Agent for suppressing decrease in muscle mass and / or muscle strength

Abstract

Provided are a pharmaceutical product, a food product and a material that can be blended with said products, which suppress a decrease in muscle mass and are useful for improving / maintaining muscle strength and muscle function.　This agent for suppressing a decrease in muscle mass and / or muscle strength contains bacterial cells or a bacterial cell culture of Phocaeicola vulgatus as an active ingredient.

Description

Agents for suppressing muscle mass and / or muscle strength decline

[0001] The present disclosure relates to pharmaceutical or food materials that are useful for suppressing a decline in muscle mass and muscle strength.

[0002] In Japan, where approximately 30% of the total population is aged 65 or older, extending the healthy lifespan of the elderly is an important social issue. To extend healthy lifespan, it is considered essential to prevent and improve frailty and pre-frailty, which are the stages before the need for nursing care, and there is a need to maintain and improve physical functions such as physical strength and muscle strength in the elderly.

[0003] Aging and various diseases disrupt the balance between anabolism and catabolism in skeletal muscle, leading to a progressive decrease in skeletal muscle mass and an increased risk of movement disorders, falls, and fractures. Sarcopenia is known as a syndrome that causes this. As sarcopenia progresses, falls and decreased activity levels become more likely, leading to increased frailty and a need for care. This not only reduces the motor and physical functions of elderly people, but also reduces their life expectancy and ADL (Activities of Daily Living), necessitating the need for countermeasures.

[0004] It has been reported that certain types of lactic acid bacteria and their cultures have the effect of building muscle. For example, Patent Document 1 discloses that Bifidobacterium bacteria such as Bifidobacterium breve have a muscle-building effect, Patent Document 2 discloses that Lactobacillus gasseri has a muscle mass-increasing effect, and Patent Document 3 discloses that Lactobacillus paracasei has an effect of maintaining muscle mass or inhibiting its decline.

[0005] Meanwhile, intestinal bacterial analysis of patients with coronary artery disease has reported a reduction in bacteria of the genus Phocaeicola (formerly the genus Bacteroides). Furthermore, it has been reported that oral administration of two bacterial species, Phocaeicola vulgatus and Phocaeicola dorei, to arteriosclerosis model mice can suppress the formation of arteriosclerosis (Non-Patent Document 1).

[0006] However, the effects of Phocaeicola vulgatus on muscle have not been known until now.

[0007] International Publication No. 2019 / 87280 JP 2016-84358 A JP 2021-42142 A

[0008] Yoshida et al., Circulation. 2018;138:2486-2498

[0009] The present disclosure relates to providing pharmaceuticals, foods, and materials that can be incorporated therein that are useful for inhibiting a decrease in muscle mass and maintaining and improving muscle strength and muscle function.

[0010] In view of the above-mentioned problems, the present inventors have searched for materials useful for maintaining and improving muscle strength and muscle function. As a result, when a culture fraction containing extracellular membrane vesicles (MVs) of Phocaeicola vulgatus was administered to nematodes in mixed feed, it was found to have the effect of suppressing the age-related decline in motor ability and the decline in the expression level of myosin heavy chains, and thus Phocaeicola vulgatus fungus or fungus cultures are useful for suppressing declines in muscle mass and strength.

[0011] That is, the present disclosure relates to the following 1) to 22). 1) An agent for suppressing muscle mass and / or muscle strength decline, which contains Phocaeicola vulgatus fungus cells or a fungus cell culture as an active ingredient. 2) An agent for suppressing motor function decline, which contains Phocaeicola vulgatus fungus cells or a fungus cell culture as an active ingredient. 3) An agent for suppressing myosin heavy chain expression decline, which contains Phocaeicola vulgatus fungus cells or a fungus cell culture as an active ingredient. 4) Any of the agents 1) to 3), wherein the fungus cell culture is an extracellular membrane vesicle fraction. 5) Any of the agents 1) to 4), wherein Phocaeicola vulgatus is Phocaeicola vulgatus ATCC 8482 strain. 6) A food for suppressing muscle mass and / or muscle strength decline, which contains Phocaeicola vulgatus fungus cells or a fungus cell culture as an active ingredient. 7) A food for suppressing motor function decline, which contains Phocaeicola vulgatus fungus cells or a fungus cell culture as an active ingredient. 8) The food according to 6) or 7), wherein the bacterial cell culture is an extracellular membrane vesicle fraction. 9) The food according to any of 6) to 8), wherein the Phocaeicola vulgatus is Phocaeicola vulgatus ATCC 8482 strain. 10) Use of Phocaeicola vulgatus fungus cells or a bacterial cell culture for producing an agent for suppressing muscle mass and / or muscle strength decline. 11) Use of Phocaeicola vulgatus fungus cells or a bacterial cell culture for producing an agent for suppressing motor function decline. 12) Use of Phocaeicola vulgatus fungus cells or a bacterial cell culture for producing an inhibitor of myosin heavy chain expression decline. 13) Use of Phocaeicola vulgatus fungus cells or a bacterial cell culture for producing a food for suppressing muscle mass and / or muscle strength decline. 14) Use of Phocaeicola vulgatus fungus cells or a bacterial cell culture for producing a food for suppressing motor function decline. 15) A fungus or a fungus culture of Phocaeicola vulgatus for inhibiting a decline in muscle mass and / or muscle strength. 16) A fungus or a fungus culture of Phocaeicola vulgatus for inhibiting a decline in motor function. 17) A fungus or a fungus culture of Phocaeicola vulgatus for inhibiting a decline in myosin heavy chain expression. 18) Non-therapeutic use of a fungus or a fungus culture of Phocaeicola vulgatus for inhibiting a decline in muscle mass and / or muscle strength. 19) Non-therapeutic use of a fungus or a fungus culture of Phocaeicola vulgatus for inhibiting a decline in motor function.20) A method for suppressing a decline in muscle mass and / or muscle strength, comprising administering or ingesting Phocaeicola vulgatus fungus or a fungus cell culture to a subject in need thereof. 21) A method for suppressing a decline in motor function, comprising administering or ingesting Phocaeicola vulgatus fungus or a fungus cell culture to a subject in need thereof. 22) A method for suppressing a decline in myosin heavy chain expression, comprising administering or ingesting Phocaeicola vulgatus fungus or a fungus cell culture to a subject in need thereof.

[0012] According to the present disclosure, it is possible to suppress the decline in muscle mass and muscle strength due to aging, and to improve activities of daily living (ADL), particularly ADL for elderly people.

[0013] Microscope photograph of a nematode (Caenorhabditis elegans). Method for measuring nematode motility. Effect of Phocaeicola vulgatus-derived MVs on nematode motility. Confirmation of uptake of fluorescently labeled Phocaeicola vulgatus-derived MVs into the nematode body. Changes in the expression levels of muscle-related genes due to aging. Changes in the expression levels of muscle-related genes due to administration of Phocaeicola vulgatus-derived MVs.

[0014] Phocaeicola vulgatus, as used in this disclosure, is an obligately anaerobic bacillus belonging to the Bacteroidetes family and is the dominant bacterial species in the human intestine. In 2020, some members of the genus Bacteroides were classified into the genus Phocaeicola, and Phocaeicola vulgatus was previously named Bacteroides vulgatus (Aharon Oren, and George M. Garrity, Int. J. Syst. Evol. Microbiol. 2020;70:2960-2966).

[0015] Phocaeicola vulgatus can be isolated from the human intestine, and although the strain is not particularly limited in the present disclosure, a preferred example is the Phocaeicola vulgatus ATCC 8482 strain, which has been deposited with the American Type Culture Collection (ATCC).

[0016] In the present disclosure, the "mycelia" of Phocaeicola vulgatus may refer to either wet or dry mycelia of Phocaeicola vulgatus. Furthermore, it may refer to not only live mycelia but also dead mycelia. The mycelia also encompasses treated mycelia obtained by treating mycelia with enzymes or physical means, as well as parts of mycelia components such as cytoplasm and cell wall fractions.

[0017] Examples of "bacterial cell cultures" of Phocaeicola vulgatus include cultures containing medium components, bacterial cells, and extracellular membrane vesicles (MVs), but are not particularly limited as long as they contain bacterial cells or MVs. Specific examples include culture fractions containing bacterial cells and MVs obtained by removing medium components from the culture, culture fractions containing medium components and MVs obtained by removing bacterial cells from the culture, and culture fractions containing MVs obtained by removing medium components and bacterial cells from the culture. A preferred example is a culture fraction containing MVs obtained by removing medium components and bacterial cells from the culture (also referred to as an "extracellular membrane vesicle fraction (MV fraction)"). Note that the bacterial cells and medium components removed from the culture may partially remain in the culture fraction, provided that the effects of the present disclosure are not impaired. In addition, extracellular membrane vesicles (MVs) are a general term for vesicles with a heterogeneous lipid bilayer structure secreted from living cells, and are also sometimes called outer membrane vesicles (OMVs) or bacterial extracellular vesicles (BEVs).

[0018] The bacterial cells or bacterial cell culture of the present disclosure can be prepared in any form depending on the intended use, such as a freeze-dried powder, a spray-dried powder, or a suspension in a liquid, as desired.

[0019] The medium for culturing Phocaeicola vulgatus of the present disclosure is not particularly limited as long as it allows Phocaeicola vulgatus to grow well, but preferably contains, for example, a nutrient source such as a nitrogen source (e.g., meat extract, peptone, yeast extract, liver extract), a carbon source (e.g., glucose, dextrose, starch), etc. More preferred media include, for example, glucose-supplemented modified GAM medium, modified GAM medium, and enriched Clostridium medium, with glucose-supplemented modified GAM medium being particularly preferred.

[0020] The culture method is not particularly limited as long as the conditions are such that the fungus cells of Phocaeicola vulgatus grow well. Anaerobic conditions are preferred, and examples include static culture at 37°C for 10 to 48 hours, preferably 20 to 28 hours, and preferably 24 hours.

[0021] In the present disclosure, when the above-mentioned extracellular membrane vesicle fraction (PvMV) is used as the fungal culture of Phocaeicola vulgatus, the fraction can be obtained by centrifuging a culture medium of Phocaeicola vulgatus (e.g., 4°C, 1,940 × g, 15 minutes), filtering the supernatant through a cellulose acetate membrane filter (pore size: 0.2 μm), and ultracentrifuging the obtained culture supernatant (e.g., 100,000 × g to 200,000 × g, 4°C to 25°C, 1 hour to 5 hours), and pelleting the obtained supernatant.

[0022] As shown in the Examples below, when administered to nematodes in feed, a culture of Phocaeicola vulgatus cells has the effect of suppressing a decrease in motility and also suppresses a decrease in the expression of myosin heavy chain, a major subunit of the myosin molecule. Therefore, Phocaeicola vulgatus cells or a culture of these cells can be used as an agent for suppressing a decrease in muscle mass and / or muscle strength, a decrease in myosin heavy chain expression, or a decrease in motor function.

[0023] Furthermore, the fungus or fungus culture of Phocaeicola vulgatus not only suppresses the decline in muscle mass and / or muscle strength but also suppresses the decrease in myosin heavy chain expression, and therefore can be used to suppress the decline in motor function. Here, such use may be administered to humans or non-human animals or used in specimens derived therefrom, and may be therapeutic or non-therapeutic.

[0024] In this disclosure, "muscle mass" refers to the weight of muscle tissue or the area and circumference of muscle fibers, and its decline refers to a decrease in the weight of muscle tissue or a decrease in the area and circumference of muscle fibers. "Muscle strength" refers to the force generated when a muscle contracts, and its decline refers to a weakening of the force generated when a muscle contracts. Inhibiting the decline of muscle mass and / or muscle strength refers to preventing or delaying the decline of either muscle mass or muscle strength, or both muscle mass and muscle strength.

[0025] "Myosin heavy chain" is a major subunit of myosin, a protein that constitutes myosin filaments, and is responsible for muscle contraction. Suppressing the decrease in myosin heavy chain expression typically includes suppressing the decrease in transcription of the myosin heavy chain gene or suppressing the decrease in translation of myosin heavy chain mRNA into myosin heavy chain protein.

[0026] In the present disclosure, "decline in motor function" means a decline in walking ability, grip strength, balance function, etc. due to, for example, a decline in muscle strength in the lower limbs or trunk caused by aging, and "inhibiting" this decline refers to preventing or delaying the decline in motor function.

[0027] The inhibitors of muscle mass and / or muscle strength decline, inhibitors of myosin heavy chain expression decline, and inhibitors of motor function decline disclosed herein can themselves be used as pharmaceuticals, quasi-drugs, or foods for suppressing decline in motor function because they suppress decline in muscle mass and / or muscle strength or suppress decline in myosin heavy chain expression, or can be used as materials or preparations to be incorporated into such pharmaceuticals, quasi-drugs, or foods. The term "foods" includes foods based on the concept of suppressing decline in motor function, etc., and labeled as such as necessary, foods with nutrient function claims, foods with functional claims, foods with health claims including foods for specified health uses, foods for patients, and supplements.

[0028] When used as a pharmaceutical, the composition may be administered orally or parenterally, with oral administration being preferred. For administration, a composition containing the active ingredient may be mixed with a solid or liquid non-toxic pharmaceutical carrier suitable for oral, rectal, or injection administration, and administered in the form of a conventional pharmaceutical formulation. Examples of such formulations include solid formulations such as tablets, granules, powders, and capsules; liquid formulations such as solutions, suspensions, and emulsions; and lyophilized formulations. These formulations can be prepared by conventional pharmaceutical techniques. Examples of such non-toxic pharmaceutical carriers include glucose, lactose, sucrose, starch, mannitol, dextrin, fatty acid glycerides, polyethylene glycol, hydroxyethyl starch, ethylene glycol, polyoxyethylene sorbitan fatty acid esters, amino acids, gelatin, albumin, water, and physiological saline. Conventional additives such as stabilizers, wetting agents, emulsifiers, binders, isotonicity agents, and excipients can also be added as needed.

[0029] The form of the food product is not particularly limited, but is preferably a fermented product, and may be a supplement in the same form as the oral pharmaceutical formulations described above (tablets, capsules, syrup, etc.). Examples of fermented products include fermented milk, soy milk, fruit juice, and plant liquid, with fermented milk being more preferred. Examples of fermented milk products include beverages such as fermented milk and dairy lactic acid bacteria beverages, as well as hard yogurt, soft yogurt, and plain yogurt, as specified by the Ministerial Ordinance on Milk, etc. The food product can be prepared according to standard methods by appropriately combining the fungus or culture of the fungus Phocaeicola vulgatus with any food material, other active ingredients, or additives acceptable for food (e.g., solvents, softeners, oils, emulsifiers, preservatives, acidulants, sweeteners, bittering agents, pH adjusters, stabilizers, colorants, UV absorbers, antioxidants, humectants, thickeners, adhesives, dispersants, flowability improvers, humectants, flavorings, seasonings, flavor adjusters, etc.).

[0030] In the above-mentioned medicines, foods, etc., the amount of Phocaeicola vulgatus cells administered or ingested can be appropriately determined depending on various conditions of the subject, such as the body weight, age, sex, and symptoms. For example, when expressed in terms of the number of Phocaeicola vulgatus cells, the amount administered or ingested per day per adult (60 kg) is preferably 1 x 10 2 or more, more preferably 1×10 6 10 or more, and preferably 1 x 10 15 or less, more preferably 1 x 10 11 The amount of membrane vesicles (MVs) of Phocaeicola vulgatus administered or ingested per person per day is preferably 0.1 μg or more, more preferably 1.0 μg or more, and is preferably 30 g or less, more preferably 15 g or less.

[0031] The subjects to which the muscle mass and / or muscle strength decline inhibitor, myosin heavy chain expression decline inhibitor, motor function decline inhibitor, etc. disclosed herein are administered or ingested are not particularly limited as long as they are subjects in need thereof, and examples include humans, companion animals, etc., and further include mammals including humans with age-related decline in muscle mass or muscle strength, mammals including humans in whom prevention of age-related decline in muscle mass or muscle strength is desired, mammals including humans with age-related decline in motor function, mammals including humans in whom prevention of age-related decline in motor function is desired, etc. Furthermore, they are particularly suitable for use in humans.

[0032] 1. Preparation of Phocaeicola vulgatus (P. vulgatus)-derived membrane vesicle (PvMV) solution. Phocaeicola vulgatus ATCC 8482 was anaerobically cultured at 37°C for 24 hours in 1% glucose-modified GAM medium. The culture was centrifuged (4°C, 1,980 x g, 15 minutes), and the supernatant was filtered through a 0.2 µm pore size cellulose acetate membrane filter (IWAKI) to obtain the culture supernatant. The resulting culture supernatant was subjected to ultracentrifugation at 200,000 × g at 4°C for 2 hours to pellet the MVs, which were then suspended in 1 / 20 volume of phosphate buffered saline (PBS) of the ultracentrifuged culture supernatant [20× PvMV stock solution].

[0033] The control (20x control stock solution) was prepared by centrifuging, filtering, and ultracentrifuging 1% glucose-added modified GAM medium without any bacterial strain in the same manner, and suspending the precipitate in PBS. Each solution was stored at -30°C until use, and then diluted to 1x with M9 Buffer [42.2 mM NaHPO, 22 mM KHPO, 85.6 mM NaCl, 2 mM MgSO].

[0034] 2. Rearing of Nematodes Caenorhabditis elegans N2 strain (FIG. 1) provided by the National BioResource Project (NBRP) was used as the nematode, and Escherichia coli OP50-1 strain (hereinafter referred to as OP50 strain) was used as the E. coli to feed the nematodes.

[0035] The normal subculture of nematodes is performed by culturing the OP50 strain (cultured in LB medium at 37°C for 16 hours with aerobically shaking, and then subcultured until the OD was reached) on Nematode Growth Medium (NGM agar medium) [0.3% (w / v) Peptone, 0.3% (w / v) NaCl, 25 mM KHPO / KHPO, 1 mM CaCl, 1 mM MgSO, 0.0005% (w / v) cholesterol, 1.7% (w / v) agar]. 600 The test was carried out at 16°C on a medium (90 mm dish) on which 1 mL of a suspension of γ-ray sterilized OP50 strain (OD = 0.8) was applied and dried. 600 Nematodes were subcultured for two generations (the live OP50 strain was washed out) on an NGM agar medium (90 mm dish) that had been coated with 1 mL of a suspension of OP50 (pH 7.0 or less, 0.8 or less, OP50-M9 suspension) and dried. After that, the larvae and adults were washed out with M9 buffer, and only the eggs were left on the agar medium to hatch. After 3 days, synchronized nematodes at the L4 stage were used.

[0036] Test Example 1: Effect of membrane vesicles (PvMV) derived from Phocaeicola vulgatus on motility of nematodes 1. Experiment of feeding MV to nematodes OP50-M9 suspension (OD 600= 0.8) was further diluted 2-fold with M9 Buffer, and OP50-M9-2-fold diluted solution was used. L4 stage synchronized nematodes were seeded individually onto NGM agar medium (40 mm dish) that had been coated with 25 μL of OP50-M9-2-fold diluted solution + 25 μL of 1x Control solution or 25 μL of OP50-M9-2-fold diluted solution + 25 μL of 1x PvMV solution and dried, and cultured at 20 ° C (control group, PvMV-treated group, n = 20 for each group). The medium was changed every 2 to 3 days by moving the nematodes by picking them using a 2 mm diameter platinum wire.

[0037] The effect of PvMV administration on the age-related decline in motility of C. elegans was assessed using a motility index known as body bending (reference: Anne C. Hart, ed. Behavior-WormBook, ed. The C. elegans Research Community, WormBook, doi / 10.1895 / wormbook.1.87.1). Motility (body bending count / 30 seconds) of C. elegans was measured on the first observation day, the day C. elegans were seeded on control or PvMV-treated medium, and measurements were performed upon medium change. The number of body bending events was measured by lightly touching a nematode on NGM agar medium with a platinum wire and then counting the number of body twists for 30 seconds (Figure 2; one body bending event was counted when the nematode's head bent in the opposite direction to the direction it had been facing after being touched once with the platinum wire. However, a body bending event was not counted when the nematode only shook its head from side to side without coordinated whole-body movement).

[0038] 3. Statistical Analysis Statistical analysis software R (Ver. 3.6) was used for various statistical analyses, and the significance level was set at P<0.05 without multiplicity correction. Individuals that died during the test period were excluded from the analysis. The motor abilities of the control group and PvMV-administered group at each measurement time point were tested for homogeneity of variance using an F-test, and then compared using Student's t-test between groups where homogeneity of variance was confirmed, and using Aspin-Welch's t-test between groups where unequal variance was confirmed.

[0039] 4. Results and Discussion The number of body bending episodes in nematodes decreased significantly with age. On the other hand, the number of body bending episodes in the PvMV-treated group was significantly higher than in the control group on the seventh day of observation (P = 0.005), and tended to be higher on the 14th and 16th days of observation (Figure 3, P = 0.05, 0.07, respectively). These results demonstrated an inhibitory effect on the decline in motor function in the PvMV-treated group, demonstrating that PvMV has an anti-frailty effect in vivo.

[0040] Test Example 2: Effect of Phocaeicola vulgatus-derived membrane vesicles (PvMV) on the expression of muscle-related genes in nematodes (1) Fluorescent labeling of PvMV Fluorescent labeling of PvMV was performed using a standard method using the red fluorescent cell linker kit PKH26 (Mini26-1KT, Merck), which binds to lipid bilayer membranes. That is, 1 mL of Diluent C and 4 μL of fluorescent reagent (PKH26) from the kit were added to 45 μL of 20×PvMV stock solution, and the PvMV was fluorescently labeled. Then, 1 mL of 1% bovine serum albumin solution (hereinafter referred to as BSA solution) was added, and unreacted PKH26 was bound to BSA. Subsequently, the BSA that had reacted with PKH26 was removed by washing four times with 10 mL of PBS using an ultrafiltration filter (Viva spin 20-100 kDa, Cytiva) with a molecular weight cutoff of 100 kDa, and the fluorescently labeled PvMV suspended in PBS was purified.

[0041] As a positive control, 50 μL of a 2-fold diluted solution of OP50-M9 was fluorescently labeled and washed with PBS in the same manner as PvMV, and then fluorescently labeled OP50 strain was finally suspended in 50 μL of PBS.

[0042] (2) Confirmation of uptake of fluorescently labeled PvMV into nematodes following dietary administration. NGM agar plates (40 mm dishes) were prepared by applying 50 μL of unlabeled OP50-M9 2-fold diluted solution (negative control), 25 μL of OP50-M9 2-fold diluted solution + 25 μL of fluorescently labeled OP50 strain solution (positive control), or 25 μL of OP50-M9 2-fold diluted solution + 25 μL of fluorescently labeled PvMV solution (PvMV group). Five L4 stage nematodes were seeded onto each agar plate. After culturing for 20 hours in a 20°C incubator, the nematodes were recovered onto a glass slide and allowed to stand at -30°C for 1 hour to immobilize them. The nematodes were photographed using a fluorescence microscope (BZ-X710, Keyence) at 10x magnification and an exposure time of 1 / 60 seconds.

[0043] (3) Effect of dietary administration of PvMV on the expression of muscle-related genes in nematodes. NGM agar medium (40 mm dishes) was prepared by applying 25 μL of 2-fold diluted OP50-M9 + 25 μL of 1× Control solution (control group) or 25 μL of 2-fold diluted OP50-M9 + 25 μL of 1× PvMV solution (PvMV group) and drying. Five L4 stage synchronized nematodes were seeded on three NGM agar plates and cultured at 20 ° C (n = 15 for each group). The medium was changed every 2 to 3 days by picking the nematodes using a 2 mm diameter platinum wire, and nematodes were collected from each group on days 5, 7, and 11 of administration.

[0044] Total RNA was extracted from the recovered nematodes according to the standard protocol of ISOGEN II (Nippon Gene). 500 ng of the extracted RNA was used as a template for reverse transcription (37°C for 10 minutes, 85°C for 120 minutes) using a High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific) to synthesize cDNA, which was then stored at -30°C until use. Using TB Green® Premix Ex Taq™ II (Tli RNase H Plus) (Takara) and the primer set shown below (Table 1), the expression levels of muscle-related genes in C. elegans were measured by real-time PCR (95°C for 30 seconds, {95°C for 5 seconds, 55°C for 30 seconds, 72°C for 31 seconds} x 40 cycles).

[0045] The series of experiments, from administering PvMV to nematodes to analyze gene expression levels, were repeated three times. The expression level of each gene was corrected for the expression level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in each experiment, and then expressed as a ratio to the average expression level of the control group in three experiments.

[0046]

[0047] (4) Statistical Analysis The changes in gene expression levels with aging in the control group were analyzed by linear regression analysis of the trends on days 5, 7, and 11 of administration (P trend Comparisons between the control group and the PvMV group were performed using an F-test to test for homogeneity of variance, followed by a Student's t-test between groups where homogeneity of variance was confirmed, and an Aspin-Welch's t-test between groups where unequal variance was confirmed. The significance level for both analyses was set at P<0.05.

[0048] (5) Results 1) Fluorescently labeled PvMV was administered to nematodes and observed under a fluorescent microscope. As a result, 20 hours after the start of administration, fluorescent coloration was observed in the digestive tract of nematodes that had been fed fluorescently labeled PvMV, similar to when the fluorescently labeled OP50 strain was administered (positive control), confirming the uptake of PvMV (Figure 4).

[0049] 2) Effect of feeding PvMV on muscle-related gene expression in nematodes: With the exception of actin, the expression levels of all muscle protein genes decreased with age (Figure 5). trend The graphs show the results of a linear trend test for the expression levels at three time points: days 5, 7, and 11 after administration. Regarding the expression levels of each gene due to PvMV administration, the expression levels of myosin heavy chain B and paramyosin genes were significantly higher in the PvMV-administered group on days 5 and 11 compared to the control group (Figure 6). The values ​​on the graph in Figure 6 show the results of Student's t-test or Aspin-Welch's t-test for the expression levels in the control and PvMV groups. In particular, on day 11, the expression levels of myosin heavy chain B gene and paramyosin gene were 1.19-fold and 1.27-fold higher in the PvMV group compared to the control group (P = 0.024 and 0.015, respectively).

[0050] Myosin heavy chain B and paramyosin are proteins that make up the myosin filaments of body wall muscles and are thought to be involved in the generalized movement of nematodes (Gieseler K, Qadota H, Benian GM. Development, structure, and maintenance of C. elegans body wall muscle. WormBook: 2005-2018.). Myosin heavy chain B accounts for 75% of myosin heavy chains in body wall muscles, and immunohistochemistry of muscle tissues has revealed that it is expressed at both ends of myosin filaments (Miller DM 3rd, Ortiz I, Berliner GC, Epstein HF. Differential localization of two myosins within nematode thick filaments. Cell. 1983 Sep; 34(2): 477-90.). Paramyosin is a core protein that forms the center of myosin filaments. This suggests that the inhibitory effect of PvMV administration on the age-related decline in motility of nematodes observed in Test Example 1 is mediated by the suppression of the decline in expression of myosin heavy chain B and paramyosin, which are the main constituent proteins of myosin filaments.

Claims

1. An agent for suppressing muscle mass and / or muscle strength decline, the active ingredient of which is a fungus or a culture of the fungus, Phocaeicola vulgatus.

2. A motor function decline inhibitor containing the fungus or culture of the fungus, Phocaeicola vulgatus, as an active ingredient.

3. An inhibitor of the decrease in myosin heavy chain expression, containing as an active ingredient the fungus or culture of the fungus, Phocaeicola vulgatus.

4. The agent according to any one of claims 1 to 3, wherein the bacterial cell culture is an extracellular membrane vesicle fraction.

5. The agent according to any one of claims 1 to 4, wherein the Phocaeicola vulgatus is the Phocaeicola vulgatus ATCC 8482 strain.

6. A food for suppressing muscle mass and / or muscle strength decline, containing as an active ingredient the fungus or culture of the fungus, Phocaeicola vulgatus.

7. A food for suppressing motor function decline, containing as an active ingredient the fungus or culture of the fungus, Phocaeicola vulgatus.

8. The food product according to claim 6 or 7, wherein the bacterial culture is an extracellular membrane vesicle fraction.

9. The food according to any one of claims 6 to 8, wherein the Phocaeicola vulgatus is Phocaeicola vulgatus ATCC 8482 strain.

10. Use of a fungus or a fungus culture of Phocaeicola vulgatus for producing an agent for inhibiting muscle mass and / or muscle strength decline.

11. Use of a fungus or a fungus culture of Phocaeicola vulgatus for producing an agent for suppressing hypomotility.

12. Use of a fungus or a culture of fungus cells of Phocaeicola vulgatus for producing an inhibitor of decreased myosin heavy chain expression.

13. Use of a fungus or a fungus culture of Phocaeicola vulgatus for producing a food for suppressing muscle mass and / or muscle strength loss.

14. Use of a fungus or a fungus culture of Phocaeicola vulgatus for producing a food for suppressing motor dysfunction.

15. A fungus or fungus culture of Phocaeicola vulgatus for inhibiting loss of muscle mass and / or muscle strength.

16. A fungus or fungus culture of Phocaeicola vulgatus for suppressing motor function decline.

17. A fungus or a fungus culture of Phocaeicola vulgatus for suppressing decreased expression of myosin heavy chain.

18. Non-therapeutic use of a fungus or fungal culture of Phocaeicola vulgatus to inhibit loss of muscle mass and / or strength.

19. Non-therapeutic use of a fungus or a fungus culture of Phocaeicola vulgatus for inhibiting motor function decline.

20. A method for suppressing a decrease in muscle mass and / or muscle strength, comprising administering or ingesting a fungus or a fungus culture of Phocaeicola vulgatus to a subject in need thereof.

21. A method for suppressing motor function decline, comprising administering or ingesting a fungus or a fungus culture of Phocaeicola vulgatus to a subject in need thereof.

22. A method for suppressing a decrease in myosin heavy chain expression, comprising administering or ingesting a fungus or a culture of the fungus cells of Phocaeicola vulgatus to a subject in need thereof.

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