Novel strain having endurance-enhancing activity

Abstract

The present invention relates to a novel strain of Allobaculums (KCCM13405P) or Bifidobacterium animalis (KCCM13404P) having anti-fatigue or endurance-enhancing activity, wherein the strain has the benefits of increasing endurance through an increase in exercise ability, increasing type 1 myofibers, biogenisis of mitochondria, and muscle development through biogenisis of muscle, and can be used for anti-fatigue, endurance enhancement, and exercise performance enhancement, and can also be used for preventing or treating muscle diseases.

Description

A new strain with endurance-enhancing activity

[0001] This application claims the benefit of Korean Patent Application No. 10-2023-0161520, filed on November 20, 2023, and Korean Patent Application No. 10-2024-0020587, filed on February 13, 2024, the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to a new strain having endurance-enhancing activity, a composition for anti-fatigue or endurance-enhancing comprising the same, and a composition for preventing or treating muscle diseases.

[0003] Muscles are broadly divided into skeletal, cardiac, and visceral muscles. Skeletal muscle accounts for a significant portion of the human body, accounting for 40% of body weight. Together with bones, it supports the body's shape and enables movement. These bones and skeletal muscles are collectively referred to as the musculoskeletal system. Muscle cells are composed of myoblasts. When muscle tissue is injured or torn due to excessive exercise, satellite cells, the stem cells of muscle tissue, are activated, promoting their division. Some of these satellite cells retain their stem cell function, while others begin to differentiate into muscle cells. At this time, various transcription factors involved in muscle cell differentiation are activated, and satellite cells differentiate into myoblasts, myocytes, and myotubes, in that order, replenishing injured and damaged muscle tissue with new muscle fibers. Muscles are largely composed of two types of muscle fibers. Type 1 muscle fibers, of which type 1 muscle fibers contain many mitochondria and are essential for endurance exercise. However, chronic metabolic diseases such as obesity and diabetes, which have increased due to Western-style diets and inactive lifestyles, cause a decrease in type 1 muscle fibers, leading to decreased endurance. Muscle growth can be broadly divided into two types: hyperplasia, which increases the number of muscle cells and growth, and hypertrophy, which increases cell size and growth. Hyperplasia primarily occurs during the growth period, from infancy to secondary sexual characteristics, while hypertrophy refers to muscle size increase through exercise. Furthermore, sustained moderate exercise stimulates muscle cells, leading to the continuous expression of factors involved in muscle development. In addition to the increase in the size and number of muscle fibers, muscle development, and muscle mass, it also improves the biogenesis and function of mitochondria, which synthesize ATP, which is essential for muscle motility.Maintaining muscle homeostasis is essential for maintaining a healthy life. However, in modern society, a lack of exercise is emerging due to factors such as aging and changes in occupational distribution. This leads to decreased muscle use, resulting in decreased muscle mass and function, which can lead to fatigue and even serious injuries from minor collisions. Furthermore, unhealthy behaviors such as irregular eating habits, stress, alcohol, and smoking can also contribute to weakened muscle function. Muscle strength refers to the ability of a muscle to exert maximum force at one time. Exercise performance refers to the ability to perform exercise using muscle strength. Weakened muscle function and muscle weakness due to the aforementioned problems can lead to decreased muscle strength and exercise performance, which can lead to physical decline, increased risk of adult diseases, and a diminished quality of life.

[0004] Fatigue is defined as a state of reduced physical and mental activity capacity. Physical fatigue, which refers to a state of reduced exercise performance capacity (Fatigue, Establishment of a Functional Evaluation System for Fatigue Recovery of Health Functional Foods, 2004 Ministry of Food and Drug Safety Research Report), is caused by the accumulation of fatigue-causing substances such as lactic acid in the muscles (Fitts et al., 1976; Savitski et al., 1979). While adequate sleep and rest can help recover from fatigue, unrecoverable fatigue can lead to various acute and chronic diseases. Fatigue can sometimes be defined as "a perception of reduced capacity for physical and mental activity due to an imbalance in the availability, utilization, and recovery of resources required to perform an activity." Fatigue is primarily a state of reduced work capacity due to physical fatigue, while stress refers to a state of mental fatigue that disrupts homeostasis. Fatigue is categorized into central nervous system fatigue, neuromuscular junction fatigue, and peripheral fatigue of the limbs. Fatigue is a comprehensive physiological process involving various physiological and biochemical factors. It is a psychological and physiological phenomenon that inevitably occurs when the body's mental or physical activity reaches a certain level. It signifies a temporary decline in the body's natural work capacity and can be a symptom indicating the body's development into a state of damage. If fatigue persists and becomes chronic, it can develop into a disease called Chronic Fatigue Syndrome (CFS). Currently, the main symptom of CFS is prolonged fatigue, a cluster of symptoms that manifest as nonspecific symptoms such as low-grade fever, headache, sore throat, muscle and joint pain, attention deficit disorder, memory loss, sleep disturbance, and depression. Physical examinations typically reveal no significant abnormalities. Causes of fatigue include sleep deprivation, lack of exercise, an unbalanced diet, bad habits such as alcoholism, mental stress, a poor work environment, and the workplace environment.There are many ways to alleviate fatigue, including rest, finding satisfying work, regular exercise, changing to a healthier diet, and getting enough sleep. Furthermore, the market for functional products that suppress the accumulation of fatigue-causing substances in the body and enhance exercise performance is booming. However, while products containing compounds such as steroids, caffeine, sodium bicarbonate, and sodium citrate can significantly enhance exercise performance when taken in high doses, they can also carry serious side effects and ultimately pose a risk of harming one's health. Recently, research is actively underway to develop products using safe lactic acid bacteria to alleviate fatigue and enhance exercise performance.

[0005] The present invention was devised to solve the above problems, and the inventors of the present invention named a newly discovered new strain among the Allobaculum strains as Allobaculum lactocepinia, and sought to elucidate the characteristics of the Allobaculum strain and Bifidobacterium animalis.

[0006] The purpose of the present invention is to provide a new strain of Allobaculum genus (KCCM13405P) or Bifidobacterium animalis (KCCM13404P) having anti-fatigue or endurance-enhancing activity.

[0007] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating muscle disease caused by muscle dysfunction, muscle wasting or muscle degeneration, comprising at least one selected from the group consisting of Allobaculum genus strain (KCCM13405P) or Bifidobacterium animalis (KCCM13404P), a culture of the strain, a lysate thereof and an extract thereof.

[0008] Another object of the present invention relates to a method for preventing or treating a muscle disease caused by muscle dysfunction, muscle wasting or muscle degeneration, comprising a step of administering or ingesting to a subject a composition comprising at least one selected from the group consisting of a strain of the genus Allobaculum (KCCM13405P) or Bifidobacterium animalis (KCCM13404P), a culture of the strain, a lysate thereof and an extract thereof.

[0009] Another object of the present invention relates to the use of a composition comprising at least one selected from the group consisting of a strain of the genus Allobaculum (KCCM13405P) or Bifidobacterium animalis (KCCM13404P), a culture of the strain, a lysate thereof, and an extract thereof, for the prevention or treatment of muscle disease caused by decreased muscle function, muscle wasting, or muscle degeneration.

[0010] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating at least one muscle disease selected from the group consisting of atony, muscular atrophy, muscular dystrophy, muscle degeneration, myasthenia, cachexia, and sarcopenia, comprising at least one selected from the group consisting of Allobaculum genus strain (KCCM13405P) or Bifidobacterium animalis (KCCM13404P), a culture of the strain, a lysate thereof, and an extract thereof.

[0011] Another object of the present invention is to provide a method for preventing or treating at least one muscle disease selected from the group consisting of atony, muscular atrophy, muscular dystrophy, muscle degeneration, myasthenia, cachexia, and sarcopenia, comprising a step of administering or ingesting to a subject a composition comprising at least one selected from the group consisting of Allobaculum genus strain (KCCM13405P) or Bifidobacterium animalis (KCCM13404P), a culture of the strain, a lysate thereof, and an extract thereof.

[0012] Another object of the present invention relates to a use of a composition comprising at least one selected from the group consisting of Allobaculum genus strain (KCCM13405P) or Bifidobacterium animalis (KCCM13404P), a culture of the strain, a lysate thereof, and an extract thereof, for the prevention or treatment of at least one muscle disease selected from the group consisting of atony, muscular atrophy, muscular dystrophy, muscle degeneration, myasthenia, cachexia, and sarcopenia.

[0013] One aspect of the present invention for achieving the above-mentioned purpose relates to a new strain of Allobaculum genus (KCCM13405P) or Bifidobacterium animalis (KCCM13404P) having anti-fatigue or endurance-enhancing activity.

[0014] In the present invention, the term Allobaculum lactocepinia strain (KCCM13405P) is a novel Allobaculum strain that was specifically isolated and identified by the inventors from the feces of mice treated with CBD, and is newly named Allobaculum lactocepinia in this patent, and can be used interchangeably with “strain of the genus” or “Allobaculum lactocepinia strain”.

[0015] In the present invention, the Allobaculum strain (KCCM13405P) or Bifidobacterium animalis (KCCM13404P) has 16S rDNA consisting of the base sequences of SEQ ID NO: 1 and SEQ ID NO: 2. The strain was named Allobaculum strain (KCCM13405P) or Bifidobacterium animalis (KCCM13404P) and deposited at the Korean Culture Centre of Microorganisms (KCCM) on October 17, 2023, and assigned the accession numbers KCCM13405P and KCCM13404P, respectively.

[0016] When mice were treated with the above strain, endurance significantly increased, and the size of the gastrocnemius and soleus muscles increased. In the group treated with the above strain, the ratio of MyHC-Ⅱ, a specific gene associated with slow muscle fibers, significantly increased. In the Bifidobacterium animalis-administered group, the ratio of MyHC-Ⅱ, associated with fast muscle fibers, significantly decreased. Groups A and B showed a significant increase in SDH-positive muscle fibers (Fig. 7). These results were also confirmed by mRNA expression, and were consistent with changes in the expression of Type-Ⅱ, a marker protein for fast muscle fibers, and Type-Ⅱ, a marker protein for slow muscle fibers (Fig. 8). Through this, it was confirmed that the two strains improved endurance.

[0017] The lactate level of Bifidobacterium animalis significantly decreased, and the blood ketone body level significantly increased in both strains. Since an increase in the blood ketone body concentration is an indicator of energy efficiency, and the lactate concentration is an indicator of an increase in muscle fatigue, it can be seen that the two strains contributed to an increase in energy efficiency, and in particular, Bifidobacterium animalis contributed to a decrease in muscle fatigue (Fig. 8).

[0018] In the present invention, the strain can increase the proportion of type 1 muscle fibers in muscle tissue.

[0019] In the present invention, the strain can increase the biogenesis of mitochondria in muscle tissue.

[0020] In the present invention, the strain can develop muscles through muscle neogenesis.

[0021] In the present invention, the strain can increase phosphorylation of AMPK or CREB and increase expression of PGC-1α.

[0022] Another aspect of the present invention relates to a pharmaceutical composition for preventing or treating muscle disease caused by muscle dysfunction, muscle wasting or muscle degeneration, comprising any one selected from Allobaculum genus strain (KCCM13405P) and Bifidobacterium animalis (KCCM13404P), a culture of the strain, a lysate thereof and an extract thereof.

[0023] In the present invention, the culture is a product obtained by culturing a microorganism in a medium, and the medium may be selected from known liquid media or solid media, and may be, for example, MRS liquid media, MRS agar media, or BL agar media.

[0024] The term "composition" used in the present invention means a material in which two or more components are uniformly mixed, and is a concept that includes not only a finished product but also an intermediate material for manufacturing a finished product.

[0025] The terms “pharmaceutically acceptable” and “foodtically acceptable” as used in the present invention mean that they do not significantly stimulate the organism and do not inhibit the biological activity and properties of the administered active substance.

[0026] The term “prevention” as used in the present invention means any act of suppressing symptoms or delaying progression of a specific disease by administering the composition of the present invention.

[0027] The term "treatment" as used in the present invention means any act of improving or beneficially altering the symptoms of a specific disease by administering the composition of the present invention.

[0028] The term "improvement" as used herein means any action that at least reduces or alleviates a parameter related to the condition being treated, for example, the severity of a symptom.

[0029] The term "administration" used in the present invention means providing a predetermined substance to an individual or patient by any appropriate method, and may be administered parenterally (for example, intravenously, subcutaneously, intraperitoneally, or locally in the form of an injection) or orally depending on the intended method, and the dosage range varies depending on the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of the disease. Liquid preparations for oral administration of the composition of the present invention include suspensions, oral solutions, emulsions, syrups, etc., and may include various excipients such as wetting agents, sweeteners, fragrances, preservatives, etc. in addition to commonly used simple diluents such as water and liquid paraffin. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories, etc. The pharmaceutical composition of the present invention may be administered by any device that allows the active substance to move to target cells. Preferred administration methods and formulations include intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, and drip injection. Injections can be manufactured using aqueous solvents such as saline solution and Ringer's solution, non-aqueous solvents such as vegetable oil, higher fatty acid ester (e.g., ethyl oleate, etc.), alcohols (e.g., ethanol, benzyl alcohol, propylene glycol, glycerin, etc.), and pharmaceutical carriers such as stabilizers to prevent deterioration (e.g., ascorbic acid, sodium bisulfite, sodium pyrosulfite, BHA, tocopherol, EDTA, etc.), emulsifiers, buffers to adjust pH, and preservatives to inhibit microbial growth (e.g., phenylmercuric nitrate, thimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol, etc.).

[0030] The term "subject" as used in the present invention refers to any animal, including humans, monkeys, cows, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits, or guinea pigs, that has developed or may develop the muscle disease, and muscle disease can be effectively prevented or treated by administering the pharmaceutical composition of the present invention to the subject. The pharmaceutical composition of the present invention can be administered in combination with existing therapeutic agents.

[0031] The term "pharmaceutically effective amount" in the present invention means an amount sufficient to treat a disease at a reasonable benefit or risk ratio applicable to medical treatment, which may be determined based on factors including the type and severity of the disease of the subject, the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and the excretion rate, the duration of treatment, concurrently used drugs, and other factors well known in the medical field.

[0032] In another aspect of the present invention, the present invention relates to a pharmaceutical composition for preventing or treating at least one muscle disease selected from the group consisting of atony, muscular atrophy, muscular dystrophy, muscle degeneration, myasthenia, cachexia, and sarcopenia, comprising any one selected from Allobaculum genus strain (KCCM13405P) and Bifidobacterium animalis (KCCM13404P), cultures of the strains, lysates thereof, and extracts thereof.

[0033] The above "atony" is a disease also called flaccidity or atonia, and refers to a state of decreased or lost muscle tone.

[0034] The above-mentioned "muscular atrophy" refers to the loss of muscle tissue resulting from muscle disuse, disease of the muscle itself, or damage to the nerves that control the muscle. It encompasses both progressive muscle atrophy and muscle weakness. In general, disuse can lead to a significant loss of muscle strength, gradually progressing to muscle atrophy. Furthermore, in people living in environments without gravity, or due to a decrease in calcium and muscle strength, symptoms of muscle weakness can also appear.

[0035] In addition, it can include myasthenia gravis, Duchenne, Becker, limb-girdle, and facioscapulohumeral types, which are muscle atrophy caused by disease of the muscle itself, and inflammation that occurs in the muscle itself, and it can include spinal muscular amyotrophy, Berdnig-Hoffmann type, Kugelberg-Welander disease, amyotrophic lateral sclerosis (ALS), Lou Gehrig's disease, and spinobular muscular atrophy, Kennedy's disease, which are muscle atrophy caused by damage to the nerves that control the muscle.

[0036] The above "muscular dystrophy" is a type of degenerative muscle disease that manifests as necrosis of muscle fibers and causes muscle weakness and atrophy through necrosis and degeneration of muscle fibers due to damage to the muscle cell membrane. These include Duchenne muscular dystrophy, Becker muscular dystrophy, Limb-girdle muscular dystrophy, Emery-Dreifuss muscular dystrophy, Facioscapulohumeral muscular dystrophy, Myotonic muscular dystrophy, Oculopharyngeal muscular dystrophy, Distal muscular dystrophy, and Congential muscular dystrophy, and can appear in various forms depending on the location.

[0037] In addition, the above muscle wasting or muscle degeneration may be caused by factors such as genetic factors, acquired factors, and aging, and may include all of the gradual loss of muscle mass, weakening and degeneration of muscles, especially skeletal muscles or voluntary muscles and cardiac muscles, and all of the resulting muscle weakness.

[0038] The term "muscle weakness" refers to a state in which the strength of one or more muscles is reduced. This muscle weakness may be limited to a single muscle, one side of the body, the upper or lower extremities, or may be systemic. Furthermore, subjective symptoms of muscle weakness, including muscle fatigue and muscle pain, can be objectively quantified through a medical examination. Causes of muscle weakness include, but are not limited to, muscle damage, decreased muscle mass due to decreased muscle cell differentiation, and muscle aging.

[0039] The aforementioned "myasthenia" is a neurological disorder that causes muscle weakness. Initially, mild symptoms such as drooping eyelids (ptosis) and poor eye movement may appear. Furthermore, patients may experience slurred speech and difficulty swallowing. Facial muscles also become weak. As myasthenia worsens, symptoms spread throughout the body, leading to decreased strength in the arms and legs, difficulty lifting objects, and an increased risk of falls. Dangerous conditions such as shortness of breath and respiratory paralysis may also occur.

[0040] The above "cachexia" refers to a state that brings about a marked general debility, and the causes include malignant tumor, Basedow's disease, hypopituitarism, malaria, hypoadrenocorticism, and thymic insufficiency. Clinically, the main symptoms are general debility, anemia, and edema. It is known that anemia from a malignant tumor and malnutrition caused by the local effects of the tumor itself occur, and systemically, the metabolites of the tumor compete with the host's metabolism and cause host metabolic disorders, resulting in cachexia.

[0041] The pharmaceutical composition of the present invention may include a carrier, diluent, excipient, or a combination of two or more thereof commonly used in biological preparations. The term "pharmaceutically acceptable" as used herein means that the composition exhibits a property of not being toxic to cells or humans exposed to the composition. The carrier is not particularly limited as long as it is suitable for delivering the composition in vivo, and for example, compounds described in Merck Index, 13th ed., Merck & Co. Inc., saline solution, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components may be mixed and used, and other common additives such as antioxidants, buffers, and bacteriostatic agents may be added as necessary. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate the composition into an injection form such as an aqueous solution, suspension, or emulsion, or into a pill, capsule, granule, or tablet. Furthermore, it can be preferably formulated according to each disease or ingredient using an appropriate method in the field or the method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).

[0042] In the present invention, the pharmaceutical composition may be one or more dosage forms selected from the group including oral dosage forms, topical preparations, suppositories, sterile injectable solutions, and sprays.

[0043] The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable additive. At this time, the pharmaceutically acceptable additive may include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose, mannitol, maltose, 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, dextrose, sorbitol, and talc. The pharmaceutically acceptable additive according to the present invention is preferably included in the composition in an amount of 0.1 to 90 parts by weight, but is not limited thereto.

[0044] Another aspect of the present invention relates to a composition for improving exercise performance, comprising any one selected from a culture of the strain, a lysate thereof, and an extract thereof.

[0045] In the present invention, the improvement in exercise performance may have one or more effects selected from the group consisting of increasing exercise endurance, strengthening muscle strength, improving balance, and improving exercise adaptation.

[0046] In the present invention, the “improvement of exercise performance” may mean preventing or treating one or more diseases selected from the group consisting of mitochondrial abnormalities, neurodegenerative diseases, decreased endurance, and decreased power output.

[0047] In the present invention, the “exercise performance ability” refers to the degree to which a person can perform physical movements that can be seen in daily life or sports, such as running, jumping, throwing, and swimming, quickly, strongly, accurately, for a long time, and skillfully. Exercise performance ability is defined by factors such as muscle strength, agility, and endurance, and “improvement in exercise performance ability” means improving or enhancing exercise performance ability.

[0048] In the present invention, the above-mentioned "mitochondrial disease" is a disease caused by a dysfunction of mitochondria, and may include all diseases caused by oxidative stress due to phosphate swelling, reactive oxygen species or free radicals, etc. due to abnormal mitochondrial membrane potential, dysfunction due to genetic factors such as mutations in genes related to mitochondrial function in mitochondrial DNA or nucleus, and diseases caused by defects in oxidative phosphorylation function for energy generation in mitochondria.

[0049] Mitochondria are essential cell organelles that produce ATP, the cellular energy. If mitochondrial dysfunction occurs, the energy produced within the cell gradually decreases, and cell damage or even cell death follows. Accordingly, mitochondrial dysfunction impairs the function of all cells that contain mitochondria, except for red blood cells that do not have mitochondria, and is particularly fatal to organs with high energy demands such as muscles and the brain. Mitochondrial dysfunction can affect virtually any tissue, and a wide variety of symptoms can exist depending on the degree of tissue involvement. Specific examples of mitochondrial dysfunction diseases include Friedreich's ataxia (FRDA), Leber's Hereditary Optic Neuropathy (LHON), and Dominant Optic Atrophy (DOA); Mitochondrial diseases include Mitochondrial Myopathy, Encephalopathy, Lactacidosis, Stroke (MELAS), Myoclonus Epilepsy Associated with Ragged-Red Fibers (MERRF) syndrome, Leigh syndrome, and oxidative phosphorylation disorders. Most mitochondrial diseases are known to cause neurodegenerative diseases, stroke, blindness, hearing impairment, diabetes, and heart failure.

[0050] Another aspect of the present invention relates to a food composition for preventing or improving muscle disease, comprising any one selected from a culture of the strain, a lysate thereof, and an extract thereof.

[0051] When the composition of the present invention is used as a food composition, the strain can be added as is or used together with other foods or food ingredients, and can be used appropriately according to conventional methods. In addition to the active ingredient, the composition can include a food-related acceptable food additive, and the amount of the active ingredient mixed can be appropriately determined depending on the intended use (prevention, health, or therapeutic treatment).

[0052] The term "food supplement additive" used in the present invention means a component that can be added to food as an auxiliary, and can be appropriately selected and used by those skilled in the art as added in the manufacture of health functional foods of each formulation. Examples of food supplement additives include various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and fillers, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc., but the types of food supplement additives of the present invention are not limited by the above examples.

[0053] The food composition of the present invention may include a health functional food. The term "health functional food" as used herein refers to a food manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. using raw materials or ingredients with useful functionality for the human body. Here, "functionality" means obtaining a beneficial effect for health purposes, such as regulating nutrients for the structure and function of the human body or physiological functions. The health functional food of the present invention can be manufactured by methods commonly used in the art, and during the manufacturing process, raw materials and ingredients commonly added in the art can be added. In addition, the formulation of the health functional food can be manufactured without limitation as long as it is a formulation recognized as a health functional food. The food composition of the present invention can be manufactured in various forms, and unlike general drugs, it has the advantage of being made from natural materials, thus avoiding side effects that may occur with long-term administration of drugs. Furthermore, due to its excellent portability, the health functional food of the present invention can be consumed as a supplement to enhance the effectiveness of muscle disease treatment agents.

[0054] In addition, there is no limitation on the type of health food in which the composition of the present invention can be used. In addition, a composition comprising the strain of the present invention can be prepared by mixing other appropriate auxiliary ingredients that can be included in health functional foods and known additives according to the selection of a person skilled in the art. Examples of foods to which it can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and it can be prepared by adding it to juice, tea, jelly, and juice made with the extract according to the present invention as a main ingredient.

[0055] The Allobaculum strain (KCCM13405P) and Bifidobacterium animalis (KCCM13404P) strain of the present invention increase endurance by increasing exercise capacity of mice, increase type 1 muscle fibers, enhance / improve mitochondrial regeneration and function, and develop muscles through muscle regeneration, and therefore can be used for anti-fatigue, endurance enhancement, and exercise performance enhancement, and can also be used for the prevention or treatment of muscle diseases.

[0056] Figure 1 is a result showing increased exercise capacity in mice according to treatment with cannabidiol, (A) is a schematic diagram showing endurance exercise capacity measured on a treadmill by administering 30 mg / kg of cannabidiol for 4 weeks to 20-week-old C57 / BL6 mice of the present invention, and (B-D) are the results of confirming endurance on a treadmill after 4 weeks of exercise training on a treadmill, (B) showing the results of measuring running time, (C) the running distance, and (D) the running time to fatigue.

[0057] Figure 2 shows the results measured using an indirect calorimeter after changing the intestinal microbial community by administering cannabidiol or antibiotics and then treating with cannabidiol or the strain of the present invention.

[0058] Figure 3 shows the results of confirming the change in the muscle fiber composition of the gastrocnemius muscle of mice according to cannabidiol administration. (A) is a representative skeletal muscle result of a 20-week-old mouse treated with a solvent or cannabidiol, (B) is the result of immunofluorescence staining with MyHC-I, MyHC-IIa, and MyHC-IIb targeting the gastrocnemius muscle, (C), (D), and (E) are the results of quantifying the composition and cross-sectional area of ​​the muscle fibers using the images of the immunofluorescence staining, (F) is the result of confirming the change in markers of slow and fast muscle fibers using q-PCR, (G) is the result of succinate dehydratase (SDH) staining of the gastrocnemius muscle, and (H) is the result of quantifying SDH-positive muscle fibers.

[0059] Figure 4 shows the results of confirming the change in mitochondrial biogenesis according to cannabidiol administration. (A) is the result of quantifying mitochondrial DNA (mtDNA) standardized to nuclear DNA (nDNA) by qPCR, (B) is the result of confirming genes related to mitochondrial biogenesis and its oxidative phosphorylation in the gastrocnemius muscle by qPCR, (C) and (D) are the results of representative Western blot analysis of the OxPhos complex and the results of quantifying the results, and (E) and (F) are the results of Western blot analysis of genes related to mitochondrial biogenesis in the gastrocnemius muscle and the results of quantifying the results.

[0060] Figure 5 shows the results of confirming the change in the composition of gut microbiota according to the administration of cannabidiol. (A) is the result of analyzing the effect of cannabidiol administration on the composition of gut microbiota at the phylum level using synthetic sequence analysis, (B) is the result of analyzing the effect of cannabidiol administration on the composition of gut microbiota at the family level using synthetic sequence analysis, (C) is the result of measuring beta diversity by analyzing the principal coordinate analysis (PCoA) plot using generalized weighted UniFrac as a distance measure, (D) is the result of measuring alpha diversity using Shannon, (E) is the result of measuring alpha diversity using the ACE index, and (F) is an outline of a study to confirm the effect of the gut microbiota community on cannabidiol-mediated exercise performance improvement. 20-week-old C57BL / 6 mice were administered 40 mg / kg of doxycycline for 5 weeks after the treadmill adaptation period, and cannabidiol was administered for 4 weeks to measure endurance exercise performance on the treadmill. 30 mg / kg was administered, and (G-I) are the results of checking the endurance on the treadmill after 4 weeks of exercise training on the treadmill, where (G) is the running time, (H) is the running distance, and (I) is the running time to fatigue, (J) is the result of representative immunofluorescence staining for MyHC-I, MyHC-IIa, and MyHC-IIb / x of the gastrocnemius (GAS) muscle, (K) is the result of quantifying the muscle fiber composition of the mice in each group, (L) is a representative photograph of representative succinate dehydrogenase (SDH) staining of the gastrocnemius muscle, and (M) is the result of quantifying it.

[0061] Figure 6 shows the results of confirming the change in mitochondrial biogenesis according to the administration of cannabidiol and antibiotics. (A) is the result of confirming the change in markers of slow and fast muscle fibers in the gastrocnemius muscle by q-PCR after treatment with a solvent, cannabidiol, doxycycline, and the combined administration of cannabidiol and doxycycline, respectively. (B) is the result of representative Western blot analysis of the OxPhos complex in each group. (C) is the result of Western blot analysis of genes related to mitochondrial biogenesis in the gastrocnemius muscle, and (D) is the result of quantifying the result. (E) and (F) are the results of confirming the genes related to mitochondrial biogenesis and its oxidative phosphorylation in the gastrocnemius muscle by qPCR. (G) is the result of analyzing the gut microbiota composition at the phylum level in each group by synthetic sequence analysis, and (H) is the result of analyzing the gut microbiota composition at the family level by synthetic sequence analysis. (I) is the result of measuring alpha diversity using Shannon, (J) is the result of measuring beta diversity using ACE index, and (K) is the result of measuring beta diversity using principal coordinate analysis (PCoA) plot using generalized weighted UniFrac as a distance measure.

[0062] FIG. 7 shows the results of confirming the improvement in exercise capacity of mice after treatment with solvent or cannabidiol or bacteria A, B, or F, (A) is a schematic diagram of a study to confirm the improvement in exercise capacity according to the treatment of each strain, (B-D) are the results of confirming endurance on a treadmill after exercise training on a treadmill for 4 weeks, in which (B) is the running time, (C) is the running distance, and (D) is the running time to fatigue, (E) is the results of representative skeletal muscles of mice (gastrocnemius and soleus), (F) is the results of immunofluorescence staining of gastrocnemius muscles with MyHC-I, MyHC-IIa, and MyHC-IIb, (G) is the result of quantifying the muscle fibers, composition, and cross-sectional area of ​​the immunofluorescence staining image, (H) is the result of succinate dehydratase (SDH) staining of gastrocnemius muscles, and (I) is the result of quantifying SDH-positive muscle fibers.

[0063] Figure 8 shows the results of measuring changes in mice after treatment with solvent, cannabidiol, or bacteria A, B, or F. (A) shows the results of body weight changes according to treatment with each strain, (B) shows the results of analyzing the changes in blood short-chain fatty acid concentrations in mice according to treatment with each strain, and (C) shows the results of confirming the changes in markers of slow and fast muscle fibers in the gastrocnemius muscle using q-PCR.

[0064] Figure 9 shows the results of measuring changes in mice after treatment with solvent, cannabidiol, or bacteria A, B, or F, where (A) is the result of measuring changes in blood glucose levels, (B) is the result of measuring blood lactate concentration, (C) is the result of measuring blood ketone body concentration, (D) is the result of measuring alpha diversity using Shannon, (E) is the result of measuring ACE index, (F) is the result of analyzing gut microbiota composition at the phylum level by synthetic sequence analysis for each group, (G) is the result of analyzing gut microbiota composition at the family level by synthetic sequence analysis, (H) is the result of measuring beta diversity by analyzing principal coordinate analysis (PCoA) plot using generalized weighted UniFrac as a distance measure, (I, J) are the results of showing the predicted chromosomes from whole genome analysis of bacteria A and B, which are colored differently according to their composition and function, (I) is bacteria B, named bacteria KBP-1, (J) is the result of strain A named KBP-1.

[0065] Figure 10 shows the results for identifying and analyzing bacteria A. (A) is the morphological result, (B) is a phylogenetic analysis graph performed based on the base sequence information of bacteria A, and (C) is the result of taxonomic comparison through whole genome analysis.

[0066] Figure 11 shows the results for identifying and analyzing bacteria B. (A) is the result of performing the base sequence of bacteria B through NCBI BLAST, and (B) is a phylogenetic analysis graph performed based on the base sequence information of bacteria A.

[0067] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0068] Example 1. Confirmation of increases in exercise capacity, composition of oxidative muscle fiber components, and mitochondrial biogenesis following cannabidiol treatment.

[0069] Example 1.1 Confirmation of exercise capacity according to cannabidiol treatment

[0070] The present invention examined the exercise capacity of mice administered cannabidiol orally on a single-lane treadmill. Mice were administered a solvent (corn oil) or cannabidiol orally and then subjected to involuntary exercise training using a single-lane treadmill for 4 weeks (Figure 1A). Specifically, 20-week-old C57 / BL6 mice were orally administered cannabidiol dissolved in either solvent or corn oil at a concentration of 30 mg / ml six times a week. All animal experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (NIH Publication No. 85-23, revised 2011). The research protocol of the present invention was approved by the Institutional Animal Care and Use Committee of Chonbuk National University (Permit No. JBNU 2022-048). Before assessing exercise capacity, mice were acclimated to a single-lane treadmill for 30 minutes daily at a speed of 10 m / min for 7 days. Mice were tested for exercise capacity on a single-rail treadmill at moderate intensity daily for 4 weeks. Moderate-intensity running was measured starting at 10 m / min for 10 minutes, increasing by 2 m / min every 10 minutes, up to a maximum of 16 m / min until exhaustion. Exhaustion was defined as the inability to return to the single-rail treadmill despite gentle stimulation with a wooden cane. Running time and distance were recorded for each mouse during the last 2 weeks of the running test period. Results confirmed that cannabidiol-treated mice exhibited significantly increased endurance capacity, as measured by average running time, running distance, and running time to exhaustion (Figures 1B-1D).

[0071] Metabolic capacity (energy intake and expenditure) was measured using an indirect caloric calculation system (Oxymax / CLAMS metabolic cage system). Before the experiment, mice were acclimated to the metabolic chamber for 2 days. Afterwards, the oxygen consumption rate (VO2; mL / kg / h), carbon dioxide production rate (VCO2; mL / kg / h), and respiratory exchange ratio (RER; VO2 / VCO2) were measured. In mice treated with cannabidiol, the RER (VO2 / VCO2) was significantly reduced. This confirmed a shift in mitochondrial substrate preference from glucose to fatty acids (Fig. 2).

[0072] 1.2 Confirmation of changes in oxidative muscle fiber components following cannabidiol treatment

[0073] Enhanced endurance capacity is generally associated with increased oxidative muscle fibers. Therefore, to investigate the composition and cross-sectional area of ​​muscle fiber types, we analyzed various types of hindlimb muscles in mice. Twenty-week-old C57 / BL6 mice were orally administered cannabidiol (30 mg / mL dissolved in a solvent (corn oil) or corn oil) six times a week, and sacrificed after four weeks. Macroscopic observation after sacrifice revealed that the hindlimb muscles of cannabidiol-treated mice were red (Fig. 3A).

[0074] For staining of myosin heavy chain isomers, serial muscle sections were preincubated in a blocking solution containing stock goat serum. Primary MyHC antibodies (MyHC I (#BA-D5), MyHCIIa (#SC-71), and MyHCIIb (#BF-F3); DSHB, Iowa City, IA, USA) were incubated overnight at 4°C. After washing, secondary antibodies (Alexa Fluor 350-conjugated goat anti-mouse IgG2b (#A21140), Alexa Fluor 488-conjugated goat anti-mouse IgG1 (#A21121), and Alexa Fluor 594-conjugated goat anti-mouse IgM (#A21044)) were used for 1 h at 37°C. For the specification of muscle fiber types, sections containing approximately 200 myofibers were selected. Images were acquired using a Leica DM750 microscope (Leica, Wetzlar, Germany). The muscle fibers were then manually classified as immunopositive or immunonegative. Furthermore, the cross-sectional area of ​​the muscle fibers was calculated using iSolution DT 36 software (Carl Zeiss, Oberkochen, Germany). Immunofluorescence staining for myosin heavy chain (MyHC) isoforms in the gastrocnemius (GAS) muscle revealed that cannabidiol administration increased oxidative-myofiber density and decreased the size of the corresponding muscle fibers (Figs. 3B–3E).

[0075] Total RNA was extracted from skeletal muscle tissue using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). cDNA was generated using primers provided in a cDNA synthesis kit (Applied Biosystems, Foster City, CA, USA). Specific primers for each gene (Table 1) were designed using PrimerBank (https: / pga.mgh.harvard.edu / primerbank).

[0076] Gene sequenceMyHC-I(Myh7)ForwardACAAGCTGCAGCTGAAGGTGReverseTCATTCAGGCCCTTGGCACMyHC-IIa(Myh2)ForwardCCAGCTGCACCTTCTCGTTTGCCAGReverseCATGGGGAAGATCTGGTCTTCTTMy HC-IIb(Myh4)ForwardCCTGGAACAGACAGAGAGGAGCAGGAGAGReverseGTGAGTTCCTTCACTCTGCGCTCGTGCMyHC-IId(Myh1)ForwardTGCAACAGTTCTTCAACCACReverseGCCAGGTCCATCCCAAAGT

[0077] [Table 1] qPCR reactions were performed in a final volume of 10 μL containing 10 ng of reverse-transcribed total RNA, 200 nM forward and reverse primers, and PCR master mix. qPCR was performed in 384-well plates using an ABI Prism 7900HT Sequence Detection System (Applied Biosystems). mRNA levels of each target gene were normalized to Gapdh (for nuclear-encoded genes) or 16S rRNA (for mtDNA-encoded genes). For mitochondrial DNA content analysis, total DNA was extracted using a genomic DNA purification kit (Qiagen, Hiaden, Germany). Relative mtDNA was quantified by qPCR using primers for the mitochondrially encoded gene cytochrome oxidase 2 (Cox2) normalized to the nuclear-encoded gene cyclophilin A (Ppia).

[0078] Comparing mRNA expression with previous results, cannabidiol treatment significantly increased MyHC-I (Myh7) and MyHC-IIa (Myh2), genes specific to slow muscle fibers, in the gastrocnemius muscle. Simultaneously, MyHC-IIb (Myh4), a gene specific to fast muscle fibers, was significantly decreased, while MyHC-IId (Myh1) remained unchanged (Fig. 3F).

[0079] After sacrifice, skeletal muscle tissues were placed in a 30% sucrose solution and embedded in liquid nitrogen-cooled isopentane. For succinate dehydrogenase (SDH) staining, cryosections (10 μm) of tissues were incubated in 0.2 M sodium phosphate buffer (pH 7.6) containing 0.6 mM nitro blue tetrazolium and 50 mM sodium succinate at 37°C for 30 min. Slides were washed with distilled water and mounted in aqueous mounting media. Immunostaining of muscle sections with succinate dehydrogenase (SDH), an indicator of mitochondrial activity, revealed that on average, more than 70% of muscle fibers were SDH-positive in cannabidiol-treated mice compared to an average of 60% in solvent-treated mice (Figs. 3G-3H).

[0080] 1.3 Confirmation of increased mitochondrial biosynthesis following cannabidiol treatment

[0081] Since increased oxidative capacity of muscle fibers is associated with mitochondrial biogenesis and function, we examined mitochondrial content and related gene expression in gastrocnemius muscles of cannabidiol-treated mice. Furthermore, we confirmed the expression of key transcription factors and regulatory factors involved in mitochondrial biogenesis.

[0082] Mitochondrial and nuclear DNA content was quantified to confirm an increase in mitochondria in the gastrocnemius muscle (Fig. 4A). Mitochondrial content was determined by the ratio of mitochondrial DNA to nuclear DNA (mtDNA / nDNA). To quantify the copy number of mitochondrial DNA (mtDNA), the method was used as described in "The AMPK-PPARGC1A pathway is required for antimicrobial host defense through activation of autophagy." Autophagy. 2014; 10:785-802. Pyruvate kinase (Pklr) was used as a marker for nDNA, and NADH dehydrogenase subunit 1 was used as a marker for mtDNA. Real-time PCR reactions were performed according to the manufacturer's instructions (QuantiFast SYBR Green PCR Master Mix; Qiagen, 204052), and temperature cycling was performed on an ABI Prism 7900HT Sequence Detection System. The mtDNA content was normalized to the nucleotide DNA content. Treatment with cannabidiol significantly increased mitochondrial DNA (mtDNA) content (Fig. 4A). This was also confirmed by qPCR of related genes. In gastrocnemius muscle, cannabidiol treatment increased the expression of genes related to mitochondrial biogenesis and oxidative phosphorylation (Fig. 4B).

[0083] Western blot analysis was also performed to measure the changes in the expression of C1-V (ATP synthase) and related signaling proteins, which correspond to various components of the electron transport chain complex in mitochondria. Cell or tissue homogenates (20 ug) were separated using 10% SDS-PAGE and transferred to PVDF membranes. After blocking with 5% skim milk, the membranes were incubated with primary antibodies against CREB (#9197), p-CREB (#9198) (Cell Signaling Technology, Beverly, MA, USA), T-OxPhos (ab110413) (Abcam, Cambridge, UK), HSP90 (#ADI-SPA-836-F, Enzo Life Sciences, Plymouth Meeting, PA, USA), and PGC-1α (#AB-3242, Millipore, Danvers, MA, USA). HSP90 was used as a loading control. After washing with PBS, the cells were incubated for 1 hour with horseradish peroxidase-conjugated IgG (Zymed, South San Francisco, CA, USA), and antibody signals were detected using a Las-4000 imager (GE Healthcare Life Science, Pittsburgh, PA, USA). As a result, a significant increase was confirmed in response to cannabidiol administration (Fig. 4C to Fig. 4D).

[0084] Western blotting was performed for AMP-activated protein kinase (AMPK), Sirt1, and CREB, which are involved in the transcriptional activation of PGC-1α associated with increased mitochondrial mass. Results showed that cannabidiol administration significantly increased the expression of Sirt1, p-AMPK, and p-CREB, along with PKA activation, leading to a significant increase in PGC-1α expression (Figures 4E to 4F). In summary, cannabidiol treatment was confirmed to enhance muscle endurance by inducing a shift to a slow muscle fiber type and mitochondrial biogenesis.

[0085] Example 2. Confirmation of changes in the composition of intestinal microorganisms following administration of cannabidiol.

[0086] The fecal microbiota composition of cannabidiol-treated mice was determined using 16S rRNA amplicon sequencing. Twenty-week-old C57 / BL6 mice were orally administered cannabidiol (30 mg / mL) dissolved in a solvent (corn oil) or corn oil six times a week, and feces were collected 4 weeks later. Genomic DNA (gDNA) was extracted from the fecal samples using a commercial DNA isolation kit (QIAamp DNA Stool Mini Kit). Amplification of gDNA was performed using a barcoded forward primer (515F: 5'-GTGCCAGCMGCCGCGGTAA-3') and a reverse primer (806R: 5'-GGACTACHVGGGTWTCTAAT-3') targeting the V4 region of the bacterial 16S rDNA gene, and amplicon sequencing was performed on an Illumina iSeq 100 (San Diego, CA). Additionally, a barcoded forward primer (341F: 5'-CCTACGGGGNGGCWGCAG-3') and a reverse primer (805R: 5'-GACTACHVGGGTATCTAATCC-3') targeting the V3-V4 region of the bacterial 16S rDNA gene were used, and amplicon sequencing was performed. Adapter sequences were removed using TRIMMOMATIC (ver. 0.39), and data quality control analysis was performed using Quantitative Insights Into Microbial Ecology (QIIME2, ver. 2022.02.). Chimeric sequences were removed by DADA2 using the q2-dada2 plugin, and diversity analysis was performed using the q2-diversity plugin. Taxonomies were identified for amplicon sequence variants (ASVs), a feature classifier in the RDP database. Microbial composition graphs, α-diversity plots, and principal coordinate analysis (PCoA) were visualized using R (ver. 4.2.2) and the ggplot2 package (ver. 3.4.2).

[0087] At the phylum level, cannabidiol treatment significantly increased the diversity of Bacillota and Actinomycetota (Fig. 5A). At the family level, cannabidiol treatment significantly increased the diversity of Erycipelotrichaceae and Bifidobacteriaceae, and decreased the diversity of Oscillopiraceae, Bacteroidaceae, and Prevotellaceae (Fig. 5B). Shannon and Gini-Simpson indices representing α-diversity did not show significant changes, but principal coordinates analysis (PCoA) using a distance measure such as Uni-Frac weighting as a measure of β-diversity confirmed that the two groups were significantly separated (Figs. 5C-5E). This confirmed that cannabidiol treatment affected the partitioning of the microbial community.

[0088] Example 3. Confirmation of a direct correlation between the exercise performance-enhancing effect of cannabidiol and changes in the gut microbiome.

[0089] To investigate the direct correlation between the exercise-enhancing effects of cannabidiol and the resulting changes in the gut microbiome, we conducted an antibiotic treatment intervention. By comparing various antibiotics, we selected an antibiotic that caused minimal changes in the gut microbiome while eliminating Erycipelotrichaceae and Bifidobacteriaceae, which increase with cannabidiol administration. To test how the gut microbiome changes when administered concurrently with cannabidiol on exercise performance, we selected doxycycline (Figure 5F). As a result, mice treated with cannabidiol showed significant increases in average running time, running distance, and running time to fatigue, whereas no changes were observed in the group treated with both antibiotics (doxycycline) and cannabidiol (Figures 5G to 5I).

[0090] Indirect calorimetric analysis of energy metabolism also showed a significant decrease in RER (VCO2 / VO2) in mice administered cannabidiol, but no change was observed in the group administered antibiotics and cannabidiol together. To further clarify the above results, immunofluorescence staining of MyHC isomers was performed in gastrocnemius muscle. Unlike the cannabidiol-only group, the combined treatment with antibiotics and cannabidiol did not show a decrease in oxidized muscle fiber density and consequently fiber size (Figs. 5J to 5L). Comparing mRNA expression with previous results, cannabidiol treatment significantly increased Type-I, a specific gene associated with slow muscle fibers, in the gastrocnemius muscle, while significantly decreased Type-IIx, and antibiotics counteracted all of these effects (Fig. 5K). Immunostaining of muscle cross-sections using SDH results also showed no increase in SDH-positive muscle fibers in the group administered antibiotics and cannabidiol together (Figs. 5L to 5M).

[0091] Cannabidiol administration antagonized the oxidation (Fig. 6A) and mitochondrial C1 to CV amounts (Fig. 6B) in the gastrocnemius muscle. It also antagonized the increased expression of PGC-1α and its associated genes, Sirt1, AMPK, and p-CREB (Figs. 6C-D). Antibiotics antagonized the expression of genes related to mitochondrial oxidation induced by cannabidiol administration (Figs. 6E-F). Antibiotics controlled the proportion of the increased microbial community composition induced by cannabidiol administration and did not restore the gut microbiota pattern (Figs. 6G-K). In summary, we confirmed that changes in the gut microbiota induced by cannabidiol administration significantly contribute to the enhanced exercise performance.

[0092] Example 4. Confirmation of the exercise capacity of bacteria A (KPB-2) and B (KPB-1) and confirmation of increase in oxidative muscle fibers

[0093] Based on the hypothesis that the exercise performance-enhancing effect of cannabidiol may be mediated through the direct effect of altered gut microbiota, specific microorganisms that showed a significant increase after cannabidiol administration were selectively isolated. These included Allobaculum and Faecalibabulum Rodentium belonging to the Erysipelotrichaceae family and Bifidobacterium animalis belonging to the Bifidobacteriaceae family. Each microorganism was administered individually or as a consortium in equal proportions and compared with the exercise performance-enhancing effect of cannabidiol (Fig. 7A). The bacteria-only treatment groups were named after the initials of each strain. Group A was treated with Allobaculum strain (KBP-2), group B with Bifidobacterium animalis (KBP-1), and group F with Faecalibabulum Rodentium. The mixed (MIX) group was treated with a consortium of the three bacteria in equal proportions.

[0094] As a result, endurance represented by average running time, running distance, and running time to fatigue significantly increased in groups A (Allobaculum spp.; KBP-2 treatment group) and B (Bifidobacterium animalis; KBP-1 treatment group), but not in groups F (Faecalibabulum Rodentium treatment group) and MIX (Fig. 7B-D). Comparison of the size and color of the gastrocnemius and soleus muscles upon visual observation after sacrifice revealed that redness and size increased in groups A (Allobaculum spp.; KBP-2 treatment group), B (Bifidobacterium animalis; KBP-1 treatment group), and cannabidiol treatment (Fig. 7E).

[0095] The MyHC isomers of gastrocnemius muscle were compared using fluorescent staining of slow and fast muscle fibers. The proportion of MyHC-Ⅱ associated with slow muscle fibers (type I muscle) significantly increased in groups A, B, or cannabidiol treated, whereas the proportion of MyHC-Ⅱ associated with fast muscle fibers (type II muscle) significantly decreased in groups B or cannabidiol treated. The proportion of MyHC-Ⅰ increased only in the cannabidiol treated group (Figs. 7F and 7G). SDH immunostaining showed a significant increase in SDH-positive muscle fibers in groups A and B, although not as much as in the cannabidiol treated group (Figs. 7H and 7I). These results suggest that A (Allobaculum spp.; KBP-2 treated group) and B (Bifidobacterium animalis; KBP-1 treated group) promoted the development of type I muscle (slow muscle fibers; oxidative muscle fibers), thereby enhancing endurance exercise performance such as running.

[0096] These results were also confirmed by mRNA expression, and were consistent with changes in the expression of Type-II, a marker protein of fast muscle fibers, and Type-II, a marker protein of slow muscle fibers. Among other slow fiber marker proteins, Tnni1 and Tnnt1 were significantly increased only in the cannabidiol-treated group, and among fast fiber marker proteins, Type-ⅠⅠTnni2, Tnnc2, and Tnnt3 were decreased, particularly only in the cannabidiol-treated group (Fig. 8C).

[0097] Example 5. Confirmation of changes in energy metabolism and gut microbiota of bacteria A and B.

[0098] Although there was no significant difference in blood glucose levels, lactate levels significantly decreased in group B and the cannabidiol-administered group, and blood ketone bodies significantly increased in groups A, B, and cannabidiol (Figures 9A-9C). When the increase in blood ketone body concentration is evaluated as an increase in energy efficiency, and the lactate concentration is evaluated as an increase in muscle fatigue, it can be seen that bacteria B and cannabidiol contributed to the increase in energy efficiency and the decrease in muscle fatigue.

[0099] Analysis of changes in gut microbiota composition revealed that alpha diversity was not affected (Figures 9D and 9E), but there were differences in composition (Figures 9F and 9G), and PCoA using weighted UniFrac showed a significant distance between groups (Figure 9H). At the family level, Erysipelotrichaceae increased in treatment group A, Bifidobacteriaceae increased in treatment group B, and both increased in the cannabidiol treatment group (Figures 9F and 9G). Bacteria A and B, which showed a significant effect of improving muscle endurance, were subjected to whole-genome analysis (Figures 9I and 9J). It was confirmed that gut microbiota species A and B increased by cannabidiol administration affected exercise performance and energy metabolism improvement through changes in the microbiota community and microbial effects even without the effect of absorbed cannabidiol.

[0100] Example 6. Identification through base sequence analysis of bacteria A and B.

[0101] In the above Examples 4 and 5, Allobaculum species belonging to the Erysipelotrichaceae family and Bifidobacterium animalis strain belonging to the Bifidobacteriaceae family, which were proven to be effective in anti-fatigue and enhancing endurance, were analyzed and identified.

[0102] 6.1 Analysis and identification of the genus Allobaculum belonging to the family Erysipelotrichaceae

[0103] To isolate Allobaculum genus, belonging to the Erysipelotrichaceae family, feces (0.1 g) from CBD-treated mice were immediately collected, suspended and diluted in 0.9 mL of general anaerobic medium, and streaked onto glucose-based blood liver agar plates containing 5% sheep blood. After streaking, the plates were incubated anaerobically at 37°C for 2 days in an anaerobic chamber containing a GasPak EZ anaerobic pouch. Surface colonies were convex, translucent, and smooth. As previously reported, bacteria cultured in GAM broth at 37°C for 2 days under anaerobic conditions were subjected to Gram staining to observe their morphology. Morphological observation using an optical microscope revealed Gram-positive, rod-shaped characteristics (Fig. 10A). The width was estimated to be approximately 1 μm and the length 1–2 μm.

[0104] Phylogenetic analysis of the above strains was performed based on 16S rRNA sequences. The 16S rRNA sequence-based phylogenetic tree was constructed based on the description of Paster et al. The Neighbor Join method, also known as the Agglomerative Clustering method, was used in MEGA 11.0.8 software, and the bootstrap test was performed based on 1,000 resamples. The 16S rDNA sequence was obtained from the whole genome sequencing results in the fasta file, and the list of comparative strains was obtained from the GeneBank database using the nucleotide BLAST program of the National Center for Biotechnology Information (Fig. 10B). The 16S rRNA sequence is deposited in GenBank under the National Center for Biotechnology Information (NCBI) accession number PP082459.

[0105] Genomic DNA (gDNA) of the strains was extracted using the MagAttract HMW DNA Kit according to the manufacturer's instructions. The size-, purity-, and quality-controlled gDNA was purified with AMpureXP beads and constructed into a SMRTbell library using the SMRTbell Express Template Prep Kit (PacBio, catalog number 100-93-8-9-00). After AMPure purification, sequence information was stored on a SMRT Cell 1M v2 sequel (Pacific Biosciences) platform using the Sequel Sequencing Kit v3.0. Sequencing of the genus Allobaculum, a member of the Erysipelotrichaceae family, was performed by CJ Bioscience, Inc. using PacBio sequencing data, and the data were assembled with SMRT Link using the Microbial Assembly protocol (Pacific Biosciences, USA). Gene prediction and annotation, such as protein coding sequence (CDS) analysis, coding or non-coding RNA, and heterologous group classification, were performed using the UBLAST program with Prodigal 2.6.2, tRNAscan-SE 1.3.1, Rfam 12.0, EggNOG 4.5, Swissprot, KEGG, and SEED databases. Whole genome sequencing was analyzed to understand the genetic characteristics of the genus Allobaculum, which belongs to the family Erysipelotrichaceae. To analyze the similarity with other strains at the whole genome level, the EzBioCloud Whole Genome public database and the OrthoANI (Orthologous Average Nucleotide Identity) analysis algorithm were used to select five strains putatively similar to the genus Allobaculum, which belongs to the family Erysipelotrichaceae.The similarity between matching sequences was color-coded in a pairwise ortholog matrix (POM) table using the phylogenetic results from OrthoANI analysis and the full calculation of pairwise ortholog detection of selected strains (Fig. 10C).

[0106] The whole genome of the genus Allobaculum belonging to the family Erysipelotrichaceae was analyzed and homology searched. The strain showed the highest homology to the strains of the genus Allobaculum [phylum Bacillota; class Erysipelotrichia; order Erysipelotrichales; family Erysipelotrichidae], and it was found to form a single phylogeny with them. However, the similarity with the 16S rRNA sequence of previously known strains was less than 95%, so it was determined to be a new strain and was newly named (Fig. 10B). Based on these results, it was confirmed that it was a new strain belonging to the genus Allobaculum and was named Allobaculum lactocepinia.

[0107] 6.2 Analysis and identification of Bifidobacterium animalis

[0108] To isolate Bifidobacterium animalis, feces (0.1 g) from CBD-treated mice were immediately collected, suspended and diluted in 0.9 mL of general anaerobic medium, and streaked onto glucose-based blood liver agar plates containing 5% sheep blood. After streaking, the plates were incubated anaerobically at 37°C for 2 days in an anaerobic chamber with a GasPak EZ anaerobic pouch. Surface colonies were convex, translucent, and smooth. As previously reported, bacteria cultured in GAM broth at 37°C for 2 days under anaerobic conditions were subjected to Gram staining to observe their morphology. Morphological observation using a light microscope revealed Gram-positive, rod-shaped characteristics. The width was estimated to be approximately 1 μm and the length 1–2 μm.

[0109] Phylogenetic analysis of the above strains was performed based on 16S rRNA sequences. The 16S rRNA sequence-based phylogenetic tree was constructed based on the description of Paster et al. The Neighbor Join method, also known as the Agglomerative Clustering method, was used in MEGA 11.0.8 software, and the bootstrap test was performed based on 1,000 resamples. The 16S rDNA sequence was obtained from the whole genome sequencing results in the fasta file, and the list of comparative strains was obtained from the GeneBank database using the nucleotide BLAST program of the National Center for Biotechnology Information (Fig. 11). As a result, the bacterium B was confirmed to be Bifidobacterium animalalis.

[0110]

[0111] While specific aspects of the present invention have been described in detail above, it should be apparent to those skilled in the art that these specific descriptions are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

[0112]

[0113] [Accession number]

[0114] Name of depositor: Korea Center for Microbiological Conservation (KCCM)

[0115] Accession number: KCCM13405P

[0116] Date of acceptance: 20231017

[0117]

[0118] Name of depositor: Korea Center for Microbiological Conservation (KCCM)

[0119] Accession number: KCCM13404P

[0120] Date of acceptance: 20231017

[0121]

Claims

1. A new strain of Allobaculums genus (KCCM13405P) or Bifidobacterium animalis (KCCM13404P) having anti-fatigue or endurance-enhancing activity.

2. In the first paragraph, the strain is a new strain that increases mitochondrial biogenesis.

3. In the first paragraph, the strain is a new strain that increases the proportion of type 1 muscle fibers in muscle tissue.

4. A pharmaceutical composition for preventing or treating muscle disease caused by muscle dysfunction, muscle wasting or muscle degeneration, comprising at least one selected from the group consisting of the strain of claim 1, a culture of the strain, a lysate thereof and an extract thereof.

5. A pharmaceutical composition for preventing or treating at least one muscle disease selected from the group consisting of atony, muscular atrophy, muscular dystrophy, muscle degeneration, myasthenia, cachexia and sarcopenia, comprising at least one selected from the group consisting of the strain of claim 1, a culture of the strain, a lysate thereof and an extract thereof.

6. A pharmaceutical composition for preventing or treating muscle disease, which develops muscles through muscle regeneration, according to claim 4 or 5.

7. A composition for improving exercise performance, comprising any one selected from the strain of paragraph 1, a culture of the strain, a fragment thereof, and an extract thereof.

8. A composition for improving exercise performance in claim 7, wherein the improvement in exercise performance has at least one effect selected from the group consisting of increasing exercise endurance, strengthening muscle strength, improving balance, and improving exercise adaptation.

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