Pharmaceutical composition, food composition and food additive for preventing, alleviating or treating muscle loss, weakness and atrophy, containing, as active ingredient, <i>Enterococcus faecalis</i>, culture liquid thereof or dead cells thereof

Abstract

The present invention relates to a pharmaceutical composition, a food composition, and a food additive for the prevention and treatment or improvement of muscle loss, muscle weakness, and muscle atrophy, which contain Enterococcus faecalis, particularly Enterococcus faecalis EF-2001 among others, a culture solution thereof, or a killed body thereof as an active ingredient. Killed Enterococcus faecalis EF-2001 of the present invention exhibits a remarkable treatment effect for muscle loss, muscle weakness, and muscle atrophy by inhibiting the damage to muscle cells induced by oxidative stress, and thus killed lactic acid bacterium Enterococcus faecalis EF-2001 of the present invention or a culture solution thereof can be usefully used as an active ingredient in a pharmaceutical composition, a food composition, and a food additive for prevention of muscle atrophy and sarcopenia.

Claims

1. A method for treating a muscle weakness-related disease comprising administering a killed body of Enterococcus faecalis EF-2001 to an individual suffering from a muscle weakness-related disease, thereby treating said muscle weakness-related disease in said individual.

2. The method for treating a muscle weakness-related disease according to claim 1, wherein the killed body of Enterococcus faecalis EF-2001 is obtained by subjecting Enterococcus faecalis EF-2001 to starter culture, then subjecting the Enterococcus faecalis EF-2001 to main culture in a neutral or acidic medium in a temperature range of 20° C. to 40° C., and then performing a heat treatment.

3. The method for treating a muscle weakness-related disease according to claim 1, wherein the killed body of Enterococcus faecalis EF-2001 increases expression of HSP70 protein or SOD1 protein in a muscle cell.

4. The method for treating a muscle weakness-related disease according to claim 1, wherein the disease is sarcopenia or muscle atrophy.

5. The method for treating a muscle weakness-related disease according to claim 4, wherein the muscle atrophy is a disease selected from the group consisting of disuse atrophy, myasthenia gravis, spinal muscular amyotrophy, amyotrophic lateral sclerosis, and spinobulbar muscular atrophy.

6. The method for treating a muscle weakness-related disease according to claim 1, wherein the killed body of Enterococcus faecalis EF-2001 is prepared in a form selected from the group consisting of a capsule, a tablet, a powder, a granule, a liquid, a pill, a flake, a paste, a syrup, a gel, a jelly, and a bar.

7. The method of claim 1, wherein the killed body of Enterococcus faecalis EF-2001 is administered to the individual in an amount ranging from 0.1 mg/kg and 100 mg/kg once a day or every other day.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 is a diagram for confirming cytotoxicity of Enterococcus faecalis EF-2001.

(2) FIG. 2 is a diagram for confirming an effect of Enterococcus faecalis EF-2001 on damage to muscle cells induced by oxidative stress. NAC denotes a N-acetyl cysteine-treated group which is a positive control group.

(3) FIG. 3 is a diagram for confirming a change in expression of HSP70 (Heat Shock Protein) in muscle cells in an oxidative stress environment by Enterococcus faecalis EF-2001.

(4) FIG. 4 is a diagram illustrating time and experimental plan of oral administration of Enterococcus faecalis EF-2001 at two different concentrations of 2 mg/kg and 30 mg/kg after surgery to induce muscle atrophy by excising the right hind limb arch of a mouse and thus limiting muscle movement.

(5) FIG. 5 is a diagram illustrating, after surgery to induce muscle atrophy by excising the right hind limb arch of a mouse and thus limiting muscle movement, muscle images of a prevented group which has ingested Enterococcus faecalis EF-2001 every day from one week before and a treated group which has ingested Enterococcus faecalis EF-2001 every day for three weeks after the surgery taken using a micro tomography apparatus in three weeks after the surgery.

(6) FIG. 6 is a diagram illustrating results of comparative analysis on influence of Enterococcus faecalis EF-2001 ingestion on muscle atrophy and differences based on the images illustrated in FIG. 5.

(7) FIG. 7 is a diagram for confirming an effect of killed Enterococcus faecalis EF-2001 on H.sub.2O.sub.2-induced cell death.

(8) FIG. 8 is a diagram for confirming a change in expression of SOD1 protein in muscle cells in an oxidative stress environment by Enterococcus faecalis EF-2001.

DESCRIPTION OF EMBODIMENTS

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

(10) The present invention provides a pharmaceutical composition for prevention and treatment of muscle weakness-related diseases containing Enterococcus faecalis, a culture solution thereof, or a killed body thereof as an active ingredient.

(11) The microorganisms of the genus Enterococcus are widely present in nature and utilize carbohydrates aerobically. In general, it is known that bacteria, such as microorganisms of the genus Enterococcus, prevent damage caused by pathogenic microorganisms by antagonism or secreted antibacterial substances in vivo.

(12) As the Enterococcus faecalis, a culture solution thereof, or a killed body thereof, both commercially available products and those prepared by known methods for preparing killed bacteria may be used, and these do not exhibit toxicity and are harmless to the human body.

(13) The culture solution refers to a culture solution in which Enterococcus faecalis have been cultured in a culture medium, a concentrated culture solution, a dried product of a culture solution, a culture filtrate, a concentrated culture filtrate, or a dried product of a culture filtrate and may be a culture solution containing the cell strain or a culture filtrate obtained by removing the cell strain after culture.

(14) The killed bacteria can be prepared by subjecting the corresponding live bacteria to a heat treatment or treating the corresponding live bacteria with formalin or other fungicides. The killed bacteria can be used even when they are substantially killed. In addition, the killed bacteria used in the present invention may be prepared by the following method, but the method is not limited to this.

(15) 1) A step of subjecting Enterococcus faecalis to starter culture and then subjecting the Enterococcus faecalis to main culture in a neutral or weakly acidic medium in a temperature range of 20° C. to 40° C.

(16) 2) A step of subjecting the culture solution to a heat treatment after the main culture of 1) and then drying the culture solution.

(17) The “muscle weakness” means a state in which the strength of one or more muscles have decreased. The muscle weakness may be limited to any one muscle, one side of the body, the upper limbs or the lower extremities or may occur throughout the body. In addition, subjective muscle weakness symptoms including muscle fatigue or muscle pain can be quantified by an objective method through physical examination.

(18) The muscle weakness-related diseases mean all diseases which can be developed due to muscle weakness, and examples thereof include, but are not limited to, sarcopenia or muscle atrophy.

(19) The sarcopenia refers to a gradual decrease in skeletal muscle mass with aging and means a state in which a decrease in muscle strength is directly caused and, as a result, various physical functions may be diminished and disorders may occur.

(20) The muscle atrophy is preferably muscle atrophy due to the loss of muscle tissue caused as muscles are not used, muscle atrophy due to the disease of muscle itself, or muscle atrophy due to the damage to nerves which dominate muscles. The muscle atrophy due to the loss of muscle tissue caused as muscles are not used is more preferably disuse atrophy, the muscle atrophy due to the disease of muscle itself is more preferably myasthenia gravis or dystrophy, and the muscle atrophy due to the damage to nerves which dominate muscles is more preferably spinal muscular amyotrophy, amyotrophic lateral sclerosis, or spinobulbar muscular atrophy, but the muscle atrophy is not limited to these.

(21) In specific Experimental Examples of the present invention, the present inventors have confirmed that killed Enterococcus faecalis EF-2001 is not cytotoxic (see FIG. 1) and have confirmed the effect that the survival rate of muscle cells is restored depending on the concentration of killed Enterococcus faecalis EF-2001 when the muscle cells are treated with the killed Enterococcus faecalis EF-2001 in the damage to muscle cells induced by oxidative stress (see FIG. 2). In addition, the change in the amounts of HSP70 and SOD1 proteins in cells to which killed Enterococcus faecalis EF-2001 have been added are measured, and as a result, it has been found that killed Enterococcus faecalis EF-2001 induces muscle atrophy inhibition by increasing the expression levels of HSP70 and SOD1 proteins in muscle cells (see FIGS. 3 and 8). It has been confirmed that the decreased muscle mass is restored again after sarcopenia-induced mice have ingested killed Enterococcus faecalis EF-2001 (see FIGS. 4 to 6). In addition, it has been confirmed that killed Enterococcus faecalis EF-2001 is resistant to cell death, which is oxidative damage, and structural changes of cells by temporarily eliminating cell death induced by oxidative stress and increased radicals in muscle cells (see FIG. 7).

(22) Consequently, killed Enterococcus faecalis EF-2001 of the present invention does not exhibit cytotoxicity and exhibits a remarkable muscle atrophy treatment effect by inhibiting the damage to muscle cells induced by oxidative stress, and thus Enterococcus faecalis EF-2001 of the present invention, a culture solution thereof, or a killed body thereof can be usefully used as an active ingredient in a pharmaceutical composition for prevention and treatment of muscle weakness-related diseases such as muscle atrophy and sarcopenia.

(23) The composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, the “pharmaceutically effective amount” refers to an amount sufficient to treat a disease at a reasonable benefit or risk ratio applicable to medical treatment, and the effective dose level can be determined depending on the kind of patient's disease, severity, drug activity, sensitivity to drug, time of administration, route of administration and rate of excretion, duration of treatment, factors including co-drugs, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered in a single or multiple manner. It is important to administer the composition in an amount so that the maximum effect can be attained in a minimal amount without side effects in consideration of all the above factors, and the amount can be easily determined by those skilled in the art.

(24) Specifically, the effective amount of the composition according to the present invention may vary depending on the patient's age, sex, and weight. In general, the composition may be administered in an amount of 0.1 mg to 100 mg, preferably 0.2 mg to 17 mg per 1 kg weight daily or every other day or one to three times a day. However, the effective amount may increase or decrease depending on the route of administration, severity of obesity, sex, weight, age, and the like, and thus the dosage does not limit the scope of the present invention in any way.

(25) When formulating the composition, the composition is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants which are commonly used.

(26) Solid preparations for oral administration include tablets, pills, powders, granules, capsules, troches, and the like. Such solid preparations are prepared by mixing at least one or more excipients, for example, starch, calcium carbonate, and sucrose or lactose or gelatin with killed lactic acid bacterium Enterococcus faecalis EF-2001 of the present invention. In addition, lubricants such as magnesium stearate and talc are also used in addition to simple excipients. Liquid preparations for oral administration include suspensions, intravenous solutions, emulsions or syrups, and the like. The liquid preparations may contain various excipients, for example, wetting agents, sweeteners, fragrances, and preservatives in addition to water and liquid paraffin which are commonly used simple diluents.

(27) Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspension solvents, emulsions, freeze-dried preparations, suppositories, and the like.

(28) As non-aqueous solvents and suspension solvents, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate, and the like may be used. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin-based oil, glycerol, gelatin, and the like may be used.

(29) The present invention also provides a food composition for prevention and improvement of muscle weakness-related diseases containing Enterococcus faecalis, a culture solution thereof, or a killed body thereof as an active ingredient.

(30) In the present invention, the muscle weakness-related diseases mean all diseases which can be developed due to muscle weakness, and examples thereof include, but are not limited to, sarcopenia or muscle atrophy.

(31) The sarcopenia refers to a gradual decrease in skeletal muscle mass with aging and means a state in which a decrease in muscle strength is directly caused and, as a result, various physical functions may be diminished and disorders may occur.

(32) The muscle atrophy is preferably muscle atrophy due to the loss of muscle tissue caused as of muscles are not used, muscle atrophy due to the disease of muscle itself, or muscle atrophy due to the damage to nerves which dominate muscles. The muscle atrophy due to the loss of muscle tissue caused as muscles are not used is more preferably disuse atrophy, the muscle atrophy due to the disease of muscle itself is more preferably myasthenia gravis or dystrophy, and the muscle atrophy due to the damage to nerves which dominate muscles is more preferably spinal muscular amyotrophy, amyotrophic lateral sclerosis, or spinobulbar muscular atrophy, but the muscle atrophy is not limited to these.

(33) Consequently, killed Enterococcus faecalis EF-2001 of the present invention or a culture solution thereof does not exhibit cytotoxicity and exhibits a remarkable sarcopenia or muscle atrophy prevention and treatment effect by inhibiting the damage to muscle cells induced by oxidative stress, and thus Enterococcus faecalis EF-2001 of the present invention, a culture solution thereof, or a killed body thereof can be used as an active ingredient in a food composition for prevention and improvement of muscle weakness-related diseases.

(34) There is no particular limitation on the kind of food to which the killed lactic acid bacterium Enterococcus faecalis EF-2001 of the present invention is added. Examples of the food to which the substance can be added include health drinks, meat, sausage, bread, biscuits, rice cakes, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, alcoholic beverages and vitamin complexes, and dairy products and processed dairy products and include both processed food and health functional food in the ordinary sense.

(35) When the food composition according to the present invention is a beverage composition, there is no particular limitation on other ingredients except that the essential ingredient contains the compound at a predetermined proportion, and the beverage composition may contain various flavoring agents, natural carbohydrates, or the like as additional ingredients as ordinary beverages. Examples of the natural carbohydrates described above include monosaccharides, for example, glucose and fructose; disaccharides, for example, maltose and sucrose; and polysaccharides, for example, conventional sugars such as dextrin and cyclodextrin and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (taumatin, stevia extract, rebaudioside A, glycyrrhizine and the like) and synthetic flavoring agents (saccharin, aspartame, and the like) may be advantageously used. The proportion of 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.

(36) The present invention also provides a food additive for the prevention and improvement of muscle weakness-related diseases containing Enterococcus faecalis, a culture solution thereof, or a killed body thereof as an active ingredient.

(37) The food additive according to the present invention may contain various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and appetizers (cheese, chocolate and the like), pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, antiseptics, glycerin, alcohols, and carbonating agents used in carbonated beverages. The food additive may contain natural fruit juice and pulp for the preparation of fruit juice beverages and vegetable beverages in addition to these. These ingredients may be used independently or in combination. The proportion of these additives added is not limited but is generally selected from a range of 0.1 to 20 parts by weight per 100 parts by weight of linoleic acid of the present invention.

(38) Killed Enterococcus faecalis EF-2001 of the present invention may be added to food as it is or used with other foods or food ingredients and may be suitably used according to conventional methods. The amount of the active ingredient added may be appropriately determined depending on the purpose of use (for prevention or improvement). In general, the amount of the composition of the present invention in the health functional food may be 0.1 to 90 parts by weight per part by weight of the entire food. However, in the case of long-term ingestion for health and hygiene purposes or for health control purposes, the amount may be equal to or less than the above range, and the active ingredient may be used in an amount equal to or more than the above range since the active ingredient does not have any problem in terms of safety.

(39) In addition, the present invention provides a method for treating or preventing muscle weakness-related diseases including administering a pharmaceutically effective amount of Enterococcus faecalis, a culture solution, or a killed body thereof to an individual suffering from a muscle weakness-related disease or an individual.

(40) The individual means human or non-human organisms, for example, non-human mammals such as cows, monkeys, birds, cats, mice, rats, hamsters, pigs, dogs, rabbits, sheep, and horses, and the treatment may be utilized for individuals with wounds or damaged skin tissues.

(41) In addition, the treatment may be performed through oral administration or parenteral administration (for example, intramuscularly, intravenously, intraperitoneally, subcutaneously, intradermally, or topically applied) depending on the intended method. The dosage depends on the patient's condition and weight, the degree of disease, the drug form, and the route and time of administration but can be appropriately selected by those skilled in the art.

EMBODIMENTS

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

(43) However, the following Examples, Experimental Examples, and Preparation Examples are merely illustrative of the present invention, and the contents of the present invention are not limited to the following Examples, Experimental Examples, and Preparation Examples.

<Example 1> Preparation of Killed Enterococcus faecalis EF-2001

(44) Killed Enterococcus faecalis EF-2001 used in the present invention were produced and prepared as follows.

(45) Specifically, Enterococcus faecalis EF-2001 was subjected to aerobic or anaerobic culture in a medium used for general lactic acid bacteria culture. After the starter culture, main culture was performed. The culture was performed for one to three days while maintaining a neutral or weakly acidic pH and a culture temperature of 20° C. to 40° C. When the fermentation proceeds so that the number of bacteria per g (gram) reaches 7.5 trillion or more based on the final raw material, the culture solution was powdered through a heat treatment and then a drying process.

<Example 2> Culture of Cell

(46) Normal muscle cells (C2C12) were cultured in DMEM medium containing fetal bovine serum (FBS) at 5%. In other words, normal skeletal muscle cells were cultured in DMEM medium containing 10% FBS, 150 μg/ml of 7.5% NaHCO.sub.3, 58.4 μg/ml of glutamine, and 4.4 μl/ml of antibiotic/antimycotics in a 75 cm.sup.2 plastic flask (SPL life science Co., Ltd. Korea) under conditions of 37° C. and 5% CO.sub.2. The cell strain was maintained through secondary culture one time every two to three days.

<Example 3> Quantification of Cell

(47) The medium was removed from the 75 cm.sup.2 plastic flask in which the cells were grown, and then the cells were washed with CMF-PBS (calcium magnesium free-phosphate buffered saline, pH 7.2), treated with 0.25% trypsin/EDTA to be removed from the bottom of the flask, neutralized with cell culture solution, and then centrifuged (1200 rpm, 5 min). The culture solution was added to the pellet of the remaining cells, and then inhalation with a sterile pipette was repeatedly performed to prepare a single cell suspension. The prepared cell suspension and trypan blue were mixed at a ratio of 1:1 and subjected to the measurement using a hemocytometer under an optical microscope.

<Experimental Example 1> Confirmation of Cytotoxicity (Cell Viability) of Killed Enterococcus faecalis EF-2001

(48) To measure the toxicity of killed Enterococcus faecalis EF-2001 to muscle cells, the EzCytox kit was used. Specifically, normal muscle cells (C2C12) were dispensed in a 96 well plate to be 2×10.sup.4 cells/well. This was cultured in an incubator under conditions of 37° C. and 5% CO.sub.2, then linoleic acid at various concentrations of 0, 25, 50, 100, 250, and 500 μg/ml was added thereto, and the cells were cultured for 24 hours. The cytotoxicity of killed Enterococcus faecalis EF-2001 was determined by calculating the survival rate of the normal muscle cells cultured for 24 hours using the EzCytox kit.

(49) As a result, as illustrated in FIG. 1, the muscle cells had a survival rate of 100% when killed Enterococcus faecalis EF-2001 was not added and the groups to which killed Enterococcus faecalis EF-2001 was added at various concentrations also had similar survival rates to that of the group to which linoleic acid was not added. Consequently, it has been confirmed that killed Enterococcus faecalis EF-2001 of the present invention does not exhibit cytotoxicity to muscle cells (FIG. 1).

<Experimental Example 2> Confirmation of Effect of Killed Enterococcus faecalis EF-2001 on Damage to Muscle Cell Induced by Oxidative Stress

(50) In order to confirm the effect of killed EF-2001 at various concentrations on H.sub.2O.sub.2-induced apoptosis, muscle cells were first placed in a 96 well plate to be 2×10.sup.4 cells/well and cultured for 24 hours under conditions of 37° C. and 5% CO.sub.2. Thereafter, killed Enterococcus faecalis EF-2001 was added thereto at concentrations of 0, 25, 50, 100, 250, and 500 μg/ml, respectively. The total volume was adjusted to 195 μl with 10% FBS-DMEM medium and the muscle cells were cultured in an incubator for 24 under conditions of 37° C. and 5% CO.sub.2. H.sub.2O.sub.2 was added to this culture solution at a concentration of 1 mM so that the total volume was 200 μl, and then the muscle cells were cultured for another 120 minutes. After culture, 10 μl of the EzCytox kit was added to the culture solution, and the absorbance was measured after 1 hour to determine the effect.

(51) As a result, it has been confirmed that the muscle cells had a survival rate of 100% when H.sub.2O.sub.2 was not added and the survival rate of muscle cells decreases when the muscle cells were treated with H.sub.2O.sub.2 as illustrated in FIG. 2. In addition, the effect has been confirmed that the survival rate of muscle cells is restored depending on the concentration of killed Enterococcus faecalis EF-2001 when the muscle cells are treated with the killed Enterococcus faecalis EF-2001 (FIG. 2).

(52) Consequently, it has been confirmed that killed Enterococcus faecalis EF-2001 significantly restores the damage to muscle cells induced by oxidative stress in a concentration-dependent manner.

<Experimental Example 3> Comparison of Change in Expression of HSP70 (Heat Shock Protein) and SOD1 Proteins

(53) Changes in the amounts of HSP70 (Heat Shock Protein) and SOD1 proteins in the cells of the addition group to which killed Enterococcus faecalis EF-2001 was added were measured using a western blot. In other words, normal muscle cells (C2C12) were dispensed in a 96 well plate to be 5×10.sup.4 cells/well and then cultured in an incubator under conditions of 37° C. and 5% CO.sub.2. Thereafter, 25 μg/ml of killed Enterococcus faecalis EF-2001 was added thereto, then the total volume was adjusted to 2 ml with 10% FBS-DMEM medium, and the cells were cultured for 24 under conditions of 37° C. and 5% CO.sub.2. H.sub.2O.sub.2 was added to this culture solution at a concentration of 1 mM so that the total volume was 2 ml, then the cells were cultured for another 60 minutes, and then the cells were crushed to obtain proteins. The expression levels of HSP70 protein and SOD1 protein were detected using HSP70, SOD1, and beta-actin antibodies using western blot.

(54) As a result, the expression levels of HSP70 and SOD1 proteins in the addition group to which killed Enterococcus faecalis EF-2001 was added were restored to those in normal cells as illustrated in FIGS. 3 and 8. In other words, it has been confirmed that the effect of protecting muscle cells from oxidative stress is exerted through the expression of HSP70. In addition, it has been confirmed that killed Enterococcus faecalis EF-2001 decreases free radicals of reactive oxygen which is oxidative stress to restore the expression of SOD1 which appears with respect to reactive oxygen.

(55) Consequently, it has been confirmed that the present killed Enterococcus faecalis EF-2001 can prevent muscle atrophy by causing increased expression of HSP70 and SOD1 proteins in muscle cells (FIGS. 3 and 8).

<Experimental Example 4> Comparison of Restoring Ability of Sarcopenia-Induced Mice from Sarcopenia after Ingestion of Killed Enterococcus faecalis EF-2001

(56) In order to confirm the effect of preventing and restoring muscle atrophy by killed Enterococcus faecalis EF-2001, muscle images were acquired using a micro tomography apparatus to measure the initial muscle mass before induction of muscle atrophy.

(57) Specifically, in order to induce muscle atrophy, the right hind limb arch of a mouse was excised to limit muscle movement and thus to induce muscle atrophy. In the design illustrated in FIG. 4, Enterococcus faecalis EF-2001 was ingested to the mouse by an oral administration method at two concentrations of 2 mg/kg and 30 mg/kg. The prevented group ingested Enterococcus faecalis EF-2001 every day from one week before the surgery to induce muscle atrophy and the treated group ingested Enterococcus faecalis EF-2001 every day for three weeks after the surgery to induce muscle atrophy. In 3 weeks after the surgery, muscle images were acquired using a microscopic tomography apparatus and illustrated in FIG. 5, and the comparative analysis on the influence of Enterococcus faecalis EF-2001 ingestion on muscle atrophy and differences was performed based on the photographed images.

(58) As a result, it has been confirmed that the muscle mass of the Enterococcus faecalis EF-2001-ingested group was restored as compared to the muscle atrophy group (denervation) when Enterococcus faecalis EF-2001 was ingested for three to four weeks as illustrated in FIG. 6 (FIGS. 5 and 6).

<Experimental Example 5> Confirmation of Effect of Killed Enterococcus faecalis EF-2001 on Muscle Cell Apoptosis Induced by Oxidative Stress

(59) The effect of killed Enterococcus faecalis EF-2001 on H.sub.2O.sub.2-induced cell death was confirmed through changes in chromatin condensation and fragmentation morphology by DAPI fixation staining.

(60) Specifically, the number of cells in the chromatin condensation and fragmentation morphology in the experimental group treated with H.sub.2O.sub.2 was higher than in the control group which was not treated with H.sub.2O.sub.2, and it has been confirmed that the number of cells in the chromatin condensation and fragmentation morphology decreases in a concentration-dependent manner in the group treated with killed Enterococcus faecalis EF-2001 at various concentrations (FIG. 7). In the case of muscle cells induced with H.sub.2O.sub.2, the protein or DNA is damaged as a high radical concentration in the cells is maintained, and this induces cell death by causing oxidation in the outer cell membrane, intracellular structural protein, nuclear DNA and the like and thus causing damage due to structural changes.

(61) Consequently, it has been confirmed that killed Enterococcus faecalis EF-2001 is resistant to cell death, which is oxidative damage, and structural changes of cells by temporarily eliminating cell death induced by oxidative stress and increased radicals in muscle cells through this experiment.

<Preparation Example 1> Preparation of Medicine

(62) 1. Preparation of Powder

(63) Killed Enterococcus faecalis EF-2001 of the present invention: 1 mg to 10 g

(64) Lactose: 1 g

(65) A powder was prepared by mixing the above ingredients together and filling the mixture in a gas-tight bag.

(66) 2. Preparation of Tablet

(67) Killed Enterococcus faecalis EF-2001 of the present invention: 1 mg to 10 g

(68) Corn starch: 100 mg

(69) Lactose: 100 mg

(70) Magnesium stearate: 2 mg

(71) A tablet was prepared by mixing the above ingredients together and then tableting the mixture according to a conventional method for preparing tablets.

(72) 3. Preparation of Capsule

(73) Killed Enterococcus faecalis EF-2001 of the present invention: 1 mg to 10 g

(74) Corn starch: 100 mg

(75) Lactose: 100 mg

(76) Magnesium stearate: 2 mg

(77) A capsule was prepared by mixing the above ingredients together and then filling the mixture in a gelatin capsule according to a conventional method for preparing capsules.

(78) 4. Preparation of Injection

(79) Killed Enterococcus faecalis EF-2001 of the present invention: 1 mg to 10 g

(80) Mannitol: 180 mg

(81) Na.sub.2.HPO.sub.4.2H.sub.2O: 26 mg

(82) Distilled water: 2974 mg

(83) An injection containing the above ingredients in predetermined amounts was prepared according to a conventional method for preparing an injection.

<Production Example 2> Preparation of Health Food

(84) 1. Preparation of Health Food

(85) Killed Enterococcus faecalis EF-2001 of the present invention: 1 mg to 10 g

(86) Vitamin mixture: suitable amount

(87) Vitamin A acetate: 70 μg

(88) Vitamin E: 1.0 mg

(89) Vitamin: 0.13 mg

(90) Vitamin B2: 0.15 mg

(91) Vitamin B6: 0.5 mg

(92) Vitamin B12: 0.2 μg

(93) Vitamin C: 10 mg

(94) Biotin: 10 μg

(95) Nicotinic acid amide: 1.7 mg

(96) Folic acid: 50 mg

(97) Calcium Pantothenate: 0.5 mg

(98) Inorganic mixture: suitable amount

(99) Ferrous sulfate: 1.75 mg

(100) Zinc oxide: 0.82 mg

(101) Magnesium carbonate: 25.3 mg

(102) Potassium dihydrogen phosphate: 15 mg

(103) Calcium hydrogen phosphate: 55 mg

(104) Potassium citrate: 90 mg

(105) Calcium carbonate: 100 mg

(106) Magnesium chloride: 24.8 mg

(107) The composition ratio of the vitamin and mineral mixtures was adjusted by mixing ingredients relatively suitable for health food in a preferred embodiment, but the mixing ratio may be arbitrarily modified. The above ingredients may be mixed together according to a conventional method for preparing health food, then prepared into granules, and used in the preparation of a health food composition according to a conventional method.

(108) 2. Preparation of Health Drink

(109) Killed Enterococcus faecalis EF-2001 of the present invention: 1 mg to 10 g

(110) Citric acid: 1000 mg

(111) Oligosaccharide: 100 g

(112) Plum concentrate: 2 g

(113) Taurine: 1 g

(114) Total with purified water: 900 ml

(115) A solution obtained by mixing the above ingredients together according to a conventional method for preparing a health drink and then stirring and heating the mixture at 85° C. for about 1 hour was filtered, collected in a sterilized container, sealed and sterilized, then stored in a refrigerator, and then used in the preparation of a health drink composition.

(116) The composition ratio was adjusted by mixing ingredients relatively suitable for favorite beverages in a preferred embodiment, but the mixing ratio may be arbitrarily modified according to regional and ethnic preferences such as class of demand, country of use, and usage.