JP7555439B2 - Composition for slowing down skeletal muscle - Google Patents

Description

IPOD FERM BP-11175

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

Muscles are the basis for everyday movements such as standing, walking, and maintaining posture. Therefore, in order to maintain quality of life, people of all ages and genders desire to maintain muscle mass as much as possible and, if possible, increase muscle mass. In addition, people who actively enjoy exercise and athletes (e.g., athletes, sports enthusiasts, competitors, etc.) desire to increase muscle mass as much as possible in order to improve their athletic performance.

Furthermore, as health consciousness grows, quality of life (QO)
When trying to improve physical fitness (L), improve frailty (a state between a healthy state and a state requiring nursing care), or combat locomotive syndrome, the challenge is how to maintain declining endurance and muscle strength.

Muscles are composed of bundles of muscle fibers. Based on properties such as contractile tension, contractile speed, and fatigue resistance, muscle fibers are classified into slow-twitch fibers (Type I) and fast-twitch fibers (Type II). Muscles with many slow-twitch fibers have low contractile force and contractile speed but are less prone to fatigue, which contributes to endurance. On the other hand, muscles with many fast-twitch fibers have high contractile force and contractile speed but are easily fatigued. Muscles with many slow-twitch fibers also act as antigravity muscles. In patients with disuse muscular atrophy, the muscles change from slow-twitch dominant to fast-twitch dominant, and fast-twitch muscles are more likely to atrophy due to inactivity than slow-twitch muscles, leading to a state that interferes with daily life, such as difficulty walking or becoming bedridden. By maintaining or strengthening slow-twitch muscles, endurance and muscle strength can be maintained, preventing or improving lack of exercise and difficulty in daily activities.

Ginger extract and the like are known to be substances that can promote slow-twitch skeletal muscles (Patent Document 1).

It has also been reported that administration of Bifidobacterium breve B-3 increases muscle mass (Non-Patent Document 1).

Patent Publication No. 2014-101346

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

The objective of the present invention is to provide a composition that can be used for specific purposes, such as a composition for slowing down skeletal muscle.

As a result of intensive research, the inventors discovered that administration of Bifidobacterium bacteria can have effects such as slowing down skeletal muscles, and thus completed the present invention.

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

Figure showing the percentage of slow-twitch muscle fibers in each group. Data were obtained from seven rats in each group and shown as mean ± standard error. Asterisks (*) indicate significant differences (P<0.05) by Dunnett's method. Stained image of slow muscle fibers in the control group (photo). Stained image of slow muscle fibers in the group administered live Bifidobacterium breve FERM BP-11175 (photo). Stained images of slow muscle fibers in the group administered heat-killed Bifidobacterium breve FERM BP-11175 (photo). Figure showing the relative expression level of PGC-1α in each group. Data were obtained from 12 or 13 rats in each group and shown as mean ± standard error. Asterisks (*) indicate significant differences (P<0.05) by Dunnett's method. Figure showing the relative amount of phosphorylated AMPK in each group. Data were obtained from six rats in each group and shown as mean ± standard error. Asterisks (*) indicate significant differences (P<0.05) by Dunnett's method.

The present invention will be described in detail below. Note that, unless otherwise specified, the following descriptions of the present invention may be used alone or in appropriate combination.

<1> Active Ingredient In the present invention, the following component (A) is used as an active ingredient:
(A) Bifidobacterium and/or products produced by said bacteria.

In other words, the above component (A) is also called the "active component."

By utilizing an active ingredient, specifically by administering the active ingredient to a subject, the skeletal muscle of the subject can be slow-twitched, that is, the effect of slow-twitching the skeletal muscle of the subject can be obtained. This effect is also called "skeletal muscle slow-twitching effect". Examples of skeletal muscle include trunk muscles (i.e., skeletal muscles of the trunk) and limb muscles (i.e., skeletal muscles of the upper and lower limbs). Examples of skeletal muscles of the lower limbs include gastrocnemius muscles and soleus muscles. The skeletal muscle slow-twitching effect may be obtained in all skeletal muscles (i.e., skeletal muscles of the entire body) or in a part of skeletal muscles. The skeletal muscle slow-twitching effect may be obtained, for example, in a region with a high ratio of slow muscles, in a region with a high ratio of fast muscles, or in both. The skeletal muscle slow-twitching effect may be obtained, for example, at least in the gastrocnemius and/or soleus muscles. "Slow-twitching of skeletal muscle" means an increase in the amount of slow muscles in skeletal muscles. In other words, "slow muscle conversion of skeletal muscle" means that the amount of slow muscle in skeletal muscle is larger when the active ingredient is administered compared to when the active ingredient is not administered. "When the active ingredient is administered" may mean after the active ingredient has been administered sufficiently, and specifically, after the active ingredient is administered under the conditions described in the method of the present invention described later. "When the active ingredient is not administered" may simply mean before the active ingredient is administered. However, "when the active ingredient is not administered" may more precisely mean a hypothetical condition that the same period as the administration period of the active ingredient has passed without administering the active ingredient. That is, for example, in the case where the amount of slow muscle in skeletal muscle is reduced by non-administration of the active ingredient, "slow muscle conversion of skeletal muscle" is sufficient if the degree of the reduction is reduced by administration of the active ingredient, and does not require the amount of slow muscle in skeletal muscle to be larger after administration of the active ingredient compared to before administration of the active ingredient. An example of such a case is when the onset or progression of muscle atrophy of slow muscle is reduced by administration of the active ingredient. "Amount of slow muscle in skeletal muscle" may mean the absolute amount of slow muscle in skeletal muscle, the relative amount of slow muscle in skeletal muscle (i.e., the ratio of the amount of slow muscle to the amount of skeletal muscle), or both. That is, the absolute amount and/or the relative amount of slow muscle may be increased by the use of the active ingredient. The relative amount of slow muscle may be increased at least by the use of the active ingredient. The total amount of skeletal muscle may be increased with the increase in the amount of slow muscle. The amount of slow muscle includes the number of slow muscle fibers, the cross-sectional area of slow muscle fibers, and the volume of slow muscle fibers. That is, for example, the relative amount of slow muscle in skeletal muscle includes the ratio of the number of slow muscle fibers to the number of muscle fibers in skeletal muscle, the ratio of the cross-sectional area of slow muscle fibers to the cross-sectional area of skeletal muscle, and the ratio of the volume of slow muscle fibers to the volume of skeletal muscle. "Slow muscle fiber" means Type I muscle fiber. The use of the active ingredient may increase, for example, one or more of these parameters. The use of the active ingredient may increase, for example, at least the number of slow muscle fibers. The amount of slow muscle fibers can be measured by known methods, for example. Methods for measuring the amount of slow muscle fibers include muscle biopsy, magnetic resonance imaging (MRI), and the like.
Imaging (MRI), and field tests. The ratio of the number of slow muscle fibers to the number of muscle fibers in skeletal muscle can be specifically measured, for example, by the method described in the Examples. The amount of slow muscle fibers in skeletal muscle when the active ingredient is administered may be, for example, 101% or more, 103% or more, 105% or more, 110% or more, 115% or more, 120% or more, 125% or more, or 130% or more, when the amount when the active ingredient is not administered is taken as 100%. In addition, slow muscle fiberization of skeletal muscle can also be measured, for example, using improved fatigue resistance, improved oxidative enzyme activity in skeletal muscle, or improved number of mitochondria in skeletal muscle as an index.

In addition, by utilizing the active ingredient, specifically by administering the active ingredient to a subject, the activation of AMP-activated protein kinase (AMPK) may be promoted in the subject, that is, the effect of promoting the activation of AMPK in the subject may be obtained. This effect is also referred to as "AMPK activation promoting effect". The activation of AMPK may be promoted, particularly in skeletal muscle. The promotion of the activation of AMPK may result in a slow-muscle skeletal muscle effect. "Promotion of AMPK activation" means an increase in the amount of active AMPK (i.e., phosphorylated AMPK). In other words, "promotion of AMPK activation" means that the amount of active AMPK is larger when the active ingredient is administered compared to when the active ingredient is not administered. "Amount of active AMPK" may mean a value standardized based on the total amount of AMPK, unless otherwise specified. The amount of active AMPK can be measured, for example, by a known method. An example of a method for measuring the amount of active AMPK is Western blotting. The amount of active AMPK can be measured, for example, by a method described in the Examples. The amount of active AMPK when the active ingredient is administered may be, for example, 101% or more, 103% or more, 105% or more, 110% or more, 115% or more, 120% or more, 125% or more, or 130% or more, relative to 100% when the active ingredient is not administered.

In addition, by utilizing an active ingredient, specifically by administering the active ingredient to a subject, the expression of peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) may be promoted in 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 a "PGC-1α expression promoting effect." The expression of PGC-1α may be promoted, particularly in skeletal muscle. The promotion of the expression of PGC-1α may provide a skeletal muscle slowing effect. "Promotion of PGC-1α expression" means an increase in the expression level of PGC-1α. In other words, "promotion of PGC-1α expression" means that the expression level of PGC-1α is higher when the active ingredient is administered compared to when the active ingredient is not administered. "Expression level of PGC-1α" may mean a standardized value based on the expression level of a housekeeping gene such as GAPDH, unless otherwise specified. The expression level of PGC-1α can be measured, for example, by a known method for measuring the expression level of a gene. Methods for measuring gene expression levels include methods for measuring the amount of mRNA and methods for measuring the amount of protein. Specific examples of methods for measuring gene expression levels include Northern hybridization, microarrays, RT-PCR, RNA-seq,
Examples of such methods include Western blotting. The expression level of PGC-1α can be specifically measured by, for example, the method described in the Examples. The expression level of PGC-1α when the active ingredient is administered may be, for example, 101% or more, 103% or more, 105% or more, 110% or more, 115% or more, 120% or more, 125% or more, or 130% or more, when the expression level when the active ingredient is not administered is 100%.

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

It is expected that slow-twitch skeletal muscle can improve the endurance of a subject, for example. That is, an effect based on slow-twitch skeletal muscle can be an effect of improving the endurance of a subject (endurance improvement effect). "Endurance" means endurance for physical exercise. That is, "endurance" may specifically mean the ability to continue physical exercise. The physical exercise is not particularly limited as long as it is an action that puts a load on the muscles. The physical exercise can be sports, physical training, physical labor, or daily movements.

By converting skeletal muscles to slow-twitch muscles, it is expected that, for example, the muscle strength of a subject can be improved. 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 a subject can be improved. In other words, an effect based on converting skeletal muscles to slow-twitch muscles is the effect of improving the muscle strength of a subject (muscle strength improvement effect).

It is expected that the slow-twitching of skeletal muscles can prevent, improve, and/or treat symptoms associated with muscle atrophy and/or muscle weakness. That is, the effects based on the slow-twitching of skeletal muscles include the effects of preventing, improving, and/or treating symptoms associated with muscle atrophy and/or muscle weakness. Muscle atrophy refers to a decrease in muscle mass. Examples of muscle atrophy include atrophy of skeletal muscles. Specific examples of muscle atrophy include atrophy of slow and/or fast muscles. Examples of muscle atrophy include atrophy of slow muscles. Muscle atrophy may result in, for example, a decrease in muscle strength. In other words, the muscle weakness may be due to, for example, muscle atrophy. Symptoms associated with muscle atrophy and/or muscle weakness may be a disease or may not be a disease. Symptoms associated with muscle atrophy and/or muscle weakness may develop or progress due to any of genetic factors, acquired factors, aging, and the like. Symptoms related to muscle atrophy and/or muscle weakness include symptoms caused by muscle atrophy and/or muscle weakness and symptoms accompanied by muscle atrophy and/or muscle weakness. Symptoms related to muscle atrophy and/or muscle weakness include muscle atrophy itself and muscle weakness itself. Specific examples of symptoms related to muscle atrophy and/or muscle weakness include disuse muscle atrophy, neurogenic muscle atrophy, and myogenic muscle atrophy. Examples of disuse muscle atrophy include muscle atrophy due to aging (sarcopenia) and muscle atrophy due to lack of use of muscles. Examples of muscle lack of use include being bedridden, not exercising, flying in zero gravity, and immobilizing the body. Examples of immobilizing the body include immobilizing the limbs performed in the treatment of injuries. Neurogenic muscular atrophy includes amyotrophic lateral sclerosis (ALS), primary lateral sclerosis (PLS), spinal muscular atrophy (SMA), spinal and bulbar muscular atrophy (SBMA), poliomyelitis (polio), and Guillain-Barre syndrome (GBS).
Examples of myogenic muscle atrophy include muscular dystrophy and polymyositis (PM). Specific examples of conditions related to muscle atrophy and/or muscle weakness include myasthenia gravis (MG), cachexia, frailty, and locomotive syndrome.
Other examples include idiopathic encephalopathy syndrome.

The active ingredient may or may not be used in combination with other techniques that can provide the above-mentioned effects, such as the skeletal muscle slow-twitching effect. Such techniques include physical exercise other than daily movements. Physical exercise other than daily movements includes sports and physical training. By using the active ingredient in combination with such techniques, it is expected that the desired effects, such as the skeletal muscle slow-twitching effect, provided by such techniques will be enhanced.

The present invention also provides the use of the active ingredient in applications associated with the above-exemplified effects such as the skeletal muscle slowing effect. That is, the present invention may provide, for example, the use of the active ingredient for slowing skeletal muscle, the use of the active ingredient for promoting AMPK activation, the use of the active ingredient for promoting PGC-1α expression, the use of the active ingredient for improving endurance, the use of the active ingredient for improving muscle strength, and the use of the active ingredient for preventing, improving, and/or treating symptoms associated with muscle atrophy and/or muscle weakness. The present invention may also provide, for example, the use of the active ingredient for the manufacture of the composition of the present invention, such as a composition for slowing skeletal muscle, a composition for promoting AMPK activation, a composition for promoting PGC-1α expression, and a composition for preventing, improving, and/or treating symptoms associated with muscle atrophy and/or muscle weakness.

The present invention also provides an active ingredient for use in applications associated with the above-exemplified effects such as the skeletal muscle slowing effect. That is, the present invention may provide, 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 the expression of PGC-1α, an active ingredient for use in improving endurance, an active ingredient for use in improving muscle strength, and an active ingredient for use in preventing, improving, and/or treating symptoms associated with muscle atrophy and/or muscle weakness. The present invention may also provide, for example, an active ingredient for use in the manufacture of the composition of the present invention, such as a composition for slowing skeletal muscle, a composition for promoting AMPK activation, a composition for promoting the expression of PGC-1α, and a composition for preventing, improving, and/or treating symptoms associated with muscle atrophy and/or muscle weakness.

The active ingredient may be used for therapeutic purposes or for non-therapeutic purposes. In other words, all of the effects exemplified above may be obtained for therapeutic purposes or for non-therapeutic purposes, unless otherwise specified. "Therapeutic purposes" may mean, for example, including treatment of the human body for treatment, and may particularly mean being performed as a medical procedure. "Non-therapeutic purposes" may mean, for example, not including treatment of the human body for treatment, and may particularly mean being performed as a non-medical procedure. Non-therapeutic purposes include purposes such as health promotion and beauty.

"Prevention of a symptom" may mean, for example, preventing or delaying the onset of a symptom, or reducing the likelihood of onset of a symptom. "Amelioration of a symptom" or "treatment of a symptom" may mean, for example, improvement of a symptom, preventing or delaying worsening of a symptom, or preventing or delaying progression of a symptom. "Amelioration of a symptom" may specifically refer to these events, but is not intended for therapeutic purposes. "Treatment of a symptom" may specifically refer to these events, but is intended for therapeutic purposes.

The Bifidobacterium bacteria are not particularly limited as long as the desired effects, such as the effect of slowing down skeletal muscle, are obtained. As the Bifidobacterium bacteria, one type of bacteria may be used, or two or more types of bacteria may be used in combination.

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

Bifidobacterium bacteria include, in particular, Bifidobacterium breve, Bifidobacterium longum,
Bifidobacterium bifidum, Bifidobacterium adolescentis, Bifidobacterium dentium, Bifidobacterium animalis, Bifidobacterium pseudolongum
olongum), Bifidobacterium thermophilum
Examples of the genus Bifidobacterium include, more particularly, Bifidobacterium breve and Bifidobacterium longum. Examples of the genus Bifidobacterium include, more particularly, Bifidobacterium breve.

Bifidobacterium longum includes strains classified into any subspecies of Bifidobacterium longum, such as B. longum subsp. longum, B. longum subsp. infantis, B. longum subsp. suis, etc. Bifidobacterium animalis includes strains classified into any subspecies of Bifidobacterium animalis, such as B. animalis subsp. lactis. Bifidobacterium pseudolongum includes strains classified into any subspecies of Bifidobacterium pseudolongum, such as B. pseudolongum subsp. globosum and B. pseudolongum subsp. pseudolongum.

Specific examples of Bifidobacterium breve include 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, and R0070. Specific examples of Bifidobacterium breve include FERM BP-11175.

Bifidobacterium breve FERM BP-11175 has been internationally deposited on August 25, 2009, under the accession number FERM BP-11175, at the National Institute of Advanced Industrial Science and Technology (currently the National Institute of Technology and Evaluation Patent Organism Depositary (NITE-IPOD), postal code: 292-0818, address: Room 120, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) based on the Budapest Treaty.

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).

Bifidobacterium bifidum, specifically, ATCC 29521, OLB6378, BF-1
Examples of Bifidobacterium adolescentis include ATCC 15703, and examples of Bifidobacterium dentium include DSM 20436.
Examples of Bifidobacterium animalis include DSM 10140, Bb-12, DN-173 010, GCL2505, and CNCM I-3446. Examples of Bifidobacterium pseudolongum include JCM 5820 and ATCC 25526. Examples of Bifidobacterium thermophilum include ATCC 25525.

These strains are available from, for example, the 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, 3-1-1 Takanodai, Tsukuba, Ibaraki, Japan, 305-0074, Japan), or from the depository institutions to which the respective strains have been deposited.

The strains specified by the above-mentioned strain names are not limited to the strains deposited or registered with a given institution under the above-mentioned strain names (hereinafter, for convenience of explanation, also referred to as "deposited strains"), but also include substantially equivalent strains thereto (also referred to as "derived strains" or "derived strains"). That is, for example, "Bifidobacterium breve FERM BP-11175" is not limited to the strain deposited with the above-mentioned depository institution under the deposit number FERM BP-11175, but also includes substantially equivalent strains thereto. For each strain, "a strain substantially equivalent to the deposited strain" refers to a strain that belongs to the same species as the deposited strain, that can obtain a desired effect such as a skeletal muscle slowing effect by its use, and that further has an identity of preferably 99.86% or more, more preferably 99.93% or more, and even more preferably 100% of the base sequence of the 16SrRNA gene to the base sequence of the 16SrRNA gene of the deposited strain, and preferably has the same mycological properties as the deposited strain. For each strain, the strain substantially equivalent to the deposited strain may be one that can obtain a desired effect such as a skeletal muscle slowing effect equivalent to or greater than that of the deposited strain. "An effect equivalent to or greater than that of the deposited strain" may mean an effect of 80% or more, 90% or more, 95% or more, or 100% or more of the effect obtained by the use of the deposited strain. For each strain, the strain substantially equivalent to the deposited strain may be, for example, a derivative strain with the deposited strain as a parent strain. The derivative strains include strains bred from the deposited strain and strains naturally occurring from the deposited strain. Breeding methods include modification by genetic engineering techniques and modification by mutation treatment. Mutation treatments include irradiation with X-rays, irradiation with ultraviolet light, and treatment with mutagens such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), ethyl methanesulfonate (EMS), and methyl methanesulfonate (MMS). Strains naturally occurring from the deposited strain include strains naturally occurring during use of the deposited strain. Such strains include mutant strains naturally occurring by culturing (e.g., subculturing) the deposited strain. The derivative strains may be constructed by one type of modification, or by two or more types of modifications.

The term "bifidobacterium bacteria" as an active ingredient may refer to the bacterial cells of the genus Bifidobacterium.

The term "product of Bifidobacterium bacteria" as an active ingredient may mean a component produced by culturing Bifidobacterium bacteria. The product of Bifidobacterium bacteria is not particularly limited as long as it can produce the desired effect, such as a skeletal muscle slowing effect. Examples of products of Bifidobacterium bacteria include components that accumulate in the medium when the bacteria are cultured, components that accumulate within the bacteria when the bacteria are cultured, and components that constitute the bacterial body of the bacteria. Examples of components that constitute the bacterial body include cell walls and peptidoglycan. Note that the bacterial body of Bifidobacterium bacteria itself does not necessarily fall under the category of "product of Bifidobacterium bacteria".

As the active ingredient, a commercially available product may be used, or one obtained by appropriate production may be used. For example, an example of a commercially available product of the genus Bifidobacterium is Bifidobacterium breve FERM BP-11175 manufactured by Morinaga Milk Industry Co., Ltd. Also, the active ingredient can be easily obtained by culturing the genus Bifidobacterium. The culture method is not particularly limited as long as the genus Bifidobacterium can grow. As the culture method, for example, a method normally used for culturing the genus Bifidobacterium can be used as is or with appropriate modification. The culture temperature may be, for example, 25 to 50°C, preferably 35 to 42°C. The culture may be preferably performed under anaerobic conditions, for example, while aerating with anaerobic gas such as carbon dioxide gas. The culture may also be performed under microaerobic conditions such as liquid stationary culture. The culture may be performed, for example, until the genus Bifidobacterium grows to a desired extent.

The medium used for the culture is not particularly limited as long as it allows the proliferation of Bifidobacterium bacteria. As the medium, for example, a medium normally used for the culture of Bifidobacterium bacteria can be used as is or after appropriate modification. The medium may contain, for example, a carbon source, a nitrogen source, an inorganic salt, an organic component, a milk component, or a combination thereof. As the carbon source, for example, sugars such as glucose, galactose, lactose, arabinose, mannose, sucrose, starch, starch hydrolysate, and blackstrap molasses can be used depending on the assimilation ability. As the nitrogen source, for example, ammonium salts such as ammonia, ammonium sulfate, ammonium chloride, and ammonium nitrate, and nitrates can be used. As the inorganic salt, for example, sodium chloride, potassium chloride, potassium phosphate, magnesium sulfate, calcium chloride, calcium nitrate, manganese chloride, ferrous sulfate, etc. can be used. As the organic component, for example, peptone, soybean flour, defatted soybean meal, meat extract, yeast extract, etc. can be used. In addition, milk components that can be used include, for example, milk proteins. Examples of milk proteins include casein, whey, and hydrolyzates thereof. These components may be used alone or in appropriate combination. In addition, specific examples of media that are commonly used to culture Bifidobacterium bacteria include Reinforced Clostridial medium, de Man, Rogosa, and Sharpe medium (MRS medium), modified MRS medium (mMRS medium), TOS propionate medium, and TOS propionate mupirocin medium (TOSP Mup medium).

As the active ingredient, the active ingredient itself or a fraction containing it can be used without any particular limitation. That is, as the Bifidobacterium bacteria, the cells of the Bifidobacterium bacteria or a fraction containing it can be used without any particular limitation. The fraction containing the cells of the Bifidobacterium bacteria may contain a product produced by the Bifidobacterium bacteria in addition to the cells. In addition, as the product produced by the Bifidobacterium bacteria, a component produced by culturing the Bifidobacterium bacteria or a fraction containing it can be used without any particular limitation. The fraction containing the component produced by culturing the Bifidobacterium bacteria may contain the Bifidobacterium bacteria in addition to the component. As the active ingredient, for example, the culture obtained by culturing may be used as it is, or the cells or culture supernatant recovered from the culture may be used, or they may be appropriately treated before use. Specifically, the active ingredient may be a culture of Bifidobacterium bacteria, cells collected from the culture, culture supernatant collected from the culture, or a processed product thereof. The treatment is not particularly limited as long as it does not impair the desired effect, such as the effect of slowing skeletal muscle. Examples of the treatment include concentration, dilution, drying, sterilization, crushing, and fractionation. These treatments may be performed alone or in appropriate combination. All of these treatments can be performed, for example, by known techniques. Drying can be performed, for example, by freeze-drying.

For example, the cells may be disrupted and the disrupted cells may be used as an active ingredient. Methods for disrupting the cells include ultrasonic treatment and homogenization. The disrupted cells may be used as is or after being appropriately subjected to the above-mentioned treatments as needed, as an active ingredient.

Also, for example, from the above-mentioned culture, bacterial cells, culture supernatant, or processed products thereof (e.g., bacterial cell disruption), a fraction containing the active ingredient may be fractionated and used as the active ingredient. Specifically, for example, from the bacterial cell disruption, a fraction such as a supernatant fraction or a precipitate fraction may be fractionated and used as the active ingredient. Also, for example, from the above-mentioned culture, bacterial cells, culture supernatant, or processed products thereof (e.g., bacterial cell disruption), an active ingredient may be fractionated (i.e., separated and purified) and used. Separation and purification of the active ingredient may be performed to a desired extent. Separation and purification of the active ingredient may be performed, for example, by a known method used for separation and purification of compounds. Examples of such methods include centrifugation, salting out, solvent extraction, gel filtration, HPLC, and membrane separation. For example, a polar solvent, a nonpolar solvent, or a mixture thereof may be used for solvent extraction.

As the active ingredient, in particular, Bifidobacterium bacteria (specifically, Bifidobacterium bacteria cells or a fraction containing the same) may be used. In other words, the active ingredient may in particular be Bifidobacterium bacteria (specifically, Bifidobacterium bacteria cells or a fraction containing the same). Specific examples of Bifidobacterium bacteria include cultures of Bifidobacterium bacteria, cells recovered from the cultures, and their dilutions, concentrates, dried products, sterilized products, and other processed products. The cells may be live or killed cells. The cells may be used in a form that contains killed cells, in particular. The cells may be, for example, dead cells, or a mixture of dead 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 cells. Killed bacteria can be prepared, for example, by subjecting live bacteria to a sterilization treatment. Examples of sterilization treatments include heat treatment, spray drying, retort sterilization, freeze drying, UHT sterilization, pressure sterilization, high-pressure steam sterilization, dry heat sterilization, circulating steam disinfection, 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 electrolyzed water treatment. Examples of heat treatments include 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 above-mentioned active ingredient.

That is, the composition of the present invention is a composition containing bacteria of the genus Bifidobacterium and/or a product produced by said bacteria.

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

By utilizing the composition of the present invention, specifically by administering the composition of the present invention to a subject, it is possible to slow-induce skeletal muscle in the subject, i.e., a skeletal muscle slow-induction effect can be obtained. In other words, the composition of the present invention may be 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, activation of AMPK in the subject may be promoted, i.e., an AMPK activation promoting effect may be obtained. In other words, the composition of the present invention may be a composition for promoting AMPK activation. One embodiment of the composition for promoting AMPK activation may be, for example, a composition for slow-twitching skeletal muscle.

Furthermore, by utilizing the composition of the present invention, specifically by administering the composition of the present invention to a subject, the expression of PGC-1α in the subject may be promoted, i.e., a PGC-1α expression promoting 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 slow-twitching skeletal muscle.

Furthermore, by utilizing the composition of the present invention, specifically by administering the composition of the present invention to a subject, the effects based on the slow-twitching of skeletal muscles as exemplified above may be obtained in the subject. That is, the composition of the present invention may be a composition for improving endurance. The composition of the present invention may also be a composition for improving muscle strength. The composition of the present invention may also be a composition for preventing, improving, and/or treating symptoms associated with muscle atrophy and/or muscle weakness. Any of these compositions may be, for example, one embodiment of a composition for slow-twitching of skeletal muscles.

The description of the subject to which the active ingredient in the "method of the present invention" is administered can be applied mutatis mutandis to the subject to which the composition of the present invention is administered.

The composition of the present invention may be, for example, a food or beverage composition, a pharmaceutical composition, or a feed composition. The composition of the present invention may in particular be a food or beverage composition or a pharmaceutical composition. That is, the present invention may provide, for example, a food or beverage composition for the above-exemplified uses such as for slow-twitching skeletal muscle, a pharmaceutical composition for the above-exemplified uses such as for slow-twitching skeletal muscle, or a feed composition for the above-exemplified uses such as for slow-twitching skeletal muscle. The composition of the present invention that is a food or beverage composition, a pharmaceutical composition, or a feed composition is also referred to as the "food or beverage composition of the present invention," the "pharmaceutical composition of the present invention," and the "feed composition of the present invention," respectively.

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

The components 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 skeletal muscle. As the components other than the active ingredient, any component that is acceptable depending on the mode of use of the composition of the present invention can be used. As the components other than the active ingredient, for example, components that are blended and used in foods and beverages, pharmaceuticals, or feed can be used. Specific examples of the components other than the active ingredient include the components exemplified for the food and beverage compositions, pharmaceutical compositions, and feed compositions described below. As the components other than the active ingredient, one type of component may be used, or two or more types of components may be used in combination.

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

The content and content ratio of each component (i.e., the active ingredient and optionally other ingredients) 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 muscle, is obtained. The content and content ratio of each component in the composition of the present invention can be appropriately set depending on various conditions such as the type of active ingredient, the type of other ingredients, the type of composition, the shape (dosage form), and the method of use, the type, age, and health condition of the subject to be administered.

The content of the active ingredient in the composition of the present invention, converted into the content of bacterial cells, may be, for example, 1 x 10 ⁴ cells/g or more, 1 x 10 ⁵ cells/g or more, 1 x 10 ⁶ cells/g or more, 1 x 10 ⁷ cells/g or more, or 1 x 10 ⁸ cells/g or more, or 10 ¹³ cells/g or less, 10 ¹² cells/g or less, or 1 x 10 ¹¹ cells/g or less, or any combination of these ranges. The content of the active ingredient in the composition of the present invention may be, for example, in terms of the content of bacterial cells, 1×10 ⁴ cells/mL or more, 1×10 ⁵ cells/mL or more, 1×10 ⁶ cells/mL or more, 1×10 ⁷ cells/mL or more, or 1×10 ⁸ cells/mL or more, or 10 ¹³ cells/mL or less, 10 ¹² cells/mL or less, or 1×10 ¹¹ cells/mL or less, or a combination of these. Specifically, the content of the active ingredient in the composition of the present invention may be, for example, in terms of the content of bacterial cells, 1×10 ⁶ to 1×10 ¹² cells/g or 1×10 ⁶ to 1×10 ¹² cells/mL, preferably 1×10 ⁷ to 1×10 ¹¹ cells/g or 1×10
The concentration may be 1× ^{10 7} to 1×10 ¹¹ cells/mL, more preferably 1×10 ⁸ to 1×10 ¹⁰ cells/g or 1×10 ⁸ to 1×10 ¹⁰ cells/mL. When the active ingredient is a live bacterial cell, "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 may be, for example, 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, or 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) or less, 70% (w/w) or less, 50% (w/w) or less, 30% (w/w) or less, 10% (w/w) or less, 5% (w/w) or less, or 1% (w/w) or less, or any compatible combination thereof, calculated based on the content of the original culture. Specifically, the content of the active ingredient in the composition of the present invention may be, for example, 1 to 10,000% (w/w), preferably 10 to 1,000% (w/w), calculated based on the content of the original culture. That is, for example, the composition of the present invention may contain a culture of Bifidobacterium bacteria or a derivative thereof (culture supernatant, etc.) in the content as exemplified above. The "original culture" refers to a culture in which no concentration change such as concentration or dilution has occurred after culture, and specifically may be a culture immediately after culture (i.e., the culture of Bifidobacterium bacteria itself). Such a culture may be, for example, a culture cultured using a medium as exemplified above so that the Bifidobacterium bacteria are present at 1 x 10 ⁷ to 1 x 10 ¹¹ cfu/mL, preferably 1 x 10 ⁸ to 1 x 10 ¹⁰ cfu/mL. A content of more than 100% (w/w) means that the culture or a product derived therefrom (such as culture supernatant) 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 a product derived therefrom (such as culture supernatant) is concentrated 10 times and contained in the composition of the present invention.

The content of the active ingredient in the composition of the present invention can be set so as to obtain, for example, the dosage of the active ingredient as described below. That is, the dosage of the composition of the present invention may be, for example, 1×10 ⁶ to 1×10 ¹² 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, calculated as the dosage of the active ingredient (based on the bacterial cells). The dosage of the composition of the present invention may be, for example, 1×10 ⁷ to 1×10 ¹⁴ 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, calculated as the dosage of the active ingredient (based on the bacterial cells). When the active ingredient is a live bacterial cell, "cells" may be read as "cfu." The dosage of the composition of the present invention may be, for example, 0.01 to 100 mL/kg body weight/day, preferably 0.1 to 10 mL/kg body weight/day, calculated as the dosage of the active ingredient (based on the original culture).

The shape of the composition of the present invention is not particularly limited. Any shape that is acceptable depending on the mode of use of the composition of the present invention can be adopted as the shape 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 beverage compositions, pharmaceutical compositions, and feed compositions described below.

<Food and drink composition>
The food and drink composition of the present invention is not particularly limited as long as it contains an active ingredient. The food and drink composition of the present invention may be provided in any form, such as a liquid, a paste, a gel-like solid, or a powder.

The food and drink composition may be, for example, a food and drink itself, or may be a material used in the manufacture of a food and drink. Examples of such materials include seasonings, food additives, and other food and drink ingredients. Specific examples of the food and drink composition include flour products, instant foods, processed agricultural products, processed marine products, processed livestock products, dairy products (fermented milk, cheese, infant formula, etc.), oils and fats, basic seasonings, complex seasonings, frozen foods, confectioneries, beverages, and other commercially available foods and drinks. Specific examples of the food and drink composition include health foods, functional foods, enteral nutritional foods, special purpose foods, health functional foods (specified health foods, nutritional functional foods, functional foods, etc.), nutritional supplements, and pharmaceutical quasi-drugs. The food and drink composition may be, for example, a supplement, and specifically, a tablet-shaped supplement.

The food and drink composition of the present invention can be produced by combining the active ingredient with other ingredients. The operation of combining the active ingredient with other ingredients is also referred to as "addition of the active ingredient". The method for producing the food and drink composition of the present invention is not particularly limited. The food and drink composition of the present invention can be produced, for example, by the same method as that of ordinary foods and drinks using the same raw materials as those of ordinary foods and drinks, except for the addition of the active ingredient. The same applies when the food and drink composition of the present invention is produced as a material used in the production of foods and drinks such as seasonings, food additives, and other food and drink ingredients. The active ingredient may be added at any stage of the production process of the food and drink composition. The active ingredient may be added, for example, during or after the production of the food and drink composition. That is, for example, the active ingredient may be added to a food and drink prepared in advance to produce the food and drink composition of the present invention. The food and drink composition may also be produced through a fermentation process using the added active ingredient (especially Bifidobacterium bacteria). Examples of food and drink compositions produced through a fermentation process include fermented products such as lactic acid bacteria drinks and fermented milk. That is, the active ingredient (particularly the Bifidobacterium genus bacteria) may be used, for example, as a starter for the production of a fermented product. The active ingredient may also be added later to the produced fermented product.

Furthermore, the food and beverage composition of the present invention can be used to produce yet another food and beverage composition. That is, for example, when the food and beverage composition of the present invention is provided as a material used in the production of food and beverage products, such as seasonings, food additives, and other food and beverage ingredients, another food and beverage composition can be produced by adding the food and beverage composition of the present invention to the food and beverage ingredients. Another food and beverage composition produced in this way is also one aspect of the food and beverage composition of the present invention. With regard to the addition of the food and beverage composition of the present invention, the description regarding the addition of active ingredients in the production of a food and beverage composition can be applied mutatis mutandis.

The food and drink composition of the present invention is intended to be used for slowing down skeletal muscle, promoting AMPK activation, promoting PGC-1α expression,
It is possible to provide and sell the food and drink composition of the present invention with a label indicating its intended use (including health use) for the prevention, improvement, and/or treatment of symptoms associated with muscle atrophy and/or muscle weakness. The food and drink composition of the present invention can be provided and sold with a label indicating its intended use for "athletes,""people who want to build muscle,""people who are concerned about muscle weakness due to lack of exercise,""people who are concerned about muscle weakness due to aging,""people who are concerned about muscle weakness due to aging,""people who want to improve frailty," and the like.

"Representation" includes all acts aimed at informing consumers of the aforementioned uses, and any expression that can recall or infer the aforementioned uses falls under "representation", regardless of the purpose of the representation, the content of the representation, the object and medium on which it is displayed, etc. It is particularly preferable that the representation be made using expressions that enable consumers to directly recognize the aforementioned uses.

Specific examples of the indication include the act of transferring, delivering, displaying for transfer or delivery, or importing a product related to the food and beverage composition of the present invention or a product package on which the above-mentioned use is indicated, displaying or distributing the product with the above-mentioned use indicated in an advertisement, price list, or transaction document related to the product, or providing the information containing the above-mentioned use by electromagnetic means (Internet, etc.), etc. In particular, examples of the indication include indication on promotional materials at the sales site such as packaging, containers, catalogs, pamphlets, and POP, as well as other documents, etc.

In addition, the labeling is preferably a labeling approved by the government or the like (for example, a labeling approved based on various systems established by the government and made in a manner based on such approval). In addition, examples of the labeling include labeling as health food, functional food, enteral nutritional food, special purpose food, health functional food (for example, specified health food, nutrient functional food, functional food), dietary supplement, medical drug, etc. Examples of the labeling approved by the government or the like include labeling approved by the Consumer Affairs Agency. Examples of the labeling approved by the Consumer Affairs Agency include labeling approved under a specified health food or similar system. Examples of the labeling approved by the Consumer Affairs Agency specifically include labeling as a specified health food, labeling as a conditional specified health food, labeling that affects the structure or function of the body, labeling that reduces the risk of disease, labeling of functionality based on scientific evidence, etc., and more specifically, labeling as a specified health food (especially labeling of health uses) and similar labels as set forth in the Cabinet Office Ordinance on the Permission of Special Purpose Labeling Provided in the Health Promotion Act (Cabinet Office Ordinance No. 57 of August 31, 2009).

The content of the active ingredient in the food and drink composition of the present invention may be, for example, within the above-mentioned range. That is, the content of the active ingredient in the food and drink composition of the present invention may be, specifically, for example, 1×10 ⁶ to 1×10 ¹² cells/g or 1×10 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 cells/g or 1×10 ⁸ to 1×10 ¹⁰ cells/mL, when converted into the content of the bacterial cells. When the active ingredient is a live bacterial cell, "cells" may be read as "cfu". Furthermore, the content of the active ingredient in the food and beverage composition of the present invention may be, specifically, for example, 1 to 10,000% (w/w), preferably 10 to 1,000% (w/w), calculated based on the content of the original culture.

The dosage of the food and drink composition of the present invention may be, for example, within the above-mentioned range. That is, the dosage of the food and drink composition of the present invention may be, for example, 1×10 ⁶ to 1×10 ¹² 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, converted into the dosage of the active ingredient (based on the amount of the bacterial body). The dosage of the food and drink composition of the present invention may be, for example, 1×10 ⁷ to 1×10 ¹⁴ 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, converted into the dosage of the active ingredient (based on the amount of the bacterial body). When the active ingredient is a live bacterial cell, "cells" may be read as "cfu." The dosage of the food and drink composition of the present invention may be, specifically, for example, 0.01 to 100 mL/kg body weight/day, preferably 0.1 to 10 mL/kg body weight/day, calculated as the dosage of the active ingredient (based on the amount of the original culture).

<Pharmaceutical Composition>
The pharmaceutical composition of the present invention is not particularly limited as long as it contains an active ingredient. 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 appropriately formulated and used.

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 depending on various conditions such as, for example, the administration method. The pharmaceutical composition of the present invention may be for oral administration or for parenteral administration. The pharmaceutical composition of the present invention may be for oral administration in particular. In the case of oral administration, the dosage form specifically includes solids such as powders, granules, tablets, and capsules, and liquids such as solutions, syrups, suspensions, and emulsions. In the case of parenteral administration, the dosage form specifically includes suppositories and ointments. In the formulation, physiologically acceptable additives can be used. Examples of additives include various organic and inorganic components. Examples of additives specifically include excipients, binders, disintegrants, lubricants, stabilizers, flavorings, diluents, surfactants, and solvents. These additives can be appropriately selected depending on various conditions such as, for example, the dosage form.

Examples of excipients include sugar derivatives such as lactose, sucrose, glucose, mannitol, and sorbitol; starch derivatives such as corn starch, potato starch, α-starch, dextrin, and carboxymethyl starch; cellulose derivatives such as crystalline cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, carboxymethyl cellulose, and calcium carboxymethyl cellulose; gum arabic; dextran; pullulan; silicate derivatives such as light anhydrous silicic acid, synthetic aluminum silicate, and magnesium aluminometasilicate; phosphate derivatives such as calcium phosphate; carbonate derivatives such as calcium carbonate; and sulfate derivatives such as calcium sulfate.

Examples of binders include the above excipients, gelatin, polyvinylpyrrolidone, macrogol, etc.

Disintegrants include, for example, the above-mentioned excipients as well as chemically modified starch or cellulose derivatives such as croscarmellose sodium, sodium carboxymethyl starch, and cross-linked polyvinylpyrrolidone.

Examples of lubricants include talc; stearic acid; metal stearates such as calcium stearate and magnesium stearate; colloidal silica; waxes such as pea gum and geranium stearate; boric acid; glycol; carboxylic acids such as fumaric acid and adipic acid; sodium carboxylates such as sodium benzoate; sulfates such as sodium sulfate; leucine; lauryl sulfates such as sodium lauryl sulfate and magnesium lauryl sulfate; silicic acids such as silicic anhydride and silicic acid hydrate; and starch derivatives.

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

Flavors and flavorings include, for example, sweeteners, acidulants, and fragrances.

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 may be, for example, 1×10 ⁶ to 1×10 ¹² cells/g or 1×10 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 cells/g or 1×10 ⁸ to 1×10 ¹⁰ cells/mL, when converted into the content of bacterial cells. When the active ingredient is live bacterial cells, "cells" may be read as "cfu". Furthermore, the content of the active ingredient in the pharmaceutical composition of the present invention may be specifically, for example, 1 to 10,000% (w/w), preferably 10 to 1,000% (w/w), calculated based on the content of the original culture.

The dosage of the pharmaceutical composition of the present invention may be, for example, within the range described above. Specifically, the dosage of the pharmaceutical composition of the present invention is, for example, converted into the dosage of the active ingredient (based on the amount of bacterial cells), 1×10 ⁶ to 1×10 ¹² cells/kg body weight/day, preferably 1×
The dosage of the pharmaceutical composition of the present invention may be, specifically, for example, 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, calculated as the dosage of the active ingredient (based on the amount of the bacterial cells), and may be, specifically, for example, 1×10 ⁷ to 1×10 ¹⁴ 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 a live bacterial cell, "cells" may be read as "cfu". The dosage of the pharmaceutical composition of the present invention may be, specifically, for example, 0.01 to 100 mL/kg body weight/day, preferably 0.1 to 10 mL/kg body weight/day, calculated as the dosage of the active ingredient (based on the amount of the original culture).

<Feed composition>
The feed composition of the present invention is not particularly limited as long as it contains an active ingredient. Examples of the feed composition include pet food and livestock feed. The feed of the present invention may be provided in any form such as powder, granule, crumble, pellet, cube, paste, liquid, etc.

The feed composition of the present invention can be produced by combining the active ingredient with other ingredients. The operation of combining the active ingredient with other ingredients is also referred to as "addition of the active ingredient". The method of 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 normal feed and by the same method as normal feed, except for adding the active ingredient. The active ingredient may be added at any stage of the production process of the feed composition. The active ingredient may be added, for example, during or after the production of the feed composition. That is, for example, the active ingredient may be added to a previously prepared feed to produce the feed composition of the present invention. The feed composition may also be produced through a fermentation process using the added active ingredient (particularly Bifidobacterium bacteria). An example of a feed composition produced through a fermentation process is silage.

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

That is, the method of the present invention is a method comprising the step of administering Bifidobacterium bacteria and/or products produced by said bacteria to a subject.

According to the method of the present invention, specifically by administering an active ingredient to a subject, it is possible to slow-twitch the skeletal muscles of the subject, i.e., a skeletal muscle slow-twitch effect can be obtained. In other words, the method of the present invention may be a method for slow-twitching skeletal muscles.

In addition, the method of the present invention, specifically by administering an active ingredient to a subject, may promote activation of AMPK in the subject, i.e., an effect of promoting AMPK activation may be obtained. In other words, the method of the present invention may be a method of promoting AMPK activation. One aspect of the method of promoting AMPK activation may be, for example, a method of slow-twitching skeletal muscle.

In addition, the method of the present invention, specifically by administering an active ingredient to a subject, may promote the expression of PGC-1α in the subject, i.e., may provide a PGC-1α expression promoting effect. In other words, the method of the present invention may be a method for promoting the expression of PGC-1α. One embodiment of the method for promoting the expression of PGC-1α may be, for example, a method for slow-twitching skeletal muscle.

Furthermore, the method of the present invention, specifically by administering an active ingredient to a subject, may provide an effect based on slow-twitching of skeletal muscles in the subject as exemplified above. That is, the method of the present invention may be a method for improving endurance. The method of the present invention may be a method for improving muscle strength. The method of the present invention may be a method for preventing, improving, and/or treating symptoms associated with muscle atrophy and/or muscle weakness. Any of these methods may be, for example, an embodiment of a method for slow-twitching of skeletal muscles.

Note that "administering an active ingredient to a subject" may be synonymous with "allowing a subject to ingest the active ingredient." Ingestion may be voluntary (free intake) or forced (forced intake). That is, the administration step may specifically be, for example, a step of blending the active ingredient with food, drink, or feed and supplying it to the subject, thereby allowing the subject to ingest the active ingredient freely. Administration may be oral or parenteral. Administration may usually be oral. Parenteral administration includes tube administration and rectal administration.

The mode of administration of the active ingredient (e.g., the subject, the timing of administration, the duration of administration, the number of administrations, the dosage, and other conditions related to administration) is not particularly limited as long as the desired effect, such as the effect of slowing down skeletal muscle, is obtained. The mode of administration of the active ingredient can be appropriately set depending on various conditions such as the type of active ingredient, the type, age, and health condition of the subject.

The subject to which the active ingredient is administered is not particularly limited as long as the desired effect, such as the skeletal muscle slowing effect, can be obtained. The subject to which the active ingredient is administered is a mammal. Examples of mammals include humans and pets. Examples of pets include dogs and cats. Examples of mammals include humans in particular. Mammals may be male or female. The condition of the human to which the active ingredient is administered is not particularly limited. The active ingredient may be administered to any human who desires the desired effect, such as the skeletal muscle slowing effect. The human may be of any age, such as an infant, a child, an adult, a middle-aged person, or an elderly person. The human may be, for example, a healthy person or an unhealthy person. Examples of unhealthy people include those who exhibit symptoms related to muscle atrophy and/or muscle weakness. The human may be, for example, an athlete, a sports enthusiast, or another person who performs physical exercise other than daily movements.

The dosage of the active ingredient may be, for example, in terms of the dosage of the bacterial cells, 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/kg body weight/day or more, or 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 may be a combination of these ranges. Specifically, the dosage of the active ingredient may be, for example, in terms of the dosage of the bacterial cells, 1×10 ⁶ to 1×10 ¹² 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. When the active ingredient is a live bacterial cell, "cells" may be read as "cfu".

The dosage of the active ingredient may be, for example, in terms of 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, or 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 may be a combination of these. Specifically, the dosage of the active ingredient may be, for example, in terms of the dosage of bacterial cells, 1×10 ⁷ to 1×10 ¹⁴ 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 a live bacterial cell, "cells" may be read as "cfu".

The dosage 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, 100 mL/kg body weight/day or less, or 10 mL/kg body weight/day or less, or a combination of these ranges, calculated as the dosage of the original culture. Specifically, the dosage of the active ingredient may be, for example, 0.01 to 100 mL/kg body weight/day, preferably 0.1 to 10 mL/kg body weight/day, calculated as the dosage of the original culture. That is, for example, a culture of Bifidobacterium bacteria or a derivative thereof (culture supernatant, etc.) may be administered at the dosage exemplified above.

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

For example, the active ingredient may be administered to a subject as it is, or may be prepared as a composition containing the active ingredient, such as a food and drink composition, a pharmaceutical composition, or a feed composition, and then administered to a subject. For compositions containing an active ingredient, the description of the composition of the present invention can be applied mutatis mutandis. In addition, the active ingredient may be administered alone or in combination with other ingredients. Examples of other ingredients include medicines and pharmaceutical compositions, food and drink and food and drink compositions, feed and feed compositions, and ingredients contained therein. These other ingredients may be intended for use in the above-mentioned applications, such as for slow-twitching skeletal muscles, or may not be intended for such uses.

The active ingredient can be administered to a subject, for example, by using the composition of the present invention (i.e., by administering the composition of the present invention). That is, one aspect of the method of the present invention may be a method including a step of administering the composition of the present invention to a subject. That is, "administration of an active ingredient" also includes administration of the composition of the present invention. The administration mode of the composition of the present invention (e.g., the subject to be administered, the administration time, the administration period, the number of administrations, the dosage, and other conditions related to administration) is not particularly limited as long as the desired effect such as the skeletal muscle slowing effect is obtained. The administration mode of the composition of the present invention can be appropriately set according to various conditions such as the type and content of the active ingredient, the type and content of other ingredients, the type and form (dosage form) of the composition, the type of the subject to be administered, the age, and the health condition of the subject to be administered. The above-mentioned description of the administration mode of the active ingredient can be applied mutatis mutandis to the case where the composition of the present invention is administered to a subject. That is, the composition of the present invention can be administered to, for example, a subject as exemplified above. In addition, the dosage of the composition of the present invention can be set so that, for example, the dosage of the active ingredient as exemplified above is obtained. In addition, the composition of the present invention may be administered alone or in combination with other components, such as medicines or pharmaceutical compositions, food and drink or food and drink compositions, feed or feed compositions, or components contained therein.

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

[Production Example 1]
Bifidobacterium breve FERM BP-11175 is added to 3 mL of MRS liquid medium, and cultured anaerobically at 37°C for 16 hours. The culture solution is concentrated and freeze-dried to obtain a freeze-dried powder (bacteria powder) of the bacteria. The bacteria powder and whey protein concentrate (WPC) are mixed uniformly 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 a food or drink. By ingesting the composition, the effects exemplified above, such as the effect of slowing skeletal muscle, can be expected.

[Production Example 2]
Bifidobacterium breve FERM BP-11175 is added to 3 mL of MRS liquid medium, and cultured anaerobically at 37°C for 16 hours. The culture solution is concentrated and freeze-dried to obtain a freeze-dried powder (bacteria powder). The bacteria powder is mixed uniformly with a dry powder of milk protein concentrate (MPC480, Fonterra, protein content 80% by mass, casein protein: whey protein = about 8:2) 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 a food or drink. By ingesting the composition, the effects exemplified above, such as the effect of slowing skeletal muscle, can be expected.

[Example]
Preparation of the drug to be administered
The freeze-dried powder of Bifidobacterium breve FERM BP-11175 was diluted with saline for 5 h.
The live cell-administered sample of Bifidobacterium breve FERM ^BP -11175 was heated at 90° C. for 30 minutes to obtain a heat-sterilized sample.

<Animal testing>
Crl:CD(SD) rats (Japan Charles River: healthy rats) were used as experimental animals to evaluate the effects of four weeks of continuous ingestion of live and heat-sterilized Bifidobacterium breve FERM BP-11175 on muscle fiber types in muscle tissue.

After purchasing the experimental animals at 7 weeks of age, they were allowed to freely consume Labo MR Stock Feed (manufactured by Nosan Kogyo Co., Ltd.) and tap water. After 1 week of acclimation, the experimental animals were divided into a control group (a group administered with 5 mL/kg body weight/day of saline), a group administered with live Bifidobacterium bacteria (a group administered with 2 mL/day/animal of live bacteria administered product (1×10 ⁹ cfu/day/animal in terms of bacterial count)), and a group administered with heat-sterilized Bifidobacterium bacteria (a group administered with 2 mL/day/animal of heat-sterilized Bifidobacterium administered product (1×10 ⁹ cells/day/animal in terms of bacterial count)), and each mouse was orally administered the administered product using an oral sonde once a day for 4 weeks. One hour after the last oral administration was completed, the experimental animals were treated under sevoflurane anesthesia, and the soleus and gastrocnemius muscles were removed.

The gastrocnemius muscle was used to measure the proportion of slow-twitch muscle fibers using the following procedure. Specifically, the excised gastrocnemius muscle was thinly sliced, and the slow-twitch muscle fibers were stained by immunohistochemical staining using an anti-SERCA2 ATPase antibody (Abcam). For each group, three fields of view in the center of the medial head of the gastrocnemius muscle were observed under a microscope, and the staining rate (slow-twitch muscle fiber proportion) was measured for a total of 450 fibers.

The slow muscle fiber ratio for each group is shown in Figure 1. Images of the stained slow muscle fibers for each group are shown in Figures 2 to 4. In Figures 2 to 4, the dark areas are slow muscle fibers and the light areas are fast muscle fibers. The administration of Bifidobacterium breve FERM BP-11175 increased the slow muscle fiber ratio compared to the control. In particular, a significant increase in the slow muscle fiber ratio was confirmed in the heat-sterilized group compared to the live bacteria group. Specifically, compared to the control, the slow muscle fiber ratio increased by 3.80% in the live bacteria group and by 12.70% in the heat-sterilized group. As described above, it was confirmed that administration of Bifidobacterium breve FERM BP-11175 resulted in slow muscle fiber formation in skeletal muscles.

The soleus muscles were used to evaluate the signal pathways involved in the slow-twitching of skeletal muscles (Zhang et al., Int J Biol Sci, 13(9), 1152-1162(2017); Suwa et al., J. Appl. Physiol, 95, 960-968(2003)) using the following procedure. Regarding the signal pathways involved in the slow-twitching of skeletal muscles, the amount of active AMP-activated protein kinase (AMPK) was measured by Western blotting, and the expression level of peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) was measured by real-time PCR.

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

The expression level of PGC-1α is shown in Figure 5. The administration of Bifidobacterium breve FERM BP-11175 increased the expression level of PGC-1α compared to the control. In particular, a significant increase in the expression level of PGC-1α was confirmed in the heat-killed group compared to the live cell group. Specifically, the expression level of PGC-1α increased 1.31-fold in the live cell group and 1.90-fold in the heat-killed group compared to the control.

Proteins were extracted from the excised soleus muscle using RIPA buffer (CST).
The amount of phosphorylated AMPK (active AMPK) was quantified by Western blotting. The amount of phosphorylated AMPK was normalized based on the amount of total AMPK and calculated as a relative value to the control.

The amount of phosphorylated AMPK is shown in Figure 6. The 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 live cell-administered group and 1.48-fold in the heat-sterilized cell-administered group compared to the control.

These results suggest that administration of Bifidobacterium breve FERM BP-11175 activates the signaling pathway involved in promoting slow muscle contraction, resulting in an increase in the proportion of slow muscle contractions in skeletal muscles. This phenomenon was more pronounced in the heat-sterilized group than in the live group.

<Explanation of sequence listing>
SEQ ID NOs: 1 to 4: Primers

The present invention provides a composition that can be used for specific purposes, such as a composition for slowing down skeletal muscle.

Claims (7)

A composition for slowing down skeletal muscle, comprising the following component (A) as an active ingredient:
(A) Viable cells of Bifidobacterium breve FERM BP-11175 . A composition for improving endurance, comprising the following component (A) as an active ingredient:
(A) Viable cells of Bifidobacterium breve FERM BP-11175 . A composition for preventing, improving, and/or treating symptoms associated with muscle atrophy and/or muscle weakness, comprising the following component (A) as an active ingredient:
(A) Viable cells of Bifidobacterium breve FERM BP-11175 . A composition for promoting AMP-activated protein kinase activation, comprising the following component (A) as an active ingredient:
(A) Viable cells of Bifidobacterium breve FERM BP-11175 . The composition according to any one of claims 1 to 4, wherein the subject to which the composition is administered is a healthy individual. The composition according to any one of claims 1 to 5 , wherein the composition is a pharmaceutical composition. The composition according to any one of claims 1 to 5 , which is a food or drink composition.

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