Composition for enhancing or protecting muscles, comprising fermented salmon roe product

Abstract

The present invention relates to a composition for enhancing or protecting muscles, comprising a fermented salmon roe product. According to the present invention, if salmon roe is fermented, the amino acid composition of proteins changes, and the effects of promoting muscle synthesis, inhibiting muscle degradation and damage, and protecting muscles are greatly improved. Therefore, the fermented salmon roe product of the present invention can be applied to food to exhibit effects of enhancing and protecting muscles, and can also be used as a pharmaceutical composition for preventing or treating sarcopenia or amyotrophic diseases through the effects.

Description

Composition for muscle strengthening or protection comprising fermented salmon roe

[0001] The present invention relates to a composition for muscle enhancement or protection comprising fermented salmon roe, and more particularly, to a functional food composition for muscle enhancement or protection comprising fermented salmon roe and a pharmaceutical composition for preventing or treating sarcopenia or muscular dystrophy.

[0002] Skeletal muscle cells mature from myoblasts into muscle fibers (myotubes) through a process called myogenesis. Muscle differentiation is regulated by myogenic regulatory factors (MRFs), such as MyoD and Myf5. After differentiation is complete, muscle size increases through muscle hypertrophy, a process in which muscle fiber diameter and length increase.

[0003] Skeletal muscle is the largest organ in the body, accounting for 50% of total body weight. Sarcopenia, a condition caused by a decline in muscle development, significantly impacts quality of life, diminishing physical performance and increasing the risk of falls and fractures. Muscle loss, which occurs with age, was once considered a simple part of the aging process. However, in November 2016, the U.S. Centers for Disease Control and Prevention (CDC) became the first in the world to classify sarcopenia as a disease, assigning it a disease code (M62.84). This indicates that sarcopenia is closely related to a decline in quality of life and a shortened healthy lifespan. Furthermore, muscle loss can lower basal metabolic rate and increase insulin resistance, which can lead to diseases such as diabetes and hypertension. Therefore, strengthening and protecting muscle is considered a crucial element in health management.

[0004] In relation to substances exhibiting such muscle enhancing effects, Korean Patent Publication No. 10-2022-0084527 describes a composition for muscle enhancing, improving exercise capacity, and recovering from exercise fatigue, containing a starfish extract, a ginkgo extract, and / or a burdock extract as active ingredients, and Korean Patent Publication No. 10-2023-0034144 describes a health functional food composition for muscle strengthening, muscle strengthening, or preventing or improving muscle loss, containing a mixed extract of bamboo leaves and goldenrod as active ingredients. However, all of the above technologies used natural extracts, and thus had limitations in the level of muscle enhancing effects.

[0005] Therefore, there is a need to develop food materials that not only have high protein content but also have excellent muscle-building effects and can effectively prevent sarcopenia.

[0006] The purpose of the present invention is to provide a functional food composition having excellent muscle strengthening or protection effects.

[0007] Another object of the present invention is to provide a pharmaceutical composition that exhibits a preventive or therapeutic effect on sarcopenia or muscular dystrophy.

[0008] To achieve the above purpose, the present invention provides a functional food composition for muscle enhancement or protection containing fermented salmon roe.

[0009] In the present invention, the salmon roe fermentation product can be obtained by fermenting salmon roe with microorganisms.

[0010] In the present invention, the microorganism may be at least one selected from the group consisting of microorganisms of the genus Saccharomyces (Saccharomyces spp.), microorganisms of the genus Aspergillus (Aspergillus spp.), microorganisms of the genus Bacillus (Bacillus spp.), and microorganisms of the genus Lactobacillus (Lactobacillus spp.).

[0011] In the present invention, the salmon roe fermentation product can be produced through the steps of: preparing a salmon roe mixture containing salmon roe and water; stirring the mixture, allowing it to settle to separate layers, and then recovering the supernatant; and adding microorganisms to the mixture and fermenting it.

[0012] In the present invention, the fermentation can be performed at 20 to 40°C for 12 to 60 hours.

[0013] In the present invention, the expression level of one or more factors selected from the group consisting of MyoD, Myf5, Myf6, Myogenin, MEF2 (myocyte enhancer factor 2), and IGF-1 (insulin-like growth factor 1) can be increased by the fermented salmon roe.

[0014] In the present invention, the expression level of one or more factors selected from the group consisting of Atrogin-1, MuRF1 (muscle RING-finger protein-1), Myostatin, TNF-α, IL-1, and IL-6 can be reduced by the fermented salmon roe.

[0015]

[0016] The present invention also provides a pharmaceutical composition for preventing or treating sarcopenia or muscular dystrophy, comprising a fermented salmon roe product.

[0017] In the present invention, the sarcopenia or muscular dystrophy may include sarcopenic obesity, myopathy, muscular dystrophy, muscular injury, myasthenia, myoneural conductive disease, nerve injury, amyotrophic lateral sclerosis (ALS), atony, myotonia, or cachexia.

[0018] In the present invention, the dosage of the fermented salmon roe may be 0.01 to 100 mg per 1 kg of body weight.

[0019] According to the present invention, fermenting salmon roe changes the amino acid composition of protein, significantly enhancing muscle synthesis promotion, muscle breakdown and damage inhibition, and muscle protection. Therefore, the fermented salmon roe product of the present invention can be applied to foods to not only enhance and protect muscles, but also, through these effects, can be used as a pharmaceutical composition for the prevention or treatment of sarcopenia or muscular dystrophy.

[0020] Figure 1 shows the results of a cytotoxicity test of a fermented salmon roe product according to one embodiment of the present invention.

[0021] Figure 2 shows the results of an experiment to confirm the morphology of myotube cells according to treatment with salmon roe fermentation in one embodiment of the present invention.

[0022] Figure 3 shows the results of measuring biomarker expression levels according to salmon roe fermentation treatment in one embodiment of the present invention.

[0023] Figure 4 shows the results of measuring the expression level of biomarkers according to the type of microorganism used for fermentation in one embodiment of the present invention.

[0024] Figures 5a, 5b and 5c respectively show the results of measuring body weight (a), quadriceps femoris weight (b) and gastrocnemius weight (c) according to treatment with fermented salmon roe in one embodiment of the present invention.

[0025] Figures 6a and 6b each show the results of calculating the ratio of the weight of the quadriceps femoris (a) and the gastrocnemius muscle (b) according to the treatment of salmon roe fermentation in one embodiment of the present invention.

[0026] Figures 7a and 7b each show the muscle mass reduction ratio of the quadriceps femoris (a) and gastrocnemius (b) according to treatment with fermented salmon roe in one embodiment of the present invention.

[0027] Figures 8a and 8b each show the muscle mass recovery rate of the quadriceps femoris (a) and gastrocnemius (b) according to treatment with fermented salmon roe in one embodiment of the present invention.

[0028] Figures 9a and 9b respectively show a photograph (a) of the volume measurement of calf muscles according to salmon roe fermentation treatment in one embodiment of the present invention and a graph (b) of the calculation results.

[0029] Figures 10a and 10b respectively show a photograph (a) of the thickness measurement of calf muscles according to salmon roe fermentation treatment in one embodiment of the present invention and a graph (b) of the calculation results.

[0030] Figures 11a and 11b respectively show the results of measuring factors related to muscle fiber formation (a) and muscle breakdown (b) in the quadriceps femoris muscle of the thigh according to treatment with salmon roe fermentation in one embodiment of the present invention.

[0031] Figures 12a and 12b respectively show the results of measuring factors related to muscle fiber formation (a) and muscle breakdown (b) in the gastrocnemius muscle according to treatment with fermented salmon roe in one embodiment of the present invention.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In general, the nomenclature used herein is well known and commonly used in the art.

[0033]

[0034] The present invention relates to a composition for muscle enhancement or protection comprising fermented salmon roe.

[0035] According to the present invention, fermenting salmon roe changes the amino acid composition of protein, significantly enhancing muscle synthesis promotion, muscle breakdown and damage inhibition, and muscle protection. Therefore, the fermented salmon roe product of the present invention can be applied to foods to not only enhance and protect muscles, but also, through these effects, can be used as a pharmaceutical composition for the prevention or treatment of sarcopenia or muscular dystrophy.

[0036] While the consumption of high-quality proteins, such as salmon, is known to increase muscle mass and help maintain physical performance and function, it was not known that the amino acid composition of salmon roe undergoes significant changes during the microbial fermentation process, significantly enhancing the muscle-building effect. Under these circumstances, the present invention confirmed that salmon roe fermentation significantly increases the amount of constituent amino acids, particularly branched-chain amino acids such as leucine, isoleucine, and valine. Furthermore, it was confirmed that when this was administered or directly to muscle cells, it had the effect of preventing and treating sarcopenia.

[0037] The salmon roe used in the present invention may refer to salmon roe itself or salmon placenta combined with the placenta before separation of the roe, and may be used in its original state or after removing the oil component.

[0038] In the present invention, the salmon roe fermentation product can be obtained by inoculating a microorganism into salmon roe and fermenting it. The microorganism may be a yeast or a lactic acid bacterium, and may be, for example, at least one microorganism selected from the group consisting of a Saccharomyces genus microorganism (Saccharomyces pp.) such as Saccharomyces cerevisiae, an Aspergillus spp. such as Aspergillus oryzae, a Bacillus spp. such as Bacillus subtilis, and a Lactobacillus spp. such as Lactobacillus plantarum. Preferably, a Saccharomyces genus microorganism can be used as the microorganism, and Saccharomyces cerevisiae can be particularly preferably used.

[0039] In this regard, in an embodiment of the present invention, it was confirmed that the content of constituent amino acids was higher in fermented salmon roe than in unfermented salmon roe, the expression level of biomarkers related to prevention of sarcopenia was increased, and there was an effect of increasing muscle mass when actually administered. It was confirmed that the effect of increasing the content of constituent amino acids was the most outstanding in fermented salmon roe fermented with Saccharomyces cerevisiae.

[0040] In the present invention, the fermented salmon roe product may be obtained by inoculating a salmon roe mixture containing salmon roe and water with microorganisms and then fermenting the mixture. Specifically, the fermented salmon roe product may be produced through the steps of: preparing a salmon roe mixture containing salmon roe and water; stirring the mixture, allowing it to settle to separate layers, and then recovering the supernatant; and adding microorganisms to the mixture and fermenting the mixture.

[0041] The above salmon roe mixture may be obtained by mixing salmon roe and water and then stirring. Specifically, after stirring, the mixture may be allowed to settle to perform layer separation, and then the supernatant (washing liquid) may be recovered and the pellet layer may be used. Preferably, the layer separation may be performed 1 to 5 times, for example, 2 to 3 times. In addition, the salmon roe mixture may be mixed in a ratio of 10 to 500 g of salmon roe, preferably 100 to 300 g, per 1 L of water.

[0042] Microorganisms can be added to the above salmon roe mixture and fermented at 20 to 40°C, preferably room temperature to 35°C, for 12 to 60 hours, preferably 36 to 60 hours, to obtain a fermented salmon roe product. The microorganisms can be inoculated at 1 to 20 wt%, preferably 5 to 15 wt%, based on the volume of the salmon roe mixture as a culture medium. The fermented product can be used after post-processing such as drying, purification, filtration, and drying after fermentation.

[0043] In the present invention, microorganisms may be used after pre-culture in a medium. The medium used for the pre-culture may contain a carbon source, a nitrogen source, inorganic salts, and other components that enable efficient microbial culture. For example, one or more nutritional components such as yeast, glucose, dextrose, and peptone may be used as the medium components. The pre-culture of the microorganisms may be performed by adding the microorganisms at 0.01 to 1 wt% relative to the volume of the pre-culture medium and culturing for 6 to 48 hours.

[0044] Additionally, to facilitate microbial fermentation in the salmon roe mixture, a medium component may be added to the salmon roe mixture. The medium component added to the salmon roe mixture may be any of the medium components exemplified in the pre-culture medium components described above, such as yeast, peptone, glucose, etc.

[0045] In the present invention, by using a fermented product obtained by fermenting salmon roe, the content of amino acids constituting salmon roe can be increased, and in particular, the content of leucine, isoleucine, and valine, which correspond to branched-chain amino acids (BCAAs) known as essential amino acids for muscle formation, can be greatly improved. In addition, the fermented salmon roe has the effect of inhibiting muscle loss when treated together with cells that have induced sarcopenia, and relatedly, when administered to actual animals, it can exhibit the effect of significantly increasing muscle mass and increasing the expression level of biomarkers related to the prevention of sarcopenia.

[0046] Specifically, the fermented salmon roe product fermented with Saccharomyces cerevisiae may have a leucine content of about 2 times or more, preferably about 2.5 times or more, and more preferably about 3 times or more, an isoleucine content of about 2 times or more, preferably about 2.5 times or more, and more preferably about 3 times or more, and a valine content of about 1.5 times or more, preferably about 2 times or more, and more preferably about 3 times or more, compared to unfermented salmon roe.

[0047] In an embodiment of the present invention, it was confirmed that the fermented product of salmon roe fermented with Saccharomyces cerevisiae had a leucine, isoleucine, and valine content increased by 3 times or more, 3 times or more, and 2.5 times or more, respectively, compared to unfermented salmon roe.

[0048] In addition, when fermented salmon roe is processed or administered, the expression levels of MyoD, Myf5, Myf6, Myogenin, and MEF2 (myocyte enhancer factor 2), which are genes that form muscle fibers and inhibit muscle cell death, may increase, and the expression level of IGF-1 (insulin-like growth factor 1), a gene that synthesizes muscle proteins, may increase. Meanwhile, the expression levels of Atrogin-1, MuRF1 (muscle RING-finger protein-1), and Myostatin, which are genes that decompose muscle proteins, and the expression levels of TNF-α, IL-1, and IL-6, which are inflammatory cytokines related to the decrease in muscle protein synthesis, may decrease. In this way, fermented salmon roe increases the expression levels of biomarkers related to muscle synthesis and enhancement, while decreasing the expression levels of biomarkers related to muscle synthesis inhibition and degradation, so it has excellent muscle enhancement and protection effects.

[0049] In an embodiment of the present invention, it was confirmed that when fermented salmon roe was treated to myotube cells, the expression levels of MyoD, Myogenin, and IGF-1 increased, and the expression levels of Atrogin-1, MuRF1, and Myostatin decreased. In particular, it was confirmed that fermented salmon roe using Saccharomyces cerevisiae increased the expression levels of MyoD, Myogenin, and IGF-1 the most, and decreased the expression levels of Atrogin-1, MuRF1, and Myostatin the most.

[0050] Accordingly, the salmon roe fermentation product can be applied not only to functional foods that exhibit muscle strengthening and protective effects, but also to pharmaceutical compositions for preventing or treating muscle diseases.

[0051] Accordingly, the present invention also provides a functional food composition comprising fermented salmon roe.

[0052] Specifically, when the fermented salmon roe product according to the present invention is applied to food, muscle strengthening and muscle protection effects are exerted upon consumption, thereby promoting health.

[0053] The above food may be food, beverage, snack, seasoning, health supplement, etc., and more specifically, it may be any one selected from the group consisting of processed food, grain product, pasta, soup, sauce, dressing, confectionery product, oil, tea, soy dairy-like product such as soy milk, frozen food, prepared food, alternative food, meat product, processed seafood product, fermented food, dairy product, confectionery, seasoning, beverage and drink preparation, snack, candy, jelly, ice cream, frozen dessert, breakfast cereal, nutrition bar, chocolate product, and nutritional supplement.

[0054] The food of the present invention may include food and feed additives for not only humans but also animals. Such animals may include dogs, cats, cows, pigs, sheep, goats, deer, chickens, ducks, geese, pheasants, and the like. Furthermore, the term "feed additive" refers to a substance added to feed to improve the productivity of livestock, promote and / or maintain the health of companion animals, or maintain their physical condition. Using the fermented salmon roe product of the present invention in food and feed additives for animals can improve the health of animals through muscle strengthening and protective effects.

[0055]

[0056] In addition, the present invention can provide a pharmaceutical composition for preventing or treating muscle disease comprising a fermented salmon roe product.

[0057] In the present invention, the muscle disease may be sarcopenia or muscular dystrophy. In the present invention, the sarcopenia or muscular dystrophy may be interpreted to include not only sarcopenia or muscular atrophy in the dictionary sense, but also muscle diseases that cause or are induced by sarcopenia or muscular atrophy, such as sarcopenic obesity, myopathy, muscular dystrophy, muscular injury, myasthenia, myoneural conductive disease, nerve injury, amyotrophic lateral sclerosis (ALS), atony, myotonia, cachexia, etc.

[0058] For example, the above muscular dystrophy may include diabetic amyotrophy, spinal muscular atrophy, etc., and the above muscular dystrophy may include Duchenne muscular dystrophy, Becker muscular dystrophy, limb girdle muscular dystrophy, fascioscapulohumeral muscular dystrophy, oculopharyngeal muscular dystrophy, myotonic dystrophy, etc., and the above myopathy may include inflammatory myopathy such as polymyositis, dermatomyositis, endocrine myopathy, toxic myopathy, metabolic myopathy, mitochondrial It may mean including mitochondrial myopathy, congenital myopathy, etc.

[0059] The above pharmaceutical composition contains fermented salmon roe as an active ingredient, and may additionally contain an appropriate pharmaceutically acceptable carrier, excipient, or diluent according to a conventional method. The pharmaceutically acceptable carrier is one commonly used in formulations, and includes, but is not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0060] The pharmaceutical composition of the present invention may further include, in addition to the above ingredients, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc. With regard to suitable pharmaceutically acceptable carriers and formulations, each ingredient can be preferably formulated according to the method disclosed in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0061] The pharmaceutical composition of the present invention can be administered either orally or parenterally, and parenteral administration includes intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, etc.

[0062] Oral dosage forms include, for example, tablets, pills, hard and soft capsules, solutions, suspensions, emulsions, syrups, and granules. These dosage forms may further contain, in addition to the active ingredient, diluents (e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, and / or glycine), lubricants (e.g., silica, talc, stearic acid and its magnesium or calcium salts, and / or polyethylene glycol). In addition, the tablets may contain binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidine, and, if desired, disintegrants or effervescent mixtures such as starch, agar, alginic acid or its sodium salt, and / or absorbents, coloring agents, flavoring agents, and sweetening agents. The above formulation can be prepared by conventional mixing, granulating or coating methods.

[0063] Furthermore, representative parenteral administration formulations include injectable preparations, and solvents for injectable preparations include water, Ringer's solution, isotonic saline solution, or suspensions. Sterile fixed oils for the injectable preparations can be used as solvents or suspending media, and any non-irritating fixed oil, including mono- and di-glycerides, can be used for this purpose. Furthermore, the injectable preparations may utilize fatty acids such as oleic acid.

[0064] The composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment. The effective dosage level can be determined based on the type and severity of the patient's disease, the activity and sensitivity of the drug, the time of administration, the route of administration, and the excretion rate, the duration of treatment, concomitant drugs, and other factors well known in the medical field.

[0065] The composition according to the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents. It can be administered sequentially or simultaneously with conventional therapeutic agents, or in single or multiple doses. Taking all of the above factors into account, it is important to administer the amount that achieves maximum effect with the minimum amount possible without causing side effects. This can be readily determined by those skilled in the art.

[0066] Specifically, the effective dose of the composition according to the present invention may vary depending on the patient's age, sex, and weight, and is generally 0.01 to 100 mg per 1 kg of human body weight, preferably 1 to 50 mg, administered daily or every other day, or divided into 1 to 3 times a day. However, since the dosage may increase or decrease depending on the route of administration, sex, body weight, age, etc., the above dosage does not limit the scope of the present invention in any way.

[0067]

[0068] Example

[0069]

[0070] Hereinafter, the present invention will be described in more detail through examples. It will be apparent to those skilled in the art that these examples are intended solely to illustrate the present invention and are not intended to limit the scope of the present invention.

[0071]

[0072] Manufacturing example: Manufacturing of fermented salmon roe

[0073]

[0074] 200g of salmon roe (Salmon Placenta) was diluted with distilled water to 1L, stirred for 3 minutes, and allowed to settle. After the layers separated, the supernatant (approximately 150mL) was recovered, its volume checked, and the same amount of distilled water was added. This process was repeated three times, and the pellet layer, excluding the supernatant, was used for fermentation.

[0075] As a pre-culture medium, 100 mL each of a 2 wt% glucose medium and a medium (pH 7.0) containing 1 wt% yeast extract and 2 wt% peptone were prepared. Saccharomyces cerevisiae yeast powder was added at 0.1% of the culture volume and pre-cultured for 24 hours.

[0076] Additionally, prior to the main culture, 0.1 wt% of yeast extract and 0.2 wt% of peptone were added to the salmon roe mixture, and 1 wt% of glucose was added.

[0077] After sterilization at 121°C for 30 minutes, Saccharomyces cerevisiae was inoculated at a ratio of 10% of the volume of the main culture, and main fermentation was performed at 30°C for 2 days. After sterilization at 100°C for 30 minutes, the powder was obtained by freeze-drying.

[0078]

[0079] Experimental Example 1: Analysis of amino acid composition by microorganism type

[0080]

[0081] To determine the differences in amino acid composition of unfermented salmon roe and salmon roe according to the type of fermented bacteria, the contents of constituent amino acids and free amino acids were measured.

[0082] Salmon roe was fermented using the manufacturing method, but Saccharomyces cerevisiae, Aspergillus oryzae, Lactobacillus plantarum, and Bacillus subtilis were used for the fermentation, respectively, and the content of constituent amino acids and free amino acids was measured for the fermented salmon roe according to the Publication Test Method for Health Functional Foods using high-performance liquid chromatography (HPLC). For comparison, the same experiment was performed on salmon roe meal before fermentation, and the results of the measurement of constituent amino acids and free amino acids are shown in Tables 1 and 2 below (unit: mg / 100g).

[0083]

[0084]

[0085]

[0086]

[0087]

[0088] The amino acid composition analysis results showed that the amino acid content of fermented salmon roe tended to be higher than that of unfermented salmon roe in all items. In particular, the contents of leucine, isoleucine, and valine, which correspond to branched-chain amino acids (BCAAs) known as essential amino acids that constitute muscles, were all measured to be higher in F-Sal, and this difference was most evident in the fermented product fermented with Saccharomyces cerevisiae. Specifically, it was confirmed that the contents of leucine, isoleucine, and valine in the fermented product fermented with Saccharomyces cerevisiae increased by approximately 3 times, 3 times, and 2.5 times, respectively, compared to the unfermented product.

[0089] From these experimental results, it was confirmed that the content of amino acids that constitute muscles significantly increases during salmon roe fermentation, and in particular, it was found that Saccharomyces cerevisiae was the most effective fermentation strain.

[0090]

[0091] Experimental Example 2: Cytotoxicity Analysis of Fermented Salmon Roe

[0092]

[0093] C2C12 myoblasts were cultured in Dulbecco's modified Eagle (DMEM) medium supplemented with 10% fetal bovine serum (FBS) and 1% P / S (100 U / ml penicillin, 100 μg / ml streptomycin) at 37°C and 5% CO2.

[0094] To determine the cytotoxicity of the unfermented (Sal) and fermented (F-Sal) products on C2C12 cells, 5,000 cells / well of C2C12 cells were seeded in 96-well plates and cultured for 24 hours. The medium was then replaced with Sal and F-Sal supplemented from 5 μg / mL to 500 μg / mL, and cultured for another 24 hours. Afterwards, the absorbance at 490 nm was measured using the CCK-8 assay (Dojindo) to compare the proliferation of C2C12 cells, and the results are shown in Fig. 1.

[0095] Cytotoxicity measurements showed that cell viability was over 83% even at a high concentration of 500 μg / mL. To conduct the experiment within a range that does not show cell toxicity, the cell experiment was conducted at 10 μg / mL, which is the no-observed-adverse-effect-level (NOAEL) and has a cell viability of over 90%.

[0096]

[0097] Experimental Example 3: Analysis of the Sarcopenia Prevention Effect of Fermented Salmon Roe

[0098]

[0099] C2C12 myoblasts were cultured in Dulbecco's modified Eagle (DMEM) medium supplemented with 10% fetal bovine serum (FBS) and 1% P / S (100 U / ml penicillin, 100 μg / ml streptomycin) at 37°C and 5% CO2. To induce differentiation, they were cultured in DMEM supplemented with 2% horse serum and 1% P / S at 37°C and 5% CO2.

[0100] In order to confirm the sarcopenia prevention effect of unfermented (Sal) and fermented (F-Sal) products on differentiated myotubes, which are myoblasts, the experimental group, excluding the control group, was treated with 100 μM concentration of Dexamethasone (DEX), which induces sarcopenia, in a culture medium containing 1% P / S in DMEM, and under the conditions, non-cytotoxic concentrations of Sal and F-Sal were simultaneously treated for 24 hours each to confirm the morphology of differentiated myotubes.

[0101] Figure 2 shows photographs of the experimental results. When treated with DEX, myotube cells were observed to fail to maintain myotubes and to discontinuously detach into individual cells. On the other hand, when salmon roe or fermented products were treated together with DEX, it was found that they maintained a morphology similar to the control group that did not induce muscle loss.

[0102] Accordingly, it was confirmed that salmon roe and its fermented products exhibited a sarcopenia prevention effect.

[0103]

[0104] Experimental Example 4: Biomarker Analysis Related to the Sarcopenia Prevention Effect of Fermented Salmon Roe

[0105]

[0106] The expression level of biomarkers related to sarcopenia was measured using myotube cells tested under the conditions of Experimental Example 3.

[0107] RNA extraction (Quiagen) and cDNA synthesis (Takara) from cells were performed according to the respective manufacturers' instructions, and real-time RT-PCR (Bio-rad) was performed using SYBR Green PCR master mix (Genetbio) to measure the mRNA expression of each target. Primers were manufactured and purchased from Cosmo Genetech, and the sequences are shown in Table 3 below. After the reaction was completed, the amplification curve and CT value were checked, and the CT value was converted to data after confirming a single amplification curve. The mRNA expression level was corrected by relative quantification with the CT value of GAPDH, and the measurement results for each marker are shown in Figure 3.

[0108]

[0109]

[0110]

[0111] MyoD and Myogenin are genes that inhibit muscle cell death, and IGF-1 (Insulin-like growth factor 1) is a gene that synthesizes muscle protein. When only DEX, a sarcopenic substance, was treated, the expression levels of the three genes that inhibit muscle cell death significantly decreased compared to the control group. On the other hand, in the experimental group treated with Sal or F-Sal together with DEX, the expression levels of muscle protein synthesis genes increased and did not show a significant difference from the control group. In particular, the expression levels of MyoD and IGF-1 tended to increase in the DEX+F-Sal experimental group compared to the DEX+Sal experimental group.

[0112] Meanwhile, Atrogin-1, MuRF1 (muscle RING-finger protein-1), and Myostatin are genes that decompose muscle proteins. When only DEX, a sarcopenic substance, was treated, the expression levels of the three genes that decompose muscle proteins increased compared to the control group, but in the experimental group treated with Sal or F-Sal together with DEX, the expression levels of genes that decompose muscle proteins significantly decreased compared to the experimental group treated with DEX alone. In particular, it was confirmed that the expression levels of Atrogin-1, MuRF1, and Myostatin tended to decrease more in the DEX+F-Sal experimental group than in the DEX+Sal experimental group.

[0113] Accordingly, it was found that fermented salmon roe had an excellent effect of increasing the expression of biomarkers related to muscle growth while decreasing the expression of biomarkers that induce sarcopenia.

[0114]

[0115] Experimental Example 5: Biomarker Analysis Related to Sarcopenia Prevention Effects by Microorganism Type

[0116]

[0117] Salmon roe was fermented using the manufacturing method, but Saccharomyces cerevisiae, Aspergillus oryzae, Lactobacillus plantarum, and Bacillus subtilis were used to ferment the salmon roe, and a biomarker analysis related to the sarcopenia prevention effect was performed in the same manner as in Experimental Example 4, and the results are shown in Fig. 4.

[0118] Salmon roe fermented using Saccharomyces cerevisiae, Aspergillus oryzae, Lactobacillus plantarum, and Bacillus subtilis were expressed as F-Sal(a), F-Sal(b), F-Sal(c), and F-Sal(d), respectively.

[0119] As shown in Figures 4a to 4c, when salmon roe fermentation was treated, the expression levels of MyoD and Myogenin, which inhibit muscle cell death, and the expression levels of IGF-1, a muscle protein synthesis gene, did not show significant differences from the control group, and in particular, the expression levels when F-Sal(a) fermented using Saccharomyces cerevisiae was treated were confirmed to be higher than when other microorganisms were treated.

[0120] In addition, as shown in Figures 4d to 4f, when salmon roe fermentation was treated, the expression levels of Atrogin-1, MuRF1, and Myostatin, which are genes that decompose muscle proteins, decreased, and in particular, it was confirmed that the expression level when F-Sal(a) fermented using Saccharomyces cerevisiae was treated was lower than when other microorganisms were treated.

[0121] Through this, it was confirmed that, compared to other microorganisms, treatment with Saccharomyces cerevisiae had the greatest effect of reducing the expression of biomarkers that induce sarcopenia while increasing the expression of biomarkers related to muscle growth.

[0122]

[0123] Experimental Example 6: In vivo efficacy analysis of fermented salmon roe (1)

[0124]

[0125] The efficacy of fermented salmon roe was evaluated in an animal model of sarcopenia.

[0126] Six-week-old male C57BL / 6 mice were divided into groups of eight and intraperitoneally administered dexamethasone (DEX) at a dose of 25 mg / kg daily for two weeks during the sarcopenia induction period. In the control group, saline (0.9% NaCl) was administered orally daily during the experimental period, and in the experimental group, the human standard dose was set to 500 mg / 60 kg, and the human equivalent dose (HED) was applied. Saline, whey protein isolate (WPI), whey protein hydrolysate (WPH), goat milk protein (GMP), soy protein isolate (ISP), salmon roe (Sal), and fermented salmon roe (F-Sal) were administered orally daily for 2 weeks after inducing muscle loss for 2 weeks at a dose of 102 mg / kg each.

[0127] For the above animal model, individual and tissue weights were measured. Individual weights were measured after 2 weeks of sarcopenia induced by DEX and 2 weeks of test substance administration. Tissue weights were measured after 2 weeks of administration, with the quadriceps muscle in each thigh and the gastrocnemius muscle in each calf weighing the same. Furthermore, the ratios of the quadriceps and gastrocnemius muscle weights to the individual weights were calculated and compared.

[0128] Figures 5a, 5b, and 5c show the results of body weight, quadriceps femoris weight, and gastrocnemius weight measurements, respectively. The experimental group administered saline for two weeks after inducing sarcopenia due to DEX administration showed a significant decrease in body weight, quadriceps femoris weight, and gastrocnemius weight compared to the control group.

[0129] In addition, the experimental group administered protein preparations also showed a decrease in body weight and tissue weight compared to the control group, and in the experimental group administered unfermented salmon roe, the weight of the quadriceps femoris was similar to that of the control group, but the body weight and gastrocnemius weight were significantly lower than that of the control group.

[0130] However, in the experimental group administered fermented salmon roe, body weight, quadriceps femoris weight, and gastrocnemius muscle weight all showed similar values ​​to the control group, with no significant differences. Therefore, we confirmed that fermented salmon roe administration can prevent body weight and tissue weight loss in animal models.

[0131] Figures 6a and 6b show the results of calculating the ratio of the weight of the quadriceps femoris and gastrocnemius muscle to the weight of the individual, respectively. After inducing sarcopenia due to DEX administration, the ratio tended to decrease compared to the control group in the DEX experimental group administered saline for 2 weeks and the experimental group administered protein preparations such as WPI, WPH, GMP or ISP. However, when salmon roe or its fermented product was administered, the ratio increased as much as the control group.

[0132] Figures 7a and 7b show the muscle mass reduction ratio of the quadriceps femoris and gastrocnemius, respectively. In the experimental group administered saline or protein preparations after sarcopenia induction, the quadriceps femoris tended to decrease by about -30 to -15% and the gastrocnemius tended to decrease by about -25 to -20% compared to the control group. A decreasing trend was also observed when salmon roe was administered. On the other hand, in the experimental group administered fermented salmon roe, the quadriceps femoris muscle mass ratio was actually higher than the control group, and the gastrocnemius muscle mass ratio was also confirmed to be almost similar to the control group.

[0133] Figures 8a and 8b show the muscle mass recovery rates of the quadriceps femoris and gastrocnemius muscle, respectively. In the experimental group administered saline or protein preparations after inducing sarcopenia, the quadriceps femoris tended to recover to a small extent. However, when salmon roe was administered, the recovery amount increased, and it was found that the fermented salmon roe product had a very excellent recovery effect.

[0134]

[0135] Experimental Example 7: In vivo efficacy analysis of fermented salmon roe (2)

[0136]

[0137] For the animal model of Experimental Example 6, measurements of the volume and thickness of the calf muscles were performed.

[0138] In the calf muscle measurement experiment, the sagittal section and transverse section of the calf muscle of each individual were photographed using an X-ray micro-computed tomography (Micro-CT) device, and the volume and thickness were measured.

[0139] Figures 9a and 9b show photographs of the volume measurement of the calf muscle and graphs of the calculation results, respectively, and Figures 10a and 10b show photographs of the thickness measurement of the calf muscle and graphs of the calculation results, respectively.

[0140] As a result of the experiment, after sarcopenia was induced by DEX administration, the DEX experimental group administered saline for 2 weeks showed a significant decrease in muscle mass compared to the control group, and the calf muscle mass of the other experimental groups was higher than that of the DEX experimental group. In particular, the experimental group administered salmon roe and its fermented product showed an increase in muscle volume compared to the other protein preparation experimental groups, and the experimental group administered salmon roe fermented product showed the largest muscle volume. A similar trend was observed in the calf thickness measurement experiment, and it was confirmed that the calf thickness of the experimental group administered salmon roe fermented product was the thickest.

[0141] Accordingly, it was confirmed that fermented salmon roe was excellent in improving the size of calf muscles.

[0142]

[0143] Experimental Example 8: In vivo efficacy analysis of fermented salmon roe (3)

[0144]

[0145] For the animal model of Experimental Example 6, biomarker measurements related to prevention of sarcopenia in the quadriceps femoris and gastrocnemius were performed.

[0146] For biomarkers, RNA extraction (Quiagen) and cDNA synthesis (Takara) were performed from the quadriceps femoris and gastrocnemius tissues of each individual according to the manufacturer's instructions. Real-time RT-PCR (Bio-rad) was performed using SYBR Green PCR master mix (Genetbio) to measure the mRNA expression of each target. Primers with the sequences in Table 3 above were used. After completion of the reaction, the amplification curve and CT value were confirmed, and the CT value was converted to data after confirming a single amplification curve. The mRNA expression level was corrected by relative quantification with the CT value of GAPDH.

[0147] Figures 11a and 11b show the results of measuring myofibrillar formation-related factors (a) and muscle breakdown-related factors (b) in the quadriceps femoris. As a result of the experiment, the expression levels of myogenic regulatory factors related to myofibrillar formation, MyoD, Myf5, Myf6, Myogenin, and MEF2 (myocyte enhancer factor 2), and IGF1 (Insulin-like growth factor-1), a factor related to increasing muscle protein synthesis, increased in the experimental group administered the preparation, and among them, the experimental group administered fermented salmon roe showed a very high result in the expression level of biomarkers. In addition, the expression levels of MuRF1, Atrogin-1, and Myostatin, which are factors related to muscle protein breakdown, and TNF-α, IL-1, and IL-6, which are factors related to decreasing muscle protein synthesis, tended to decrease, and in particular, the expression levels were confirmed to be the lowest when fermented salmon roe was administered.

[0148] Figures 12a and 12b illustrate the measurement results of factors related to muscle fiber formation (a) and muscle breakdown (b) in the gastrocnemius muscle. As a result of the experiment, similar to the measurement results in the quadriceps femoris, the expression levels of factors related to increased muscle production / protein synthesis significantly increased in the salmon roe fermentation experimental group, while the expression levels of factors related to decreased muscle breakdown / protein synthesis were significantly reduced.

[0149] From this, it was found that fermented salmon roe has an excellent effect in promoting muscle fiber formation and muscle synthesis, while inhibiting muscle breakdown and protein synthesis reduction.

[0150]

[0151] While specific aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A functional food composition for muscle enhancement or protection, comprising fermented salmon roe.

2. In paragraph 1, A functional food composition for muscle enhancement or protection, wherein the above salmon roe fermentation product is obtained by fermenting salmon roe with microorganisms.

3. In paragraph 2, A functional food composition for muscle enhancement or protection, wherein the microorganism is at least one selected from the group consisting of microorganisms of the genus Saccharomyces (Saccharomyces spp.), microorganisms of the genus Aspergillus (Aspergillus spp.), microorganisms of the genus Bacillus (Bacillus spp.), and microorganisms of the genus Lactobacillus (Lactobacillus spp.).

4. In paragraph 2, A functional food composition for muscle enhancement or protection, which is manufactured through the steps of: preparing a salmon roe mixture containing salmon roe and water; stirring the mixture, allowing it to settle to separate layers, and then recovering the supernatant; and adding microorganisms to the pellet layer obtained by removing the supernatant from the mixture and fermenting the pellet layer.

5. In paragraph 2, A functional food composition for muscle enhancement or protection, wherein the above fermentation is performed at 20 to 40°C for 12 to 60 hours.

6. In paragraph 1, A functional food composition for muscle enhancement or protection, wherein the expression level of at least one factor selected from the group consisting of MyoD, Myf5, Myf6, Myogenin, MEF2 (myocyte enhancer factor 2) and IGF-1 (insulin-like growth factor 1) is increased by the above-mentioned salmon roe fermentation product.

7. In paragraph 1, A functional food composition for muscle enhancement or protection, wherein the expression level of at least one factor selected from the group consisting of Atrogin-1, MuRF1 (muscle RING-finger protein-1), Myostatin, TNF-α, IL-1, and IL-6 is reduced by the above-mentioned salmon roe fermentation product.

8. A pharmaceutical composition for preventing or treating sarcopenia or muscular atrophy, comprising fermented salmon roe.

9. In paragraph 8, A pharmaceutical composition for preventing or treating sarcopenia or muscular dystrophy, wherein the sarcopenia or muscular dystrophy includes sarcopenic obesity, myopathy, muscular dystrophy, muscular injury, myasthenia, myoneural conductive disease, nerve injury, amyotrophic lateral sclerosis (ALS), atony, myotonia or cachexia.

10. In paragraph 8, A pharmaceutical composition for preventing or treating sarcopenia or muscular atrophy, wherein the dose of the above-mentioned fermented salmon roe is 0.01 to 100 mg per 1 kg of body weight.

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