WO2022215568A1 - Muscle synthesis promoting agent, and agent for promoting phosphorylation of p70s6k protein - Google Patents

Muscle synthesis promoting agent, and agent for promoting phosphorylation of p70s6k protein Download PDF

Info

Publication number
WO2022215568A1
WO2022215568A1 PCT/JP2022/014842 JP2022014842W WO2022215568A1 WO 2022215568 A1 WO2022215568 A1 WO 2022215568A1 JP 2022014842 W JP2022014842 W JP 2022014842W WO 2022215568 A1 WO2022215568 A1 WO 2022215568A1
Authority
WO
WIPO (PCT)
Prior art keywords
lipoteichoic acid
muscle
gram
acid
promoting
Prior art date
Application number
PCT/JP2022/014842
Other languages
French (fr)
Japanese (ja)
Inventor
大輔 河田
健 浮辺
泰幸 瀬戸
Original Assignee
雪印メグミルク株式会社
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 雪印メグミルク株式会社 filed Critical 雪印メグミルク株式会社
Priority to JP2023512947A priority Critical patent/JPWO2022215568A1/ja
Publication of WO2022215568A1 publication Critical patent/WO2022215568A1/en

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/116—Heterocyclic compounds
    • A23K20/121—Heterocyclic compounds containing oxygen or sulfur as hetero atom
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • 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
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7032—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a polyol, i.e. compounds having two or more free or esterified hydroxy groups, including the hydroxy group involved in the glycosidic linkage, e.g. monoglucosyldiacylglycerides, lactobionic acid, gangliosides
    • 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
    • A61K35/742—Spore-forming bacteria, e.g. Bacillus coagulans, Bacillus subtilis, clostridium or Lactobacillus sporogenes
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor

Definitions

  • the present invention relates to a muscle synthesis promoter for muscles.
  • the present invention also relates to promoters of p70S6K protein phosphorylation.
  • Muscles are essential organs for human physical movement. Since increasing muscle mass and strengthening muscle strength leads to improvement in athletic performance, it is a matter of great interest to people who play sports regardless of whether they are professionals or amateurs. Also, for people who do not usually exercise, maintaining and improving muscle mass is an important factor in maintaining good health and leading a smooth daily life.
  • Non-Patent Document 1 Whole-body muscle mass is maintained by a balance between muscle synthesis and muscle breakdown. Therefore, preventing muscle atrophy by promoting muscle synthesis and maintaining muscle mass are essential factors for extending healthy life expectancy and improving the QOL of the elderly. Even young people, for example, become temporarily bedridden due to injury or disease, and muscle atrophy easily progresses when the load on muscles decreases, adversely affecting rehabilitation and prognosis. Therefore, preventing muscle atrophy or maintaining muscle mass by promoting muscle synthesis will lead to improved QOL not only for the elderly but also for young people.
  • Patent Document 1 discloses that a physical activity promoter containing Lactobacillus gasseri strain OLL2809 as an active ingredient promotes physical activity and increases muscle mass.
  • Patent Document 2 discloses a lactobacillus strain of the genus Lactobacillus that promotes myoblast proliferation and muscle repair.
  • Patent Document 3 discloses that lipoteichoic acid derived from Lactobacillus carbatus CP2998 strain suppresses muscle degradation.
  • Patent document 1 increases muscle mass as a secondary effect associated with promotion of physical activity, but does not directly promote muscle synthesis.
  • Patent Document 2 also promotes muscle repair of damaged muscles, and does not promote muscle synthesis in a normal, undamaged state.
  • Patent Document 3 discloses that lipoteichoic acid is used as an active ingredient in the same manner as the present invention, but its effect is only to suppress muscle degradation and does not promote differentiation into skeletal muscle. Synthetic acceleration is not disclosed. Also, with regard to muscle degradation inhibition, only the action of lipoteichoic acid derived from a limited strain of lactic acid bacteria has been disclosed. Furthermore, the technique is disclosed as suppressing the degree of muscle breakdown when muscle breakdown is induced, and is intended for patients with advanced muscle atrophy. On the other hand, the present invention is different in that it is effective for muscle cells under normal conditions and is effective not only for sick people but also for healthy people.
  • the present invention is to provide a novel technique that contributes to promoting muscle synthesis and preventing muscle atrophy, characterized by directly promoting muscle synthesis without physical activity.
  • Muscle synthesis is promoted by activation of the Akt-mTOR (mechanistic target of rapamycin) pathway in muscle cells.
  • the p70S6K protein is downstream of the Akt-mTOR pathway and is activated by phosphorylation. Therefore, the amount of phosphorylated p70S6K is used as an indicator of activation of muscle synthesis, and an increase in the amount of phosphorylated p70S6K means activation of muscle synthesis.
  • the inventors have found that adding the cell wall of Gram-positive bacteria to muscle cells increases the amount of phosphorylated p70S6K in muscle cells and promotes muscle synthesis.
  • the present invention relates to the following inventions [1] to [8].
  • [1] A muscle synthesis promoter containing lipoteichoic acid or Gram-positive bacteria containing lipoteichoic acid as an active ingredient.
  • [2] The muscle synthesis promoter according to [1], wherein the lipoteichoic acid is derived from lactic acid bacteria or Bacillus subtilis, or the Gram-positive bacteria containing lipoteichoic acid is lactic acid bacteria or Bacillus subtilis.
  • the lipoteichoic acid is derived from a lactic acid bacterium belonging to the genus Lactobacillus or Bifidobacterium, or the Gram-positive bacterium containing lipoteichoic acid is a lactic acid bacterium belonging to the genus Lactobacillus or Bifidobacterium, [1] ] The muscle synthesis promoter described in ].
  • [6] A method of promoting muscle synthesis by causing lipoteichoic acid or Gram-positive bacteria containing lipoteichoic acid to act on muscle cells, excluding medical procedures.
  • [7] A p70S6K protein phosphorylation promoter containing lipoteichoic acid or Gram-positive bacteria containing lipoteichoic acid as an active ingredient.
  • p70S6K according to [7], wherein the lactic acid bacterium from which lipoteichoic acid is derived is Lactobacillus gasseri or Lactobacillus delbrechskyi, or the lactic acid bacterium containing lipoteichoic acid is Lactobacillus gasseri or Lactobacillus delbrechskyi Promoter of protein phosphorylation.
  • Use of lipoteichoic acid or Gram-positive bacteria containing lipoteichoic acid for the production of a muscle synthesis promoter.
  • Lipoteichoic acid or Gram-positive bacteria containing lipoteichoic acid for use in promoting muscle synthesis.
  • a method for promoting muscle synthesis comprising ingesting or administering an effective amount of lipoteichoic acid or Gram-positive bacteria containing lipoteichoic acid to a subject in need thereof.
  • muscle synthesis is promoted by ingesting Gram-positive bacterial cell walls or lipoteichoic acid, or by directly acting lipoteichoic acid on muscle cells in vivo by a method such as intramuscular injection. can be made In addition, it is possible to increase the muscle mass of livestock by ingesting them as feed.
  • 1 is a photograph of Western blotting using an anti-lipoteichoic acid antibody on a fraction obtained in the process of purifying lipoteichoic acid from lactic acid bacteria.
  • a strong band was observed in the 25% and 35% propanol fractions, and in the 25% fraction of the extraction from Lactobacillus delbrecci, lipoteichoic acid was purified in this fraction.
  • 1 is a Western blot photograph and a quantitative graph showing that Gram-positive bacterial cell walls have an effect of promoting muscle synthesis in Test Example 1.
  • FIG. 1 is a Western blot photograph and a quantitative graph showing that a lipoteichoic acid-purified fraction of Lactobacillus gasseri has an effect of promoting muscle synthesis, according to Test Example 1.
  • FIG. 1 is a Western blot photograph and a quantitative graph showing that a lipoteichoic acid-purified fraction of Lactobacillus delbreeckii has an effect of promoting muscle synthesis, according to Test Example 1.
  • FIG. 2 is a Western blot photograph and a quantitative graph showing that Bacillus subtilis-derived lipoteichoic acid has an effect of promoting muscle synthesis in Test Example 2.
  • FIG. FIG. 10 is a Western blot photograph and a quantitative graph showing that the muscle synthesis-promoting effect is mediated by TLR2, according to Test Example 3.
  • the present invention provides a muscle synthesis promoter containing Gram-positive bacterial cell wall or lipoteichoic acid as an active ingredient.
  • a drug, food, drink, or feed containing gram-positive bacterial cell wall or lipoteichoic acid for promoting muscle synthesis or preventing muscle atrophy, and provide a method for promoting muscle synthesis by causing lipoteichoic acid to act on muscle cells.
  • Lipoteichoic acid is one of the cell wall constituents of Gram-positive bacteria and is generally included in Gram-positive bacteria. Moreover, not only the cell wall but also a part thereof is contained in the cell membrane. Lipoteichoic acid consists of long chains of glycerophosphate and ribitol phosphate. Since Gram-positive bacteria generally contain lipoteichoic acid, lipoteichoic acid can be obtained by extraction from suitable Gram-positive bacteria, and lipoteichoic acid derived from Bacillus subtilis is sold as a commercial reagent. In addition, lactic acid bacteria belong to Gram-positive bacteria. Therefore, it is possible to obtain inexpensive and safe lipoteichoic acid by extracting it from lactic acid bacteria that have been used in yogurt, lactic acid beverages, and the like and have been eaten.
  • the method for extracting and purifying lipoteichoic acid from Gram-positive bacteria is shown below.
  • the medium used for culturing the Gram-positive bacterium is not particularly limited as long as the bacterium can grow, and the bacterium can be cultured by a conventional method.
  • the cultured bacteria can be collected by a method such as centrifugation.
  • the obtained microbial cells may be used as they are, or the microbial cells subjected to concentration, drying, freeze-drying, and crushing treatment may be used. Cells killed by heat drying or the like can be used.
  • After extracting lipoteichoic acid from these cells using water, saline, organic solvents such as butanol, ethyl acetate, chloroform, or a mixture thereof, gel filtration chromatography, hydrophobic chromatography, etc. can be purified by
  • lactic acid bacteria belonging to the genus Lactobacillus or Bifidobacterium, or Bacillus subtilis are preferable, and lactic acid bacteria belonging to the genus Lactobacillus or Bifidobacterium are more preferable.
  • lactic acid bacteria belonging to the genus Lactobacillus include Lactobacillus acidophilus, Lactobacillus amylovorus, Lactobacillus gasseri, Lactobacillus delbrechky, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus paracasei, and Lactobacillus planus.
  • Lactic acid bacteria belonging to the genus Bifidobacterium include Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium longum, Bifidobacterium animalis, Bifidobacterium breve, and Bifidobacterium.
  • Catenulatum Bifidobacterium mongoliense, Bifidobacterium pseudolongum, Bifidobacterium pseudocatenulatum, Bifidobacterium ficari or Bifidobacterium thermophilum are more preferred.
  • Most preferred Gram-positive bacteria are Lactobacillus gasseri SBT1848 (NITE-BP03075) or Lactobacillus delbreeckii SBT2002 (NITE-BP03280).
  • lipoteichoic acid lipoteichoic acid contained in Lactobacillus gasseri SBT1848 (NITE-BP03075) or Lactobacillus delbrecci SBT2002 (NITE-BP03280) is most preferable.
  • the muscle synthesis promoter of the present embodiment may contain lipoteichoic acid, and may be lipoteichoic acid extracted by the method described above, or may be a method using commercially available lipoteichoic acid.
  • a method of using Gram-positive bacteria as they are without performing extraction and purification as lipoteichoic acid may be used.
  • Gram-positive bacteria When Gram-positive bacteria are used as they are, the Gram-positive bacteria may be concentrated, dried, freeze-dried, or crushed, or killed by heat drying or the like. Therefore, it can be widely used as a composition contained in pharmaceuticals, quasi-drugs, food and drink, feed, and the like.
  • the muscle synthesis promoter of this embodiment can be in any form as long as the effects of the present invention can be obtained. That is, it may be in the form of fermented milk, or in other dosage forms.
  • Dosage forms include solid or powder formulations such as powders, fine granules, granules, tablets, capsules, and pills, and liquid formulations such as suspensions, emulsions, syrups, and extracts. can be mentioned.
  • the administration subject and the daily intake of the agent for preventing muscle atrophy according to the present embodiment are not particularly limited. It can be administered to elderly people, people under the age of 65, and the like. It can also be administered to subjects who have already developed locomotive syndrome or sarcopenia for treatment or alleviation, and can also be administered to healthy subjects who do not yet have such symptoms for prophylaxis.
  • the daily intake is also not particularly limited because it varies depending on age, symptoms, body weight, and purpose. 5g.
  • locomotive syndrome refers to the functional impairment of one or more locomotive organs such as bones, joints, cartilage, intervertebral discs, and muscles due to aging, resulting in decreased functions such as “standing” and “walking”. It means the state of being
  • sarcopenia refers to a syndrome characterized by age-related skeletal muscle atrophy, accompanied by a decrease in skeletal muscle mass and skeletal muscle strength, or a decrease in physical function.
  • the terms “promote muscle synthesis” or “promote muscle synthesis” refer to cases in which the agent for promoting muscle synthesis of the present invention is ingested by the subject, and cases in which the agent for promoting muscle synthesis of the present invention is not ingested by the subject. In comparison, it means that there is more synthesis of skeletal muscle. Whether “promoting muscle synthesis” or “promoting muscle synthesis” means that phosphorylation of the p70S6K protein is promoted when the active ingredient is administered to the subject, compared to the subject to which the active ingredient is not administered.
  • Examples of the myosynthesis promoter of the present invention include “maintain muscle”, “inhibit muscle atrophy”, “prevent muscle weakening”, “suppress muscle weakening”, “build muscle”, “ Increases muscle”, “builds muscle”, “promotes muscle synthesis”, “supports the ability to build muscle”, “helps prevent loss of muscle mass and strength”, “maintains muscle that declines with age” It is possible to display functions that are useful for” or that can be equated with these.
  • the above-mentioned “indication” includes everything that informs consumers of the above functions, and the object to be displayed is regardless of the medium such as the muscle synthesis promoter itself, packaging, containers, catalogs, pamphlets, and Internet homepages. .
  • p70S6K protein phosphorylation accelerator As used herein, "p70S6K protein” means the p70 ribosomal S6 kinase protein.
  • p70S6K p70 ribosomal protein S6 kinase
  • the p70S6K protein is downstream of the Akt-mTOR pathway and is activated by phosphorylation.
  • the p70S6K protein phosphorylation promoting agent of the present invention can be labeled with a function that is exhibited by promoting p70S6K protein phosphorylation.
  • the food or drink for promoting muscle synthesis or preventing muscle atrophy of the present invention may be any food or drink containing lipoteichoic acid or Gram-positive bacteria containing lipoteichoic acid, and may be general food or drink, or food for specified health use. , foods with function claims, dietary supplements, and supplements. Lactic acid beverages, cheese, yogurt and the like are examples of foods and drinks containing Gram-positive bacteria containing lipoteichoic acid.
  • Specific examples of the food and drink for promoting muscle synthesis or preventing muscle atrophy of the present invention include carbonated drinks, various fruit juices, fruit juice drinks, soft drinks containing fruit juice, fruit drinks, fruit drinks containing fruit grains, and vegetables including various vegetables.
  • beverages soymilk/soymilk drinks, coffee drinks, tea drinks, powdered drinks, concentrated drinks, sports drinks, nutritional drinks, and other alcoholic beverages; caramel/candy, chewing gum, chocolate, cookies/biscuits, cakes/pies, snacks Confectionery such as crackers, Japanese confectionery, rice confectionery, bean confectionery, dessert confectionery; Butter, margarine, mayonnaise, vegetable oil and other fats and oils; Milk, processed milk, milk drinks, yogurts, lactic acid beverages, cheese, ice cream Milk and dairy products such as powdered milk, cream, processed agricultural products such as cereals (processed grains), baby food, and other commercially available foods such as liquid diet.
  • the food and drink for promoting muscle synthesis or preventing muscle atrophy of the present invention may optionally contain antioxidants, flavoring agents, acidulants, coloring agents, emulsifiers, preservatives, seasonings, sweeteners, spices, pH adjusters, and stabilizers. agents, vegetable oils, animal oils, sugars and sugar alcohols, vitamins, organic acids, fruit juice extracts, vegetable extracts, grains, beans, vegetables, meats, seafood, and other additives and materials alone or in combination of two or more. can be blended together. The blending amounts of these materials and additives can be determined as appropriate.
  • the form of the food and drink for promoting muscle synthesis or preventing muscle atrophy of the present invention is not particularly limited. , semi-liquid, gel form, solid, bar, powder form.
  • the amount of lipoteichoic acid for promoting muscle synthesis or preventing muscle atrophy in these foods and drinks is not particularly limited because it varies depending on the form, dosage form, symptoms, body weight, application, etc. of the subject to be administered. It can be blended at, for example, 0.001 to 100 (w/w)%, 0.01 to 10 (w/w), or 0.1 to 1 (w/w)% on the basis of food and drink.
  • the above compounding amount is the compounding amount as lipoteichoic acid.
  • the feed for promoting muscle synthesis or preventing atrophy may be obtained by mixing the above-mentioned active ingredient with a normal feed, or by administering lipoteichoic acid or Gram-positive bacteria containing lipoteichoic acid.
  • the present embodiment can provide a novel muscle synthesis promoter.
  • This muscle synthesis accelerator contains lipoteichoic acid, which is generally contained in Gram-positive bacteria, as an active ingredient. If positive bacteria are used, highly safe products can be provided.
  • the mixture was separated into an aqueous layer, an intermediate layer and a butanol layer.
  • the aqueous layer was collected and the solvent was removed by lyophilization.
  • the resulting extract was dissolved in 5 mL of 15% propanol/0.1 M sodium acetate aqueous solution to obtain a crude lipoteichoic acid fraction.
  • the lipoteichoic acid crude fraction was then centrifuged and the supernatant filtered through a 0.22 ⁇ m filter.
  • a 1 mL Hitrap Octyl FF column (GE Healthcare) was equilibrated with a 15% propanol/sodium acetate aqueous solution, and 1 mL of the crude fraction supernatant was applied to the column.
  • lipoteichoic acid was purified to fractions eluted with 25% and 35% propanol/sodium acetate aqueous solutions in the extraction from Lactobacillus gasseri, and in the 25% fraction in the extraction from Lactobacillus delbrecci. (Fig. 1).
  • each fraction obtained in the process of lipoteichoic acid purification from lactic acid bacteria cell walls or strains was added.
  • the cells were washed with PBS and lysed using Alpha SureFire Ultra Lysis Buffer (PerkinElmer).
  • the cell lysate was collected and centrifuged to obtain the supernatant to obtain the sample for subsequent Western blotting.
  • Each sample was mixed with Laemmli sample buffer and heated at 90°C for 10 minutes for denaturation. The heated sample was applied to a 4-20% Criterion TGX gel (Bio-Rad) and electrophoresed at 200V constant pressure for 40 minutes.
  • the membrane was washed with TBST, immersed in HRP-labeled secondary antibody (Anti-rabbit IgG HRP-linked Antibody; Cell Signaling Technology) diluted 10,000 times with blocking buffer, and shaken for 1 hour. After secondary antibody reaction, the membrane was washed with TBST and reacted for 5 minutes using ECL Prime Western Blotting Detection Reagent or Clarity Max Western ECL Substrate (Bio-Rad). After completion of the reaction, signals were detected with Chemi Doc MP (Bio-Rad). After quantifying the signal intensity, the muscle synthesis promoting effect was evaluated by dividing the phosphorylated p70S6K value by the total p70S6K value.
  • HRP-labeled secondary antibody Anti-rabbit IgG HRP-linked Antibody; Cell Signaling Technology
  • propanol fractions which are purified fractions of lactic acid bacteria cell walls and lipoteichoic acid, were found to have muscle synthesis promoting activity (Figs. 2, 3 and 4). Since lipoteichoic acid is one of the cell wall constituents, it is contained in both the sediment (cell wall fraction) and the supernatant (soluble fraction) after centrifugation of the disrupted cells.
  • the control group was supplemented with the supernatant of the disrupted bacterial cells, which was found to have muscle synthesis-promoting activity. After recovering the cells, Western blotting was performed to evaluate the effect of promoting muscle synthesis. As a result, Bacillus subtilis-derived lipoteichoic acid was found to promote muscle synthesis at concentrations of 100 ng/mL or higher (Fig. 5).
  • TLR2 Toll-like receptor 2
  • TLR2 Toll-like receptor 2
  • muscle synthesis can be promoted or muscle atrophy can be prevented by Gram-positive bacterial cell walls or lipoteichoic acid.
  • muscle strength can be maintained and improved, healthy life expectancy can be extended, and QOL can be improved.
  • SBT1848 strain B Name and address of the depositary institution that deposited the biological material National Institute of Technology and Evaluation Patent Microorganism Depositary Center (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture (zip code 292-0818) ) Date of depositing the biological material at Roy's depository: November 25, 2019 (original date of deposit) September 10, 2021 (the date of transfer from the original deposit to the deposit under the Budapest Treaty) Accession number NITE BP-03075 assigned to the deposit by the depositary institution of Hai (2) SBT2002 strain A.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Microbiology (AREA)
  • Engineering & Computer Science (AREA)
  • Mycology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Polymers & Plastics (AREA)
  • Molecular Biology (AREA)
  • Zoology (AREA)
  • Epidemiology (AREA)
  • Organic Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Nutrition Science (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Animal Husbandry (AREA)
  • Physiology (AREA)
  • General Engineering & Computer Science (AREA)
  • Virology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Neurology (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Physical Education & Sports Medicine (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

The purpose of the present invention is to provide a novel technique for directly promoting muscle synthesis without physical activity and preventing muscle atrophy.　Provided is: a muscle synthesis promoting agent or muscle atrophy preventing agent that contains a gram-positive bacterial cell wall or lipoteichoic acid as an active ingredient; or a method for promoting muscle synthesis by allowing a gram-positive bacterial cell wall or lipoteichoic acid to act on muscle cells.

Description

筋合成促進剤及びｐ７０Ｓ６Ｋタンパク質のリン酸化促進剤Muscle synthesis promoter and p70S6K protein phosphorylation promoter
　本発明は、筋肉の筋合成促進剤に関する。本発明は、またp70S6Kタンパク質のリン酸化促進剤に関する。 The present invention relates to a muscle synthesis promoter for muscles. The present invention also relates to promoters of p70S6K protein phosphorylation.
　筋肉はヒトの身体運動に必須の器官である。筋量を増加させ、筋力を強化することは、運動能力の向上につながることから、プロやアマチュアを問わずスポーツを行う人々にとって関心が高い事柄である。また、普段運動を行わない人々にとっても、筋量の維持、向上は、健康を維持し、円滑な日常生活を営む上で重要な要素である。 Muscles are essential organs for human physical movement. Since increasing muscle mass and strengthening muscle strength leads to improvement in athletic performance, it is a matter of great interest to people who play sports regardless of whether they are professionals or amateurs. Also, for people who do not usually exercise, maintaining and improving muscle mass is an important factor in maintaining good health and leading a smooth daily life.
　また、超高齢化が進展する日本において、平均寿命と健康寿命の乖離は喫緊の課題となっている。とりわけ加齢に伴う運動器の機能低下、いわゆるロコモティブシンドロームは高齢者のQOL（Quality of Life）を低下させる大きな要因の一つであり、介護負担や医療費増大の観点からも大きな問題となっている。運動器の内、特に筋肉は、筋肉自身が持つ運動機能に加え、衝撃吸収作用や姿勢安定化作用を有しており、他の運動器の障害を予防するという観点からも重要な運動器である。しかし、筋量は30歳頃から減少を開始し、全身の骨格筋量が低下した状態であるサルコペニアに至るヒトの割合は加齢に伴って増大する。その割合は75歳～79歳の高齢者で25%程度、80歳以上では35%を超えるとする報告もなされている（非特許文献１）。全身の筋量は筋合成と筋分解のバランスによって維持されている。そのため、筋合成を促進することで筋委縮を予防し、筋量を維持することは、健康寿命を延伸し、高齢者のQOLを向上させるうえで必要不可欠な要素である。
　若年者であっても、例えば外傷や疾病によって一時的な寝たきり状態となり筋肉への負荷が低下すると、筋委縮は容易に進行し、リハビリテーションや予後に悪影響を及ぼす。したがって、筋合成を促進することで筋委縮を予防し、もしくは筋量を維持することは高齢者に限らず若年者のQOL向上にもつながる。 In Japan, where the population is rapidly aging, the gap between average life expectancy and healthy life expectancy is becoming an urgent issue. In particular, age-related declines in locomotor function, so-called locomotive syndrome, are one of the major factors in lowering the quality of life (QOL) of the elderly, and have also become a major problem from the perspective of increasing nursing care burden and medical expenses. there is Among locomotor organs, muscles in particular have not only their own motor functions, but also shock-absorbing and posture-stabilizing effects. be. However, muscle mass begins to decrease around the age of 30, and the proportion of humans who develop sarcopenia, which is a state in which the skeletal muscle mass of the whole body is reduced, increases with age. It has also been reported that the rate is about 25% in elderly people aged 75 to 79, and exceeds 35% in people aged 80 and over (Non-Patent Document 1). Whole-body muscle mass is maintained by a balance between muscle synthesis and muscle breakdown. Therefore, preventing muscle atrophy by promoting muscle synthesis and maintaining muscle mass are essential factors for extending healthy life expectancy and improving the QOL of the elderly.
Even young people, for example, become temporarily bedridden due to injury or disease, and muscle atrophy easily progresses when the load on muscles decreases, adversely affecting rehabilitation and prognosis. Therefore, preventing muscle atrophy or maintaining muscle mass by promoting muscle synthesis will lead to improved QOL not only for the elderly but also for young people.
　以上のように、筋合成を促進することで、筋量を増加させ、維持し、又は筋委縮を予防することは幅広い層の人々にとって有用な技術となりうる。 As described above, increasing and maintaining muscle mass or preventing muscle atrophy by promoting muscle synthesis can be a useful technique for a wide range of people.
　さらに、家畜の筋合成を促進することができれば、家畜の肉付きを向上させ、あるいは運動能力を向上させることが可能となることから、筋合成を促進する技術は畜産にとっても有用な技術となりうる。 Furthermore, if it is possible to promote muscle synthesis in livestock, it will be possible to improve the fleshiness of livestock or improve their athletic performance, so technology that promotes muscle synthesis can also be a useful technology for livestock.
　ここで、筋量を増加させ、又は筋委縮を予防する方法として、筋力トレーニングを行い、それに加え、筋肉の原料となるタンパク質や、筋肉の合成シグナルを刺激する分岐鎖アミノ酸を摂取することが一般に推奨されている。また、以下に記載の技術が存在する。例えば特許文献１には、ラクトバチルス・ガセリ OLL2809株を有効成分とする身体活動促進剤によって、身体活動が促進され、筋肉量が増加することについて開示されている。
　特許文献２には、筋芽細胞の増殖を促進し筋修復を促進するラクトバチルス属の乳酸菌株が開示されている。特許文献３には、ラクトバチルス・カルバタスCP2998株に由来するリポテイコ酸が筋分解を抑制することが開示されている。 Here, as a method for increasing muscle mass or preventing muscle atrophy, it is common to perform muscle strength training and, in addition, to ingest protein that is the raw material for muscle and branched-chain amino acids that stimulate muscle synthesis signals. Recommended. Also, there is a technique described below. For example, Patent Document 1 discloses that a physical activity promoter containing Lactobacillus gasseri strain OLL2809 as an active ingredient promotes physical activity and increases muscle mass.
Patent Document 2 discloses a lactobacillus strain of the genus Lactobacillus that promotes myoblast proliferation and muscle repair. Patent Document 3 discloses that lipoteichoic acid derived from Lactobacillus carbatus CP2998 strain suppresses muscle degradation.
　しかし、高齢者や寝たきり状態の患者が継続的に筋力トレーニングに取り組むことは現実的ではない。また、タンパク質は筋合成においては専ら原料として働くのであって、タンパク質の摂取量を増やしたとしても、筋細胞における筋合成促進経路が活性化しなければその効果は限定的である。とりわけ高齢者においては分岐鎖アミノ酸に対する筋合成応答が減弱するとされており、筋委縮を予防するためには別途筋合成促進経路を活性化させる必要がある。
　特許文献１は身体活動の促進に伴う副次的な効果として筋肉量を増加させるものであるが、直接筋合成を促進するものではない。特許文献２についても、損傷した筋の筋修復を促進させるものであって、損傷を受けていない正常な状態において筋合成を促進するものではない。
　特許文献３は、本発明と同様にリポテイコ酸を有効成分とするものであるが、その効果は筋分解を抑制するのみで、骨格筋への分化を促進しないことが開示されている一方、筋合成の促進については開示されていない。また、筋分解抑制についても、限定的な1菌株の乳酸菌に由来するリポテイコ酸の作用が開示されているのみである。さらに、当該技術は筋分解を誘導した際に、その筋分解の程度を抑制するものとして開示されており、筋委縮が進行した病者を対象とするものである。一方、本発明は正常な状態での筋細胞に対して効果が認められ、病者のみならず健常人にも有効である点で異なっている。 However, it is not realistic for the elderly and bedridden patients to continuously engage in strength training. In addition, since protein acts exclusively as a raw material in muscle synthesis, even if the intake of protein is increased, its effect is limited unless the muscle synthesis-promoting pathway in muscle cells is activated. Especially in elderly people, the muscle synthesis response to branched chain amino acids is said to be attenuated, and in order to prevent muscle atrophy, it is necessary to activate the muscle synthesis promotion pathway separately.
Patent document 1 increases muscle mass as a secondary effect associated with promotion of physical activity, but does not directly promote muscle synthesis. Patent Document 2 also promotes muscle repair of damaged muscles, and does not promote muscle synthesis in a normal, undamaged state.
Patent Document 3 discloses that lipoteichoic acid is used as an active ingredient in the same manner as the present invention, but its effect is only to suppress muscle degradation and does not promote differentiation into skeletal muscle. Synthetic acceleration is not disclosed. Also, with regard to muscle degradation inhibition, only the action of lipoteichoic acid derived from a limited strain of lactic acid bacteria has been disclosed. Furthermore, the technique is disclosed as suppressing the degree of muscle breakdown when muscle breakdown is induced, and is intended for patients with advanced muscle atrophy. On the other hand, the present invention is different in that it is effective for muscle cells under normal conditions and is effective not only for sick people but also for healthy people.
特開2016-84358JP 2016-84358 国際公開2019-230957International publication 2019-230957 特許6762855号公報Patent No. 6762855
　本発明は、身体活動を介さずに直接筋合成を促進させることを特徴とする、筋合成促進及び筋委縮予防に資する新規な技術を提供することである。 The present invention is to provide a novel technique that contributes to promoting muscle synthesis and preventing muscle atrophy, characterized by directly promoting muscle synthesis without physical activity.
　筋合成は筋細胞内におけるAkt-mTOR (mechanistic target of rapamycin)経路の活性化によって促進される。p70S6Kタンパク質はAkt-mTOR経路の下流にあり、リン酸化によって活性化される。したがってリン酸化p70S6K量が筋合成活性化の指標として用いられており、リン酸化p70S6K量の増大は筋合成の活性化を意味する。
　発明者らは鋭意研究の結果、グラム陽性菌の細胞壁を筋細胞に添加すると、筋細胞のリン酸化p70S6K量が増加し筋合成が促進されること、及びグラム陽性菌の細胞壁構成成分の１つであるリポテイコ酸を筋細胞に添加すると、筋細胞のリン酸化p70S6K量が増加し筋合成が促進されることを見出した。さらに、この効果がリポテイコ酸一般を認識する細胞表面上の受容体タンパク質TLR2(Toll-like receptor 2)を介して惹起されるものであり、リポテイコ酸の由来する菌種に依存しないことを見出すことで、本発明を完成させた。 Muscle synthesis is promoted by activation of the Akt-mTOR (mechanistic target of rapamycin) pathway in muscle cells. The p70S6K protein is downstream of the Akt-mTOR pathway and is activated by phosphorylation. Therefore, the amount of phosphorylated p70S6K is used as an indicator of activation of muscle synthesis, and an increase in the amount of phosphorylated p70S6K means activation of muscle synthesis.
As a result of intensive research, the inventors have found that adding the cell wall of Gram-positive bacteria to muscle cells increases the amount of phosphorylated p70S6K in muscle cells and promotes muscle synthesis. It was found that the addition of lipoteichoic acid to muscle cells increased the amount of phosphorylated p70S6K in muscle cells and promoted muscle synthesis. Furthermore, to discover that this effect is mediated by the receptor protein TLR2 (Toll-like receptor 2) on the cell surface that recognizes lipoteichoic acid in general, and does not depend on the bacterial species from which lipoteichoic acid is derived. Thus, the present invention was completed.
　本発明は以下[１]～[８]の発明に係るものである。
[１]　リポテイコ酸、又はリポテイコ酸を含有するグラム陽性細菌を有効成分とする筋合成促進剤。
[２]　リポテイコ酸が乳酸菌もしくは枯草菌に由来し、又はリポテイコ酸を含有するグラム陽性細菌が乳酸菌もしくは枯草菌である、[１]に記載の筋合成促進剤。
[３]　リポテイコ酸が、ラクトバチルス属もしくはビフィドバクテリウム属に属する乳酸菌に由来し、又はリポテイコ酸を含有するグラム陽性細菌がラクトバチルス属もしくはビフィドバクテリウム属に属する乳酸菌である、[１]に記載の筋合成促進剤。
[４]　リポテイコ酸の由来する乳酸菌がラクトバチルス・ガセリもしくはラクトバチルス・デルブレッキーであり、又はリポテイコ酸を含有する乳酸菌がラクトバチルス・ガセリもしくはラクトバチルス・デルブレッキーである[１]に記載の筋合成促進剤。
[５]　リポテイコ酸又はリポテイコ酸を含有するグラム陽性細菌を含有し筋合成を促進させることを特徴とする筋合成促進用又は筋委縮予防用医薬品、飲食品及び飼料。
[６]　リポテイコ酸又はリポテイコ酸を含有するグラム陽性細菌を筋細胞に作用させることによって筋合成を促進する方法であって医療行為を除くもの。
[７]　リポテイコ酸、又はリポテイコ酸を含有するグラム陽性細菌を有効成分とする、p70S6Kタンパク質のリン酸化促進剤。
[８]　リポテイコ酸が乳酸菌もしくは枯草菌に由来し、又はリポテイコ酸を含有するグラム陽性細菌が乳酸菌もしくは枯草菌である[７]に記載のp70S6Kタンパク質のリン酸化促進剤。
[９]　リポテイコ酸の由来する乳酸菌がラクトバチルス・ガセリもしくはラクトバチルス・デルブレッキーであり、又はリポテイコ酸を含有する乳酸菌がラクトバチルス・ガセリもしくはラクトバチルス・デルブレッキーである[７]に記載のp70S6Kタンパク質のリン酸化促進剤。
[１０]　筋合成促進剤の製造のための、リポテイコ酸、又はリポテイコ酸を含有するグラム陽性細菌の使用。
[１１]　筋合成促進に使用するための、リポテイコ酸、又はリポテイコ酸を含有するグラム陽性細菌。
[１２]　有効量のリポテイコ酸、又はリポテイコ酸を含有するグラム陽性細菌を、それを必要としている対象に摂取させるか、又は投与することを含む、筋合成促進方法。 The present invention relates to the following inventions [1] to [8].
[1] A muscle synthesis promoter containing lipoteichoic acid or Gram-positive bacteria containing lipoteichoic acid as an active ingredient.
[2] The muscle synthesis promoter according to [1], wherein the lipoteichoic acid is derived from lactic acid bacteria or Bacillus subtilis, or the Gram-positive bacteria containing lipoteichoic acid is lactic acid bacteria or Bacillus subtilis.
[3] The lipoteichoic acid is derived from a lactic acid bacterium belonging to the genus Lactobacillus or Bifidobacterium, or the Gram-positive bacterium containing lipoteichoic acid is a lactic acid bacterium belonging to the genus Lactobacillus or Bifidobacterium, [1] ] The muscle synthesis promoter described in ].
[4] The muscle according to [1], wherein the lactic acid bacterium from which lipoteichoic acid is derived is Lactobacillus gasseri or Lactobacillus delbrechskyi, or the lactic acid bacterium containing lipoteichoic acid is Lactobacillus gasseri or Lactobacillus delbrechskyi Synthetic accelerator.
[5] A drug, food, drink and feed for promoting muscle synthesis or preventing muscle atrophy, characterized by containing lipoteichoic acid or Gram-positive bacteria containing lipoteichoic acid and promoting muscle synthesis.
[6] A method of promoting muscle synthesis by causing lipoteichoic acid or Gram-positive bacteria containing lipoteichoic acid to act on muscle cells, excluding medical procedures.
[7] A p70S6K protein phosphorylation promoter containing lipoteichoic acid or Gram-positive bacteria containing lipoteichoic acid as an active ingredient.
[8] The p70S6K protein phosphorylation promoter of [7], wherein the lipoteichoic acid is derived from lactic acid bacteria or Bacillus subtilis, or the Gram-positive bacterium containing lipoteichoic acid is lactic acid bacteria or Bacillus subtilis.
[9] p70S6K according to [7], wherein the lactic acid bacterium from which lipoteichoic acid is derived is Lactobacillus gasseri or Lactobacillus delbrechskyi, or the lactic acid bacterium containing lipoteichoic acid is Lactobacillus gasseri or Lactobacillus delbrechskyi Promoter of protein phosphorylation.
[10] Use of lipoteichoic acid or Gram-positive bacteria containing lipoteichoic acid for the production of a muscle synthesis promoter.
[11] Lipoteichoic acid or Gram-positive bacteria containing lipoteichoic acid for use in promoting muscle synthesis.
[12] A method for promoting muscle synthesis, comprising ingesting or administering an effective amount of lipoteichoic acid or Gram-positive bacteria containing lipoteichoic acid to a subject in need thereof.
　筋合成を促進することで筋委縮を予防し筋量を維持することは高齢者に限らず、若年者のQOL向上にもつながる。また、筋合成を促進することで筋量を増加させることは一般の健常人やスポーツに取り組む人々にとっても有用である。本発明によれば、グラム陽性菌細胞壁もしくはリポテイコ酸を摂取することによって筋合成を促進させ、又は生体内の筋細胞に筋肉注射等の方法によって直接リポテイコ酸を作用させることによって、筋合成を促進させることができる。
　また、これらを飼料として家畜に摂取させることで家畜の筋量増加を図ることができる。 Preventing muscle atrophy and maintaining muscle mass by promoting muscle synthesis leads to improved QOL not only for the elderly but also for young people. In addition, increasing muscle mass by promoting muscle synthesis is also useful for healthy people in general and people who engage in sports. According to the present invention, muscle synthesis is promoted by ingesting Gram-positive bacterial cell walls or lipoteichoic acid, or by directly acting lipoteichoic acid on muscle cells in vivo by a method such as intramuscular injection. can be made
In addition, it is possible to increase the muscle mass of livestock by ingesting them as feed.
乳酸菌からリポテイコ酸を精製する過程で得られた画分に抗リポテイコ酸抗体を用いてウエスタンブロットを実施した写真である。ラクトバチルス・ガセリからの抽出ではプロパノール濃度25%、35%の画分に、ラクトバチルス・デルブレッキーからの抽出では25%の画分に濃いバンドが認められ、リポテイコ酸がこの画分に精製されていることを示している。1 is a photograph of Western blotting using an anti-lipoteichoic acid antibody on a fraction obtained in the process of purifying lipoteichoic acid from lactic acid bacteria. In the extraction from Lactobacillus gasseri, a strong band was observed in the 25% and 35% propanol fractions, and in the 25% fraction of the extraction from Lactobacillus delbrecci, lipoteichoic acid was purified in this fraction. indicates that 試験例１に係り、グラム陽性菌細胞壁が筋合成促進効果を有することを示したウエスタンブロット写真と定量グラフである。1 is a Western blot photograph and a quantitative graph showing that Gram-positive bacterial cell walls have an effect of promoting muscle synthesis in Test Example 1. FIG. 試験例１に係り、ラクトバチルス・ガセリのリポテイコ酸精製画分が筋合成促進効果を有することを示したウエスタンブロット写真と定量グラフである。1 is a Western blot photograph and a quantitative graph showing that a lipoteichoic acid-purified fraction of Lactobacillus gasseri has an effect of promoting muscle synthesis, according to Test Example 1. FIG. 試験例１に係り、ラクトバチルス・デルブレッキーのリポテイコ酸精製画分が筋合成促進効果を有することを示したウエスタンブロット写真と定量グラフである。1 is a Western blot photograph and a quantitative graph showing that a lipoteichoic acid-purified fraction of Lactobacillus delbreeckii has an effect of promoting muscle synthesis, according to Test Example 1. FIG. 試験例２に係り、枯草菌由来リポテイコ酸が筋合成促進効果を有することを示したウエスタンブロット写真と定量グラフである。2 is a Western blot photograph and a quantitative graph showing that Bacillus subtilis-derived lipoteichoic acid has an effect of promoting muscle synthesis in Test Example 2. FIG. 試験例３に係り、筋合成促進効果がTLR2を介していることを示したウエスタンブロット写真と定量グラフである。FIG. 10 is a Western blot photograph and a quantitative graph showing that the muscle synthesis-promoting effect is mediated by TLR2, according to Test Example 3. FIG.
　本発明はグラム陽性菌細胞壁又はリポテイコ酸を有効成分とする筋合成促進剤を提供する。また、グラム陽性菌細胞壁又はリポテイコ酸を含有する筋合成促進用又は筋委縮予防用の医薬品、飲食品又は飼料を提供するとともに、リポテイコ酸を筋細胞に作用させることで筋合成を促進する方法を提供する。 The present invention provides a muscle synthesis promoter containing Gram-positive bacterial cell wall or lipoteichoic acid as an active ingredient. In addition, we provide a drug, food, drink, or feed containing gram-positive bacterial cell wall or lipoteichoic acid for promoting muscle synthesis or preventing muscle atrophy, and provide a method for promoting muscle synthesis by causing lipoteichoic acid to act on muscle cells. offer.
　リポテイコ酸はグラム陽性菌の細胞壁構成成分の１つであって、グラム陽性菌一般に含まれる。また、細胞壁のみならず一部は細胞膜にも含まれる。リポテイコ酸はグリセロリン酸やリビトールリン酸の長鎖からなる。グラム陽性菌であれば一般にリポテイコ酸を有しているため、適当なグラム陽性菌から抽出することでリポテイコ酸を得ることができるほか、市販試薬として枯草菌由来のリポテイコ酸が販売されている。
　また、乳酸菌はグラム陽性菌に属している。したがって、ヨーグルトや乳酸菌飲料等に利用され喫食経験のある乳酸菌等から抽出することによって、安価かつ安全なリポテイコ酸を得ることができる。 Lipoteichoic acid is one of the cell wall constituents of Gram-positive bacteria and is generally included in Gram-positive bacteria. Moreover, not only the cell wall but also a part thereof is contained in the cell membrane. Lipoteichoic acid consists of long chains of glycerophosphate and ribitol phosphate. Since Gram-positive bacteria generally contain lipoteichoic acid, lipoteichoic acid can be obtained by extraction from suitable Gram-positive bacteria, and lipoteichoic acid derived from Bacillus subtilis is sold as a commercial reagent.
In addition, lactic acid bacteria belong to Gram-positive bacteria. Therefore, it is possible to obtain inexpensive and safe lipoteichoic acid by extracting it from lactic acid bacteria that have been used in yogurt, lactic acid beverages, and the like and have been eaten.
　以下ではグラム陽性菌からリポテイコ酸を抽出、精製する方法を示す。グラム陽性菌の培養に用いる培地は当該細菌が生育可能なものであれば特に限定されず、当該細菌の培養法の常法によって行うことができる。 The method for extracting and purifying lipoteichoic acid from Gram-positive bacteria is shown below. The medium used for culturing the Gram-positive bacterium is not particularly limited as long as the bacterium can grow, and the bacterium can be cultured by a conventional method.
　培養した細菌は遠心分離等の方法によって集菌することができる。リポテイコ酸の抽出には得られた菌体をそのまま用いてもよいし、濃縮、乾燥、凍結乾燥、破砕処理に供した菌体を用いてもよい。菌体は加熱乾燥等により死菌体にしたものを用いることができる。
　これらの菌体から水や生理食塩水又はブタノールや酢酸エチル、クロロホルム等の有機溶媒もしくはそれらの混合液を用いてリポテイコ酸を抽出した後、ゲル濾過クロマトグラフィーや疎水性クロマトグラフィー等の手法を用いて精製することができる。 The cultured bacteria can be collected by a method such as centrifugation. For the extraction of lipoteichoic acid, the obtained microbial cells may be used as they are, or the microbial cells subjected to concentration, drying, freeze-drying, and crushing treatment may be used. Cells killed by heat drying or the like can be used.
After extracting lipoteichoic acid from these cells using water, saline, organic solvents such as butanol, ethyl acetate, chloroform, or a mixture thereof, gel filtration chromatography, hydrophobic chromatography, etc. can be purified by
　グラム陽性菌としては、ラクトバチルス属又はビフィドバクテリウム属に属する乳酸菌、もしくは枯草菌が好ましく、ラクトバチルス属又はビフィドバクテリウム属に属する乳酸菌がより好ましい。ラクトバチルス属に属する乳酸菌としては、ラクトバチルス・アシドフィルス、ラクトバチルス・アミロボラス、ラクトバチルス・ガセリ、ラクトバチルス・デルブレッキー、ラクトバチルス・ヘルベティカス、ラクトバチルス・ジョンソニー、ラクトバチルス・パラカゼイ、ラクトバチルス・プランタラム、ラクトバチルス・ラムノーサス、ラクトバチルス・ムコーサエ、ラクトバチルス・ファーメンタム、ラクトバチルス・カルバタス、ラクトバチルス・サリバリウス又はラクトバチルス・ロイテリがさらに好ましく、ラクトバチルス・ガセリ又はラクトバチルス・デルブレッキーが最も好ましい。ビフィドバクテリウム属に属する乳酸菌としては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・ビフィダム、ビフィドバクテリウム・ロンガム、ビフィドバクテリウム・アニマリス、ビフィドバクテリウム・ブレーベ、ビフィドバクテリウム・カテヌラータム、ビフィドバクテリウム・モンゴリエンセ、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・シュードカテヌラータム、ビフィドバクテリウム・フィーカリ又はビフィドバクテリウム・サーモフィラムがさらに好ましい。
　グラム陽性菌としては、ラクトバチルス・ガセリSBT1848（NITE-BP03075）又はラクトバチルス・デルブレッキー SBT2002（NITE-BP03280）が最も好ましい。リポテイコ酸としては、ラクトバチルス・ガセリSBT1848（NITE-BP03075）又はラクトバチルス・デルブレッキー SBT2002（NITE-BP03280）に含まれるリポテイコ酸が最も好ましい。 As Gram-positive bacteria, lactic acid bacteria belonging to the genus Lactobacillus or Bifidobacterium, or Bacillus subtilis are preferable, and lactic acid bacteria belonging to the genus Lactobacillus or Bifidobacterium are more preferable. Examples of lactic acid bacteria belonging to the genus Lactobacillus include Lactobacillus acidophilus, Lactobacillus amylovorus, Lactobacillus gasseri, Lactobacillus delbrechky, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus paracasei, and Lactobacillus planus. More preferred are Tallum, Lactobacillus rhamnosus, Lactobacillus mucosae, Lactobacillus fermentum, Lactobacillus carbatas, Lactobacillus salivarius or Lactobacillus reuteri, and most preferably Lactobacillus gasseri or Lactobacillus delbrecci. Lactic acid bacteria belonging to the genus Bifidobacterium include Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium longum, Bifidobacterium animalis, Bifidobacterium breve, and Bifidobacterium. Catenulatum, Bifidobacterium mongoliense, Bifidobacterium pseudolongum, Bifidobacterium pseudocatenulatum, Bifidobacterium ficari or Bifidobacterium thermophilum are more preferred.
Most preferred Gram-positive bacteria are Lactobacillus gasseri SBT1848 (NITE-BP03075) or Lactobacillus delbreeckii SBT2002 (NITE-BP03280). As lipoteichoic acid, lipoteichoic acid contained in Lactobacillus gasseri SBT1848 (NITE-BP03075) or Lactobacillus delbrecci SBT2002 (NITE-BP03280) is most preferable.
　本実施形態の筋合成促進剤は、リポテイコ酸を含有していればよく、上述の方法によって抽出したリポテイコ酸でもよいし、市販のリポテイコ酸を使用する方法でもよい。また、リポテイコ酸として抽出、精製を行わずグラム陽性菌をそのまま利用する方法でもよい。グラム陽性菌をそのまま利用する場合、そのグラム陽性菌は濃縮、乾燥、凍結乾燥、破砕処理に供した菌体でもよいし、加熱乾燥等により死菌体にしたものを用いることもできる。したがって医薬品、医薬部外品、飲食品、飼料等に含まれる組成物として広く利用できる。製剤化に際しては、賦形剤、結合剤、崩壊剤、矯味剤等、一般に用いられる添加物を適宜混合しても良い。
　すなわち、本実施形態の筋合成促進剤は、本発明の効果が得られる限り、任意の形態であることができる。すなわち、発酵乳の形態であってもよく、その他の剤形であってもよい。剤形としては、散剤、細粒剤、顆粒剤、錠剤、カプセル剤、及び丸剤等の固形状又は粉末状製剤、並びに懸濁液、エマルジョン剤、シロップ剤、及びエキス剤等の液状製剤を挙げることができる。 The muscle synthesis promoter of the present embodiment may contain lipoteichoic acid, and may be lipoteichoic acid extracted by the method described above, or may be a method using commercially available lipoteichoic acid. Alternatively, a method of using Gram-positive bacteria as they are without performing extraction and purification as lipoteichoic acid may be used. When Gram-positive bacteria are used as they are, the Gram-positive bacteria may be concentrated, dried, freeze-dried, or crushed, or killed by heat drying or the like. Therefore, it can be widely used as a composition contained in pharmaceuticals, quasi-drugs, food and drink, feed, and the like. At the time of formulation, commonly used additives such as excipients, binders, disintegrants, corrigents and the like may be appropriately mixed.
That is, the muscle synthesis promoter of this embodiment can be in any form as long as the effects of the present invention can be obtained. That is, it may be in the form of fermented milk, or in other dosage forms. Dosage forms include solid or powder formulations such as powders, fine granules, granules, tablets, capsules, and pills, and liquid formulations such as suspensions, emulsions, syrups, and extracts. can be mentioned.
　本実施形態に係る筋委縮予防剤の投与対象や一日あたりの摂取量についても、特に限定されず、例えば投与対象がヒトである場合は、20歳未満の未成年、成人、65歳以上の高齢者、65歳未満の者などに投与することができる。また、ロコモティブシンドロームやサルコペニアをすでに発症している対象に治療又は緩和のために投与することもでき、そのような症状を未だ有しない健康な対象に予防のために投与することもできる。
　一日の摂取量についても、年齢、症状、体重、目的によって異なるため、特に限定されないが、あえて挙げるとすれば、例えば、菌体又は菌体処理物の重量として0.001～10g、好ましくは0.01～5gが挙げられる。 The administration subject and the daily intake of the agent for preventing muscle atrophy according to the present embodiment are not particularly limited. It can be administered to elderly people, people under the age of 65, and the like. It can also be administered to subjects who have already developed locomotive syndrome or sarcopenia for treatment or alleviation, and can also be administered to healthy subjects who do not yet have such symptoms for prophylaxis.
The daily intake is also not particularly limited because it varies depending on age, symptoms, body weight, and purpose. 5g.
　本明細書において、「ロコモティブシンドローム」とは、骨、関節、軟骨、椎間板、筋肉といった運動器のいずれか、あるいは複数に加齢による機能障害が起こり、「立つ」、「歩く」といった機能が低下している状態を意味する。
　本明細書において、「サルコペニア」とは、加齢に伴い骨格筋が萎縮し、骨格筋量及び骨格筋力の低下又は身体機能の低下を伴うことを特徴とする症候群を意味する。 As used herein, the term “locomotive syndrome” refers to the functional impairment of one or more locomotive organs such as bones, joints, cartilage, intervertebral discs, and muscles due to aging, resulting in decreased functions such as “standing” and “walking”. It means the state of being
As used herein, "sarcopenia" refers to a syndrome characterized by age-related skeletal muscle atrophy, accompanied by a decrease in skeletal muscle mass and skeletal muscle strength, or a decrease in physical function.
（筋合成促進剤）
　本明細書において「筋合成促進」又は「筋肉の合成を促進する」とは、本発明の筋合成促進剤を対象が摂取した場合に、本発明の筋合成促進剤を対象が摂取しない場合と比較して、骨格筋の合成が多くみられることを意味する。
　「筋合成促進」又は「筋肉の合成を促進する」かどうかは、対象に有効成分を投与した場合に、有効成分を投与していない対象と比較して、p70S6Kタンパク質のリン酸化が促進されるかどうかを評価することにより、決定することができる。
　本発明の筋合成促進剤には、例えば、「筋肉を維持する」、「筋委縮を抑制する」、「筋肉の衰えを防ぐ」、「筋肉の衰えを抑える」、「筋肉をつける」、「筋肉を増やす」、「筋肉をつくる」、「筋肉の合成を促進する」、「筋肉をつくる力をサポートする」、「筋肉量及び筋力の低下抑制に役立つ」、「加齢によって衰える筋肉の維持に役立つ」、或いは、これらと同視できる機能の表示をすることができる。上記の「表示」は、消費者に前記の機能を知らしめる全てが含まれ、表示される対象は、筋合成促進剤自体、包装、容器、カタログ、パンフレット、及びインターネットホームページなどその媒体を問わない。 (muscle synthesis accelerator)
As used herein, the terms "promote muscle synthesis" or "promote muscle synthesis" refer to cases in which the agent for promoting muscle synthesis of the present invention is ingested by the subject, and cases in which the agent for promoting muscle synthesis of the present invention is not ingested by the subject. In comparison, it means that there is more synthesis of skeletal muscle.
Whether "promoting muscle synthesis" or "promoting muscle synthesis" means that phosphorylation of the p70S6K protein is promoted when the active ingredient is administered to the subject, compared to the subject to which the active ingredient is not administered. can be determined by evaluating whether
Examples of the myosynthesis promoter of the present invention include "maintain muscle", "inhibit muscle atrophy", "prevent muscle weakening", "suppress muscle weakening", "build muscle", " Increases muscle", "builds muscle", "promotes muscle synthesis", "supports the ability to build muscle", "helps prevent loss of muscle mass and strength", "maintains muscle that declines with age" It is possible to display functions that are useful for" or that can be equated with these. The above-mentioned "indication" includes everything that informs consumers of the above functions, and the object to be displayed is regardless of the medium such as the muscle synthesis promoter itself, packaging, containers, catalogs, pamphlets, and Internet homepages. .
（p70S6Kタンパク質のリン酸化促進剤）
　本明細書において「p70S6Kタンパク質」とは、p70リボソームS6キナーゼタンパク質を意味する。p70S6K（p70 ribosomal protein S6 kinase）は、タンパク質の合成を制御する細胞内シグナル伝達物質であり、筋肥大の重要な調節因子である。p70S6Kタンパク質はAkt-mTOR経路の下流にあり、リン酸化によって活性化される。
　本発明のp70S6Kタンパク質のリン酸化促進剤には、p70S6Kタンパク質のリン酸化促進により発揮される機能の表示を付することができる。p70S6Kタンパク質のリン酸化促進により発揮される機能の表示の内容については、上記の「筋合成促進剤」の場合に加えて、「p70S6Kタンパク質のリン酸化を促進して筋肉の合成を助ける」、「p70S6Kタンパク質のリン酸化を促進して筋肉を維持する」、「p70S6Kタンパク質のリン酸化を促進して筋委縮を抑制する」、或いは、これらと同視できる機能の表示を含む。
　p70S6Kタンパク質のリン酸化が促進されるかどうかは、例えば、後述の試験例１に記載の手法で評価することができる。 (p70S6K protein phosphorylation accelerator)
As used herein, "p70S6K protein" means the p70 ribosomal S6 kinase protein. p70S6K (p70 ribosomal protein S6 kinase) is an intracellular signaling substance that controls protein synthesis and is an important regulator of muscle hypertrophy. The p70S6K protein is downstream of the Akt-mTOR pathway and is activated by phosphorylation.
The p70S6K protein phosphorylation promoting agent of the present invention can be labeled with a function that is exhibited by promoting p70S6K protein phosphorylation. Regarding the content of the indication of the function exhibited by promoting phosphorylation of p70S6K protein, in addition to the above-mentioned "muscle synthesis promoter", "promoting phosphorylation of p70S6K protein to aid muscle synthesis", " It includes indications of functions equivalent to these, such as "promoting phosphorylation of p70S6K protein to maintain muscle", "suppressing muscle atrophy by promoting phosphorylation of p70S6K protein".
Whether the phosphorylation of the p70S6K protein is promoted can be evaluated, for example, by the method described in Test Example 1 below.
　本発明の筋合成促進用又は筋委縮予防用の飲食品としては、リポテイコ酸又はリポテイコ酸を含有するグラム陽性菌が含まれる飲食品であればよく、一般の飲食品のほか、特定保健用食品、機能性表示食品、栄養補助食品、サプリメント等があげられる。乳酸菌飲料、チーズ、ヨーグルト等はリポテイコ酸を含有するグラム陽性菌が含まれる飲食品の例である。 The food or drink for promoting muscle synthesis or preventing muscle atrophy of the present invention may be any food or drink containing lipoteichoic acid or Gram-positive bacteria containing lipoteichoic acid, and may be general food or drink, or food for specified health use. , foods with function claims, dietary supplements, and supplements. Lactic acid beverages, cheese, yogurt and the like are examples of foods and drinks containing Gram-positive bacteria containing lipoteichoic acid.
　本発明の筋合成促進用又は筋委縮予防用飲食品としては、具体的には、炭酸飲料、各種果汁、果汁飲料、果汁入り清涼飲料、果肉飲料、果粒入り果実飲料、各種野菜を含む野菜系飲料、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、栄養飲料などの一般飲料又はアルコール飲料；キャラメル・キャンディー、チューイングガム、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、デザート菓子などの菓子類；バター、マーガリン類、マヨネーズ類、植物油などの油脂類；牛乳・加工乳、乳飲料、ヨーグルト類、乳酸菌飲料、チーズ、アイスクリーム類、調製粉乳類、クリームなどの乳・乳製品、シリアル（穀物加工品）などの農産加工品、ベビーフード、流動食などのその他の市販食品などが挙げられる。 Specific examples of the food and drink for promoting muscle synthesis or preventing muscle atrophy of the present invention include carbonated drinks, various fruit juices, fruit juice drinks, soft drinks containing fruit juice, fruit drinks, fruit drinks containing fruit grains, and vegetables including various vegetables. beverages, soymilk/soymilk drinks, coffee drinks, tea drinks, powdered drinks, concentrated drinks, sports drinks, nutritional drinks, and other alcoholic beverages; caramel/candy, chewing gum, chocolate, cookies/biscuits, cakes/pies, snacks Confectionery such as crackers, Japanese confectionery, rice confectionery, bean confectionery, dessert confectionery; Butter, margarine, mayonnaise, vegetable oil and other fats and oils; Milk, processed milk, milk drinks, yogurts, lactic acid beverages, cheese, ice cream Milk and dairy products such as powdered milk, cream, processed agricultural products such as cereals (processed grains), baby food, and other commercially available foods such as liquid diet.
　本発明の筋合成促進用又は筋委縮予防用飲食品には、所望により、酸化防止剤、香料、酸味料、着色料、乳化剤、保存料、調味料、甘味料、香辛料、ｐＨ調整剤、安定剤、植物油、動物油、糖及び糖アルコール類、ビタミン、有機酸、果汁エキス類、野菜エキス類、穀類、豆類、野菜類、肉類、魚介類等の添加物及び素材を単独で又は２種以上組み合わせて配合することができる。これらの素材及び添加物の配合量は、適宜決定することができる。
　本発明の筋合成促進用又は筋委縮予防用飲食品の形態は特に限定されるものではなく、例えば、飲料や流動食のような液状、流動性の形態であっても、ゼリー状、ペースト状、半液体、ゲル状の形態であっても、固形、バー、粉末の形態であってもよい。 The food and drink for promoting muscle synthesis or preventing muscle atrophy of the present invention may optionally contain antioxidants, flavoring agents, acidulants, coloring agents, emulsifiers, preservatives, seasonings, sweeteners, spices, pH adjusters, and stabilizers. agents, vegetable oils, animal oils, sugars and sugar alcohols, vitamins, organic acids, fruit juice extracts, vegetable extracts, grains, beans, vegetables, meats, seafood, and other additives and materials alone or in combination of two or more. can be blended together. The blending amounts of these materials and additives can be determined as appropriate.
The form of the food and drink for promoting muscle synthesis or preventing muscle atrophy of the present invention is not particularly limited. , semi-liquid, gel form, solid, bar, powder form.
　筋合成促進用又は筋委縮予防用のリポテイコ酸のこれらの飲食品への配合量は、形態、剤型、投与対象の症状、体重、用途などによって異なるため、特に限定されないが、あえて挙げるなら、飲食品基準で、例えば0.001～100（w/w）％、0.01～10（w/w）、又は0.1～1（w/w）％で配合することができる。上記配合量は、リポテイコ酸としての配合量である。
　筋合成促進用又は委縮予防用の飼料としては、通常の飼料に前記有効成分を混合すればよいほか、リポテイコ酸又はリポテイコ酸を含有するグラム陽性菌を投与する方法によってもよい。 The amount of lipoteichoic acid for promoting muscle synthesis or preventing muscle atrophy in these foods and drinks is not particularly limited because it varies depending on the form, dosage form, symptoms, body weight, application, etc. of the subject to be administered. It can be blended at, for example, 0.001 to 100 (w/w)%, 0.01 to 10 (w/w), or 0.1 to 1 (w/w)% on the basis of food and drink. The above compounding amount is the compounding amount as lipoteichoic acid.
The feed for promoting muscle synthesis or preventing atrophy may be obtained by mixing the above-mentioned active ingredient with a normal feed, or by administering lipoteichoic acid or Gram-positive bacteria containing lipoteichoic acid.
　以上、本実施形態によって新規な筋合成促進剤を提供することができる。本筋合成促進剤はグラム陽性菌一般に含まれるリポテイコ酸を有効成分とするものであり、グラム陽性菌を原料にすることによって安価かつ大量に製造が可能であって、乳酸菌等の喫食経験のあるグラム陽性菌を利用すれば安全性の高い製品を提供することができる。 As described above, the present embodiment can provide a novel muscle synthesis promoter. This muscle synthesis accelerator contains lipoteichoic acid, which is generally contained in Gram-positive bacteria, as an active ingredient. If positive bacteria are used, highly safe products can be provided.
　以下、本発明の実施例として、グラム陽性菌である乳酸菌細胞壁が有する筋合成促進効果、乳酸菌から抽出したリポテイコ酸及びグラム陽性菌である枯草菌由来の市販のリポテイコ酸が有する筋合成促進効果を具体的に説明する。しかし、本発明の実施形態はこれに限定されるものではない。 Hereinafter, as examples of the present invention, the effect of promoting muscle synthesis possessed by the cell wall of lactic acid bacteria, which is a Gram-positive bacterium, the effect of promoting muscle synthesis possessed by lipoteichoic acid extracted from lactic acid bacteria and commercially available lipoteichoic acid derived from Bacillus subtilis, which is a Gram-positive bacterium. A specific description will be given. However, embodiments of the invention are not so limited.
[乳酸菌破砕物の調製]
　ラクトバチルス・ガセリSBT1848（NITE-BP03075）及びラクトバチルス・デルブレッキー SBT2002（NITE-BP03280）を使用する例を記載する。しかし上述のように、これら以外のグラム陽性菌を使用することもできる。各菌株のグリセロールストックをMRS平板培地に画線し、37℃で3晩（64時間）培養した。次いで平板培地で得られた1コロニーを10 mLのMRS培地に植菌し、37℃で1晩培養（16時間）した。この菌体培養液300μL を新しいMRS培地10 mLに植え継ぎ、再度37℃で1晩培養（16時間）した。次に、菌体培養液3 mLをMRS培地100 mLに植菌し、37℃で1晩培養（16時間）した。得られた菌体培養液を遠心分離し、沈殿した菌体をPBS、超純水で洗浄した。菌体は4 mLの超純水に再懸濁して-80℃で凍結させた。これを凍結乾燥機（東京理化器械）を用いて凍結乾燥させることで、凍結乾燥菌体を得た。得られた凍結乾燥菌体をPBS又はDMEM培地（gibco）に懸濁し、マルチビーズショッカー（安井機械）を用いて破砕することで乳酸菌破砕物を調製した。 [Preparation of crushed lactic acid bacteria]
An example using Lactobacillus gasseri SBT1848 (NITE-BP03075) and Lactobacillus delbreeckii SBT2002 (NITE-BP03280) is described. However, as mentioned above, Gram-positive bacteria other than these can also be used. Glycerol stocks of each strain were streaked onto MRS plates and incubated at 37° C. for 3 nights (64 hours). Then, one colony obtained on the plate medium was inoculated into 10 mL of MRS medium and cultured overnight (16 hours) at 37°C. 300 μL of this cell culture was transferred to 10 mL of new MRS medium, and cultured again at 37° C. overnight (16 hours). Next, 3 mL of the cell culture was inoculated into 100 mL of MRS medium and cultured overnight at 37°C (16 hours). The obtained cell culture was centrifuged, and the precipitated cells were washed with PBS and ultrapure water. Cells were resuspended in 4 mL of ultrapure water and frozen at -80°C. This was freeze-dried using a freeze dryer (Tokyo Rika Kikai Co., Ltd.) to obtain freeze-dried cells. The resulting freeze-dried cells were suspended in PBS or DMEM medium (gibco) and crushed using a multi-bead shocker (Yasui Kikai Co., Ltd.) to prepare crushed lactic acid bacteria.
[細胞壁の調製]
　乳酸菌破砕物を遠心分離し、上清を除去した。得られた沈渣を滅菌水で洗浄し、再度遠心分離によって上清を除去、この操作を数回繰り返すことで細胞壁を得た。
[乳酸菌からのリポテイコ酸の精製]
　凍結乾燥菌体80mgから上述の方法により乳酸菌破砕物を調製した。これを遠心分離し、上清を0.22μmフィルター（Merck）で濾過した。この濾過液を滅菌水で6mLにメスアップし、1-ブタノールを6mL添加して、抽出操作を行った。この操作によって混合液は水層、中間層、ブタノール層に分離した。水層を採取し、凍結乾燥によって溶媒を除去した。得られた抽出物を15%プロパノール/0.1M酢酸ナトリウム水溶液5mLに溶解したものをリポテイコ酸粗精製画分とした。
　次にリポテイコ酸粗精製画分を遠心分離し、上清を0.22μmフィルターで濾過した。容量1mLのHitrap Octyl FFカラム（GE Healthcare）を15%プロパノール/酢酸ナトリウム水溶液で平衡化し、粗精製画分上清1mLをカラムに供した。続いて、プロパノール濃度を15%、25%、35%、45%と段階的に上げた溶出液を2.5mLずつカラムに通し、サンプル添加時のフロースルー及び各プロパノール濃度の画分を2.5mLずつ得た。
　続いてフロースルー、15%画分あわせて5mLをカラムに供し、溶出された液をフロースルー（2回目）として5mL回収した。次にプロパノール濃度を15%、25%、35%、45%と段階的に上げた溶出液を2.5mLずつカラムに通し、各プロパノール濃度の画分を2.5mLずつ得た。
　再度フロースルー（2回目）5mLをカラムに供し、溶出された液をフロースルー（3回目）として5mLを回収した。これまでと同様プロパノール濃度を15%、25%、35%、45%と段階的に上げた溶出液を2.5mLずつカラムに通し、各プロパノール濃度の画分を2.5mLずつ得た。この操作をもう一度繰り返した。
　以上の操作によって、フロースルー（4回目）計5mL、15%画分（2～4回目）計7.5mL及び25%画分、35%画分、45%画分（1～4回目）計10mLを得た。
　回収した各画分を遠心エバポレーターによって濃縮し、滅菌水でメスアップすることで2mLの濃縮溶液とした。これらをMicrosep Advance with 1K Omega（Pall Corporation）及びVivaspin 500, 3 kDa MWCO（Cytiva）により濃縮、脱塩し、DMEM培地でメスアップすることで各200μLの画分を得た。 [Preparation of cell wall]
The lactic acid bacteria lysate was centrifuged and the supernatant was removed. The obtained sediments were washed with sterilized water, centrifuged again to remove the supernatant, and this operation was repeated several times to obtain cell walls.
[Purification of lipoteichoic acid from lactic acid bacteria]
A lactic acid bacteria crushed product was prepared from 80 mg of freeze-dried cells by the method described above. This was centrifuged and the supernatant filtered through a 0.22 μm filter (Merck). This filtrate was made up to 6 mL with sterilized water, and 6 mL of 1-butanol was added to carry out an extraction operation. By this operation, the mixture was separated into an aqueous layer, an intermediate layer and a butanol layer. The aqueous layer was collected and the solvent was removed by lyophilization. The resulting extract was dissolved in 5 mL of 15% propanol/0.1 M sodium acetate aqueous solution to obtain a crude lipoteichoic acid fraction.
The lipoteichoic acid crude fraction was then centrifuged and the supernatant filtered through a 0.22 μm filter. A 1 mL Hitrap Octyl FF column (GE Healthcare) was equilibrated with a 15% propanol/sodium acetate aqueous solution, and 1 mL of the crude fraction supernatant was applied to the column. Subsequently, 2.5 mL of the eluate with a stepwise increase in propanol concentration of 15%, 25%, 35%, and 45% was passed through the column, and 2.5 mL of the flow-through at the time of sample addition and fractions with each propanol concentration were collected. Obtained.
Subsequently, 5 mL of the combined flow-through and 15% fraction was applied to the column, and 5 mL of the eluted liquid was collected as a flow-through (second time). Next, 2.5 mL of the eluate with a stepwise increase in propanol concentration of 15%, 25%, 35%, and 45% was passed through the column to obtain 2.5 mL of each propanol concentration fraction.
5 mL of the flow-through (second time) was applied to the column again, and 5 mL of the eluted liquid was collected as the flow-through (third time). As before, 2.5 mL of the eluate with the propanol concentration increased stepwise to 15%, 25%, 35%, and 45% was passed through the column to obtain 2.5 mL of each propanol concentration fraction. This operation was repeated once more.
By the above operation, flow-through (4th time) total 5mL, 15% fraction (2nd to 4th time) total 7.5mL and 25% fraction, 35% fraction, 45% fraction (1st to 4th time) total 10mL got
Each collected fraction was concentrated by a centrifugal evaporator and diluted with sterilized water to obtain a 2 mL concentrated solution. These were concentrated and desalted using Microsep Advance with 1K Omega (Pall Corporation) and Vivaspin 500, 3 kDa MWCO (Cytiva), and diluted with DMEM medium to obtain fractions of 200 μL each.
 [ウエスタンブロットによるリポテイコ酸の検出]
　精製時に得た画分にリポテイコ酸が含有されているかはウエスタンブロットにより確認した。
　各画分をLaemmli sample bufferと混合し、90℃で10分間加熱、変性を行った。加熱したサンプルを4-20% Mini Protean TGXゲルにアプライして150V定圧で40分間電気泳動した。泳動後Criterionプレートトランスブロットセル（Bio-Rad）を使用して、50V定圧60分でPVDFメンブレンに転写した。転写後、メンブレンはTBSTで洗浄し、5% BSA(岩井化学薬品)/TBSTのブロッキングバッファー中で1時間振盪することでブロッキングを行った。ブロッキングバッファーで希釈した抗LTA一次抗体(Lipoteichoic acid clone 55 ; Hycult Biotech 2000倍希釈)に浸し、10°Cで1晩振盪した。一次抗体反応後、TBSTでメンブレンを洗浄後、ブロッキングバッファーで2000倍希釈したHRP標識二次抗体（Anti-mouse IgG HRP-linked Antibody；Cell Signaling Technology）に浸し、1時間振盪した。二次抗体反応後、TBSTでメンブレンを洗浄し、ECL Prime Western Blotting Detection Reagent（GE healthcare）を使用し5分間反応させた。反応終了後、Chemi Doc MP（Bio-Rad）でシグナルの検出を行った。
　この結果、リポテイコ酸はラクトバチルス・ガセリからの抽出では25%及び35%プロパノール/酢酸ナトリウム水溶液で溶出させた画分に、ラクトバチルス・デルブレッキーからの抽出では25%の画分に精製されていることが確認された（図１）。 [Detection of lipoteichoic acid by Western blot]
It was confirmed by Western blotting whether the fraction obtained during purification contained lipoteichoic acid.
Each fraction was mixed with Laemmli sample buffer and heated at 90°C for 10 minutes for denaturation. The heated samples were applied to a 4-20% Mini Protein TGX gel and electrophoresed at 150V constant pressure for 40 minutes. After electrophoresis, using a Criterion plate transblot cell (Bio-Rad), it was transferred to a PVDF membrane at 50V constant pressure for 60 minutes. After transfer, the membrane was washed with TBST, and blocked by shaking in a 5% BSA (Iwai Chemicals)/TBST blocking buffer for 1 hour. It was immersed in anti-LTA primary antibody (Lipoteichoic acid clone 55; Hycult Biotech, 2000-fold dilution) diluted with blocking buffer, and shaken overnight at 10°C. After primary antibody reaction, the membrane was washed with TBST, immersed in HRP-labeled secondary antibody (Anti-mouse IgG HRP-linked Antibody; Cell Signaling Technology) diluted 2000-fold with blocking buffer, and shaken for 1 hour. After secondary antibody reaction, the membrane was washed with TBST and reacted for 5 minutes using ECL Prime Western Blotting Detection Reagent (GE healthcare). After completion of the reaction, signals were detected with Chemi Doc MP (Bio-Rad).
As a result, lipoteichoic acid was purified to fractions eluted with 25% and 35% propanol/sodium acetate aqueous solutions in the extraction from Lactobacillus gasseri, and in the 25% fraction in the extraction from Lactobacillus delbrecci. (Fig. 1).
[試験例１]乳酸菌細胞壁、乳酸菌から精製したリポテイコ酸の筋合成促進効果の評価
　マウス筋芽細胞であるC2C12細胞を、10% FBS、1% penicillin-streptomycin含有DMEM培地を添加した48well培養プレート(IWAKI)に1.8×104 cell/wellとなるよう播種した。これを37℃、5%CO2条件下で2日間、80%～90%コンフルエントまで培養した。
　次に培地をFBSを含まない1% penicillin-streptomycin含有DMEM培地に交換し、4時間培養した。培養後、乳酸菌細胞壁又は菌株からのリポテイコ酸精製の過程で得られた各画分を添加した。このC2C12細胞をさらに2時間培養後、PBSで洗浄し、Alpha SureFire Ultra Lysis Buffer(PerkinElmer)を用いて細胞を溶解した。細胞溶解液を回収し、遠心分離して上清を得ることで、次のウエスタンブロットで用いるサンプルを得た。
　各サンプルをLaemmli sample bufferと混合し、90℃で10分間加熱、変性を行った。加熱したサンプルは4-20% Criterion TGXゲル（Bio-Rad）にアプライして200V定圧で40分間電気泳動した。泳動後、Criterionプレートトランスブロットセル（Bio-Rad）を使用して、50V定圧60分でPVDFメンブレンに転写した。転写後、メンブレンをTBSTで洗浄し、5% BSA(岩井化学薬品)/ TBSTのブロッキングバッファー中で1時間振盪することでブロッキングを行った。ブロッキングバッファーで希釈した一次抗体(Phospho-p70 S6 Kinase (Thr389) (108D2) Rabbit mAb；Cell Signaling Technology 1000倍希釈、p70 S6 Kinase (49D7) Rabbit mAb；Cell Signaling Technology 2000倍希釈)にメンブレンを浸し、10℃で1晩振盪した。一次抗体反応後、TBSTでメンブレンを洗浄後、ブロッキングバッファーで10000倍希釈したHRP標識二次抗体（Anti-rabbit IgG HRP-linked Antibody；Cell Signaling Technology）に浸し、1時間振盪した。二次抗体反応後、TBSTでメンブレンを洗浄し、ECL Prime Western Blotting Detection Reagent又はClarity Max Western ECL Substrate（Bio-Rad）を使用し5分間反応させた。反応終了後、Chemi Doc MP（Bio-Rad）でシグナルの検出を行った。シグナル強度を定量後、リン酸化p70S6Kの値を総p70S6Kの値で除すことで、筋合成促進効果を評価した。
　その結果、乳酸菌細胞壁とリポテイコ酸の精製画分であるプロパノール25%及び35%画分に筋合成促進活性が認められた（図２、図３、図４）。なお、リポテイコ酸は細胞壁構成成分の一つであるため菌体破砕物を遠心処理した後の沈渣（細胞壁画分）、上清（可溶画分）のいずれにも含まれる。 [Test Example 1] Evaluation of muscle synthesis promoting effect of lactic acid bacteria cell wall and lipoteichoic acid purified from lactic acid bacteria C2C12 cells, which are mouse myoblasts, were added to a 48-well culture plate (10% FBS, 1% penicillin-streptomycin-containing DMEM medium) IWAKI) was seeded at 1.8×10 4 cells/well. This was cultured at 37°C and 5% CO2 for 2 days until 80% to 90% confluency.
Next, the medium was changed to DMEM medium containing 1% penicillin-streptomycin without FBS, and cultured for 4 hours. After culturing, each fraction obtained in the process of lipoteichoic acid purification from lactic acid bacteria cell walls or strains was added. After culturing the C2C12 cells for an additional 2 hours, the cells were washed with PBS and lysed using Alpha SureFire Ultra Lysis Buffer (PerkinElmer). The cell lysate was collected and centrifuged to obtain the supernatant to obtain the sample for subsequent Western blotting.
Each sample was mixed with Laemmli sample buffer and heated at 90°C for 10 minutes for denaturation. The heated sample was applied to a 4-20% Criterion TGX gel (Bio-Rad) and electrophoresed at 200V constant pressure for 40 minutes. After electrophoresis, they were transferred to a PVDF membrane at 50V constant pressure for 60 minutes using a Criterion plate transblot cell (Bio-Rad). After transfer, the membrane was washed with TBST, and blocked by shaking in a 5% BSA (Iwai Chemicals)/TBST blocking buffer for 1 hour. Immerse the membrane in primary antibody (Phospho-p70 S6 Kinase (Thr389) (108D2) Rabbit mAb; Cell Signaling Technology 1000-fold dilution, p70 S6 Kinase (49D7) Rabbit mAb; Cell Signaling Technology 2000-fold dilution) diluted in blocking buffer. Shake overnight at 10°C. After primary antibody reaction, the membrane was washed with TBST, immersed in HRP-labeled secondary antibody (Anti-rabbit IgG HRP-linked Antibody; Cell Signaling Technology) diluted 10,000 times with blocking buffer, and shaken for 1 hour. After secondary antibody reaction, the membrane was washed with TBST and reacted for 5 minutes using ECL Prime Western Blotting Detection Reagent or Clarity Max Western ECL Substrate (Bio-Rad). After completion of the reaction, signals were detected with Chemi Doc MP (Bio-Rad). After quantifying the signal intensity, the muscle synthesis promoting effect was evaluated by dividing the phosphorylated p70S6K value by the total p70S6K value.
As a result, 25% and 35% propanol fractions, which are purified fractions of lactic acid bacteria cell walls and lipoteichoic acid, were found to have muscle synthesis promoting activity (Figs. 2, 3 and 4). Since lipoteichoic acid is one of the cell wall constituents, it is contained in both the sediment (cell wall fraction) and the supernatant (soluble fraction) after centrifugation of the disrupted cells.
[試験例２]枯草菌由来リポテイコ酸の筋合成促進効果の評価
　見出されたリポテイコ酸の筋合成促進効果が乳酸菌に限らずグラム陽性菌一般に認められるか検討した。市販されている枯草菌由来のリポテイコ酸を用い、試験例１と同様に筋合成促進効果が認められるか確認を行った。
　試験例１と同様にC2C12細胞を培養、枯草菌由来リポテイコ酸（InvivoGen）を各終濃度となるよう添加した。コントロール群には筋合成促進活性が認められた菌体破砕物上清を添加した。細胞を回収後、ウエスタンブロットにより筋合成促進効果を評価した。その結果、枯草菌由来リポテイコ酸では 100ng/mL以上の濃度で筋合成促進効果が認められた（図５）。 [Test Example 2] Evaluation of muscle synthesis-promoting effect of lipoteichoic acid derived from Bacillus subtilis It was examined whether the found muscle synthesis-promoting effect of lipoteichoic acid is recognized not only in lactic acid bacteria but also in Gram-positive bacteria in general. Using commercially available lipoteichoic acid derived from Bacillus subtilis, it was confirmed in the same manner as in Test Example 1 whether or not a muscle synthesis promoting effect was observed.
C2C12 cells were cultured in the same manner as in Test Example 1, and Bacillus subtilis-derived lipoteichoic acid (InvivoGen) was added to each final concentration. The control group was supplemented with the supernatant of the disrupted bacterial cells, which was found to have muscle synthesis-promoting activity. After recovering the cells, Western blotting was performed to evaluate the effect of promoting muscle synthesis. As a result, Bacillus subtilis-derived lipoteichoic acid was found to promote muscle synthesis at concentrations of 100 ng/mL or higher (Fig. 5).
[試験例３]筋合成促進効果に対するTLR2の関与の評価
　一般にグラム陽性菌のリポテイコ酸はTLR2(Toll-like receptor 2)によって認識され、下流にシグナルが伝達される。そこで、本発明における筋合成促進効果がこのTLR2を介したものであるか否か検討を行った。試験例１と同様にC2C12細胞を培養し、リポテイコ酸が含まれる菌体破砕物上清とTLR2のシグナル伝達を阻害するペプチド（TIRAP Inhibitor Peptide; NOVUS BIOLOGICALS）を終濃度が50μMとなるよう添加した。対照群には阻害配列を持たないペプチド（Controlペプチド）を添加した。細胞を回収後、ウエスタンブロットにより筋合成促進効果を評価した。その結果、TLR2のシグナル伝達を阻害すると筋合成促進効果が消失すること、すなわち筋合成促進効果がTLR2を介して惹起されることが明らかとなった（図６）。TLR2はグラム陽性細菌一般のリポテイコ酸を認識するとされている。
　したがってこのことから、本発明の筋合成促進効果がリポテイコ酸に一般的な効果であり、リポテイコ酸の由来する菌種や菌株に依存しないことが裏付けられる。 [Test Example 3] Evaluation of involvement of TLR2 in promoting muscle synthesis Lipoteichoic acid of Gram-positive bacteria is generally recognized by TLR2 (Toll-like receptor 2), and signals are transmitted downstream. Therefore, it was examined whether or not the effect of promoting muscle synthesis in the present invention is mediated by this TLR2. C2C12 cells were cultured in the same manner as in Test Example 1, and lipoteichoic acid-containing cell lysate supernatant and a peptide that inhibits TLR2 signaling (TIRAP Inhibitor Peptide; NOVUS BIOLOGICALS) were added to a final concentration of 50 μM. . A peptide without an inhibitory sequence (Control peptide) was added to the control group. After recovering the cells, Western blotting was performed to evaluate the effect of promoting muscle synthesis. As a result, it was revealed that inhibition of TLR2 signaling abolishes the muscle synthesis promoting effect, ie, the muscle synthesis promoting effect is induced via TLR2 (Fig. 6). TLR2 is believed to recognize lipoteichoic acid in Gram-positive bacteria in general.
Therefore, this proves that the muscle synthesis-promoting effect of the present invention is a general effect of lipoteichoic acid and does not depend on the bacterial species or strain from which lipoteichoic acid is derived.
　本発明によれば、グラム陽性菌細胞壁又はリポテイコ酸によって筋合成を促進し又は筋委縮を予防することができる。特に既に乳製品等に利用され喫食経験のある乳酸菌を利用することで、安全かつ安価に本発明の筋合成促進剤又は筋委縮予防剤を提供することが可能である。以上、本発明によって人々の筋量を増加させ、あるいは筋委縮を予防することで、筋力の維持向上や健康寿命の延伸がなされ、QOL向上を実現することができる。
　さらに飼料として家畜に摂取させることで、家畜の筋量増加を実現することも可能である。 According to the present invention, muscle synthesis can be promoted or muscle atrophy can be prevented by Gram-positive bacterial cell walls or lipoteichoic acid. In particular, it is possible to safely and inexpensively provide the agent for promoting muscle synthesis or the agent for preventing muscle atrophy of the present invention by using lactic acid bacteria that have already been used in dairy products and the like and have been eaten. As described above, by increasing the muscle mass of people or preventing muscle atrophy according to the present invention, muscle strength can be maintained and improved, healthy life expectancy can be extended, and QOL can be improved.
Furthermore, it is possible to increase the muscle mass of livestock by ingesting it as feed.
[寄託生物材料への言及]
（１）ＳＢＴ１８４８株
イ　当該生物材料を寄託した寄託機関の名称及び住所
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（千葉県木更津市かずさ鎌足２-５-８（郵便番号２９２-０８１８））
ロ　イの寄託機関に生物材料を寄託した日付
令和１年１１月２５日（原寄託日）
令和３年９月１０日（原寄託によりブタペスト条約に基づく寄託への移管日）ハ　イの寄託機関が寄託について付した受託番号
ＮＩＴＥ　ＢＰ－０３０７５
（２）ＳＢＴ２００２株
イ　当該生物材料を寄託した寄託機関の名称及び住所
上記（１）と同じ
ロ　イの寄託機関に生物材料を寄託した日付
令和２年９月１５日（原寄託日）
令和３年９月１０日（原寄託によりブタペスト条約に基づく寄託への移管日）ハ　イの寄託機関が寄託について付した受託番号
ＮＩＴＥ　ＢＰ－０３２８０  [Reference to deposited biological material]
(1) SBT1848 strain B. Name and address of the depositary institution that deposited the biological material National Institute of Technology and Evaluation Patent Microorganism Depositary Center (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture (zip code 292-0818) )
Date of depositing the biological material at Roy's depository: November 25, 2019 (original date of deposit)
September 10, 2021 (the date of transfer from the original deposit to the deposit under the Budapest Treaty) Accession number NITE BP-03075 assigned to the deposit by the depositary institution of Hai
(2) SBT2002 strain A. Name and address of the depositary institution where the biological material was deposited September 15, 2020 (original date of deposit) Date the biological material was deposited at the same Roy depository as in (1) above
September 10, 2021 (the date of transfer from the original deposit to the deposit under the Budapest Treaty) Accession number NITE BP-03280 assigned to the deposit by the depositary institution of Hai

Claims (12)

  1. 　リポテイコ酸、又はリポテイコ酸を含有するグラム陽性細菌を有効成分とする筋合成促進剤。 A muscle synthesis accelerator containing lipoteichoic acid or Gram-positive bacteria containing lipoteichoic acid as an active ingredient.
  2. 　リポテイコ酸が、乳酸菌もしくは枯草菌に由来し、又はリポテイコ酸を含有するグラム陽性細菌が乳酸菌もしくは枯草菌である、請求項１に記載の筋合成促進剤。 The muscle synthesis promoter according to claim 1, wherein the lipoteichoic acid is derived from lactic acid bacteria or Bacillus subtilis, or the Gram-positive bacteria containing lipoteichoic acid is lactic acid bacteria or Bacillus subtilis.
  3. 　リポテイコ酸が、ラクトバチルス属もしくはビフィドバクテリウム属に属する乳酸菌に由来し、又はリポテイコ酸を含有するグラム陽性細菌がラクトバチルス属もしくはビフィドバクテリウム属に属する乳酸菌である、請求項１に記載の筋合成促進剤。 2. The method according to claim 1, wherein the lipoteichoic acid is derived from lactic acid bacteria belonging to the genus Lactobacillus or Bifidobacterium, or the Gram-positive bacterium containing lipoteichoic acid is a lactic acid bacterium belonging to the genus Lactobacillus or Bifidobacterium. muscle synthesis promoter.
  4. 　リポテイコ酸の由来する乳酸菌が、ラクトバチルス・ガセリもしくはラクトバチルス・デルブレッキーであり、又はリポテイコ酸を含有する乳酸菌がラクトバチルス・ガセリもしくはラクトバチルス・デルブレッキーである請求項１に記載の筋合成促進剤。 The muscle synthesis-promoting agent according to claim 1, wherein the lactic acid bacterium from which lipoteichoic acid is derived is Lactobacillus gasseri or Lactobacillus delbrechskyi, or the lactic acid bacterium containing lipoteichoic acid is Lactobacillus gasseri or Lactobacillus delbrechskyi. agent.
  5. 　リポテイコ酸、又はリポテイコ酸を含有するグラム陽性細菌を含有し筋合成を促進させることを特徴とする筋合成促進用又は筋委縮予防用医薬品、飲食品及び飼料。 A drug, food, drink, and feed for promoting muscle synthesis or preventing muscle atrophy, characterized by containing lipoteichoic acid or Gram-positive bacteria containing lipoteichoic acid and promoting muscle synthesis.
  6. 　リポテイコ酸、又はリポテイコ酸を含有するグラム陽性細菌を筋細胞に作用させることによって筋合成を促進する方法であって医療行為を除くもの。 A method of promoting muscle synthesis by allowing lipoteichoic acid or Gram-positive bacteria containing lipoteichoic acid to act on muscle cells, excluding medical procedures.
  7. 　リポテイコ酸、又はリポテイコ酸を含有するグラム陽性細菌を有効成分とする、p70S6Kタンパク質のリン酸化促進剤。 A p70S6K protein phosphorylation accelerator containing lipoteichoic acid or Gram-positive bacteria containing lipoteichoic acid as an active ingredient.
  8. 　リポテイコ酸が乳酸菌もしくは枯草菌に由来し、又はリポテイコ酸を含有するグラム陽性細菌が乳酸菌もしくは枯草菌である請求項７に記載のp70S6Kタンパク質のリン酸化促進剤。 The p70S6K protein phosphorylation accelerator according to claim 7, wherein the lipoteichoic acid is derived from lactic acid bacteria or Bacillus subtilis, or the Gram-positive bacterium containing lipoteichoic acid is lactic acid bacteria or Bacillus subtilis.
  9. 　リポテイコ酸の由来する乳酸菌がラクトバチルス・ガセリもしくはラクトバチルス・デルブレッキーであり、又はリポテイコ酸を含有する乳酸菌がラクトバチルス・ガセリもしくはラクトバチルス・デルブレッキーである請求項７に記載のp70S6Kタンパク質のリン酸化促進剤。 8. The phosphor of the p70S6K protein according to claim 7, wherein the lactic acid bacterium from which lipoteichoic acid is derived is Lactobacillus gasseri or Lactobacillus delbrechskyi, or the lactic acid bacterium containing lipoteichoic acid is Lactobacillus gasseri or Lactobacillus delbrechskyi. Prooxidant.
  10. 　筋合成促進剤の製造のための、リポテイコ酸、又はリポテイコ酸を含有するグラム陽性細菌の使用。 The use of lipoteichoic acid or Gram-positive bacteria containing lipoteichoic acid for the production of muscle synthesis promoters.
  11. 　筋合成促進に使用するための、リポテイコ酸、又はリポテイコ酸を含有するグラム陽性細菌。 Gram-positive bacteria containing lipoteichoic acid or lipoteichoic acid for use in promoting muscle synthesis.
  12. 　有効量のリポテイコ酸、又はリポテイコ酸を含有するグラム陽性細菌を、それを必要としている対象に摂取させるか、又は投与することを含む、筋合成促進方法。 A method for promoting muscle synthesis, comprising ingesting or administering an effective amount of lipoteichoic acid or Gram-positive bacteria containing lipoteichoic acid to a subject in need thereof.
PCT/JP2022/014842 2021-04-08 2022-03-28 Muscle synthesis promoting agent, and agent for promoting phosphorylation of p70s6k protein WO2022215568A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2023512947A JPWO2022215568A1 (en) 2021-04-08 2022-03-28

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2021065572 2021-04-08
JP2021-065572 2021-04-08

Publications (1)

Publication Number Publication Date
WO2022215568A1 true WO2022215568A1 (en) 2022-10-13

Family

ID=83545422

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2022/014842 WO2022215568A1 (en) 2021-04-08 2022-03-28 Muscle synthesis promoting agent, and agent for promoting phosphorylation of p70s6k protein

Country Status (3)

Country Link
JP (1) JPWO2022215568A1 (en)
TW (1) TW202304470A (en)
WO (1) WO2022215568A1 (en)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2013502456A (en) * 2009-08-26 2013-01-24 アールエヌエー アイエヌシー Glycolipid derived from lipoteichoic acid and composition containing the same
JP2013047192A (en) * 2011-08-29 2013-03-07 Meiji Co Ltd Lactobacillus promoting physical activity
WO2015068809A1 (en) * 2013-11-07 2015-05-14 サントリーホールディングス株式会社 Intestinal barrier function enhancer containing lactic acid bacteria
JP2018083761A (en) * 2016-11-21 2018-05-31 アサヒグループホールディングス株式会社 Agent for inhibiting decomposition of muscle

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2013502456A (en) * 2009-08-26 2013-01-24 アールエヌエー アイエヌシー Glycolipid derived from lipoteichoic acid and composition containing the same
JP2013047192A (en) * 2011-08-29 2013-03-07 Meiji Co Ltd Lactobacillus promoting physical activity
WO2015068809A1 (en) * 2013-11-07 2015-05-14 サントリーホールディングス株式会社 Intestinal barrier function enhancer containing lactic acid bacteria
JP2018083761A (en) * 2016-11-21 2018-05-31 アサヒグループホールディングス株式会社 Agent for inhibiting decomposition of muscle

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
AISO, CHISATO: "Effects of Lactococcus lactis subsp. cremoris FC-fermented milk on muscle mass and protein metabolism signaling in aged mice", THE JOURNAL OF PHYSICAL FITNESS AND SPORTS MEDICINE, vol. 9, no. 6, 25 November 2020 (2020-11-25), pages 305, XP009535208, ISSN: 2186-8131 *
SIJTSMA L, WOUTERS J T, HELLINGWERF K J: "Isolation and characterization of lipoteichoic acid, a cell envelope component involved in preventing phage adsorption, from Lactococcus lactis subsp. cremoris SK110", JOURNAL OF BACTERIOLOGY, AMERICAN SOCIETY FOR MICROBIOLOGY, US, vol. 172, no. 12, 1 December 1990 (1990-12-01), US , pages 7126 - 7130, XP055976557, ISSN: 0021-9193, DOI: 10.1128/jb.172.12.7126-7130.1990 *

Also Published As

Publication number Publication date
TW202304470A (en) 2023-02-01
JPWO2022215568A1 (en) 2022-10-13

Similar Documents

Publication Publication Date Title
US8501169B2 (en) Microorganisms for improving the health of individuals with disorders related to gluten ingestion
AU2019243093B2 (en) Nutritional composition, food/drink composition using nutritional composition, and modified milk powder using nutritional composition
JP5923238B2 (en) Vagus nerve activator
US20140369965A1 (en) Bifidobacterium cect 7765 and use thereof in the prevention and/or treatment of overweight, obesity and associated pathologies
US11607435B2 (en) Probiotics Bifidobacteria strains
NO327792B1 (en) Bifidobacterium strain, formulation comprising said strain, and its use in food products or drugs in the treatment of disease.
SG177743A1 (en) Novel lactobacillus plantarum and composition comprising same
KR102224072B1 (en) Bifidobacterium longum subsp. longum having both abilities of reducing total cholesterol in serum and immune regulation and its application
JP6923883B2 (en) Compositions for use in improving nutritional status
Lau et al. Probiotics: Health benefits, food application, and colonization in the human gastrointestinal tract
JP2018177703A (en) Toll-like receptor 2 activating composition
WO2022215568A1 (en) Muscle synthesis promoting agent, and agent for promoting phosphorylation of p70s6k protein
JP5950993B2 (en) Vagus nerve activator
JP2021180619A (en) Fermented tea composition for controlling intestinal function and method of producing the same
AU2017287987B2 (en) Cartilage regeneration facilitating composition
da Motta et al. Probiotics in milk and dairy foods
TW201138651A (en) Agent for controlling the increase and decrease of lactobacillus bifidus in colon
Lavermicocca et al. Table olives: a carrier for delivering probiotic bacteria to humans
WO2017057307A1 (en) Antibody production controller
AU2013201442B2 (en) Treatment of IBD and IBS using both probiotic bacteria and fermented cereal as treatment effectors
WO2020116511A1 (en) Composition for suppressing norovirus infection
Ibrahim et al. Dietary modulation of gut microbiota by cultured products
Pal et al. Probiotics: beneficial microbes for health and the food industry
EP4289485A1 (en) Composition having immunostimulatory effect
US20220387522A1 (en) Composition for enhancing or improving immune system comprising bifidobacterium bifidum

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22784553

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2023512947

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 22784553

Country of ref document: EP

Kind code of ref document: A1