KR102329853B1 - Pharmaceutical composition for preventing or treating muscular atrophy comprising akkermansia muciniphila strain - Google Patents

Abstract

The present invention relates to a pharmaceutical composition effective for preventing or treating muscular atrophy, and a health functional food for preventing or alleviating muscular atrophy, comprising an Akkermansia muciniphila EB-AMDK19 strain or a culture or dried product thereof. The pharmaceutical composition of the present invention suppresses the mRNA expression of muscular atrophy proteins (myostatin, atrogin-1, and MuRF-1), activates energy metabolism in the muscle, and activates GLP-1 to exhibit an excellent effect of preventing or treating muscular atrophy.

Description

A pharmaceutical composition for preventing or treating muscular atrophy comprising Akkermansia muciniphila strain {PHARMACEUTICAL COMPOSITION FOR PREVENTING OR TREATING MUSCULAR ATROPHY COMPRISING AKKERMANSIA MUCINIPHILA STRAIN}

The present invention relates to a muscular dystrophy prevention or treatment a pharmaceutical composition comprising, but more specifically, a cyano marker Mu shinny pillar EB-AMDK19 strain relates to a pharmaceutical composition for the prevention or treatment of muscular dystrophy, as an active ingredient.

Muscular atrophy is caused by a gradual decrease in muscle mass, and refers to muscle weakness and degeneration. Muscular atrophy is promoted by inactivity, oxidative stress, and chronic inflammation, and weakens muscle function and athletic performance. The most important factor that determines muscle function is muscle mass, which is maintained by a balance between protein synthesis and breakdown. Muscular atrophy occurs when protein breakdown occurs more than synthesis. Muscle size is regulated by intracellular signaling pathways that induce anabolism or catabolism within the muscle, and signaling reactions that induce synthesis rather than breakdown of muscle proteins occur more frequently. In this case, muscle protein synthesis is increased, which is indicated by an increase in muscle size or an increase in the number of muscle fibers according to an increase in muscle protein.

As an example of the current therapeutic agent for muscular dystrophy, WO 2007/088123 discloses a therapeutic agent for muscular atrophy comprising a nitroxy derivative as an active ingredient, and WO 2006/081997 discloses a therapeutic agent for muscular atrophy including atralic acid or a derivative thereof as an active ingredient. . However, since these therapeutic agents containing the compound as an active ingredient act not only on the skeletal muscle with muscle atrophy, but also on the visceral muscle or myocardium not related to muscular atrophy, various side effects, large and small, may be induced, and thus have not been used in actual treatment.

Decreased motor function due to muscular atrophy causes fractures or falls, resulting in long-term inactivity, leading to a vicious cycle of additional muscle atrophy and decreased motor function, and also increases the frequency of complications of metabolic disorders or infections. In terms of worsening the quality of life (QOL) as well as the prognosis of the underlying disease, the development of an effective treatment for the muscular atrophy layer is urgently required.

WO 2007/088123 A WO 2006/081997 A KR 10-1869400 B

The present invention is to solve the above technical problem, one object of the present invention is to provide a pharmaceutical composition for preventing or treating muscular dystrophy.

It is an object of the present invention to provide a pharmaceutical composition for preventing or treating muscular atrophy, which is more effective in preventing or treating muscular atrophy than a pharmaceutical composition comprising the Akkermansia muciniphila BAA-835 standard strain.

One object of the present invention is to provide a functional health food composition for preventing or improving muscular atrophy.

Another object of the present invention is to provide a method for treating muscular dystrophy using the pharmaceutical composition.

One aspect of the present invention for achieving the above object is achieved by the deposition No. KCTC 13761BP markers only cyano mu shinny pillar EB-AMDK19 strain pharmaceutical composition for muscular dystrophy the prevention and treatment, including culture or a dried form of the strain of it's about

Another aspect of the present invention for achieving the above object is, only the markers of the present invention cyano mu shinny pillar EB-AMDK19 strain (KCTC 13761BP), the food on for muscular dystrophy prevention or improvement containing culture or dry product of the strain will be.

The present invention Acker only cyano mu shinny pillar EB-AMDK19 strain muscular atrophy prevention or treatment a pharmaceutical composition is the expression of the gene to destroy a reduced weight due to muscular dystrophy, the weight of the skeletal muscle, grip strength and muscle protein comprising as an active ingredient of By inhibiting it, it may be developed as an effective therapeutic agent for muscular atrophy.

The pharmaceutical composition for the prevention or treatment of muscular atrophy of the present invention has high safety and can be safely taken for a long period of time. can be improved

1 is only the markers of the present invention cyano mu shinny pillar EB-AMDK19 strain (KCTC 13761BP) and type strain only the marker (type strain) cyano mu shinny pillar ATCC BAA-835 is a microscopic observation of the strain.
2 is only a cyano mu shinny pillar ATCC BAA-835 is a PCR analysis of the strains, only markers cyano mu shinny pillar EB-AMDK19 strains and markers of the present invention.
Figure 3 is a marker of the present invention, only a cyano mu shinny pillar EB-AMDK19 strains and only markers cyano mu shinny pillar ATCC BAA-835 in the genomic DNA of the strain RAPD (Random Amplified Polymorphic DNA) analysis.
Figure 4 is Akkermansia muciniphila of the present invention EB-AMDK19 Akermansia muciniphila different from strains It shows a comparison of the phylogenetic relationship of the strains.
5 is only a cyano mu shinny pillar EB-AMDK19 only strains with marker Mu is cyano hemolytic activity results of shinny pillar ATCC BAA-835 strain marker of the present invention.
6 is a photograph of a state in which a plaster cast is applied to induce muscular atrophy in mice in an animal experiment according to an embodiment of the present invention.
7 is the control group, muscular atrophy induced group (Imm), positive control (Livamax group), markers only cyano mu shinny pillar ATCC BAA-835 type strain treated group, and only the markers of the present invention cyano mu shinny pillar EB-AMDK19 mouse strains group As a graph showing the change in body weight measured while rearing for 5 weeks, it is a graph showing the rate of change in body weight for the normal control group.
8 is a photograph for explaining a grip test method performed in an embodiment of the present invention.
Figure 9 is a graph showing the grip strength (grip strength) changes in the markers of the present invention, only a cyano mu shinny pillar EB-AMDK19 mouse strain by the administration.
10 is a normal control group, muscular atrophy induction group, positive control group (LVM), Akkermansia muciniphila AT CC BAA-835 strain administration group, and Akermansia muciniphila EB-AMDK19 strain administration group of the present invention. It is a graph showing the result of comparing the weight of the liver tissue obtained from the mouse, the tibialis anterior muscle (TA), and the gastrocnemius muscle (GA).
11 is a normal control group, muscular atrophy induction group, positive control group (LVM), Akkermansia muciniphila AT CC BAA-835 strain administration group, and Akermansia muciniphila EB-AMDK19 strain administration group of the present invention. It is a graph showing the result of comparing the concentration of Myostatin in the serum of mice.
12 is a normal control group, muscular atrophy induction group, positive control group (LVM), Akkermansia muciniphila AT CC BAA-835 strain administration group, and Akermansia muciniphila EB-AMDK19 strain administration group of the present invention in the tibialis anterior muscle (TA, tibialis anterior) and gastrocnemius (GA) is a graph showing the results of comparing the expression levels of genes (Atrogin-1 and MuRF1) related to muscular atrophy in muscle cells.
13 is a graph showing the results of comparing the expression level of the autophage index cathepsin (Cathepsin) L in muscle tissue.
14 is a graph showing the results of comparing the expression level of PGC-1α related to energy metabolism in muscle tissue.
15 is a graph showing the results of comparing the expression levels of GPR43 and GPR119, which are SCFA receptors in colon tissue.
16 is a graph showing the results of comparing the expression level of the anti-inflammatory cytokine IL-10.
17 is a graph showing the results of comparing the expression level of the pro-inflammatory cytokine IL-6.
18 is a graph showing the result of measuring the expression change according to the administration of the strain of the present invention of ZO-1, a tight junction protein in the colonic mucosa.

Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

As used herein, the term “about,” when used in reference to a particular recited numerical value, means that the numerical value may vary by no more than 1% from the recited value. For example, as used herein, the expression “about 100” includes 99 and 101 and all numbers in between (eg, 99.1, 99.2, 99.3, 99.4, etc.).

In the present specification, when a part 'includes' a certain component, it means that other components may be further included, rather than excluding other components, unless otherwise stated.

As used herein, the term “muscular atrophy” refers to a disease in which the muscles of the extremities are gradually atrophied symmetrically left and right, and may accompany the induction of cancer, kidney disease, hereditary disease, and various chronic diseases. , amyotrophic lateral sclerosis (Lou Gehrig's disease), and progressive progressive muscular atrophy of the spinal cord.

As used herein, "Myostatin" is a protein belonging to the TGF-β superfamily, and is specifically expressed in skeletal muscle, myocardium, and adipose tissue as a muscle inhibitory factor. Myostatin is converted into an activator in the cell and inhibits Akt through phosphorylation/activation of Smad (small mothers against decapentaplegic), thereby promoting muscle degradation and also inhibiting muscle synthesis.

As used herein, the term “prevention” refers to any action that suppresses or delays the onset of muscular dystrophy by administration of the pharmaceutical composition according to the present invention.

As used herein, the terms "treat", "treatment" and the like mean to temporarily or permanently alleviate symptoms, eliminate the cause of symptoms, or prevent or slow the onset of symptoms of the disease or condition. do.

As used herein, the term “improvement” refers to any action that reduces a parameter associated with an abnormal condition, for example, the severity of a symptom.

As used herein, the term "pharmaceutically acceptable" means that the benefit/risk ratio is reasonable without undue toxicity, irritation, allergic reaction, or other problems or complications, so that it is suitable for use in contact with the tissues of a subject (eg, a human) and means a composition that is within the scope of sound medical judgment.

Pharmabiotics are defined as human-derived bacteria or their products that have a proven pharmacological role for health or disease (“Probiotics and pharmabiotics,” Bioeng Bugs. 2010 Mar-Apr; 1 (2): 79-84.).

One aspect of the invention relates to markers only cyano mu shinny pillar EB-AMDK19 strain (Akkermansia muciniphila EB-AMDK19) muscular atrophy prevention or treatment a pharmaceutical composition comprising (KCTC13761BP) as an active ingredient.

Muscle wasting and degeneration occurs due to genetic factors, acquired factors, degenerative changes, etc., and muscle wasting is characterized by a gradual loss of muscle mass, weakness and degeneration of muscles, especially skeletal or voluntary muscles and cardiac muscles. Examples of related diseases include atony, muscular atrophy, muscular dystrophy, muscle degeneration, myasthenia gravis, cachexia, and the like.

Muscular atrophy refers to loss of muscle mass and/or progressive muscle weakness and degeneration. In some cases, loss of muscle mass and/or gradual muscle weakness and degeneration results from a high rate of protein breakdown, a low rate of protein synthesis, or a combination of the two. In some cases, the high rate of muscle protein breakdown is due to muscle protein catabolism (ie, the breakdown of muscle proteins to use amino acids as substrates for gluconeogenesis).

Muscular atrophy refers to a significant loss of muscle strength. A significant loss of muscle strength means a decrease in strength in diseased, injured or unused muscle tissue in a subject compared to the same muscle tissue in a control subject. In another embodiment, muscular atrophy refers to a decrease in muscle volume in diseased, injured or unused muscle tissue in a subject compared to the same muscle tissue in a control subject.

In the present invention, the marker, only a cyano mu shinny pillar EB-AMDK19 has is used as an active ingredient of a pharmaceutical composition for muscular dystrophy treatment, the marker only cyano mu shinny pillar EB-AMDK19 increased weight against muscular dystrophy patients, skeletal muscle increased weight , suppression of expression of genes that destroy muscle proteins, and activation of energy metabolism in muscle. At this time, the gene that destroys the muscle protein is not particularly limited thereto, but as an example, a protein encoding a protein such as Myostatin, Atrogin-1, MuRF1 (Muscle RING-finger protein-1) It may be a gene, and the gene for generating muscle protein may be a gene encoding a protein such as MyoD or Myogenin.

The composition according to the present invention may increase the expression of PGC-1α and GPR119, or decrease the expression of Atrogin-1, Muscle Ring-Finger Protein (MuRF1) and Myostatin genes. Atrogin-1 and MuRF1 are muscle-specific ubiquitin-ligases, and are representative proteins that induce muscle reduction by attaching ubiquitin to the lysine site of the target protein to promote protein degradation. The pharmaceutical composition of the present invention can inhibit muscle loss by reducing the expression of Atrogin-1 and MuRF-1, and increase muscle mass by increasing the amount of muscle protein.

According to one embodiment of the invention, each processing, and use them for the vehicle (PBS) or a cyano mu shinny pillar EB-AMDK19 only markers on by a plaster cast as an animal model of muscular dystrophy, this induced mice and the diseases induced mouse the markers only cyano mu if a shinny pillar EB-AMDK19 to handle the muscular dystrophy result of verifying the effect of the mouse models, only markers cyano mu shinny pillar EB-AMDK19 to the mouse, the weight loss is restored, the reduced skeletal muscle The expression level of genes (Myostatin, Atrogin-1, and MuRF1) that destroys muscle proteins increased by the plaster cast is reduced, while the weight of the muscle is restored, the reduced grip strength is restored, and the energy metabolism in the muscle is activated. It was confirmed that the expression level of PGC-1α was increased. From this, the inventors have confirmed that only the marker cyano mu shinny pillar EB-AMDK19 can be effectively used for the prevention and treatment of muscular dystrophy. In addition, these effects are only markers cyano mu shinny only pillars cyano mu shinny pillar was confirmed to be significantly better than the type strain BAA-835 strain of markers of the present invention.

Only markers of deposition No. KCTC 13761BP according to the present invention cyano mu shinny pillar (Akkermansia muciniphila) EB-AMDK19 strain has a 16s rRNA gene of SEQ ID NO: 1.

The Ackermansia muciniphila EB-AMDK19 strain used in the present invention was isolated from the feces of healthy Koreans, was oval cells of 0.5-1 μm in size, was monococci or dicocci, anaerobic, non-motile, and Gram-negative. and mucin-degrading bacteria that do not form endospores. Only markers cyano mu shinny pillar EB-AMDK19 strain may be used as a carbon and nitrogen source for the mucus produced by some of the mucus degrading enzymes, glucose, galactose, N- acetyl can metabolize a variety of carbon sources, including glucosamine and lactose, and , short-chain fatty acids such as propionic acid and acetic acid are produced as major metabolites.

Thus there marker only cyano mu shinny pillars strain known to secrete short-chain fatty acids (Short chain fatty acid, SCFA) , G protein-coupled GLP-1 a receptor of GPR43 and GPR119 receptor of short chain fatty acids (SCFA) ( It plays a role in stimulating the secretion of Glucagon-like peptide 1) (Shah, Current Opinion in Drug Discovery & Development, (2009) 12:519-532; Jones, et al., Ann. Rep. Med. Chem., ( 2009) 44:149-170; see Schwartz et al., Cell Metabolism, 2010, 11:445-447). For GLP-1 is to enhance the insulin sensitivity of muscle using the energy source of glucose, to only the markers of the present invention is a cyano mu shinny pillar strains increase the level of expression of GPR43 and GPR119 has been found to facilitate this path (Fig. 16 Reference).

Muscular dystrophy prevention or treatment a pharmaceutical composition of the present invention may comprise only the cyano mu shinny pillar EB-AMDK19 probiotic strain marker or include pasteurized strain. Only markers cyano mu shinny pillar EB-AMDK19 strain of the present invention is cultured, and collected by the separation process such as centrifugation, dried, for example, may be used to prepare probiotics in the form by lyophilization. Pasteurization of Akermansia muciniphila strains means heating at a temperature of 50 °C or higher and less than 100 °C for at least 10 minutes. For example, it can be pasteurized at 70 °C for 30 minutes.

The pharmaceutical composition of the present invention may contain, as an active ingredient, the Akermansia muciniphila EB-AMDK19 strain in an amount of 10 ⁸ to 10 ¹² CFU, or a culture having an equivalent number of live cells, based on the total weight of the composition. can The strain is the cell of the strain, the lysate of the cell, the culture of the strain, the culture solution from which the cell is removed from the culture of the strain, the cell extract of the strain, the extract of the culture of the strain, or the extract of the culture solution from which the cell is removed from the culture of the strain. can be selected.

In one embodiment, the marker only cyano mu shinny pillar pharmaceutical composition comprising an EB-AMDK19 strains, according to a conventional method, each powder, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, It may be formulated and used in the form of oral dosage forms, such as oral preparations, external preparations, suppositories, or sterile injection solutions, but is not necessarily limited thereto.

In some embodiments, the pharmaceutical compositions of the present invention are aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, liposome dispersions, aerosols, solid bodies, powders, immediate release formulations, controlled release formulations, fast melt formulations, tablets, capsules, pills , delayed release formulations, sustained release formulations, pulsating release formulations, multiparticulate formulations, and immediate and controlled release formulations upon mixing.

The pharmaceutical composition of the present invention may be formulated as a product for enteral or oral administration. In addition, the pharmaceutical composition of the present invention can be enteric-coated and commercialized so that the microorganism, which is an active ingredient, is rapidly released into the intestine after passing through the stomach and reaching the small intestine using a known method.

In one embodiment, the pharmaceutical composition according to the invention may also advantageously comprise other active agents having a supplemental or synergistic effect. When the pharmaceutical composition according to the present invention is used in combination with another second active agent, it may be administered sequentially or simultaneously, and may be administered singly or multiple times.

Such active agents may be one or more medicaments or food supplements or antibodies commonly used for the prevention or treatment of muscle dysfunction or loss of muscle mass, which, depending on the circumstances, may be used using the Ackermansia muciniphila strain of the present invention. useful pharmacological synergies.

Such active agents include proteins or amino acids (e.g., lysine, arginine, leucine, beta-hydroxy-beta-methylbutyrate, citrulline, etc.), vitamins (vitamin D, vitamin B, etc.), minerals (magnesium, calcium, etc.), or other nutritional agents (such as omega-3, polyunsaturated fatty acids (DHA, EPA)), or phospholipids such as phosphatidylcholine, phosphatidylserine.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier and/or excipient in addition to the active ingredient, and in addition to this, various additives commonly used pharmaceutically such as binders, disintegrants, coating agents, lubricants, etc. It can be formulated and prepared with

The pharmaceutically acceptable carrier may further include, for example, a carrier for oral administration or a carrier for parenteral administration. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. In addition, various drug delivery materials used for oral administration may be included. In addition, carriers for parenteral administration may include water, suitable oils, saline, aqueous glucose and glycols, and the like. Stabilizers and preservatives may be further included. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, and the like, in addition to the above components. For other pharmaceutically acceptable carriers and agents, reference may be made to those described in the following literature (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).

Excipients usable in the present invention include sugars such as sucrose, lactose, mannitol, and glucose, and starches such as corn starch, potato starch, rice starch, and partially pregelatinized starch. The binder may include polysaccharides such as dextrin, sodium alginate, carrageenan, guar gum, acacia and agar, naturally-occurring macromolecular substances such as tragacanth, gelatin, gluten, hydroxypropylcellulose, methylcellulose, hydroxypropylmethyl Cellulose derivatives such as cellulose, ethylcellulose, hydroxypropylethylcellulose, and carboxymethylcellulose sodium, and polyvinylpyrrolidone, polyvinyl alcohol, polyvinyl acetate, polyethylene glycol, polyacrylic acid, polymethacrylic acid, and vinyl acetate resin. contains polymers.

The disintegrating agent usable in the present invention includes cellulose derivatives such as carboxymethyl cellulose, carboxymethyl cellulose calcium, low-substituted hydroxypropyl cellulose, sodium carboxymethyl starch, hydroxypropyl starch, corn starch, potato starch, rice starch, and partially starch. Starch, such as gelatinized starch, can be used.

Examples of lubricants usable in the present invention include talc, stearic acid, calcium stearate, magnesium stearate, colloidal silica, hydrosilicone dioxide, various types of waxes and hydrogenated oils.

As a coating agent, dimethylaminoethylmethacrylate-methacrylic acid copolymer, polyvinylacetaldiethylaminoacetate, ethylacrylate-methacrylic acid copolymer, ethylacrylate-methylmethacrylate-chlorotrimethylammoniumethylmethacrylate Copolymer, water-insoluble polymer such as ethyl cellulose, methacrylic acid-ethyl acrylate copolymer, hydroxypropylmethylcellulose phthalate, hydroxypropylmethyl cellulose acetate succinate, etc. tonicity polymer and methylcellulose, hydroxypropylmethylcellulose , polyvinylpyrrolidone, water-soluble polymers such as polyethylene glycol, but is not necessarily limited thereto.

In the pharmaceutical composition for the prevention or treatment of muscular atrophy of the present invention, the dosage of the strains as an active ingredient is various types of diseases, the age, weight, sex, medical condition of the patient, severity of the condition, sensitivity to drugs, administration time , route of administration and excretion rate, duration of treatment, factors including concomitant drugs, and other factors well known in the medical field. Therefore, although the dosage regimen can vary widely, it is important to administer the amount that can obtain the maximum effect with the minimum amount without side effects, taking all of the above factors into account, which can be readily determined by one of ordinary skill in the art using standard methods. .

In general, for an adult patient, 1Х10 ⁸ or more live cells or pasteurized bacteria, preferably 1Х10 ⁸ to 1Х10 ¹² live cells or pasteurized bacteria can be administered once or in divided doses as needed. In one embodiment, the SMA pharmaceutical composition for preventing or treating only the marker cyano mu shinny pillar EB-AMDK19 long as it comprises a strain does not specially limit the content of, for example, only the marker cyano The Muciniphila EB-AMDK19 strain may ^{be included at a concentration of 1×10 8} cells/ml to 1×10 ¹⁰ cells/ml, but is not necessarily limited thereto. For example, only the marker cyano mu shinny concentration of pillars EB-AMDK19 strain is 1 × 10 ⁸ cells / ㎖ to 1 × ¹⁰ 10 cells / ㎖, 2 × 10 ⁸ cells / ㎖ to 1 × ¹⁰ 10 cells / ㎖, 3×10 ⁸ cells/mL to 1×10 ¹⁰ cells/mL, 5×10 ⁸ cells/mL to 1×10 ¹⁰ cells/mL, 1×10 ⁸ cells/mL to 5×10 ⁹ cells/mL, 2× 10 ⁸ cells/ml to 5×10 ⁹ cells/ml, 3×10 ⁸ cells/ml to 5×10 ⁹ cells/ml, 5×10 ⁸ cells/ml to 5×10 ⁹ cells/ml, but must be It is not limited to these.

Another aspect of the present invention relates to a food or health functional food comprising the Ackermansia muciniphila EB-AMDK19 strain, a culture or a dried product thereof. In another aspect, the composition of the present invention may be prepared as a health functional formula for preventing or improving muscular atrophy. The health functional food for preventing or improving muscular atrophy of the present invention can be used for preventing or improving muscle diseases caused by muscle wasting or degeneration.

The functional health food of the present invention includes all forms such as functional food, nutritional supplement, health food, food additives and feed. The above type of health functional food can be prepared in various forms according to conventional methods known in the art.

The above type of health functional food can be prepared in various forms according to conventional methods known in the art. Common foods include, but are not limited to, beverages (including alcoholic beverages), fruits and their processed foods (eg, canned fruit, canned fruit, jam, marmalade, etc.), fish, meat and their processed foods (eg, ham, sausages) corn beef, etc.), breads and noodles (eg udon noodles, soba noodles, ramen, spagate, macaroni, etc.), fruit juice, various drinks, cookies, syrup, dairy products (eg butter, cheese, etc.), edible vegetable oils and fats, margarine It can be produced by adding a marker, only a cyano mu shinny pillar EB-AMDK19 strain or the like: (miso, soy sauce, sauce, etc.), plant proteins, retort foods, frozen foods, various seasonings.

In addition to the above, the health functional food of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, colorants, pectic acid, pectic acid salts, alginic acid, alginic acid salts, organic acids, protective colloidal thickeners, pH regulators, stabilizers, preservatives , glycerin, alcohol, or a carbonation agent.

Another aspect of the invention relates to a method of treating muscular atrophy in a subject. In the method of the present invention only the markers described herein, a therapeutically effective amount to a subject comprises the step of administering a cyano mu shinny pillar EB-AMDK19 strain.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following examples.

Example

Example 1: Isolation and identification of Ackermansia muciniphila strains

1.1. Isolation and identification of strains

To healthy Korean remove the cyano mu shinny pillars only markers from the feces of the (female, age 35, BMI 23.3), mucin medium according to the process of _{Derrien (0.4 g KH 2 PO 4} ; 0.53 g Na 2 HPO 4; 0.3 g NaCl 0.1 g MgCl ₂ 6 (H ₂ O); 0.11 g CaCl ₂ 0.4 g/L, 1 ml acid trace element solution, 1 ml alkaline trace element solution, 1 ml vitamin solution, 2.5 g/L pig stomach mucus (type III )), and 0.25 g/L sodium sulfide nonhydrate), and the strain was isolated after selective culture (Derrien et al., 2004).

In order to confirm that the isolated strain is an Acermansia muciniphila strain, the isolated strain was observed under a microscope and the results are shown in FIG. 1, and AM-specific primers (SEQ ID NO: 2 and SEQ ID NO: 3 in Table 1 below) ) was subjected to PCR analysis, and the results are shown in FIG. 2 .

In Figure 1 A is Akkermansia muciniphila ATCC BAA-835 strain, B is Acermansia muciniphila It is a micrograph of the EB-AMDK19 strain at a magnification of 1000 times. In FIG. 2, lane M is a DNA size marker, lane 1 is a positive control (ATCC BAA-835), lane 2 is Akkermansia muciniphila EB-AMDK19 strain, and lane 3 is a result of a negative control (distilled water). As a result, as shown in Figure 2, the strain of the present invention is a positive control strain Acermansia muciniphila It was confirmed that the result was obtained with a band similar to that of the ATCC BAA-835 strain.

Designation Direction Sequence (5'→3') Amplicon size SEQ ID NO: AM1 Forward CAG CAC GTG AAG GTG GGG AC 327 bp SEQ ID NO: 2 AM2 Reverse CCT TGC GGT TGG CTT CAG AT SEQ ID NO: 3

1.2. Confirmation of sugar availability of isolated Akermansia muciniphila strains

In order to determine the sugar availability of the Ackermansia muciniphila EB-AMDK19 strain of the present invention isolated above, the API50CH kit (Biomerieux, France) was applied and cultured, followed by growth using each sugar as a standard strain (ATCC BAA- 835), and the results are shown in Table 2 below.

No Carbohydrates ATCC BAA-835 EB-AMDK19 No Carbohydrates ATCC BAA-835 EB-AMDK19 0 negative control - - 25 Esculine - - One Glycerol - - 26 Salicine - - 2 Erythritol - - 27 D-Cellobiose - - 3 D-Arabinose w w 28 D-Maltose - - 4 L-Arabinose w w 29 D-Lactose (bovine origin) + + 5 Ribose + w 30 D-Melibiose - - 6 D-Xylose w w 31 D-Saccharose (sucrose) - - 7 L-Xylose w w 32 D-Trehalose - - 8 adonitol - - 33 Inuline - - 9 β-Methyl-xyloside - - 34 D-Melezitose - - 10 D-Galactose w + 35 D-Raffinose - - 11 D-Glucose + Mucin + + 36 Amidon (starch) - - 12 D-Fructose w - 37 Glycogene - - 13 D-Mannose + w 38 Xylitol - - 14 L-Sorbose - - 39 Gentiobiose - - 15 L-Rhamnose - - 40 D-Turanose - - 16 Dulcitol - - 41 D-Lyxose w w 17 Inositol - - 42 D-Tagatose - - 18 D-Mannitol - - 43 D-Fucose - - 19 D-Sorbitol - - 44 L-Fucose + + 20 Methyl-α D-mannopyranoside - - 45 D-Arabitol - - 21 Methyl-α D-glucopyranoside - - 46 L-Arabitol - - 22 N-Acetylglucosamine + + 47 Potassium Gluconate - - 23 Amygdaline - - 48 Potassium 2-Ketogluconate - - 24 Arbutine - - 49 Potassium 5-Ketogluconate w w Ketogluconate + : growth, w : weak growth, - : no growth,

As can be seen in Table 2, only the markers of the present invention cyano mu shinny pillar EB-AMDK19 strain is compared to the mu cyano shinny pillar ATCC BAA-835 strain only markers of type strain, ribose, D- galactose, D- It was confirmed that there is a difference in the ability to use fructose and D-mannose.

1.3. Jeon body genome sequencing (Whole genome sequencing )

Discrete markers as described above, only a cyano mu shinny only pillars EB-AMDK19 strains with marker cyano mu shinny pillar ATCC and BAA-835 using a PacBio techniques variations between strains to analyze the dielectric level and analyze the entire nucleotide sequence of the genome Compared with the Akkermansia muciniphila ATCC BAA-835 standard strain, the results are shown in Tables 3 and 4 below.

genomic statistics strain ATCC BAA-835 EB-AMDK19 Accseeion no. CP001071 CP025834 Assembly level Complete Complete Seq. category Chromosome Chromosome Total size (Mb) 2.6641 2.7242 GC (%) 55.8 55.3 Protein 2,246 2,140 Gene 2,321 2,357 CDS 2,257 2,293 Coding 2,246 2,250 rRNA 9 9 tRNA 52 52 Other RNA 3 3 Pseudogene 11 44 Symmetrical identity (%) - 90.1368 *Symmetrical identity is similar to ATCC BAA-835

gene strain ATCC BAA-835 EB-AMDK19 Amuc_1098 sequence length 901 900 Align identity (%) 100 99.3 Mismatch no. 0 5 Insertion no. 0 0 Deletion no. 0 One Amuc_1099 sequence length 337 337 Align identity (%) 100 100 Mismatch no. 0 0 Insertion no. 0 0 Deletion no. 0 0 Amuc_1100 sequence length 316 316 Align identity (%) 100 99.4 Mismatch no. 0 2 Insertion no. 0 0 Deletion no. 0 0 Amuc_1101 sequence length 612 612 Align identity (%) 100 99.2 Mismatch no. 0 5 Insertion no. 0 0 Deletion no. 0 0 Amuc_1102 sequence length 237 237 Align identity (%) 100 98.7 Mismatch no. 0 3 Insertion no. 0 0 Deletion no. 0 0 Gene_cluster sequence length 2403 2402 Align identity (%) 100 99.3 Mismatch no. 0 15 Insertion no. 0 0 Deletion no. 0 One *Length of 5 genes for Gene_cluster

As can be seen from Table 3 and Table 4, only the markers of the present invention cyano mu look shinny pillar EB-AMDK19 strains with marker only compare the cyano mu shinny pillar ATCC entire genome statistics of BAA-835 type strain is significantly different there was.

1.4. Random Amplified Polymorphic DNA (RAPD) Analysis

Only discrete markers as described above, to verify whether a cyano mu shinny pillar EB-AMDK19 not strain the only already markers of the reported homologous equal to a cyano mu shinny pillar ATCC BAA-835 strains, subjected to a type of RAPD of molecular typing did. To this end, the genomic DNA extracted from the cells was amplified using the universal primers in Table 5 below, and then electrophoresed on a 1% agarose gel for 1 hour and 30 minutes, and the DNA segmentation pattern was compared on a UV perforator. The results are shown in FIG. 3 .

Designation direction sequence (5'→3') SEQ ID NO: ERIC-1 Forward ATG TAA GCT CCT GGG GAT TCA C SEQ ID NO: 4 ERIC-2 Reverse AAG TAA GTG ACT GGG GTG AGC G SEQ ID NO: 5 (GTG) ₅ Forward/Reverse GTG GTG GTG GTG GTG SEQ ID NO: 6

As will be confirmed by the Figure 3, only the markers of the present invention cyano mu shinny pillar EB-AMDK19 strain showed, different RAPD band pattern compared with when only a cyano mu shinny pillar ATCC BAA-835 type strain marker. Therefore, the Acermansia muciniphila EB-AMDK19 strain of the present invention belongs to the same species as the Acermansia muciniphila ATCC BAA-835 standard strain, but it was confirmed that it is a different strain.

1.5. Phylogenetic tree analysis using full-length 16S rRNA gene sequencing

Discrete markers as described above, only a cyano mu shinny pillar EB-AMDK19 to the full-length (full-length) 16S rRNA gene nucleotide sequence analysis of the strain, and then to use the 27F and 1541R primers shown in Table 6 amplify the 16S rRNA gene 3730xl The nucleotide sequence was determined using a DNA analyzer. Thus obtained only marker cyano mu shinny pillar EB-AMDK19 and already using the 16S rRNA gene sequences of other strains of the published homologous shown in Figure 4 to create the phylogenetic tree (phylogenetic tree).

Designation Direction Sequence (5'→3') Amplicon size SEQ ID NO: 27F Forward AGA GTT TGA TCM TGG CTC AG 1,505 bp SEQ ID NO: 7 1541R Reverse AAG GAG GTG ATC CAG CCG CA SEQ ID NO: 8

A, 16s rRNA gene sequence analysis of phylogenetic trees for evolutionary flexibility relationship through the analysis via the (phylogenetic tree), only the markers of the present invention cyano mu shinny pillar EB-AMDK19 strain as shown in Fig. 4 is a genetic marker it was confirmed that Mansiysk Ah Mu shinny Pilar (A. muciniphila) strains belonging to the species. Only markers isolated from feces of man cyano mu shinny Pillar the EB-AMDK19 strains only marker cyano mu shinny pillar ^T (ATCC BAA-835) a biochemical method for the control group (API), and molecular biological methods (16s rRNA Sequence Analysis , RAPD, and full-length screening), and confirmed that it is a safe strain capable of having the function of probiotics through an antibiotic resistance test to be described later. These results only markers separated on the basis of Asian MU naming shinny pillar strains as "markers only Asian Mu shinny Pilar EB-AMDK19 'strains and the accession to the Korea Research Institute of Bioscience and Biotechnology, Microbial Resources Center (KCTC) given an accession number KCTC13761BP received.

Example 2: Safety test of Ackermansia muciniphila EB-AMDK19 strain

2.1. antibiotic resistance

Piperacillin-tazobactam (PTZ), an antibacterial agent for anaerobic bacteria, according to the broth microdilution method of the Clinical & Laboratory Standard Institute (CLSI) guidelines in order to determine the antimicrobial susceptibility of the Ackermansia muciniphila EB-AMDK19 strain isolated as described above. ), ceftidoxime (CTZ), chloramphenicol (CHL), clindamycin (CLI), meropenem (MEM), moxifloxacin (MXF), metronidazole (MTZ), ciprofloxacin (CIP)) concentration, MIC) was determined (CLSI, 2017), and the results are shown in Table 7 below.

Antibiotic MIC ^a Breakpoints (μg/ml) QC test strain S I R ATCC 29741 ^b ATCC BAA-835 EB-AMDK19 PTZ ≤32/4 64/4 ≥128/4 8/4 ≤0.5/4 (S) ≤0.5/4 (S) CTZ ≤32 64 ≥128 16 2 (S) 1 (S) CHL ≤8 16 ≥32 8 4 (S) 4 (S) CLI ≤2 4 ≥8 4 ≤0.125(S) 4 (I) MEM ≤4 8 ≥16 0.5 1 (S) 0.5 (S) MXF ≤2 4 ≥8 8 >32 (R) >32 (R) MTZ ≤8 16 ≥32 2 ≤0.25 (S) 0.5 (S) CIP ≤1 2 ≥4 >32 >32 (R) 32 (R) PTZ: Piperacillin-tazobactam, CTZ: ceftizoxime (3 rd gen), CHL: chloramphenicol,
CLI: clindamycin, MEM: meropenem, MXF: moxifloxacin (4 ^th gen),
MTZ: metronidazole, CIP: ciprofloxacin ( 2 nd gen),
^a MIC : minimal inhibitory concentration, ^b Bacteroides thetiotaomicron ATCC 29741

As can be seen in Table 7, only the markers of the present invention cyano mu shinny pillar EB-AMDK19 strain exhibited moderate resistance to clindamycin, an antibiotic of the quinolone family fluoro visual proxy reaper and ciprofloxacin has showed the resistance, It showed sensitivity to all antibacterial agents except for this. Compared with the standard strain Akermansia muciniphila ATCC BAA-835, there was a slight difference in the antimicrobial resistance pattern. It can be confirmed that the Ackermansia muciniphila EB-AMDK19 strain according to the present invention is a safe strain without resistance to most antibiotics.

2.2. Confirmation of hemolytic activity

To verify the safety of the Ackermansia muciniphila EB-AMDK19 strain isolated as described above, it was evaluated whether hemolytic activity was retained. For this purpose tryptic soy agar (17.0 g/L casein pancreatic digest, 3.0 g/L soybean pancreatic digest, 2.5 g/L dextrose, 5.0 g/L sodium chloride, 2.5 g/L potassium phosphate, 15 g/L L agar) was cultured using a blood agar medium prepared by adding 5% w/v defibrinated sheep blood, and the results are shown in FIG. 5 .

As can be seen from FIG. 5 , it was confirmed that the Ackermansia muciniphila EB-AMDK19 strain of the present invention did not cause β-hemolysis related to pathogenicity because a completely transparent part around the colony did not appear.

Example 3: Confirmation of the treatment efficacy of Akkermansia muciniphila strains for muscular atrophy

3.1. strain sample

Markers used in this experiment only a cyano mu shinny pillar ATCC BAA-835 strain (control) and markers only cyano mu shinny pillar EB-AMDK19 (KCTC13761BP) live cells was ^{1x10 8 CFU / 150μl PBS (25} % glycerol, 0.05% cysteine / PBS) concentration was prepared.

3.2. animal testing

Animal experiments were conducted in accordance with the Animal use and Care Protocol of the Institutional Animal Care and Use Committee (IACUC). To induce muscular atrophy, 6-week-old male C57BL/6 mice were purchased, and after an adaptation period for a week, breeding was carried out for 5 weeks. The breeding environment maintained a constant temperature (22℃) and relative humidity (40-60%), and was reared while controlling the light and shade every 12 hours.

3.3. Sample administration and experimental group setting

Each of the drugs shown in Table 8 below was orally administered to mice every day for 5 weeks. As a positive control group, Rivamax suspension (Talcon RF Pharmaceuticals), a branched chain amino acid (BCAA; Leucine, Isoleucine, Valine) formulation, was orally administered at 600 mg/kg each.

For each group of mice, a plaster cast was applied to the hind legs of the mice after 4 weeks of oral administration of the drug. At this time, the cast was wrapped from the leg to the foot with the ankle folded as shown in FIG. 6 , and the cast was maintained for one week.

group experimental group drug administered I normal control PBS administration II Muscular atrophy induction group (plaster cast) PBS administration III Positive control group (Livamax administered group) 600 mg/kg, 150 μl (BCAA formulation) IV ATCC BAA-835 standard strain administration group BAA-835 live cells, 1x10 ⁸ CFU V EB-AMDK19 strain administered group EB-AMDK19 live cells, 1x10 ⁸ CFU

Example 3.4: Effect of Akkermansia muciniphila EB-AMDK19 strain on body weight of animals induced by muscular atrophy

When the wake up muscular atrophy was to verify for only the effect of the cyano mu shinny pillar AMDK19 EB-strain marker of the present invention. While breeding each mouse in the 5 groups of Table 8 for 5 weeks, the change in body weight was measured, and it is shown as a graph in FIG. 7 . Body weight was measured once a week during the 5-week experimental period to compare the differences between each group.

Referring to FIG. 7 , from the time point 11 days elapsed, compared to mice treated with Rivamax alone, the weight of mice treated with Rivamax and the Acermansia muciniphila EB-AMDK19 strain of the present invention increased. confirmed

After 4 weeks of administration of the strain, it was observed that the weight was reduced by limiting the movement of the hind legs with a plaster cast. The experimental group to which the strain was administered also showed a difference of about 2 g from the control group due to a decrease in body weight. Plaster in the positive control group (Riva Max) or a test group, only one group administered a cyano mu shinny pillar EB-AMDK19 markers of the present invention as compared to the parking right after the 5 weight results given by loosening the cast was decreased the least weight. Therefore, only the markers of the present invention cyano mu shinny pillar EB-AMDK19 strain was found to represent the effect of improving the symptoms in the muscular dystrophy-induced mouse.

Example 4: Effect of the Ackermansia muciniphila EB-AMDK19 strain of the present invention on the grip test of an animal induced by muscular atrophy

Normal control group, mice in which muscular atrophy was induced by treatment with Rivamax (positive control group), control group treated with the standard strain Akermansia muciniphila BAA-836 strain after Rivamax treatment, Akkermansia muciniphila EB of the present invention - By measuring the grip strength of the mice of the group treated with the AMDK19 strain, it was attempted to confirm whether the muscle function was restored.

After releasing the plaster cast maintained for a week, the grip strength of the hind legs was measured. To measure the grip strength, a Grip Strength Meter JD-A-22 from Jeongdo BNP Co., Ltd. was used. At this time, as shown in FIG. 8 , the mouse's hind foot was placed on the wire mesh to which the instrument panel capable of monitoring the strength of the force was attached, and the force (N) for the mouse to hold the wire mesh was measured while grabbing the tail and pulling it backward. The test was measured three times consecutively, and the maximum value was determined as the grip force. To standardize the experimental results, the grip force (N) was calculated by dividing the weight (mg).

Figure 9 is a normal control group (Normal), muscular atrophy induced group (Imm), positive control (LVM), type strain treated group (BAA-835) and the grip strength measured in only markers of the present invention cyano mu shinny pillar EB-AMDK19 strain group It is a graph showing the comparison result. 9, the mice (Imm) with muscular atrophy induced the most, while showing a low level of grip, only the markers of the present invention cyano mu shinny pillar EB-AMDK19 strains treated in a significantly higher level than mice muscular atrophy induced grip to check the indicated, only the markers of the present invention was confirmed cyano mu shinny pillar EB-AMDK19 Sikkim strain recover the grip weakened by muscular dystrophy. Only markers of the present invention cyano mu grip strength improving effect of shinny pillar EB-treated AMDK19 strain appeared to be better than the positive control group and the type strain.

Example 5: Effect of the Akermansia muciniphila EB-AMDK19 strain of the present invention on the muscle weight of an animal induced by muscular atrophy

The next day of grip strength measurement, mice were anesthetized with _{CO 2 and sacrificed for the end of the experiment.} After blood collection, in order to check the change in muscle weight, the leg muscles, tibialis anterior (TA), gastrocnemius (GA, gastrocnemius), and liver tissue were excised and the weights were measured, and the weights are shown in FIG. 10 . In addition, in order to compare the changes in other organs according to the body weight, the weight of the liver tissue was also checked. Colonic tissues were also extracted and stored for analysis.

As can be seen in FIG. 10 , the muscle mass of the mice induced by muscular atrophy was decreased, but in the group administered with the Ackermansia muciniphila EB-AMDK19 strain of the present invention, it was confirmed that the muscle weight was increased to a level similar to that of the positive control group.

Example 6: Measurement of Myostatin Concentration in Serum

The blood collected from the mouse was centrifuged at 10,000 rpm for 5 minutes to separate serum, and the concentration of Myostatin in the serum was measured, which is shown as a graph in FIG. 12 . In this case, the Myostatin concentration was measured using an ELISA kit (GDF-8/Myostatin Quantikine ELISA Kit, DGDF80, R&D systems, MN, USA).

Referring to FIG. 11 , myostatin is a regulatory protein that restricts muscle development and growth, and the myostatin concentration in the serum increased in the group in which exercise was restricted with a plaster cast. Positive control group Max Leva, including a marker, only a cyano mu shinny pillar BAA-835 type strain and the marker only cyano mu shinny pillar is Myostatin serum concentration was decreased in both the EB-AMDK19. The EB-AMDK19 administration group had a lower serum myostatin concentration than the standard strain BAA-835 administration group. This demonstrates that the effect to accelerate the development and growth of the muscles in a condition the only marker cyano mu shinny pillar EB-AMDK19 of the present invention limits the movement is superior compared to the type strain (BAA-835).

Example 7: Analysis of indicators related to muscular atrophy in muscles

To check gene expression in muscle tissue and colon tissue, total RNA was extracted using TRI reagent (Sigma, USA). 1 μg of RNA was quantified and cDNA was synthesized using the M-MLV cDNA synthesis kit (Enzynomics, Korea). Real-time PCR was performed using the synthesized cDNA and SYBR Green TOPreal ^TM qPCR 2X PreMIX (Enzynomics, Korea) to check the gene expression in the tissue. This was performed using the Quant Studio 3 real time PCR system (Applied Biosystems, USA). In muscle tissue, the expression of Atrogin-1 and MuRF, which are indicators of muscular atrophy, and CathepsinL, which is an autophage indicator, were confirmed.

It is known that when muscular atrophy occurs, the expression of genes that destroy muscle proteins (Myostatin, Atrogin-1, and MuRF1) increases, and the expression of genes that produce muscle proteins (MyoD and Myogenin) decrease. Cathepsins have also been implicated in muscular dystrophy.

Figure 12 is a gene destroys muscle protein in the muscles of the control group, muscular atrophy induced group, a positive control, a standard strain group (BAA-835) and a marker, only a cyano mu shinny pillar EB-AMDK19 strain group of the present invention (Atrogin-1) It is a graph showing the result of comparing the expression level of Referring to FIG. 12 , it was found that the expression of Atrogin-1 was increased in both the tibialis anterior muscle and gastrocnemius muscle in the muscle tissue of the muscle atrophy-induced. On the other hand, only the marker, including the positive control group, Max Leva the cyano mu shinny pillar type strain BAA-835 treated groups and markers only treated group, a cyano mu shinny pillar EB-AMDK19 decreased the amount of expression of Atrogin-1. In addition, the expression of CathepsinL in the tibialis anterior muscle was increased in the muscular atrophy induction group, but the expression of CathepsinL in the tibialis anterior muscle was decreased by administration of Rivamax or EB-AMDK19 strain. In particular, in the group administered with the strain of the present invention, Atrogin-1, a gene that destroys muscle protein, was expressed at a much lower level than in the group administered with the standard strain. This haejueo prevent only markers of the present invention cyano mu shinny pillar EB-AMDK19 isolates that muscle caused by the movement limiting decomposition indicates that the effect on muscular atrophy.

Example 8: Analysis of indicators related to energy metabolism in muscle

The mitochondria in the muscle oxidize energy by producing ATP, and PGC-1α acts as an important regulator of mitochondrial biosynthesis. In general, PGC-1α is known to be activated in muscles due to exercise. Therefore, it was attempted to confirm the effect of the strain in a state where exercise was restricted.

In a method similar to Example 7, the expression of PGC-1α related to energy metabolism was measured in muscle tissue and shown in FIG. 14 .

14, the tibialis anterior (TA) in the positive control of only the marker, including the Riva Max group cyano mu shinny pillar BAA-835 type strain treated with markers only cyano mu shinny pillar EB-AMDK19 group PGC-1α expression in both It can be seen that the amount increases. In gastrocnemius (GA), only the Acermansia muciniphila EB-AMDK19 group showed a significant increase. This is presumed to prevent muscular atrophy by inducing mitochondrial biosynthesis and activating energy metabolism by increasing the expression level of PGC-1α in the EB-AMDK19 strain even when exercise is restricted.

Example 9: Confirmation of expression of SCFA receptor in the colon

Acermansia muciniphila is known to secrete SCFA (Short Chain Fatty Acid). G protein-coupled receptors GPR43 and GPR119 act as receptors for these SCFAs to activate GLP-1 (glucagon-like peptide 1). The active GLP-1 was to determine how to enable the energy source to glucose increase insulin sensitivity in muscle, only a cyano marker Mu was shinny pillar promotes these paths.

Colon of mice whose muscular atrophy was improved by normal control group, muscular atrophy induction group, positive control group (LVM), Akkermansia muciniphila AT CC BAA-835 strain administration group, and Akkermansia muciniphila EB-AMDK19 strain administration group of the present invention In the tissue, the expression levels of GPR43 and GPR119, which are SCFA reporters, were measured, and the comparison results are shown as a graph in FIG. 15 .

Referring to Figure 15, the expression of GPR43 was not statistically significant, but only the positive control (LVM) and markers increases due to a cyano mu shinny pillar strains administration. However, only the markers of the present invention in a GPR119 expression showed a shinny cyano mu pillar EB-AMDK19 strain group was significantly high expression. This marker only presumably cyano mu shinny pillar EB-AMDK19 strain of the present invention in helping the activity of GLP-1.

Example 10: Analysis of anti-inflammatory and pro-inflammatory indicators in the colon

The expression of IL-10, an anti-inflammatory cytokine, IL-6, a pro-inflammatory cytokine, and ZO-1 related to tight junctions were confirmed in colon tissue.

16 is a normal control group, muscular atrophy induction group, positive control group (LVM), Akkermansia muciniphila AT CC BAA-835 strain administration group, and Akermansia muciniphila EB-AMDK19 strain administration group of the present invention. It is a graph showing the result of comparing the expression level of the anti-inflammatory cytokine IL-10 in the mouse, Figure 17 is a graph showing the result of comparing the expression level of the pro-inflammatory cytokine IL-6.

16 and 17 , the expression of IL-6 as an anti-inflammation index and IL-10 as a pro-inflammation index in the colon was confirmed. As the muscular atrophy induced group limit the movement, while the expression of IL-6 increased, positive control only markers including Riva Max group cyano mu shinny pillar BAA-835 type strain treated with markers only cyano mu shinny pillar EB-AMDK19 group A decrease in expression was observed in all. On the other hand IL-10 expression level compared to experiment, muscular atrophy induced in group and determined that to decrease the amount of IL-10 expression, as limiting the movements, markers only cyano mu shinny pillars only on par with the control group treated group, the EB-AMDK19 increased again. It was confirmed that Akermansia muciniphila EB-AMDK19 was effective in alleviating the inflammatory response induced by exercise restriction. There was no significant change in IL-10 expression level in the Rivamax-administered group and the Akkermansia muciniphila BAA-835 standard strain group.

Example 11: Analysis of indicators related to close junctions of colonic epithelial cells

Colon in mice whose muscular atrophy was improved by normal control group, muscular atrophy induction group, positive control group (LVM), Akkermansia muciniphila AT CC BAA-835 strain administration group and Akkermansia muciniphila EB-AMDK19 strain administration group A change in the expression of ZO-1, a tight junction protein, was measured in the mucosa, and is shown as a graph in FIG. 18 .

The spaces between epithelial or endothelial cells must be tightly sealed so that molecules transported from one layer to another cannot diffuse back through these spaces. Tight junctions create barriers in epithelial and endothelial cells, which regulate the movement of water and solutes through the paracellular space. Tight junctions also function to maintain cell polarity by forming a fence to prevent mixing of molecules on the apical membrane with molecules on the lateral membrane.

Epithelial cells present in the mucosal layer of the colon form a barrier by tight junction, and an important protein is ZO-1. Referring to FIG. 19 , in the muscular atrophy induction group, the expression of ZO-1 was reduced as exercise was restricted, and the positive control group administered with Rivamax, the group administered with the Akermansia muciniphila BAA-835 standard strain, and the group administered with the Akkermansia muciniphila It was confirmed that the expression of ZO-1 was increased again in all of the EB-AMDK19 strains. In particular, it was confirmed that when the strain of the present invention was administered, the expression of ZO-1 was higher than that of the standard strain administered group. Therefore, it can be inferred that the Ackermansia muciniphila EB-AMDK19 strain of the present invention prevents toxic substances from penetrating into intestinal cells by strongly improving the barrier.

According to one embodiment of the present invention, there is provided an animal model each process a cyano mu shinny pillar EB-AMDK19 strains only muscular dystrophy-induced mice and the the vehicle (PBS) in the induced mouse disease or markers by Riva max, and this If the use of the marker, only a cyano mu shinny pillar EB-AMDK19 strain the result of verifying the effect of the mouse models, only markers cyano mu shinny pillar handle EB-AMDK19 strains in the mouse, the weight loss is restored, reduced Genes that destroy muscle proteins increased by Rivamax (Myostatin, Atrogin-1, and MuRF1) are reduced in expression, while reduced grip strength is restored, and energy metabolism in the muscle is activated. It was confirmed that the expression level of PGC-1α increased. When the synthesis result of this, only the markers of the present invention confirmed that a cyano mu shinny pillar EB-AMDK19 strain may be used in the prevention and treatment of muscular dystrophy.

The specific examples described herein are merely illustrative of preferred embodiments of the present invention and should not be construed as limiting the present invention. The present invention can be practiced with various modifications and variations without departing from the spirit and scope of the present invention, and it will be apparent to those skilled in the art. The protection scope of the present invention should be defined by the appended claims, and various modifications and changes described above are intended to be included in the protection scope of the present invention.

Korea Institute of Bioscience and Biotechnology KCTC13761BP 20181205

<110> Enterobiome Co., Ltd. <120> PHARMACEUTICAL COMPOSITION FOR PREVENTING OR TREATING MUSCULAR ATROPHY DISEASE COMPRISING AKKERMANSIA MUCINIPHILA STRAIN <130> P20-ETB-06 <160> 8 <170> KoPatentIn 3.0 <210> 1 <211> 1464 <212> DNA <213> Unknown <220> <223> Akkermansia muciniphila <400> 1 aacgaacgct ggcggcgtgg ataagacatg caagtcgaac gagagaattg ctagcttgct 60 aataattctc tagtggcgca cgggtgagta acacgtgagt aacctgcccc cgagagcggg 120 atagccctgg gaaactggga ttaataccgc atagtatcga aagattaaag cagcaatgcg 180 cttggggatg ggctcgcggc ctattagtta gttggtgagg taacggctca ccaaggcgat 240 gacgggtagc cggtctgaga ggatgtccgg ccacactgga actgagacac ggtccagaca 300 cctacgggtg gcagcagtcg agaatcattc acaatggggg aaaccctgat ggtgcgacgc 360 cgcgtggggg aatgaaggtc ttcggattgt aaacccctgt catgtgggag caaattaaaa 420 agatagtacc acaagaggaa gagacggcta actctgtgcc agcagccgcg gtaatacaga 480 ggtctcaagc gttgttcgga atcactgggc gtaaagcgtg cgtaggctgt ttcgtaagtc 540 gtgtgtgaaa ggcgcgggct caacccgcgg acggcacatg atactgcgag actagagtaa 600 tggaggggga accggaattc tcggtgtagc agtgaaatgc gtagatatcg agaggaacac 660 tcgtggcgaa ggcgggttcc tggacattaa ctgacgctga ggcacgaagg ccaggggagc 720 gaaagggatt agatacccct gtagtcctgg cagtaaacgg tgcacgcttg gtgtgcgggg 780 aatcgacccc ctgcgtgccg gagctaacgc gttaagcgtg ccgcctgggg agtacggtcg 840 caagattaaa actcaaagaa attgacgggg acccgcacaa gcggtggagt atgtggctta 900 attcgatgca acgcgaagaa ccttacctgg gcttgacatg taatgaacaa catgtgaaag 960 catgcgactc ttcggaggcg ttacacaggt gctgcatggc cgtcgtcagc tcgtgtcgtg 1020 agatgtttgg ttaagtccag caacgagcgc aacccctgtt gccagttacc agcacgtgaa 1080 ggtggggact ctggcgagac tgcccagatc aactgggagg aaggtgggga cgacgtcagg 1140 tcagtatggc ccttatgccc agggctgcac acgtactaca atgcccagta cagagggggc 1200 cgaagccgcg aggcggagga aatcctgaaa actgggccca gttcggactg taggctgcaa 1260 cccgcctaca cgaagccgga atcgctagta atggcgcatc agctacggcg ccgtgaatac 1320 gttccccgggt cttgtacaca ccgcccgtca catcatggaa gccggtcgca cccgaagtat 1380 ctgaagccaa ccgcaaggag gcagggtcct aaggtgagac tggtaactgg gatgaagtcg 1440 taacaaggta gccgtagggg aacc 1464 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> AM1 primer forward <400> 2 cagcacgtga aggtggggac 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> AM2 primer revere <400> 3 ccttgcggtt ggcttcagat 20 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> ERIC-1 primer Forward <400> 4 atgtaagctc ctggggattc ac 22 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> ERIC-2 primer Reverse <400> 5 aagtaagtga ctggggtgag cg 22 <210> 6 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> (GTG)5 Forward/Reverse <400> 6 gtggtggtgg tggtg 15 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 27F primer Forward <400> 7 agagtttgat cmtggctcag 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 1541R primer Reverse <400> 8 aaggaggtga tccagccgca 20

Claims (7)

Only markers cyano mu shinny pillar EB-AMDK19 strain (Akkermansia muciniphila) (KCTC13761BP) preventing or treating a muscular dystrophy pharmaceutical composition comprising as an active ingredient.
The method of claim 1, wherein only the marker cyano mu shinny pillar EB-AMDK19 strain pharmaceutical for muscular dystrophy the prevention and treatment, comprising a step of reducing the Antrogin-1, MuRF1 (Muscle Ring -Finger Protein), or the expression of myostatin composition.
The method of claim 1, wherein the pharmaceutical composition comprises only the marker cyano mu shinny pillar EB-AMDK19 muscular atrophy prevention or treatment a pharmaceutical composition comprising a probiotic strain or strains include pasteurized.
According to claim 1, wherein the strain is a strain cell, a lysate of the cell, the culture of the strain, the culture solution from which the cell is removed from the culture of the strain, the cell extract of the strain, the extract of the culture of the strain or the cell from the culture of the strain A pharmaceutical composition for the prevention or treatment of muscular atrophy comprising one selected from the extracts of the removed culture solution.
The method according to claim 1, wherein the pharmaceutical composition for preventing or treating muscular atrophy contains, as an active ingredient, the Akkermansia muciniphila EB-AMDK19 strain in an amount of 10 ⁸ to 10 ¹² CFU, or an equivalent number based on the total weight of the composition. A pharmaceutical composition for preventing or treating muscular atrophy, comprising a culture with live cells or pasteurized of
According to claim 1, wherein the muscular atrophy is muscular atrophy, muscular dystrophy, dystonia, muscular dystrophy, muscular dystrophy, and myasthenia gravis, characterized in that any one of muscular dystrophy prevention or treatment pharmaceutical composition.
Acker million mu Asian shinny pillar EB-AMDK19 strains (Akkermansia muciniphila EB-AMDK19) muscular dystrophy prevention or health supplements for improved including the (KCTC13761BP) as an active ingredient.

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