Composition for muscle strengthening containing fermented product of Lactobacillus microorganisms from Tenebrio molitor extract and use thereof

Abstract

The present specification relates to a muscle-strengthening composition comprising a fermented product of Lactobacillus genus microorganisms of mealworm extract and the use thereof. The muscle-strengthening composition contains a high degree of hydrolysis and a high content of BCAA, and when administered to an individual, it is easily digested and has the effect of inducing muscle growth in the individual.

Description

Technology Field

[0001] This specification relates to a muscle-strengthening composition comprising a fermented product of Lactobacillus genus microorganisms of mealworm extract and the use thereof. Background Technology

[0002] Compared to livestock farming, insect farming has the advantages of being environmentally and economically advantageous, as it produces 3 to 20 times less greenhouse gas emissions such as carbon dioxide (CO2), methane (CH4), and nitrous oxide (NO2), consumes 360 to 1,500 times less water, and consumes 2 to 12 times less feed, while having 7 to 13 times higher rearing efficiency per unit area. In addition, the protein content of insects is 31 to 65%, which is richer than plant-based proteins such as soybeans (23.5%), lentils (26.7%), and soybeans (41.1%), and it has been confirmed that the protein content per gram is similar to or higher than that of chickens, pigs, and cattle.

[0003] Currently, the mealworm larva (*Gosoae*) is recognized as a domestic edible insect by the Ministry of Food and Drug Safety. Edible insects are an excellent food source, rich in vitamins and minerals in addition to protein, unsaturated fatty acids, and carbohydrates.

[0004] Among edible insects, the brown mealworm ( Tenebrio molitor ; T. molitor The brown mealworm belongs to the family Tenebrionidae of the order Coleoptera and is a holometabolous insect that undergoes egg, larva, pupa, and adult stages, distributed worldwide including Korea. Its nutritional composition includes 9.3% carbohydrates, 50.3% protein, and 33.7% fat. Furthermore, it has a protein content similar to meat and contains a large amount of minerals; it is also recognized for its efficacy in treating liver diseases, making it rich in nutritional value as a medicinal and food product (Kwon et al. 2013). In particular, the brown mealworm is known to be a high-protein material, and when hydrolyzed, it exhibits antioxidant activities such as free radical and hydrogen peroxide scavenging activity.

[0005] To utilize insects in food products, it is necessary to ensure safety through heavy metal content and toxicity assessments, prepare for contamination and spoilage caused by microorganisms such as bacteria and viruses, and conduct research on safe long-term storage, distribution, and preservation methods, as well as on their functions and immune enhancement. Meanwhile, in the case of fermentation methods using microorganisms, new efficacy distinct from the existing benefits of the raw materials can be imparted, and numerous harmful components can be transformed into safe ones. Furthermore, by breaking down nutritional components into low-molecular-weight compounds through the action of decomposing enzymes, absorption can be increased, irritation can be reduced, and allergenic components can be decreased.

[0006] There is a high need for research regarding fermented edible insect extracts, which are produced by generating natural antimicrobial substances using bioconversion technology with lactic acid bacteria and applying them to edible insects to enhance antimicrobial, antioxidant, and preservative functions for storage, distribution, and preservation; and regarding fermented edible insect extracts that improve bioavailability and functionality by breaking down amino acids, peptides, fats, and carbohydrates using the enzymes of the lactic acid bacteria themselves. Furthermore, fermentation by beneficial intestinal microorganisms generates short-chain fatty acids such as acetic acid, propionic acid, and butyric acid, which serve as major energy sources for the intestinal microbiome and the human body. Short-chain fatty acids (SCFAs) in the intestines influence the viable cell count and ratio of the intestinal microbial community, pH, gene expression for cell proliferation, and serve as nutrients for intestinal epithelial cells (Cook 1998). Short-chain fatty acids have functions to improve irritable bowel syndrome and inflammatory bowel disease, as well as to prevent cardiovascular disease and obesity (Kumar 2012).

[0007] In humans, muscle mass decreases with aging, impairing normal walking and the performance of daily activities. Consequently, reduced physical activity capacity leads to a 1.5 to 2-fold increase in the incidence of other diseases such as diabetes and arthritis, and a more than 3-fold increase in the incidence of metabolic diseases. Sarcopenia is caused by a deficiency in the intake and absorption of essential amino acids (9 types for adults, 10 types for children). It was classified as a disease under the 2016 disease code (M62.84), and its prevalence is recently increasing regardless of gender or age, in addition to the elderly population. Muscles serve as a major storage site for amino acids that maintain protein synthesis in life-sustaining tissues and organs in the absence of amino acid absorption from the intestines; they also replenish glucogenic precursors in the liver and play a central role in systemic protein metabolism.

[0008] Proteins influence various aspects of the body, including hormone formation, metabolic regulation, and bone and joint health. During the digestive process, proteins break down into amino acid subunits as peptide bonds are severed, resulting in physiological activities distinct from their original functions. Free amino acids, oligopeptides, and low-molecular-weight proteins produced during this breakdown are representative physiologically active materials; research results have reported that certain proteins and peptides enhance antioxidant function, exhibit anti-inflammatory effects, and possess antibacterial properties. The production of physiologically active peptides is generally achieved through enzymatic hydrolysis, and the specific enzyme can be selected according to the intended purpose. However, peptide production may be limited by the protein structure during enzymatic hydrolysis; to address this, protein denaturation can be induced using heat, pH, ions, or physical pressure.

[0009] Branched-chain amino acids (BCAAs) such as leucine, isoleucine, and valine, which are known to be essential for maintaining normal protein metabolic balance and synthesis processes in humans, can reduce muscle damage and promote muscle protein synthesis. In particular, leucine has been reported to restore muscle protein synthesis, increase metabolic responses, and enhance skeletal muscle performance when consumed in sufficient quantities after exercise (Dickinson, 2014). Whey protein concentrate (WPC), which is high in BCAA content, has a high protein content of 70–85%, but it causes gastrointestinal disorders in people with lactose intolerance due to the 5% lactose content. Soy protein, a plant-based protein, is lacking in improving muscle strength due to concerns regarding phytate anti-nutrients and the promotion of female hormone secretion, as well as a low amount of essential amino acids for muscle strength such as leucine.

[0010] There is a need for research on the muscle-strengthening effects of hydrolysates produced by fermenting protein-rich mealworms with lactic acid bacteria, as these materials can complement the strengths and weaknesses of animal proteins (such as whey) and plant proteins (such as soybeans). The problem to be solved

[0012] Against the background described above, the inventors [discuss] Lactobacillus plantarum (brown mealworm extract) Lactobacillus plantarum ) or Lactobacillus brevis ( Lactobacillus brevis The present invention was completed by confirming the muscle-increasing effect of the fermented product of ).

[0013] This specification provides a muscle-strengthening composition comprising a microbial fermented product of mealworm extract.

[0014] Another example provides a composition for preventing, improving, and / or treating muscle loss-related diseases, comprising a microbial fermented product of mealworm extract.

[0015] Another example provides a muscle strengthening method comprising the step of administering and / or feeding the above-mentioned microbial fermentation product and / or composition to a subject requiring muscle strengthening.

[0016] Another example provides a method for preventing, improving, and / or treating a muscle loss-related disease, comprising the step of administering or feeding the above-mentioned microbial fermentation product and / or composition to a subject who requires prevention, improvement, and / or treatment of a muscle loss-related disease. means of solving the problem

[0017] This specification describes a muscle-strengthening composition comprising a microbial fermented product of mealworm extract, specifically Lactobacillus ( Lactobacillus A muscle-strengthening composition comprising a microbial fermentation product is provided.

[0018] Another example is a composition for the prevention, improvement, and / or treatment of muscle loss-related diseases comprising a microbial fermentation product of mealworm extract, specifically Lactobacillus ( Lactobacillus A composition for preventing, improving, and / or treating muscle loss-related diseases, comprising a fermented microbial product of the genus )

[0019] The above Lactobacillus ( Lactobacillus Microorganisms of the genus ) are Lactobacillus plantarum ( Lactobacillus plantarum ) or Lactobacillus brevis ( Lactobacillus brevis It may be, but is not limited to.

[0020] The above composition may be a feed composition, a food composition, a pharmaceutical composition, or a beverage composition, but is not limited thereto.

[0021] Another example provides a muscle strengthening method comprising the step of administering and / or feeding the above-mentioned microbial fermentation product and / or composition to a subject requiring muscle strengthening.

[0022] Another example provides a method for preventing, improving, and / or treating a muscle loss-related disease, comprising the step of administering or feeding the above-mentioned microbial fermentation product and / or composition to a subject who requires prevention, improvement, and / or treatment of a muscle loss-related disease.

[0023] Another example provides the use of the above microbial fermented product and / or composition for muscle strengthening and / or the use of the above microbial fermented product and / or composition for preparing a muscle strengthening composition.

[0024] Another example provides the use of the microbial fermented product and / or composition for the prevention, improvement, and / or treatment of muscle loss-related diseases and for the preparation of a composition for the prevention, improvement, and / or treatment of muscle loss-related diseases.

[0025] The above composition may have a muscle-increasing effect, and individuals administered and / or fed the microbial fermented product and / or composition of the brown mealworm extract may have a higher amount of muscle increase and / or muscle loss due to disease, etc., compared to individuals not administered and / or fed the microbial fermented product and / or composition of the brown mealworm extract.

[0027] The present invention will be described in more detail below.

[0029] Mealworm extract and its fermented product

[0030] In this specification, "mealworm ( Tenebrio molitor ; T. molitorThe brown mealworm belongs to the family Tenebrionidae of the order Coleoptera and is a holometabolous insect that undergoes egg, larva, pupa, and adult stages, and is distributed worldwide, including in Korea. The brown mealworm is known to be effective for liver disease as a high-protein material, and when hydrolyzed, it exhibits excellent antioxidant activity, such as scavenging free radicals and hydrogen peroxide. The brown mealworm may refer to one or more selected from the group consisting of the eggs, larvae, pupae, and adults of the brown mealworm, and specifically, it may be a brown mealworm larva.

[0031] The above mealworm extract may be used with an appropriate extraction solvent selected as needed, and the extraction solvent may be one or more selected from the group consisting of alcohols having 1 to 4 carbon atoms and water. For example, it may be water, straight-chain or branched-chain alcohols having 1 to 4 carbon atoms, and mixtures thereof. The extraction method for preparing the above extract may use a conventional extraction method, for example, extraction with an extraction solvent, supercritical fluid extraction, ultrasonic extraction, hot water extraction, etc.

[0032] In this specification, "extract" may be a hot water extract, solvent extract, distillation extract, ultrasonic extract, press extract, supercritical extract, and / or ultra-high pressure extract, and may be extracted by methods of hot water extraction, solvent extraction, distillation extraction, ultrasonic extraction, press extraction, supercritical extraction, and / or ultra-high pressure extraction, but is not limited thereto.

[0033] The above mealworm extract comprises mealworm powder in an amount of 1 to 10 (w / v)%, 1 to 8 (w / v)%, 1 to 6 (w / v)%, 1 to 5 (w / v)%, 1 to 4.7 (w / v)%, 3 to 10 (w / v)%, 3 to 8 (w / v)%, 3 to 6 (w / v)%, 3 to 5 (w / v)%, 3 to 4.7 (w / v)%, 4 to 10 (w / v)%, 4 to 8 (w / v)%, 4 to 6 (w / v)%, 4 to 5 (w / v)%, 4 to 4.7 (w / v)%, 4.5 to 10 (w / v)%, 4.5 to 8 (w / v)%, or 4.5 to 6 based on the total volume of the extract. It may be included at a concentration of (w / v)%, 4.5 to 4.7 (w / v)%, for example, 4.5 (w / v)%, but is not limited thereto.

[0034] The mealworm, which serves as the raw material for the above-mentioned fermented product, can be powdered by one or more methods selected from the group consisting of hot air drying, natural drying, microwave drying, and freeze-drying, and can be used in powder form. If necessary, additional washing and fasting steps may be performed prior to the drying step. The fasting step is a step for emptying the internal organs of the mealworm, and within the scope of the purpose, it may be carried out by stopping the feeding of the mealworm or by feeding glutinous rice flour, but is not limited thereto.

[0035] The above pulverization process can be performed by appropriately utilizing grinding and resting times as needed, and by having a resting time, it has the advantage of minimizing damage to the mealworm larva sample caused by heat generated in the grinder. In one embodiment, mealworm larva powder can be produced by repeating a grinding cycle of grinding for 1 minute followed by resting for 3 minutes five times.

[0036] The above mealworm extract can be prepared using mealworms, dried mealworms, and / or freeze-dried mealworms, and if necessary, mealworms, dried mealworms, and / or freeze-dried mealworms can be powdered and used.

[0037] The above mealworm extract may be sterilized.

[0038] The above sterilization treatment may be heat treatment at a temperature of 100 to 150°C, 100 to 130°C, 100 to 125°C, 110 to 150°C, 110 to 130°C, 110 to 125°C, 120 to 150°C, 120 to 130°C, 120 to 125°C, for example, 121°C, but is not limited thereto. The above sterilization treatment may be heat treatment for 5 to 30 minutes, 5 to 20 minutes, 5 to 15 minutes, 10 to 30 minutes, 10 to 20 minutes, 10 to 15 minutes, 15 to 30 minutes, 15 to 20 minutes, for example, 15 minutes, but is not limited thereto.

[0039] The above microbial fermentation product is Lactobacillus ( Lactobacillus Microorganisms of the genus ) (specifically, Lactobacillus plantarum ( Lactobacillus plantarum ) and / or Lactobacillus brevis ( Lactobacillus brevis It may be a fermented product by )), but is not limited thereto.

[0040] The microbial fermented product of the above mealworm extract can be used as a raw material for fermentation in its original form, or as a fermentation raw material after undergoing post-processing steps such as centrifugation or filtration. To minimize quantitative loss in order to utilize the active substances of the mealworm, the entire mealworm extract may be used, or only the supernatant of the mealworm extract may be used.

[0041] The above-mentioned Lactobacillus plantarum microorganism may be the microorganism with deposit number KCCM13068P, deposited with the Korean Culture Collection Center (KCCM) on October 27, 2021.

[0042] The above-mentioned Lactobacillus brevis microorganism may be the microorganism with deposit number KCCM13069P, deposited with the Korean Culture Collection of Microorganisms (KCCM) on October 27, 2021.

[0043] The above microbial fermentation product can be produced through a process of inoculating the above mealworm extract with microorganisms of the genus Lactobacillus and fermenting it.

[0044] The above microbial fermented product may be fermented for 10 to 100 hours, 10 to 80 hours, 10 to 60 hours, 10 to 50 hours, 10 to 40 hours, 10 to 30 hours, 15 to 100 hours, 15 to 80 hours, 15 to 60 hours, 15 to 50 hours, 15 to 40 hours, 15 to 30 hours, 20 to 100 hours, 20 to 80 hours, 20 to 60 hours, 20 to 50 hours, 20 to 40 hours, 20 to 30 hours, for example, 24 hours, but is not limited thereto.

[0045] The above microbial fermentation product can be prepared through a process of adding a basic substance to the brown mealworm extract and inoculating it with microorganisms of the genus Lactobacillus to ferment it. Specifically, it can be prepared by adding a basic substance to the brown mealworm extract and culturing it, and the pH of the fermentation product can be adjusted and / or controlled (e.g., increased or decreased pH) due to the addition of the basic substance.

[0046] The above microbial fermented product has a pH of 6.7 to 8.0, 6.7 to 7.8, 6.7 to 7.6, 6.7 to 7.4, 6.7 to 7.2, 6.7 to 7.1, 6.7 to 7.0, 6.9 to 8.0, 6.9 to 7.8, 6.9 to 7.6, 6.9 to 7.4, 6.9 to 7.2, 6.9 to 7.1, 6.9 to 7.0, 7 to 8.0, 7 to 7.8, 7 to 7.6, 7 to 7.4, 7 to 7.2, 7 to 7.1, 7.1 to 8.0, 7.1 to 7.8, 7.1 to 7.6, 7.1 to 7.4, or 7.1 to 7.2, For example, it may be 7.0 or 7.2, but is not limited thereto.

[0047] The above microbial fermented product may include a basic substance, and specifically, the basic substance may be included at a concentration of 0.1 to 10 (v / v)%, 0.1 to 7 (v / v)%, 0.1 to 5 (v / v)%, 0.1 to 4 (v / v)%, 0.1 to 3 (v / v)%, 1 to 10 (v / v)%, 1 to 7 (v / v)%, 1 to 5 (v / v)%, 1 to 4 (v / v)%, 1 to 3 (v / v)%, 2 to 10 (v / v)%, 2 to 7 (v / v)%, 2 to 5 (v / v)%, 2 to 4 (v / v)%, 2 to 3 (v / v)%, for example, 3 (v / v)% relative to the total volume of the mealworm extract, but is not limited thereto.

[0048] The above basic substances may be one or more selected from the group consisting of sodium hydroxide (NaOH) and potassium hydroxide (KOH), but are not limited thereto.

[0050] Uses for muscle strengthening and / or prevention, improvement, and / or treatment of conditions related to muscle loss

[0051] This specification provides a muscle-strengthening composition comprising a microbial fermented product of mealworm extract.

[0052] In this specification, "muscle strengthening" may refer to any action that exhibits a beneficial effect on the quantity and / or function of muscles, such as increasing muscle mass, increasing muscle strength, inhibiting muscle loss (muscle reduction), recovering, improving, and / or enhancing lost or reduced muscle exercise capacity, inhibiting muscle reduction caused by diseases that induce muscle loss, and / or strengthening muscle exercise capacity.

[0053] In this specification, "muscle increase" means an increase in muscle mass, mass, volume, and / or density of skeletal muscle cells or skeletal muscle compared to before administration of a Lactobacillus genus microbial ferment of mealworm extract and / or a composition containing said ferment.

[0054] This specification provides a composition for preventing, improving, and / or treating muscle loss-related diseases, comprising a microbial fermented product of mealworm extract.

[0055] The "muscle loss-related disease" in this specification may be one or more diseases selected from the group consisting of sarcopenia, muscular atrophy, muscular dystrophy, hypotonia, myasthenia gravis, myosophagosis, and amyotrophic lateral sclerosis (ALS, Lou Gehrig's disease), but is not limited thereto.

[0056] In this specification, "sarcopenia" refers to a symptom or disease resulting from a gradual decrease in skeletal muscle mass due to certain factors, such as aging, and may refer to a pathological condition that directly causes a decline in muscle strength and, as a result, may cause a decrease and / or impairment of various physical functions.

[0057] In this specification, "muscular atrophy" refers to the progressive atrophy of the muscles of the limbs, which may cause progressive degeneration of motor nerve fibers and cells in the spinal cord, leading to Amyotrophic Lateral Sclerosis (ALS) and / or Spinal Progressive Muscular Atrophy (SPMA). In one example, the muscular atrophy may be age-related muscular atrophy or obesity-related muscular atrophy. The "age-related muscular atrophy" may be caused by chronic low-grade inflammation, which involves a progressive loss of muscle mass and / or muscle strength due to aging and / or an increase in visceral fat accompanied by an increase in inflammatory cytokines. The aforementioned "obesity-induced muscular atrophy" refers to a phenomenon in which obesity, such as obesity in old age, further exacerbates muscle loss. It may be caused by the accumulation of fat within muscles due to obesity, which leads to a decrease in muscle mass, an increase in mast cell-derived inflammatory factors, and a decline in the function of mitochondria within the muscles, which further weakens muscle function; additionally, if abnormalities in insulin secretion occur due to obesity, the inability to properly supply energy to cells leads to impaired muscle development.

[0058] In this specification, "muscular dystrophy" is also referred to as muscular degenerative atrophy and is a disease characterized by progressive muscle atrophy and muscle weakness, and is one of the degenerative myopathy pathologically characterized by necrosis of muscle fibers.

[0059] In this specification, "atony" is also referred to as hypotonia and may mean a state in which muscle tension is reduced or lost, or a pathological condition resulting therefrom. Muscle tension is regulated by reflexes in addition to central control; when this regulatory function is disrupted, it may be a disease caused by increased or lost tension.

[0060] In this specification, "myasthenia" refers to a disease in which muscle contractility is reduced and muscles become easily fatigued and lose strength, such as myasthenia gravis, myasthenia gravis, and myasthenia gravis, but generally it may refer to myasthenia gravis. Myasthenia gravis is a disease caused by impaired neurotransmission at the neuromuscular junction and may exhibit pathological findings such as changes in the morphology of the postsynaptic membrane at the neuromuscular junction and a decrease in the number of nicotinic acetylcholine receptors.

[0061] In this specification, "muscle rigidity" refers to a condition in which the muscle stretch reflex is excessively excited due to central nervous system diseases such as brain and spinal cord diseases and injuries, resulting in increased muscle tone in proportion to the speed of movement.

[0062] In this specification, "amyotrophic lateral sclerosis (ALS, Lou Gehrig's disease)" refers to a disease characterized by the selective death of motor neurons, specifically the progressive destruction of upper motor neurons in the cerebral cortex and lower motor neurons in the brainstem and spinal cord.

[0063] The above microbial fermentation product and / or composition may contain free amino acids and / or constituent amino acids in a higher amount than in a mealworm extract in which lactic acid fermentation has not taken place.

[0064] Specifically, the microbial fermentation product and / or composition may contain one or more amino acids selected from the group consisting of methionine, cysteine, leucine, isoleucine, and valine in a higher amount than the brown mealworm extract, but is not limited thereto.

[0065] In this specification, "free amino acid" refers to an amino acid that exists as a single molecule, unlike bound amino acids such as proteins or peptides, and "constituent amino acid" refers to an amino acid that constitutes a protein.

[0066] In this specification, "methionine" may have functions of skeletal muscle formation, growth, antioxidant effect, reduction of high-temperature stress, inhibition of fatty liver, and liver detoxification.

[0067] In this specification, "cysteine" may have protein biosynthetic functions and efficacy against angina, cardiovascular disease, bronchitis, diabetes, influenza, arthritis, etc.

[0068] The total content of leucine, isoleucine, and valine of the above microbial fermented product and / or composition may be 2 to 20 times, 2 to 15 times, 2 to 12 times, 2 to 11.5 times, 2 to 11.1 times, 5 to 20 times, 5 to 15 times, 5 to 12 times, 5 to 11.5 times, 5 to 11.1 times, 6 to 20 times, 6 to 15 times, 6 to 12 times, 6 to 11.5 times, 6 to 11.1 times, 6.1 to 20 times, 6.1 to 15 times, 6.1 to 12 times, 6.1 to 11.5 times, or 6.1 to 11.1 times compared to the mealworm extract in which lactic acid fermentation has not proceeded, but It is not limited.

[0069] The total protein content of the above microbial fermentation product and / or composition may be 1.01 times or more, 1.05 times or more, 1.1 times or more, 1.2 times or more, 1.3 times or more, 1.4 times or more, 1.01 times to 10 times, 1.05 times to 10 times, 1.1 times to 10 times, 1.2 times to 10 times, 1.3 times to 10 times, or 1.4 times to 10 times compared to the mealworm extract in which lactic acid fermentation has not taken place, for example, 1.06 times or 1.4 times, but is not limited thereto.

[0070] In this specification, "degree of hydrolysis" refers to the degree of fragmentation of a mixture of peptides and / or amino acids produced by the combined activity of proteases, peptidases, and / or amidases. A high degree of hydrolysis allows proteins to be effectively broken down into peptides and amino acids without the need for digestive enzymes produced in the digestive tract, salivary glands, gastric juice, pancreas, etc., and promotes absorption into the body, thereby helping to improve the function and activity of cells and tissues in a short period of time. The degree of hydrolysis may be measured using a hydrolysis measurement method generally performed using a TNBS (trinitrobenzene sulfonic acid; G-Bio science, USA) solution, but is not limited thereto.

[0071] The degree of hydrolysis of the above microbial fermented product and / or composition is 1.01 times or more, 1.03 times or more, 1.05 times or more, 1.07 times or more, 1.1 times or more, 1.15 times or more, 1.19 times or more, 2 times or more, 2.3 times or more, 3 times or more, 5 times or more, 7 times or more, 1.01 to 10 times, 1.03 to 10 times, 1.05 to 10 times, 1.07 to 10 times, 1.1 to 10 times, 1.15 to 10 times, 1.09 to 10 times, 2 to 10 times, 2.3 to 10 times, 3 to 10 times, 5 to 10 times, or 7 to 10 times, for example, compared to a mealworm extract in which lactic acid fermentation has not proceeded. It may be 1.07 times, 1.20 times, 5.8 times, or 7.6 times, but is not limited thereto.

[0072] In this specification, "branched-chain amino acid (BCAA)" refers to a combination of three amino acids—leucine, isoleucine, and valine—that is involved in muscle building and helps strengthen muscles.

[0073] The BCAA content of the above microbial fermented product and / or composition is at least 1.1 times, at least 1.2 times, at least 1.3 times, at least 1.4 times, at least 1.5 times, at least 5 times, at least 7 times, at least 10 times, at least 12 times, at least 13 times, at least 15 times, at least 17 times, at least 20 times, 1.1 to 50 times, 1.2 to 50 times, 1.3 to 50 times, 1.4 to 50 times, 1.5 to 50 times, 5 to 50 times, 7 to 50 times, 10 to 50 times, 12 to 50 times, 13 to 50 times, 15 to 50 times, 17 to 50 times, or 20 to 50 times, for example, compared to the content of the mealworm extract in which lactic acid fermentation has not taken place. It may be 1.29 times, 1.35 times, 14 times, or 20 times, but is not limited thereto.

[0074] Unlike the mealworm extract in which lactic acid fermentation has not proceeded, the above microbial fermentation product and / or composition may have a higher proportion of peptides with relatively lower molecular weights due to internal hydrolysis, and specifically, may include peptides with molecular weights of 80 kDa or less, 70 kDa or less, 60 kDa or less, 50 kDa or less, 40 kDa or less, 30 kDa or less, 20 kDa or less, 15 kDa or less, 10 kDa or less, 1 to 80 kDa, 1 to 70 kDa, 1 to 60 kDa, 1 to 50 kDa, 1 to 40 kDa, 1 to 30 kDa, 1 to 20 kDa, 1 to 15 kDa, or 1 to 10 kDa, but are not limited thereto.

[0075] The above microbial fermentation product and / or composition may reduce the expression of related factors that inhibit muscle growth, muscle differentiation, etc., and induce muscle atrophy compared to a mealworm extract that has not undergone lactic acid fermentation, and specifically, may reduce the expression of one or more genes selected from the group consisting of Myostatin and Atrogin, but is not limited thereto. The "Myostatin" may inhibit muscle differentiation and proliferation, and the "Atrogin" may induce muscle atrophy.

[0076] The lean body mass of an individual with induced muscle atrophy to which the above-mentioned microbial fermentation product and / or composition is administered and / or fed may be 1.02 times or more, 1.04 times or more, 1.06 times or more, 1.07 times or more, 1.09 times or more, 1.1 times or more, 1.02 times to 10 times, 1.04 times to 10 times, 1.06 times to 10 times, 1.07 times to 10 times, 1.09 times to 10 times, or 1.1 times to 10 times, for example, 1.07 times or 1.1 times, compared to the lean body mass of an individual with induced muscle atrophy to which the above-mentioned fermentation product and / or composition is not administered and / or fed, but is not limited thereto.

[0077] The body fat mass of an individual with induced muscle atrophy to which the above-mentioned microbial fermentation product and / or composition is administered and / or fed may be 0.9 times or less, 0.85 times or less, 0.81 times or less, 0.8 times or less, 0.75 times or less, 0.73 times or less, 0.1 times to 0.9 times, 0.1 times to 0.85 times, 0.1 times to 0.81 times, 0.1 times to 0.8 times, 0.1 times to 0.75 times, or 0.1 times to 0.73 times, for example, 0.72 times or 0.8 times, compared to the body fat mass of an individual with induced muscle atrophy to which the above-mentioned fermentation product and / or composition is not administered and / or fed, but is not limited thereto.

[0078] The quadriceps / body weight ratio of an individual with induced muscle atrophy to which the above-mentioned microbial fermentation product and / or composition is administered and / or fed may be 1.5 times or more, 1.7 times or more, 1.9 times or more, 2 times or more, 2.1 times or more, 2.2 times or more, 2.3 times or more, 1.5 times to 10 times, 1.7 times to 10 times, 1.9 times to 10 times, 2 times to 10 times, 2 times to 10 times, 2 times to 10 times, 2 times to 10 times, 2 times to 10 times, or 2.3 times to 10 times, e.g., 2.02 times or 2.35 times, but is not limited thereto.

[0079] The calf muscle / body weight ratio of an individual with induced muscle atrophy to which the above-mentioned microbial fermentation product and / or composition is administered and / or fed may be 1.05 times or more, 1.1 times or more, 1.15 times or more, 1.2 times or more, 1.05 times to 10 times, 1.1 times to 10 times, 1.15 times to 10 times, or 1.2 times to 10 times, e.g., 1.14 times or 1.26 times, compared to the calf muscle / body weight ratio of an individual with induced muscle atrophy to which the above-mentioned fermentation product and / or composition is not administered and / or fed, but is not limited thereto.

[0080] "Lean body mass" in this specification refers to the remaining weight after subtracting the proportion of fat from total body weight.

[0081] "Body fat mass" in this specification refers to the proportion of fat in the total body weight.

[0082] In this specification, "muscular endurance exercise performance" is evaluated by having experimental animals (e.g., mice) run on an incline for a certain period of time with increasing speed, specifically by running on a 10% incline starting at a speed of 10 m / min and increasing the speed by 2 m / min every 2 minutes for 20 minutes, and when the experimental animal does not respond to electrical stimulation for 10 seconds, it is considered exhausted and that point is set as the "exercise end time related to muscular endurance exercise performance" (minute).

[0083] The exercise end time related to the muscle endurance exercise performance of an individual with induced muscle atrophy to whom the above-mentioned microbial fermentation product and / or composition has been administered and / or fed may be 1.01 times or more, 1.03 times or more, 1.05 times or more, 1.07 times or more, 1.09 times or more, 1.1 times or more, 1.01 times to 10 times, 1.03 times to 10 times, 1.05 times to 10 times, 1.07 times to 10 times, 1.09 times to 10 times, or 1.1 times to 10 times, e.g., 1.05 or 1.16 times longer than the exercise end time of an individual with induced muscle atrophy to whom the above-mentioned fermentation product and / or composition has not been administered and / or fed, but is not limited thereto.

[0084] In this specification, "sense of balance" is evaluated by rotating an experimental animal (e.g., a mouse) on a Rota-rod at a specific rotational speed and then increasing the rotational speed to a certain speed, specifically rotating the experimental animal at a rotational speed of 4 rpm on the Rota-rod and gradually accelerating the rotational speed to 30 rpm, and then rotating the experimental animal to reach a final speed of 30 rpm within a maximum duration of 5 minutes, and then the time at which the experimental animal falls to the bottom of the equipment due to fatigue or loss of balance, and that time is defined as the "point of end of balance-related exercise" (minute).

[0085] The end point of balance-related exercise in an individual with muscle atrophy to whom the above-mentioned microbial fermentation product and / or composition has been administered and / or fed may be 1.1 times or more, 1.2 times or more, 1.3 times or more, 1.4 times or more, 1.5 times or more, 1.1 to 10 times, 1.2 to 10 times, 1.3 to 10 times, 1.4 to 10 times, or 1.5 to 10 times, for example, 1.4 times or 1.5 times longer than the end point of exercise in an individual with muscle atrophy to whom the above-mentioned fermentation product and / or composition has not been administered and / or fed, but is not limited thereto.

[0086] The above microbial fermentation product and / or composition may have a superior muscle-strengthening effect, an effect of enhancing muscle endurance exercise performance, and / or an effect of enhancing balance compared to a mealworm extract not fermented by the above lactic acid bacteria.

[0087] Another example provides an antioxidant composition comprising a microbial fermentation product of the above-mentioned mealworm extract.

[0088] Another example provides a composition for alleviating inflammation comprising a microbial fermentation product of the above-mentioned mealworm extract.

[0089] The above microbial fermentation product and / or composition may have antioxidant activity, specifically free radical scavenging activity.

[0090] In this specification, "antioxidant" refers to the action of inhibiting oxidation. Although the human body maintains a balance between prooxidants and antioxidants, various factors can disrupt this balance, causing it to shift toward oxidation promotion. This leads to oxidative stress, which induces potential cell damage and pathological diseases. Reactive oxygen species (ROS), which are the direct cause of such oxidative stress, are unstable and highly reactive. They readily react with various biomaterials and attack macromolecules within the body, causing irreversible damage to cells and tissues, or resulting in mutations, cytotoxicity, and cancer.

[0091] The above microbial fermentation product and / or composition may have an anti-inflammatory effect. Specifically, nitric oxide is a substance involved in the generation of inflammation, and this can be confirmed by adding the above microbial fermentation product and / or composition to cells and measuring the change in nitric oxide (NO) production in the cells; in particular, the anti-inflammatory effect can be confirmed by confirming the inhibition of nitric oxide production.

[0093] Methods for strengthening muscles and / or preventing, improving, and / or treating conditions related to muscle loss

[0094] Another example provides a muscle strengthening method comprising the step of administering and / or feeding the above-mentioned microbial fermentation product and / or composition to a subject requiring muscle strengthening.

[0095] Another example provides a method for preventing, improving, and / or treating a muscle loss-related disease, comprising the step of administering and / or feeding the above-mentioned microbial fermentation product and / or composition to a subject who requires prevention, improvement, and / or treatment of a muscle loss-related disease.

[0096] The subject may be an individual whose muscle mass has decreased due to a muscle loss-inducing disease and / or a muscle loss-related disease, specifically, an individual with induced muscle atrophy, an individual with induced muscle loss, an individual with induced muscle damage, and / or an individual with a weak-exercise syndrome (specifically, a weak-exercise syndrome of muscle tissue caused by repetitive movements, excessive training, or prolonged fatigue), but is not limited thereto.

[0097] The above subject may be an animal, and the animal may be fish (e.g., cherry salmon, flounder (spotted flounder, stone flounder), halibut, rockfish, trout (rainbow trout), mullet, yellowtail, eel, carp, ornamental fish, catfish, herring, sea bass, cod, anglerfish, pufferfish, scorpionfish, horse mackerel, skate, smelt, etc.), crustaceans (e.g., shrimp, etc.), birds (e.g., chicken, duck, goose, etc.), cattle (e.g., cow, etc.), pigs (e.g., pig, etc.), canines (e.g., dog, wolf, fox, coyote, jackal, etc.), felines (e.g., lion, tiger, domestic cat, wild cat, other big cats, cheetah, lynx, etc.), equine (e.g., horse, etc.), sheep (e.g., sheep, goat, lamb, bison, etc.), primates The animals may include reptiles, amphibians, etc. (e.g., humans, prosimians, tarsiers, monkeys, gibbons, apes, etc.), reptiles, amphibians, etc., and specifically may include birds such as chickens (e.g., broiler chickens, laying hens, etc.), ducks, geese, turkeys, quails, pheasants, parrots, finches, hawks, crows, ostriches, emus, and cassowaries, but are not limited thereto. The animals may include individual animals at all developmental stages, such as embryonic and fetal stages, but are not limited thereto. Additionally, humans may be excluded from the animals.

[0098] The above-mentioned subject may be a part of a living organism (e.g., a myoblast), specifically fibrous tissue and / or organ found in the muscle tissue, skeletal muscle, cardiac muscle, and visceral muscle of animals, and / or tissue taken from organs, tissues and / or mother cells of animals and plants, but is not limited thereto.

[0100] Types of compositions

[0101] The compositions provided herein may be intended for use in animals, and may be used in fish (e.g., cherry salmon, flounder (spotted flounder, stone flounder), halibut, rockfish, trout (rainbow trout), mullet, yellowtail, eel, carp, ornamental fish, catfish, herring, sea bass, cod, anglerfish, pufferfish, scorpionfish, horse mackerel, skate, smelt, etc.), crustaceans (e.g., shrimp, etc.), birds (e.g., chicken, duck, goose, etc.), cattle (e.g., cow, etc.), pigs (e.g., pig, etc.), canids (e.g., dog, wolf, fox, coyote, jackal, etc.), felines (e.g., lion, tiger, domestic cat, wild cat, other large cats, cheetah, lynx, etc.), equine (e.g., horse, etc.), sheep (e.g., sheep, goat, lamb, bison, etc.), primates The animals may include reptiles, amphibians, etc. (e.g., humans, prosimians, tarsiers, monkeys, gibbons, apes, etc.), reptiles, amphibians, etc., and specifically may include birds such as chickens (e.g., broiler chickens, laying hens, etc.), ducks, geese, turkeys, quails, pheasants, parrots, finches, hawks, crows, ostriches, emus, and cassowaries, but are not limited thereto. The animals may include individual animals at all developmental stages, such as embryonic and fetal stages, but are not limited thereto. Additionally, humans may be excluded from the animals.

[0102] The compositions provided in this specification may be adjuvants, additives, and / or compositions of differentiation promoters for myoblasts—stem cells capable of differentiating into muscle cells—to fully differentiate into specific cells or to a pre-differentiation stage, but are not limited thereto. Specifically, they may be differentiation adjuvants and / or promoters for primary cultures in which cells are cultured from fibrous tissues or organs found in most animal muscle tissues, skeletal muscle, cardiac muscle, and visceral muscle; tissue cultures in which tissues are cultured from organs, tissues, or parent cells of animals and plants; and organ cultures in which a part of an organism is isolated and cultured while preserving its three-dimensional structure and function, but are not limited thereto.

[0103] In this specification, the "fermented product" and / or "composition" may be administered by various routes to a selected subject among humans, primates such as monkeys, rodents such as mice, rats, and rabbits, mammals including dogs, cats, cattle, pigs, sheep, horses, goats, etc., birds including chickens, ducks, geese, etc., reptiles including snakes, lizards, turtles, crocodiles, etc., amphibians, and vertebrates such as fish, or animals other than humans (primates, vertebrates, etc.).

[0104] The microbial fermentation product and / or composition of the above extract can inhibit muscle loss caused by muscle loss-related diseases.

[0105] The above composition may be a feed composition, a food composition, a pharmaceutical composition, or a food composition, but is not limited thereto.

[0106] In this specification, the term "feed" may refer to food given to livestock (animals, etc.), but is not limited thereto.

[0107] In the food compositions provided in this specification, the term "food" refers to an edible natural or processed product containing one or more nutrients, and in a conventional sense, may be used to refer to one or more selected from the group consisting of various general foods, health functional foods, beverages, food additives, and beverage additives. The term "food composition" may refer to a combination of materials for manufacturing said food.

[0108] The content of the herbal ingredient in the above food composition can be appropriately adjusted according to the form of food use, purpose, etc., and, for example, may be 0.00001 to 99.9 weight%, 0.0001 to 99.9 weight%, 0.001 to 99.9 weight%, or 0.001 to 50 weight% based on the weight of the solid content, but is not limited thereto.

[0109] The above-mentioned health functional food refers to any food manufactured using ingredients (hereinafter referred to as "functional ingredients") that have functions useful to the human body or nutrients that are easily deficient in daily meals, and which helps maintain health or prevent and / or improve (alleviate) certain diseases or symptoms, and there are no special restrictions on the final product form. For example, the above-mentioned health functional food may be selected from a group consisting of various ordinary foods, food supplements, beverages, food additives, etc., but is not limited thereto. The above-mentioned health functional food may be in solid, semi-solid, or liquid form, and specifically may be formulated as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., but is not limited thereto.

[0110] The above-mentioned health functional food may additionally contain one or more selected from the group consisting of various nutritional supplements, vitamins, minerals (electrolytes), flavoring agents such as synthetic or natural flavoring agents, coloring agents, thickening agents (cheese, chocolate, etc.), pectic acid or its salt, alginic acid or its salt, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. The proportion of such additives is generally selected in the range of 0.001 to about 20 parts by weight per 100 parts by weight of the total health functional food, but is not limited thereto.

[0111] The mode of administration of the pharmaceutical composition provided in this specification may be any commonly used mode, and may be administered, for example, by oral administration, or by a non-parenteral route such as intravenous administration, intramuscular administration, subcutaneous administration, intraperitoneal administration, local administration to a lesion site (e.g., a site requiring prevention, treatment or improvement of inflammation, antibacterial action, etc.), or intraperitoneal injection.

[0112] The above pharmaceutical composition may be administered in a pharmaceutically effective amount. The dosage of the above pharmaceutical composition may be prescribed differently depending on factors such as the formulation method, method of administration, patient's age, weight, sex, pathological condition, food, time of administration, interval of administration, route of administration, excretion rate, and response sensitivity. The dosage may vary depending on the patient's age, weight, sex, form of administration, health condition, and degree of disease, and may be divided into one to several doses per day at regular intervals at the discretion of a physician or pharmacist. For example, a single dose or daily dose of a pharmaceutical composition is 0.001 to 10,000 mg / kg based on the weight of the solid content of the active ingredient (plant extract), specifically, 0.01 to 10,000 mg / kg, 0.01 to 5,000 mg / kg, 0.01 to 3,000 mg / kg, 0.01 to 2,500 mg / kg, 0.01 to 2,000 mg / kg, 0.01 to 1,800 mg / kg, 0.1 to 10,000 mg / kg, 0.1 to 5,000 mg / kg, 0.1 to 3,000 mg / kg, 0.1 to 2,500 mg / kg, 0.1 to 2,000 mg / kg, 0.1 to 1,800 mg / kg, 1 to 10,000 mg / kg, 1 to The range may be 5000 mg / kg, 1 to 3000 mg / kg, 1 to 2500 mg / kg, 1 to 2000 mg / kg, 1 to 1800 mg / kg, 10 to 10000 mg / kg, 10 to 5000 mg / kg, 10 to 3000 mg / kg, 10 to 2500 mg / kg, 10 to 2000 mg / kg, 10 to 1800 mg / kg, 100 to 10000 mg / kg, 100 to 5000 mg / kg, 100 to 3000 mg / kg, 100 to 2500 mg / kg, 100 to 2000 mg / kg, or 100 to 1800 mg / kg, but is not limited thereto. The above single dose or daily dose may be formulated as a single preparation in the form of a unit dose, formulated in appropriate quantities, or manufactured by being contained in a multi-dose container.The above dosage is an example of an average case, and the dosage may be higher or lower depending on individual differences.

[0113] The above pharmaceutical compositions may each be formulated and used according to conventional methods into oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, or parenteral formulations such as sterile injectable solutions.

[0114] In addition to the active ingredient described above, the pharmaceutical composition may be administered by mixing with one or more adjuvants selected from the group consisting of pharmaceutically and / or physiologically acceptable carriers, excipients, and diluents, selected in consideration of the method of administration and standard pharmaceutical practice. For example, the pharmaceutically and / or physiologically acceptable carrier may include one or more selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil, but is not limited thereto, and may be any carrier commonly used in the pharmaceutical field.

[0115] The above pharmaceutically and / or physiologically acceptable diluents and / or excipients may be one or more selected from the group consisting of all fillers, extenders, binders, wetting agents, disintegrants, lubricants, surfactants, etc., commonly used in the proper formulation of pharmaceutical compositions.

[0116] For example, in the case of solid formulations for oral administration such as tablets, pills, powders, granules, or capsules, the excipient may be one or more selected from the group consisting of at least one substance for the formulation of such solid formulations, such as starch, calcium carbonate, sucrose, lactose, gelatin, etc. In addition, lubricants such as magnesium styrate talc may also be used. Furthermore, in the case of formulation of liquid formulations for oral administration such as suspensions, liquid formulations, emulsions, or syrups, one or more selected from the group consisting of commonly used simple diluents such as water and liquid paraffin may be used, and in addition, various commonly used excipients, such as humectants, sweeteners, flavorings, and / or preservatives, may be included, but are not limited thereto.

[0117] For example, the above pharmaceutical composition may be administered orally, intraorally, or sublingually in the form of tablets containing starch or lactose, in the form of capsules containing suitable excipients, or in the form of elixirs or suspensions containing chemicals for flavoring or coloring. These liquid formulations may be formulated with pharmaceutically acceptable additives such as suspensions (e.g., glyceride mixtures such as methylcellulose, semi-synthetic glycerides such as Witepsol, or mixtures of apricot kernel oil and PEG-6 esters, or mixtures of PEG-8 and caprylic / capric glycerides), but are not limited thereto. Effects of the invention

[0118] The present invention relates to a muscle-strengthening composition comprising a fermented product of Lactobacillus genus microorganisms of mealworm extract and the use thereof. The muscle-strengthening composition contains a high degree of hydrolysis and a high content of BCAA, and when administered to an individual, it is easily digested and has the effect of inducing muscle growth in the individual. Brief explanation of the drawing

[0119] Figure 1 is a graph showing the results of measuring the molecular weight of peptides contained in the mealworm extract, Lactobacillus plantarum fermented product, and Lactobacillus brevis fermented product. Figure 2 is a photograph showing the Western blot results confirming changes in muscle-related factors due to the intake of Lactobacillus plantarum ferment. Figure 3 is a photograph showing the results of an image analysis of the muscle mass of experimental animals when a fermented product of Lactobacillus microorganisms from mealworm extract was added to experimental animals. Specific details for implementing the invention

[0120] The present invention will be explained in more detail below through examples. However, the following examples are intended to illustrate the present invention and do not limit the scope of the invention.

[0122] Example 1. Preparation of mealworm powder

[0123] brown mealworm ( Tenebrio molitor) The larvae were raised for 15 to 18 hours at a temperature of 27°C and a humidity of 60%, and then fasted for 3 days to clean the internal organs of the mealworm larvae. Next, the mealworm larvae were washed under running water and then dried with a microwave to treat them for insecticide.

[0124] The above-mentioned dried mealworm larvae were prepared into powder using a grinder. Specifically, to prevent heat generation caused by the use of the grinder, a grinding cycle of grinding for 1 minute followed by a 3-minute rest was repeated 5 times to produce mealworm larva powder ( Tenebrio molitor ; Tm) was manufactured.

[0126] Example 2. Isolation and identification of lactic acid bacteria for the preparation of fermented product

[0127] Example 2-1. Isolation of lactic acid bacteria

[0128] For the preparation of a fermented product using lactic acid bacteria, Lactobacillus plantarum, a lactic acid bacteria strain having functions such as antibacterial activity and enzymatic activity ( Lactobacillus plantarum ) and Lactobacillus brevis ( Lactobacillus brevis ) was isolated and identified from seawater and used. Specifically, Lactobacillus plantarum and Lactobacillus brevis were isolated from sediment in a seawater aquaculture farm at the Gyeongbuk Fisheries Research Institute in June 2018.

[0129] The strain isolated from the above-mentioned seawater aquaculture sediment was plated onto DE MAN, ROGOSA and SHARPE (MRS) agar medium supplemented with 0.1% (v / v) bromocresol purple (BCP) and cultured at 37°C for 1 day. Afterward, a single species was isolated by repeatedly selecting yellow colonies formed by reaction with BCP.

[0130] The isolated strain was cultured in MRS liquid medium, mixed with 50% (v / v) glycerol to achieve a final glycerol concentration of 25%, and then stored at -70℃.

[0132] Example 2-2. Identification of Lactic Acid Bacteria

[0133] The strain isolated in Example 2-1 above was identified using 16S rRNA sequencing analysis. To identify the strain, the genomic DNA of the strain was isolated using a mini genomic DNA extraction kit (Promega).

[0134] Using the isolated genomic DNA above as a template, the 16S rRNA gene region of the strain was amplified using a TaKaRa PCR Thermal Cycler (Japan) with the following 27F (Sequence No. 1) and 1492R primer (Sequence No. 2) sets and the AccPower PCR premix kit (Bioneer, Korea), and the information of the primer sets is shown in Table 1 below.

[0135] primer Nucleotide sequence (5' → 3') Sequence number 27F primer AGAGTTTGATCMTGGCTCAG 1 1492R primer GGTTACCTTGTTACGACTT 2

[0136] The above PCR products were purified using PCR clean-up Gel extraction (Macherey Nagel, USA) and their gene sequences were analyzed using an ABI3730 DNA analyzer (Applied Bioxyxtems, USA). The 16S rDNA sequences of the isolated Lactobacillus plantarum and Lactobacillus brevis strains are shown in SEQ ID NOs 3 and 4, respectively, and the information is shown in Table 2 below.

[0137] In addition, 16S rRNA sequence similarity analysis was performed using the 16S rRNA sequence information of the above strain, and the results are shown in Table 3 below, the close species used ( Lactobacillus plantarum MT597760.1 and Lactobacillus brevis , MT597799.1) 16S rRNA information is shown in Table 2 below.

[0138] strain Nucleotide sequence (5' → 3') Sequence number Lactobacillus plantarum MIRE_TS55 GGGCGAAGTGCCGGGGGTGCCTGATACATGCAAGTCGAACGAACTCTGGTATTGATTGGTGCTTGCATCATGATTTACATTTGAGTGAGTGGCGAACTGGTGAGTAACACGTGGGAAACCTGCCCAGAAGCGGGGGATAACACCTGGAAACAGATGCTAATACCGCATAACAACTTGGACCGCATGGTCCGAGCTTGAAAGATGGCTTCGGCTATCACTTTTGGATGGTCCCGCGGCGTATTAGCTAGATGGTGGGGTAACGGCTCACCATGGCAATGATACGTAGCCGACCTGAGAGGGTAATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAACTCTGTTGTTAAAGAAGAACATATCTGAGAGTAACTGTTCAGGTATTGACGGTATTTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCTTCGGCTCAACCGAAGAAGTGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGTATGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCATACCGTAAACGATGAATGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATACTATGCAAATCTAAGAGATTAGACGTTCCCTTCGGGGACATGGATACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTATCAGTTGCCAGCATTAAGTTGGGCACTCTGGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAACGAGTTGCGAACTCGCGAGAGTAAGCTAATCTCTTAAAGCCATTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCAAAGTCGGTGGGGTAACCTTTAGGAACCAGCCGCTTAAGTGTACAGGATTCC 3 Lactobacillus brevis MIRE_TS66 CGGGAATGGGGGGCTGCCGTATACATGCAAGTCGAACGAGCTTCCGTTGAATGACGTGCTTGCACTGATTTCAACAATGAAGCGAGTGGCGAACTGGTGAGTAACACGTGGGGAATCTGCCCAGAAGCAGGGGATAACACTTGGAAACAGGTGCTAATACCGTATAACAACAAAATCCGCATGGATTTTGTTTGAAAGGTGGCTTCGGCTATCACTTCTGGATGATCCCGCGGCGTATTAGTTAGTTGGTGAGGTAAAGGCCCACCAAGACGATGATACGTAGCCGACCTGAGAGGGTAATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGAAAGTCTGATGGAGCAATGCCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAACTCTGTTGTTAAAGAAGAACACCTTTGAGAGTAACTGTTCAAGGGTTGACGGTATTTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCTTCGGCTTAACCGGAGAAGTGCATCGGAAACTGGGAGACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGGAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTAGTCTGTAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAGTGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATCTTCTGCCAATCTTAGAGATAAGACGTTCCCTTCGGGGACAGAATGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTATCAGTTGCCAGCATTCAGTTGGGCACTCTGGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGGTACAACGAGTTGCGAAGTCGTGAGGCTAAGCTAATCTCTTAAAGCCGTTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCAAAGCCGTTGAGATAACCTTCGGGAGTCAGCAGTCTAAGTGACAGGGGATCGCCG 4 Lactobacillus plantarum MT597760.1 CCGCAAGTGGCGGGGGTGCCTATACATGCAAGTCGAACGAACTCTGGTATTGATTGGTGCTTGCATCATGATTTACATTTGAGTGAGTGGCGAACTGGTGAGTAACACGTGGGAAACCTGCCCAGAAGCGGGGGATAACACCTGGAAACAGATGCTAATACCGCATAACAACTTGGACCGCATGGTCCGAGTTTGAAAGATGGCTTCGGCTATCACTTTTGGATGGTCCCGCGGCGTATTAGCTAGATGGTGGGGTAACGGCTCACCATGGCAATGATACGTAGCCGACCTGAGAGGGTAATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAACTCTGTTGTTAAAGAAGAACATATCTGAGAGTAACTGTTCAGGTATTGACGGTATTTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCTTCGGCTCAACCGAAGAAGTGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGTATGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCATACCGTAAACGATGAATGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATACTATGCAAATCTAAGAGATTAGACGTTCCCTTCGGGGACATGGATACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTATCAGTTGCCAGCATTAAGTTGGGCACTCTGGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAACGAGTTGCGAACTCGCGAGAGTAAGCTAATCTCTTAAAGCCATTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCAAAGTCGGTGGGGTAACCTTTAGGAACCAGCCGCCTAAGGTTGAACCGAATA 5 Lactobacillus brevis MT597799.1 CGGGGGGGGATGCCTATACATGCAAGTCGAACGAGCTTCCGTTGAATGACGTGCTTGCACTGATTTCAACAATGAAGCGAGTGGCGAACTGGTGAGTAACACGTGGGGAATCTGCCCAGAAGCAGGGGATAACACTTGGAAACAGGTGCTAATACCGTATAACAACAAAATCCGCATGGATTTTGTTTGAAAGGTGGCTTCGGCTATCACTTCTGGATGATCCCGCGGCGTATTAGTTAGTTGGTGAGGTAAAGGCCCACCAAGACGATGATACGTAGCCGACCTGAGAGGGTAATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGAAAGTCTGATGGAGCAATGCCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAACTCTGTTGTTAAAGAAGAACACCTTTGAGAGTAACTGTTCAAGGGTTGACGGTATTTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCTTCGGCTTAACCGGAGAAGTGCATCGGAAACTGGGAGACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGGAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTAGTCTGTAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAGTGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATCTTCTGCCAATCTTAGAGATAAGACGTTCCCTTCGGGGACAGAATGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTATCAGTTGCCAGCATTCAGTTGGGCACTCTGGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGGTACAACGAGTCGCGAAGTCGTGAGGCTAAGCTAATCTCTTAAAGCCGTTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCAAAGCCGGTGAGATAACCTTCGGGAGTCAGCCGTCTAAGTGACAGAATG 6

[0139] Lactic acid bacteria origin isolated strain Proximate 16S rRNA sequence identity Trustee number sea ​​water Lactobacillus plantarum MIRE_TS55(16S rRNA of sequence number 3) Lactobacillus plantarum MT597760.1 (Sequence No. 5) 99.6% KCCM13068P sea ​​water Lactobacillus brevis MIRE_TS66 (16S rRNA of sequence number 4) Lactobacillus brevis MT597799.1 (Sequence No. 6) 99.7% KCCM13069P

[0140] As shown in Table 3 above, Lactobacillus plantarum and Lactobacillus brevis isolated in Example 2-1 showed sequence similarities of 99.6% and 99.7%, respectively, with Lactobacillus plantarum strain (Accession No. GenBank accession no. MT597760.1) and Lactobacillus brevis strain (Accession No. GenBank accession no. MT597799.1) as a result of 16S rRNA analysis.

[0141] The strains isolated from the above seawater, respectively Lactobacillus plantarum MIRE_TS55 and Lactobacillus brevis It was named MIRE_TS66, and both strains were deposited with the Korean Culture Collection of Microorganisms (KCCM) on October 27, 2021, and received accession numbers KCCM13068P and KCCM13069P, respectively.

[0143] Example 3. Measurement of the antioxidant activity of lactic acid bacteria fermented mealworm extract

[0144] To confirm the antioxidant activity of the lactic acid bacteria fermented product of mealworm extract, a free radical scavenging activity analysis was performed using DPPH (2,2-diphenyl-1-picrylhydrazyl).

[0145] Specifically, a sample (Tm) was prepared by adding 45g (4.5 w / v%) of the mealworm larva powder prepared in Example 1 to 1L of water, sterilizing at 121℃ for 15 minutes, and cooling, and 100mL of the above sample was added to Example 2-2 Lactobacillus plantarum MIRE_TS55 1x10 8 A sample (TmLp) was prepared by inoculating 1 mL at a concentration of cfu / ml (1 v / v%) and incubating at 30°C for 24 hours to hydrolyze the protein in the sample, and a sample (TmLp_NaOH) was prepared by adding 3 mL (3%; v / v) of 1M NaOH before incubation and performing incubation, and 10 μL of each of the above samples was dispensed into a 96-well plate.

[0146] DPPH in methanol (Sigma, USA) (Sigma, USA; C 18 H 12 0.039432 g of N5O6 (394.32 g / mol) was dissolved to prepare 1 mM DPPH, and 50 μL of the 1 mM DPPH was mixed with 50 μL of 100 mM Tris-HCl (pH 7.4) to achieve a final DPPH concentration of 100 μM, which was used as the stock solution. 100 μL of the prepared stock solution was added to well plates containing the sample, and after being shielded from light, the plates were left at room temperature for 10 to 30 minutes. Subsequently, the absorbance was measured and verified by irradiating with light at a wavelength of 517 nm using a microplate spectrophotometer.

[0147] As a negative control, zero treatment was performed by treating methanol with 100 μL of DPPH and measuring the absorbance. The experiment was conducted in triplicate for each sample, and the antioxidant activity was calculated using Equation 1 below to obtain the average value. The results are shown in Table 4 below.

[0148] [Mathematical Formula 1]

[0149] Scavenging activity (%) = (1 - OD sample / OD blind) x 100

[0150] (In the above Equation 1, OD sample refers to the absorbance value at 517 nm of the mixture of 100 μM DPPH and each sample, and OD blind refers to the absorbance at 517 nm of 100 μM DPPH without sample addition.)

[0151] Sample types Scavenging activity (%) Tm 28.94 TmLp 35.58 TmLp_NaOH 37.1

[0152] As a result of confirming antioxidant activity, Lactobacillus plantarum It was confirmed that the antioxidant activity of the lactic acid bacteria fermented brown mealworm extract fermented by MIRE_TS55 is high.

[0154] Example 4. Preparation of Lactobacillus plantarum fermented mealworm extract and analysis of viable cell count, protein, and survival-related characteristics

[0155] Example 4-1. Measurement of changes in viable cell count according to pH adjustment of Lactobacillus plantarum fermented mealworm extract

[0156] Samples Tm, TmLp, and TmLp_NaOH were prepared by adding NaOH to the brown mealworm extract of Example 3, the lactic acid bacteria fermented product thereof, and NaOH before fermentation, and the pH and the number of viable cells in the samples were measured after preparation.

[0157] Specifically, the pH of the above Tm was measured immediately after preparation using a pH meter (Fisher Scientific, USA), and the pH of the above experimental samples TmLp and TmLp_NaOH was measured at the time when 24 hours of fermentation were completed. In addition, the above samples were diluted to 1 (v / v)% in DE MAN, ROGOSA and SHARPE (MRS) agar medium (BD Difco, USA), 1 ml of each diluted sample was plated on a LAB (Lactic Acid Bacteria) plate and incubated at 30°C for 24 hours to measure the number of colonies formed to determine the LAB (Lactic Acid Bacteria) Counts (log cfu / ml), and the results of the pH and LAB Counts measurements are shown in Table 5 below.

[0158] Sample name Final pH Viable cell (log cfu / ml) Tm 7.1 0.00E+00 TmLp 6.4 1.96E+09 TmLp_NaOH 7.2 1.90E+10

[0159] As a result of measuring pH and viable cell count, the pH of the fermented product was induced to neutral upon the addition of NaOH. Since the viable cell count of lactic acid bacteria did not decrease, it was confirmed that the addition did not have a significant effect on the growth or viable cell count of the bacteria. Furthermore, as expected, no lactic acid bacteria were detected at Tm, where no culture was performed.

[0161] Example 4-2. Measurement of protein hydrolysis degree of Lactobacillus plantarum fermented mealworm extract according to pH adjustment

[0162] A sample (fermented product) was prepared in substantially the same manner as in Example 4-1 above, and the degree of protein hydrolysis in the sample was measured.

[0163] Specifically, pH was measured in substantially the same manner as in Example 4-1 above, and 0.01% TNBS was prepared by mixing 10 μL of 1 (v / v)% TNBS (trinitrobenzene sulfonic acid; G-Bio science, USA) with 990 μL of 100 mM sodium bicarbonate (pH 8.5).

[0164] 100 μL each of the hydrolyzed fermented product prepared above was mixed with 50 μL of 0.01% TNBS and reacted at 37°C for 2 hours. After the reaction was completed, 50 μL of 10% SDS and 25 μL of 1N HCl were added in sequence to stop the reaction.

[0165] The TNBS standard value was determined by measuring the absorbance at 335 nm using a microplate spectrophotometer (BIOTek USA), preparing L-tyrosine at concentrations of 0.0001, 0.001, 0.01, 0.1, or 1 mg / mL, and constructing a standard curve according to the standard curve construction method included in the microplate spectrophotometer to obtain the formula in Equation 2 below. The absorbance of each measured sample was substituted into Equation 2 below to calculate the amino acid content concentration, and the degree of hydrolysis was confirmed by measuring the TNBS concentration. The results are shown in Table 6 below.

[0166] [Mathematical Formula 2]

[0167] Y = 0.1428X + 0.0896 (R 2 =0.9843)

[0168] (Y represents the measured absorbance value of each sample, and X represents the TNBS content)

[0169] Sample name Final pH TNBS (mg / mL) Tm 7.1 5.03 TmLp 6.4 11.63 TmLp_NaOH 7.2 29.21

[0170] As a result of measuring the degree of hydrolysis, the degree of hydrolysis of the TmLp_NaOH sample, which was adjusted to a final pH of 7.2 through a culture process with added NaOH, was measured to be higher than that of TmLp without added NaOH, confirming that the hydrolysis of mealworm protein proceeded more significantly due to pH adjustment.

[0172] Example 4-3. Analysis of Branched-Chain Amino Acids (BCAAs) in Lactobacillus plantarum Fermented Mealworm Extract According to pH Adjustment

[0173] A sample (fermented product) was prepared in substantially the same manner as in Example 4-1 above, and branched-chain amino acid analysis was performed on the sample.

[0174] Specifically, the quantification of branched-chain amino acids (BCAAs), which are a combination of three amino acids—leucine, isoleucine, and valine—in the prepared fermented peptide and are involved in muscle growth, was performed according to the Branched Chain Amino Acid assay kit (BCAA kit; Cell Biolabs, USA). Specifically, 100 μL of the prepared fermented hydrolysate peptide was rapidly homogenized in 100 μL of the BCAA assay buffer in the kit, centrifuged at 13,000 rpm at 4°C for 10 minutes, and only the supernatant was collected and used.

[0175] 50 μL of each prepared supernatant, 50 μL of the buffer solution (BCAA assay buffer) of the BCAA kit, 50 μL of 1x leucine dehydrogenase solution, and 50 μL of the sample reaction mixture (WST, NAD+, PBS) were placed in a 96-well plate and mixed thoroughly. The mixture was left in the dark at 25°C (room temperature) for 30 minutes until the reaction was complete, after which the absorbance was measured at 450 nm using a microplate spectrophotometer (BIOTek USA). At this time, a sample without the BCAA enzyme mix was used as a blank sample for measurement.

[0176] The BCAA standard values ​​were obtained by preparing L-Leucine at concentrations of 0, 2, 4, 6, 8, and 10 nM / mL, constructing a calibration curve according to the calibration curve construction method included in the BCAA kit, obtaining the equation in Equation 3 below, and substituting the absorbance of each sample into Equation 3 below to obtain nM unit results, which were then converted to μg values ​​using the method of the ENDMEMO website (https: / / endmemo.com / chem / massmolarconcentration.php?l=ng / mL&s=nmol / mL), and the concentrations obtained by dividing the converted values ​​by the volume of the samples used are shown in Table 7 below.

[0177] [Mathematical Formula 3]

[0178] Y = 0.1254X + 0.0462 (R 2 =0.971)

[0179] (Y represents the measured absorbance value of each sample, and X represents the BCAA content)

[0180] Sample name Final pH BCAA (μg / mL) Tm 7.1 2.69 TmLp 6.4 37.47 TmLp_NaOH 7.2 56.23

[0181] As a result of measuring the BCAA value, the BCAA content of the TmLp_NaOH sample, with the final pH adjusted to 7.2, was measured to be higher than that of TmLp without added NaOH, thereby confirming the increase in BCAA content due to pH adjustment.

[0183] Example 5. Analysis of viable cell count, protein, and survival-related characteristics in lactic acid bacteria fermented product of mealworm extract in powder form

[0184] Example 5-1. Measurement of viable cell count, pH value, protein quantification, degree of protein hydrolysis, and branched-chain amino acid measurement in lactic acid bacteria fermented product of mealworm extract in powder form

[0185] Since the lactic acid bacteria fermented mealworm extract is susceptible to external factors when in liquid form, it is appropriate to use it in powder form for actual application; therefore, the degree of protein hydrolysis and branched-chain amino acids of the powdered fermented product were analyzed.

[0186] Specifically, a TmLp_NaOH sample containing Tm and culture medium was prepared in substantially the same manner as in Example 4-1, and instead of the Lactobacillus plantarum of Example 3, the sample of Example 2-2 Lactobacillus brevis A sample (TmLb_NaOH) was prepared in substantially the same manner as the TmLp_NaOH of Example 4, except that it was cultured using MIRE_TS66. The TmLp_NaOH and TmLb_NaOH samples were prepared as powder samples obtained by freeze-drying to include the hydrolysate of the supernatant.

[0187] For Tm samples other than culture medium, a powder sample was prepared by drying the sample itself with a microwave. As a comparative example, 4.5 g (4.5% w / w) of the mealworm larva powder prepared in Example 1 was added to 100 mL of 10 mM sodium phosphate buffer (pH 7.0) and treated at 90°C for 20 minutes to undergo a self-enzyme inactivation process. Then, alcalase (Sigma, USA) was added at a concentration of 1% (v / w), treated at 55°C for 24 hours, and treated at 90°C for 20 minutes to inactivate any remaining enzymes. The supernatant obtained by centrifuging the enzyme-inactivated material at 13,000 rpm for 10 minutes was freeze-dried to prepare a powder sample (TmAlc).

[0188] For samples prepared by freeze-drying each of the above samples and adding the powder to distilled water at 10% (w / v), the viable cell count, final pH value, TNBS, and BCAA values ​​were measured in substantially the same manner as in Examples 4-1 to 4-3 above, and the results are shown in Table 8 below.

[0189] Additionally, protein was quantified using the TaKaRa BCA Protein assay kit (TaKaRa, Japan) for protein quantification using BCA reagents. Specifically, the bovine serum albumin (BSA) standard and BCA working agent (WR) from the kit were prepared, and each standard sample and the Tm, TmLp_NaOH, and TmLb_NaOH samples were mixed separately in their respective microplates by shaking well, then covered with a cover plate and incubated at 37°C for 30 minutes.

[0190] After keeping the plate warm, the plate was cooled to room temperature, and the absorbance at 562 nm was measured using a plate reader. The average absorbance of the control sample (no addition) was subtracted from the absorbances of the standard sample (BSA) and the Tm, TmLp_NaOH, and TmLb_NaOH samples, and then the average absorbance of each BSA standard and the μg / μL concentration were plotted to form a standard curve to determine the protein concentration of each unknown sample. The results are shown in Table 8 below.

[0191] However, unlike the above Examples 4-1 to 4-3, experiments were conducted on "powder" samples, and each result was calculated based on the total weight of the powder sample.

[0192] Sample name Final pH Viable cell (log cfu / g) Protein quantification (mg / g) TNBS (mg / g) BCAA (ug / g) Tm 6.63 - 11.71 354.32 435.82 TmLp_Alc 7.01 - - 134.24 509.32 TmLp_NaOH 7.23 1.09E+09 12.39 380.75 562.35 TmLb_NaOH 7.00 3.01E+09 16.45 423.80 589.17

[0193] As a result of measuring the final pH and viable cell count, it was confirmed that the pH of TmLp_NaOH and TmLb_NaOH was adjusted by the addition of NaOH, and that a large number of strains survived, unlike in the unfermented Tm. Additionally, protein quantification revealed that the protein content of TmLp_NaOH and TmLb_NaOH was higher compared to the unfermented Tm.

[0194] As a result of measuring TNBS and BCAA levels, the TNBS and BCAA levels of both the TmLp_NaOH sample and TmLb_NaOH were measured to be higher than those of TmLp_Alc, which is the hydrolysate of mealworm powder by alkaline enzymes, confirming that both the degree of protein hydrolysis and the BCAA content are high.

[0196] Example 5-2. Analysis of free amino acids and constituent amino acids in powder form of lactic acid bacteria fermented mealworm extract

[0197] Powdered Tm, TmLp_NaOH, and TmLb_NaOH obtained by substantially the same method as in Example 5-1 above were prepared to analyze the composition of constituent amino acids in the powdered sample, and the composition of free amino acids in the liquid sample was prepared by adding the powdered sample to distilled water at 10% (w / v).

[0198] Specifically, a Biochrom 20 amino acid analyzer from Biochrom (UK) was used. The prepared powder or liquid samples were dispensed using an automatic dispenser, and the amino acids reacted with ninhydrin were analyzed at 570 nm and 440 nm using an automated post-column method; the detection sensitivity was less than 15 pmole. The analysis conditions for the 40 constituent and free amino acids were as follows:

[0199] All buffers and ninhydrin were reagents specifically designed for Biochrom 20, with a flow rate of 20 mL / hr, a buffer pressure of 75 bar, and a ninhydrin pressure of 14 bar. 0.2 M lithium citrate buffer at pH 2.8 was used as Buffer 1, 0.3 M lithium citrate buffer at pH 3.0 as Buffer 2, 0.5 M lithium citrate buffer at pH 3.15 as Buffer 3, 0.9 M lithium citrate buffer at pH 3.5 as Buffer 4, 1.65 M lithium citrate buffer at pH 3.55 as Buffer 5, and 0.3 M lithium hydroxide as Buffer 6.

[0200] Standard materials were purchased from Sigma (USA) and prepared for use. The results are shown in Tables 9 and 10 below, respectively. The unit of content for the components in Tables 9 and 10 below is mg / 100 g.

[0201] Composition of free amino acids Tm TmLp_NaOH TmLb_NaOH essential amino acids Threonine 0.67 8.49 5.05 Valine 3.15 23.68 20.65 Methionine - 0.35 0.43 Isoleucine 1.83 12.99 10.97 Leucine 1.1 12.04 7.72 Phenylalanine 0.7 6.90 4.84 Histidine 4.33 26.68 26.28 Lysine 1.22 12.04 14.56 Tryptophan 1.95 16.73 13.92 Conditionally Essential Amino Acids Arginine 6.78 - - Cysteine - 　 0.3 Glycine 0.94 7.97 8 Proline 17.67 162.91 143.37 Tyrosine 5.08 48.28 27.66 non-essential amino acids Aspartic acid 0.51 2.98 4.04 Serine 0.59 4.94 4.63 Glutamic acid 3.6 21.88 26.53 Alanine 6.08 19.22 36.66 Total BCAA (Leucine, Isoleucine, and Valine) Content 6.08 48.71 39.34

[0202] Composition of constituent amino acids Tm TmLp_NaOH TmLb_NaOH essential amino acids Threonine 4.3 48.99 44.33 Valine 7.18 64.25 53.58 Methionine 1.99 14.36 12.37 Isoleucine 3.21 40.27 34.46 Leucine 4.28 58.79 55.18 Phenylalanine 4.15 33.56 30.81 Histidine 6 51.98 46.1 Lysine 4.22 69.78 63.35 Tryptophan - - Conditionally Essential Amino Acids Arginine 7.11 57.17 37.1 Cysteine - - Glycine 3.78 67.44 60.32 Proline 12.9 204.55 159.86 Tyrosine 5.19 48.28 58.73 non-essential amino acids Aspartic acid 5.3 103.84 102.16 Serine 5.06 54.77 51.14 Glutamic acid 18.77 207.13 203.38 Alanine 4.77 87.22 69.41 Total BCAA (Leucine, Isoleucine, and Valine) Content 14.67 163.31 143.22

[0203] As a result of analyzing the above composition, it was confirmed that both the free amino acids and constituent amino acids of TmLp_NaOH and TmLb_NaOH increased compared to Tm.

[0204] Specifically, in the free amino acids of TmLp_NaOH and TmLb_NaOH, methionine, which is used for protein biosynthesis not identified in Tm and has functions of skeletal muscle formation, growth, antioxidant effect, reduction of high-temperature stress, inhibition of fatty liver, and liver detoxification, was identified at 0.35 (mg / 100 g) and 0.43 (mg / 100 g), respectively, and in TmLb_NaOH, cysteine, which has protein biosynthetic function and efficacy against angina, cardiovascular disease, bronchitis, diabetes, influenza, arthritis, etc., was identified at 0.3 (mg / 100 g).

[0205] The total content of leucine, isoleucine, and valine (BCAA), which help improve muscle function, in both free amino acids and constituent amino acids of TmLp_NaOH and TmLb_NaOH increased by 6.4 to 11.1 times compared to Tm. In addition, glutamic acid, which provides umami flavor in food and functions such as improving cognitive function, brain growth, muscle growth, and immune enhancement, also increased by 6.1 to 11.1 times in the fermented product compared to Tm.

[0207] Example 5-3. Analysis of the molecular weight (SDS-PAGE) of hydrolysates in the lactic acid bacteria fermented product of mealworm extract

[0208] Powder samples of Tm, TmLp_Alc, TmLp_NaOH, and TmLb_NaOH were prepared in substantially the same manner as in Example 5-1 above, and the hydrolysis pattern of each sample was confirmed using SDS-PAGE (Sodium dodecyl sulfate-polyacrylaminde gel electrophoresis) to confirm the hydrolysis characteristics of the samples.

[0209] Specifically, a 15% SDS-PAGE acrylamide gel (BioRad, UAS) was used, and 100 μL of the mixture was prepared by adding each of the above-prepared samples to distilled water at a concentration of 10 (w / v)%. 16 μL of the supernatant obtained by centrifuging at 8,000 rpm for 10 minutes was loaded onto the gel and electrophoresed at 100 V for 1 hour and 30 minutes. The gel was then stained with Coomassie brilliant blue (CBB) and decolorized with a decolorizing solution containing 30 (v / v)% methanol and 10 (v / v)% acetic acid.

[0210] Decolorization was performed, and the molecular weight was confirmed using a protein marker (Bio-Rad, USA; M) to analyze the hydrolysis pattern. The results are shown in Figure 1 below. In the figure, the number on the left represents the molecular weight of the marker (M), and the number on the right represents 15 kDa, which is the starting point of the upper band, middle band, and the dark drag band of the sample Tm.

[0211] As a result of examining the hydrolysis pattern, the 80 and 40 kDa bands of the untreated group were hydrolyzed, and in the TmLp_NaOH and TmLb_NaOH hydrolysates, most of the bands were found to be dragging at 40 kDa or lower.

[0212] In particular, a dark band was observed at 10–15 kDa or less, and a hydrolyzed peptide drag band of 10 kDa or less, which was not observed in the control group, was observed.

[0213] The low molecular weight peptides of TmLp_NaOH and TmLb_NaOH identified above, with a molecular weight of 15 kDa or less, have a faster absorption rate in the body compared to high molecular weight proteins when ingested, and can enhance the activity of BCAA amino acids that help improve muscle, such as leucine, isoleucine, and valine, as well as other active peptides and amino acids that help with strength training.

[0215] Example 5-4. Analysis of human intestinal cell viability of Lactobacillus plantarum fermented mealworm extract

[0216] Tm, TmLp_Alc, and TmLp_NaOH powder samples (fermented products) obtained by freeze-drying in substantially the same manner as in Example 5-1 above were prepared, and it was confirmed whether the fermented products were helpful for cell survival.

[0217] Specifically, 1 x 10⁶ Caco-2 cells (Korean Cell Line Bank; http: / / cellbank.snu.ac.kr) were placed in a 96-well plate. 5 Cells were seeded into wells and incubated in a CO2 incubator at 37°C for 24 hours with Dulbecco's Modified Eagles Medium (DMEM) (Gibco, USA) containing 10% FBS (Gibco, USA). Afterward, the medium was replaced with Minimum Essential Medium (MEM) containing 0.5% FBS (Gibco, USA) and left for 12 hours.

[0218] 100 μL of a mixture was prepared by adding the above Tm, TmLp_Alc, and TmLp_NaOH samples to distilled water at a concentration of 10 (w / v)%. Each mixture was diluted to a concentration of 10 or 100 μL / mL and applied to Caco-2 cells in the medium. After incubation for 24 hours, 10 μL of Cell Counting Kit-8 (CCK-8; Dojindo, KOREA) reagent was added per well, and the cells were incubated for 2 hours in a 5% CO2, 37°C incubator. Absorbance at 450 nm was measured using an ELISA reader (BioTek Instruments Inc., USA) as a relative percentage to the 100% viability of a control group (CON) without the diluted samples. The results are shown in Table 11 below. In Table 11 below, the unit of cell viability is %.

[0219] Sample name Concentration (uL / mL) 10 100 Tm 134.30 140.63 TmLp_Alc 121.79 116.44 TmLp_NaOH 151.95 149.67

[0220] As a result of confirming the cell viability according to the concentration of each sample added to the above human intestinal cells (Caco-2), it was confirmed that the TmLp_NaOH sample had a higher cell viability than the TmLp_Alc sample treated with 1% alkaline in the mealworm extract, thus confirming that it helps with cell viability.

[0222] Example 6. Analysis of the Nitric Oxide (NO) Production Inhibitory Efficacy of Lactobacillus brevis Fermented Extract from Mealworms

[0223] To confirm the anti-inflammatory efficacy of the mealworm extract Lactobacillus brevis fermented mucilage, the efficacy of inhibiting nitric oxide production was confirmed.

[0224] Specifically, Raw 264.7 (ATCC TIB-71) and MC3T3-E1 (ATCC 2595) cell lines were subcultured five times at 37°C and 5% CO2 in Dulbecco's modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% (w / v) antimycotics (GIBOCO, USA), respectively.

[0225] After subculture, 5 x 10⁶ in DMEM medium containing 10% fetal bovine plasma without phenyl red 4 The cells were suspended in a cell / well, dispensed at a rate of 100 μL into a 96-well plate, and cultured for 24 hours.

[0226] For cultured cells, samples prepared by adding Tm and TmLb_NaOH to distilled water, respectively, in substantially the same manner as in Example 5-1, were treated at a concentration of 0.1 or 1 μg / mL, respectively, and after 1 hour, lipopolysaccharide (LPS), a bacterial inflammation-inducing component, was treated at 1 μg / mL and cultured for 24 hours.

[0227] As a comparison group, cells treated only with LPS without the above Tm or TmLb_NaOH were prepared (nCONT), and as a control group, cells not treated with either the above Tm or TmLb_NaOH and LPS were prepared (CONT). After 24 hours of treatment, the efficacy of inhibiting nitric oxide production was measured on the supernatant using the EZ-Nitric Oxide assay kit (DogenBio, KOREA).

[0228] Specifically, absorbance was measured at 540 nm using Greiss reagent (mixed in a ratio of 0.2% N-(1-naphthyl)ethylene diamine solution to 0.2% sulfanilamide solution = 1:1 (v:v)). To construct a calibration curve, 100 μL of nitrite ion standard solution (DoGenBio, KOREA) (0, 5, 10, 15, 20, 30, 50 μM) was mixed with an equal amount of Greiss reagent, the mixture was sealed with foil at room temperature for 15 minutes, and then the absorbance was measured at 540 nm. After sample treatment, NO concentration was calculated using the standard curve obtained from the nitrite ion standard solution, and the results are shown in Table 12 below.

[0229] Sample name NO concentration (μM) CONT 6.67 nCONT 17.98 Tm 1 μg / mL 9.98 0.1 μg / mL 9.44 TmLb_NaOH 1 μg / mL 8.23 0.1 μg / mL 7.74

[0230] Upon checking the NO concentrations, it was confirmed that the NO concentrations of both Tm and TmLb_NaOH decreased compared to the inflammation-induced negative control group. In particular, it was confirmed that the NO concentration of the fermented product TmLb was significantly reduced compared to Tm, which did not undergo fermentation, thereby confirming its anti-inflammatory effect.

[0232] Example 7. Analysis of the muscle strength improvement effect of lactic acid bacteria fermented mealworm extract

[0233] Example 7-1. Preparation of experimental animals and dietary supply

[0234] To confirm the muscle strength-improving effect of the fermented mealworm extract, experimental animals were prepared under the use guidelines approved by the KFRI-IACUC (Korea Food Research Institute, Institutional Animal Care and Use Committee) (KFRI-M-23027). Specifically, 5-week-old male C57BL / 6 mice weighing 19–22 g were purchased from Orient Bio (Seong-nam, Korea) and acclimatized to the environment for one week in experimental animal rearing cages. The rearing conditions for the experimental animals were maintained at a temperature of 22±1℃ and a humidity of 55±5%, with a light-dark cycle of 12 hours (illumination 300 Lux), an ammonia concentration of 20 ppm or less, and a ventilation rate of 10–15 times per hour.

[0235] The experiment was conducted by dividing the above experimental animals into a normal control (CONT) with no treatment, a control group (nCONT) in which muscle atrophy was induced with dexamethasone, a positive control group (BCAA) in which muscle atrophy was induced with dexamethasone and BCAA was administered at a concentration of 930 μg / kg, and a group in which muscle atrophy was induced with dexamethasone and powdered samples of Tm, TmLp_NaOH, and TmLn_NaOH from Example 5-1 were administered at a concentration of 2,000 mg / kg each. Five experimental animals were prepared for each group.

[0236] The above muscle atrophy was induced by intraperitoneal administration of dexamethasone (D2915, Sigma Aldrich) prepared at a concentration of 25 mg / kg in sterile drinking water, and the normal group was administered the same amount of sterile drinking water as the solvent intraperitoneally. In addition, the BCAA, Tm, TmLp_NaOH, and TmLb_NaOH groups were orally administered the BCAA, sample, and culture, respectively, for 8 weeks prior to the induction of muscle atrophy, followed by 6 weeks of muscle atrophy induction. The samples were also orally administered in the same manner during the induction period, and the total experimental period was set to 14 weeks.

[0238] Example 7-2. Measurement of changes in muscle-related factors following intake of fermented mealworm extract

[0239] To measure changes in muscle-related factors caused by the intake of Lactobacillus plantarum fermented mealworm extract, the presence or absence of Atrogin expression following the intake of the fermented product was checked.

[0240] Specifically, in a 6-well plate, the myoblast cell line C2C12 (ATCC cells (1 × 10⁶ 5 Cells / well) were cultured in 10% FBS supplemented medium for 24 hours, then replaced with 2% horse serum medium to induce differentiation, and subsequently cultured for 5 days with the medium replaced every 2 days.

[0241] Differentiated cells were treated with 200 mM / mL Dexamethasone for 1 hour, and then treated with the powder form of the Tm or TmLp_NaOH samples from Example 5-1 at concentrations of 50 or 200 µg / mL, respectively, for 24 hours. After washing the cells with PBS, lysis buffer was dispensed onto the cells to induce lysis, and then centrifuged to separate only the protein supernatant. Cells with only Dexamathasone added and no Tm or TmLp_NaOH samples added (DEXA) were prepared as a comparison group, and cells with neither Dexamathasone nor Tm or TmLp_NaOH samples added (CONT) were prepared as a control group.

[0242] Protein concentration was quantified using the Bradford protein assay, and the quantified protein was electrophoresed on a 10–12% polyacrylamaide gel and then transferred to a poly-vinylidene difluoride (PVDF) membrane using the Trans-Blot Turbo Transfer (Bio-rad, USA) method.

[0243] The protein-transferred membrane was placed in EveryBlot Blocking Buffer and blocked at room temperature. Subsequently, the primary antibody was diluted 1:1000 in Tris-Buffered Saline and Tween 20 (TBST) and incubated with the membrane. The primary antibody reaction was performed using Atrogin antibody, Myostatin antibody, and GAPDH antibody, incubated overnight at 4°C, and washed three times with TBST. Then, the secondary antibody was diluted 1:5000 in TBST and reacted for two hours, followed by three washes with TBST. Proteins were measured using the ECL™ Select Western Blotting Detection Reagent via a Chemidoc imaging system, and the results are shown in Figure 2.

[0244] Western blot analysis was performed to determine the expression of Myostatin, a proteolytic factor that acts on skeletal muscle cells to inhibit muscle differentiation and proliferation, and Atrogin, which induces muscle atrophy, in C2C12 cells. The results confirmed that the expression of both genes decreased when the fermented product TmLp_NaOH was added compared to when the control, Dexamethasone, was added.

[0246] Example 7-3. Measurement of body muscle mass following intake of fermented mealworm extract

[0247] The lean mass of each group of experimental animals in Example 7-1 was measured at week 14 based on the date of oral administration. To prevent errors caused by movement during measurement, inhalation anesthesia was administered with isoflurane at a concentration of 2% (v / v). After inhalation anesthesia, the animals were placed in the measurement area in a prone position with their arms and legs extended to the sides.

[0248] The lean body mass and body fat mass of the above experimental animals were measured using an InAnalyzer (Medikors Inc., Korea) of the Dual Energy X-ray Absorptiometry (DEXA) method, and the analysis conditions were set to Pbgh 80 kV and 1.0 mA, low 55 kV and 1.25 mA, and the average values ​​of lean body mass and body fat mass for each group measured using the above equipment are shown in Table 13 below.

[0249] After measurement, muscle mass information and images were obtained using InAnalyzer software. Based on the muscle mass information, the average values ​​of the quadriceps / body weight ratio and gastrocnemius / body weight ratio for each group are shown in Table 13 below, and the image information is shown in Figure 3. The unit of each value in Table 13 below is %.

[0250] lean body mass body fat mass Quadriceps / body weight ratio Calf muscle / body weight ratio CONT 81.30 16.40 1.15 1.18 nCONT 69.80 28.23 0.51 0.93 BCAA 69.39 30.10 0.70 1.07 Tm 73.65 24.30 0.97 1.02 TmLp_NaOH 75.38 22.68 1.03 1.06 TmLb_NaOH 77.61 20.35 1.20 1.14

[0251] It was confirmed that lean body mass, corresponding to muscle mass, was measured to be significantly higher in the TmLp_NaOH or TmLb_NaOH groups with added fermented product than in the nCONT, BCAA, and Tm groups with induced muscle atrophy, thereby confirming that the administration of fermented product helps improve muscle after muscle atrophy, and that body fat mass was also measured to be lower.

[0252] In addition, when measuring the ratio of the quadriceps or gastrocnemius muscles to body weight, it was confirmed that the TmLp_NaOH or TmLb_NaOH groups were measured at a level similar to the normal group (CONT) that received no treatment, and were measured higher than the nCONT, BCAA, and Tm groups in which muscle atrophy was induced, thus confirming that the administration of the fermented product helps improve the quadriceps and gastrocnemius muscles.

[0254] Example 7-4. Measurement of muscle endurance exercise performance following intake of fermented mealworm extract

[0255] For each group of experimental animals in Example 7-1 above, an evaluation of muscular endurance exercise performance was performed using a treadmill (Cat. No. 47300, Ugo Basile, Italy). Specifically, for each group in Example 7-1 above, for 2 days prior to measurement, an adaptation exercise was conducted by starting running on a 10% incline at a speed of 10 m / min and increasing the speed by 2 m / min every 2 minutes for 20 minutes. Subsequently, in the same manner as the adaptation exercise, running on a 10% incline at a speed of 10 m / min was started and the speed was gradually increased by 2 m / min every 2 minutes.

[0256] When mice in each group did not respond to electrical stimulation for 10 seconds, the experiment was considered to be exhausted and stopped. That point was set as the exercise end time (min) and the evaluation was conducted. The average value of the exercise end time for each group was calculated, and the measured results are shown in Table 14 below.

[0257] Exercise end time (minutes) CONT 29.03 nCONT 22.78 BCAA 25.16 Tm 24.35 TmLp_NaOH 25.40 TmLb_NaOH 24.10

[0258] As a result of measuring muscle endurance performance, exercise time was maintained for longer in all Tm, Tm, TmLp_NaOH, and TmLb_NaOH groups compared to the control group (Ncont) with induced muscle atrophy. In particular, the average value at the end of exercise in the TmLp_NaOH group with added fermented product was measured to be longer than that of the Tm and TmLb_NaOH groups with induced muscle atrophy, confirming that the administration of fermented product, especially TmLp_NaOH, helps improve muscle endurance after muscle atrophy.

[0260] Example 7-5. Measurement of sense of balance following ingestion of fermented mealworm extract

[0261] A balance assessment was performed on each group of experimental animals in Example 7-1 using a Rota-rod (Panlab LE 8505, Panlab, Spain). Specifically, for each group in Example 7-1, adaptation practice was performed on the Rota-rod for 2 days starting at a rotational speed of 4 rpm and reaching 30 rpm after 5 minutes. Then, for each group, the initial rotational speed was gradually accelerated from 4 rpm to 30 rpm, and the process was conducted by setting the final speed to reach 30 rpm within a maximum duration of 5 minutes.

[0262] When an animal fell to the bottom of the equipment due to fatigue or loss of balance, the test was stopped and that point was set as the exercise end time (min) for evaluation. The average value of the exercise end time for each group was calculated, and the measurement results are shown in Table 15 below.

[0263] Exercise end time (minutes) CONT 2.01 nCONT 1.02 BCAA 1.32 Tm 1.18 TmLp_NaOH 1.55 TmLb_NaOH 1.45

[0264] As a result of measuring the sense of balance, it was confirmed that the average value at the end of exercise for the TmLp_NaOH and TmLb_NaOH groups, to which the brown mealworm fermentation product was added, was longer than that of the group with induced muscle atrophy, thus confirming that the administration of the fermentation product helps improve the sense of balance.

[0266] Name of depositing institution: Korean Culture Collection of Microorganisms Trustee Number: KCCM13068P Date of Deposit: 2021-10-27 Name of depositing institution: Korean Culture Collection of Microorganisms Trustee Number: KCCM13069P Date of Deposit: 2021-10-27

Claims

Claim 1 Lactobacillus in mealworm extract ( Lactobacillus A muscle-strengthening composition comprising a fermented product of a genus of microorganisms, wherein the genus of Lactobacillus microorganism is (i) a Lactobacillus plantarum MIRE_TS55 strain having accession number KCCM13068P; or (ii) a Lactobacillus brevis MIRE_TS66 strain having accession number KCCM13069P. Claim 2 A muscle-strengthening composition according to claim 1, wherein the brown mealworm extract is a solvent extract obtained by extracting brown mealworms with water, a straight-chain or branched-chain alcohol having 1 to 4 carbon atoms, or a mixture thereof. Claim 3 delete Claim 4 A muscle-strengthening composition according to claim 1, wherein the pH of the fermented product is 6.7 to 8.

0. Claim 5 A muscle-strengthening composition according to claim 4, wherein the fermented product comprises a basic substance. Claim 6 delete Claim 7 A muscle-strengthening composition having a muscle-increasing effect in any one of claims 1, 2, 4 and 5. Claim 8 Lactobacillus in mealworm extract ( Lactobacillus A composition for preventing, improving, or treating muscle loss-related diseases comprising a fermented product of a genus of microorganisms, wherein the genus of Lactobacillus microorganism is (i) the Lactobacillus plantarum MIRE_TS55 strain having accession number KCCM13068P; or (ii) the Lactobacillus brevis MIRE_TS66 strain having accession number KCCM13069P.

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