Use of bifidobacterium animalis lactis MG741 strain promoting GLP-1 secretion for prevention, improvement, and treatment of muscle loss due to metabolic disease

Abstract

The present invention relates to a use of a Bifidobacterium animalis lactis MG741 strain promoting GLP-1 secretion for preventing, improving, or treating muscle loss or muscle atrophy. The Bifidobacterium animalis lactis MG741 strain according to the present invention has been confirmed to have the capability to promote GLP-1 secretion and to have excellent cell proliferation capability and cell protection capability for muscle cells. Furthermore, the Bifidobacterium animalis lactis MG741 strain also has been confirmed to inhibit muscle damage and promote muscle regeneration through improvement of oxidative stress. Therefore, the Bifidobacterium animalis lactis MG741 strain of the present invention can be usefully utilized in the field of treating muscle loss disease and in fields related to muscle growth.

Description

Therapeutic use of the GLP-1 secretion-promoting Bifidobacterium animalis lactis MG741 strain for the prevention and improvement of muscle loss caused by metabolic diseases

[0001] The present invention relates to the use of the Bifidobacterium animalis lactis MG741 strain, which promotes GLP-1 secretion, for the prevention, improvement, or treatment of muscle loss or muscle atrophy.

[0002] Muscle accounts for approximately 40–50% of a person's body weight. Around the age of 50, the rate of muscle protein synthesis within muscle cells slows down compared to the rate of breakdown, causing muscles to begin degenerating rapidly and making individuals susceptible to muscle loss diseases. Sarcopenia, a disease characterized by a gradual decline in muscle mass and strength, primarily manifests as muscle loss and weakness. The significant reduction in physical activity not only impairs mental health but also lowers life satisfaction; furthermore, it can lead to easy injuries even during simple daily activities, potentially resulting in serious complications. While sarcopenia primarily affects the elderly, it is now occurring across all generations. Currently in Korea, one in six people is aged 65 or older, and the number of sarcopenia patients is on the rise due to the aging population. Recently, muscle loss in the elderly has been recognized as a disease rather than natural aging, and it is already being managed with a disease code assigned in the United States (2016), the WHO (2017), and Japan (2018), and a disease code was also assigned in Korea in 2021. It is known that in the presence of sarcopenia, the risk of death increases fivefold due to metabolic syndrome, including obesity, diabetes, and heart disease, as well as osteoporosis and fractures. Although a disease code has been assigned for sarcopenia, there is currently no cure available.

[0003] Accordingly, while researching the prevention, improvement, or treatment of muscle loss or muscle atrophy, the inventors completed the present invention by confirming that the Bifidobacterium animalis lactis MG741 strain promotes GLP-1 secretion and not only inhibits muscle damage but also regenerates it through the improvement of oxidative stress.

[0004] Accordingly, the object of the present invention is to provide a composition for preventing, improving, or treating diseases caused by muscle loss or muscle atrophy, comprising Bifidobacterium animalis subsp. lactis MG741 (accession number: KCTC13453BP), a culture medium thereof, a concentrate thereof, a lysate thereof, a dried product thereof, or a combination thereof.

[0005] Another object of the present invention is to provide a muscle-enhancing composition comprising Bifidobacterium animalis lactis MG741 (accession number: KCTC13453BP), a culture medium thereof, a concentrate thereof, a mash thereof, a dried product thereof, or a combination thereof.

[0006] Another object of the present invention is to provide a method for treating a disease caused by muscle loss or muscle atrophy, comprising the step of administering Bifidobacterium animalis lactis MG741 (accession number: KCTC13453BP), its culture medium, its concentrate, its lysate, its dried form, or a combination thereof to an individual in need thereof.

[0007] Another object of the present invention is to provide a method for muscle enhancement, muscle strength enhancement, muscle differentiation promotion, or muscle regeneration, comprising the step of administering Bifidobacterium animalis lactis MG741 (accession number: KCTC13453BP), its culture medium, its concentrate, its lysate, its dried form, or a combination thereof to an individual in need thereof.

[0008] To achieve the above objective, the present invention provides a composition for preventing or treating diseases caused by muscle loss or muscle atrophy, comprising Bifidobacterium animalissubsp.lactis MG741 (accession number: KCTC13453BP), a culture medium thereof, a concentrate thereof, a lysate thereof, a dried product thereof, or a combination thereof.

[0009] In addition, the present invention provides a food composition for preventing or improving diseases caused by muscle loss or muscle atrophy, comprising Bifidobacterium animalis lactis MG741 (accession number: KCTC13453BP), its culture medium, its concentrate, its mash, its dried form, or a combination thereof.

[0010] In addition, the present invention provides a health functional food composition for preventing or improving diseases caused by muscle loss or muscle atrophy, comprising Bifidobacterium animalis lactis MG741 (accession number: KCTC13453BP), its culture medium, its concentrate, its mash, its dried form, or a combination thereof.

[0011] In addition, the present invention provides a feed composition for preventing or improving diseases caused by muscle loss or muscle atrophy, comprising Bifidobacterium animalis lactis MG741 (accession number: KCTC13453BP), its culture medium, its concentrate, its crushed product, its dried product, or a combination thereof.

[0012] In addition, the present invention provides a muscle-enhancing composition comprising Bifidobacterium animalis lactis MG741 (accession number: KCTC13453BP), its culture medium, its concentrate, its mash, its dried form, or a combination thereof.

[0013] In addition, the present invention provides a method for treating a disease caused by muscle loss or muscle atrophy, comprising the step of administering Bifidobacterium animalis lactis MG741 (accession number: KCTC13453BP), its culture medium, its concentrate, its lysate, its dried form, or a combination thereof to an individual in need thereof.

[0014] In addition, the present invention provides a method for muscle enhancement, muscle strength enhancement, promotion of muscle differentiation, or muscle regeneration, comprising the step of administering Bifidobacterium animalis lactis MG741 (accession number: KCTC13453BP), its culture medium, its concentrate, its lysate, its dried form, or a combination thereof to an individual in need thereof.

[0015] It was confirmed that the Bifidobacterium animalis lactis MG741 strain according to the present invention has the ability to promote GLP-1 secretion and exhibits excellent cell proliferation and cell protective abilities against muscle cells. In addition, it was confirmed that the Bifidobacterium animalis lactis MG741 strain inhibits muscle damage and promotes muscle regeneration by improving oxidative stress. Therefore, the Bifidobacterium animalis lactis MG741 strain of the present invention can be utilized in various fields related to the treatment of muscle loss diseases and muscle growth.

[0016] Figure 1 is a phylogenetic tree of the Bifidobacterium animalis lactis MG741 strain according to the present invention.

[0017] Figure 2A is a figure showing the results of confirming the C2C12 cell proliferation ability of the Bifidobacterium animalis lactis MG741 strain according to the present invention (p<0.05).

[0018] Figure 2B shows the results of confirming the C2C12 cytoprotective ability of the Bifidobacterium animalis lactis MG741 strain according to the present invention (p<0.05).

[0019] Figure 3A shows the results of analyzing GLP-1 in serum in a high-fat diet animal model (HFD) administered with the Bifidobacterium animalis lactis MG741 strain according to the present invention (p<0.01).

[0020] Figure 3B shows the results of analyzing creatine kinase (CK) in serum in a high-fat diet animal model (HFD) administered with the Bifidobacterium animalis lactis MG741 strain according to the present invention (p<0.01).

[0021] Figure 3C shows the results of analyzing LDH (lactate dehydrogenase) in serum in a high-fat diet animal model (HFD) administered with the Bifidobacterium animalis lactis MG741 strain according to the present invention (p<0.05).

[0022] Figure 4 is a figure showing the results of observing muscle tissue in an animal model administered with the Bifidobacterium animalis lactis MG741 strain according to the present invention and the results of calculating the muscle fiber cross-sectional area therefrom (p<0.05).

[0023] Figure 5 shows the results of analyzing the protein expression of MnSOD, Catalase, and Gpx in a high-fat diet animal model (HFD) administered with the Bifidobacterium animalis lactis MG741 strain according to the present invention (p<0.05).

[0024] The present invention will be described in detail below.

[0025] According to an aspect of the present invention, the present invention provides a pharmaceutical composition for the prevention or treatment of diseases caused by muscle loss or muscle atrophy, comprising Bifidobacterium animalis subsp. lactis MG741 (accession number: KCTC13453BP), a culture medium thereof, a concentrate thereof, a lysate thereof, a dried product thereof, or a combination thereof. The composition of the present invention may be a pharmaceutical composition, a food composition, a health functional food composition, or a feed composition.

[0026] In a specific embodiment of the present invention, the Bifidobacterium animalis lactis MG741 strain preferably comprises 16S rRNA represented by the nucleotide sequence of SEQ ID NO. 1. The 16S rRNA sequence is the sequence used to identify the Bifidobacterium animalis lactis MG741 strain of the present invention. The Bifidobacterium animalis lactis MG741 strain is a strain isolated from the feces of an infant, which was deposited at the National Institute of Biotechnology and Bioengineering (NIBB) Biological Resource Center (Jeongeup-si, Jeollabuk-do, Korea) on January 4, 2018, and was assigned accession number KCTC13453BP. The Bifidobacterium animalis lactis MG741 strain of the present invention is a human-derived strain and is characterized as being safe for ingestion; in particular, since it was isolated from an infant, it is a lactic acid bacterium that can be safely consumed by infants as well as adults.

[0027] In the present invention, as long as the purpose of the present invention can be achieved, the Bifidobacterium animalis lactis MG741 strain itself, the culture medium thereof, or the cell-free supernatant thereof deposited under accession number KCTC13453BP may be used.

[0028] Furthermore, it is clear to those skilled in the art that any method known in the art may be used to obtain the culture solution of the above strain, and is not limited thereto.

[0029] In a specific embodiment of the present invention, the strain preferably has one or more activities selected from the group consisting of muscle regeneration, promotion of muscle cell proliferation, protective effect on muscle cells, promotion of GLP-1 secretion, and increase in muscle cross-sectional area, but the scope of the present invention is not limited thereto.

[0030] In the present invention, the culture medium refers to a medium containing by-products generated through the intake of nutrients and metabolism of a strain and the strain itself, obtained by culturing the strain in a medium; specifically, it may refer to a culture medium or culture product of the strain. Furthermore, a concentrated or diluted solution of the culture medium, a dried or freeze-dried product obtained by drying the culture medium, a modified or purified product of the culture medium, a crushed product obtained by crushing the culture medium, or a mixture thereof may also be included as an active ingredient in the composition of the present invention.

[0031] In a specific embodiment of the present invention, the strain may be included in the form of a live cell, a dead cell, or a combination thereof.

[0032] In the present invention, the cell refers to the strain of the present invention itself and includes the strain itself or a strain isolated from a culture medium by culturing said strain, and may be a live cell or a dead cell. The cell may be obtained by centrifuging the culture medium and taking the portion that settles to the bottom layer, or by letting it sit for a certain period of time and removing the liquid from the top, as it sinks to the bottom layer of the culture medium due to gravity.

[0033] In a specific embodiment of the present invention, the disease caused by muscle loss or muscle atrophy is preferably one or more selected from the group consisting of sarcopenia, atony, muscular atrophy, muscular dystrophy, muscle degeneration, myotonic dystrophy, amyotrophic lateral sclerosis, myasthenia, and cachexia, and is applicable without limitation to any symptom, disease, or illness caused by muscle loss or muscle atrophy.

[0034] In a specific embodiment of the present invention, the prevention or improvement of muscle loss or muscle atrophy may be muscle strengthening, muscle augmentation, promotion of muscle differentiation, or muscle regeneration.

[0035] In the present invention, muscle differentiation refers to the process in which mononuclear myoblasts form multinuclear myotubes through fusion, and refers to the induction of such a process. Cells in the differentiation stage forming myotubes are identified through the expression of markers such as Pax7, MyoD, and MyoG. By promoting the differentiation of myoblasts, the number of muscle cells increases, which can ultimately lead to an increase in muscle mass in the individual.

[0036] In the present invention, muscle regeneration refers to the ability of a damaged muscle to recover to a normal state. The muscle regeneration may be an effect resulting from the promotion of muscle cell differentiation, which increases the absolute amount of muscle cells, or from an increase in the diameter of individual muscle canals.

[0037] In the present invention, prevention refers to any act of suppressing or delaying the onset of a disease caused by muscle loss or muscle atrophy through the administration of a pharmaceutical composition according to the present invention. Furthermore, in the present invention, treatment refers to any act of improving or beneficially altering the symptoms of a disease caused by muscle loss or muscle atrophy through the administration of a pharmaceutical composition according to the present invention.

[0038] The pharmaceutical composition of the present invention may further include pharmaceutically acceptable additives, wherein the pharmaceutically acceptable additives may include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose, mannitol, malt syrup, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, carnauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, sucrose, etc. The pharmaceutically acceptable additive according to the present invention is preferably included in an amount of 0.1 to 90 parts by weight with respect to the composition, but is not limited thereto.

[0039] In addition, the pharmaceutical composition of the present invention may be administered in various oral or parenteral formulations during actual clinical administration. When formulating, it may be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants, and it is preferable to use suitable formulations known in the art. Carriers, excipients, and diluents that may be included in the composition include lactose, dextrose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, etc.

[0040] The above-mentioned solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid dosage forms are prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium styrate and talc are also used. Furthermore, the above-mentioned liquid dosage forms for oral administration include suspensions, liquid formulations, emulsions, syrups, etc., and may include various excipients, such as humectants, sweeteners, flavorings, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin.

[0041] The above-mentioned preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspension solvents. Witepsol, Macrogol, Tween 61, cacao oil, laurin oil, glycerogelatin, etc. may be used as bases for suppositories. The above-mentioned parenteral administration may be performed using external application to the skin or by intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection.

[0042] The dosage of the pharmaceutical composition of the present invention varies depending on the patient's weight, age, gender, health condition, diet, time of administration, method of administration, excretion rate, and severity of the disease, and may be administered once a day or divided into several doses.

[0043] The pharmaceutical composition of the present invention can be administered to an individual via various routes.

[0044] The pharmaceutical composition of the present invention may be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers for the prevention or treatment of diseases caused by muscle loss or muscle atrophy.

[0045]

[0046] When the composition of the present invention is a food composition or a health functional food composition, the food according to the present invention includes, for example, various types of food, beverages, chewing gum, tea, vitamin complexes, functional foods, etc. In addition, the food includes, but is not limited to, special nutritional foods (e.g., infant formula, baby food, etc.), processed meat products, fish products, tofu products, jelly products, noodles (e.g., ramen, noodles, etc.), bread products, health supplements, seasoning products (e.g., soy sauce, soybean paste, red pepper paste, mixed sauce, etc.), sauces, confectionery products (e.g., snacks), candies, chocolates, chewing gum, ice cream, dairy products (e.g., fermented milk, cheese, etc.), other processed foods, kimchi, pickled foods (various types of kimchi, pickled vegetables, etc.), beverages (e.g., fruit beverages, vegetable beverages, soy milk, fermented beverages, etc.), natural seasonings (e.g., ramen soup, etc.), and food additives. The above food, beverage, or food additive can be manufactured by conventional manufacturing methods.

[0047] In the present invention, a health functional food refers to a group of foods to which added value has been imparted by utilizing physical, biochemical, or biotechnological methods to enable the function of the food to act or manifest for a specific purpose, or to a food processed by designing it to sufficiently express in the body the in vivo regulatory functions regarding the regulation of biological defense rhythms, disease prevention, and recovery possessed by the food composition. For the purposes of the present invention, the health functional food refers to one intended to promote muscle strength enhancement, muscle augmentation, muscle differentiation, or muscle regeneration activity.

[0048] When the composition of the present invention is used as an additive to food or health functional food, the composition may be added as is or used together with other ingredients, or it may be used appropriately according to conventional methods. The amount of the active ingredient can be appropriately determined according to the purpose of use. Generally, when manufacturing food or beverages, the composition of the present invention may be added to the raw material in an amount preferably 50 parts by weight or less, more preferably 25 parts by weight or less. However, in the case of long-term consumption for the purpose of health control and hygiene, the above 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 greater than the above range.

[0049] The food or health functional food composition of the present invention may contain various ordinary flavoring agents or natural carbohydrates, etc., as additional ingredients that are active ingredients. Examples of the above-mentioned natural carbohydrates are monosaccharides, e.g., glucose, fructose, etc.; disaccharides, e.g., maltose, sucrose, etc.; polysaccharides, e.g., dextrin, cyclodextrin, etc., and ordinary sugars, and sugar alcohols such as xylitol, sorbitol, erythritol, etc. The above-mentioned flavoring agents may advantageously include natural flavoring agents (thaumatin), stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.), and synthetic flavoring agents (saccharin, aspartame, etc.).

[0050] In the present invention, the term "feed" refers to any natural or artificial meal, single-meal food, etc., consumed by an animal, or the components of said single-meal food. The feed composition according to the present invention can be prepared in various forms of feed known in the art, and preferably may include concentrated feed, roughage, and / or special feed, but is not limited thereto.

[0051] The feed composition of the present invention may be a feed additive composition. The feed additive composition includes a substance added to the feed for the purpose of various effects, such as alleviating disease symptoms in animals, supplementing nutrients and preventing weight loss, improving the digestibility and utilization of fiber in the feed, improving milk quality, preventing reproductive disorders and improving conception rates, and preventing high-temperature stress during the summer.

[0052] The feed additive composition of the present invention corresponds to an auxiliary feed under the Feed Management Act and may additionally include mineral preparations such as sodium bicarbonate, bentonite, magnesium oxide, and complex minerals; mineral preparations that are trace minerals such as zinc, copper, cobalt, and selenium; vitamin preparations such as carotene, vitamins A, E, nicotinic acid, and vitamin B complex; protected amino acid preparations such as methionine and lysine; protected fatty acid preparations such as calcium salts of fatty acids; probiotics (lactic acid bacteria preparations); probiotics such as yeast cultures and mold fermentation products; yeast preparations, etc.

[0053] Among the above feeds, concentrated feeds include, but are not limited to, seed grains such as wheat, oats, and corn; bran, which is a byproduct obtained by refining grains and includes rice bran, wheat bran, and barley bran; oilseed meal, which is a byproduct obtained by extracting oil from soybeans, rapeseed, sesame, flaxseed, coconut, etc.; residues such as residual starch, which is the main component of starch residue remaining after removing starch from sweet potatoes, potatoes, etc.; fish meal, fish residue; fish soluble, which is a concentrated fresh liquid obtained from fish; meat meal, blood meal, feather meal, skim milk powder; dried whey, which is the residue obtained when making cheese from milk or casein from skim milk; yeast, Chlorella, and seaweed.

[0054] Among the above feeds, roughage includes fresh grass feeds such as wild grass, pasture grass, and green cuts; root vegetables such as feed turnips, feed beets, and a type of turnip called lutebread; silage, which is a stored feed made by filling a silo with fresh grass, green cut crops, and grains and fermenting them with lactic acid; hay made by cutting and drying wild grass and pasture grass; straw of livestock crops; and leaves of legumes, but is not limited thereto. Special feeds include mineral feeds such as oyster shells and rock salt; urea feeds such as urea or its derivatives such as diuretic isobutane; feed additives and dietary supplements, which are substances added in trace amounts to compound feed to supplement components that are prone to being deficient when only natural feed ingredients are mixed, or to improve the shelf life of the feed, but are not limited thereto.

[0055] The feed additive composition according to the present invention can be prepared by adding one or more selected from the group consisting of ppGpp (Guanosine 5'-diphosphate 3'-diphosphate) deficient variant enteropathogenic E. coli (EPEC), lysed products thereof, and cultures of said strains, in an appropriate effective concentration range according to various feed manufacturing methods known in the art.

[0056] The feed additive according to the present invention can be applied without limitation to any individual intended for the prevention or improvement of muscle loss or muscle atrophy. For example, it can be applied to any individual, such as non-human animals like monkeys, dogs, cats, rabbits, guinea pigs, rats, mice, cattle, sheep, pigs, goats, etc., as well as birds and fish.

[0057]

[0058] According to another aspect of the present invention, the present invention provides a muscle-enhancing composition comprising Bifidobacterium animalis lactis MG741 (accession number: KCTC13453BP), a culture medium thereof, a concentrate thereof, a mash thereof, a dried product thereof, or a combination thereof.

[0059] In the present invention, the muscle enhancement and muscle strength improvement are preferably due to muscle regeneration, promotion of muscle cell proliferation, protective efficacy of muscle cells, promotion of GLP-1 secretion, or an increase in muscle cross-sectional area.

[0060] In an embodiment of the present invention, the Bifidobacterium animalis lactis MG741 strain was orally administered to mice. As a result, it was confirmed that the cross-sectional area of ​​the muscle fibers in the mice increased. In addition, it was confirmed that the Bifidobacterium animalis lactis MG741 strain promotes the proliferation of muscle cells. The above results indicate that the Bifidobacterium animalis lactis MG741 of the present invention has an excellent muscle-enhancing effect and can be usefully utilized as a composition for muscle enhancement.

[0061] The composition for muscle enhancement or muscle strength strengthening of the present invention can be manufactured in various forms such as food compositions, health functional food compositions, and food additive compositions, and can be used as an adjuvant for muscle growth and strength strengthening in the general public as well as for muscle loss due to aging or disease.

[0062]

[0063] According to another aspect of the present invention, the present invention provides a method for treating a disease caused by muscle loss or muscle atrophy, comprising the step of administering Bifidobacterium animalis lactis MG741 (accession number: KCTC13453BP), its culture medium, its concentrate, its lysate, its dried form, or a combination thereof to an individual in need thereof.

[0064] In a specific embodiment of the present invention, the individual may be an individual expected to develop a disease due to muscle loss or muscle atrophy; an individual that has developed the disease; or an individual that has been determined to be cured, but is not limited thereto.

[0065]

[0066] According to another aspect of the present invention, the present invention provides a method for muscle enhancement, muscle strength enhancement, promotion of muscle differentiation, or muscle regeneration, comprising the step of administering Bifidobacterium animalis lactis MG741 (accession number: KCTC13453BP), its culture medium, its concentrate, its lysate, its dried form, or a combination thereof to an individual in need thereof.

[0067] In a specific embodiment of the present invention, the individual may be an individual in whom muscle reduction or muscle atrophy is expected or has occurred.

[0068] Bifidobacterium animalis lactis MG741 (accession number: KCTC13453BP), its culture medium, its concentrate, its lysate, its dried form, or a combination thereof used in the method of the present invention may be administered in the form of a powder dried by mixing with a freeze-drying agent or in the form of a rehydrated powder, or may be administered together with a filler, extender, binder, wetting agent, disintegrant, surfactant, diluent, excipient, or carrier.

[0069]

[0070] Redundant content is omitted out of consideration for the complexity of this specification, and terms not otherwise defined in this specification have the meanings commonly used in the technical field to which this invention belongs.

[0071] The present invention will be described in more detail below through examples. These examples are intended solely to illustrate the present invention, and it will be obvious to those skilled in the art that the scope of the present invention is not to be interpreted as being limited by these examples.

[0072]

[0073] Example 1. Preparation of Bifidobacterium animalis lactis MG741 strain

[0074] Bifidobacterium animalis subsp. lactis MG741 strain is a strain isolated from the feces of infants and was provided by Mediogen Co., Ltd. (Jecheon, Korea).

[0075] A phylogenetic analysis was performed using the 16S rRNA (Sequence No. 1) of the above-mentioned Bifidobacterium animalis lactis MG741 strain. Specifically, a phylogenetic analysis of the above-mentioned Bifidobacterium animalis lactis MG741 strain based on the sequence homology of the 16S rRNA was performed using the neighbor-joining method, and the constructed phylogenetic tree is shown in Figure 1.

[0076] As shown in Figure 1, it was confirmed that the Bifidobacterium animalis lactis MG741 strain is different from strains known in the field.

[0077] The strain Bifidobacterium animalis lactis MG741 was deposited at the National Institute of Biotechnology and Bioengineering (Jeongeup-si, Jeollabuk-do, Korea) on January 4, 2018, and was assigned accession number KCTC13453BP.

[0078] The Bifidobacterium animalis lactis MG741 strain was activated by culturing in De Man, Rogosa and Sharpe (MRS) liquid medium at 37°C for 18 hours. The culture was subjected to cold centrifugation (4,000 xg for 15 minutes at 4°C) to harvest the cell-free supernatant and pellet. The supernatant was filtered using a 0.2-μm polytetrafluoroethylene (PTFE) membrane (ADVANTEC, Tokyo, Japan) for use in cell experiments, and the pellet was mixed with a cryoprotectant, dried, and powdered. The strain powder was stored at 4°C until use in animal experiments.

[0079]

[0080] Example 2. Characterization of Bifidobacterium animalis lactis MG741 strain

[0081] 2-1. Carbohydrate Fermentation Profile Analysis

[0082] The carbohydrate fermentation profile of the Bifidobacterium animalis lactis MG741 strain was confirmed. The carbohydrate fermentation profile analysis was performed using the API 50CHL kit (Biomeriux, France) according to the manufacturer's manual. In addition, the control for the analysis was the same strain, Bifidobacterium animalis lactis KCTC5854 (DSM10140). The results of the mycological characterization evaluation are shown in Table 1.

[0083] CarbohydratesBi. lactisMG741Bi. lactisKCTC5854CarbohydratesBi. lactisMG741Bi. lactisKCTC5854Control--Esculin++Glycerol--Salicin+-Erythritol--D-Cellobiose--D-Arabinose--D-Maltose++L-Arabinose+-D-Lactose+-D-Ribose+-D-Melibiose++D-Xylose +-D-Sucrose++L-Xylose--D-Trehalose--D-Adonitol--Inulin--Methyl-β-D-xyloside--D-Melezitose--D-Galactose++D-Raffinose++D-Glucose++Starch+-D-Fructose--Glycogen --D-Mannose+-Xylitol--L-Sorbose+-Gentiobiose+-L-Rhamnose--D-Turanose--Dulcitol--D-Lyxose+-Inositol--D-Tagatose--D-Mannitol--D-Fucose--D-Sorbitol--L-Fucose-- Methyl-α-D-mannoside--D-Arabitol--Methyl-α-D-glucoside+-L-Arabitol--N-Acetylglucosamine--Gluconate--Amygdalin+-2-keto-gluconate--Arbutin+-5-keto-gluconate--

[0084] As shown in Table 1, it was confirmed that the Bifidobacterium animalis lactis MG741 strain had different sugar utilization capabilities compared to the control strain. Specifically, the Bifidobacterium animalis lactis MG741 strain utilized L-Arabinose, D-ribose, D-Mannose, L-Sorbose, Methyl-α-D-glucoside, Salicin, D-Lactose, Starch, Genitombiose, and D-Lyxose, whereas the control strain could not utilize the aforementioned sugars.

[0085]

[0086] 2-2. Evaluation of C2C12 Cell Proliferative and Cytoprotective Ability of Bifidobacterium animalis lactis MG741 Strain

[0087] The cell proliferation ability of C2C12 cells following treatment with the Bifidobacterium animalis lactis MG741 strain was evaluated. Specifically, C2C12 cells were placed in a 96-well plate at a ratio of 1 x 10⁶ 4 Cells were seeded at a cell / well density and cultured for 24 hours. Subsequently, 5% of the cell-free supernatant was treated for 48 hours, followed by the incubation of each well with MTT (3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) solution at a concentration of 0.1 mg / mL for 2 hours. Afterward, the cell supernatant treated with the MTT solution was removed, and formazan crystals were dissolved using DMSO (150 μL). Cell proliferation was evaluated by measuring absorbance (550 nm) using a microplate reader (Bi-otek, Winooski, VT, USA). The control for the analysis was the same species, Bifidobacterium animalis lactis KCTC5854 (DSM10140). The results of evaluating the cell proliferation of the control in this experiment are shown in Figure 2A.

[0088] As shown in Figure 2A, it was confirmed that the group administered with the Bifidobacterium animalis lactis MG741 strain had a higher C2C12 cell proliferation ability compared to the control and the control strain (KCTC5854). In particular, the Bifidobacterium animalis lactis MG741 strain had a significantly higher C2C12 cell proliferation ability than KCTC5854, which is of the same species.

[0089]

[0090] In addition, to evaluate cytoprotective ability, C2C12 cells were treated with hydrogen peroxide and the Bifidobacterium animalis lactis MG741 strain. Subsequently, cell viability was evaluated using the same method as above. The results of evaluating cytoprotective ability on C2C12 cells are shown in Figure 2B.

[0091] As shown in Figure 2B, it was confirmed that C2C12 cells showed a decrease in cell viability upon treatment with hydrogen peroxide, meaning that muscle cells were damaged due to oxidative stress. However, it was confirmed that cell viability increased when C2C12 cells were treated with either the Bifidobacterium animalis lactis MG741 strain or the KCTC5854 strain along with hydrogen peroxide. In particular, it was confirmed that the cell viability of C2C12 cells in the group treated with the Bifidobacterium animalis lactis MG741 strain was significantly higher than that of the group treated with the same strain, KCTC5854.

[0092] The above results indicate that the Bifidobacterium animalis lactis MG741 strain can not only effectively proliferate C2C12 cells but also has the ability to protect against muscle cell damage caused by oxidative stress.

[0093]

[0094] Example 3. Preparation of Animal Model

[0095] Forty-five 6-week-old mice (C57BL / 6J, male) were obtained from the Central Experimental Animal Center and acclimatized for one week. Subsequently, the mice were divided into a normal diet group (ND), an experimental control group (HFD), and a sample group (HFD+MG741), and each substance was orally administered for 12 weeks. For the sample group, the Bifidobacterium animalis lactis MG741 strain isolated in Example 1 was dissolved in physiological saline and 10 9 It was administered in CFU amounts. All groups except the normal group were provided with a high-fat diet.

[0096]

[0097] Example 4. Serological analysis of an animal model administered with Bifidobacterium animalis lactis MG741 strain

[0098] The animal model prepared as in Example 3 was fasted for 18 hours, then anesthetized, and blood was collected from the vena cava. Serum was separated by centrifugation at 3,000 rpm for 15 minutes. The separated serum was stored in a -70°C freezer until analysis. The GLP-1 content in the serum was measured using an ELISA kit (AFG Scientific, USA), and CK (creatine kinase) and LDH (lactate dehydrogenase) were measured using a biochemical analyzer (Hitachi 3500, HITACHI, JAPAN). The results of the analysis of GLP-1, CK, and LDH in the serum are shown in Figures 3A to 3C, respectively.

[0099] As shown in Figure 3A, it was confirmed that the serum GLP-1 concentration in the group administered the Bifidobacterium animalis lactis MG741 strain was significantly increased compared to the normal group (ND) and the control group (HFD). In particular, while the serum GLP-1 concentration significantly decreased when a high-fat diet was provided, it was confirmed that the serum GLP-1 concentration increased significantly compared to the normal group upon administration of the Bifidobacterium animalis lactis MG741 strain. The above results indicate that the Bifidobacterium animalis lactis MG741 strain has the ability to promote GLP-1 secretion.

[0100] As shown in Figures 3B and C, it was confirmed that the concentrations of CK and LDH in the serum of the Bifidobacterium animalis lactis MG741 strain administered were significantly reduced compared to the normal group (ND) and the control group (HFD). The above results indicate that the Bifidobacterium animalis lactis MG741 strain has an inhibitory effect on muscle damage.

[0101]

[0102] Example 5. Histological analysis of an animal model administered with Bifidobacterium animalis lactis MG741 strain

[0103] Muscle tissue was obtained by sacrificing an animal model prepared as in Example 3 above. The muscle tissue was fixed in 10% formalin for at least 24 hours and embedded in paraffin, after which 4 μm muscle tissue sections stained with hematoxylin and eosin (H&E) were obtained. The muscle tissue sections were observed under a microscope, and the muscle fiber cross-sectional area was calculated based on the observation results. The results of the microscopic observation and the calculation of the muscle fiber cross-sectional area are shown in Figure 4.

[0104] As shown in Figure 4, it was confirmed that the muscle fiber cross-sectional area of ​​the group administered with the Bifidobacterium animalis lactis MG741 strain was significantly increased compared to the normal group (ND) and the control group (HFD). In particular, while the muscle fiber cross-sectional area was significantly reduced when a high-fat diet was provided, it was confirmed that the muscle fiber cross-sectional area increased significantly compared to the normal group upon administration of the Bifidobacterium animalis lactis MG741 strain.

[0105]

[0106] Example 6. Analysis of protein expression in an animal model administered with Bifidobacterium animalis lactis MG741 strain

[0107] The expression of antioxidant enzymes following the administration of the Bifidobacterium animalis lactis MG741 strain was analyzed. Specifically, femur muscle tissue was obtained by sacrificing mice. A lysis buffer containing a protease inhibitor was added to the obtained tissue, homogenized, and centrifuged at 10,000 xg for 5 minutes to obtain the supernatant. After quantifying the protein, an equal amount of protein was electrophoresed using a 10% SDS-polyacrylamide gel (Bio-Rad, CA, USA). Subsequently, the protein was transferred to a PVDF membrane (Millipore, MA, USA). After blocking non-specific binding sites in a 5% BSA (0.1% Tween 20 containing TBS, TBST) solution, the samples were incubated overnight at 4°C with primary antibodies (anti-β-actin, anti-MnSOD, anti-Catalase, anti-Gpx) and for 1 hour at room temperature with secondary antibodies (anti-rabbit IgG or anti-mouse IgG linked with horseradish peroxidase, Cell Signaling Technology). Proteins were then detected using WestGlow™ FEMTO ECL Chemiluminescent substrate (BIOMAX Co., Ltd., Korea) and LuminoGraph III Lite (ATTO, Tokyo, Japan). The Western blotting results are shown in Figure 5.

[0108] As shown in Figure 5, it was confirmed that the protein expression of MnSOD, Catalase, and Gpx in the group administered with the Bifidobacterium animalis lactis MG741 strain was significantly increased compared to the normal group (ND) and the control group (HFD). The above results indicate that the Bifidobacterium animalis lactis MG741 strain inhibits muscle damage by improving oxidative stress, and that its efficacy is excellent.

[0109]

[0110] Overall, the inventors confirmed that the Bifidobacterium animalis lactis MG741 strain has a carbohydrate fermentation profile different from that of the same strain, and possesses excellent cell proliferation and cell protective abilities against muscle cells. In addition, it was experimentally confirmed that the Bifidobacterium animalis lactis MG741 strain exhibits excellent GLP-1 secretion-promoting ability and increases the cross-sectional area of ​​muscle fibers in animal models. This implies that the Bifidobacterium animalis lactis MG741 strain inhibits muscle damage and regenerates muscle by improving oxidative stress; thus, the strain of the present invention can be utilized in various fields related to the treatment of muscle loss diseases and muscle growth.

[0111]

[0112] Foregoing, specific parts of the present invention have been described in detail. It will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents.

[0113]

[0114] [Consignment Number]

[0115] Name of Depositing Institution: Korea Research Institute of Bioscience and Biotechnology Biological Resource Center

[0116] Trustee Number: KCTC13453BP

[0117] Date of Trust: 20180104

[0118]

[0119]

Claims

1. Bifidobacterium animalissubsp.lactis MG741 (accession number: KCTC13453BP), comprising its culture medium, its concentrate, its lysate, its dried form, or a combination thereof, A pharmaceutical composition for the prevention or treatment of diseases caused by muscle loss or muscle atrophy.

2. A composition according to claim 1, wherein the strain has one or more activities selected from the group consisting of muscle regeneration, promotion of muscle cell proliferation, protective effect on muscle cells, promotion of GLP-1 secretion, and increase in muscle cross-sectional area.

3. A composition according to claim 1, wherein the disease resulting from muscle loss or muscle atrophy is one or more selected from the group consisting of sarcopenia, atony, muscular atrophy, muscular dystrophy, muscle degeneration, myotonic dystrophy, amyotrophic lateral sclerosis, myasthenia, and cachexia.

4. Bifidobacterium animalissubsp.lactis MG741 (accession number: KCTC13453BP), comprising its culture medium, its concentrate, its lysate, its dried form, or a combination thereof, Food composition for strengthening muscles, promoting muscle differentiation, or regenerating muscles.

5. In paragraph 4, the prevention or improvement of muscle loss or muscle atrophy is a food composition that strengthens muscle, increases muscle mass, promotes muscle differentiation, or regenerates muscle.

6. Bifidobacterium animalissubsp.lactis MG741 (accession number: KCTC13453BP), comprising its culture medium, its concentrate, its lysate, its dried form, or a combination thereof, A health functional food composition for preventing or improving diseases caused by muscle loss or muscle atrophy.

7. Bifidobacterium animalissubsp.lactis MG741 (accession number: KCTC13453BP), comprising its culture medium, its concentrate, its lysate, its dried form, or a combination thereof, Feed composition for preventing or improving diseases caused by muscle loss or muscle atrophy.

8. A muscle-enhancing composition comprising Bifidobacterium animalissubsp.lactis MG741 (accession number: KCTC13453BP), a culture solution thereof, a concentrate thereof, a mash thereof, a dried product thereof, or a combination thereof.

9. A method for treating a disease caused by muscle loss or muscle atrophy, comprising the step of administering Bifidobacterium animalissubsp.lactis MG741 (accession number: KCTC13453BP), a culture medium thereof, a concentrate thereof, a lysate thereof, a dried product thereof, or a combination thereof to an individual in need thereof.

10. A method for muscle enhancement, muscle strength enhancement, promotion of muscle differentiation, or muscle regeneration, comprising the step of administering Bifidobacterium animalis subsp. lactis MG741 (accession number: KCTC13453BP), a culture medium thereof, a concentrate thereof, a lysate thereof, a dried product thereof, or a combination thereof to an individual in need thereof.

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