WO2022215568A1 - Agent favorisant la synthèse musculaire et agent favorisant la phosphorylation de la protéine p70s6k - Google Patents

Agent favorisant la synthèse musculaire et agent favorisant la phosphorylation de la protéine p70s6k Download PDF

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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
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lipoteichoic acid
muscle
gram
acid
promoting
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PCT/JP2022/014842
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Japanese (ja)
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大輔 河田
健 浮辺
泰幸 瀬戸
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雪印メグミルク株式会社
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    • 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 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
    • 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
    • A61K31/70—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.

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Abstract

Le but de la présente invention est de fournir une nouvelle technique pour favoriser directement la synthèse musculaire sans activité physique et prévenir l'atrophie musculaire.　L'invention concerne : un agent favorisant la synthèse musculaire ou un agent de prévention de l'atrophie musculaire contenant une paroi cellulaire bactérienne à gram positif ou un acide lipotéichoïque en tant que principe actif; ou un procédé pour favoriser la synthèse musculaire en permettant à une paroi cellulaire bactérienne à gram positif ou à un acide lipotéichoïque d'agir sur des cellules musculaires.
PCT/JP2022/014842 2021-04-08 2022-03-28 Agent favorisant la synthèse musculaire et agent favorisant la phosphorylation de la protéine p70s6k WO2022215568A1 (fr)

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JP2013047192A (ja) * 2011-08-29 2013-03-07 Meiji Co Ltd 身体活動を促進させる乳酸菌
WO2015068809A1 (fr) * 2013-11-07 2015-05-14 サントリーホールディングス株式会社 Agent d'amélioration de la fonction de barrière intestinale contenant un ferment lactique
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