fruit juice, fruit drinks, fruit drinks containing fruit, vegetable drinks, soy milk, soy milk drinks, coffee drinks, tea drinks, powdered drinks, concentrated drinks, sports drinks, nutritional drinks, alcoholic beverages, etc.
  • Pleasant beverages such as beverages, other commercially available foods such as baby food, furikake, ochazuke seaweed, etc.; powdered milk for infants; enteral nutrition food; ) and the like.
  • the feed can also be set as feed as one aspect
  • the feed include pet food, livestock feed, fish feed, and the like.
  • the form of the feed is not particularly limited, and in addition to threonic acid, the bacteria and/or culture thereof, grains such as corn, wheat, barley, rye, and milo; soybean meal, rapeseed meal, coconut oil meal, and linseed meal.
  • Rice brans such as wheat bran, wheat bran, rice bran, and defatted rice bran; Manufacturing lees such as corn gluten meal and corn jam meal; Animal feeds such as fish meal, skimmed milk powder, casein, yellow grease, and tallow.
  • Yeasts such as torula yeast and brewer's yeast; mineral feed such as tribasic calcium phosphate and calcium carbonate; oils and fats; simple amino acids;
  • composition of the present invention is in the form of a food or drink (including feed), it can be provided and sold as a food or drink labeled with uses related to increasing muscle mass.
  • Such "display” acts include all acts for informing consumers of the above-mentioned use. Regardless of the object, medium, etc. to be displayed, all of them fall under the act of "display” in the present invention.
  • the "indication” be expressed in such a way that the consumer can directly recognize the use.
  • the act of transferring, handing over, displaying for the purpose of transfer or delivery, importing products related to food and beverages or product packaging that describes the above-mentioned use, advertisements related to products, price lists or transaction documents Examples include the act of displaying or distributing information describing the use, or providing information containing such information using an electromagnetic method (such as the Internet) after describing the use.
  • the content of the display is a display approved by the government (for example, a display that is approved based on various systems established by the government and performed in a manner based on such approval).
  • a display that is approved based on various systems established by the government and performed in a manner based on such approval it is preferable to attach such display contents to packaging, containers, catalogs, pamphlets, POP and other advertising materials at sales sites, other documents, and the like.
  • labeling includes labeling as health food, functional food, enteral nutrition food, food for special dietary use, food with health claims, food for specified health use, food with nutrient function claims, food with function claims, quasi-drugs, etc. is also mentioned.
  • the labeling approved by the Consumer Affairs Agency for example, the labeling approved by the system related to food for specified health use, food with nutrient function claims, or food with function claims, or similar system.
  • labeling as a food for specified health use labeling as a food for specified health use with certain conditions, labeling to the effect that it affects the structure and function of the body, labeling to reduce the risk of disease, labeling for functionality based on scientific evidence. Labeling, etc.
  • Such indications include, for example, “Increase muscle”, “For those who want to build muscle”, “For prevention of frailty”, “For improvement of QOL”, “Support for increasing muscle strength of exercisers”, and the like. mentioned.
  • One or more bacteria selected from bacteria belonging to the genus Bacillus, bacteria belonging to the genus Levilactobacillus, bacteria belonging to the genus Regilactobacillus, and bacteria belonging to the genus Lactobacillus, and bacteria belonging to the genus Lactobacillus, and one or more selected from cultures of the bacteria is a composition containing Another aspect of the present invention is the use of threonic acid and Bifidobacterium, Lactobacillus, Lacticaseibacillus, Lactipplantibacillus, Remocilactobacillus in the manufacture of a muscle building composition.
  • One or two or more bacteria selected from the genus bacteria, Levilactobacillus bacteria, Regilactobacillus bacteria, and Rachilactobacillus bacteria, and one or two or more bacteria selected from the cultures of the bacteria is used.
  • Another aspect of the present invention is the combination of threonic acid with Bifidobacterium, Lactobacillus, Lacticaseibacillus, Lactipplantibacillus, Remocilactobacillus, used for muscle gain.
  • Bacteria one or more bacteria selected from the genus Levilactobacillus, bacteria of the genus Regilactobacillus, and bacteria of the genus Lachilactobacillus, and one or more bacteria selected from cultures of the bacteria It is a composition that Another aspect of the present invention is the use of threonic acid with bacteria of the genus Bifidobacterium, Lactobacillus, Lacticaseibacillus, Lactipplantibacillus, Remocilactobacillus, Levilactobacillus, muscle, including administering to a subject one or more bacteria selected from bacteria belonging to the genus Rezilactobacillus and bacteria belonging to the genus Lachilactobacillus, and one or more bacteria selected from the cultures of said bacteria is a method of increasing the amount of Another aspect of the present invention is Bifidobacterium, Lactobacillus, Lacticaseibacillus, Lactipplantibacillus, Remosilactobacillus, Reviract used for muscle
  • Another aspect of the present invention is the use of Bifidobacterium, Lactobacillus, Lacticaseibacillus, Lactipplantibacillus, Remocilactobacillus, Levi in the manufacture of a muscle building composition.
  • Another aspect of the present invention is to provide bacteria of the genus Bifidobacterium, bacteria of the genus Lactobacillus, bacteria of the genus Lacticaseibacillus, bacteria of the genus Lactiplantibacillus, bacteria of the genus Remosilactobacillus, bacteria of the genus Levilactobacillus, and genus Lyzilactobacillus.
  • bacteria of the genus Bifidobacterium including administering to a subject one or more selected from bacteria and bacteria of the genus Lachilactobacillus, and one or more selected from cultures of said bacteria, said bacteria and/or said A method for increasing muscle bulk wherein the culture contains 1 ⁇ g/g or more of threonic acid per total dry weight thereof.
  • This specification also discloses, as a fourth aspect, a muscle building composition containing threonic acid as an active ingredient.
  • threonic acid activates protein synthesis-related genes in skeletal muscle cells, promotes muscle hypertrophy, and exerts a muscle-increasing effect.
  • Such an invention can also be rephrased as threonic acid used for increasing muscle mass.
  • Such an invention can also be translated into the use of threonic acid in the manufacture of a muscle building composition.
  • This invention can also be translated into a method for increasing muscle mass, including administering threonic acid to a subject.
  • the pharmaceutical composition of the present invention can increase muscle mass, it prevents and/or improves symptoms such as muscle atrophy and diseases caused by decreased muscle mass, and prevents decreased muscle mass due to lack of exercise and aging. and/or are expected to make improvements.
  • Diseases accompanied by symptoms such as muscle atrophy include, for example, hypotonia, muscle atrophy, muscle dystrophy, muscle degeneration, inflammatory muscle disease, myasthenia and sarcopenia.
  • Diseases caused by decreased muscle mass include osteoporosis, bone fracture, diabetes, chronic obstructive pulmonary disease, chronic kidney disease, dementia, and the like.
  • lack of exercise and age-related loss of muscle mass can cause sarcopenia, frailty, locomotive syndrome, and the like, so it is also expected to prevent and/or improve these.
  • the subject (administrator) to whom the pharmaceutical compositions of the second to fourth aspects of the present invention are administered (administrator) and the subject (ingester) to be ingested are not particularly limited as long as they are animals, but are usually humans. Moreover, any of adults, children, infants, newborns (including low-weight infants), and the like may be used. Moreover, sex is not specifically limited.
  • the timing of ingestion (administration) of the pharmaceutical composition of the present invention is not particularly limited, and can be appropriately selected according to the condition of the subject of ingestion (administration).
  • the intake (administration) amount of the pharmaceutical composition of the present invention is appropriately selected depending on the age, sex, condition, other conditions, etc. of the intake (administration) target.
  • the ingestion (administration) amount of the pharmaceutical composition of the present invention is preferably in the range of 100 ⁇ g/day to 10 g/day, and 1 mg/day to 1 mg/day in terms of solid content of the bacteria and/or culture thereof. A range of 1 g/day is more preferred, and 10 mg/day to 500 mg/day is even more preferred.
  • the range for adults is preferably 1 ng/day to 100 ⁇ g/day, more preferably 1 ng/day to 1 ⁇ g/day, and even more preferably 10 ng/day to 500 ng/day.
  • the pharmaceutical composition of the present invention can be ingested (administered) once a day or divided into multiple times a day, regardless of the amount and duration of ingestion (administration).
  • the timing of ingesting (administering) the drug of the present invention is not particularly limited, such as before meals, after meals, between meals, and before bedtime.
  • the intake (administration) period of the pharmaceutical composition of the present invention is not particularly limited. In addition, there is no particular upper limit for the intake (administration) period, and continuous long-term intake (administration) is possible.
  • the route of intake (administration) of the drug may be oral or parenteral, but oral is preferred.
  • parenteral intake (administration) includes percutaneous, intravenous, rectal administration, inhalation, and the like.
  • the pharmaceutical form it can be appropriately formulated into a desired dosage form depending on the intake (administration) method.
  • solid formulations such as powders, granules, tablets and capsules; and liquid formulations such as solutions, syrups, suspensions and emulsions can be formulated.
  • parenteral intake (administration) it can be formulated into suppositories, ointments, injections, and the like.
  • ingredients such as excipients, pH adjusters, colorants, and corrigents that are commonly used for formulation can be used.
  • other medicinal ingredients in combination, such as other medicinal ingredients, ingredients known or discovered in the future that have muscle-enhancing effects.
  • formulation can be appropriately carried out by a known method depending on the dosage form.
  • a carrier that is usually used for formulation may be blended as appropriate to form the formulation.
  • Such carriers include excipients, binders, disintegrants, lubricants, stabilizers, flavoring agents and the like.
  • Excipients include, for example, sugar derivatives such as lactose, sucrose, glucose, mannitol, sorbitol; starch derivatives such as corn starch, potato starch, ⁇ -starch, dextrin, carboxymethyl starch; crystalline cellulose, hydroxypropyl cellulose, Hydroxypropylmethylcellulose, carboxymethylcellulose, cellulose derivatives such as carboxymethylcellulose calcium; gum arabic; dextran; pullulan; silicate derivatives such as light silicic anhydride, synthetic aluminum silicate, and magnesium aluminometasilicate; phosphate derivatives such as calcium phosphate; carbonate derivatives such as calcium; sulfate derivatives such as calcium sulfate;
  • binders examples include gelatin; polyvinylpyrrolidone; macrogol, etc., in addition to the aforementioned excipients.
  • disintegrants include, in addition to the excipients mentioned above, chemically modified starch or cellulose derivatives such as croscarmellose sodium, carboxymethyl starch sodium, and crosslinked polyvinylpyrrolidone.
  • Lubricants include, for example, talc; stearic acid; metal stearates such as calcium stearate and magnesium stearate; colloidal silica; waxes such as Veegum and Geiro; ; carboxylic acid sodium salts such as sodium benzoate; sulfates such as sodium sulfate; leucine; lauryl sulfates such as sodium lauryl sulfate and magnesium lauryl sulfate; silicic acid anhydride and silicic acid hydrate; be done.
  • stabilizers include paraoxybenzoic acid esters such as methylparaben and propylparaben; alcohols such as chlorobutanol, benzyl alcohol and phenylethyl alcohol; benzalkonium chloride; acetic anhydride; and sorbic acid.
  • Flavoring agents include, for example, sweeteners, acidulants, flavoring agents, and the like.
  • examples of carriers used include solvents such as water.
  • Rat myoblast cell line L6 cells obtained from the American Type Culture Collection (ATCC) were adjusted to 1.5 ⁇ 10 4 cells/cm 2 .
  • the cells were seeded on a 6-well plate and cultured in DMEM medium (containing 10% Fetal Bovine Serum and 1% Penicillin Streptomycin) under 5% CO 2 at 37° C. for 24 hours.
  • DMEM medium containing 10% Horse Serum and 1% Penicillin Streptomycin; hereinafter referred to as "differentiation medium” was replaced with fresh medium every 2 days and cultured for 7 days.
  • the 1 M threonic acid sample prepared in (1) above was added to a final concentration of 1 or 10 ⁇ M, cultured for 1 hour, and then a lysate was prepared using RIPA buffer.
  • Activation (phosphorylation) of the protein synthesis-related gene p70S6K was assessed by Western blotting. Specifically, the fluorescence intensity of phosphorylated p70S6K and total p70S6K bands was measured using ChemiDoc (registered trademark) MP Imaging System (Bio-Rad Laboratories), and the ratio of phosphorylated p70S6K to total p70S6K was calculated. The activity value of p70S6K was used. As a control, the same procedure as described above was performed except that the threonic acid sample was not added.
  • HE staining hematoxylin and eosin staining
  • Tables 1 and 2 show the activity values of the protein synthesis-related gene (p70S6K) in L6 to which threonic acid was added.
  • the activity (degree of phosphorylation) of p70S6K increased 3.37-fold in the group treated with 1 ⁇ M threonate and increased 3.03-fold in the group treated with 10 ⁇ M threonate compared to the control.
  • Table 3 shows the diameters (average values) of myotube cells obtained by the procedure in (3) above. It was confirmed that the diameter of myotube cells increased by 2.58 ⁇ m or more in the 1 ⁇ M threonic acid-treated group and by 1.85 ⁇ m or more in the 10 ⁇ M threonic acid-treated group, compared to the control, and muscle mass increased.
  • 1% (v/v) of each was seeded in the MRS liquid medium and anaerobically cultured at 37°C (preculture). After 16 hours of culture, 1% (v/ v) were inoculated individually and anaerobically cultured at 37°C (main culture). After 16 hours of culture, the culture supernatant was collected by centrifugation. Bacteria were cultured in the same manner as a control, except that ascorbic acid was not added.
  • Test Example 3 Investigation of muscle volume-increasing effect by probiotic culture supernatant Using probiotic culture supernatant cultured in the presence of ascorbic acid, promotion of activity of protein synthesis-related gene (p70S6K) in L6 was evaluated, and the muscle volume-increasing effect was examined.
  • Test Example 2 The three strains prepared in Test Example 2 (1) (Bifidobacterium breve FERM BP-11175, Bifidobacterium pseudolongum subspecies pseudolongum JCM1205T, Limo Sodium hydroxide was added to the culture supernatant of Silactobacillus reuteri JCM1112T) to adjust the pH to 7.0 ⁇ 0.05, followed by filter sterilization.
  • Results Table 5 shows the activity value of the protein synthesis-related gene (p70S6K) in L6 of each group.
  • p70S6K activity values were higher when cultured in the presence of ascorbic acid than when cultured in the absence of ascorbic acid.
  • Culture supernatant of Bifidobacterium breve FERM BP-11175, Bifidobacterium pseudolongum subspecies pseudolongum JCM1205T, and Remocilactobacillus reuteri JCM1112T cultured in the presence of ascorbic acid was found to have a stimulatory effect on muscle protein synthesis.
  • ⁇ Test Example 4 Investigation of intracellular threonic acid production by probiotics (1) Culture of probiotics and quantification of threonic acid In the same procedure as in Test Example 2, each of the probiotics shown in Table 6 was cultured. bottom. The culture was centrifuged to collect the cells, washed with ultrapure water, suspended at a concentration of 10 mg/mL (wet weight of bacteria/ultrapure water), and treated with FastPrep-24 5G Homogenizer (manufactured by MP Biomedicals)). The resulting crude lysate was centrifuged to collect the supernatant (hereinafter referred to as "supernatant of cell lysate"). After that, in the same procedure as in Test Example 2, the amount of threonic acid in the supernatant of cell disruption was measured.

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Abstract

A problem addressed is to provide a useful means for increasing muscle mass. A composition containing threonic acid, one or more bacteria selected from Bifidobacterium spp., Lactobacillus spp., Lacticaseibacillus spp., Lactiplantibacillus spp., Limosilactobacillus spp., Levilactobacillus spp., Ligilactobacillus spp., and Latilactobacillus spp., and one or more selected from cultures of the aforementioned bacteria is used to increase muscle mass. Such a composition is produced through, e.g., a method that includes a step for culturing one or more bacteria selected from the aforementioned bacteria in ascorbic-acid-containing medium and a step for recovering a threonic-acid-containing fraction from the culture after culturing.

Description

トレオン酸含有組成物及びその製造方法Composition containing threonic acid and method for producing the same
　本発明は、トレオン酸含有組成物及びその製造方法に関する。 The present invention relates to a threonic acid-containing composition and a method for producing the same.
　筋肉は、立つ、歩く、姿勢を維持するといった日常動作の基盤となるため、日常生活の質を維持したり向上させたりするために、筋肉量を維持したり筋肉を増量させたいという要望が老若男女を問わず存する。また、運動を好む人やスポーツ競技者等は、成績を上げるため、筋肉を増量させたいという要望がある。
　さらに、健康志向が強まるなか、クオリティ・オブ・ライフ（quality of life：ＱＯＬ）の向上、フレイルの改善、ロコモティブシンドローム（locomotive syndrome：運動器症候群）対策において、いかに筋肉を増量させるかが社会的な課題となっている。なお、「フレイル」とは、高齢期に生理的予備能が低下することでストレスに対する脆弱性が亢進し、健常な状態と生活機能に障害がある状態又は要介護状態との中間にある虚弱化状態を指す語である（非特許文献１）。 Muscles are the foundation of daily activities such as standing, walking, and maintaining posture. It exists regardless of gender. In addition, people who like to exercise, athletes, and the like desire to increase their muscle mass in order to improve their performance.
Furthermore, as people become more health-conscious, how to increase muscle mass is becoming a social issue in improving quality of life (QOL), improving frailty, and preventing locomotive syndrome. It has become a challenge. In addition, “frailty” is a weakened state between a healthy state and a state in which life functions are impaired or a state requiring nursing care due to increased vulnerability to stress due to a decrease in physiological reserve in old age. It is a word indicating a state (Non-Patent Document 1).
　筋肉は主にタンパク質と遊離アミノ酸とで構成される。また、分岐鎖アミノ酸は、タンパク質合成を促進する刺激になることが知られている。そのため、従来、種々のタンパク質やアミノ酸を含有する飲食品が、筋肉量を増加させるために使用されている（非特許文献２）。
　また、近年、ビフィドバクテリウム属細菌を摂取すると、筋管が太くなり、筋肉を増量させることができることが報告されており、筋肉量を増加させるための新たな有効成分として期待されている（特許文献１）。 Muscle is mainly composed of protein and free amino acids. Also, branched-chain amino acids are known to act as stimuli to promote protein synthesis. Therefore, conventionally, foods and drinks containing various proteins and amino acids have been used to increase muscle mass (Non-Patent Document 2).
In recent years, it has also been reported that ingestion of Bifidobacterium bacteria can thicken myotubes and increase muscle mass, and is expected to be a new active ingredient for increasing muscle mass ( Patent document 1).
国際公開２０１９／０８７２８０号WO2019/087280
　従来の筋肉増量用組成物では、必ずしも十分な効果が得られるものではなかったり、日常において継続的に摂取しにくい場合があったりした。また、筋肉が増量するメカニズムには、種々の栄養素やタンパク質合成系が複雑に関与しているため、依然として明らかでない部分がある。そのため、筋肉を増量させるための新たなアプローチを模索する余地がある。
　かかる状況に鑑みて、本発明は筋肉を増量させるために有用な手段を提供することを課題とする。 Conventional compositions for increasing muscle volume do not necessarily provide sufficient effects, and in some cases, it is difficult to continuously ingest them in daily life. In addition, some aspects of the muscle mass-increasing mechanism are still unclear because various nutrients and protein synthesis systems are involved in a complex manner. Therefore, there is room for exploring new approaches to increasing muscle mass.
In view of such circumstances, an object of the present invention is to provide useful means for increasing muscle mass.
　本発明者らは、前記の課題を解決すべく鋭意研究を行った結果、トレオン酸が筋タンパク質合成関連遺伝子を活性化させ、筋肥大を促進することを見出した。また、多くのビフィドバクテリウム属細菌や乳酸菌がトレオン酸を産生すること、さらにこれらの細菌をアスコルビン酸存在下で培養するとトレオン酸産生が促進されることをも見出した。これらの知見から、トレオン酸やトレオン酸産生細菌が、筋肉増量用組成物の有効成分とし得ることに想到し、本発明を完成するに至った。 As a result of intensive research aimed at solving the above problems, the present inventors found that threonic acid activates muscle protein synthesis-related genes and promotes muscle hypertrophy. They also found that many Bifidobacterium bacteria and lactic acid bacteria produce threonic acid, and that culturing these bacteria in the presence of ascorbic acid promotes threonic acid production. Based on these findings, the inventors have conceived that threonic acid and threonic acid-producing bacteria can be used as an active ingredient in a composition for increasing muscle mass, and have completed the present invention.
　すなわち、本発明の第一の態様は、ビフィドバクテリウム（Bifidobacterium）属細菌、ラクトバチルス（Lactobacillus）属細菌、ラクチカゼイバチルス（Lacticaseibacillus）属細菌、ラクチプランティバチルス（Lactiplantibacillus）属細菌、リモシラクトバチルス（Limosilactobacillus）属細菌、レヴィラクトバチルス（Levilactobacillus）属細菌、リジラクトバチルス（Ligilactobacillus）属細菌、及びラチラクトバチルス(Latilactobacillus)属細菌から選択される一種又は二種以上の細菌を、アスコルビン酸類含有培地で培養する工程、並びに培養後の培養物からトレオン酸含有画分を回収する工程を含む、トレオン酸含有組成物、又は筋肉増量用組成物の製造方法である。
　本発明の製造方法において前記細菌は、好ましくはビフィドバクテリウム・ブレーベ（Bifidobacterium breve）であり、より好ましくはビフィドバクテリウム・ブレーベ ＦＥＲＭ ＢＰ－１１１７５である。
　本発明の第二の態様は、トレオン酸と、ビフィドバクテリウム属細菌、ラクトバチルス属細菌、ラクチカゼイバチルス属細菌、ラクチプランティバチルス属細菌、リモシラクトバチルス属細菌、レヴィラクトバチルス属細菌、リジラクトバチルス属細菌、及びラチラクトバチルス属細菌から選択される一種又は二種以上の細菌、並びに前記細菌の培養物から選択される一種又は二種以上とを含有する、筋肉増量用組成物である。
　本発明の第三の態様は、ビフィドバクテリウム属細菌、ラクトバチルス属細菌、ラクチカゼイバチルス属細菌、ラクチプランティバチルス属細菌、リモシラクトバチルス属細菌レヴィラクトバチルス属細菌、リジラクトバチルス属細菌、及びラチラクトバチルス属細菌から選択される一種又は二種以上の細菌、並びに前記細菌の培養物から選択される一種又は二種以上を含有する組成物であって、前記細菌及び／又は前記培養物が、その総乾燥重量あたりトレオン酸を１μｇ／ｇ以上含有する、組成物である。第三の態様にかかる組成物は、筋肉増量用とすることが好ましい。
　本発明の第二の態様及び第三の態様にかかる組成物は、以降、「本発明の組成物」とも記す。
　本発明の組成物において前記細菌は、好ましくはビフィドバクテリウム・ブレーベであり、より好ましくはビフィドバクテリウム・ブレーベ ＦＥＲＭ ＢＰ－１１１７５である。
　本発明の組成物は、好ましくは、飲食品である。
　本発明の組成物は、好ましくは、医薬品である。 That is, the first aspect of the present invention provides bacteria of the genus Bifidobacterium, bacteria of the genus Lactobacillus, bacteria of the genus Lacticaseibacillus, bacteria of the genus Lactiplantibacillus, rimosi One or two or more bacteria selected from bacteria of the genus Limosilactobacillus, bacteria of the genus Levilactobacillus, bacteria of the genus Ligilactobacillus, and bacteria of the genus Latilactobacillus, ascorbic acids A method for producing a threonic acid-containing composition or a composition for building muscle, comprising the steps of culturing in a medium containing threonic acid and recovering a threonic acid-containing fraction from the culture after culturing.
In the production method of the present invention, the bacterium is preferably Bifidobacterium breve, more preferably Bifidobacterium breve FERM BP-11175.
A second aspect of the present invention is a combination of threonic acid and bacteria of the genus Bifidobacterium, Lactobacillus, Lacticaseibacillus, Lactipplantibacillus, Remocilactobacillus, and Levilactobacillus. , Rezilactobacillus, and Lactylactobacillus, and one or more selected from cultures of said bacteria. is.
A third aspect of the present invention relates to bacteria belonging to the genus Bifidobacterium, bacteria belonging to the genus Lactobacillus, bacteria belonging to the genus Lacticaseibacillus, bacteria belonging to the genus Lactiplantibacillus, bacteria belonging to the genus Remosilactobacillus, bacteria belonging to the genus Levilactobacillus, and bacteria belonging to the genus Lysilactobacillus. A composition containing one or more bacteria selected from bacteria and bacteria of the genus Lactobacillus, and one or more bacteria selected from cultures of said bacteria, wherein said bacteria and/or said A composition wherein the culture contains 1 μg/g or more of threonic acid per total dry weight thereof. The composition according to the third aspect is preferably used for increasing muscle mass.
Compositions according to the second and third aspects of the present invention are hereinafter also referred to as "compositions of the present invention".
In the composition of the present invention, the bacterium is preferably Bifidobacterium breve, more preferably Bifidobacterium breve FERM BP-11175.
The composition of the present invention is preferably food or drink.
The compositions of the invention are preferably pharmaceuticals.
　本発明によれば、継続的に摂取することができ、安全性が高い、筋肉を効率的に増量させることができる組成物を提供することができる。そのため、筋肉を増量させることにより、日常生活や運動を向上させたり、フレイルやロコモティブシンドロームを改善したりすることができるため、幅広い年代における健康的な生活をサポートすることができる。 According to the present invention, it is possible to provide a composition that can be taken continuously, is highly safe, and can effectively increase muscle mass. Therefore, by increasing muscle mass, daily life and exercise can be improved, and frailty and locomotive syndrome can be improved, so it is possible to support a healthy life in a wide range of age groups.
　次に、本発明を詳細に説明する。ただし、本発明は以下の実施形態に限定されず、本発明の範囲内で自由に変更することができるものである。 Next, the present invention will be described in detail. However, the present invention is not limited to the following embodiments, and can be freely modified within the scope of the present invention.
＜本発明の製造方法＞
　本発明はトレオン酸産生細菌をアスコルビン酸類含有培地で培養する工程、並びに培養後の培養物からトレオン酸含有画分を回収する工程を含む、筋肉増量用組成物の製造方法である。すなわち、本発明の第一の態様は、ビフィドバクテリウム属細菌、ラクトバチルス属細菌、ラクチカゼイバチルス属細菌、ラクチプランティバチルス属細菌、リモシラクトバチルス属細菌、レヴィラクトバチルス属細菌、リジラクトバチルス属細菌、及びラチラクトバチルス属細菌から選択される一種又は二種以上の細菌を、アスコルビン酸類含有培地で培養する工程、並びに培養後の培養物からトレオン酸含有画分を回収する工程を含む、トレオン酸含有組成物、又は筋肉増量用組成物の製造方法である（以降、「本発明の製造方法」とも記す）。
　本明細書において「トレオン酸含有組成物」とは、トレオン酸を含有する限りにおいて限定されないが、菌体そのもの、培養上清、培地、精製されたトレオン酸のいずれかを含有するものであってよく、これらの混合物であってもよい。また、本明細書において「トレオン酸含有組成物」とは、後述するトレオン酸含有画分を含有するものであってよい。
　なお、前述したビフィドバクテリウム属細菌以外の細菌は、いずれも従来ラクトバチルス（Lactobacillus）属に分類されていたが、２０２０年に国際原核生物命名委員会（ＩＣＳＰ）による規則（ＩＣＮＰ）に則りInternational Journal of Systematic and Evolutionary Microbiology（ＩＪＳＥＭ）誌において再分類されたものである。 <Manufacturing method of the present invention>
The present invention is a method for producing a composition for muscle building, comprising the steps of culturing a threonic acid-producing bacterium in an ascorbic acid-containing medium and recovering a threonic acid-containing fraction from the culture after culturing. That is, the first aspect of the present invention provides bacteria belonging to the genus Bifidobacterium, bacteria belonging to the genus Lactobacillus, bacteria belonging to the genus Lacticaseibacillus, bacteria belonging to the genus Lactiplantibacillus, bacteria belonging to the genus Remocilactobacillus, bacteria belonging to the genus Levilactobacillus, bacterium A step of culturing one or more bacteria selected from bacteria belonging to the genus Lactobacillus and bacteria belonging to the genus Lactobacillus in an ascorbic acid-containing medium, and recovering a threonic acid-containing fraction from the culture after culturing. (hereinafter also referred to as "the production method of the present invention").
As used herein, the term "threonic acid-containing composition" is not limited as long as it contains threonic acid, but contains any of bacterial cells, culture supernatant, medium, and purified threonic acid. Well, it may be a mixture of these. Further, the "threonic acid-containing composition" as used herein may contain a threonic acid-containing fraction described later.
In addition, all bacteria other than the Bifidobacterium genus described above were conventionally classified into the genus Lactobacillus, but in 2020, according to the rules (ICNP) by the International Committee for Prokaryotic Nomenclature (ICSP) It has been reclassified in the International Journal of Systematic and Evolutionary Microbiology (IJSEM).
　本明細書に開示された受託番号が付与された菌株については、それぞれ後記の寄託機関から入手可能である。
　「ＮＰＭＤ」とは独立行政法人製品評価技術基盤機構特許微生物寄託センターの略称であり、所在地は〒２９２－０８１８ 千葉県木更津市かずさ鎌足２－５－８ １２２号室である。本明細書において、「ＮＩＴＥ」で始まる受託番号が付された菌株については、ＮＰＭＤに寄託されたものである。
　「ＩＰＯＤ」とは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の略称である。２０１２年度に独立行政法人産業技術総合研究所から独立行政法人製品評価技術基盤機構へ国際寄託の地位が継承され、特許微生物寄託業務についてＮＰＭＤに一元化されている。本明細書において「ＦＥＲＭ」で始まる受託番号が付与された菌株については、ＩＰＯＤに寄託され、その後ＮＰＭＤにその管理が移管されたものである。
　「ＡＴＣＣ」とはＡｍｅｒｉｃａｎ Ｔｙｐｅ Ｃｕｌｔｕｒｅ Ｃｏｌｌｅｃｔｉｏｎの略称であり、所在地は米国、２０１１０、ヴァージニア州、マナサス、ユニバーシティ ブルバード１０８０１（１０８０１ Ｕｎｉｖｅｒｓｉｔｙ Ｂｏｕｌｅｖａｒｄ， Ｍａｎａｓｓａｓ， ＶＡ ２０１１０，Ｕｎｉｔｅｄ Ｓｔａｔｅｓ ｏｆ Ａｍｅｒｉｃａ）である。本明細書において「ＡＴＣＣ」で始まる受託番号が付与された菌株については、ＡＴＣＣに寄託されたものである。
　「ＪＣＭ」とは国立研究開発法人理化学研究所バイオリソース研究センター微生物材料開発室のＪａｐａｎ Ｃｏｌｌｅｃｔｉｏｎ ｏｆ Ｍｉｃｒｏｏｒｇａｎｉｓｍｓの略称である。所在地は〒３０５－００７４　茨城県つくば市高野台３－１－１である。本明細書において「ＪＣＭ」で始まる受託番号が付与された菌株については、ＪＣＭに寄託されたものである。
　「ＤＳＭＺ」とはＤｅｕｔｓｃｈｅ Ｓａｍｍｌｕｎｇ ｖｏｎ Ｍｉｋｒｏｏｒｇａｎｉｓｍｅｎ ｕｎｄ Ｚｅｌｌｋｕｌｔｕｒｅｎ ＧｍｂＨの略称である。所在地はＩｎｈｏｆｆｅｎｓｔｒ. ７Ｂ　３８１２４ Ｂｒａｕｎｓｃｈｗｅｉｇ Ｇｅｒｍａｎｙである。本明細書において「ＤＳＭ」で始まる受託番号が付与された菌株については、ＤＳＭＺに寄託されたものである。 The accession numbered strains disclosed herein are available from the respective depositary institutions listed below.
"NPMD" is an abbreviation for the Patent Microorganism Depositary Center, National Institute of Technology and Evaluation, located at 2-5-8-122 Kazusa Kamatari, Kisarazu City, Chiba Prefecture 292-0818. As used herein, strains with accession numbers beginning with "NITE" have been deposited with NPMD.
"IPOD" is an abbreviation for the National Institute of Advanced Industrial Science and Technology Patent Organism Depositary Center. In 2012, the National Institute of Advanced Industrial Science and Technology inherited the international deposit status from the National Institute of Advanced Industrial Science and Technology to the National Institute of Technology and Evaluation, and the patent microorganism deposit business has been integrated into NPMD. Strains with accession numbers beginning with "FERM" herein have been deposited with IPOD and subsequently transferred to NPMD.
"ATCC" is an abbreviation for American Type Culture Collection, located at 10801 University Boulevard, Manassas, VA 20110, United States of America. Strains with accession numbers beginning with "ATCC" herein have been deposited with the ATCC.
"JCM" is an abbreviation for Japan Collection of Microorganisms, Microbial Material Development Section, RIKEN BioResource Research Center. The location is 3-1-1 Takanodai, Tsukuba City, Ibaraki Prefecture, 305-0074. Strains with accession numbers beginning with "JCM" herein have been deposited with the JCM.
"DSMZ" is an abbreviation for Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH. The address is Inhoffenstr. 7B 38124 Braunschweig Germany. Strains with accession numbers beginning with "DSM" herein have been deposited with the DSMZ.
　本発明の製造方法において、菌体を含むトレオン酸含有組成物を製造する場合は、下記の細菌を使用することが可能である。また本発明の製造方法において、培養上清、培地、精製されたトレオン酸のいずれかを含むトレオン酸含有組成物を製造する場合にも下記の細菌を使用することが可能である。ビフィドバクテリウム属細菌としては、ビフィドバクテリウム・ロンガム・サブスピーシーズ・ロンガム、ビフィドバクテリウム・ロンガム・サブスピーシーズ・インファンティス、ビフィドバクテリウム・ブレーベ、ビフィドバクテリウム・アニマリス・サブスピーシーズ・アニマリス、ビフィドバクテリウム・アニマリス・サブスピーシーズ・ラクティス、ビフィドバクテリウム・シュードロンガム、ビフィドバクテリウム・シュードカテニュラタム、ビフィドバクテリウム・アドレセンティス、及びビフィドバクテリウム・ビフィダム等が挙げられる。 In the production method of the present invention, the following bacteria can be used when producing a threonic acid-containing composition containing bacterial cells. In the production method of the present invention, the following bacteria can also be used when producing a threonic acid-containing composition containing any of culture supernatant, medium, and purified threonic acid. Bifidobacterium bacteria include Bifidobacterium longum subspecies longum, Bifidobacterium longum subspecies infantis, Bifidobacterium breve, Bifidobacterium animalis sub Species animalis, Bifidobacterium animalis subspecies lactis, Bifidobacterium pseudolongum, Bifidobacterium pseudocatenulatum, Bifidobacterium adrecentis, and Bifidobacterium bifidum etc.
　ビフィドバクテリウム・ロンガム・サブスピーシーズ・ロンガムとしては、例えばビフィドバクテリウム・ロンガム・サブスピーシーズ・ロンガムＮＩＴＥ ＢＰ－０２６２１（別名：ＢＢ５３６又はＢｉｆｉｄｏｂａｃｔｅｒｉｕｍ ｌｏｎｇｕｍ ｓｕｂｓｐ. ｌｏｎｇｕｍ ＡＴＣＣ ＢＡＡ－９９９、例えば特開２０１２－２２３１３４号公報等参照）、
ビフィドバクテリウム・ロンガム・サブスピーシーズ・ロンガムＡＴＣＣ １５７０７、ビフィドバクテリウム・ロンガム・サブスピーシーズ・ロンガム ＪＣＭ１２１７等を用いることができる。 Bifidobacterium longum subspecies longum, for example, Bifidobacterium longum subspecies longum NITE BP-02621 (alias: BB536 or Bifidobacterium longum subsp. longum ATCC BAA-999, for example, JP-A-2012- 223134, etc.),
Bifidobacterium longum subspecies longum ATCC 15707, Bifidobacterium longum subspecies longum JCM1217 and the like can be used.
　ビフィドバクテリウム・ロンガム・サブスピーシーズ・インファンティスとしては、例えばビフィドバクテリウム・ロンガム・サブスピーシーズ・インファンティス Ｍ－６３（受託番号ＮＩＴＥ ＢＰ－０２６２３）、ビフィドバクテリウム・ロンガム・サブスピーシーズ・インファンティスＡＴＣＣ １５６９７を用いることができる。 Bifidobacterium longum subspecies infantis, for example, Bifidobacterium longum subspecies infantis M-63 (accession number NITE BP-02623), Bifidobacterium longum subspecies Species Infantis ATCC 15697 can be used.
　ビフィドバクテリウム・ブレーベとしては、例えばビフィドバクテリウム・ブレーベ ＭＣＣ１２７４（受託番号ＦＥＲＭ ＢＰ－１１１７５、）ビフィドバクテリウム・ブレーベ Ｍ－１６Ｖ（受託番号ＮＩＴＥ ＢＰ－０２６２２、市販品として入手可能な、例えば、森永乳業社製の「ビフィドバクテリウム・ブレーベ Ｍ－１６Ｖ」を用いてもよい。）、ビフィドバクテリウム・ブレーベＡＴＣＣ １５７００等を用いることができる。 As Bifidobacterium breve, for example, Bifidobacterium breve MCC1274 (accession number FERM BP-11175,) Bifidobacterium breve M-16V (accession number NITE BP-02622, commercially available, For example, "Bifidobacterium breve M-16V" manufactured by Morinaga Milk Industry Co., Ltd. may be used.), Bifidobacterium breve ATCC 15700, etc. may be used.
　ビフィドバクテリウム・アニマリス・サブスピーシーズ・アニマリスとしては、例えばビフィドバクテリウム・アニマリス・サブスピーシーズ・アニマリス ＡＴＣＣ２５５２７を用いることができる。 As the Bifidobacterium animalis subspecies animalis, for example, Bifidobacterium animalis subspecies animalis ATCC25527 can be used.
　ビフィドバクテリウム・シュードロンガムとしては、例えばビフィドバクテリウム・シュードロンガム・サブスピーシーズ・シュードロンガム ＪＣＭ１２０５Ｔを用いることができる。 As the Bifidobacterium pseudolongum, for example, Bifidobacterium pseudolongum Subspecies pseudolongum JCM1205T can be used.
　ビフィドバクテリウム・シュードカテニュラタムとしては、例えばビフィドバクテリウム・シュードカテニュラタムＤＳＭ２０４３８を用いることができる。 As the Bifidobacterium pseudocatenulatum, for example, Bifidobacterium pseudocatenulatum DSM20438 can be used.
　ビフィドバクテリウム・アニマリス・サブスピーシーズ・ラクティスとしては、例えばビフィドバクテリウム・アニマリス・サブスピーシーズ・ラクティス ＤＳＭ１０１４０を用いることができる。 As the Bifidobacterium animalis subspecies lactis, for example, Bifidobacterium animalis subspecies lactis DSM10140 can be used.
　ビフィドバクテリウム・アドレセンティスとしては、ビフィドバクテリウム・アドレセンティス ＪＣＭ１２７５を用いることができる。 As Bifidobacterium adolescentis, Bifidobacterium adolescentis JCM1275 can be used.
　ビフィドバクテリウム・ビフィダムとしては、例えば、ビフィドバクテリウム・ビフィダムＭＣＣ１０９２（受託番号ＮＩＴＥ ＢＰ－０２４２９）、ビフィドバクテリウム・ビフィダムＭＣＣ１３１９（受託番号ＮＩＴＥ ＢＰ－０２４３１）、ビフィドバクテリウム・ビフィダムＭＣＣ１８６８（受託番号ＮＩＴＥ ＢＰ－０２４３２）、
　ビフィドバクテリウム・ビフィダムＭＣＣ１８７０（受託番号ＮＩＴＥ ＢＰ－０２４３３）、ビフィドバクテリウム・ビフィダムＪＣＭ１２５５等を用いることができる。 Bifidobacterium bifidum includes, for example, Bifidobacterium bifidum MCC1092 (accession number NITE BP-02429), Bifidobacterium bifidum MCC1319 (accession number NITE BP-02431), Bifidobacterium bifidum MCC1868 (Accession number NITE BP-02432),
Bifidobacterium bifidum MCC1870 (accession number NITE BP-02433), Bifidobacterium bifidum JCM1255, etc. can be used.
　本発明の製造方法における新たな分類であるラクトバチルス属（旧分類においてもラクトバチルス属に分類されていた）としては、ラクトバチルス・アシドフィラス（Lactobacillus acidophillus）、ラクトバチルス・デルブリッキイ・サブスピーシーズ・ブルガリクス（Lactobacillus delbrueckii subsp. bulgaricus）、ラクトバチルス・クリスパタス（Lactobacillus crispatus）、ラクトバチルス・ガセリ（Lactobacillus gasseri）、ラクトバチルス・ジョンソニイ（Lactobacillus johnsonii）、ラクトバチルス・ヘルベティカス（Lactobacillus helveticus）、ラクトバチルス・アミロヴォラス（Lactobacillus amylovorus）が挙げられる。
　ラクトバチルス・アシドフィラスとしては、ラクトバチルス・アシドフィラス ＭＣＣ１８４７（ＮＩＴＥ ＢＰ－０１６９５）を用いることができる。
　ラクトバチルス・ガセリとしては、ラクトバチルス・ガセリ ＭＣＣ１８４６（ＮＩＴＥ ＢＰ－０１６６９）、ラクトバチルス・ガセリＪＣＭ１１３１等を用いることができる。
　ラクトバチルス・ヘルベティカスとしては、ラクトバチルス・ヘルベティカス ＪＣＭ１１２０、ラクトバチルス・ヘルベティカス ＭＣＣ１８４８（ＮＩＴＥ ＢＰ－０１６７１）、ラクトバチルス・ヘルベティカス ＭＣＣ１８４４（ＮＩＴＥ ＢＰ－０２１８５）等を用いることができる。 Lactobacillus acidophillus, Lactobacillus acidophillus, Lactobacillus delbrueckii subsp. (Lactobacillus delbrueckii subsp. bulgaricus), Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus johnsonii, Lactobacillus helveticus, Lactobacillus amylovorus amylovorus).
As Lactobacillus acidophilus, Lactobacillus acidophilus MCC1847 (NITE BP-01695) can be used.
As Lactobacillus gasseri, Lactobacillus gasseri MCC1846 (NITE BP-01669), Lactobacillus gasseri JCM1131, and the like can be used.
As Lactobacillus helveticus, Lactobacillus helveticus JCM1120, Lactobacillus helveticus MCC1848 (NITE BP-01671), Lactobacillus helveticus MCC1844 (NITE BP-02185) and the like can be used.
　本発明の製造方法における新たな分類であるラクチカゼイバチルス属（旧分類においてラクトバチルス属に分類されていた）としては、ラクチカゼイバチルス・カゼイ（Lacticaseibacillus casei、旧分類名はラクトバチルス・カゼイ）、ラクチカゼイバチルス・パラカゼイ（Lacticaseibacillus paracasei、旧分類名はラクトバチルス・パラカゼイ）、ラクチカゼイバチルス・ラムノーサス（Lacticaseibacillus rhamnosus、旧分類名はラクトバチルス・ラムノーサス）等が挙げられる。
　ラクチカゼイバチルス・カゼイとしては、例えばラクトバチルス・カゼイＪＣＭ１１３４、ラクトバチルス・カゼイ ＡＴＣＣ３９３、等を用いることができる。
　ラクチカゼイバチルス・パラカゼイとしては、ラクチカゼイバチルス・パラカゼイＪＣＭ８１３０、ラクトバチルス・パラカゼイ ＭＣＣ１８４９（ＮＩＴＥ ＢＰ－０１６３３）、ラクトバチルス・パラカゼイ ＭＣＣ１３７５（ＮＩＴＥ ＢＰ－１１３１３）等を用いることができる。
　ラクチカゼイバチルス・ラムノーサスとしては、ラクトバチルス・ラムノーサスＪＣＭ１１３６、ラクトバチルス・ラムノーサス ＭＣＣ１８５５（ＬＣＳ７４２、市販品として入手可能な、例えば、森永乳業社製の「ＬＣＳ－７４２」を用いることができる。）、ラクトバチルス・ラムノーサス ＡＴＣＣ５３１０３、ラクトバチルス・ラムノーサス ＡＴＣＣ５３１０３、等を用いることができる。 The new classification of the genus Lactobacillus in the production method of the present invention (classified in the genus Lactobacillus in the old classification) includes Lacticaseibacillus casei (old classification name is Lactobacillus casei), Lacticaseibacillus paracasei (formerly classified as Lactobacillus paracasei), Lacticaseibacillus rhamnosus (formerly classified as Lactobacillus rhamnosus), and the like.
As Lactobacillus casei, for example, Lactobacillus casei JCM1134, Lactobacillus casei ATCC393, etc. can be used.
As Lactobacillus paraceasi JCM8130, Lactobacillus paraceasi MCC1849 (NITE BP-01633), Lactobacillus paraceasi MCC1375 (NITE BP-11313) and the like can be used as Lactobacillus paraceasi.
As Lactobacillus rhamnosus, Lactobacillus rhamnosus JCM1136, Lactobacillus rhamnosus MCC1855 (LCS742, commercially available, for example, "LCS-742" manufactured by Morinaga Milk Industry Co., Ltd. can be used), lacto Bacillus rhamnosus ATCC53103, Lactobacillus rhamnosus ATCC53103, etc. can be used.
　本発明の製造方法における新たな分類であるラクチプランティバチルス属（旧分類においてラクトバチルス属に分類されていた）に含まれるものとしては、ラクチプランチバチルス・プランタラム（Lactiplantibacillus plantarum、旧分類名はラクトバチルス・プランタラム）、ラクチプランチバチルス・ペントーサス（Lactiplantibacillus pentosus、旧分類名はラクトバチルス・ペントーサス）が挙げられる。
　ラクチプランチバチルス・プランタラムとしては、ラクチプランチバチルス・プランタラム ＡＴＣＣ１４９１７を用いることができる。 Included in the genus Lactibacillus (classified in the genus Lactobacillus in the old classification), which is a new classification in the production method of the present invention, is Lactiplantibacillus plantarum (old classification name is Lactiplantibacillus plantarum). Lactobacillus plantarum), Lactiplantibacillus pentosus (formerly known as Lactiplantibacillus pentosus).
Lactiprantivibacillus plantarum ATCC14917 can be used as Lactiprantivibacillus plantarum.
　本発明の製造方法における新たな分類であるリモシラクトバチルス属（旧分類においてラクトバチルス属に分類されていた）に含まれるものとしては、リモシラクトバチルス・ファーメンタム（Limosilactobacillus fermentum、旧分類名はラクトバチルス・ファーメンタム）、リモシラクトバチルス・ロイテリ（Limosilactobacillus reuteri、旧分類名はラクトバチルス・ロイテリ）が挙げられる。
　リモシラクトバチルス・ファーメンタムとしては、リモシラクトバチルス・ファーメンタム ＳＢＳ―１を用いることができる。
　リモシラクトバチルス・ロイテリとしては、リモシラクトバチルス・ロイテリ ＪＣＭ１１１２、リモシラクトバチルス・ロイテリ ＤＳＭ１７９３８、リモシラクトバチルス・ロイテリ ＡＴＣＣ ＰＴＡ ６４７５、リモシラクトバチルス・ロイテリ ＡＴＣＣ ＰＴＡ ５２８９等を用いることができる。 Included in the genus Limosilactobacillus (classified in the genus Lactobacillus in the old classification), which is a new classification in the production method of the present invention, is Limosilactobacillus fermentum (old classification name) is Lactobacillus fermentum), and Limosilactobacillus reuteri (formerly classified as Lactobacillus reuteri).
As the Remosilactobacillus fermentum, Remosilactobacillus fermentum SBS-1 can be used.
As Remocilactobacillus reuteri, Remocilactobacillus reuteri JCM1112, Remocilactobacillus reuteri DSM17938, Remocilactobacillus reuteri ATCC PTA 6475, Remocilactobacillus reuteri ATCC PTA 5289, etc. can be used. .
　本発明の製造方法における新たな分類であるレヴィラクトバチルス属（旧分類においてラクトバチルス属に分類されていた）に含まれるものとしては、レヴィラクトバチルス・ブレヴィス（Levilactobacillus brevis、旧分類名はラクトバチルス・ブレヴィス）が挙げられる。 Included in the genus Levilactobacillus (classified in the genus Lactobacillus in the old classification), which is a new classification in the production method of the present invention, is Levilactobacillus brevis (old classification name is Lactobacillus)・Brevis).
　本発明の製造方法における新たな分類であるリジラクトバチルス属（旧分類においてラクトバチルス属に分類されていた）に含まれるものとしては、リジラクトバチルス・サリヴァリウス（Ligilactobacillus salivarius、旧分類名はラクトバチルス・サリヴァリウス）が挙げられる。 Ligilactobacillus salivarius (formerly classified as lacto Bacillus salivarius).
　本発明の製造方法における新たな分類であるラチラクトバチルス属細菌（旧分類においてラクトバチルス属に分類されていた）に含まれるものとしては、ラチラクトバチルス・クルヴァトゥス(Latilactobacillus curvatus、旧分類名はラクトバチルス・クルヴァトゥス)が挙げられる。 Lactobacillus curvatus (formerly classified as Lactobacillus curvatus).
　本発明の製造方法においては、前記の細菌の任意の一種又は二種以上を組み合わせて用いることができる。これらのうち好ましくはビフィドバクテリウム・ブレーベ、ビフィドバクテリウム・ロンガム・サブスピーシーズ・ロンガム、ビフィドバクテリウム・ロンガム・サブスピーシーズ・インファンティス、ビフィドバクテリウム・アニマリス・サブスピーシーズ・アニマリス、ビフィドバクテリウム・シュードロンガム、ビフィドバクテリウム・シュードカテニュラタム、ビフィドバクテリウム・アドレセンティス、ビフィドバクテリウム・アニマリス・サブスピーシーズ・ラクティス、ラクトバチルス・ガセリ、ラクトバチルス・ヘルベティカス、ラクチカゼイバチルス・カゼイ、ラクチカゼイバチルス・パラカゼイ、ラクチカゼイバチルス・ラムノーサス、リモシラクトバチルス・ロイテリから選択される一種又は二種以上である。より好ましくはビフィドバクテリウム・ブレーベ、ビフィドバクテリウム・ロンガム・サブスピーシーズ・ロンガム、ビフィドバクテリウム・ロンガム・サブスピーシーズ・インファンティス、ラクトバチルス・ガセリ、ラクトバチルス・ヘルベティカス、ラクチカゼイバチルス・パラカゼイ、ラクチカゼイバチルス・ラムノーサスから選択される一種又は二種以上である。さらにより好ましくはビフィドバクテリウム・ブレーベ、又はビフィドバクテリウム・ロンガム・サブスピーシーズ・ロンガムである。最も好ましくはビフィドバクテリウム・ブレーベ ＦＥＲＭ ＢＰ－１１１７５、又はビフィドバクテリウム・ロンガムＮＩＴＥ ＢＰ－０２６２１である。 In the production method of the present invention, any one or a combination of two or more of the above bacteria can be used. Among these, preferably Bifidobacterium breve, Bifidobacterium longum subspecies longum, Bifidobacterium longum subspecies infantis, Bifidobacterium animalis subspecies animalis, Bifidobacterium pseudolongum, Bifidobacterium pseudocatenulatum, Bifidobacterium adrecentis, Bifidobacterium animalis subspecies lactis, Lactobacillus gasseri, Lactobacillus helveticus, Lacti It is one or two or more selected from Bacillus casei, Lacticaseibacillus paracasei, Lacticaseibacillus rhamnosus, and Remocilactobacillus reuteri. More preferably, Bifidobacterium breve, Bifidobacterium longum subspecies longum, Bifidobacterium longum subspecies infantis, Lactobacillus gasseri, Lactobacillus helveticus, Lacticaseibacillus It is one or two or more selected from Paracasei and Lacticaceae Bacillus rhamnosus. Even more preferred is Bifidobacterium breve or Bifidobacterium longum subspecies longum. Most preferred is Bifidobacterium breve FERM BP-11175 or Bifidobacterium longum NITE BP-02621.
　なお、前記例示した細菌名で特定される細菌には、当該細菌名で所定の機関に寄託や登録がなされている株そのもの（以下、説明の便宜上、「寄託株」ともいう）に限られず、それと実質的に同等な株（「派生株」または「誘導株」ともいう）も包含される。すなわち、前記受託番号で前記寄託機関に寄託されている株そのものに限られず、それと実質的に同等な株も包含される。各細菌について、「前記寄託株と実質的に同等の株」とは、前記寄託株と同一の種に属し、さらにその１６ＳｒＲＮＡ遺伝子の塩基配列が、前記寄託株の１６ＳｒＲＮＡ遺伝子の塩基配列に対して、好ましくは９７％以上、より好ましくは９８％以上、よりさらに好ましくは９９％以上、よりさらに好ましくは１００％の同一性を有し、かつ、好ましくは前記寄託株と同一の菌学的性質を有する株をいう。各細菌について、前記寄託株と実質的に同等の株は、例えば、当該寄託株を親株とする派生株であってよい。派生株としては、寄託株から育種された株や寄託株から自然に生じた株が挙げられる。育種方法としては、遺伝子工学的手法による改変や、突然変異処理による改変が挙げられる。突然変異処理としては、Ｘ線の照射、紫外線の照射、ならびにＮ－メチル－Ｎ'－ニトロ－Ｎ－ニトロソグアニジン、エチルメタンスルフォネート、およびメチルメタンスルフォネート等の変異剤による処理が挙げられる。寄託株から自然に生じた株としては、寄託株の使用の際に自然に生じた株が挙げられる。そのような株としては、寄託株の培養（例えば継代培養）により自然に生じた変異株が挙げられる。派生株は、一種の改変により構築されてもよく、二種またはそれ以上の改変により構築されてもよい。 In addition, the bacterium specified by the exemplified bacterial name is not limited to the strain itself (hereinafter also referred to as "deposited strain" for convenience of explanation) that has been deposited or registered with a predetermined institution under the bacterial name, Strains substantially equivalent thereto (also referred to as "derivative strains" or "derived strains") are also included. That is, it is not limited to the strain itself deposited with the depositary institution under the above accession number, but also includes substantially equivalent strains. For each bacterium, a "strain substantially equivalent to the deposited strain" means that it belongs to the same species as the deposited strain, and the nucleotide sequence of the 16S rRNA gene is different from the nucleotide sequence of the 16S rRNA gene of the deposited strain. , preferably 97% or more, more preferably 98% or more, even more preferably 99% or more, even more preferably 100% identity, and preferably the same mycological properties as the deposited strain. refers to the shares held. For each bacterium, a strain substantially equivalent to the deposited strain may be, for example, a derivative of the deposited strain as a parent strain. Derivative strains include strains bred from the deposited strain and strains that arise naturally from the deposited strain. Breeding methods include modification by genetic engineering techniques and modification by mutation treatment. Mutagenesis treatments include X-ray irradiation, ultraviolet irradiation, and treatment with mutating agents such as N-methyl-N'-nitro-N-nitrosoguanidine, ethyl methanesulfonate, and methyl methanesulfonate. be done. Strains naturally occurring from the deposited strain include strains naturally occurring during use of the deposited strain. Such strains include mutants naturally occurring by culturing (eg, subculturing) the deposited strain. Derivative strains may be constructed with one modification, or may be constructed with two or more modifications.
　本発明の製造方法において、前記細菌を培養する際に培地にアスコルビン酸類を添加する。本明細書において「アスコルビン酸類」とは、アスコルビン酸、アスコルビン酸誘導体、及びこれらの塩から選択される一種又は二種以上を意味する。アスコルビン酸類は通常Ｌ体を用いることができる。
　アスコルビン酸としてはＬ－アスコルビン酸を用いることができ、Ｌ－アスコルビン酸の立体異性体であるエリソルビン酸も含まれる。
　アスコルビン酸誘導体としては、Ｌ－アスコルビン酸リン酸エステル、Ｌ－アスコルビン酸硫酸エステル等のＬ－アスコルビン酸の無機酸エステルの塩や、Ｌ－アスコルビン酸－２－グルコシド等のＬ－アスコルビン酸の配糖体等を好ましく挙げられる。
　アスコルビン酸又はその誘導体の塩としては、特に限定されないが、例えば、ナトリウム塩、カリウム塩等のアルカリ金属塩、カルシウム、マグネシウム等のアルカリ土類金属塩、アンモニウム塩、トリエタノールアミン塩、トリエチルアミン塩等の有機アミン塩類、リジン塩、アルギニン塩等の塩基性アミノ酸塩等を好ましく挙げられる。
　本発明におけるアスコルビン酸類の具体例としては、Ｌ－アスコルビン酸ナトリウム、Ｌ－アスコルビン酸カリウム、Ｌ－アスコルビン酸カルシウム、Ｌ－アスコルビン酸マグネシウム、エリソルビン酸ナトリウム、エリソルビン酸カリウム、エリソルビン酸カルシウム、エリソルビン酸マグネシウム、Ｌ－アスコルビン酸－２－グルコシド等を好ましく挙げられる。
　アスコルビン酸類の添加量としては、特に限定されないが、培地に対して好ましくは１ｍｇ／Ｌ～１００ｇ／Ｌ、より好ましくは１０ｍｇ／Ｌ～１０ｇ／Ｌ、さらに好ましくは１００ｍｇ／Ｌ～１ｇ／Ｌとすることができる。また、アスコルビン酸類を添加するタイミングは、特に限定されないが、通常は培養開始時から添加する。 In the production method of the present invention, ascorbic acids are added to the medium when culturing the bacteria. As used herein, “ascorbic acids” means one or more selected from ascorbic acid, ascorbic acid derivatives, and salts thereof. L-isomers of ascorbic acids can usually be used.
L-ascorbic acid can be used as ascorbic acid, and erythorbic acid, which is a stereoisomer of L-ascorbic acid, is also included.
Examples of ascorbic acid derivatives include salts of inorganic acid esters of L-ascorbic acid such as L-ascorbic acid phosphate and L-ascorbic acid sulfate, and L-ascorbic acid derivatives such as L-ascorbic acid-2-glucoside. Preferred examples include saccharides and the like.
Salts of ascorbic acid or derivatives thereof are not particularly limited, but examples include alkali metal salts such as sodium salts and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, ammonium salts, triethanolamine salts, triethylamine salts, and the like. organic amine salts, lysine salts, basic amino acid salts such as arginine salts, and the like.
Specific examples of ascorbic acids in the present invention include sodium L-ascorbate, potassium L-ascorbate, calcium L-ascorbate, magnesium L-ascorbate, sodium erythorbate, potassium erythorbate, calcium erythorbate, and magnesium erythorbate. , L-ascorbic acid-2-glucoside, and the like.
The amount of ascorbic acid added is not particularly limited, but is preferably 1 mg/L to 100 g/L, more preferably 10 mg/L to 10 g/L, and still more preferably 100 mg/L to 1 g/L, relative to the medium. be able to. Also, the timing of adding ascorbic acids is not particularly limited, but it is usually added from the start of culture.
　本発明の製造方法における培養工程は、前記細菌が増殖できる限り、特に制限されない。培養方法としては、例えば、前記細菌の培養に通常用いられる方法を、そのまま、あるいは適宜修正して用いることができる。培養温度は、例えば、２５～５０℃であってよく、３５～４２℃であることが好ましい。培養は、好ましくは嫌気条件下で実施することができ、例えば、炭酸ガス等の嫌気ガスを通気しながら実施することができる。また、培養は、液体静置培養等の微好気条件下で実施することもできる。培養は、例えば、トレオン酸が所望の程度に菌体内に蓄積及び／又は菌体外に分泌するまで、あるいは前記細菌が所望の程度に増殖するまで実施することができる。たとえば、１２～７２時間であってよい。 The culture step in the production method of the present invention is not particularly limited as long as the bacteria can grow. As the culturing method, for example, the method normally used for culturing the bacteria can be used as it is or after being modified as appropriate. The culture temperature may be, for example, 25-50°C, preferably 35-42°C. Culturing is preferably carried out under anaerobic conditions, for example, while passing anaerobic gas such as carbon dioxide. Cultivation can also be performed under microaerobic conditions such as liquid stationary culture. Cultivation can be carried out, for example, until threonic acid accumulates within and/or is secreted outside the cells to a desired extent, or until the bacteria proliferate to a desired extent. For example, it may be 12-72 hours.
　培養に用いる培地は、アスコルビン酸類を含有し、また前記細菌が増殖できる限り、特に制限されない。培地としては、例えば、前記細菌の培養に通常用いられる培地を、そのまま、あるいは適宜修正して、用いることができる。すなわち、炭素源としては、例えば、ガラクトース、グルコース、フルクトース、マンノース、セロビオース、マルトース、ラクトース、スクロース、トレハロース、デンプン、デンプン加水分解物、廃糖蜜等の糖類を資化性に応じて用いることができる。窒素源としては、例えば、アンモニア、硫酸アンモニウム、塩化アンモニウム、硝酸アンモニウム等のアンモニウム塩類や硝酸塩類を用いることができる。また、無機塩類としては、例えば、塩化ナトリウム、塩化カリウム、リン酸カリウム、硫酸マグネシウム、塩化カルシウム、硝酸カルシウム、塩化マンガン、硫酸第一鉄等を用いることができる。また、ペプトン、大豆粉、脱脂大豆粕、肉エキス、酵母エキス等の有機成分を用いてもよい。また、ビフィドバクテリウム属細菌の培養に通常用いられる培地として、具体的には、強化クロストリジア培地（Reinforced Clostridial medium）、ＭＲＳ培地（de Man, Rogosa, and Sharpe medium）、ｍＭＲＳ培地（modified MRS medium）、ＴＯＳＰ培地（TOS propionate medium）、ＴＯＳＰ Ｍｕｐ培地（TOS propionate mupirocin medium）が挙げられる。 The medium used for culture is not particularly limited as long as it contains ascorbic acids and the bacteria can grow. As the medium, for example, a medium commonly used for culturing the bacteria can be used as it is or after being appropriately modified. That is, as carbon sources, for example, sugars such as galactose, glucose, fructose, mannose, cellobiose, maltose, lactose, sucrose, trehalose, starch, starch hydrolysate, blackstrap molasses, etc. can be used depending on the assimilation. . As the nitrogen source, for example, ammonia, ammonium salts such as ammonium sulfate, ammonium chloride and ammonium nitrate, and nitrates can be used. Examples of inorganic salts that can be used include sodium chloride, potassium chloride, potassium phosphate, magnesium sulfate, calcium chloride, calcium nitrate, manganese chloride, and ferrous sulfate. Organic ingredients such as peptone, soybean flour, defatted soybean meal, meat extract and yeast extract may also be used. In addition, as media commonly used for culturing Bifidobacterium bacteria, specifically, reinforced Clostridial medium (Reinforced Clostridial medium), MRS medium (de Man, Rogosa, and Sharpe medium), mMRS medium (modified MRS medium ), TOSP medium (TOS propionate medium), and TOSP Mup medium (TOS propionate mupirocin medium).
　本発明の製造方法においては、培養工程の後に培養物からトレオン酸含有画分を回収する工程を含む。本明細書において「トレオン酸含有画分」とは、トレオン酸を含有する限りにおいて、菌体そのもの、培養上清、培地のいずれであってもよく、これらの混合物であってもよい。
後記実施例の通り、トレオン酸は筋肉増量作用を発揮する有効成分となる。また、培地へアスコルビン酸類を添加し、トレオン酸産生細菌を培養することで、培養上清、菌体破砕上清にトレオン酸の存在が確認されたことから、菌体によりアスコルビン酸がトレオン酸に代謝され、菌体内に蓄積され、また菌体外に分泌もされることが明らかとなった。
　本発明の製造方法において、菌体を含むトレオン酸含有組成物を製造する場合は、回収されたトレオン酸含有画分を遠心分離、破砕、洗浄、凍結乾燥、又は噴霧乾燥等で処理する工程を含んでいてもよい。
　本発明の製造方法において、菌体を含むトレオン酸含有組成物を製造する場合は、破砕された組成物、洗浄された組成物、凍結乾燥された組成物、又は噴霧乾燥された組成物を得る工程を含んでいてもよい。 The production method of the present invention includes a step of collecting a threonic acid-containing fraction from the culture after the culturing step. As used herein, the term "threonic acid-containing fraction" may be any of bacterial cells, culture supernatant, medium, or a mixture thereof, as long as it contains threonic acid.
As shown in the examples below, threonic acid is an active ingredient that exerts a muscle-increasing effect. In addition, by adding ascorbic acids to the medium and culturing threonic acid-producing bacteria, the presence of threonic acid was confirmed in the culture supernatant and bacterial cell crushing supernatant. It was clarified that it was metabolized, accumulated inside the cells, and also secreted outside the cells.
In the production method of the present invention, when producing a threonic acid-containing composition containing bacterial cells, a step of treating the collected threonic acid-containing fraction by centrifugation, crushing, washing, freeze drying, spray drying, or the like is performed. may contain.
In the production method of the present invention, when producing a threonic acid-containing composition containing bacterial cells, a crushed composition, washed composition, freeze-dried composition, or spray-dried composition is obtained. A process may be included.
　本発明の製造方法において、培養上清を含むトレオン酸含有組成物を製造する場合は、回収されたトレオン酸含有画分を遠心分離、上清回収、濃縮、凍結乾燥、又は噴霧乾燥等で処理する工程を含んでいてもよい。
　本発明の製造方法において、培養上清を含むトレオン酸含有組成物を製造する場合は、濃縮された組成物、凍結乾燥された組成物、又は噴霧乾燥された組成物を得る工程を含んでいてもよい。 In the production method of the present invention, when producing a threonic acid-containing composition containing a culture supernatant, the recovered threonic acid-containing fraction is treated by centrifugation, supernatant recovery, concentration, freeze drying, spray drying, or the like. may include a step of
In the production method of the present invention, when producing a threonic acid-containing composition containing a culture supernatant, it includes a step of obtaining a concentrated composition, a freeze-dried composition, or a spray-dried composition. good too.
　本発明の製造方法において、培地を含むトレオン酸含有組成物を製造する場合は、回収されたトレオン酸含有画分を遠心分離、培地回収、濃縮、凍結乾燥、又は噴霧乾燥等で処理する工程を含んでいてもよい。
　本発明の製造方法において、培地を含むトレオン酸含有組成物を製造する場合は、濃縮された組成物、凍結乾燥された組成物、又は噴霧乾燥された組成物を得る工程を含んでいてもよい。 In the production method of the present invention, when producing a threonic acid-containing composition containing a medium, a step of treating the recovered threonic acid-containing fraction by centrifugation, medium recovery, concentration, freeze drying, spray drying, or the like is included. may contain.
In the production method of the present invention, when producing a threonic acid-containing composition containing a medium, it may include a step of obtaining a concentrated composition, a freeze-dried composition, or a spray-dried composition. .
　本発明の製造方法において、精製されたトレオン酸を含むトレオン酸含有組成物を製造する場合は、トレオン酸含有画分に含まれるトレオン酸を精製する工程を含んでいてもよい。トレオン酸を精製する工程としては、トレオン酸を精製することができれば特に限定されないが、菌体の破砕、遠心分離、膜分離、溶媒抽出離等で処理する工程が可能である。
　本発明の製造方法において、トレオン酸含有画分からトレオン酸を精製する工程を含む場合、本発明の製造方法はトレオン酸の製造方法とすることができる。本発明の製造方法をトレオン酸の製造方法とする場合は、回収されたトレオン酸含有画分を含んでいてもよい。
　このようにして回収されたトレオン酸含有画分は、通常はその総乾燥重量あたりトレオン酸を好ましくは０．０１μｇ／ｇ以上含み、より好ましくは０．１μｇ／ｇ以上含み、さらに好ましくは１μｇ／ｇ以上含み、さらにより好ましくは５μｇ／ｇ以上含み、さらにより好ましくは１０μｇ／ｇ以上含む。
　なお、本明細書において「トレオン酸」とは通常Ｌ体のものを意味する。本発明の組成物中においてトレオン酸は、トレオン酸塩の態様で含有されてもよく、かかる塩としてはナトリウム塩、カリウム塩、カルシウム塩等が挙げられるが特に限定されない。
　トレオン酸の同定、定量方法は、公知の分析手法を用いて適宜行うことができる。例えば、後記実施例に示すように、液体クロマトグラフィー質量分析（ＬＣ／ＭＳ）により行うことが可能であり、例えば、ＨＰＬＣにＰｒｏｍｉｎｅｎｃｅ（島津製作所社製）を、質量分析にＴＳＱ Ｑｕａｎｔｕｍ Ｄｉｓｃｏｖｅｒｙ ＭＡＸ（Thermo社製）を用いることが可能である。 In the production method of the present invention, when producing a threonic acid-containing composition containing purified threonic acid, a step of purifying threonic acid contained in the threonic acid-containing fraction may be included. The step of purifying threonic acid is not particularly limited as long as it can purify threonic acid, but it is possible to use a step of crushing cells, centrifugation, membrane separation, solvent extraction, or the like.
When the production method of the present invention includes a step of purifying threonic acid from the threonic acid-containing fraction, the production method of the present invention can be a method for producing threonic acid. When the production method of the present invention is used as a method for producing threonic acid, it may contain a collected threonic acid-containing fraction.
The threonic acid-containing fraction recovered in this manner usually contains threonic acid of preferably 0.01 μg/g or more, more preferably 0.1 μg/g or more, and still more preferably 1 μg/g of threonic acid per total dry weight. g or more, still more preferably 5 μg/g or more, even more preferably 10 μg/g or more.
In the present specification, "threonic acid" usually means L-form. In the composition of the present invention, threonic acid may be contained in the form of threonate, and examples of such salts include, but are not limited to, sodium salts, potassium salts, calcium salts, and the like.
The identification and quantification of threonic acid can be appropriately carried out using known analysis methods. For example, as shown in Examples below, it can be performed by liquid chromatography mass spectrometry (LC/MS). For example, Prominence (manufactured by Shimadzu Corporation) is used for HPLC, and TSQ Quantum Discovery MAX (Thermo company) can be used.
　上記のとおり、本発明の製造方法において、トレオン酸含有画分を回収する工程の後に、回収されたトレオン酸含有画分を処理する工程を含んでいてもよい。トレオン酸含有画分を処理する方法は、筋肉増量作用を損なわない限り特に限定されないが、希釈、濃縮、加熱、凍結乾燥、噴霧乾燥、破砕、分画等が挙げられる。
　すなわち、本発明の製造方法において、回収されたトレオン酸含有画分を処理する工程として、菌体を分離回収する工程、培養上清を分離回収する工程、培地を分離回収する工程、トレオン酸含有画分からトレオン酸を精製する工程であってよい。
　さらに、本発明の製造方法において、トレオン酸含有画分からトレオン酸を精製する工程を含む場合、本発明の製造方法はトレオン酸の製造方法とすることができる。 As described above, the production method of the present invention may include a step of treating the recovered threonic acid-containing fraction after the step of recovering the threonic acid-containing fraction. The method of treating the threonic acid-containing fraction is not particularly limited as long as it does not impair the muscle-increasing action, and includes dilution, concentration, heating, freeze-drying, spray-drying, crushing, fractionation, and the like.
That is, in the production method of the present invention, the steps of treating the recovered threonic acid-containing fraction include a step of separating and recovering bacterial cells, a step of separating and recovering the culture supernatant, a step of separating and recovering the medium, and a step of separating and recovering the medium containing threonic acid. It may be a step of purifying threonic acid from the fraction.
Furthermore, when the production method of the present invention includes a step of purifying threonic acid from the threonic acid-containing fraction, the production method of the present invention can be a method for producing threonic acid.
本発明の製造方法において、回収されたトレオン酸含有画分と食品原料とを混合する工程を含んでいてもよい。
本発明の製造方法において、回収されたトレオン酸含有画分を、粉末、顆粒、ペースト、エマルション、湿潤剤、カプセル、タブレット等の形態にする工程を含んでいてもよい。 The production method of the present invention may include a step of mixing the collected threonic acid-containing fraction with the food raw material.
The production method of the present invention may include a step of converting the recovered threonic acid-containing fraction into a form such as powder, granules, paste, emulsion, wetting agent, capsule, tablet, or the like.
　本発明の製造方法において、トレオン酸含有画分を回収する工程の後に、回収されたトレオン酸含有画分を殺菌処理する工程を含んでいてもよい。トレオン酸含有画分を殺菌処理する工程は、筋肉増量作用を損なわない限り特に限定されないが、加熱処理、破砕処理、加圧処理、薬剤による処理等が挙げられる。特に、トレオン酸含有画分として菌体を回収する場合、菌体を殺菌処理する工程を含んでいてもよく、その殺菌処理は加熱処理、破砕処理、加圧処理であってよい。すなわち、得られる筋肉増量用組成物にトレオン酸含有画分として菌体が含まれる場合、菌体は生菌体であってもよく、死菌体であってもよく、生菌体と死菌体の混合物であってもよい。　これらの回収されたトレオン酸含有画分を処理する工程、食品原料と混合する工程、液体等の形態にする工程、殺菌処理する工程は任意の工程であり、その工程順は適宜変更することが可能である。 The production method of the present invention may include a step of sterilizing the recovered threonic acid-containing fraction after the step of recovering the threonic acid-containing fraction. The step of sterilizing the threonic acid-containing fraction is not particularly limited as long as it does not impair the muscle volume-increasing action, and includes heat treatment, crushing treatment, pressure treatment, treatment with chemicals, and the like. In particular, when the cells are collected as the threonic acid-containing fraction, a step of sterilizing the cells may be included, and the sterilization may be heat treatment, crushing treatment, or pressure treatment. That is, when the resulting composition for increasing muscle mass contains bacterial cells as the threonic acid-containing fraction, the bacterial cells may be viable cells or dead cells. It may be a mixture of bodies. The process of treating the recovered threonic acid-containing fraction, the process of mixing it with food ingredients, the process of making it into a liquid or the like, and the process of sterilizing are optional steps, and the order of the steps can be changed as appropriate. It is possible.
＜本発明の組成物＞
　本発明の第二の態様の組成物は、トレオン酸と、ビフィドバクテリウム属細菌、ラクトバチルス属細菌、ラクチカゼイバチルス属細菌、ラクチプランティバチルス属細菌、リモシラクトバチルス属細菌、レヴィラクトバチルス属細菌、リジラクトバチルス属細菌、及びラチラクトバチルス属細菌から選択される一種又は二種以上の細菌、並びに前記細菌の培養物から選択される一種又は二種以上とを含有し、筋肉増量用途に供される。
　本発明の第二の他の態様の組成物は、精製されたトレオン酸、又はトレオン酸含有画分を含有し、筋肉増量用途に供される。 <Composition of the present invention>
The composition of the second aspect of the present invention comprises threonic acid, bacteria of the genus Bifidobacterium, bacteria of the genus Lactobacillus, bacteria of the genus Lacticaseibacillus, bacteria of the genus Lactipplantibacillus, bacteria of the genus Remocilactobacillus, and Reviract. containing one or more bacteria selected from bacteria belonging to the genus Bacillus, bacteria belonging to the genus Redilactobacillus, and bacteria belonging to the genus Rachilactobacillus, and one or more bacteria selected from cultures of the above bacteria to increase muscle mass provided for use.
A composition according to a second alternative aspect of the present invention contains purified threonic acid, or a threonic acid-containing fraction, for use in muscle building.
　本発明の組成物は、前述した本発明の製造方法によって好ましく製造することができる。その場合、通常トレオン酸は、細菌及び／又はその培養物に含まれる。
　そのため、本発明の組成物は、別の観点から第三の態様として、ビフィドバクテリウム属細菌、ラクトバチルス属細菌、ラクチカゼイバチルス属細菌、ラクチプランティバチルス属細菌、リモシラクトバチルス属細菌、レヴィラクトバチルス属細菌、リジラクトバチルス属細菌、及びラチラクトバチルス属細菌から選択される一種又は二種以上の細菌、並びに前記細菌の培養物から選択される一種又は二種以上を含有する組成物であって、前記細菌及び／又は前記培養物が、その総乾燥重量あたりトレオン酸を１μｇ／ｇ以上含有するものである。かかる組成物は、筋肉増量用途に好ましく供することができる。
　本発明の組成物における前記細菌の種類は、前述した本発明の製造方法での説明に準じる。本発明の組成物に含有させる前記細菌及び／又はその培養物は、前記製造方法において回収されるトレオン酸含有画分を指すものであってよい。 The composition of the present invention can be preferably produced by the production method of the present invention described above. In that case, threonic acid is usually included in the bacteria and/or culture thereof.
Therefore, from another aspect, the composition of the present invention, as a third aspect, includes bacteria belonging to the genus Bifidobacterium, bacteria belonging to the genus Lactobacillus, bacteria belonging to the genus Lacticaseibacillus, bacteria belonging to the genus Lactipplantibacillus, and bacteria belonging to the genus Remocilactobacillus. , one or more bacteria selected from the genus Levilactobacillus, the genus Revilactobacillus, and the genus Lachilactobacillus, and one or more bacteria selected from cultures of the bacteria wherein said bacterium and/or said culture contains 1 μg/g or more of threonic acid per total dry weight thereof. Such compositions can preferably serve for muscle building applications.
The type of bacteria in the composition of the present invention conforms to the description of the production method of the present invention described above. The bacterium and/or its culture to be contained in the composition of the present invention may refer to the threonic acid-containing fraction recovered in the production method.
　本発明の組成物における前記細菌及び／又はその培養物の含有量としては特に限定されないが、例えば前記細菌の菌体量に換算して、組成物１ｇ当たり１．０×１０７ｃｆｕ以上とすることが好ましく、より好ましくは１．０×１０８ｃｆｕ以上、さらに好ましくは１．０×１０９ｃｆｕ以上の菌体を含有するとしてよい。なお、ｃｆｕはコロニー形成単位（Ｃｏｌｏｎｙ ｆｏｒｍｉｎｇ ｕｎｉｔ）を指す。本明細書においては、例えば、還元脱脂粉乳１０質量％を含む固体培地にて３８℃で培養したときの値とすることができる。また、菌体が死菌体である場合、ｃｆｕは個細胞（ｃｅｌｌｓ）と置き換えることができる。また、菌体の含有量は、例えば培養物の固形物量に換算して、組成物１ｇ当たり０．１ｍｇ以上とすることが好ましく、より好ましくは１ｍｇ以上、さらに好ましくは１０ｍｇ以上を含有するとしてよい。 The content of the bacterium and/or its culture in the composition of the present invention is not particularly limited, but is, for example, 1.0×10 7 cfu or more per 1 g of the composition in terms of the amount of the bacterium. preferably 1.0×10 8 cfu or more, and still more preferably 1.0×10 9 cfu or more. In addition, cfu refers to a colony forming unit. In this specification, for example, the value obtained when cultured at 38° C. in a solid medium containing 10% by mass of reconstituted skim milk can be used. Also, when the cells are dead cells, the cfu can be replaced with cells. In addition, the content of the bacterial cells is preferably 0.1 mg or more, more preferably 1 mg or more, and still more preferably 10 mg or more per 1 g of the composition in terms of the amount of solids in the culture. .
　本発明の組成物における、トレオン酸の含有量としては特に限定されないが、組成物１ｇ当たり０．０１μｇ以上とすることが好ましく、より好ましくは０．１μｇ以上、さらに好ましくは１μｇ以上とすることが好ましく、さらにより好ましくは５μｇ以上とすることが好ましく、さらにより好ましくは１０μｇ以上を含有するとしてよい。
　これらは、通常、経口組成物として流通するときの含有量の範囲であってよい。 Although the content of threonic acid in the composition of the present invention is not particularly limited, it is preferably 0.01 μg or more, more preferably 0.1 μg or more, and still more preferably 1 μg or more per 1 g of the composition. Preferably, it may contain 5 μg or more, and still more preferably 10 μg or more.
These may be in the range of content when they are usually distributed as an oral composition.
　本発明の組成物の形態としては特に限定されず、例えば、細菌及び／又はその培養物が、希釈された組成物、濃縮された組成物、加熱された組成物、凍結乾燥された組成物、噴霧乾燥された組成物、破砕された組成物、分画された組成物、又は殺菌された組成物であってよい。また本発明の組成物の形態としては、液体、粉末、顆粒、ペースト、エマルション、湿潤剤、カプセル、タブレット等であってよい。
　本発明の組成物を殺菌された組成物とする場合、その殺菌方法は限定されず、加熱殺菌された組成物、薬剤で殺菌された組成物、破砕処理により殺菌された組成物であってよい。 The form of the composition of the present invention is not particularly limited. It may be a spray dried composition, a crushed composition, a fractionated composition, or a sterilized composition. The form of the composition of the present invention may be liquid, powder, granules, paste, emulsion, wetting agent, capsule, tablet and the like.
When the composition of the present invention is a sterilized composition, the sterilization method is not limited, and it may be a composition sterilized by heat, a composition sterilized with a drug, or a composition sterilized by crushing. .
　本発明の組成物は、ヒトを含む動物において筋肉を増量する作用を有する。
　「筋肉増量」は、筋肉の容量及び／又は重量が増加すること、並びに筋肉の容量及び／又は重量の減少を抑制することを含む。筋肉の容量及び／又は重量の増加、並びに筋肉の容量及び／又は重量の減少の抑制は、筋肥大（筋管又は筋線維が太くなること）によるものであってもよいし、筋管又は筋線維の数が増えることによるものであってもよい。本発明の組成物の筋肉増量作用は、トレオン酸によりｐ７０Ｓ６Ｋ等のタンパク質合成関連遺伝子が活性化され、筋タンパク質の合成が促進される作用に起因すると考えられる。 The composition of the present invention has the effect of increasing muscle mass in animals, including humans.
“Muscle mass” includes increasing muscle volume and/or weight and inhibiting muscle volume and/or weight loss. Increase in muscle volume and/or weight and inhibition of decrease in muscle volume and/or weight may be due to muscle hypertrophy (thickening of myotubes or muscle fibers), It may be due to an increase in the number of fibers. The action of the composition of the present invention to increase muscle mass is believed to be due to the action of threonic acid activating protein synthesis-related genes such as p70S6K and promoting muscle protein synthesis.
　なお、骨格筋における筋肉量の維持にはアスコルビン酸が寄与していることが知られているが（S. Takisawa et al., Scientific Reports vol.9, Art. no.4702, (2019)）、トレオン酸はアスコルビン酸とは構造が大きく異なるため、トレオン酸はアスコルビン酸とは異なる機序で筋肉増量作用を発揮すると考えられる。
　また、ビフィドバクテリウム属細菌やその培養物にも筋肉増量作用があることが知られているが（特許文献１）、これにトレオン酸を組み合わせたものや、トレオン酸を産生した細菌やその培養物を有効成分とすることにより、さらに筋肉増量作用を高めることができる。 It is known that ascorbic acid contributes to the maintenance of muscle mass in skeletal muscle (S. Takisawa et al., Scientific Reports vol.9, Art. no.4702, (2019)). Since the structure of threonic acid is significantly different from that of ascorbic acid, threonic acid is thought to exert a muscle-enhancing effect through a mechanism different from that of ascorbic acid.
Bifidobacterium bacteria and their cultures are also known to have a muscle-enhancing effect (Patent Document 1). By using the culture as an active ingredient, the muscle volume-enhancing action can be further enhanced.
　本発明の組成物を経口摂取（投与）される組成物とする場合は、飲食品の態様とすることが好ましい。
　すなわち、本発明の別の側面は、筋肉増量における、トレオン酸と、ビフィドバクテリウム属細菌、ラクトバチルス属細菌、ラクチカゼイバチルス属細菌、ラクチプランティバチルス属細菌、リモシラクトバチルス属細菌、レヴィラクトバチルス属細菌、リジラクトバチルス属細菌、及びラチラクトバチルス属細菌から選択される一種又は二種以上の細菌、並びに前記細菌の培養物から選択される一種又は二種以上とを含有する組成物の使用である。
　本発明の別の側面は、筋肉増量における、ビフィドバクテリウム属細菌、ラクトバチルス属細菌、ラクチカゼイバチルス属細菌、ラクチプランティバチルス属細菌、リモシラクトバチルス属細菌、レヴィラクトバチルス属細菌、リジラクトバチルス属細菌、及びラチラクトバチルス属細菌から選択される一種又は二種以上の細菌、並びに前記細菌の培養物から選択される一種又は二種以上の使用であって、前記細菌及び／又は前記培養物が、その総乾燥重量あたりトレオン酸を１μｇ／ｇ以上含有する使用である。
　本発明の別の側面は、ビフィドバクテリウム属細菌、ラクトバチルス属細菌、ラクチカゼイバチルス属細菌、ラクチプランティバチルス属細菌、リモシラクトバチルス属細菌、レヴィラクトバチルス属細菌、リジラクトバチルス属細菌、及びラチラクトバチルス属細菌から選択される一種又は二種以上の細菌、並びに前記細菌の培養物から選択される一種又は二種以上を対象に投与することを含み、前記細菌及び／又は前記培養物が、その総乾燥重量あたりトレオン酸を１μｇ／ｇ以上含有する、筋肉を増量する方法である。 When the composition of the present invention is to be orally ingested (administered), it is preferably in the form of a food or drink.
That is, another aspect of the present invention is the combination of threonic acid and Bifidobacterium, Lactobacillus, Lacticaseibacillus, Lactipplantibacillus, Remocilactobacillus, A composition containing one or more bacteria selected from bacteria belonging to the genus Levilactobacillus, bacteria belonging to the genus Rydilactobacillus, and bacteria belonging to the genus Lachilactobacillus, and one or more bacteria selected from cultures of the bacteria It is the use of things.
Another aspect of the present invention is the use of Bifidobacterium, Lactobacillus, Lacticaseibacillus, Lactipplantibacillus, Remocilactobacillus, Levilactobacillus, in muscle building. Use of one or more bacteria selected from the genus Rezilactobacillus and bacteria of the genus Lachilactobacillus, and one or more bacteria selected from cultures of the bacteria, wherein the bacteria and/or The culture contains 1 μg/g or more of threonic acid per total dry weight.
Another aspect of the present invention is to provide bacteria of the genus Bifidobacterium, bacteria of the genus Lactobacillus, bacteria of the genus Lacticaseibacillus, bacteria of the genus Lactiplantibacillus, bacteria of the genus Remosilactobacillus, bacteria of the genus Levilactobacillus, and genus Lyzilactobacillus. including administering to a subject one or more selected from bacteria and bacteria of the genus Lachilactobacillus, and one or more selected from cultures of said bacteria, said bacteria and/or said A method for increasing muscle bulk wherein the culture contains 1 μg/g or more of threonic acid per total dry weight thereof.
　本明細書は、第四の態様として、トレオン酸を有効成分として含有する筋肉増量用組成物をも開示する。
　これは、トレオン酸が骨格筋細胞においてタンパク質合成関連遺伝子を活性化し、筋肥大を促進して筋肉増量作用を発揮することに基づく発明である。
　　また、かかる発明は、筋肉増量におけるトレオン酸の使用とも言い換えることができる。
　また、かかる発明は、トレオン酸を対象に投与することを含む、筋肉を増量する方法とも言い換えることができる。 This specification also discloses, as a fourth aspect, a muscle building composition containing threonic acid as an active ingredient.
This is an invention based on the fact that threonic acid activates protein synthesis-related genes in skeletal muscle cells, promotes muscle hypertrophy, and exerts a muscle-increasing effect.
Such invention can also be translated into the use of threonic acid in muscle mass building.
This invention can also be translated into a method for increasing muscle mass, including administering threonic acid to a subject.
　本発明の飲食品組成物は、筋肉を増量することができるため、筋肉萎縮などの症状や筋肉量低下に起因する疾患などの予防及び／又は改善、運動不足や加齢に伴う筋肉量低下の予防及び／又は改善をすることが期待される。筋肉萎縮などの症状を伴う疾患としては、例えば、緊張減退症、筋萎縮症、筋異栄養症、筋肉退化、炎症性筋疾患、筋無力症及び筋肉減少症等が挙げられる。筋肉量低下に起因する疾患としては、骨粗鬆症、骨折、糖尿病、慢性閉塞性肺疾患、慢性腎臓病、認知症等が挙げられる。
　また、運動不足や加齢に伴う筋肉量低下は、サルコペニア、フレイル、ロコモティブシンドローム等を引き起こしうるため、これらの予防及び／又は改善をすることも期待できる。
　また、健常な人や、スポーツをする人にとっても、日常生活を維持・向上したり、運動効率を向上したりできることからも、本発明の飲食品組成物は有用である。
　ここで、本明細書において症状又は疾患の「改善」とは、疾患が治癒すること、症状が緩和すること、疾患又は症状の程度が低減すること、疾患又は症状の進行が遅延することを含む。また、症状又は疾患の「予防」とは、症状又は疾患の発生を防止すること、該発生を遅延させること、該発生のリスクを低下させることを含む。 Since the food and drink composition of the present invention can increase muscle mass, it prevents and/or improves symptoms such as muscle atrophy and diseases caused by decreased muscle mass, and prevents decreased muscle mass due to lack of exercise and aging. Prevention and/or amelioration is expected. Diseases accompanied by symptoms such as muscle atrophy include, for example, hypotonia, muscle atrophy, muscle dystrophy, muscle degeneration, inflammatory muscle disease, myasthenia and sarcopenia. Diseases caused by decreased muscle mass include osteoporosis, bone fracture, diabetes, chronic obstructive pulmonary disease, chronic kidney disease, dementia, and the like.
In addition, lack of exercise and age-related loss of muscle mass can cause sarcopenia, frailty, locomotive syndrome, and the like, so it is also expected to prevent and/or improve these.
In addition, the food and drink composition of the present invention is also useful for healthy people and people who play sports because they can maintain and improve their daily lives and improve exercise efficiency.
Here, as used herein, "amelioration" of symptoms or diseases includes curing of diseases, alleviation of symptoms, reduction in severity of diseases or symptoms, and delay of progression of diseases or symptoms. . In addition, "prevention" of a symptom or disease includes preventing the occurrence of the symptom or disease, delaying the occurrence, and reducing the risk of the occurrence.
　本発明の第二乃至第四の態様の飲食品組成物を投与する対象（被投与者）及び摂取させる対象（摂取者）は、動物であれば特に限定されないが、通常はヒトである。また、成人、小児、乳児、新生児（低体重児を含む）等のいずれであってもよい。また、性別は特に限定されない。 The subject (administrator) to whom the food and drink compositions of the second to fourth aspects of the present invention are administered (administrator) and the subject (consumer) to be ingested are not particularly limited as long as they are animals, but are usually humans. Moreover, any of adults, children, infants, newborns (including low-weight infants), and the like may be used. Moreover, sex is not specifically limited.
　なお、本明細書において「対象に投与すること」は、「対象に摂取させること」と同義であってよい。摂取は、通常は自発的なもの（自由摂取）であるが、強制的なもの（強制摂取）であってもよい。 In this specification, "administering to a subject" may be synonymous with "making a subject ingest". The intake is usually voluntary (free intake), but may be forced (forced intake).
　本発明の飲食品組成物の摂取（投与）時期は、特に限定されず、摂取（投与）対象の状態に応じて適宜選択することが可能である。 The intake (administration) timing of the food and drink composition of the present invention is not particularly limited, and can be appropriately selected according to the condition of the intake (administration) subject.
　本発明の飲食品組成物の摂取（投与）量は、摂取（投与）対象の年齢、性別、状態、その他の条件等により適宜選択される。
　本発明の飲食品組成物の摂取（投与）量は、前記細菌及び／又はその培養物の固形分量に換算して、例えば、成人において１００μｇ／日～１０ｇ／日の範囲が好ましく、１ｍｇ／日～１ｇ／日の範囲がより好ましく、１０ｍｇ／日～５００ｍｇ／日がさらに好ましい。また、トレオン酸量に換算して、例えば、成人において１ｎｇ／日～１００μｇ／日の範囲が好ましく、１ｎｇ／日～１μｇ／日の範囲がより好ましく、１０ｎｇ／日～５００ｎｇ／日がさらに好ましい。
　なお、摂取（投与）の量や期間にかかわらず、本発明の飲食品組成物は１日１回又は複数回に分けて摂取（投与）することができる。 The intake (administration) amount of the food and drink composition of the present invention is appropriately selected depending on the age, sex, condition, other conditions, etc. of the intake (administration) subject.
The intake (administration) amount of the food and drink composition of the present invention is preferably in the range of 100 μg/day to 10 g/day, for example, 1 mg/day in terms of the solid content of the bacteria and/or culture thereof for adults. A range of -1 g/day is more preferred, and 10 mg/day to 500 mg/day is even more preferred. In terms of threonic acid, for example, the range for adults is preferably 1 ng/day to 100 μg/day, more preferably 1 ng/day to 1 μg/day, and even more preferably 10 ng/day to 500 ng/day.
The food and drink composition of the present invention can be ingested (administered) once a day or divided into multiple times a day, regardless of the amount and period of ingestion (administration).
　本発明の飲食品組成物の摂取（投与）期間は、特に限定されない。また、摂取（投与）期間の上限は特に設けられず、継続的な、長期の摂取（投与）が可能である。 The intake (administration) period of the food and drink composition of the present invention is not particularly limited. In addition, there is no particular upper limit for the intake (administration) period, and continuous long-term intake (administration) is possible.
　飲食品としては、本発明の効果を損なわず、経口摂取（投与）できるものであれば形態や性状は特に制限されず、トレオン酸や前記細菌及び／又はその培養物を含有させること以外は、通常飲食品に用いられる原料を用いて通常の方法によって製造することができる。 As food and drink, the form and properties are not particularly limited as long as they can be orally ingested (administered) without impairing the effects of the present invention. It can be produced by a normal method using raw materials that are usually used for food and drink.
　飲食品としては、液状、ペースト状、ゲル状固体、粉末等の形態を問わず、例えば、栄養補助食品（サプリメント）、錠菓；流動食（経管摂取用栄養食）；パン、マカロニ、スパゲッティ、めん類、ケーキミックス、から揚げ粉、パン粉等の小麦粉製品；即席めん、カップめん、レトルト・調理食品、調理缶詰め、電子レンジ食品、即席スープ・シチュー、即席みそ汁・吸い物、スープ缶詰め、フリーズ・ドライ食品、その他の即席食品等の即席食品類；農産缶詰め、果実缶詰め、ジャム・マーマレード類、漬物、煮豆類、農産乾物類、シリアル（穀物加工品）等の農産加工品；水産缶詰め、魚肉ハム・ソーセージ、水産練り製品、水産珍味類、つくだ煮類等の水産加工品；畜産缶詰め・ペースト類、畜肉ハム・ソーセージ等の畜産加工品；加工乳、乳飲料、ヨーグルト類、乳酸菌飲料類、チーズ、アイスクリーム類、調製粉乳類、クリーム、その他の乳製品等の乳・乳製品；バター、マーガリン類、植物油等の油脂類；しょうゆ、みそ、ソース類、トマト加工調味料、みりん類、食酢類等の基礎調味料；調理ミックス、カレーの素類、たれ類、ドレッシング類、めんつゆ類、スパイス類、その他の複合調味料等の複合調味料・食品類；素材冷凍食品、半調理冷凍食品、調理済冷凍食品等の冷凍食品；キャラメル、キャンディー、チューインガム、チョコレート、クッキー、ビスケット、ケーキ、パイ、スナック、クラッカー、和菓子、米菓子、豆菓子、デザート菓子、ゼリー、その他の菓子などの菓子類；炭酸飲料、天然果汁、果汁飲料、果汁入り清涼飲料、果肉飲料、果粒入り果実飲料、野菜系飲料、豆乳、豆乳飲料、コーヒー飲料、お茶飲料、粉末飲料、濃縮飲料、スポーツ飲料、栄養飲料、アルコール飲料、その他の嗜好飲料等の嗜好飲料類、ベビーフード、ふりかけ、お茶漬けのり等のその他の市販食品等；育児用調製粉乳；経腸栄養食；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）等が挙げられる。 Food and drink, regardless of the form such as liquid, paste, gel-like solid, powder, etc., for example, nutritional supplements (supplements), tablet confectionery; liquid diet (nutrition for ingestion); bread, macaroni, spaghetti Flour products such as , noodles, cake mixes, fried chicken powder, bread crumbs; instant noodles, cup noodles, retort/prepared foods, cooked canned foods, microwave oven foods, instant soups/stews, instant miso soups/soups, canned soups, freeze-dried foods , and other instant foods; canned agricultural products, canned fruits, jams and marmalades, pickled vegetables, boiled beans, dried agricultural products, processed agricultural products such as cereals (processed grains); canned marine products, fish meat hams and sausages , fish paste products, marine delicacies, processed fish products such as tsukudani; processed livestock products such as canned livestock products, pastes, meat hams and sausages; processed milk, milk drinks, yogurts, lactic acid bacteria drinks, cheese, ice creams Milk and dairy products such as , modified milk powder, cream, and other dairy products; Fats and oils such as butter, margarine, and vegetable oils; Basic seasonings such as soy sauce, miso, sauces, tomato processing seasonings, mirin, and vinegar Ingredients: Cooking mixes, curry ingredients, sauces, dressings, noodle soups, spices, and other complex seasonings and foods; Ingredients: Frozen food, half-cooked frozen food, cooked frozen food, etc. confectionery such as caramels, candies, chewing gums, chocolates, cookies, biscuits, cakes, pies, snacks, crackers, Japanese confectionery, rice confectionery, bean confectionery, dessert confectionery, jelly, and other confectionery; carbonated drinks, natural fruit juices , fruit juice drinks, soft drinks containing fruit juice, fruit drinks, fruit drinks containing fruit, vegetable drinks, soy milk, soy milk drinks, coffee drinks, tea drinks, powdered drinks, concentrated drinks, sports drinks, nutritional drinks, alcoholic beverages, etc. Pleasant beverages such as beverages, other commercially available foods such as baby food, furikake, ochazuke seaweed, etc.; powdered milk for infants; enteral nutrition food; ) and the like.
　また、飲食品の一態様として飼料とすることもできる。飼料としては、ペットフード、家畜飼料、養魚飼料等が挙げられる。
　飼料の形態としては特に制限されず、トレオン酸や前記細菌及び／又はその培養物の他に例えば、トウモロコシ、小麦、大麦、ライ麦、マイロ等の穀類；大豆油粕、ナタネ油粕、ヤシ油粕、アマニ油粕等の植物性油粕類；フスマ、麦糠、米糠、脱脂米糠等の糠類；コーングルテンミール、コーンジャムミール等の製造粕類；魚粉、脱脂粉乳、カゼイン、イエローグリース、タロー等の動物性飼料類；トルラ酵母、ビール酵母等の酵母類；第三リン酸カルシウム、炭酸カルシウム等の鉱物質飼料；油脂類；単体アミノ酸；糖類等を含有するものであってよい。 Moreover, it can also be set as feed as one aspect|mode of food-drinks. Examples of the feed include pet food, livestock feed, fish feed, and the like.
The form of the feed is not particularly limited, and in addition to threonic acid, the bacteria and/or culture thereof, grains such as corn, wheat, barley, rye, and milo; soybean meal, rapeseed meal, coconut oil meal, and linseed meal. Rice brans such as wheat bran, wheat bran, rice bran, and defatted rice bran; Manufacturing lees such as corn gluten meal and corn jam meal; Animal feeds such as fish meal, skimmed milk powder, casein, yellow grease, and tallow. Yeasts such as torula yeast and brewer's yeast; mineral feed such as tribasic calcium phosphate and calcium carbonate; oils and fats; simple amino acids;
　本発明の組成物が飲食品（飼料を含む）の態様である場合、筋肉増量に関する用途が表示された飲食品として提供・販売されることが可能である。 When the composition of the present invention is in the form of a food or drink (including feed), it can be provided and sold as a food or drink labeled with uses related to increasing muscle mass.
　かかる「表示」行為には、需要者に対して前記用途を知らしめるための全ての行為が含まれ、前記用途を想起・類推させうるような表現であれば、表示の目的、表示の内容、表示する対象物・媒体等の如何に拘わらず、全て本発明における「表示」行為に該当する。
　また、「表示」は、需要者が前記用途を直接的に認識できるような表現により行われることが好ましい。具体的には、飲食品に係る商品又は商品の包装に前記用途を記載したものを譲渡し、引き渡し、譲渡若しくは引き渡しのために展示し、輸入する行為、商品に関する広告、価格表若しくは取引書類に前記用途を記載して展示し、若しくは頒布し、又はこれらを内容とする情報に前記用途を記載して電磁気的（インターネット等）方法により提供する行為等が挙げられる。 Such "display" acts include all acts for informing consumers of the above-mentioned use. Regardless of the object, medium, etc. to be displayed, all of them fall under the act of "display" in the present invention.
In addition, it is preferable that the "indication" be expressed in such a way that the consumer can directly recognize the use. Specifically, the act of transferring, handing over, displaying for the purpose of transfer or delivery, importing products related to food and beverages or product packaging that describes the above-mentioned use, advertisements related to products, price lists or transaction documents Examples include the act of displaying or distributing information describing the use, or providing information containing such information using an electromagnetic method (such as the Internet) after describing the use.
　一方、表示内容としては、行政等によって認可された表示（例えば、行政が定める各種制度に基づいて認可を受け、そのような認可に基づいた態様で行う表示等）であることが好ましい。また、そのような表示内容を、包装、容器、カタログ、パンフレット、ＰＯＰ等の販売現場における宣伝材、その他の書類等へ付することが好ましい。 On the other hand, it is preferable that the content of the display is a display approved by the government (for example, a display that is approved based on various systems established by the government and performed in a manner based on such approval). In addition, it is preferable to attach such display contents to packaging, containers, catalogs, pamphlets, POP and other advertising materials at sales sites, other documents, and the like.
　また、「表示」には、健康食品、機能性食品、経腸栄養食品、特別用途食品、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、医薬部外品等としての表示も挙げられる。この中でも特に、消費者庁によって認可される表示、例えば、特定保健用食品、栄養機能食品、若しくは機能性表示食品に係る制度、又はこれらに類似する制度にて認可される表示等が挙げられる。具体的には、特定保健用食品としての表示、条件付き特定保健用食品としての表示、身体の構造や機能に影響を与える旨の表示、疾病リスク減少表示、科学的根拠に基づいた機能性の表示等を挙げることができ、より具体的には、健康増進法に規定する特別用途表示の許可等に関する内閣府令（平成二十一年八月三十一日内閣府令第五十七号）に定められた特定保健用食品としての表示（特に保健の用途の表示）及びこれに類する表示が典型的な例である。 In addition, “labeling” includes labeling as health food, functional food, enteral nutrition food, food for special dietary use, food with health claims, food for specified health use, food with nutrient function claims, food with function claims, quasi-drugs, etc. is also mentioned. Among these, in particular, the labeling approved by the Consumer Affairs Agency, for example, the labeling approved by the system related to food for specified health use, food with nutrient function claims, or food with function claims, or similar system. Specifically, labeling as a food for specified health use, labeling as a food for specified health use with certain conditions, labeling to the effect that it affects the structure and function of the body, labeling to reduce the risk of disease, labeling for functionality based on scientific evidence. Labeling, etc. can be mentioned, more specifically, the Cabinet Office Ordinance Concerning Permission for Special Use Labeling as stipulated in the Health Promotion Act (Cabinet Office Ordinance No. 57 of August 31, 2009) A typical example is labeling as a food for specified health use (especially labeling for health use) and similar labeling.
　かかる表示としては、例えば、「筋肉を増やす」、「筋力をつけたい人に」、「フレイルの予防に」、「ＱＯＬの改善のために」、「運動する人の筋力アップをサポート」等が挙げられる。 Such indications include, for example, “Increase muscle”, “For those who want to build muscle”, “For prevention of frailty”, “For improvement of QOL”, “Support for increasing muscle strength of exercisers”, and the like. mentioned.
　本発明の組成物は、医薬品の態様とすることもできる。
　すなわち、本発明の別の側面は、筋肉増量のために用いられる、トレオン酸と、ビフィドバクテリウム属細菌、ラクトバチルス属細菌、ラクチカゼイバチルス属細菌、ラクチプランティバチルス属細菌、リモシラクトバチルス属細菌、レヴィラクトバチルス属細菌、リジラクトバチルス属細菌、及びラチラクトバチルス属細菌から選択される一種又は二種以上の細菌、並びに前記細菌の培養物から選択される一種又は二種以上とを含有する組成物である。
　本発明の別の側面は、筋肉増量用組成物の製造における、トレオン酸と、ビフィドバクテリウム属細菌、ラクトバチルス属細菌、ラクチカゼイバチルス属細菌、ラクチプランティバチルス属細菌、リモシラクトバチルス属細菌、レヴィラクトバチルス属細菌、リジラクトバチルス属細菌、及びラチラクトバチルス属細菌から選択される一種又は二種以上の細菌、並びに前記細菌の培養物から選択される一種又は二種以上との使用である。
　本発明の別の側面は、筋肉増量のために用いられる、トレオン酸と、ビフィドバクテリウム属細菌、ラクトバチルス属細菌、ラクチカゼイバチルス属細菌、ラクチプランティバチルス属細菌、リモシラクトバチルス属細菌、レヴィラクトバチルス属細菌、リジラクトバチルス属細菌、及びラチラクトバチルス属細菌から選択される一種又は二種以上の細菌、並びに前記細菌の培養物から選択される一種又は二種以上とを含有する組成物である。
　本発明の別の側面は、トレオン酸と、ビフィドバクテリウム属細菌、ラクトバチルス属細菌、ラクチカゼイバチルス属細菌、ラクチプランティバチルス属細菌、リモシラクトバチルス属細菌、レヴィラクトバチルス属細菌、リジラクトバチルス属細菌、及びラチラクトバチルス属細菌から選択される一種又は二種以上の細菌、並びに前記細菌の培養物から選択される一種又は二種以上とを対象に投与することを含む、筋肉を増量する方法である。
　本発明の別の側面は、筋肉増量のために用いられる、ビフィドバクテリウム属細菌、ラクトバチルス属細菌、ラクチカゼイバチルス属細菌、ラクチプランティバチルス属細菌、リモシラクトバチルス属細菌、レヴィラクトバチルス属細菌、リジラクトバチルス属細菌、及びラチラクトバチルス属細菌から選択される一種又は二種以上の細菌、並びに前記細菌の培養物から選択される一種又は二種以上を含有する組成物であって、前記細菌及び／又は前記培養物が、その総乾燥重量あたりトレオン酸を１μｇ／ｇ以上含有する組成物である。
　本発明の別の側面は、筋肉増量用組成物の製造における、ビフィドバクテリウム属細菌、ラクトバチルス属細菌、ラクチカゼイバチルス属細菌、ラクチプランティバチルス属細菌、リモシラクトバチルス属細菌、レヴィラクトバチルス属細菌、リジラクトバチルス属細菌、及びラチラクトバチルス属細菌から選択される一種又は二種以上の細菌、並びに前記細菌の培養物から選択される一種又は二種以上の使用であって、前記細菌及び／又は前記培養物が、その総乾燥重量あたりトレオン酸を１μｇ／ｇ以上含有する使用である。
　本発明の別の側面は、ビフィドバクテリウム属細菌、ラクトバチルス属細菌、ラクチカゼイバチルス属細菌、ラクチプランティバチルス属細菌、リモシラクトバチルス属細菌、レヴィラクトバチルス属細菌、リジラクトバチルス属細菌、及びラチラクトバチルス属細菌から選択される一種又は二種以上の細菌、並びに前記細菌の培養物から選択される一種又は二種以上を対象に投与することを含み、前記細菌及び／又は前記培養物が、その総乾燥重量あたりトレオン酸を１μｇ／ｇ以上含有する、筋肉を増量する方法である。 The compositions of the invention can also be in the form of pharmaceuticals.
That is, another aspect of the present invention relates to threonic acid, Bifidobacterium, Lactobacillus, Lacticaseibacillus, Lactipplantibacillus, and Rimosilact, which are used for increasing muscle mass. One or more bacteria selected from bacteria belonging to the genus Bacillus, bacteria belonging to the genus Levilactobacillus, bacteria belonging to the genus Regilactobacillus, and bacteria belonging to the genus Lactobacillus, and bacteria belonging to the genus Lactobacillus, and one or more selected from cultures of the bacteria is a composition containing
Another aspect of the present invention is the use of threonic acid and Bifidobacterium, Lactobacillus, Lacticaseibacillus, Lactipplantibacillus, Remocilactobacillus in the manufacture of a muscle building composition. One or two or more bacteria selected from the genus bacteria, Levilactobacillus bacteria, Regilactobacillus bacteria, and Rachilactobacillus bacteria, and one or two or more bacteria selected from the cultures of the bacteria is used.
Another aspect of the present invention is the combination of threonic acid with Bifidobacterium, Lactobacillus, Lacticaseibacillus, Lactipplantibacillus, Remocilactobacillus, used for muscle gain. Bacteria, one or more bacteria selected from the genus Levilactobacillus, bacteria of the genus Regilactobacillus, and bacteria of the genus Lachilactobacillus, and one or more bacteria selected from cultures of the bacteria It is a composition that
Another aspect of the present invention is the use of threonic acid with bacteria of the genus Bifidobacterium, Lactobacillus, Lacticaseibacillus, Lactipplantibacillus, Remocilactobacillus, Levilactobacillus, muscle, including administering to a subject one or more bacteria selected from bacteria belonging to the genus Rezilactobacillus and bacteria belonging to the genus Lachilactobacillus, and one or more bacteria selected from the cultures of said bacteria is a method of increasing the amount of
Another aspect of the present invention is Bifidobacterium, Lactobacillus, Lacticaseibacillus, Lactipplantibacillus, Remosilactobacillus, Reviract used for muscle mass gain. A composition containing one or more bacteria selected from bacteria belonging to the genus Bacillus, bacteria belonging to the genus Regilactobacillus, and bacteria belonging to the genus Rachilactobacillus, and bacteria belonging to the genus Bacillus, and bacteria belonging to the genus Rachilactobacillus, and one or more bacteria selected from cultures of said bacteria. and the bacteria and/or the culture contain 1 μg/g or more of threonic acid per total dry weight thereof.
Another aspect of the present invention is the use of Bifidobacterium, Lactobacillus, Lacticaseibacillus, Lactipplantibacillus, Remocilactobacillus, Levi in the manufacture of a muscle building composition. One or two or more bacteria selected from bacteria of the genus Lactobacillus, bacteria of the genus Redilactobacillus, and bacteria of the genus Lactobacillus, and one or more bacteria selected from cultures of the bacteria, The use is such that the bacterium and/or the culture contains 1 μg/g or more of threonic acid per total dry weight thereof.
Another aspect of the present invention is to provide bacteria of the genus Bifidobacterium, bacteria of the genus Lactobacillus, bacteria of the genus Lacticaseibacillus, bacteria of the genus Lactiplantibacillus, bacteria of the genus Remosilactobacillus, bacteria of the genus Levilactobacillus, and genus Lyzilactobacillus. including administering to a subject one or more selected from bacteria and bacteria of the genus Lachilactobacillus, and one or more selected from cultures of said bacteria, said bacteria and/or said A method for increasing muscle bulk wherein the culture contains 1 μg/g or more of threonic acid per total dry weight thereof.
　本明細書は、第四の態様として、トレオン酸を有効成分として含有する筋肉増量用組成物をも開示する。
　これは、トレオン酸が骨格筋細胞においてタンパク質合成関連遺伝子を活性化し、筋肥大を促進して筋肉増量作用を発揮することに基づく発明である。
　かかる発明は、筋肉増量のために用いられるトレオン酸とも言い換えることができる。
　また、かかる発明は、筋肉増量用組成物の製造におけるトレオン酸の使用とも言い換えることができる。
　また、かかる発明は、トレオン酸を対象に投与することを含む、筋肉を増量する方法とも言い換えることができる。 This specification also discloses, as a fourth aspect, a muscle building composition containing threonic acid as an active ingredient.
This is an invention based on the fact that threonic acid activates protein synthesis-related genes in skeletal muscle cells, promotes muscle hypertrophy, and exerts a muscle-increasing effect.
Such an invention can also be rephrased as threonic acid used for increasing muscle mass.
Such an invention can also be translated into the use of threonic acid in the manufacture of a muscle building composition.
This invention can also be translated into a method for increasing muscle mass, including administering threonic acid to a subject.
　本発明の医薬品組成物は、筋肉を増量することができるため、筋肉萎縮などの症状や筋肉量低下に起因する疾患などの予防及び／又は改善、運動不足や加齢に伴う筋肉量低下の予防及び／又は改善をすることが期待される。筋肉萎縮などの症状を伴う疾患としては、例えば、緊張減退症、筋萎縮症、筋異栄養症、筋肉退化、炎症性筋疾患、筋無力症及び筋肉減少症等が挙げられる。筋肉量低下に起因する疾患としては、骨粗鬆症、骨折、糖尿病、慢性閉塞性肺疾患、慢性腎臓病、認知症等が挙げられる。
　また、運動不足や加齢に伴う筋肉量低下は、サルコペニア、フレイル、ロコモティブシンドローム等を引き起こしうるため、これらの予防及び／又は改善をすることも期待できる。 Since the pharmaceutical composition of the present invention can increase muscle mass, it prevents and/or improves symptoms such as muscle atrophy and diseases caused by decreased muscle mass, and prevents decreased muscle mass due to lack of exercise and aging. and/or are expected to make improvements. Diseases accompanied by symptoms such as muscle atrophy include, for example, hypotonia, muscle atrophy, muscle dystrophy, muscle degeneration, inflammatory muscle disease, myasthenia and sarcopenia. Diseases caused by decreased muscle mass include osteoporosis, bone fracture, diabetes, chronic obstructive pulmonary disease, chronic kidney disease, dementia, and the like.
In addition, lack of exercise and age-related loss of muscle mass can cause sarcopenia, frailty, locomotive syndrome, and the like, so it is also expected to prevent and/or improve these.
　本発明の第二乃至第四の態様の医薬品組成物を投与する対象（被投与者）及び摂取させる対象（摂取者）は、動物であれば特に限定されないが、通常はヒトである。また、成人、小児、乳児、新生児（低体重児を含む）等のいずれであってもよい。また、性別は特に限定されない。 The subject (administrator) to whom the pharmaceutical compositions of the second to fourth aspects of the present invention are administered (administrator) and the subject (ingester) to be ingested are not particularly limited as long as they are animals, but are usually humans. Moreover, any of adults, children, infants, newborns (including low-weight infants), and the like may be used. Moreover, sex is not specifically limited.
　本発明の医薬品組成物の摂取（投与）時期は、特に限定されず、摂取（投与）対象の状態に応じて適宜選択することが可能である。 The timing of ingestion (administration) of the pharmaceutical composition of the present invention is not particularly limited, and can be appropriately selected according to the condition of the subject of ingestion (administration).
　本発明の医薬品組成物の摂取（投与）量は、摂取（投与）対象の年齢、性別、状態、その他の条件等により適宜選択される。
　本発明の医薬品組成物の摂取（投与）量は、前記細菌及び／又はその培養物の固形分量に換算して、例えば、成人において１００μｇ／日～１０ｇ／日の範囲が好ましく、１ｍｇ／日～１ｇ／日の範囲がより好ましく、１０ｍｇ／日～５００ｍｇ／日がさらに好ましい。また、トレオン酸量に換算して、例えば、成人において１ｎｇ／日～１００μｇ／日の範囲が好ましく、１ｎｇ／日～１μｇ／日の範囲がより好ましく、１０ｎｇ／日～５００ｎｇ／日がさらに好ましい。
　なお、摂取（投与）の量や期間にかかわらず、本発明の医薬品組成物は１日１回又は複数回に分けて摂取（投与）することができる。本発明の医薬品を摂取（投与）するタイミングは、例えば食前、食後、食間、就寝前など特に限定されない。 The intake (administration) amount of the pharmaceutical composition of the present invention is appropriately selected depending on the age, sex, condition, other conditions, etc. of the intake (administration) target.
The ingestion (administration) amount of the pharmaceutical composition of the present invention is preferably in the range of 100 μg/day to 10 g/day, and 1 mg/day to 1 mg/day in terms of solid content of the bacteria and/or culture thereof. A range of 1 g/day is more preferred, and 10 mg/day to 500 mg/day is even more preferred. In terms of threonic acid, for example, the range for adults is preferably 1 ng/day to 100 μg/day, more preferably 1 ng/day to 1 μg/day, and even more preferably 10 ng/day to 500 ng/day.
The pharmaceutical composition of the present invention can be ingested (administered) once a day or divided into multiple times a day, regardless of the amount and duration of ingestion (administration). The timing of ingesting (administering) the drug of the present invention is not particularly limited, such as before meals, after meals, between meals, and before bedtime.
　本発明の医薬品組成物の摂取（投与）期間は、特に限定されない。また、摂取（投与）期間の上限は特に設けられず、継続的な、長期の摂取（投与）が可能である。 The intake (administration) period of the pharmaceutical composition of the present invention is not particularly limited. In addition, there is no particular upper limit for the intake (administration) period, and continuous long-term intake (administration) is possible.
　医薬品の摂取（投与）経路は、経口又は非経口のいずれでもよいが経口が好ましい。また、非経口摂取（投与）としては、経皮、静注、直腸投与、吸入等が挙げられる。
　医薬品の形態としては、摂取（投与）方法に応じて、適宜所望の剤形に製剤化することができる。例えば、経口摂取（投与）の場合、散剤、顆粒剤、錠剤、カプセル剤等の固形製剤；溶液剤、シロップ剤、懸濁剤、乳剤等の液剤等に製剤化することができる。また、非経口摂取（投与）の場合、座剤、軟膏剤、注射剤等に製剤化することができる。
　製剤化に際しては、通常製剤化に用いられている賦形剤、ｐＨ調整剤、着色剤、矯味剤等の成分を用いることができる。また、他の薬効成分や、公知の又は将来的に見出される筋肉増量作用を有する成分等の他の医薬を併用することも可能である。
　加えて、製剤化は剤形に応じて適宜公知の方法により実施できる。製剤化に際しては、適宜、通常製剤化に用いる担体を配合して製剤化してもよい。かかる担体としては、賦形剤、結合剤、崩壊剤、滑沢剤、安定剤、矯味矯臭剤等が挙げられる。 The route of intake (administration) of the drug may be oral or parenteral, but oral is preferred. In addition, parenteral intake (administration) includes percutaneous, intravenous, rectal administration, inhalation, and the like.
As for the pharmaceutical form, it can be appropriately formulated into a desired dosage form depending on the intake (administration) method. For example, in the case of oral ingestion (administration), solid formulations such as powders, granules, tablets and capsules; and liquid formulations such as solutions, syrups, suspensions and emulsions can be formulated. In the case of parenteral intake (administration), it can be formulated into suppositories, ointments, injections, and the like.
For formulation, ingredients such as excipients, pH adjusters, colorants, and corrigents that are commonly used for formulation can be used. In addition, it is also possible to use other medicinal ingredients in combination, such as other medicinal ingredients, ingredients known or discovered in the future that have muscle-enhancing effects.
In addition, formulation can be appropriately carried out by a known method depending on the dosage form. For formulation, a carrier that is usually used for formulation may be blended as appropriate to form the formulation. Such carriers include excipients, binders, disintegrants, lubricants, stabilizers, flavoring agents and the like.
　賦形剤としては、例えば、乳糖、白糖、ブドウ糖、マンニット、ソルビット等の糖誘導体；トウモロコシデンプン、馬鈴薯デンプン、α－デンプン、デキストリン、カルボキシメチルデンプン等のデンプン誘導体；結晶セルロース、ヒドロキシプロピルセルロース、ヒドロキシプロピルメチルセルロース、カルボキシメチルセルロース、カルボキシメチルセルロースカルシウム等のセルロース誘導体；アラビアゴム；デキストラン；プルラン；軽質無水珪酸、合成珪酸アルミニウム、メタ珪酸アルミン酸マグネシウム等の珪酸塩誘導体；リン酸カルシウム等のリン酸塩誘導体；炭酸カルシウム等の炭酸塩誘導体；硫酸カルシウム等の硫酸塩誘導体等が挙げられる。 Excipients include, for example, sugar derivatives such as lactose, sucrose, glucose, mannitol, sorbitol; starch derivatives such as corn starch, potato starch, α-starch, dextrin, carboxymethyl starch; crystalline cellulose, hydroxypropyl cellulose, Hydroxypropylmethylcellulose, carboxymethylcellulose, cellulose derivatives such as carboxymethylcellulose calcium; gum arabic; dextran; pullulan; silicate derivatives such as light silicic anhydride, synthetic aluminum silicate, and magnesium aluminometasilicate; phosphate derivatives such as calcium phosphate; carbonate derivatives such as calcium; sulfate derivatives such as calcium sulfate;
　結合剤としては、例えば、前記賦形剤の他、ゼラチン；ポリビニルピロリドン；マクロゴール等が挙げられる。 Examples of binders include gelatin; polyvinylpyrrolidone; macrogol, etc., in addition to the aforementioned excipients.
　崩壊剤としては、例えば、前記賦形剤の他、クロスカルメロースナトリウム、カルボキシメチルスターチナトリウム、架橋ポリビニルピロリドン等の化学修飾されたデンプン又はセルロース誘導体等が挙げられる。 Examples of disintegrants include, in addition to the excipients mentioned above, chemically modified starch or cellulose derivatives such as croscarmellose sodium, carboxymethyl starch sodium, and crosslinked polyvinylpyrrolidone.
　滑沢剤としては、例えば、タルク；ステアリン酸；ステアリン酸カルシウム、ステアリン酸マグネシウム等のステアリン酸金属塩；コロイドシリカ；ビーガム、ゲイロウ等のワックス類；硼酸；グリコール；フマル酸、アジピン酸等のカルボン酸類；安息香酸ナトリウム等のカルボン酸ナトリウム塩；硫酸ナトリウム等の硫酸塩類；ロイシン；ラウリル硫酸ナトリウム、ラウリル硫酸マグネシウム等のラウリル硫酸塩；無水珪酸、珪酸水和物等の珪酸類；デンプン誘導体等が挙げられる。 Lubricants include, for example, talc; stearic acid; metal stearates such as calcium stearate and magnesium stearate; colloidal silica; waxes such as Veegum and Geiro; ; carboxylic acid sodium salts such as sodium benzoate; sulfates such as sodium sulfate; leucine; lauryl sulfates such as sodium lauryl sulfate and magnesium lauryl sulfate; silicic acid anhydride and silicic acid hydrate; be done.
　安定剤としては、例えば、メチルパラベン、プロピルパラベン等のパラオキシ安息香酸エステル類；クロロブタノール、ベンジルアルコール、フェニルエチルアルコール等のアルコール類；塩化ベンザルコニウム；無水酢酸；ソルビン酸等が挙げられる。 Examples of stabilizers include paraoxybenzoic acid esters such as methylparaben and propylparaben; alcohols such as chlorobutanol, benzyl alcohol and phenylethyl alcohol; benzalkonium chloride; acetic anhydride; and sorbic acid.
　矯味矯臭剤としては、例えば、甘味料、酸味料、香料等が挙げられる。
　なお、経口摂取（投与）用の液剤の場合に使用する担体としては、水等の溶剤等が挙げられる。 Flavoring agents include, for example, sweeteners, acidulants, flavoring agents, and the like.
In the case of liquid formulations for oral ingestion (administration), examples of carriers used include solvents such as water.
　以下に、実施例を用いて本発明をさらに具体的に説明するが、本発明はこれら実施例に限定されるものではない。 The present invention will be described in more detail below using examples, but the present invention is not limited to these examples.
＜試験例１＞トレオン酸による筋肉増量作用の検討
（１）試料の調製
　Ｌ－トレオン酸カルシウム（東京化成工業社製）１５５ｇを９０ｍＬの水に懸濁し、完全に溶解するまで６Ｎの塩酸（ナカライテクス社製）を適量添加した。これを４Ｎの水酸化ナトリウム（ナカライテクス社製）を用いて中和し、水にて１００ｍＬにメスアップして１Ｍのトレオン酸試料を得た。 <Test Example 1> Investigation of muscle bulking action by threonic acid (1) Preparation of sample 155 g of calcium L-threonate (manufactured by Tokyo Kasei Kogyo Co., Ltd.) was suspended in 90 mL of water, and 6N hydrochloric acid (Nacalai (manufactured by Techs Co., Ltd.) was added in an appropriate amount. This was neutralized with 4N sodium hydroxide (manufactured by Nacalai Techs) and diluted to 100 mL with water to obtain a 1M threonic acid sample.
（２）タンパク質合成関連遺伝子活性の評価
　American Type Culture Collection（ATCC）から得たラット筋芽細胞株Ｌ６細胞（以下、「Ｌ６」という）を１．５×１０４ ｃｅｌｌｓ／ｃｍ２になるように６ウェルプレートに播種し、ＤＭＥＭ培地（１０％Fetal Bovine Serum及び１％Penicillin Streptomycin含有）にて、５％ＣＯ２下、３７℃、２４時間培養した。分化誘導のため、ＤＭＥＭ培地（２％Horse Serum及び１％Penicillin Streptomycin含有。以下、「分化用培地」という）を２日毎に新鮮な培地に交換し、７日間培養した。その後、前記（１）で調製した１Ｍのトレオン酸試料を終濃度１又は１０μＭになるように添加し、１時間培養した後、ＲＩＰＡバッファーを用いてライセート調製した。ウェスタンブロッティングによりタンパク質合成関連遺伝子ｐ７０Ｓ６Ｋの活性化（リン酸化）を評価した。具体的には、リン酸化ｐ７０Ｓ６Ｋおよび全ｐ７０Ｓ６Ｋのバンドの蛍光強度をChemiDoc（登録商標） MP Imaging System（Bio-Rad Laboratories社）を用いて測定し、全ｐ７０Ｓ６Ｋにおけるリン酸化ｐ７０Ｓ６Ｋの割合を算出してｐ７０Ｓ６Ｋの活性値とした。
　トレオン酸試料を添加しない以外は前記と同様の手順を行い、コントロールとした。 (2) Evaluation of protein synthesis-related gene activity Rat myoblast cell line L6 cells (hereinafter referred to as “L6”) obtained from the American Type Culture Collection (ATCC) were adjusted to 1.5×10 4 cells/cm 2 . The cells were seeded on a 6-well plate and cultured in DMEM medium (containing 10% Fetal Bovine Serum and 1% Penicillin Streptomycin) under 5% CO 2 at 37° C. for 24 hours. For induction of differentiation, the DMEM medium (containing 2% Horse Serum and 1% Penicillin Streptomycin; hereinafter referred to as "differentiation medium") was replaced with fresh medium every 2 days and cultured for 7 days. Thereafter, the 1 M threonic acid sample prepared in (1) above was added to a final concentration of 1 or 10 μM, cultured for 1 hour, and then a lysate was prepared using RIPA buffer. Activation (phosphorylation) of the protein synthesis-related gene p70S6K was assessed by Western blotting. Specifically, the fluorescence intensity of phosphorylated p70S6K and total p70S6K bands was measured using ChemiDoc (registered trademark) MP Imaging System (Bio-Rad Laboratories), and the ratio of phosphorylated p70S6K to total p70S6K was calculated. The activity value of p70S6K was used.
As a control, the same procedure as described above was performed except that the threonic acid sample was not added.
（３）筋管細胞の太さの評価
　Ｌ６を１．５×１０４ ｃｅｌｌｓ／ｃｍ２になるように１２ウェルプレートに播種し、ＤＭＥＭ培地（１０％Fetal Bovine Serum及び１％Penicillin Streptomycin含有）で、５％ＣＯ２下、３７℃、２４時間培養した。その後、分化用培地であるＤＭＥＭ培地に、前記（１）で調製した１Ｍのトレオン酸試料を終濃度１又は１０μＭになるように添加し、５％ＣＯ２下、３７℃で、７日間培養した。２日毎に新鮮な培地に交換した。この手順により得られた細胞を、以下、「筋管細胞」という。
　トレオン酸試料を添加しない以外は同様の手順を行い、コントロールとした。
　その後、ヘマトキシリン・エオジン染色（ＨＥ染色）を行い、光学顕微鏡 ＢＸ５３（オリンパス社製）を用いて筋管細胞の太さを５０本無作為に測定した。 (3) Evaluation of Myotube Cell Thickness L6 cells were seeded in a 12-well plate at 1.5×10 4 cells/cm 2 and treated with DMEM medium (containing 10% Fetal Bovine Serum and 1% Penicillin Streptomycin). , and cultured at 37° C. for 24 hours under 5% CO 2 . Thereafter, the 1 M threonic acid sample prepared in (1) above was added to DMEM medium, which is a medium for differentiation, to a final concentration of 1 or 10 μM, and cultured for 7 days at 37° C. under 5% CO 2 . . The medium was replaced with fresh medium every 2 days. Cells obtained by this procedure are hereinafter referred to as "myotube cells".
The same procedure was performed except that the threonic acid sample was not added, and used as a control.
Thereafter, hematoxylin and eosin staining (HE staining) was performed, and the thickness of 50 myotube cells was measured at random using an optical microscope BX53 (manufactured by Olympus).
（４）結果
（４－１）タンパク質合成関連遺伝子活性の評価
　トレオン酸を添加したＬ６におけるタンパク質合成関連遺伝子（ｐ７０Ｓ６Ｋ）の活性値を表１および表２に示す。ｐ７０Ｓ６Ｋの活性（リン酸化度）は、コントロールに対して、トレオン酸１μＭ処理群では３．３７倍増加し、トレオン酸１０μＭ処理群では３．０３倍増加した。 (4) Results (4-1) Evaluation of protein synthesis-related gene activity Tables 1 and 2 show the activity values of the protein synthesis-related gene (p70S6K) in L6 to which threonic acid was added. The activity (degree of phosphorylation) of p70S6K increased 3.37-fold in the group treated with 1 μM threonate and increased 3.03-fold in the group treated with 10 μM threonate compared to the control.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
（４－２）筋管細胞の太さの評価
　前記（３）の手順により得られた筋管細胞の直径（平均値）を表３に示す。筋管細胞の直径が、コントロールに対してトレオン酸１μＭ処理群では２．５８μｍ以上、トレオン酸１０μＭ処理群では１．８５μｍ以上太くなり、筋量が増加することが確認された。 (4-2) Evaluation of thickness of myotube cells Table 3 shows the diameters (average values) of myotube cells obtained by the procedure in (3) above. It was confirmed that the diameter of myotube cells increased by 2.58 μm or more in the 1 μM threonic acid-treated group and by 1.85 μm or more in the 10 μM threonic acid-treated group, compared to the control, and muscle mass increased.
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
（４－３）
　以上の結果から、トレオン酸は筋タンパク質合成関連遺伝子を活性化させ、筋肥大促進作用を有することが明らかとなった。 (4-3)
From the above results, it was clarified that threonic acid activates muscle protein synthesis-related genes and has a muscle hypertrophy-promoting effect.
＜試験例２＞プロバイオティクスによる菌体外へのトレオン酸産生の検討
（１）アスコルビン酸含有培地を用いたプロバイオティクスの培養
　Difco Lactobacilli MRS Broth（BD社製）５．５ｇ、及びL-Cysteine Monohydrochloride, Monohydrate（和光純薬工業社製）５０ｍｇを、純水に溶解させ１００ｍＬとした後、塩酸でｐＨ６．５に調整し、１２１℃で１５分間滅菌してＭＲＳ液体培地を調製した。脱脂粉乳１０％を含む水溶液にて凍結保存された状態のビフィドバクテリウム・ブレーベ ＦＥＲＭ ＢＰ－１１１７５、ビフィドバクテリウム・シュードロンガム・サブスピーシーズ・シュードロンガム ＪＣＭ１２０５Ｔ、及びリモシラクトバチルス・ロイテリ ＪＣＭ１１１２Ｔを解凍した後、それぞれ前記ＭＲＳ液体培地に１％（ｖ／ｖ）ずつ播種して、３７℃で嫌気培養した（前培養）。培養１６時間後、前記ＭＲＳ液体培地にＬ－アスコルビン酸（和光純薬工業社製）水溶液を終濃度１ｇ／Ｌとなるように添加したアスコルビン酸含有培地に、前培養液を１％（ｖ／ｖ）ずつ播種して、３７℃で嫌気培養した（本培養）。培養１６時間後、遠心分離により培養上清液を回収した。
　アスコルビン酸を添加しない以外は同様の手順で細菌の培養を行い、コントロールとした。 <Test Example 2> Examination of extracellular threonic acid production by probiotics (1) Culture of probiotics using ascorbic acid-containing medium Difco Lactobacilli MRS Broth (manufactured by BD) 5.5 g and L- 50 mg of Cysteine Monohydrochloride, Monohydrate (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in pure water to make 100 mL, adjusted to pH 6.5 with hydrochloric acid, and sterilized at 121° C. for 15 minutes to prepare an MRS liquid medium. Bifidobacterium breve FERM BP-11175, Bifidobacterium pseudolongum subsp. After thawing JCM1112T, 1% (v/v) of each was seeded in the MRS liquid medium and anaerobically cultured at 37°C (preculture). After 16 hours of culture, 1% (v/ v) were inoculated individually and anaerobically cultured at 37°C (main culture). After 16 hours of culture, the culture supernatant was collected by centrifugation.
Bacteria were cultured in the same manner as a control, except that ascorbic acid was not added.
（２）トレオン酸の同定及び定量
　前記（１）にて回収した培養上清液に対して等量の０．２％ギ酸水を添加し混合してフィルター（φ＝０．２２μｍ、ミリポア社製）処理をした後、トレオン酸含有量を分析した。トレオン酸の分析はＬＣ／ＭＳにより行った。ＨＰＬＣにはＰｒｏｍｉｎｅｎｃｅ（島津製作所社製）を用い、質量分析にはＴＳＱ Ｑｕａｎｔｕｍ Ｄｉｓｃｏｖｅｒｙ  ＭＡＸ（Ｔｈｅｒｍｏ社製）を用いたタンデム質量分析を行った。カラムはＸＢｒｉｄｇｅ　Ｃ１８カラム（Ｗａｔｅｒ社製）を用い、移動相は、０．１％ギ酸水と０．１％ギ酸含有アセトニトリルを用いて、サンプル注入後２５分間に２％から９５％までアセトニトリル濃度を上昇させ、トレオン酸を溶出させた。サンプル注入後７．８分前後に溶出するトレオン酸を、前駆イオンｍ／ｚ＝１３７．０５０、プロダクトイオンｍ／ｚ＝１１９．０４２、衝突エネルギー５．２５ｅＶで測定し、トレオン酸の同定及び定量を行った。なお、トレオン酸の同定及び定量については、標品を用いてＬＣ／ＭＳにて行うことが可能である。 (2) Identification and quantification of threonic acid An equal amount of 0.2% formic acid water is added to the culture supernatant collected in (1) above, mixed and filtered (φ = 0.22 μm, manufactured by Millipore). ) were analyzed for threonic acid content after treatment. Analysis of threonic acid was performed by LC/MS. Tandem mass spectrometry was performed using Prominence (manufactured by Shimadzu Corporation) for HPLC and TSQ Quantum Discovery MAX (manufactured by Thermo) for mass spectrometry. An XBridge C18 column (manufactured by Water) was used as the column, and the mobile phase was acetonitrile containing 0.1% formic acid and 0.1% formic acid. It was raised to elute the threonic acid. Threonic acid eluted around 7.8 minutes after sample injection was measured at precursor ion m/z = 137.050, product ion m/z = 119.042, and collision energy 5.25 eV to identify and quantify threonic acid. did The identification and quantification of threonic acid can be performed by LC/MS using a standard.
（３）結果
　各菌株の培地中のトレオン酸含有量を表４に示す。全ての菌株においてアスコルビン酸添加により培養上清中のトレオン酸量が増加することが確認された。 (3) Results Table 4 shows the threonic acid content in the medium of each strain. It was confirmed that the addition of ascorbic acid increased the amount of threonic acid in the culture supernatant for all strains.
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
＜試験例３＞プロバイオティクスの培養上清液による筋肉増量作用の検討
　アスコルビン酸存在下で培養したプロバイオティクスの培養上清液を用いて、Ｌ６におけるタンパク質合成関連遺伝子（ｐ７０Ｓ６Ｋ）の活性促進を評価し、筋肉増量作用を検討した。
（１）試料の調製
　試験例２の（１）で調製した３種の菌株（ビフィドバクテリウム・ブレーベＦＥＲＭ　ＢＰ－１１１７５、ビフィドバクテリウム・シュードロンガム・サブスピーシーズ・シュードロンガム ＪＣＭ１２０５Ｔ、リモシラクトバチルス・ロイテリ ＪＣＭ１１１２Ｔ）の培養上清液に、水酸化ナトリウムを添加してｐＨを７．０±０．０５となるように調整し、フィルター滅菌した。 <Test Example 3> Investigation of muscle volume-increasing effect by probiotic culture supernatant Using probiotic culture supernatant cultured in the presence of ascorbic acid, promotion of activity of protein synthesis-related gene (p70S6K) in L6 was evaluated, and the muscle volume-increasing effect was examined.
(1) Sample preparation The three strains prepared in Test Example 2 (1) (Bifidobacterium breve FERM BP-11175, Bifidobacterium pseudolongum subspecies pseudolongum JCM1205T, Limo Sodium hydroxide was added to the culture supernatant of Silactobacillus reuteri JCM1112T) to adjust the pH to 7.0±0.05, followed by filter sterilization.
（２）タンパク質合成関連遺伝子活性の評価
　１Ｍのトレオン酸試料を添加する代わりに、前記３種の菌株の培養上清を添加した以外は試験例１の（２）と同様の手順にてＬ６を培養した。
　培養上清を添加しない以外は同様の手順を行い、コントロールとした。なお、コントロールのうちアスコルビン酸添加有の群は、分化用培地に１ｇ／Ｌのアスコルビン酸水溶液を１％（ｖ／ｖ）添加して培養した。
　試験例１と同様に、ｐ７０Ｓ６Ｋの活性値を算出した。 (2) Evaluation of protein synthesis-related gene activity Instead of adding 1M threonic acid sample, L6 was added in the same procedure as in Test Example 1 (2) except that the culture supernatant of the three strains was added. cultured.
As a control, the same procedure was performed except that the culture supernatant was not added. Among the controls, the group with addition of ascorbic acid was cultured by adding 1% (v/v) of 1 g/L ascorbic acid aqueous solution to the medium for differentiation.
The activity value of p70S6K was calculated in the same manner as in Test Example 1.
（３）結果
　各群のＬ６におけるタンパク質合成関連遺伝子（ｐ７０Ｓ６Ｋ）の活性値を表５に示す。いずれの菌株においても、アスコルビン酸存在下で培養した場合の方が、アスコルビン酸非存在下で培養した場合よりもｐ７０Ｓ６Ｋの活性値が大きかった。ビフィドバクテリウム・ブレーベ ＦＥＲＭ ＢＰ－１１１７５、ビフィドバクテリウム・シュードロンガム・サブスピーシーズ・シュードロンガム ＪＣＭ１２０５Ｔ、及びリモシラクトバチルス・ロイテリ ＪＣＭ１１１２Ｔをアスコルビン酸存在下で培養した際の培養上清液には、筋タンパク質合成促進作用があることがわかった。 (3) Results Table 5 shows the activity value of the protein synthesis-related gene (p70S6K) in L6 of each group. In all strains, p70S6K activity values were higher when cultured in the presence of ascorbic acid than when cultured in the absence of ascorbic acid. Culture supernatant of Bifidobacterium breve FERM BP-11175, Bifidobacterium pseudolongum subspecies pseudolongum JCM1205T, and Remocilactobacillus reuteri JCM1112T cultured in the presence of ascorbic acid was found to have a stimulatory effect on muscle protein synthesis.
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
＜試験例４＞プロバイオティクスによる菌体内におけるトレオン酸産生の検討
（１）プロバイオティクスの培養及びトレオン酸の定量
　試験例２と同様の手順にて、表６に示すプロバイオティクスをそれぞれ培養した。培養物を遠心分離をかけて菌体を集菌し、超純水で洗浄した後、１０ｍｇ／ｍＬ（菌湿重量／超純水）の濃度になるように懸濁し、ＦａｓｔＰｒｅｐ－２４ ５Ｇ Ｈｏｍｏｇｅｎｉｚｅｒ（ＭＰ Ｂｉｏｍｅｄｉｃａｌｓ社製)を用いて破砕した。得られた粗破砕液を遠心分離にかけて上清を回収した（以下、「菌体破砕上清液」という）。その後、試験例２と同様の手順にて、菌体破砕上清液中のトレオン酸量の測定を行った。 <Test Example 4> Investigation of intracellular threonic acid production by probiotics (1) Culture of probiotics and quantification of threonic acid In the same procedure as in Test Example 2, each of the probiotics shown in Table 6 was cultured. bottom. The culture was centrifuged to collect the cells, washed with ultrapure water, suspended at a concentration of 10 mg/mL (wet weight of bacteria/ultrapure water), and treated with FastPrep-24 5G Homogenizer ( (manufactured by MP Biomedicals)). The resulting crude lysate was centrifuged to collect the supernatant (hereinafter referred to as "supernatant of cell lysate"). After that, in the same procedure as in Test Example 2, the amount of threonic acid in the supernatant of cell disruption was measured.
（２）結果
　各菌体破砕上清液中のトレオン酸含有量を、表６（アスコルビン酸添加）及び表７（アスコルビン酸未添加）にそれぞれ示す。全ての菌株において、アスコルビン酸添加によってトレオン酸が産生されることが確認された（＞５ｎｇ／ｍＬ）。ビフィドバクテリウム・ビフィダムＪＣＭ１２５５以外の菌株については、いずれも１００ ｎｇ／ｍＬ以上のトレオン酸が産生されることが確認された。アスコルビン酸添加時のトレオン酸産生量は、全菌株の中で、リモシラクトバチルス・ロイテリ ＪＣＭ１１１２Ｔが最も多かった。ビフィドバクテリウム・ビフィダムＪＣＭ１２５５においてはトレオン酸が産生されたものの、産生量は５１．２ｎｇ／ｍＬに留まった。
　アスコルビン酸未添加時は、ビフィドバクテリウム・シュードロンガム・サブスピーシーズ・シュードロンガム ＪＣＭ１２０５Ｔではトレオン酸の産生が認められたが、それ以外の全ての菌株において検出限界（＜５ng／ｍＬ）以下であった。
　前記の結果から、表６に示される細菌において培養の際にアスコルビン酸を添加することで、菌体内に産生されるトレオン酸産生量が顕著に増加することが明らかとなった。 (2) Results Table 6 (ascorbic acid added) and Table 7 (ascorbic acid not added) show the threonic acid content in each supernatant of cell disruption. All strains were confirmed to produce threonic acid (>5 ng/mL) upon addition of ascorbic acid. All of the strains other than Bifidobacterium bifidum JCM1255 were confirmed to produce 100 ng/mL or more of threonic acid. Among all strains, Remosilactobacillus reuteri JCM1112T produced the highest amount of threonic acid when ascorbic acid was added. Although threonic acid was produced in Bifidobacterium bifidum JCM1255, the production amount remained at 51.2 ng/mL.
When no ascorbic acid was added, Bifidobacterium pseudolongum subspecies pseudolongum JCM1205T produced threonic acid, but all other strains were below the detection limit (<5 ng/mL). Met.
From the above results, it was clarified that the addition of ascorbic acid during culture to the bacteria shown in Table 6 markedly increases the amount of threonic acid produced in the cells.
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000007
[規則26に基づく補充 09.12.2022]　
Figure WO-DOC-FIGURE-1

Figure WO-DOC-FIGURE-2

Figure WO-DOC-FIGURE-3
[Correction under Rule 26 09.12.2022]
Figure WO-DOC-FIGURE-1

Figure WO-DOC-FIGURE-2

Figure WO-DOC-FIGURE-3

Claims (10)

  1. 　ビフィドバクテリウム（Bifidobacterium）属細菌、ラクトバチルス（Lactobacillus）属細菌、ラクチカゼイバチルス（Lacticaseibacillus）属細菌、ラクチプランティバチルス（Lactiplantibacillus）属細菌、リモシラクトバチルス（Limosilactobacillus）属細菌、レヴィラクトバチルス（Levilactobacillus）属細菌、リジラクトバチルス（Ligilactobacillus）属細菌、及びラチラクトバチルス(Latilactobacillus)属細菌から選択される一種又は二種以上の細菌を、アスコルビン酸類含有培地で培養する工程、並びに
    　培養後の培養物からトレオン酸含有画分を回収する工程を含む、トレオン酸含有組成物の製造方法。     Bifidobacterium bacteria, Lactobacillus bacteria, Lacticaseibacillus bacteria, Lactiplantibacillus bacteria, Limosilactobacillus bacteria, Leviractobacillus A step of culturing one or more bacteria selected from bacteria of the genus Levilactobacillus, bacteria of the genus Ligilactobacillus, and bacteria of the genus Latilactobacillus in an ascorbic acid-containing medium, and after culturing A method for producing a threonic acid-containing composition, comprising a step of recovering a threonic acid-containing fraction from a culture.
  2. 　前記細菌が、ビフィドバクテリウム・ブレーベ（Bifidobacterium breve）である、請求項１に記載の製造方法。 The production method according to claim 1, wherein the bacterium is Bifidobacterium breve.
  3. 　前記細菌が、ビフィドバクテリウム・ブレーベ ＦＥＲＭ ＢＰ－１１１７５である、請求項２に記載の製造方法。 The production method according to claim 2, wherein the bacterium is Bifidobacterium breve FERM BP-11175.
  4. 　トレオン酸と、
    　ビフィドバクテリウム属細菌、ラクトバチルス属細菌、ラクチカゼイバチルス属細菌、ラクチプランティバチルス属細菌、リモシラクトバチルス属細菌、レヴィラクトバチルス属細菌、リジラクトバチルス属細菌、及びラチラクトバチルス属細菌から選択される一種又は二種以上の細菌、並びに前記細菌の培養物から選択される一種又は二種以上とを含有する、筋肉増量用組成物。     threonic acid;
    Bifidobacterium, Lactobacillus, Lacticaseibacillus, Lactiplantibacillus, Remocilactobacillus, Leviractobacillus, Lydilactobacillus, and Rachilactobacillus and one or more bacteria selected from cultures of said bacteria.
  5. 　ビフィドバクテリウム属細菌、ラクトバチルス属細菌、ラクチカゼイバチルス属細菌、ラクチプランティバチルス属細菌、リモシラクトバチルス属細菌、レヴィラクトバチルス属細菌、リジラクトバチルス属細菌、及びラチラクトバチルス属細菌から選択される一種又は二種以上の細菌、並びに前記細菌の培養物から選択される一種又は二種以上を含有する組成物であって、
    　前記細菌及び／又は前記培養物が、その総乾燥重量あたりトレオン酸を１μｇ／ｇ以上含有する、トレオン酸含有組成物。     Bifidobacterium, Lactobacillus, Lacticaseibacillus, Lactiplantibacillus, Remocilactobacillus, Leviractobacillus, Lydilactobacillus, and Rachilactobacillus A composition containing one or more bacteria selected from and one or more bacteria selected from cultures of said bacteria,
    A threonic acid-containing composition, wherein the bacterium and/or the culture contains 1 μg/g or more of threonic acid per total dry weight thereof.
  6. 　筋肉増量用である、請求項５に記載の組成物。 The composition according to claim 5, which is for increasing muscle mass.
  7. 　前記細菌が、ビフィドバクテリウム・ブレーベである、請求項４～６の何れか一項に記載の組成物。 The composition according to any one of claims 4 to 6, wherein the bacterium is Bifidobacterium breve.
  8. 　前記細菌が、ビフィドバクテリウム・ブレーベ ＦＥＲＭ ＢＰ－１１１７５である、請求項７に記載の組成物。 The composition according to claim 7, wherein the bacterium is Bifidobacterium breve FERM BP-11175.
  9. 　飲食品である、請求項４～８のいずれか一項に記載の組成物。 The composition according to any one of claims 4 to 8, which is a food or drink.
  10. 　医薬品である、請求項４～８のいずれか一項に記載の組成物。 The composition according to any one of claims 4 to 8, which is a pharmaceutical.
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JP2016166176A (en) * 2009-11-12 2016-09-15 ネステク ソシエテ アノニム Nutritional composition for promoting gut microbiota balance and health
JP2019518744A (en) * 2016-05-27 2019-07-04 ネステク ソシエテ アノニム Nutritional composition for treating or preventing movement disorders
WO2019087280A1 (en) * 2017-10-31 2019-05-09 森永乳業株式会社 Muscle-building composition

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Title
KAZUYA TODA, YAMAUCHI YUKI, TANAKA AZUSA, KUHARA TETSUYA, ODAMAKI TOSHITAKA, YOSHIMOTO SHIN, XIAO JIN-ZHONG: "Heat-Killed Bifidobacterium breve B-3 Enhances Muscle Functions: Possible Involvement of Increases in Muscle Mass and Mitochondrial Biogenesis", NUTRIENTS, vol. 12, no. 1, 15 January 2020 (2020-01-15), pages 1 - 13, XP055745828, DOI: 10.3390/nu12010219 *

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