Muscle atrophy prevention agent

Abstract

The purpose of the present invention is to provide a novel technique for preventing muscle atrophy by inhibiting muscle degradation or directly promoting muscle synthesis without involving physical activity.　Provided is a muscle atrophy prevention agent containing, as an active ingredient, lactic acid bacteria having a muscle synthesis promotion effect and / or a muscle degradation inhibition effect, a processed product of the lactic acid bacteria, or an extract thereof. A lactic acid bacterium having the muscle synthesis promotion effect is Lactobacillus gasseri or the like, and a lactic acid bacterium having the muscle degradation inhibition effect is Lactobacillus reuteri or the like.

Description

[Technical Field]

[0001] The present invention relates to a muscle atrophy prevention agent containing, as an active ingredient, a lactic acid bacterium having the effect of promoting muscle synthesis and / or inhibiting muscle degradation, a processed product of the lactic acid bacterium, or an extract thereof. The present invention also relates to a muscle atrophy prevention drug, food, drink, or feed containing the muscle atrophy prevention agent. [Background technology]

[0002] In Japan, where the super-aging population is rapidly increasing, the discrepancy between average life expectancy and healthy life expectancy has become an urgent issue. In particular, age-related decline in musculoskeletal function, commonly known as locomotive syndrome, is a major factor in reducing the quality of life (QOL) of elderly people and is a major problem in terms of increasing nursing care burdens and medical expenses. Muscle, in particular, plays an important role in preventing disorders of other musculoskeletal systems, not only providing its own motor function but also shock absorption and postural stabilization. However, muscle mass begins to decline around the age of 30, and the proportion of people who develop sarcopenia, a condition characterized by a loss of skeletal muscle mass throughout the body, increases with age. It has been reported that the proportion is approximately 25% in elderly people aged 75 to 79 and exceeds 35% in those aged 80 or older (Non-Patent Document 1). Therefore, preventing muscle atrophy and maintaining muscle mass in elderly people is essential for extending healthy life expectancy and improving their QOL.

[0003] Even in young people, if they become temporarily bedridden due to injury or illness and the load on their muscles is reduced, muscle atrophy can easily progress, adversely affecting rehabilitation and prognosis. Therefore, preventing muscle atrophy and maintaining muscle mass can improve the quality of life not only for the elderly but also for young people.

[0004] Muscle mass is regulated by the balance between muscle synthesis and muscle breakdown. Therefore, to prevent muscle atrophy, it is necessary to either promote muscle synthesis or inhibit muscle breakdown. In particular, if muscle synthesis can be promoted while inhibiting muscle breakdown at the same time, muscle atrophy can be prevented more effectively.

[0005] Here, resistance training is generally recommended as a method for preventing muscle atrophy and increasing muscle mass, in addition to ingesting protein, which is the raw material for muscle, and branched-chain amino acids, which stimulate muscle synthesis signals. Patent Document 1 also describes a technology that promotes physical activity and increases muscle mass using a physical activity promoter containing Lactobacillus gasseri OLL2809 as an active ingredient. Patent Document 2 also discloses a Lactobacillus lactic acid bacteria strain that promotes myoblast proliferation and muscle repair. Furthermore, Patent Document 3 discloses that Lactobacillus curvatus or Lactobacillus amylovorus has the effect of suppressing muscle breakdown caused by the expression of Atrogin-1. Furthermore, Non-Patent Document 2 (Nutirients) discloses that bifidobacteria have the effect of increasing muscle mass.

[0006] However, it is not realistic for elderly people or bedridden patients to engage in continuous strength training. Protein acts solely as a raw material for muscle synthesis, and even if protein intake is increased, the effect will be limited unless the muscle synthesis-promoting pathway and degradation-inhibiting pathway in muscle cells are activated. In particular, the muscle synthesis response to branched-chain amino acids is thought to be weakened in elderly people, so in order to prevent muscle atrophy, it is necessary to activate the muscle synthesis-promoting pathway and degradation-inhibiting pathway separately. Furthermore, Patent Document 1 increases muscle mass as a secondary effect of promoting physical activity, but does not directly promote muscle synthesis or inhibit muscle breakdown. Patent Document 2 also promotes muscle repair of damaged muscles, but does not promote muscle synthesis or inhibit muscle breakdown. Patent Document 3 inhibits muscle breakdown, but only discloses two specific types of lactic acid bacteria. Furthermore, Non-Patent Document 2 only discloses that certain bifidobacteria have a muscle-building effect, but does not mention lactic acid bacteria. Furthermore, none of the above documents discloses lactic acid bacteria that have both the effects of promoting muscle synthesis and inhibiting muscle breakdown. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Patent Publication No. 2016-84358 [Patent Document 2] International Publication No. 2019-230957 [Patent Document 3] Patent No. 6339526 [Non-patent literature]

[0008] [Non-Patent Document 1] JAMDA 2013, 14, 911-915 [Non-patent document 2] Nutrients 2020, 12, 219 Summary of the Invention [Problem to be solved by the invention]

[0009] The present invention aims to provide a novel technique for preventing muscle atrophy by directly promoting muscle synthesis or suppressing muscle breakdown without the intervention of physical activity, or a novel technique for effectively preventing muscle atrophy by promoting muscle synthesis and simultaneously suppressing muscle breakdown. [Means for solving the problem]

[0010] Muscle synthesis is promoted by activation of the Akt-mTOR (mechanistic target of rapamycin) pathway in muscle cells. The p70S6K protein is downstream of the Akt-mTOR pathway and is activated by phosphorylation. Therefore, the amount of phosphorylated p70S6K is used as an indicator of muscle synthesis activation, and an increase in the amount of phosphorylated p70S6K indicates activation of muscle synthesis. On the other hand, muscle degradation is primarily mediated by the ubiquitin-proteasome system, and Atrogin-1 and MuRF1 (muscle ring finger 1) are known to be muscle-specific ubiquitin ligases. Specifically, increased expression of Atrogin-1 and MuRF1 promotes muscle degradation, whereas decreased expression of these genes inhibits muscle degradation. As a result of extensive research, the inventors have found that adding Streptococcus thermophilus, Lactococcus lactis subsp. cremoris, Lactobacillus rhamnosus, or Lactococcus lactis subsp. lactis to muscle cells increases the amount of phosphorylated p70S6K in the muscle cells, that adding Lactobacillus mucosae, Lactobacillus fermentum, and Pediococcus acidilactici reduces the gene expression levels of Atrogin-1 and MuRF1, and that adding Lactobacillus delbreckii subsp. lactis, Lactobacillus gasseri, or Lactobacillus reuteri not only increases the amount of phosphorylated p70S6K in muscle cells, but also reduces the gene expression levels of Atrogin-1 and MuRF1, thereby completing the present invention.

[0011] The present invention relates to the following inventions [1] to

[10] . [1] A muscle atrophy prevention agent containing, as an active ingredient, a lactic acid bacterium having a muscle synthesis promoting effect and / or a muscle degradation inhibiting effect, a processed product of the lactic acid bacterium, or an extract thereof, The lactic acid bacteria having a muscle synthesis promoting effect are at least one selected from the group consisting of Lactobacillus gasseri, Streptococcus thermophilus, Lactobacillus rhamnosus, Lactococcus lactis, Lactobacillus delbreckii, and Lactobacillus reuteri; The muscle atrophy prevention agent, wherein the lactic acid bacteria having muscle degradation inhibitory effect is at least one selected from the group consisting of Lactobacillus reuteri, Lactobacillus mucosae, Lactobacillus fermentum, Pediococcus acidilactici, Lactobacillus delbreckii, and Lactobacillus gasseri. [2] A muscle atrophy prevention agent according to [1], wherein the lactic acid bacteria having the muscle synthesis promoting effect are Lactococcus lactis subsp. cremoris or Lactococcus lactis subsp. lactis. [3] The muscle atrophy prevention agent according to [1] or [2], wherein the lactic acid bacteria having a muscle synthesis promoting effect are Lactobacillus gasseri SBT1848 strain (NITE BP-03075), Streptococcus thermophilus SBT1021A strain (FERM P-10658), Lactococcus lactis subsp. cremoris SBT1393 strain (NITE P-03278), Lactobacillus rhamnosus SBT2299 strain (NITE BP-02994), Lactococcus lactis subsp. lactis SBT2397 strain (NITE P-03080), or Lactobacillus reuteri SBT2970 strain (NITE BP-03282). [4] Lactic acid bacteria with muscle degradation inhibitory effects were identified as Lactobacillus reuteri SBT2970 (NITE BP-03282), Lactobacillus mucosae SBT2958 (NITE P-02803), Lactobacillus mucosae SBT10038 (NITE P-03283), Lactobacillus mucosae SBT10043 (NITE BP-03187), Lactobacillus fermentum SBT1846 (NITE AP-03279), Lactobacillus fermentum SBT1859 (NITE P-02996), Pediococcus acidilactici SBT3331 (NITE BP-02991), and Lactobacillus gasseri SBT1848 (NITE AP-03279). BP-03075) according to [1]. [5] A muscle atrophy prevention agent containing, as an active ingredient, a lactic acid bacterium having a muscle synthesis promoting effect and a muscle degradation inhibiting effect, a processed product of the lactic acid bacterium, or an extract thereof. [6] A muscle atrophy prevention agent according to [5], wherein the lactic acid bacteria having the effect of promoting muscle synthesis and inhibiting muscle degradation are Lactobacillus delbreckii, Lactobacillus gasseri, or Lactobacillus reuteri. [7] A muscle atrophy prevention agent according to [6], wherein the lactic acid bacteria having the effect of promoting muscle synthesis and inhibiting muscle breakdown is Lactobacillus delbreckii subsp. lactis. [8] The muscle atrophy prevention agent according to [6], wherein the lactic acid bacteria having the effect of promoting muscle synthesis and inhibiting muscle degradation are Lactobacillus delbreckii subsp. lactis SBT1371 strain (NITE BP-03277), Lactobacillus delbreckii subsp. lactis SBT2002 strain (NITE BP-03280), Lactobacillus delbreckii subsp. lactis SBT2080 strain (NITE BP-03281), Lactobacillus gasseri SBT1848 strain (NITE BP-03075), or Lactobacillus reuteri SBT2970 strain (NITE BP-03282). [9] A pharmaceutical, food or drink, functional food, food for specified health uses, nutritional supplement, supplement, or feed for preventing muscle atrophy, comprising the muscle atrophy preventive agent according to any one of [1] to [8].

[10] Novel lactic acid bacteria: Lactobacillus delbreckii subsp. lactis SBT1371 (NITE BP-03277), Lactobacillus delbreckii subsp. lactis SBT2002 (NITE BP-03280), Lactobacillus delbreckii subsp. lactis SBT2080 (NITE BP-03281), Lactococcus lactis subsp. cremoris SBT1393 (NITE P-03278), Lactobacillus reuteri SBT2970 (NITE BP-03282), Lactobacillus mucosae SBT10038 (NITE P-03283), and Lactobacillus fermentum SBT1846 (NITE P-03279). The present invention further includes the following features

[11] to

[18] .

[11] A method for preventing muscle atrophy, comprising administering to a subject lactic acid bacteria having a muscle synthesis promoting effect and / or a muscle degradation inhibiting effect, a processed product of the lactic acid bacteria, or an extract thereof, The lactic acid bacteria having a muscle synthesis promoting effect are at least one selected from the group consisting of Lactobacillus gasseri, Streptococcus thermophilus, Lactobacillus rhamnosus, Lactococcus lactis, and Lactobacillus delbreckii, The method for preventing muscle atrophy, wherein the lactic acid bacteria having the muscle degradation inhibitory effect are any one or more species selected from the group consisting of Lactobacillus reuteri, Lactobacillus mucosae, Lactobacillus fermentum, Pediococcus acidilactici, and Lactobacillus delbreckii.

[12] A method for preventing muscle atrophy according to

[11] , wherein the lactic acid bacteria having the effect of promoting muscle synthesis are Lactococcus lactis subsp. cremoris or Lactococcus lactis subsp. lactis.

[13] A method for preventing muscle atrophy according to

[11] or

[12] , wherein the lactic acid bacteria having a muscle synthesis-promoting effect are Lactobacillus gasseri SBT1848 strain (NITE BP-03075), Streptococcus thermophilus SBT1021A strain (FERM P-10658), Lactococcus lactis subsp. cremoris SBT1393 strain (NITE P-03278), Lactobacillus rhamnosus SBT2299 strain (NITE BP-02994), Lactococcus lactis subsp. lactis SBT2397 strain (NITE P-03080), or Lactobacillus reuteri SBT2970 strain (NITE BP-03282).

[14] Lactic acid bacteria with muscle degradation inhibitory effects were identified as Lactobacillus reuteri SBT2970 (NITE BP-03282), Lactobacillus mucosae SBT2958 (NITE P-02803), Lactobacillus mucosae SBT10038 (NITE P-03283), Lactobacillus mucosae SBT10043 (NITE BP-03187), Lactobacillus fermentum SBT1846 (NITE AP-03279), Lactobacillus fermentum SBT1859 (NITE P-02996), Pediococcus acidilactici SBT3331 (NITE BP-02991), and Lactobacillus gasseri SBT1848 (NITE AP-03279). The method for preventing muscle atrophy described in

[11] is the method for preventing muscle atrophy described in

[12] .

[15] A method for preventing muscle atrophy, comprising the step of administering to a subject lactic acid bacteria having muscle synthesis-promoting effects and muscle degradation-inhibiting effects, a processed product of the lactic acid bacteria, or an extract thereof.

[16] The method for preventing muscle atrophy according to

[15] , wherein the lactic acid bacteria having the effect of promoting muscle synthesis and inhibiting muscle degradation are Lactobacillus delbreckii, Lactobacillus gasseri, or Lactobacillus reuteri.

[17] A method for preventing muscle atrophy according to

[16] , wherein the lactic acid bacteria having the effect of promoting muscle synthesis and inhibiting muscle degradation is Lactobacillus delbreckii subsp. lactis.

[18] The method for preventing muscle atrophy according to

[16] , wherein the lactic acid bacteria having the effect of promoting muscle synthesis and inhibiting muscle degradation are Lactobacillus delbreckii subsp. lactis SBT1371 strain (NITE BP-03277), Lactobacillus delbreckii subsp. lactis SBT2002 strain (NITE BP-03280), Lactobacillus delbreckii subsp. lactis SBT2080 strain (NITE BP-03281), Lactobacillus gasseri SBT1848 strain (NITE BP-03075), or Lactobacillus reuteri SBT2970 strain (NITE BP-03282). [Effects of the Invention]

[0012] Preventing muscle atrophy and maintaining muscle mass improves the quality of life of not only the elderly but also young people. According to the present invention, muscle atrophy can be prevented by ingesting specific lactic acid bacteria that promote muscle synthesis or inhibit muscle breakdown. Furthermore, muscle atrophy can be effectively prevented by ingesting lactic acid bacteria that promote muscle synthesis and simultaneously inhibit muscle breakdown. [Brief explanation of the drawings]

[0013] [Figure 1] This graph relates to Test Example 1 and shows the relative concentration of phosphorylated p70S6K when each lactic acid bacteria was added to mouse myoblast C2C12 cells, with the mean value for the control group set at 1. (A) compares a control group to which no lactic acid bacteria lysate was added, one strain of lactic acid bacteria that showed no muscle synthesis-promoting effect, and eight strains of lactic acid bacteria that have a muscle synthesis-promoting effect. (B) compares a control group to which no lactic acid bacteria lysate was added, and three strains of lactic acid bacteria that have a muscle synthesis-promoting effect (of these, SBT1848 and 2002 were tested in duplicate with (A)). [Figure 2] This graph shows the muscle degradation inhibitory effect of each lactic acid bacteria strain based on the gene expression level of Atrogin-1 after adding dexamethasone to C2C12 cells differentiated into myotubes to induce muscle degradation, pertaining to Test Example 2. (A) shows a comparison between a group with nothing added, a group with dexamethasone only added, a group with dexamethasone and one strain of lactic acid bacteria that does not have a muscle degradation inhibitory effect added, and a group with dexamethasone and 10 strains of lactic acid bacteria that have a muscle degradation inhibitory effect added. (B) shows a comparison between a group with nothing added, a group with dexamethasone only added, and a group with dexamethasone and one strain of lactic acid bacteria that has a muscle degradation inhibitory effect added. [Figure 3]This graph shows the muscle degradation inhibitory effect of each lactic acid bacteria strain based on the gene expression level of MuRF-1 after adding dexamethasone to C2C12 cells differentiated into myotubes to induce muscle degradation, pertaining to Test Example 3. (A) compares a group with nothing added, a group with dexamethasone only added, a group with dexamethasone and one strain of lactic acid bacteria that does not have a muscle degradation inhibitory effect added, and a group with dexamethasone and 10 strains of lactic acid bacteria that have a muscle degradation inhibitory effect added. (B) compares a group with nothing added, a group with dexamethasone only added, and a group with dexamethasone and one strain of lactic acid bacteria that has a muscle degradation inhibitory effect added. DETAILED DESCRIPTION OF THE INVENTION

[0014] The present invention provides an agent for preventing muscle atrophy, which contains lactic acid bacteria, a processed product of lactic acid bacteria, or an extract thereof (hereinafter, also simply referred to as lactic acid bacteria, etc.) as an active ingredient. The lactic acid bacteria contained in the muscle atrophy prevention agent of the present invention are specific lactic acid bacteria having the effect of promoting muscle synthesis, and are preferably obtained from one or more species selected from the group consisting of Lactobacillus gasseri, Streptococcus thermophilus, Lactobacillus rhamnosus, Lactococcus lactis, Lactobacillus delbreckii, and Lactobacillus reuteri. Among these, Lactococcus lactis is more preferably Lactococcus lactis subsp. cremoris or Lactococcus lactis subsp. lactis. Furthermore, among the above-mentioned bacterial species, it is more preferable that the lactic acid bacteria etc. are obtained from Lactobacillus gasseri SBT1848 strain, Streptococcus thermophilus SBT1021A strain, Lactococcus lactis subsp. cremoris SBT1393 strain, Lactobacillus rhamnosus SBT2299 strain, Lactococcus lactis subsp. lactis SBT2397 strain, or Lactobacillus reuteri SBT2970 strain.

[0015] Furthermore, the lactic acid bacteria contained in the muscle atrophy prevention agent of the present invention are specific lactic acid bacteria having the effect of inhibiting muscle breakdown, and are preferably obtained from one or more species selected from the group consisting of Lactobacillus reuteri, Lactobacillus mucosae, Lactobacillus fermentum, Pediococcus acidilactici, Lactobacillus delbreckii, and Lactobacillus gasseri. Furthermore, among the above bacterial species, it is more preferable that the lactic acid bacteria etc. are obtained from Lactobacillus reuteri SBT2970 strain, Lactobacillus mucosae SBT2958 strain, Lactobacillus mucosae SBT10038 strain, Lactobacillus mucosae SBT10043 strain, Lactobacillus fermentum SBT1846 strain, Lactobacillus fermentum SBT1859 strain, Pediococcus acidilactici SBT3331 strain, or Lactobacillus gasseri SBT1848 strain.

[0016] Furthermore, the lactic acid bacteria contained in the muscle atrophy prevention agent of the present invention are lactic acid bacteria that have the effect of promoting muscle synthesis and inhibiting muscle breakdown, and any lactic acid bacteria that have these effects can be used. Lactic acid bacteria having the effects of promoting muscle synthesis and inhibiting muscle breakdown are preferably obtained from Lactobacillus delbreckii, Lactobacillus reuteri, or Lactobacillus gasseri. Lactobacillus delbreckii is preferably obtained from Lactobacillus delbreckii subsp. lactis. Furthermore, among the above-mentioned bacterial species, it is even more preferable that the lactic acid bacteria etc. are obtained from Lactobacillus delbreckii subsp. lactis SBT1371 strain, Lactobacillus delbreckii subsp. lactis SBT2002 strain, Lactobacillus delbreckii subsp. lactis SBT2080 strain, Lactobacillus reuteri SBT2970 strain, or Lactobacillus gasseri SBT1848 strain.

[0017] The lactic acid bacteria are preferably obtained from at least one of Lactobacillus delbreckii subsp. lactis, Lactobacillus gasseri, Streptococcus thermophilus, Lactococcus lactis subsp. cremoris, Lactobacillus rhamnosus, Lactococcus lactis subsp. lactis, Lactobacillus reuteri, Lactobacillus mucosae, Lactobacillus fermentum, and Pediococcus acidilactici, and the above lactic acid bacteria may be used alone or in combination of two or more species.

[0018] All of the lactic acid bacteria strains described above have been deposited at the National Institute of Technology and Evaluation's Patent Microorganisms Depositary (NPMD). Deposit information is provided later in the specification.

[0019] The muscle atrophy prevention agent of the present invention has the effect of preventing muscle atrophy, and the presence or absence of the muscle atrophy prevention effect can be determined by the presence or absence of the muscle synthesis promoting effect and / or muscle degradation inhibiting effect. As will be shown in the Examples below, the presence or absence of the muscle synthesis promoting effect can be determined by, for example, whether or not an increase in the amount of phosphorylated p70S6K is observed in the target lactic acid bacteria, etc., compared to when no additive is added. Furthermore, as shown in the Examples, the presence or absence of muscle degradation inhibitory action can be determined by, for example, whether the gene expression levels of Atrogin-1 and MuRF1 for the target lactic acid bacteria, etc., are reduced compared to when no additives are added.

[0020] The medium for culturing the lactic acid bacteria of the present invention is not particularly limited as long as it is a medium in which the lactic acid bacteria can be cultured, and any medium can be used.

[0021] The lactic acid bacteria according to this embodiment may be cultured according to a conventional method for culturing lactic acid bacteria and prepared in a desired amount. For example, lactic acid bacteria may be cultured in a synthetic medium such as MRS medium, and the resulting culture may be centrifuged to obtain lactic acid bacteria. The resulting bacterial cells may be used as is, or may be subjected to concentration, drying, freeze-drying, or crushing treatment. The bacterial cells may be killed by heat drying or the like before use.

[0022] As described above, the muscle atrophy prevention agent of this embodiment can use bacterial cells that have been subjected to concentration, drying, freeze-drying, or crushing treatment, or bacterial cells that have been killed by heat drying, etc., and therefore can be widely used as a composition contained in pharmaceuticals, quasi-drugs, foods and beverages, feed, etc. When preparing the formulation, commonly used additives such as excipients, binders, disintegrants, and flavoring agents may be appropriately mixed. The food and drink products for preventing muscle atrophy of the present invention may be any food and drink products containing the active ingredient, such as lactic acid bacteria, and examples thereof include functional foods, foods for specified health uses, nutritional supplements, and supplements. Examples of the food and drink products of the present invention include milk drinks, yogurt, cheese, ice cream, soft drinks, butter, condensed milk, crackers, infant formula, powdered milk, liquid milk, and seasonings. The active ingredient of the present invention may also be added to existing food and drink products at the raw material stage, during production, or after production. Furthermore, since the active ingredient of the present invention is lactic acid bacteria, fermented foods such as yogurt, cheese, and milk drinks fermented with the lactic acid bacteria of the present invention are examples of food and drink products in which the active ingredient itself is used to prevent muscle atrophy. The amount of muscle atrophy prevention agent to be added to these foods and beverages is not particularly limited, as it varies depending on the form, dosage form, symptoms, weight, and purpose of the subject, but if one were to give an example, it can be added at 0.001 to 100 (w / w)%. As a feed for preventing muscle atrophy, the active ingredient may be mixed with ordinary feed.

[0023] There are no particular limitations on the recipients of the muscle atrophy preventive agent according to this embodiment or the daily intake amount, and for example, when the recipients are humans, the agent can be administered to minors under 20 years of age, adults, elderly people over 65 years of age, etc. The daily intake amount is not particularly limited as it differs depending on age, symptoms, body weight, and purpose, but an example would be 0.001 to 10 g, preferably 0.01 to 5 g, in terms of the weight of the bacterial cells or treated bacterial cells.

[0024] As described above, this embodiment provides a novel muscle atrophy prevention agent. This muscle atrophy prevention agent contains lactic acid bacteria or the like as an active ingredient, can be produced in large quantities at low cost, and is highly safe. In particular, lactic acid bacteria or the like that can promote muscle synthesis while simultaneously suppressing muscle breakdown are highly effective in preventing muscle atrophy. [Example]

[0025] As an example of the present invention, the results of evaluating the muscle synthesis promoting effect and muscle degradation inhibiting effect using mouse myoblast C2C12 cells will be briefly described below. However, the present invention is not limited to the embodiments of the example.

[0026] [Preparation of lactic acid bacteria lysate] Glycerol stocks of each strain were streaked onto MRS plates and cultured at 37°C for three nights (64 hours). A single colony from the plate was then inoculated into 10 mL of MRS medium and cultured overnight at 37°C or 30°C for 16 hours. 300 μL of this bacterial culture was transferred to 10 mL of fresh MRS medium and cultured again overnight at 37°C or 30°C for 16 hours. Next, 3 mL of the bacterial culture was inoculated into 100 mL of MRS medium and cultured overnight at 37°C or 30°C for 16 hours. The resulting bacterial culture was centrifuged, and the precipitated bacterial cells were washed with PBS and ultrapure water. The bacterial cells were resuspended in 4 mL of ultrapure water and frozen at -80°C. The cells were then freeze-dried using a freeze dryer (Tokyo Rikakikai). Lyophilized bacterial cells were obtained. The obtained freeze-dried cells were dissolved in PBS or DMEM medium (Gibco) and disrupted using a Multi-Beads Shocker (Yasui Kikai) to prepare disrupted lactic acid bacteria.

[0027] [Test Example 1] Evaluation of the effect of enhancing p70S6K phosphorylation Mouse myoblast C2C12 cells were cultured at 1.8 × 10 cells / well in a 48-well culture plate (IWAKI) containing DMEM medium containing 10% FBS and 1% penicillin-streptomycin. 4 The cells were seeded at 100 cells / well and cultured at 37°C and 5% CO2 for 2 days until the cells reached 80% to 90% confluence. The medium was then replaced with DMEM containing 1% penicillin-streptomycin without FBS and cultured for 4 hours. After culture, lysates of each LAB strain shown in Figure 1, suspended in DMEM containing 1% penicillin-streptomycin, were added to the cells at a concentration of 100 μg / mL. An equal volume of DMEM containing 1% penicillin-streptomycin without LAB lysates was added to the control group. After culturing the C2C12 cells for an additional 2 hours, the cells were washed with PBS and lysed using Cell Extraction Buffer PTR provided with the p70S6K (pT389) SimpleStep ELISA (Abcam). The cell lysate was collected and centrifuged to obtain the supernatant, which was used to prepare the protein solution sample for the subsequent ELISA. ELISA was performed using the p70S6K (pT389) SimpleStep ELISA and a pre-coated 384-well microplate SimpleStep ELISA (Abcam), which can detect the concentration of phosphorylated p70S6K. The total protein concentration of cell lysates was quantified by the BCA method, and each sample was diluted with Cell Extraction Buffer PTR to a constant concentration. These, along with a control sample for creating a standard curve, were added to a pre-coated 384-well microplate SimpleStep ELISA at 25 μL / well, and color development was performed as described in the p70S6K (pT389) SimpleStep ELISA manual. Absorbance was measured at a dominant wavelength of 450 nm and a subordinate wavelength of 620 nm using a VARIOSKAN (Thermo Scientific), and the difference between the absorbances at the two wavelengths was calculated. A standard curve was then created using the control samples, and the relative concentration of phosphorylated p70S6K in each sample was calculated.

[0028] The results of comparing eight strains that showed activity with one strain that did not show activity are shown in Figure 1. Three strains of Lactobacillus delbreckii subsp. lactis (SBT2002, SBT2080, and SBT1371), as well as Lactobacillus gasseri (SBT1848), Streptococcus thermophilus (SBT1021A), Lactococcus lactis subsp. cremoris (SBT1393), Lactobacillus rhamnosus (SBT2299), Lactococcus lactis subsp. lactis (SBT2397), and Lactobacillus reuteri (SBT2970) increased the amount of phosphorylated p70S6K and demonstrated muscle synthesis-promoting activity. On the other hand, Leuconostoc mesenteroides subsp. mesenteroides A did not increase the amount of phosphorylated p70S6K and did not exhibit muscle synthesis-promoting activity.

[0029] [Test Example 2] Evaluation of the effect of suppressing gene expression of Atrogin-1 and MuRF1 Mouse myoblast C2C12 cells were cultured at 1.8 × 10 cells / well in a 48-well culture plate (IWAKI) containing DMEM medium containing 10% FBS and 1% penicillin-streptomycin. 4 The cells were seeded at 100 cells / well and cultured at 37°C and 5% CO2 for 2 days until the cells reached 80% to 90% confluence. The medium was then replaced with DMEM containing 2% horse serum and 1% penicillin-streptomycin. The medium was changed every 2–3 days, and the cells were cultured for 5 days to differentiate. Subsequently, dexamethasone, known to upregulate Atrogin-1 and MuRF1 gene expression, was added to the same medium at a concentration of 1 μM. The Lactobacillus lysate shown in Figures 2 and 3 was added at 100 μg / mL. After 24 hours of culture, the medium was removed, and 200 μL of Sepasol-RNA I Super G (Nacalai Tesque) was added to the cells. The cells were then disrupted by pipetting and harvested. RNA was extracted from the harvested solution, and cDNA was synthesized using approximately 500 ng of RNA as a template using ReverTra Ace qPCR RT Master Mix with gDNA remover (TOYOBO). The expression levels of Atrogin-1 and MuRF1 genes were then quantified using the ViiA7 Real-time PCR system (Thermo Fisher Scientific) with THUNDERBIRD SYBR qPCR Mix (TOYOBO) and amplification primers for various genes. The Gapdh gene was used as an endogenous control. The sequences of the primers used are shown in Table 1.

[0030] [Table 1]

[0031] The results of comparing strains that showed activity with those that did not are shown in Figures 2 and 3. Three strains of Lactobacillus delbreckii subsp. lactis (SBT2002, SBT2080, and SBT1371), Lactobacillus reuteri (SBT2970), Lactobacillus mucosae (SBT2958), Lactobacillus mucosae (SBT10038), Lactobacillus mucosae (SBT10043), Lactobacillus fermentum (SBT1846), Lactobacillus fermentum (SBT1859), Pediococcus acidilactici (SBT3331), and Lactobacillus gasseri (SBT1848) reduced the expression levels of both Atrogin-1 and MuRF1 genes, demonstrating inhibitory effects on muscle degradation. On the other hand, Lactobacillus lactis subsp. lactis A did not reduce the gene expression levels of Atrogin-1 or MuRF1, and did not exhibit any inhibitory effect on muscle breakdown.

[0032] These results demonstrate that the lactic acid bacteria described in each test example have the ability to promote muscle synthesis or inhibit muscle breakdown. In particular, the Lactobacillus delbreckii subsp. lactis, Lactobacillus reuteri, and Lactobacillus gasseri tested all exhibited both muscle synthesis and muscle breakdown inhibitory activities.

[0033] The present invention is not limited to the strains described in the examples, and is likely to be widely applicable to the same bacterial species. [Industrial Applicability]

[0034] According to the present invention, muscle atrophy can be prevented by ingesting specific lactic acid bacteria, which promote muscle synthesis or inhibit muscle breakdown. Furthermore, muscle atrophy can be effectively prevented by ingesting lactic acid bacteria that promote muscle synthesis and simultaneously inhibit muscle breakdown. The muscle atrophy prevention agent of the present invention can improve the QOL of not only elderly people but also young people. [Accession number]

[0035] [Reference to deposited biological material] (1)SBT1371 a) The name and address of the depository institution that deposited the biological material National Institute of Technology and Evaluation, Patent Microorganism Deposit Center (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture (Postal Code 292-0818)) Date of deposit of biological material in a depository institution in the Republic of Ireland September 15, 2020 (Transfer to international deposit date: September 10, 2021) The accession number assigned to the deposit by the depository institution NITE BP-03277 (2) SBT2002 a) The name and address of the depository institution that deposited the biological material Same as (1) above Date of deposit of biological material in a depository institution in the Republic of Ireland September 15, 2020 (Transfer to international deposit date: September 10, 2021) The accession number assigned to the deposit by the depository institution NITE BP-03280 (3) SBT2080 a) The name and address of the depository institution that deposited the biological material Same as (1) above Date of deposit of biological material in a depository institution in the Republic of Ireland September 15, 2020 (Transfer to international deposit date: September 10, 2021) The accession number assigned to the deposit by the depository institution NITE BP-03281 (4)SBT1393 a) The name and address of the depository institution that deposited the biological material Same as (1) above Date of deposit of biological material in a depository institution in the Republic of Ireland September 15, 2020 The accession number assigned to the deposit by the depository institution NITE P-03278 (5)SBT2970 a) The name and address of the depository institution that deposited the biological material Same as (1) above Date of deposit of biological material in a depository institution in the Republic of Ireland September 15, 2020 (Transfer to international deposit date: September 10, 2021) The accession number assigned to the deposit by the depository institution NITE BP-03282 (6)SBT10038 a) The name and address of the depository institution that deposited the biological material Same as (1) above Date of deposit of biological material in a depository institution in the Republic of Ireland September 15, 2020 The accession number assigned to the deposit by the depository institution NITE P-03283 (7)SBT1846 a) The name and address of the depository institution that deposited the biological material Same as (1) above Date of deposit of biological material in a depository institution in the Republic of Ireland September 15, 2020 The accession number assigned to the deposit by the depository institution NITE P-03279 (8)SBT10043 a) The name and address of the depository institution that deposited the biological material Same as (1) above Date of deposit of biological material in a depository institution in the Republic of Ireland March 27, 2020 (Transfer to international deposit March 23, 2021) The accession number assigned to the deposit by the depository institution NITE BP-03187 (9) SBT1848 strain a) The name and address of the depository institution that deposited the biological material Same as (1) above Date of deposit of biological material in a depository institution in the Republic of Ireland November 25, 2019 (Transfer to international deposit: September 10, 2021) The accession number assigned to the deposit by the depository institution NITE BP-03075 (10) SBT1021A strain a) The name and address of the depository institution that deposited the biological material National Institute of Technology and Evaluation, Patent Organism Deposit Center (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture (Postal Code 292-0818)) Date of deposit of biological material in a depository institution in the Republic of Ireland April 13, 1989 The accession number assigned to the deposit by the depository institution FERM P-10658 (11) SBT2299 strain a) The name and address of the depository institution that deposited the biological material Same as (1) above Date of deposit of biological material in a depository institution in the Republic of Ireland June 26, 2019 (Transfer to international deposit date: July 13, 2020) The accession number assigned to the deposit by the depository institution NITE BP-02994 (12) SBT2397 strain a) The name and address of the depository institution that deposited the biological material Same as (1) above Date of deposit of biological material in a depository institution in the Republic of Ireland November 25, 2019 The accession number assigned to the deposit by the depository institution NITE P-03080 (13) SBT2958 strain a) The name and address of the depository institution that deposited the biological material Same as (1) above Date of deposit of biological material in a depository institution in the Republic of Ireland October 31, 2018 The accession number assigned to the deposit by the depository institution NITE P-02803 (14) SBT1859 strain a) The name and address of the depository institution that deposited the biological material Same as (1) above Date of deposit of biological material in a depository institution in the Republic of Ireland June 26, 2019 The accession number assigned to the deposit by the depository institution NITE P-02996 (15) SBT3331 strain a) The name and address of the depository institution that deposited the biological material Same as (1) above Date of deposit of biological material in a depository institution in the Republic of Ireland June 26, 2019 (Transfer to international deposit date: July 13, 2020) The accession number assigned to the deposit by the depository institution NITE BP-02991

Claims

1. A muscle atrophy prevention agent containing, as an active ingredient, a lactic acid bacterium having muscle synthesis promoting effects and muscle degradation inhibiting effects, a processed product of the lactic acid bacterium, or an extract thereof, wherein the lactic acid bacterium having muscle synthesis promoting effects and muscle degradation inhibiting effects is Lactobacillus gasseri SBT1848 strain (NITE BP-03075).

2. A pharmaceutical, food or drink, functional food, food for specified health uses, nutritional supplement, supplement, or feed for preventing muscle atrophy, comprising the muscle atrophy prevention agent according to claim 1.

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