JP7261867B2 - Composition for making skeletal muscle slow-twitch - Google Patents

Description

IPOD FERM BP-11175

TECHNICAL FIELD The present invention relates to a composition that can be used for specific purposes such as a composition for slowing skeletal muscle.

Muscles are the basis of everyday actions such as standing, walking, and maintaining posture. Therefore, in order to maintain the quality of life, there is a desire to maintain muscle mass as much as possible and to increase muscle mass if possible, regardless of age or sex. In addition, people and athletes (for example, athletes, sports enthusiasts, athletes, etc.) who actively enjoy exercise desire to increase muscle mass as much as possible in order to improve their exercise performance.

Furthermore, as people become more health-conscious, it is important to improve quality of life (QOL), improve frailty (an intermediate state between a healthy state and a state requiring long-term care), or locomotive syndrome (locomotive syndrome). Syndrome), the challenge is how to maintain the decline in endurance and muscle strength.

Muscles are made up of bundles of muscle fibers. Muscle fibers are classified into slow-twitch fibers (Type I) and fast-twitch fibers (Type II) based on properties such as contraction tension, contraction speed, and fatigue resistance. Muscles with a large number of slow-twitch fibers have low contractile force and contraction speed, but are less prone to fatigue, contributing to endurance. On the other hand, muscles with a large number of fast-twitch fibers have a large contractile force and contraction speed, but are easily fatigued. In addition, muscles with many slow-twitch fibers act as anti-gravity muscles. In patients with disuse muscle atrophy, muscles change from slow-twitch to fast-twitch. It gets to the point where it hurts. Maintaining or strengthening slow-twitch muscles can prevent or improve physical inactivity and difficulties in daily activities by preserving endurance and strength.

Ginger extract and the like are known as a substance capable of promoting slow-twitching of skeletal muscle (Patent Document 1).

In addition, it has been reported that administration of Bifidobacterium breve B-3 increases muscle mass (Non-Patent Document 1).

JP 2014-101346

Minami J et al. Biosci Microbiota Food Health. 2018;37(3):67-75.

An object of the present invention is to provide a composition that can be used for specific purposes such as a composition for slowing skeletal muscle.

As a result of intensive research, the present inventors have found that the administration of bacteria belonging to the genus Bifidobacterium has the effect of slowing skeletal muscle, and completed the present invention.

That is, the present invention can be exemplified as follows.
One aspect of the present invention is a composition for slow-twitching skeletal muscle, containing the following component (A) as an active ingredient:
(A) Bifidobacterium and/or products thereof.
One aspect of the present invention is a composition for improving endurance containing the following component (A) as an active ingredient:
(A) Bifidobacterium and/or products thereof.
One aspect of the present invention is a composition for preventing, improving, and/or treating symptoms associated with muscle atrophy and/or muscle weakness, containing the following component (A) as an active ingredient:
(A) Bifidobacterium and/or products thereof.
A preferred embodiment of the composition is a pharmaceutical composition.
A preferred aspect of the composition is that it is a food and drink composition.
A preferred aspect of the composition is that the component (A) is the bacterium.
A preferred embodiment of the composition is that the component (A) contains dead cells of the bacteria.
A preferred embodiment of the composition is that the bacterium is Bifidobacterium breve.
A preferred embodiment of the composition is that the bacterium is Bifidobacterium breve FERM BP-11175.
One aspect of the present invention is a method for slowing skeletal muscle, comprising the step of administering the following component (A) to a subject:
(A) Bifidobacterium and/or products thereof.
One aspect of the present invention is a method for preventing, ameliorating, and/or treating symptoms associated with muscle atrophy and/or muscle weakness, comprising the step of administering component (A) below to a subject:
(A) Bifidobacterium and/or products thereof.
One aspect of the present invention is the use of component (A) below for the prevention, amelioration and/or treatment of symptoms associated with muscle atrophy and/or weakness:
(A) Bifidobacterium and/or products thereof.
One aspect of the present invention is the use of component (A) below for the slow-twitching of skeletal muscle:
(A) Bifidobacterium and/or products thereof.
One aspect of the present invention is the use of component (A) below for the manufacture of said composition:
(A) Bifidobacterium and/or products thereof.

The figure which shows the slow-twitch fiber ratio (%) in each group. Data were obtained from 7 rats in each group and presented as mean ± s.e.m. An asterisk (*) indicates a significant difference (P<0.05) by Dunnett's method. Slow-twitch fiber staining images (photographs) in the control group. Stained image of slow-twitch fibers in the group administered live Bifidobacterium breve FERM BP-11175 (photograph). Stained image of slow-twitch fibers in the heat-sterilized Bifidobacterium breve FERM BP-11175-administered group (photograph). A diagram showing relative values of PGC-1α expression levels in each group. Data were obtained from 12 or 13 rats in each group and presented as mean ± standard error. An asterisk (*) indicates a significant difference (P<0.05) by Dunnett's method. A diagram showing the relative value of the amount of phosphorylated AMPK in each group. Data were obtained from 6 rats in each group and presented as mean ± s.e.m. An asterisk (*) indicates a significant difference (P<0.05) by Dunnett's method.

The present invention will be described in detail below. Unless otherwise specified, any of the following descriptions relating to the present invention may be employed singly or may be employed in combination as appropriate.

<1> Active ingredient In the present invention, the following ingredient (A) is used as an active ingredient:
(A) Bifidobacterium and/or products thereof.

That is, the component (A) is also referred to as an "active ingredient".

By using an active ingredient, specifically by administering an active ingredient to a subject, skeletal muscle can be slow-moving in the subject. can get. The same effect is also referred to as "skeletal muscle slow-twitch effect". Skeletal muscles include trunk muscles (ie, trunk skeletal muscles) and limb muscles (ie, upper and lower extremity skeletal muscles). Skeletal muscles of the lower extremities include gastrocnemius muscle and soleus muscle. The skeletal muscle slow-moving effect may be obtained in all skeletal muscles (that is, skeletal muscles of the whole body) or may be obtained in some skeletal muscles. The slow-twitch skeletal muscle effect may be obtained, for example, in a site with a high proportion of slow-twitch fibers, in a site with a high proportion of fast-twitch fibers, or in both of them. A skeletal muscle slow-moving effect may be obtained, for example, at least in the gastrocnemius muscle and/or the soleus muscle. "Slow-twitch skeletal muscle" means an increase in the amount of slow-twitch muscle in skeletal muscle. In other words, "slow-twitch skeletal muscle" means that the amount of slow-twitch muscle in skeletal muscle is greater when the active ingredient is administered than when the active ingredient is not administered. "At the time of administration of the active ingredient" may mean after the active ingredient has been sufficiently administered, specifically, after the active ingredient has been administered under the conditions described in the method of the present invention described later. can mean "When not administering the active ingredient" may conveniently mean prior to administration of the active ingredient. However, "at the time of non-administration of the active ingredient" may precisely mean the assumption that after the same period of time as the administration period of the active ingredient has elapsed without administration of the active ingredient. That is, for example, when the amount of slow-twitch muscle in skeletal muscle decreases due to non-administration of an active ingredient, "slow-twitch skeletal muscle" means that the degree of decrease is reduced by administration of an active ingredient. It is not necessary that the amount of slow twitch muscle in skeletal muscle after administration of the active ingredient is greater than that before administration of the active ingredient. Such cases include cases where the onset or progression of slow-twitch muscle atrophy is alleviated by administration of the active ingredient. "Amount of slow-twitch muscle in skeletal muscle" may mean the absolute amount of slow-twitch muscle in skeletal muscle, and the relative amount of slow-twitch muscle in skeletal muscle (i.e., the amount of slow-twitch muscle relative to the amount of skeletal muscle). ratio) or both. That is, utilization of the active ingredient may increase the absolute and/or relative amount of slow twitch muscle. Utilization of the active ingredient may at least increase the relative amount of slow twitch fibers. The total skeletal muscle mass may increase with increasing slow twitch mass. The amount of slow-twitch fibers includes the number of slow-twitch fibers, the cross-sectional area of slow-twitch fibers, and the volume of slow-twitch fibers. That is, for example, the relative amount of slow-twitch fibers in skeletal muscle includes the ratio of the number of slow-twitch fibers to the number of muscle fibers in skeletal muscle, the ratio of the cross-sectional area of slow-twitch fibers to the cross-sectional area of skeletal muscle, Examples include the ratio of slow-twitch fiber volume to volume. By "slow-twitch fibers" is meant Type I muscle fibers. Utilization of active ingredients may, for example, increase one or more of these parameters. Utilization of the active ingredient may, for example, at least increase the number of slow twitch fibers. The amount of slow-twitch fibers can be measured, for example, by a known method. Techniques for measuring the amount of slow muscle tissue include muscle biopsy, magnetic resonance imaging (MRI), and field test. Specifically, the ratio of the number of slow-twitch fibers to the number of muscle fibers in skeletal muscle can be measured, for example, by the method described in Examples. The amount of slow muscle in skeletal muscle during administration of the active ingredient is, for example, 101% or more, 103% or more, 105% or more, 110% or more, 115% or more when the amount of skeletal muscle when the active ingredient is not administered is 100%. , 120% or more, 125% or more, or 130% or more. In addition, the slow muscle growth of skeletal muscle can also be measured using, for example, an improvement in fatigue resistance, an improvement in oxidase activity in skeletal muscle, or an improvement in the number of mitochondria in skeletal muscle.

Further, by using the active ingredient, specifically by administering the active ingredient to the subject, the activation of AMP-activated protein kinase (AMPK) may be promoted in the subject. That is, an effect of promoting AMPK activation in the subject may be obtained. This effect is also called "AMPK activation promoting effect". Activation of AMPK may be particularly enhanced in skeletal muscle. A skeletal muscle slow-twitching effect may be obtained by promoting AMPK activation. “Enhancement of AMPK activation” means an increase in the amount of active AMPK (ie, phosphorylated AMPK). In other words, "promotion of AMPK activation" means that the amount of active AMPK is greater when the active ingredient is administered than when the active ingredient is not administered. Unless otherwise specified, "the amount of active AMPK" may mean a value normalized based on the total amount of AMPK. The amount of active AMPK can be measured, for example, by a known method. Methods for measuring the amount of active AMPK include Western blotting. Specifically, the amount of active AMPK can be measured, for example, by the method described in Examples. The amount of active AMPK at the time of administration of the active ingredient is, for example, 101% or more, 103% or more, 105% or more, 110% or more, 115% or more, 120%, when the amount at the time of non-administration of the active ingredient is 100%. % or more, 125% or more, or 130% or more.

In addition, the expression of peroxisome proliferator-activated receptor γ-coupling factor-1α (PGC-1α) may be promoted in the subject by using the active ingredient, specifically by administering the active ingredient to the subject. That is, the effect of promoting the expression of PGC-1α in the subject may be obtained. This effect is also referred to as "PGC-1α expression promoting effect". Expression of PGC-1α may be particularly enhanced in skeletal muscle. A skeletal muscle slow-twitching effect may be obtained by promoting the expression of PGC-1α. “Promoting expression of PGC-1α” means increasing the expression level of PGC-1α. In other words, “promotion of PGC-1α expression” means that the expression level of PGC-1α is greater when the active ingredient is administered than when the active ingredient is not administered. Unless otherwise specified, the “PGC-1α expression level” may mean a value standardized based on the expression level of a housekeeping gene such as GAPDH. The expression level of PGC-1α can be measured, for example, by a known technique for measuring gene expression levels. Techniques for measuring the expression level of genes include a technique for measuring the amount of mRNA and a technique for measuring the amount of protein. Specific examples of techniques for measuring gene expression levels include Northern hybridization, microarray, RT-PCR, RNA-seq, and Western blotting. Specifically, the expression level of PGC-1α can be measured, for example, by the method described in Examples. The expression level of PGC-1α at the time of administration of the active ingredient is, for example, 101% or more, 103% or more, 105% or more, 110% or more, 115% or more, when the non-administration of the active ingredient is taken as 100%, It may be 120% or more, 125% or more, or 130% or more.

In addition, by using the active ingredient, specifically by administering the active ingredient to the subject, an effect based on slow-moving skeletal muscle may be obtained in the subject.

It is expected that, for example, the endurance of a subject can be improved by making skeletal muscles slow. That is, an effect based on slow-moving skeletal muscles includes an effect of improving the endurance of the subject (endurance-improving effect). "Endurance" means endurance for physical exertion. Thus, "endurance" may specifically mean the ability to sustain physical exertion. Physical exercise is not particularly limited as long as it is an operation that places a load on muscles. Physical exercise includes sports, physical training, manual labor, and everyday activities.

It is expected that, for example, the muscle strength of a subject can be improved by slowing down skeletal muscle. Specifically, for example, when the total amount of skeletal muscles increases with an increase in the amount of slow-twitch muscles, it is expected that the muscle strength of the subject can be improved. That is, the effect based on the slow-moving skeletal muscle includes the effect of improving the muscle strength of the subject (muscle strength-improving effect).

Skeletal muscle slowing is expected to prevent, ameliorate, and/or treat, for example, symptoms associated with muscle atrophy and/or muscle weakness. That is, the effects based on slow-moving skeletal muscles include the effects of preventing, improving, and/or treating symptoms associated with muscle atrophy and/or muscle weakness. Muscle atrophy refers to loss of muscle mass. Muscle atrophy includes atrophy of skeletal muscle. Muscle atrophy specifically includes slow and/or fast twitch atrophy. Muscle atrophy includes, in particular, atrophy of slow muscles. Muscle atrophy may, for example, reduce muscle strength. In other words, muscle weakness may be due to muscle atrophy, for example. A condition associated with muscle atrophy and/or weakness may or may not be a disease. Symptoms associated with muscle atrophy and/or muscle weakness may develop or progress due to genetic factors, acquired factors, aging, or the like. Symptoms associated with muscle atrophy and/or muscle weakness include symptoms caused by muscle atrophy and/or muscle weakness and symptoms associated with muscle atrophy and/or muscle weakness. The symptoms related to muscle atrophy and/or muscle weakness include muscle atrophy itself and muscle weakness itself. Symptoms associated with muscle atrophy and/or muscle weakness specifically include disuse muscle atrophy, neurogenic muscle atrophy, and myogenic muscle atrophy. Disuse muscle atrophy includes age-related muscle atrophy (sarcopenia) and muscle atrophy due to insufficient use of muscles. Insufficient use of muscles may be due to bedriddenness, lack of exercise, zero-gravity flight, immobilization of the body, and the like. Immobilization of the body includes immobilization of extremities carried out for treatment of injuries and the like. Neurogenic muscular atrophy includes amyotrophic lateral sclerosis (ALS), primary lateral sclerosis (PLS), spinal muscular atrophy (SMA), Spinal and bulbar muscular atrophy (SBMA), acute poliomyelitis (poliomyelitis), and Guillain-Barre syndrome (GBS). Myogenic muscle atrophy includes muscular dystrophy and polymyositis (PM). In addition, symptoms related to muscle atrophy and / or muscle weakness, specifically, myasthenia gravis (MG), cachexia, frailty, locomotive syndrome; locomotive syndrome).

The active ingredient may or may not be used in combination with other techniques for obtaining the above-exemplified effects, such as the effect of slowing muscle growth in skeletal muscle. Such techniques include performing physical exercise outside of everyday activities. Examples of physical exercise other than daily movements include sports and physical training. By using an active ingredient in combination with such techniques, it is expected that the desired effects of such techniques, such as the skeletal muscle slow-twitching effect, will be enhanced.

In addition, the present invention provides use of the active ingredient in applications associated with the above-exemplified effects such as skeletal muscle slow-moving effects. That is, the present invention provides, for example, the use of an active ingredient for slowing skeletal muscle, the use of an active ingredient for promoting AMPK activation, and the use of an active ingredient for promoting PGC-1α expression. , the use of active ingredients for improving endurance, the use of active ingredients for improving muscle strength, the use of active ingredients for the prevention, amelioration, and/or treatment of symptoms associated with muscle atrophy and/or weakness. May provide use. In addition, the present invention provides, for example, compositions for slowing skeletal muscle muscle, compositions for promoting AMPK activation, compositions for promoting PGC-1α expression, prevention and improvement of symptoms associated with muscle atrophy and/or muscle weakness, and/or the use of active ingredients for the manufacture of compositions of the invention, such as therapeutic compositions.

In addition, the present invention provides active ingredients for use in applications associated with the above-exemplified effects such as skeletal muscle slow-moving effects. That is, the present invention provides, for example, an active ingredient for use in slowing skeletal muscle, an active ingredient for use in promoting AMPK activation, an active ingredient for use in promoting expression of PGC-1α, and endurance. active ingredients for use in improving muscle strength, active ingredients for use in preventing, ameliorating, and/or treating symptoms associated with muscle atrophy and/or muscle weakness . In addition, the present invention provides, for example, compositions for slowing skeletal muscle muscle, compositions for promoting AMPK activation, compositions for promoting PGC-1α expression, prevention and improvement of symptoms associated with muscle atrophy and/or muscle weakness, and/or may provide active ingredients for use in preparing compositions of the invention, such as therapeutic compositions.

The active ingredient may be used for therapeutic or non-therapeutic purposes. That is, any of the above-exemplified effects may be obtained for therapeutic purposes or for non-therapeutic purposes, unless otherwise specified. "Therapeutic purposes" may mean, for example, the act of treating a human body for treatment, and in particular, may mean practiced as a medical act. "Non-therapeutic purpose" may mean, for example, not including treatment of the human body for treatment, and in particular, may mean performed as a non-medical action. Non-therapeutic purposes include purposes such as health promotion and beauty.

"Prophylaxis of symptoms" can mean, for example, preventing or delaying the onset of symptoms, or reducing the likelihood of developing symptoms. "Amelioration of symptoms" or "treatment of symptoms" can mean, for example, ameliorating symptoms, preventing or delaying worsening of symptoms, or preventing or delaying progression of symptoms. "Amelioration of symptoms" may specifically refer to these events and those that are not for therapeutic purposes. "Treatment of symptoms" may specifically refer to those events and which are intended for treatment.

Bifidobacterium bacteria are not particularly limited as long as desired effects such as skeletal muscle slow-moving effects are obtained. As Bifidobacterium bacteria, one type of bacteria may be used, or two or more types of bacteria may be used in combination.

Bifidobacterium genus bacteria include Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium bifidum, Bifidobacterium adolescentis ( Bifidobacterium adolescentis, Bifidobacterium angulatum, Bifidobacterium dentium, Bifidobacterium pseudocatenulatum, Bifidobacterium animalis ), Bifidobacterium pseudolongum, Bifidobacterium thermophilum.

Bifidobacterium bacteria include, in particular, Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium bifidum, and Bifidobacterium adore. Bifidobacterium adolescentis, Bifidobacterium dentium, Bifidobacterium animalis, Bifidobacterium pseudolongum, Bifidobacterium thermophilum ). Bifidobacterium bacteria include, more particularly, Bifidobacterium breve and Bifidobacterium longum. Bifidobacterium bacteria more particularly include Bifidobacterium breve.

Bifidobacterium longum includes Bifidobacterium longum subsp. longum, Bifidobacterium longum subsp. infantis Also included are strains classified in any subspecies of Bifidobacterium longum, such as B. longum subsp. suis. Bifidobacterium animalis includes strains classified into any subspecies of Bifidobacterium animalis, such as B. animalis subsp. lactis. be. Bifidobacterium pseudolongum includes Bifidobacterium pseudolongum subspecies globosum (B. pseudolongum subsp. globosum) and Bifidobacterium pseudolongum subspecies pseudolongum (B. pseudolongum subsp. pseudolongum), strains classified into any subspecies of Bifidobacterium pseudolongum are also included.

As Bifidobacterium breve, specifically M-16V (NITE BP-02622), MCC1274 (FERM BP-11175), ATCC 15700, B632 (DSM 24706), Bb99 (DSM 13692), ATCC 15698, DSM 24732, UCC2003, YIT4010, YIT4064, BBG-001, BR-03, C50, R0070. Bifidobacterium breve includes in particular FERM BP-11175.

On August 25, 2009, Bifidobacterium breve FERM BP-11175 was registered with the National Institute of Advanced Industrial Science and Technology Patent Organism Depository (currently National Institute of Technology and Evaluation Patent Organism Depositary Center (NITE-IPOD). ), Zip Code: 292-0818, Address: Room 120, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) under the Budapest Treaty under the accession number FERM BP-11175.

As Bifidobacterium longum, specifically, BB536 (NITE BP-02621), ATCC 15697, ATCC 15707, ATCC 25962, ATCC 15702, ATCC 27533, M-63, BG7, DSM 24736, SBT 2928, NCC 490 (CNCM I-2170), NCC 2705 (CNCM I-2618).

Specific examples of Bifidobacterium bifidum include ATCC 29521, OLB6378, and BF-1. Specific examples of Bifidobacterium adolescentis include ATCC 15703. Specific examples of Bifidobacterium dentium include DSM 20436. Specific examples of Bifidobacterium animalis include DSM 10140, Bb-12, DN-173010, GCL2505 and CNCM I-3446. Specific examples of Bifidobacterium pseudolongum include JCM 5820 and ATCC 25526. Specific examples of Bifidobacterium thermophilum include ATCC 25525.

These strains are, for example, American Type Culture Collection (ATCC, Address: 10801 University Boulevard Manassas, VA 20110, United States of America), Belgian Coordinated Collections of Microorganisms (BCCM, Address: Rue de la Science 8, 1000 Brussels, Belgium). ), German Collection of Microorganisms and Cell Cultures (DSMZ, Address: Inhoffenstr.7B, D38124 Braunschweig, Germany), Japan Collection of Microorganisms (JCM, Zip code: 305-0074, Address: 3- Koyadai, Tsukuba, Ibaraki, Japan) 1-1 RIKEN Bioresource Research Center Microbial Materials Development Division) or from the depositary institution where each strain was deposited.

In addition, the strains specified by the strain names exemplified above are not limited to strains themselves that have been deposited or registered with a predetermined institution under the strain name (hereinafter, for convenience of explanation, also referred to as "deposited strain"). Strains substantially equivalent thereto (also referred to as "derivative strains" or "derived strains") are also included. That is, for example, "Bifidobacterium breve FERM BP-11175" is not limited to the strain itself deposited at the above depository under the deposit number of FERM BP-11175, but also includes substantially equivalent strains. be done. For each strain, "a strain substantially equivalent to the above-deposited strain" means that it belongs to the same species as the above-deposited strain. The base sequence of the gene preferably has 99.86% or more, more preferably 99.93% or more, and still more preferably 100% identity with the base sequence of the 16S rRNA gene of the deposited strain, and It preferably refers to a strain having the same mycological properties as the above deposited strain. For each strain, a strain substantially equivalent to the above deposited strain may provide desired effects such as a skeletal muscle slow-twitch effect equal to or greater than that of the deposited strain. The term "effect equal to or greater than that of the deposited strain" may mean 80% or more, 90% or more, 95% or more, or 100% or more of the effect obtained by using the deposited strain. For each strain, a strain that is substantially equivalent to the deposited strain may be, for example, a derivative of the deposited strain as a parent strain. Derivative strains include strains bred from the deposited strain and strains that arise naturally from the deposited strain. Breeding methods include modification by genetic engineering techniques and modification by mutation treatment. Mutation treatments include X-ray irradiation, ultraviolet irradiation, and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), ethyl methanesulfonate (EMS), and methyl methanesulfonate (MMS). ) and other mutating agents. Strains naturally occurring from the deposited strain include strains naturally occurring during use of the deposited strain. Such strains include mutants naturally occurring by culturing (eg, subculturing) the deposited strain. Derivative strains may be constructed with one modification, or may be constructed with two or more modifications.

"Bifidobacterium bacterium" as an active ingredient may mean cells of the genus Bifidobacterium.

A “product of the genus Bifidobacterium” as an active ingredient may mean a component produced by culturing a bacterium of the genus Bifidobacterium. The product produced by Bifidobacterium bacteria is not particularly limited as long as the desired effect such as skeletal muscle slow-moving effect can be obtained. Products of Bifidobacterium genus bacteria include components that accumulate in the medium during cultivation of the bacteria, components that accumulate in the cells during cultivation of the bacteria, and components that make up the cells of the bacteria. mentioned. Cell walls and peptidoglycan are examples of components that constitute the microbial cell. It should be noted that the Bifidobacterium bacterium itself does not have to correspond to the "product of the Bifidobacterium genus bacterium".

As the active ingredient, a commercially available product may be used, or an appropriately manufactured product may be used. For example, commercially available Bifidobacterium bacteria include Bifidobacterium breve FERM BP-11175 manufactured by Morinaga Milk Industry Co., Ltd. In addition, the active ingredient can be easily obtained by culturing Bifidobacterium. The culture method is not particularly limited as long as the bacteria of the genus Bifidobacterium can grow. As a culture method, for example, a method commonly used for culturing Bifidobacterium bacteria can be used as it is or after being modified as appropriate. The culture temperature may be, for example, 25-50°C, preferably 35-42°C. Culturing is preferably carried out under anaerobic conditions, for example, while passing anaerobic gas such as carbon dioxide. Cultivation can also be performed under microaerobic conditions such as liquid stationary culture. Culturing can be carried out, for example, until the Bifidobacterium spp. grows to the desired extent.

The medium used for culture is not particularly limited as long as the Bifidobacterium bacteria can grow. As the medium, for example, a medium commonly used for culturing bacteria of the genus Bifidobacterium can be used as it is or after being modified as appropriate. The medium may contain, for example, carbon sources, nitrogen sources, inorganic salts, organic components, dairy components, or combinations thereof. As carbon sources, for example, sugars such as glucose, galactose, lactose, arabinose, mannose, sucrose, starch, starch hydrolysates, blackstrap molasses and the like can be used depending on the assimilation. As the nitrogen source, for example, ammonia, ammonium salts such as ammonium sulfate, ammonium chloride and ammonium nitrate, and nitrates can be used. Examples of inorganic salts that can be used include sodium chloride, potassium chloride, potassium phosphate, magnesium sulfate, calcium chloride, calcium nitrate, manganese chloride, and ferrous sulfate. Examples of organic ingredients that can be used include peptone, soybean flour, defatted soybean meal, meat extract, and yeast extract. Moreover, as a milk component, for example, milk protein or the like can be used. Milk proteins include casein, whey, and hydrolysates thereof. These components may be used alone or in combination as appropriate. In addition, as the medium commonly used for culturing Bifidobacterium bacteria, specifically, reinforced Clostridial medium, MRS medium (de Man, Rogosa, and Sharpe medium), mMRS medium (modified MRS medium) ), TOSP medium (TOS propionate medium), and TOSP Mup medium (TOS propionate mupirocin medium).

As the active ingredient, the active ingredient itself or a fraction containing it can be used without particular limitation. That is, as the Bifidobacterium bacterium, cells of the genus Bifidobacterium or fractions containing them can be used without particular limitation. The fraction containing cells of the genus Bifidobacterium may contain, in addition to the cells, a product of the bacterium of the genus Bifidobacterium. In addition, as the product of the Bifidobacterium genus bacteria, a component produced by culturing the Bifidobacterium genus bacteria or a fraction containing the component can be used without particular limitation. The fraction containing components produced by culturing Bifidobacterium bacteria may contain Bifidobacterium bacteria in addition to the components. As the active ingredient, for example, the culture obtained by culturing may be used as it is, or the cells or culture supernatant collected from the culture may be used, or they may be used after being appropriately treated. That is, specific examples of active ingredients include cultures of Bifidobacterium spp., cells collected from the cultures, culture supernatants collected from the cultures, and processed products thereof. The treatment is not particularly limited as long as it does not impair the desired effect such as the skeletal muscle slow-moving effect. Processing includes concentration, dilution, drying, sterilization, crushing and fractionation. These treatments may be performed alone or in combination as appropriate. All of these treatments can be performed, for example, by known techniques. Drying can be performed, for example, by freeze-drying.

For example, cells may be crushed and the crushed cells may be used as an active ingredient. Ultrasonic treatment and homogenization treatment are examples of techniques for disrupting the cells. The disrupted cells may be used as an active ingredient as is or after being appropriately subjected to the treatment as described above.

Alternatively, for example, a fraction containing the active ingredient may be fractionated from the above-described cultures, cells, culture supernatants, or processed products thereof (e.g., disrupted cells) and used as the active ingredient. . Specifically, for example, a fraction such as a supernatant fraction or a precipitate fraction may be fractionated from the disrupted cells and used as an active ingredient. In addition, for example, the active ingredient may be fractionated (that is, separated and purified) from the above-described cultures, cells, culture supernatants, or processed products thereof (for example, disrupted cells). Separation and purification of the active ingredient may be carried out to the desired degree. Separation and purification of the active ingredient can be carried out, for example, by known methods used for separation and purification of compounds. Such techniques include centrifugation, salting out, solvent extraction, gel filtration, HPLC, membrane separation. For solvent extraction, for example, polar solvents, non-polar solvents, or mixed solvents thereof may be used.

As an active ingredient, in particular, Bifidobacterium bacteria (specifically, cells of the Bifidobacterium genus or fractions containing them) may be used. In other words, the active ingredient may be, in particular, a Bifidobacterium bacterium (specifically, Bifidobacterium bacterium or a fraction containing it). Specific examples of Bifidobacterium bacteria include cultures of Bifidobacterium bacteria, bacterial cells recovered from the same cultures, and processed products such as their dilutions, concentrates, dried products, and sterilized products. mentioned. The cells may be viable cells or dead cells. Cells may be used particularly in a form containing dead cells. The cells may be, for example, dead cells or a mixture of dead cells and live cells. The ratio of dead cells in the cells may be, for example, 50% or more, 70% or more, 90% or more, 95% or more, 97% or more, or 99% or more, based on the number of bacteria. Killed cells can be prepared, for example, by subjecting live cells to sterilization treatment. As sterilization treatment, heat treatment, spray drying method (spray drying method), retort sterilization method, freeze drying method, UHT sterilization method, autoclave sterilization method, high pressure steam sterilization method, dry heat sterilization method, distribution steam sterilization method, electromagnetic wave Sterilization, electron beam sterilization, high-frequency sterilization, radiation sterilization, ultraviolet sterilization, ethylene oxide gas sterilization, hydrogen peroxide gas plasma sterilization, alcohol sterilization, formalin fixation, and electrolytic water treatment. Heat treatment includes heating at 70 to 100° C. for 10 to 60 minutes.

<2> Composition of the Present Invention The composition of the present invention is a composition containing the active ingredient described above.

That is, the composition of the present invention is a composition containing bacteria of the genus Bifidobacterium and/or products thereof.

The compositions of the invention can be used by administration to a subject. Specifically, the composition of the present invention can be administered to a subject in the manner described below in the method of the present invention. The composition of the present invention can be used, for example, to obtain the effects exemplified above.

By using the composition of the present invention, specifically by administering the composition of the present invention to a subject, skeletal muscle can be slowed in the subject, that is, the skeletal muscle slow-twitch effect is obtained. That is, the composition of the present invention may be a skeletal muscle slow-twitching composition.

Further, by using the composition of the present invention, specifically by administering the composition of the present invention to a subject, the activation of AMPK may be promoted in the subject, i.e., the effect of promoting AMPK activation can be obtained. That is, the composition of the present invention may be a composition for promoting AMPK activation. One aspect of the composition for promoting AMPK activation may be, for example, a composition for slow muscle stimulation of skeletal muscle.

Also, by using the composition of the present invention, specifically by administering the composition of the present invention to a subject, expression of PGC-1α may be promoted in the subject, i.e., PGC-1α expression A stimulating effect may be obtained. That is, the composition of the present invention may be a composition for promoting PGC-1α expression. One embodiment of the composition for promoting PGC-1α expression may be, for example, a composition for slowing skeletal muscle.

In addition, by using the composition of the present invention, specifically by administering the composition of the present invention to a subject, an effect based on slow-moving skeletal muscle as exemplified above can be obtained in the subject. you can That is, the composition of the present invention may be a composition for improving endurance. Moreover, the composition of the present invention may be a composition for improving muscle strength. The composition of the present invention may also be a composition for preventing, ameliorating, and/or treating symptoms associated with muscle atrophy and/or muscle weakness. Any one of these compositions may be, for example, one aspect of a composition for slow-twitch skeletal muscle.

For subjects to whom the composition of the present invention is administered, the description of subjects to which the active ingredient is administered in "Method of the present invention" can be applied mutatis mutandis.

The composition of the present invention may be, for example, a food or drink composition, a pharmaceutical composition, or a feed composition. The composition of the invention may in particular be a food or beverage composition or a pharmaceutical composition. That is, the present invention provides, for example, food and drink compositions for the above-exemplified uses such as skeletal muscle slow-twitch, pharmaceutical compositions for the above-exemplified uses such as skeletal muscle slow-twitch, or skeletal muscle slow-twitch. A feed composition for the above-exemplified uses, such as, may be provided. The compositions of the present invention, which are food and drink compositions, pharmaceutical compositions, or feed compositions, are respectively referred to as "food and drink compositions of the present invention," "pharmaceutical compositions of the present invention," and "feed compositions of the present invention." Also called "thing".

The composition of the present invention may consist of active ingredients, or may contain ingredients other than active ingredients.

Ingredients other than the active ingredient are not particularly limited as long as they do not impair the desired effect such as the effect of slowing muscle growth. As components other than the active ingredient, those that are acceptable depending on the mode of use of the composition of the present invention can be used. Ingredients other than the active ingredient can be, for example, ingredients that are used by blending in foods and beverages, pharmaceuticals, or feeds. Examples of components other than the active ingredient include, specifically, components exemplified for food and drink compositions, pharmaceutical compositions, and feed compositions to be described later. As components other than the active ingredient, one component may be used, or two or more components may be used in combination.

Specific examples of ingredients other than the active ingredient include ingredients that have a muscle-increasing effect and ingredients that assist the muscle-increasing effect. Specific examples of such components include proteins such as whey protein, casein protein, soy protein, pea protein (pea protein), mixtures thereof, decomposition products thereof; leucine, valine, isoleucine, glutamine, etc. amino acids; vitamins such as vitamin B6 and vitamin C; creatine; citric acid; and fish oil.

The content and content ratio of each component (that is, the active ingredient and optionally other components) in the composition of the present invention are not particularly limited as long as the desired effect such as the effect of slowing skeletal muscle is obtained. The content and content ratio of each component in the composition of the present invention are the type of active ingredient, the type of other ingredients, the type of composition, shape (dosage form), and usage, the type of administration subject, age, and It can be appropriately set according to various conditions such as health condition.

The content of the active ingredient in the composition of the present invention is, for example, 1×10 ⁴ cells/g or more, 1×10 ⁵ cells/g or more, or 1×10 ⁶ cells/g in terms of bacterial cell content. 1×10 ⁷ cells/g or more, or 1×10 ⁸ cells/g or more, and 10 ¹³ cells/g or less, 10 ¹² cells/g or less, or 1×10 ¹¹ cells/g or less There may be, or the range may be a combination thereof. In addition, the content of the active ingredient in the composition of the present invention is, for example, 1×10 ⁴ cells/mL or more, 1×10 ⁵ cells/mL or more, 1×10 ⁶ cells in terms of bacterial cell content. /mL or more, 1×10 ⁷ cells/mL or more, or 1×10 ⁸ cells/mL or more, 10 ¹³ cells/mL or less, 10 ¹² cells/mL or less, or 1×10 ¹¹ cells/mL It may be less than or equal to or within the range of a combination thereof. Specifically, the content of the active ingredient in the composition of the present invention is, for example, 1×10 ⁶ to 1×10 ¹² cells/g or 1×10 ⁶ to 1×10 12 cells/g in terms of bacterial cell content. 10 ¹² cells/mL, preferably 1×10 ⁷ to 1×10 ¹¹ cells/g or 1×10 ⁷ to 1×10 ¹¹ cells/mL, more preferably 1×10 ⁸ to 1×10 ¹⁰ cells/g or It may be 1×10 ⁸ to 1×10 ¹⁰ cells/mL. When the active ingredient is viable cells, "cells" may be read as "cfu". "cfu" stands for colony forming unit.

The content of the active ingredient in the composition of the present invention is, for example, converted to the content of the original culture, 0.01% (w/w) or more, 0.1% (w/w) or more, 1 % (w/w) or more, 5% (w/w) or more, or 10% (w/w) or more, 10000% (w/w) or less, 5000% (w/w) or less, 1000% (w/w) or less, 500% (w/w) or less, 300% (w/w) or less, 200% (w/w) or less, 150% (w/w) or less, 100% (w/w) w) no more than 70% (w/w), no more than 50% (w/w), no more than 30% (w/w), no more than 10% (w/w), no more than 5% (w/w), or It may be 1% (w/w) or less, or any compatible combination thereof. Specifically, the content of the active ingredient in the composition of the present invention is, for example, converted to the content of the original culture, 1 to 10000% (w / w), preferably 10 to 1000% (w /w). That is, for example, the composition of the present invention may contain a culture of Bifidobacterium or its derivative (culture supernatant, etc.) in the content as exemplified above. The "original culture" refers to a culture that has not undergone a concentration change such as concentration or dilution after culturing. ). Such a culture can be prepared, for example, by using a medium as exemplified above and containing 1×10 ⁷ to 1×10 ¹¹ cfu/mL, preferably 1×10 ⁸ to 1×10 ¹⁰ cfu of Bifidobacterium genus bacteria. /mL may be cultured. A content exceeding 100% (w/w) means that the culture or its derivative (culture supernatant, etc.) is concentrated and contained in the composition of the present invention. That is, for example, a content of 1000% (w/w) means that the culture or its derivative (culture supernatant, etc.) is 10-fold concentrated and contained in the composition of the present invention.

In addition, the content of the active ingredient in the composition of the present invention can be set, for example, so that the dose of the active ingredient as described later can be obtained. That is, the dosage of the composition of the present invention is, for example, 1×10 ⁶ to 1×10 ¹² cells/kg body weight/day, preferably 1×10 cells/kg body weight/day, in terms of the dosage of the active ingredient (based on bacterial cells). ⁷ to 1×10 ¹¹ cells/kg body weight/day, more preferably 1×10 ⁸ to 1×10 ¹⁰ cells/kg body weight/day. The dosage of the composition of the present invention is, for example, 1×10 ⁷ to 1×10 ¹⁴ cells/day/individual, preferably 1×10 ⁸ in terms of the dosage of the active ingredient (on a bacterial basis). It may be up to 1×10 ¹³ cells/day/individual, more preferably 1×10 ⁹ to 1×10 ¹² cells/day/individual. When the active ingredient is viable cells, "cells" may be read as "cfu". In addition, the dosage of the composition of the present invention is, for example, 0.01 to 100 mL/kg body weight/day, preferably 0.1 to 10 mL/day in terms of the dosage of the active ingredient (based on the original culture). kg body weight/day.

The shape of the composition of the present invention is not particularly limited. As the shape of the composition of the present invention, any acceptable shape can be adopted depending on the mode of use of the composition of the present invention. Specific examples of the shape of the composition of the present invention include shapes exemplified for food and drink compositions, pharmaceutical compositions, and feed compositions described later.

<Beverage composition>
The food and drink composition of the present invention is not particularly limited as long as it contains active ingredients. The food and drink composition of the present invention may be provided in any form such as liquid, paste, solid gel, and powder.

The food/drink composition may be, for example, the food/drink itself, or may be a material used in the production of the food/drink. Such materials include seasonings, food additives, and other ingredients for food and drink. Specific examples of food and drink compositions include wheat flour products, instant foods, processed agricultural products, processed marine products, processed livestock products, dairy products (fermented milk, cheese, powdered milk for infants, etc.), oils and fats, and basic seasonings. , compound seasonings, frozen foods, confectionery, beverages, and other commercially available food and drink. In addition, as food and drink compositions, specifically, health food, functional food, enteral nutrition food, food for special use, food with health claims (food for specified health use, food with nutrient function claims, food with function claims, etc.), Dietary supplements and quasi-drugs are also included. Moreover, the food and drink composition may be, for example, a supplement, and specifically, may be a tablet-like supplement.

The food and drink composition of the present invention can be produced by combining an active ingredient with other ingredients. The operation of combining an active ingredient with other ingredients is also referred to as "addition of an active ingredient." The method for producing the food and drink composition of the present invention is not particularly limited. The food/drink composition of the present invention can be produced, for example, by using the same raw materials as those for normal food/drinks and by the same method as for normal food/drinks, except that an active ingredient is added. The same applies when the food and drink composition of the present invention is produced as a material used in the production of food and drink, such as seasonings, food additives, and other raw materials for food and drink. Addition of the active ingredient may be carried out at any stage of the manufacturing process of the food and drink composition. Addition of the active ingredient may be carried out, for example, during or after production of the food and drink composition. That is, for example, the food and drink composition of the present invention may be obtained by adding an active ingredient to a food and drink prepared in advance. Also, the food and drink composition may be produced through a fermentation step using added active ingredients (particularly Bifidobacterium bacteria). Fermented products such as lactic acid beverages and fermented milk are examples of food and drink compositions produced through a fermentation process. Thus, active ingredients (especially Bifidobacterium) may be used, for example, as starters for the production of fermented products. The active ingredient can also be added later to the produced fermented product.

Further, another food and drink composition can be produced using the food and drink composition of the present invention. That is, for example, when the food and drink composition of the present invention is provided as a material used in the production of food and drink, such as seasonings, food additives, and other food and drink raw materials, Another food and beverage composition can be produced by adding the food and beverage composition. Another food and drink composition produced in this manner is also an aspect of the food and drink composition of the present invention. For the addition of the food and drink composition of the present invention, the description of the addition of active ingredients in the production of the food and drink composition can be applied mutatis mutandis.

The food/beverage composition of the present invention is used for making skeletal muscles slow, for promoting AMPK activation, for promoting PGC-1α expression, and for preventing, improving, and/or treating symptoms associated with muscle atrophy and/or muscle weakness. It is possible to provide and sell food and drink labeled with such uses (including health uses). In addition, the food and drink composition of the present invention can be ingested by "athletes", "those who want to increase muscle", "those who are concerned about weakening of muscle strength due to lack of exercise", and "those who are weakened by aging". It is possible to provide and sell products labeled as “Those who are concerned about muscle”, “Those who are concerned about muscle strength that declines with age”, and “Those who want to improve frailty”.

"Indication" includes all acts to inform consumers of the above-mentioned use, and if it is an expression that can remind or analogize the above-mentioned use, the purpose of the display, the content of the display, and the display Regardless of the object, medium, etc., all correspond to "display". In particular, it is preferable that the display is made by an expression that allows consumers to directly recognize the use.

Specifically, as an indication, specifically, a product or product packaging that describes the use of the food and drink composition of the present invention is transferred, delivered, displayed for transfer or delivery, imported Act of doing business, displaying or distributing the above use in advertisements related to products, price lists or transaction documents, or providing information by electromagnetic (Internet etc.) methods with the above use in information containing these actions, etc. Examples of indications include, in particular, indications on packaging, containers, catalogs, pamphlets, POP and other advertising materials at sales sites, and on other documents.

Moreover, as the display, it is preferable to use a display approved by the government (for example, a display that is approved based on various systems established by the government and performed in a manner based on such approval). In addition, as for labeling, health food, functional food, enteral nutrition food, food for special dietary use, food with health claims (e.g., food for specified health use, food with nutrient function claims, food with function claims), dietary supplement, pharmaceutical part For example, it may be displayed as an off-the-shelf product. Labeling approved by the government includes labeling approved by the Consumer Affairs Agency. The labeling approved by the Consumer Affairs Agency includes the labeling approved under the Foods for Specified Health Uses and similar systems. Specifically, the labeling approved by the Consumer Affairs Agency includes labeling as a food for specified health use, labeling as a food for specified health use with certain conditions, labeling to the effect that it affects the structure and function of the body, and reducing disease risk. Labeling, functional labeling based on scientific grounds, etc. Japanese Cabinet Office Ordinance No. 57) as food for specified health use (especially labeling for health use) and similar labeling.

The content of the active ingredient in the food and drink composition of the present invention may be, for example, within the range described above. Specifically, the content of the active ingredient in the food and drink composition of the present invention is, for example, 1×10 ⁶ to 1×10 ¹² cells/g or 1×10 12 cells/g in terms of bacterial cell content. ⁶ to 1×10 ¹² cells/mL, preferably 1×10 ⁷ to 1×10 ¹¹ cells/g or 1×10 ⁷ to 1×10 ¹¹ cells/mL, more preferably 1×10 ⁸ to 1×10 ¹⁰ It may be cells/g or 1×10 ⁸ to 1×10 ¹⁰ cells/mL. When the active ingredient is viable cells, "cells" may be read as "cfu". Further, the content of the active ingredient in the food and drink composition of the present invention is specifically, for example, 1 to 10000% (w/w), preferably 10 to 10000% (w/w) in terms of the content of the original culture. It may be 1000% (w/w).

The dosage of the food and drink composition of the present invention may be, for example, within the range described above. Specifically, the dosage of the food and drink composition of the present invention is, for example, 1×10 ⁶ to 1×10 ¹² in terms of the dosage of the active ingredient (based on the amount of bacteria). cells/kg body weight/day, preferably 1×10 ⁷ to 1×10 ¹¹ cells/kg body weight/day, more preferably 1×10 ⁸ to 1×10 ¹⁰ cells/kg body weight/day. Further, the dosage of the food and drink composition of the present invention is specifically, for example, 1×10 ⁷ to 1×10 ¹⁴ in terms of the dosage of the active ingredient (based on the amount of bacteria). cells/day/individual, preferably 1×10 ⁸ to 1×10 ¹³ cells/day/individual, more preferably 1×10 ⁹ to 1×10 ¹² cells/day/individual. When the active ingredient is viable cells, "cells" may be read as "cfu". Further, the dosage of the food and drink composition of the present invention is specifically, for example, 0.01 to 100 mL/kg in terms of the dosage of the active ingredient (based on the amount of the original culture). Body weight/day, preferably 0.1-10 mL/kg body weight/day.

<Pharmaceutical composition>
The pharmaceutical composition of the present invention is not particularly limited as long as it contains active ingredients. As the pharmaceutical composition of the present invention, for example, the active ingredient may be used as it is, or the active ingredient may be formulated and used as appropriate.

The dosage form of the pharmaceutical composition of the present invention is not particularly limited. The dosage form of the pharmaceutical composition of the present invention can be appropriately selected according to various conditions such as the administration method. The pharmaceutical composition of the present invention may be for oral administration or parenteral administration. The pharmaceutical composition of the invention may in particular be for oral administration. For oral administration, specific dosage forms include solid formulations such as powders, granules, tablets and capsules, and liquid formulations such as solutions, syrups, suspensions and emulsions. For parenteral administration, specific dosage forms include suppositories and ointments. Physiologically acceptable additives can be used for formulation. Additives include various organic and inorganic components. Specific examples of additives include excipients, binders, disintegrants, lubricants, stabilizers, corrigents, diluents, surfactants, and solvents. These additives can be appropriately selected according to various conditions such as the dosage form.

Excipients include, for example, sugar derivatives such as lactose, sucrose, glucose, mannitol, sorbitol; starch derivatives such as corn starch, potato starch, α-starch, dextrin, carboxymethyl starch; crystalline cellulose, hydroxypropyl cellulose, Hydroxypropylmethylcellulose, carboxymethylcellulose, cellulose derivatives such as carboxymethylcellulose calcium; gum arabic; dextran; pullulan; silicate derivatives such as light silicic anhydride, synthetic aluminum silicate, and magnesium aluminometasilicate; phosphate derivatives such as calcium phosphate; carbonate derivatives such as calcium; sulfate derivatives such as calcium sulfate;

Examples of binding agents include gelatin, polyvinylpyrrolidone, macrogol, etc., in addition to the excipients described above.

Examples of disintegrants include, in addition to the excipients described above, chemically modified starch or cellulose derivatives such as croscarmellose sodium, carboxymethyl starch sodium, and crosslinked polyvinylpyrrolidone.

Lubricants include, for example, talc; stearic acid; metal stearates such as calcium stearate and magnesium stearate; colloidal silica; waxes such as pea gum and gairou; ; carboxylic acid sodium salts such as sodium benzoate; sulfates such as sodium sulfate; leucine; lauryl sulfates such as sodium lauryl sulfate and magnesium lauryl sulfate; silicic acid anhydride and silicic acid hydrate; be done.

Examples of stabilizers include paraoxybenzoic acid esters such as methylparaben and propylparaben; alcohols such as chlorobutanol, benzyl alcohol and phenylethyl alcohol; benzalkonium chloride; acetic anhydride;

Flavoring agents include, for example, sweeteners, acidulants, flavoring agents, and the like.

The content of the active ingredient in the pharmaceutical composition of the present invention may be, for example, within the range described above. Specifically, the content of the active ingredient in the pharmaceutical composition of the present invention is, for example, 1×10 ⁶ to 1×10 ¹² cells/g or 1×10 ⁶ in terms of bacterial cell content. ~1 x 10 ¹² cells/mL, preferably 1 x 10 ⁷ - 1 x 10 ¹¹ cells/g or 1 x 10 ⁷ - 1 x 10 ¹¹ cells/mL, more preferably 1 x 10 ⁸ - 1 x 10 ¹⁰ cells /g or 1×10 ⁸ to 1×10 ¹⁰ cells/mL. When the active ingredient is viable cells, "cells" may be read as "cfu". Further, the content of the active ingredient in the pharmaceutical composition of the present invention is specifically, for example, 1 to 10000% (w/w), preferably 10 to 1000%, in terms of the content of the original culture. % (w/w).

The dosage of the pharmaceutical composition of the present invention may, for example, be in the ranges described above. Specifically, the dosage of the pharmaceutical composition of the present invention is, for example, 1×10 ⁶ to 1×10 ¹² cells in terms of the dosage of the active ingredient (based on the amount of bacterial cells). Cells/kg body weight/day, preferably 1×10 ⁷ to 1×10 ¹¹ cells/kg body weight/day, more preferably 1×10 ⁸ to 1×10 ¹⁰ cells/kg body weight/day. Further, the dosage of the pharmaceutical composition of the present invention is specifically, for example, 1×10 ⁷ to 1×10 ¹⁴ cells in terms of the dosage of the active ingredient (based on the amount of bacterial cells). cells/day/individual, preferably 1×10 ⁸ to 1×10 ¹³ cells/day/individual, more preferably 1×10 ⁹ to 1×10 ¹² cells/day/individual. When the active ingredient is viable cells, "cells" may be read as "cfu". Further, the dosage of the pharmaceutical composition of the present invention is specifically, for example, 0.01 to 100 mL/kg body weight in terms of the dosage of the active ingredient (based on the amount of the original culture). /day, preferably 0.1-10 mL/kg body weight/day.

<Feed composition>
The feed composition of the present invention is not particularly limited as long as it contains active ingredients. Feed compositions include pet food and livestock feed. The feed of the present invention may be provided in any form such as powder, granules, crumbles, pellets, cubes, pastes and liquids.

The feed composition of the present invention can be produced by combining the active ingredient with other ingredients. The operation of combining an active ingredient with other ingredients is also referred to as "addition of an active ingredient." The method for producing the feed composition of the present invention is not particularly limited. The feed composition of the present invention can be produced, for example, by using the same raw materials as those for ordinary feeds and by the same method as for ordinary feeds, except that the active ingredient is added. Addition of the active ingredient may be carried out at any stage of the manufacturing process of the feed composition. Addition of the active ingredient may be carried out, for example, during or after the production of the feed composition. That is, for example, the feed composition of the present invention may be obtained by adding an active ingredient to a previously prepared feed. Also, the feed composition may be produced through a fermentation process using added active ingredients (especially Bifidobacterium bacteria). A feed composition manufactured through a fermentation process includes silage.

<3> Method of the present invention The method of the present invention is a method comprising the step of administering the above active ingredient to a subject. This step is also referred to as "administration step".

That is, the method of the present invention is a method comprising the step of administering a Bifidobacterium bacterium and/or a product of the bacterium to a subject.

According to the method of the present invention, specifically, by administering an active ingredient to a subject, skeletal muscle can be slowed in the subject, that is, a skeletal muscle slow-twitch effect can be obtained. That is, the method of the present invention may be a method for making skeletal muscles slow-twitch.

In addition, according to the method of the present invention, specifically, by administering an active ingredient to a subject, AMPK activation may be promoted in the subject, that is, an AMPK activation promoting effect may be obtained. That is, the method of the present invention may be a method of promoting AMPK activation. One aspect of the method for promoting AMPK activation may be, for example, a method for slowing skeletal muscle.

In addition, by the method of the present invention, specifically by administering an active ingredient to a subject, expression of PGC-1α may be promoted in the subject, that is, a PGC-1α expression promoting effect may be obtained. That is, the method of the present invention may be a method for promoting expression of PGC-1α. One aspect of the method for promoting expression of PGC-1α may be, for example, a method for making skeletal muscle slow-twitch.

Further, by administering an active ingredient to a subject according to the method of the present invention, an effect based on slow-moving skeletal muscle as exemplified above may be obtained in the subject. That is, the method of the present invention may be a method for improving endurance. The method of the present invention may also be a method of improving muscle strength. The method of the present invention may also be a method of preventing, ameliorating, and/or treating symptoms associated with muscle atrophy and/or muscle weakness. Any of these methods may be, for example, one aspect of a method for slowing skeletal muscle.

In addition, "administering an active ingredient to a subject" may be synonymous with "making a subject ingest an active ingredient." The intake may be voluntary (free intake) or forced (forced intake). That is, specifically, the administration step may be, for example, a step of mixing the active ingredient into food, drink or feed and supplying it to the subject, thereby allowing the subject to freely ingest the active ingredient. Administration may be oral or parenteral. Administration may generally be oral administration. Parenteral administration includes tube administration and rectal administration.

The mode of administration of the active ingredient (e.g., subject of administration, timing of administration, administration period, frequency of administration, dose, and other conditions related to administration) is not particularly limited as long as the desired effect such as the effect of slowing skeletal muscle is obtained. . The mode of administration of the active ingredient can be appropriately set according to various conditions such as the type of active ingredient and the type, age, and health condition of the subject to be administered.

The subject to whom the active ingredient is administered is not particularly limited as long as the desired effect such as the effect of slowing muscle growth of skeletal muscle is obtained. Subjects to which the active ingredient is administered include mammals. Mammals include humans and pets. Pets include dogs and cats. Mammals particularly include humans. Mammals may be male or female. The human condition to which the active ingredient is administered is not particularly limited. The active ingredient may be administered to any human desiring a desired effect, such as, for example, a skeletal muscle slowing effect. Humans may be of any age, such as infants, children, adults, middle-aged and elderly people, and the elderly. A human may be, for example, a healthy person or an unhealthy person. Non-healthy subjects include those who exhibit symptoms associated with muscle atrophy and/or weakness. A human may or may not be a person who performs physical exercise other than daily activities, such as an athlete or a sports enthusiast, for example.

The dose of the active ingredient is, for example, 1×10 ⁶ cells/kg body weight/day or more, 1×10 ⁷ cells/kg body weight/day or more, or 1×10 ⁸ cells/day in terms of bacterial cell dosage. kg body weight/day or more, 1×10 ¹² cells/kg body weight/day or less, 1×10 ¹¹ cells/kg body weight/day or less, or 1×10 ¹⁰ cells/kg body weight/day or less or a range of combinations thereof. Specifically, the dose of the active ingredient is, for example, 1×10 ⁶ to 1×10 ¹² cells/kg body weight/day, preferably 1×10 ⁷ to 1×10 cells/kg body weight/day, in terms of bacterial cell dosage. ¹¹ cells/kg body weight/day, more preferably 1×10 ⁸ to 1×10 ¹⁰ cells/kg body weight/day. When the active ingredient is viable cells, "cells" may be read as "cfu".

The dosage of the active ingredient is, for example, converted to the dosage of bacterial cells, 1×10 ⁷ cells/day/individual or more, 1×10 ⁸ cells/day/individual or more, or 1×10 ⁹ cells/day/individual or more. It may be 1×10 ¹⁴ cells/day/individual or less, 1×10 ¹³ cells/day/individual or less, or 1×10 ¹² cells/day/individual or less, or combinations thereof. may be in the range of Specifically, the dosage of the active ingredient is, for example, 1×10 ⁷ to 1×10 ¹⁴ cells/day/individual, preferably 1×10 ⁸ to 1×10 ¹³ , in terms of bacterial dosage. cells/day/individual, more preferably 1×10 ⁹ to 1×10 ¹² cells/day/individual. When the active ingredient is viable cells, "cells" may be read as "cfu".

The dose of the active ingredient may be, for example, 0.01 mL/kg body weight/day or more, or 0.1 mL/kg body weight/day or more, converted to the dose of the original culture, or 100 mL/kg. Body weight/day or less, or 10 mL/kg body weight/day or less, or a range of combinations thereof. Specifically, the dose of the active ingredient is, for example, 0.01 to 100 mL/kg body weight/day, preferably 0.1 to 10 mL/kg body weight/day in terms of the dose of the original culture. There may be. That is, for example, a culture of bacteria belonging to the genus Bifidobacterium or a product derived therefrom (culture supernatant, etc.) may be administered at dosages as exemplified above.

The administration period of the active ingredient may be, for example, 4 weeks or longer, preferably 8 weeks or longer, more preferably 12 weeks or longer. The active ingredient may be administered, for example, once a day, or divided into multiple doses per day. Alternatively, the active ingredient may be administered, for example, daily or once every few days. The active ingredient may, in particular, be administered daily. The dose of active ingredient at each administration may or may not be constant.

For example, the active ingredient may be administered to the subject as it is, or may be prepared as a composition such as a food composition, a pharmaceutical composition, or a feed composition containing the active ingredient and administered to the subject. The description of the composition of the present invention can be applied mutatis mutandis to the composition containing active ingredients. Also, the active ingredient may be administered alone or in combination with other ingredients. Other components include pharmaceuticals, pharmaceutical compositions, food and drink, food and drink compositions, feeds and feed compositions, and components contained therein. These other ingredients may or may not be used for the applications exemplified above, such as for slowing skeletal muscle.

Active ingredients can also be administered to a subject, for example, using a composition of the invention (ie, by administering a composition of the invention). That is, one aspect of the method of the present invention may be a method comprising the step of administering the composition of the present invention to a subject. Thus, "administration of an active ingredient" also includes administration of the composition of the present invention. The mode of administration of the composition of the present invention (e.g., subject of administration, timing of administration, administration period, frequency of administration, dosage, and other conditions related to administration) is as long as the desired effect such as the effect of slowing skeletal muscle is obtained. There are no particular restrictions. The mode of administration of the composition of the present invention includes the type and content of active ingredients, the type and content of other ingredients, the type and shape of the composition (dosage form), the type of administration subject, age, and health condition. It can be appropriately set according to various conditions. The above-described descriptions of modes of administration of active ingredients can also be applied mutatis mutandis to administration of the composition of the present invention to a subject. That is, the composition of the present invention can be administered to subjects such as those exemplified above. In addition, the dosage of the composition of the present invention can be set, for example, so as to obtain the dosage of the active ingredient as exemplified above. In addition, the composition of the present invention may be administered alone, and other ingredients such as pharmaceuticals, pharmaceutical compositions, food and drink, food and drink compositions, feeds and feed compositions, and ingredients contained therein They may be co-administered.

The present invention will now be described in more detail with reference to non-limiting examples.

[Production Example 1]
Bifidobacterium breve FERM BP-11175 was added to 3 mL of the MRS liquid medium and cultured anaerobically at 37°C for 16 hours. get A composition is obtained by uniformly mixing the fungus powder and a whey protein concentrate (WPC). 20 g of the composition is dissolved in 200 g of water to obtain a composition that can be used for specific purposes such as a composition for slowing skeletal muscle. The composition can be ingested, for example, as food or drink. By ingesting the same composition, the above-exemplified effects such as the effect of slowing down the skeletal muscles can be expected.

[Production Example 2]
Bifidobacterium breve FERM BP-11175 was added to 3 mL of the MRS liquid medium and cultured anaerobically at 37°C for 16 hours. get The bacterial powder and dry powder of milk protein concentrate (MPC480, manufactured by Fonterra, protein content 80% by mass, casein protein: whey protein = about 8:2) are uniformly mixed to obtain a composition. 20 g of the composition is dissolved in 200 g of water to obtain a composition that can be used for specific purposes such as a composition for slowing skeletal muscle. The composition can be ingested, for example, as food or drink. By ingesting the same composition, the above-exemplified effects such as the effect of slowing down the skeletal muscles can be expected.

[Example]
<Preparation of product to be administered>
A lyophilized powder of Bifidobacterium breve FERM BP-11175 was diluted with physiological saline to a concentration of 5×10 ⁸ cfu/mL to obtain a live cell administration product. A live cell-administered product of Bifidobacterium breve FERM BP-11175 was heated at 90° C. for 30 minutes to obtain a heat-sterilized product.

<Animal test>
Using Crl:CD(SD) rats (Charles River Japan: healthy rats) as experimental animals, continuous intake of Bifidobacterium breve FERM BP-11175 with live and heat-killed products administered for 4 weeks. was evaluated for the effect of on muscle fiber types in muscle tissue.

After being purchased at 7 weeks of age, experimental animals were allowed to freely ingest Labo MR stock feed (manufactured by Nosan Kogyo Co., Ltd.) and tap water. After one week of acclimatization, the experimental animals were divided into a control group (a group administered with 5 mL/kg body weight/day of physiological saline), and a group administered with live Bifidobacterium (2 mL/day/animal of a live cell administration product (bacteria 1 × 10 ⁹ cfu / day / animal in terms of number) administration group), Bifidobacterium heat-killed body administration group (heat-killed body administration product 2 mL / day / animal (1 × 10 ⁹ cells / The mice were divided into groups (day/mouse) and administration groups), and oral administration of the administration product using an oral probe was continued to each mouse once a day for 4 weeks. One hour after the end of the last oral administration, the experimental animals were treated under sevoflurane anesthesia and the soleus and gastrocnemius muscles were removed.

The gastrocnemius muscle was used for the measurement of the slow-twitch fiber ratio by the following procedure. Specifically, the excised gastrocnemius muscle was sliced and the slow-twitch fibers were stained by immunohistochemical staining using an Anti-SERCA2 ATPase antibody (Abcam). For each group, 3 visual fields of the central part of the medial head of the gastrocnemius muscle were observed under a microscope, and the staining rate (percentage of slow-twitch fibers) was measured for a total of 450 fibers.

FIG. 1 shows the percentage of slow-twitch fibers in each group. Figures 2 to 4 show the slow-twitch fiber staining images of each group. In FIGS. 2 to 4, dark regions are slow-twitch fibers, and bright regions are fast-twitch fibers. Administration of Bifidobacterium breve FERM BP-11175 increased the percentage of slow-twitch fibers compared to controls. In particular, it was confirmed that the ratio of slow-twitch fibers was significantly increased in the heat-killed body administration group than in the live cell administration group. Specifically, compared with the control, the slow-twitch fiber ratio increased by 3.80% in the live bacterial body-administered group, and the slow-twitch fiber ratio increased by 12.70% in the heat-sterilized body-administered group. As described above, it was confirmed that administration of Bifidobacterium breve FERM BP-11175 slowed skeletal muscle.

Soleus muscle was subjected to the following procedures to identify signal pathways involved in skeletal muscle slow-twitching (Zhang et al., Int J Biol Sci, 13(9), 1152-1162 (2017); Suwa et al., J. Appl Physiol, 95, 960-968 (2003)). As a signaling pathway involved in skeletal muscle slow-twitching, the amount of activated AMP-activated protein kinase (AMPK) was measured by Western blotting, and the expression of peroxisome proliferator-activated receptor gamma-coupling factor-1α (PGC-1α) was measured. The amount was measured by real-time PCR method.

RNA was extracted from a portion of excised soleus muscle using RNeasy Mini Kit (Qiagen), and a complementary DNA (cDNA) library was prepared using PrimeScript RT Reagent Kit (Takara Bio). Using the resulting cDNA library as a template, SYBR Premix Ex Taq2 (Takara Bio Inc.) and primers for PGC-1α (SEQ ID NOS: 1 and 2) or primers for endogenous control GAPDH (SEQ ID NOS: 3 and 4) were used. PCR was performed to quantify the expression level of PGC-1α at the mRNA level. At this time, the expression level of PGC-1α was standardized based on the expression level of GAPDH and calculated as a relative value to the control.

FIG. 5 shows the expression level of PGC-1α. Administration of Bifidobacterium breve FERM BP-11175 increased the expression level of PGC-1α compared to the control. In particular, a marked increase in the expression level of PGC-1α was confirmed in the heat-killed body administration group than in the live cell administration group. Specifically, compared with the control, the expression level of PGC-1α increased 1.31 times in the group administered with live cells, and the expression level of PGC-1α increased 1.90 times in the heat-killed cells. .

Proteins were extracted from a portion of the excised soleus muscle using RIPA buffer (CST), and phosphorylated AMPK (activated AMPK) was quantified by Western blotting. At this time, the phosphorylated AMPK amount was standardized based on the total AMPK amount and calculated as a relative value to the control.

FIG. 6 shows phosphorylated AMPK levels. Administration of Bifidobacterium breve FERM BP-11175 increased the amount of active AMPK compared to the control. Specifically, the amount of active AMPK increased 1.42-fold in the group administered with viable cells, and the amount of active AMPK increased 1.48-fold in the group administered with heat-killed cells, as compared with the control.

The above results suggest that administration of Bifidobacterium breve FERM BP-11175 activates signal pathways involved in promoting slow-twitch muscle formation, resulting in an increase in the proportion of slow-twitch muscle in skeletal muscle. This phenomenon was more pronounced in the heat-killed body administration group than in the live cell administration group.

<Description of Sequence Listing>
SEQ ID NOs: 1-4: primers

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a composition that can be used for specific purposes such as a composition for slowing skeletal muscles.

Claims (7)

A composition for slow-twitching skeletal muscle, containing the following component (A) as an active ingredient:
(A) Heat-killed form of Bifidobacterium breve FERM BP-11175 . A composition for improving endurance, containing the following component (A) as an active ingredient:
(A) Heat-killed form of Bifidobacterium breve FERM BP-11175 . A composition for preventing, ameliorating, and/or treating symptoms associated with muscle atrophy and/or muscle weakness, containing the following component (A) as an active ingredient:
(A) Heat-killed form of Bifidobacterium breve FERM BP-11175 . A composition according to any one of claims 1 to 3, wherein said composition is a pharmaceutical composition. The composition according to any one of claims 1 to 3, wherein the composition is a food or beverage composition. A method for slowing skeletal muscle (excluding a method for treating humans), which comprises the step of administering the following component (A) to a subject:
(A) Heat-killed form of Bifidobacterium breve FERM BP-11175 . A method of preventing, ameliorating, and/or treating symptoms associated with muscle atrophy and/or muscle weakness (excluding methods of treating humans), comprising administering the following component (A) to a subject:
(A) Heat-killed form of Bifidobacterium breve FERM BP-11175 .

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