KR101800632B1 - Pharmaceutical composition, food composition or food additives for prevention, improvement or treatment of muscle loss, weakening, and atrophy comprising Enterococcus faecalis, it culture broth or heat killed Enterococcus faecalis as an active ingredient - Google Patents

Abstract

The present invention relates to a pharmaceutical composition, a food composition and a pharmaceutical composition for preventing, ameliorating or ameliorating muscular weakness and muscle atrophy, which contains, as an active ingredient, an enterococcus faecalis EF-2001, a culture thereof or a bacterium thereof, The present invention relates to a food additive. Since the Enterococcus faecalis EF-2001 cells of the present invention inhibit muscle cell damage caused by oxidative stress and exhibit remarkable effect on muscle decay, weakening and muscle atrophy, Of the lactic acid bacterium Enterococcus faecalis EF-2001 or a culture thereof can be effectively used as an active ingredient of a pharmaceutical composition, a food composition and a food additive for the prevention of muscle atrophy and muscle decline.

Description

TECHNICAL FIELD [0001] The present invention relates to a pharmaceutical composition, a food composition or a food additive for prevention, a composition for preventing or ameliorating muscular weakening, weakening and muscle atrophy, which contains the enterococcus faecalis, improvement or treatment of muscle loss, weakening, and atrophy by Enterococcus faecalis, it culture broth or heat killed Enterococcus faecalis as an active ingredient}

The present invention relates to a novel use of an Enterococcus faecalis, a culture thereof or a bacterium thereof, and more particularly to a novel use of Enterococcus faecalis EF-2001 The present invention relates to a pharmaceutical composition, a food composition and a food additive for preventing, treating and / or ameliorating muscle decline, weakening and muscle atrophy, which comprises a culture solution thereof or a bacterium thereof as an active ingredient.

Diseases that cause weakness of muscle strength include sarcopenia with aging, muscle atrophy caused by imbalance of protein metabolism or muscle use, starvation, wasting disease (such as cancer), aging And acardiotrophy.

Sarcopenia refers to a decrease in muscle strength with a decrease in skeletal muscle mass during aging. In addition to the decrease in muscle mass, which is the most important feature of myopenia, changes in the type of muscle fiber are also observed. Type 1 and Type 2 decrease in age at the same rate, whereas Type 2 muscle fibers do not change much in the presence of myopenia, but Type 1 muscle fiber thickness decreases significantly. This myopenic disorder has been reported to cause senescence and dysfunctions among the elderly (Roubenoff R., Can. J. Appl. Physiol. 26, 78-89, 2001).

Although myopenia is caused by various factors, research on each factor is still insignificant. The balance of growth hormone reduction or neurological change, changes in physical activity, changes in metabolism, sex hormone levels or fat or catabolic cytokines, and the synthesis and differentiation of proteins (Roubenoff R. and Hughes VA, J. Gerontol. A. Biol. Sci. Med. Sci. 55, M716-M724, 2000). One of the most important features of myopenia is the decrease in satellite cell activation. Satellite cells are small mononuclear cells located between the basement membrane and the sarcolemma of the myofiber. They are activated by stimulation, such as injury or exercise, and proliferate into myoblasts. When differentiation proceeds, they fuse with other cells to form multinuclear muscle fibers. Thus, as the activity of satellite cells decreases, the ability to regenerate damaged muscle or response to differentiation signals is reduced, resulting in decreased muscle formation.

On the other hand, muscle atrophy is caused by damage to muscle tissue due to lack of mechanical stimulation such as decrease of muscle use, destruction of muscles due to direct injury or physical factors, restoration of muscular cell by aging, , And disability of muscle use due to damage to the nerves that regulate muscle function (Booth FW, J Appl Physiol Respir Environ Exerc Physiol., 1982). In general, disuse atrophy occurs gradually due to loss of muscle strength due to the disability of the muscles of the affected part and the peripheral part due to disability or accident for a long time, and myasthenia due to the disease of the muscle itself gravis, dystrophy: progressive muscular dystrophy, myotonic muscular dystrophy, duchen type, Becker type, parietal lobe, facial shoulder brachial type, inflammation occurring in the muscle itself, muscle atrophy caused by muscle- Amyotrophic lateral sclerosis (ALS): Lou Gehrig's disease, sphinobulbar muscular atrophy: Kennedy's disease, and the like. But also in form.

Based on the quality of life and the quality of life that has been achieved today, the interests of people are increasingly focused on welfare and leisure activities as compared to the past that was concentrated in food and shelter, and they are trying to lead a healthy life through exercise. In order to carry out such a life, the free control and use of the veterinary muscle should be premised, and situations in which the use of the veterinary muscle due to muscle function abnormality is impossible may result in limiting the activity range of the person and lowering the quality of life.

There are three main treatment methods for myopenia. The first is exercise. Exercise has been reported to increase skeletal muscle protein synthesis in the short term and to increase muscular strength and motility of the elderly. However, it is inappropriate for long-term treatment (Timothy J. Doherty, J. Appl. Physiol. 95, 1717-1727, 2003). The second is the use of testosterone or anabolic steroid as a medication, but it induces menstruation in women and side effects such as prostate symptoms in men. Other approved regimens include DHEA (dehydroepiandrosterone) and growth hormone, which have been reported to be therapeutically feasible at sites that include SARMs (DD Thompson, J. Musculoskelet Neuronal Interact 7, 344-345 , 2007). Recently, stem cell therapy (stem cell therapy) which separates satellite cells and introduces them into the body after in vitro differentiation and activates satellite cells directly in the body and promotes myogenesis, (Shihuan Kuang and Michael A. Rudnicki, Trends in Molecular Medicine 14, 82-31, 2008).

In general, the best way to prevent the onset of muscular atrophy in daily life is to prevent muscle loss through continuous use of muscles, but even if muscles can not be used regardless of their will, such as injury or disability, May result in muscle atrophy caused by degeneration of the muscle. In this case, it is necessary to study countermeasures to prevent muscle atrophy by stimulating muscles or preventing muscle atrophy.

Reactive oxygen species are highly chemically reactive groups of molecules that are highly reactive for redox reactions that attempt to exchange electrons with nearby molecules. Due to the reactivity of these active oxygen species, surrounding substances such as protein molecules or DNA in the cell lose electrons and become oxidized to exhibit mutations, failing to perform their proper functions, and failing to function properly, Death can be induced. Oxidative stress caused by reactive oxygen species is also present in muscle cells, and oxidative stress may lead to muscle cell loss and muscle atrophy. Several stressors have been shown to affect various tissues in vivo as well as damage to muscle tissue, and have been attracting attention as various disease factors (MCKinnell IW., And Rudnicki MA., Cell, 2004).

Enterococcus faecalis EF-2001 ( Enterococcus faecalis EF-2001) were identified through intestinal flora screening of a 2-year-old girl. The Enterococcus faecalis EF-2001 is heat-treated to kill and the cell component recovered is the Enterococcus faecalis EF-2001 microbial cell.

Studies on the physiological activity of Enterococcus faecalis EF-2001 have shown that DSS (Dextran sulfate sodium) -deleted mice have a DSS mitigation effect in mice fed Enterococcus faecalis EF-2001 (Tadano et al., J. Japan Mibyou System association, 2011). In the present study, we examined the effect of NK cells on the proliferation of sarcoma-180 cells. Biochemically, mice fed Enterococcus faecalis EF-2001 also showed a decrease in the ratio of total cholesterol and triglycerides after ingesting high-fat diets (Ku et al., Medicine and biology, 2007). Regarding the suppression of harmful microorganisms and the effect of dressing, the Enterococcus faecalis EF-2001 was found to be a causative agent of Candida to suppress the activity of albican shows the symptoms improved and the prevention effect efficacy is started to (Ishijima et al., Med. Mycol. J, 2014), in mice by administration of antibiotics as compared to the enteric control yuikgyun is multiply rapidly and harmful bacteria is inhibited (Simohashi et al., Medicine and biology, 2002). The various physiological activities of Enterococcus faecalis EF-2001 are not influenced by heat and pH due to the nature of dead cells, so they can be processed into various forms (Kan, Food industry, 2001). It is also possible to ingest a large amount of lactic acid bacteria in a small amount with a cell content of 7.5 trillion grams per gram.

On the other hand, Korean Patent No. 1,560,799 discloses a composition for treating spinal muscular atrophy comprising, as an active ingredient, hnRNP M (Heterogeneous nuclear ribonucleoprotein M) or a polynucleotide encoding the hnRNP M, Patent No. 1,468,123 has a disease, amyotrophic for improving or therapeutic composition comprising a mammalian umbilical cord-derived stem cells as an active ingredient is disclosed, Korea Patent registration No. 1385191 discloses a chicory (Cichorium intybus ) extract as an active ingredient, and Korean Patent No. 1,349,361 discloses a pharmaceutical composition for prevention and treatment of muscle atrophy comprising Eupatorium There is chinensis . simplicifolium ) as an active ingredient. However, the effect of the composition on the atrophy of the enterococcus faecalis, the culture solution thereof or the bacterium thereof is not known at all.

Accordingly, the present inventors have made efforts to develop a novel agent for treating muscle weakness, weakness and muscular dystrophy which is free from cytotoxicity and secured to the human body. As a result, it has been found that the Enterococcus faecalis EF-2001 cells are free from cytotoxicity, The present invention has been accomplished by discovering that it is effective to treat muscular dysfunction, weakness and muscle atrophy by inhibiting muscle cell damage and cell death caused by oxidative stress.

Korean Patent No. 1560799 (registered on Aug. 2015) Korean Registered Patent No. 1349361 (Registered on April 1, 2014)

Roubenoff R., Can. J. Appl. Physiol. 26, 78-89, 2001 Roubenoff R. and Hughes V. A., J. Gerontol. A. Biol. Sci. Med. Sci. 55, M716-M724, 2000 D.D. Thompson, J. Musculoskelet Neuronal Interact 7, 344-345, 2007 Shihuan Kuang, and Michael A. Rudnicki, Trends in Molecular Medicine 14, 82-31, 2008 Timothy J. Doherty, J. Appl. Physiol. 95, 1717-1727, 2003 Tadano et al., J. Japan Mibyou System association, 2011 Ku et al., Medicine and biology, 2007 Ishijima et al., Med. Mycol. J, 2014 Simohashi et al., Medicine and biology, 2002 Kan, Food industry, 2001

Conventionally, there has been no known use for the treatment of muscle weakness related to muscles of the Enterobacteriacephalus. Accordingly, the present invention demonstrates the stability of the Enterococcus faecalis EF-2001 to the human body and inhibits muscle cell damage caused by oxidative stress, thereby remarkably inhibiting muscle weakness, weakening, and treating atrophy Respectively. Therefore, it is an object of the present invention to provide a pharmaceutical composition and a food composition for proving the efficacy of treatment of muscle wasting, weakening and muscular atrophy of Enterococcus faecalis EF-2001 dead cells and culture thereof and for preventing and treating new muscle decline, weakening and muscle atrophy And a food additive.

In order to achieve the above object,

The present invention provides a pharmaceutical composition for preventing and treating muscular weakening-related diseases comprising Enterococcus faecalis , a culture solution thereof or a bacterium thereof as an active ingredient.

 The present invention also provides a food composition for preventing and ameliorating muscular weakness related to muscular weakness comprising Enterococcus faecalis, a culture solution thereof or a bacterium thereof as an active ingredient.

The present invention also provides a food additive for preventing and ameliorating muscle weakness-related diseases of muscles containing Enterococcus faecalis, a culture solution thereof or a bacterium cell thereof as an active ingredient.

The Enterococcus faecalis EF-2001 cells of the present invention are free from cytotoxicity and inhibit damage of muscle cells due to oxidative stress, thereby remarkably suppressing muscle weakness, weakening, and treatment of muscle atrophy Therefore, the enterococcus faecalis of the present invention, the culture thereof or the microbial cells thereof can be usefully used as an effective ingredient, a food composition and a food additive for a composition for preventing or treating muscular weakness, weakening and muscle atrophy.

FIG. 1 is a view showing cytotoxicity of Enterococcus faecalis EF-2001 ( Enterococcus faecalis EF-2001).
FIG. 2 shows the effect of Enterococcus faecalis EF-2001 on oxidative stress-induced muscle cell damage. NAC is a group treated with N-acetylcysteine, which is a positive control.
FIG. 3 is a graph comparing the expression of HSP70 (Heat Shock Protein) protein in muscle cells of Enterococcus faecalis EF-2001 in an oxidative stress environment.
FIG. 4 shows the results of oral administration of two different concentrations of Enterococcus faecalis EF-2001 at 2 mg / kg and 30 mg / kg after surgery in which the right hindlimb nerve of the rat was excised to restrict muscle movement and induce atrophy. And an experimental plan.
FIG. 5 is a graph showing the results of a prophylactic treatment in which the intramuscular movement of the right hindlimb nerve of the rat was resected to induce atrophy, and then the enterococcus faecalis EF-2001 was ingested daily for 1 week before surgery, FIG. 3 is a view showing a muscle image of a muscle of a treatment group ingested with Enterococcus faecalis EF-2001 by using a micro-tomography apparatus after 3 weeks of operation.
FIG. 6 is a graph showing a comparison and analysis of the effect and the difference on the atrophy according to the ingestion of the Enterococcus faecalis EF-2001 based on the photographed image shown in FIG.

Hereinafter, the present invention will be described in more detail.

The present invention provides a pharmaceutical composition for preventing and treating muscular weakening-related diseases comprising Enterococcus faecalis , a culture solution thereof or a bacterium thereof as an active ingredient.

The enterococcus microorganisms are widely available in nature and utilize carbohydrates aerobically. In general, bacteria such as enterococcus microorganisms are known to prevent damage by pathogenic microorganisms due to in vivo antagonistic action or secreted antimicrobial substances.

The culture of Enterococcus faecalis, its culture or its microbial cells may be used either commercially or produced by known microbial cell production methods without any toxicity and harmless to human body.

The culture medium refers to a culture of Enterococcus faecalis cultured in a culture medium, a culture solution, a concentrated culture solution, a culture solution, a culture filtrate, a concentrated culture filtrate, or a culture filtrate. And may be a culture filtrate obtained by removing the strain.

The dead cells may be prepared by heat treating the corresponding live cells or treating them with formalin or other bactericides, and the dead cells may be substantially dead. In addition, the dead cells used in the present invention can be produced by the following method, but are not limited thereto:

1) seed culture of Enterococcus faecalis followed by culture at a temperature range of from 20 to 40 ° C in a neutral or slightly acidic state;

2) a step of culturing the above 1), followed by heat treatment and drying;

"Muscle weakness" of the muscle means a state in which the force of one or more muscles is reduced. The muscle weakness may be limited to one muscle, one side, upper or lower side of the body, or may appear throughout the body. In addition, subjective muscle weakness symptoms including myopia and myalgia can be quantified in an objective way through physical examination.

The muscle weakness-related disease refers to all diseases that may occur due to weakness of muscle strength, such as, for example, muscular dystrophy or muscular dystrophy.

The myopenia refers to a gradual decrease in skeletal muscle mass due to aging, which directly leads to a decrease in muscle strength, resulting in a decrease in various body functions and a disorder.

It is preferable that the atrophy is atrophy caused by muscle atrophy caused by loss of muscle tissue caused by not using muscle, muscle atrophy caused by muscle itself, or muscle damage caused by nerve damage. Muscle atrophy caused by loss of muscle tissue caused by not using the muscles is called disuse atrophy. Muscle atrophy caused by the muscle itself is caused by myasthenia gravis or dystrophy, It is preferable that the atrophy caused by the damage of the dominant nerve is spinal muscular amyotrophy, amyotrophic lateral sclerosis or sphinobulbar muscular atrophy.

In a specific experimental example of the present invention, the present inventors confirmed that the Enterococcus faecalis EF-2001 dead cells were not cytotoxic (see FIG. 1), and the enterococcus faecalis EF-2001 The effect of restoring the survival rate of muscle cells according to the concentration of the microbial cells was confirmed (see FIG. 2). As a result of measurement of the amount of HSP70 protein in cells supplemented with Enterococcus faecalis EF-2001 cells, enterococcus faecalis EF-2001 cells increased expression of HSP70 protein in muscle cells to induce inhibition of muscle atrophy , And it was confirmed that the reduced muscle mass was restored after ingesting the Enterococcus faecalis EF-2001 cells in the muscle-induced mice.

In conclusion, the Enterococcus faecalis EF-2001 microbial cells of the present invention have no cytotoxicity and exhibit a remarkable effect of treating atrophy by inhibiting oxidative stress-induced muscle cell damage. Therefore, the Enterococcus faecalis EF -2001, a pharmaceutical composition for preventing and treating muscular atrophy and muscle reduction comprising the culture liquid or its dead cells as an active ingredient can be provided.

The composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit or risk ratio applicable to medical treatment, and the effective dose level will depend on the type of disease, severity, , Sensitivity to the drug, time of administration, route of administration and rate of release, duration of treatment, factors including co-administered drugs, and other factors well known in the medical arts. The composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered singly or in multiple doses. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without side effects, which can be easily determined by those skilled in the art.

Specifically, the effective amount of the composition according to the present invention may vary depending on the age, sex, and body weight of the patient, and is generally in the range of 0.1 mg to 100 mg, preferably 0.2 mg to 17 mg per kg of body weight per day or every other day Or one to three times a day. However, the dosage may be varied depending on the route of administration, the severity of obesity, sex, weight, age, etc. Therefore, the dosage is not limited to the scope of the present invention by any means.

When the composition is formulated, it is prepared using diluents or excipients such as fillers, extenders, binders, humectants, disintegrants, surfactants and the like which are usually used.

Solid preparations for oral administration include tablets, patients, powders, granules, capsules, troches and the like. These solid preparations can be prepared by adding to the lactic acid bacterium Enterococcus faecalis EF-2001 of the present invention at least one excipient, , Starch, calcium carbonate, sucrose or lactose, or gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions or syrups. In addition to water and liquid paraffin which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances and preservatives may be included. have.

Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, and the like.

Examples of the non-aqueous solvent and the suspending agent include propylene glycol, polyethyleneglycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As a base for suppositories, witepsol, macrogol, tween 61, cacao paper, laurin, glycerol, gelatin and the like can be used.

In addition, the present invention provides a food composition for preventing and ameliorating muscular weakness related to muscular weakness comprising Enterococcus faecalis, a culture thereof or a bacterium cell thereof as an active ingredient.

In the present invention, muscle weakness related to muscle weakness refers to all diseases that may occur due to weakness of muscles, such as, for example, muscular dystrophy or muscular dystrophy.

The myopenia refers to a gradual decrease in skeletal muscle mass due to aging, which directly leads to a decrease in muscle strength, resulting in a decrease in various body functions and a disorder.

 It is preferable that the atrophy is atrophy caused by muscle atrophy caused by loss of muscle tissue caused by not using muscle, muscle atrophy caused by muscle itself, or muscle damage caused by nerve damage. Muscle atrophy caused by loss of muscle tissue caused by not using the muscles is called disuse atrophy. Muscle atrophy caused by the muscle itself is caused by myasthenia gravis or dystrophy, It is preferable that the atrophy caused by the damage of the dominant nerve is spinal muscular amyotrophy, amyotrophic lateral sclerosis, or sphinobulbar muscular atrophy.

In conclusion, the Enterococcus faecalis EF-2001 microorganism cells or culture thereof of the present invention is free from cytotoxicity and inhibits the damage of muscle cells induced by oxidative stress, thereby exhibiting remarkable muscle reduction or prevention of muscular atrophy and therapeutic effect, It is possible to provide a food composition for preventing and ameliorating muscular weakness related to muscular weakness comprising Enterococcus faecalis EF-2001 of the present invention, its culture liquid or its dead cells as an active ingredient.

There is no particular limitation on the kind of the food to which the lactic acid bacterium Enterococcus faecalis EF-2001 of the present invention is added. Examples of the foods to which the above substances can be added include dairy products including dairy products, meat, sausage, bread, biscuits, rice cakes, chocolate, candies, snacks, confectionery, pizza, ramen and other noodles, gums, ice cream, Beverages, alcoholic beverages and vitamin complexes, dairy products and dairy products, and includes processed foods and health functional foods in a conventional sense.

When the food composition according to the present invention is a beverage composition, there are no particular restrictions on the ingredients other than those containing the compound in the indicated ratios, and various flavors or natural carbohydrates, such as ordinary beverages, . Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And polysaccharides such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol, and erythritol. Natural flavors (tau martin, stevia extract, rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharine, aspartame, etc.) can be advantageously used as flavorings other than those described above. The ratio of the natural carbohydrate is generally about 1 g to 20 g, preferably about 5 g to 10 g per 100 g of the composition of the present invention.

In addition, the present invention provides a food additive for preventing and ameliorating muscular weakness related to muscular weakness comprising Enterococcus faecalis, a culture thereof or a bacterium cell thereof as an active ingredient.

The food additive according to the present invention can be used as a food additive in the form of a flavoring agent such as various nutrients, vitamins, minerals (electrolyte), synthetic flavors and natural flavors, a coloring agent and a thickening agent (cheese, chocolate etc.), a pectic acid and its salt, , Organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like. In addition, it may contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The addition ratio of such additives is not limited, but is generally selected in the range of 0.1 to 20 parts by weight per 100 parts by weight of the linoleic acid of the present invention.

The Enterococcus faecalis EF-2001 dead cells of the present invention can be added directly to food or used together with other food or food ingredients, and can be suitably used according to conventional methods. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (for prevention or improvement). In general, the amount of the composition of the present invention in a health functional food may be 0.1 to 90 parts by weight per total food weight. However, in the case of long-term intake for the purpose of health and hygiene or for the purpose of controlling health, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range.

Hereinafter, the present invention will be described in detail with reference to Examples, Experimental Examples and Preparation Examples.

However, the following Examples, Experimental Examples and Preparation Examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following Examples, Experimental Examples and Production Examples.

Example 1 Preparation of Enterococcus faecalis EF-2001 dead cells

The Enterococcus faecalis EF-2001 dead cells used in the present invention were prepared and prepared as follows.

Specifically, the Enterococcus faecalis EF-2001 was aerobically or anaerobically cultured on a medium used for culturing a general lactic acid bacterium, and then cultured after seed culture. The pH was maintained at a neutral or weakly acidic temperature of 20 to 40 ° C for 1 to 3 days. When the fermentation progressed so that the number of cells per g (gram) of the final raw material reached 7,500 billion won or more, it was pulverized through heat treatment and drying process.

≪ Example 2 > Culture of cells

Normal muscle cells (C2C12) were cultured in DMEM medium containing 5% fetal bovine serum (FBS). Normal skeletal muscle cells were cultured in a 75 ㎠ plastic flask (SPL life science Co., Ltd. Korea) supplemented with 10% FBS, 7.5% NaHCO _3, 150 μg / ml, glutamine 58.4 μg / ml, and antibiotics / antimycotics ) Was cultured in DMEM medium containing 4.4 μl / ml at 37 ° C and 5% CO 2. Cells were maintained in secondary culture once every 2-3 days.

≪ Example 3 >

Cells were removed from the bottom of the flask by treatment with 0.25% trypsin / EDTA followed by washing with CMF-PBS (calcium magnesium free-phosphate buffered saline, pH 7.2) After neutralization with the culture medium, the cells were centrifuged (1200 rpm, 5 min). The culture solution was added to the pellet of the remaining cells, and repeatedly inhaled with a sterile pipette to prepare a single cell suspension. The prepared cell suspension and trypan blue were mixed at a ratio of 1: 1 and measured using a hemocytometer on an optical microscope.

<Experimental Example 1> Confirmation of cell viability of Enterococcus faecalis EF-2001 cells

To determine the toxicity of the Enterococcus faecalis EF-2001 cells to muscle cells, an EzCytox kit was used. Specifically, normal muscle cells (C2C12) were dispensed in 96-well plates at 2 × 10 ⁴ cells / well. The cells were cultured in an incubator at 37 ° C and 5% CO 2, and then cultured for 24 hours at various concentrations of linoleic acid at 0, 25, 50, 100, 250 and 500 μg / ml. Cell viability of normal muscle cells cultured for 24 hours was calculated using EzCytox kit to determine the cytotoxicity of Enterococcus faecalis EF-2001 cells.

As a result, as shown in Fig. 1, when no Enterococcus faecalis EF-2001 cells were added, muscle cells showed a survival rate of 100%, and when Enterococcus faecalis EF-2001 cells were added at various concentrations The survival rate was similar to that of the group without addition of linoleic acid. Therefore, it was confirmed that the Enterococcus faecalis EF-2001 cells of the present invention did not show cytotoxicity on muscle cells (Fig. 1).

Experimental Example 2 Experimental Example 2 Experimental Example 2: EF-2001 Confirmation of effect of dead cells

In order to confirm the effect of EF-2001 cells on H ₂ O ₂ -induced cell death, first, muscle cells were placed in a 96-well plate at 2 × 10 ⁴ cells / well and incubated for 24 hours at 37 ° C., And cultured at 5% CO 2. Then, cells of Enterococcus faecalis EF-2001 were added at concentrations of 0, 25, 50, 100, 250 and 500 μg / ml, respectively. The total volume was adjusted to 195 μl with 10% FBS- And cultured in an incubator at 37 ° C and 5% CO ₂ for 24 hours. H ₂ O ₂ was added to the culture solution to a total volume of 200 μl at a concentration of 1 mM, and then cultured for another 120 minutes. After incubation, 10 E of EzCytox kit was added and the absorbance was measured after 1 hour to determine its effect.

As a result, as shown in FIG. 2, when H ₂ O ₂ was not added, the survival rate of muscle cells was 100%, and it was confirmed that the cell viability was decreased when H ₂ O ₂ was treated. Furthermore, it was confirmed that the survival rate of muscle cells was restored according to the concentration of Enterococcus faecalis EF-2001 cells (FIG. 2). Therefore, it was confirmed that the cells of Enterococcus faecalis EF-2001 significantly restored muscle cell damage to oxidative stress in a concentration-dependent manner.

EXPERIMENTAL EXAMPLE 3 Comparison of Expression Changes of HSP70 (Heat Shock Proein) Protein

The change in the amount of HSP70 (Heat Shock Proein) protein was measured in western blot in addition group cells to which Enterococcus faecalis EF-2001 cells were added. That is, normal muscle cells (C2C12) were dispensed in a 96-well plate at 5 × 10 ⁴ cells / well and cultured in an incubator at 37 ° C and 5% CO 2. Thereafter, 25 μg / ml of Enterococcus faecalis EF-2001 cells were added, and the total volume was adjusted to 2 ml with 10% FBS-DMEM medium and cultured at 37 ° C. and 5% CO 2 for 24 hours. H ₂ O ₂ was added to the culture solution to a total volume of 2 ml at a concentration of 1 mM, and then cultured for another 60 minutes. Cells were disrupted to obtain proteins. The expression level of HSP70 protein was detected using Western blot using HSP70 and beta actin antibody.

As a result, as shown in FIG. 3, in the addition group supplemented with Enterococcus faecalis EF-2001 cells, normal cells were restored to HSP70 protein expression level. Thus, it was found that the cells of the present Enterococcus faecalis EF-2001 can prevent the inhibition of muscle atrophy by inducing an increase in expression of HSP70 protein in muscle cells (see FIG. 3)

<Experimental Example 4> The mice that induced muscle reduction were administered Enterococcus faecalis Comparison of EF-2001 muscle cell recovery ability

To confirm the effect of prevention and recovery of muscle atrophy of Enterococcus faecalis EF-2001 cells, muscle images were obtained using a micro-tomography system to measure the initial muscle volume before muscle atrophy. In order to induce atrophy, the right hindlimb nerve of the rat was excised to restrict muscle movement and induce atrophy. In the design shown in FIG. 4, Enterococcus faecalis EF-2001 was orally administered to rats at two concentrations of 2 mg / kg and 30 mg / kg. The prophylactic group received Enterococcus faecalis EF-2001 daily for one week prior to surgery to induce atrophy, and the treatment group received Enterococcus faecalis EF-2001 for three weeks after surgery to induce atrophy. After 3 weeks of operation, the muscle images were acquired using a micro-tomography apparatus and are shown in FIG. 5. Based on the images, the difference in the effect of the ingestion of the Enterococcus faecalis EF-2001 on muscle atrophy Respectively.

As a result, as shown in FIG. 6, it was confirmed that the ingestion of Enterococcus faecalis EF-2001 from 3 weeks to 4 weeks recovered the amount of muscle in the Enterococcus faecalis EF-2001 group compared to the denervation group. (See Fig. 4)

&Lt; Preparation Example 1 > Preparation of medicine

1. Manufacturing of powder

1 mg to 10 g of the Enterococcus faecalis EF-2001 dead cells of the present invention

Lactose 1 g

The above components were mixed and packed in airtight bags to prepare powders.

2. Preparation of tablets

1 mg to 10 g of the Enterococcus faecalis EF-2001 dead cells of the present invention

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

3. Preparation of capsules

1 mg to 10 g of the Enterococcus faecalis EF-2001 dead cells of the present invention

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

After mixing the above components, the capsules were filled in gelatin capsules according to the conventional preparation method of capsules.

4. Preparation of injections

Enterococcus faecalis EF-2001 Cells 1 mg to 10 g

180 mg mannitol

_{Na₂ · HPO₄ · 2H 2 O 26} mg

Distilled water 2974 mg

According to the conventional method for preparing an injectable preparation, an injectable preparation was prepared by incorporating the aforementioned components in the amounts indicated.

&Lt; Preparation Example 2 > Preparation of health food

1. Manufacture of health food

1 mg to 10 g of the Enterococcus faecalis EF-2001 dead cells of the present invention

Vitamin mixture quantity

70 [mu] g of vitamin A acetate

Vitamin E 1.0 mg

0.13 mg of vitamin

0.15 mg of vitamin B2

Vitamin B6 0.5 mg

0.2 [mu] g vitamin B12

Vitamin C 10 mg

Biotin 10 μg

Nicotinic acid amide 1.7 mg

Folic acid 50 mg

Calcium pantothenate 0.5 mg

Mineral mixture quantity

1.75 mg of ferrous sulfate

0.82 mg of zinc oxide

Magnesium carbonate 25.3 mg

Potassium monophosphate 15 mg

Secondary calcium phosphate 55 mg

Potassium citrate 90 mg

Calcium carbonate 100 mg

Magnesium chloride 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixture is comparatively mixed with a composition suitable for health food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional method for producing healthy foods , Granules can be prepared and used in the manufacture of health food compositions according to conventional methods.

2. Manufacture of health drinks

1 mg to 10 g of the Enterococcus faecalis EF-2001 dead cells of the present invention

Citric acid 1000 mg

100 g of oligosaccharide

Plum concentrate 2 g

Taurine 1 g

Total 900 ml with purified water

The above components were mixed according to a conventional health drink manufacturing method, and the mixture was stirred and heated at 85 DEG C for about 1 hour. The resulting solution was filtered to obtain a sterilized container, which was sealed and sterilized, And used for manufacturing.

Although the compositional ratio is relatively mixed with a component suitable for a favorite drink, it is also possible to arbitrarily modify the compounding ratio according to the regional or national preference such as the demand class, the demanding country, and the use purpose.

Claims (17)

A pharmaceutical composition for the prevention and treatment of myopenia or muscular dystrophy comprising Enterococcus faecalis EF-2001, a culture thereof or a bacterium thereof as an active ingredient.
delete delete [Claim 2] The method according to claim 1, wherein the Enterococcus faecalis EF-2001 microorganism cells are cultured in the presence of a seed culture of Enterococcus faecalis, followed by culturing in a neutral or weakly acidic and temperature range of 20 to 40 DEG C, Or a pharmaceutically acceptable salt thereof.
The pharmaceutical composition according to claim 1, wherein the composition increases the expression of HSP70 protein in muscle cells.
delete The method of claim 1, wherein the muscular atrophy is selected from the group consisting of disuse atrophy, myasthenia gravis, dystrophy, spinal muscular amyotrophy, amyotrophic lateral sclerosis, Wherein said disease is selected from the group consisting of sphinobulbar muscular atrophy, and myofascial atrophy.
The method according to claim 1, wherein the pharmaceutical composition is a formulation selected from the group consisting of capsules, tablets, powders, granules, liquids, rings, flakes, pastes, syrups, gels, A pharmaceutical composition for preventing and treating muscular atrophy.
A food composition for preventing or ameliorating myopenia or muscular dystrophy comprising Enterococcus faecalis EF-2001, a culture solution thereof or a bacterium cell thereof as an active ingredient.
delete delete [Claim 11] The method according to claim 9, wherein the Enterococcus faecalis EF-2001 microorganism cells are cultured in an anaerobic microorganism culture medium after incubation with Enterococcus faecalis in a neutral or weakly acidic condition at a temperature ranging from 20 to 40 DEG C, Wherein the composition is used for preventing or ameliorating myopenia or muscular atrophy.
delete 10. The method according to claim 9, wherein the muscular atrophy is selected from the group consisting of atrophic muscular atrophy, myasthenia gravis, muscular dystrophy, spinal muscular atrophy, amyotrophic axillary sclerosis and spinal cord bulbar muscular atrophy. Food composition for improvement.
10. The method of claim 9, wherein the food is selected from the group consisting of nutrients, vitamins, minerals (electrolytes), synthetic flavors, natural flavors, colorants, wherein the composition further comprises one or more additives selected from the group consisting of a pH adjuster, a stabilizer, an antiseptic, glycerin, an alcohol, a carbonating agent and flesh.
Enterococcus faecalis EF-2001, a food additive for preventing or ameliorating myopenia or muscular dystrophy containing the culture liquid or its dead cells as an active ingredient.


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