Microbiome composition for improving muscle strength using heat-treated fermented culture complex of lactiplantibacillus plantarum km2

Abstract

To provide a microbiome composition for improving muscle strength using a heat-treated fermented culture complex of Lactiplantibacillus plantarum KM2.SOLUTION: The present invention relates to a microbiome composition for improving muscle strength using a heat-treated fermented culture complex of Lactiplantibacillus plantarum KM2. The composition according to the present invention increased synthesis of myoproteins and showed an effect of inhibiting degradation of myoproteins. In animal tests, improvements in muscle strength through increased grip strength, travel distance, and speed, as well as increases in leg muscle mass, were confirmed. In human clinical trials, improvements in hamstring muscle strength and increases in muscle mass were also confirmed. In addition, it was confirmed that beneficial bacteria increased and harmful bacteria decreased through an increase in the diversity of intestinal microflora altered by muscle reduction and through improvement of the microflora composition. Accordingly, the composition can be usefully employed as a composition for the prevention, improvement, or treatment of muscular diseases, and as a composition for improving muscle strength or increasing muscle mass.SELECTED DRAWING: Figure 19

Description

[Technical Field]

[0001] The present invention relates to a microbiome composition for improving muscle strength that utilizes a fermented, cultured, and heat-treated Lactiplantibacillus plantarum KM2 composite. [Background technology]

[0002] Muscle is the most abundant tissue in the human body, and maintaining adequate muscle mass is essential for maintaining the body's functional capabilities and preventing metabolic diseases. Muscle size is regulated by intracellular signaling processes that induce anabolic and catabolic reactions within the muscle. When signaling reactions that induce muscle protein synthesis predominate over muscle protein breakdown, muscle protein synthesis increases, resulting in muscle hypertrophy (an increase in muscle size) and an increase in the number of muscle fibers.

[0003] Muscles promote calcium influx and increase bone density. However, as the body ages, changes in composition occur, resulting in a redistribution of body fat and body protein. Around age 50, the rate of protein synthesis within muscle cells slows down compared to its breakdown, leading to rapid muscle degeneration and the risk of developing muscle loss. Sarcopenia, one of the muscle loss disorders, typically refers to a loss of approximately 13-24% of body mass and is characterized by decreased protein content, fiber diameter, muscle strength production, and fatigue resistance. Sarcopenia can occur for a variety of reasons, including sepsis, cancer, renal failure, glucocorticoid excess, denervation, muscle disuse, and the aging process.

[0004] Causes of sarcopenia include the gradual loss of skeletal muscle mass and quality due to aging, as well as weight loss, including fat and body fat components, caused by inadequate dietary energy intake. Sarcopenia is often attributed to aging and is closely correlated with age. Sarcopenia results from an imbalance between protein synthesis and degradation. Sarcopenia reduces life satisfaction and can lead to injuries even during simple daily activities. Excessive exercise can also lead to muscle fatigue and damage, reducing athletic performance. Muscle damage includes bruises, tears, local anemia, contusions, and severe damage to skeletal muscle. Such damage can cause severe pain. In cases of severe skeletal muscle damage, treatments that can reduce muscle damage or accelerate muscle tissue recovery may accelerate recovery of muscle force generation after exercise. This can also aid muscle recovery after illness.

[0005] Recently, as interest in dieting has increased, rapid weight loss can lead to sarcopenia regardless of age, and strenuous exercise can damage muscles. Therefore, research and efforts are being focused on treating muscle loss due to common sarcopenia disorders or increasing muscle mass, and research is being conducted on treating muscle disorders and strengthening muscles. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Korean J.Microbiol.2021;57(4):303-306(Published 2021.12.31.) Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention aims to provide a composition for preventing, improving or treating muscle diseases through improving muscle strength or increasing muscle mass, and a composition for improving muscle strength or increasing muscle mass, which comprises a fermented, cultured and heat-treated complex of Lactiplantibacillus plantarum KM2 strain, which is deposited under KCTC 14637BP, a concentrate thereof, a dried product thereof, a fermented metabolite thereof, or a mixture thereof as an active ingredient. [Means for solving the problem]

[0008] The present invention provides a pharmaceutical composition for preventing or treating muscle diseases, which contains as an active ingredient a fermentation-cultured heat-treated composite of Lactiplantibacillus plantarum KM2 strain, which is deposited under the accession number KCTC 14637BP, a concentrate thereof, a dried product thereof, a fermentation metabolite thereof, or a mixture thereof.

[0009] The present invention also provides a functional health food composition for preventing or improving muscle diseases, which contains as an active ingredient a fermented, cultured, and heat-treated complex of Lactiplantibacillus plantarum KM2 strain, which is deposited under KCTC 14637BP, a concentrate thereof, a dried product thereof, a fermented metabolite thereof, or a mixture thereof.

[0010] The present invention also provides a health functional food composition for improving muscle strength or increasing muscle mass, which contains as an active ingredient a fermented, cultured, and heat-treated complex of Lactiplantibacillus plantarum KM2 strain, which is deposited under KCTC 14637BP, a concentrate thereof, a dried product thereof, a fermented metabolite thereof, or a mixture thereof. [Effects of the Invention]

[0011] The present invention relates to a microbiome composition for improving muscle strength using a Lactipranchibacillus plantarum KM2 fermented, cultured, and heat-treated complex. Human studies demonstrated that 12 weeks of intake of the Lactipranchibacillus plantarum KM2 fermented, cultured, and heat-treated complex resulted in significant increases in thigh muscle strength (hamstring strength) compared to the control group, demonstrating improved muscle strength. Muscle mass analysis confirmed that muscle mass increased in the test group compared to the control group after 12 weeks of intake. In particular, analysis of subjects in their 50s, an age at which physical function begins to decline rapidly, confirmed a statistically significant increase in muscle mass in the test group compared to the control group. Furthermore, fecal flora analysis confirmed that 12 weeks of intake of the Lactipranchibacillus plantarum KM2 fermented, cultured, and heat-treated complex increased gut microbial diversity and significantly altered gut community composition in the test group compared to the control group. We also confirmed that beneficial bacteria, Veillonella spp., increased and harmful bacteria, Shigella spp., decreased. [Brief explanation of the drawings]

[0012] [Figure 1] 1 shows the results of analyzing cytotoxicity and myotube diameter in a cell experiment. [Figure 2] 1 shows the results of analyzing muscle protein degradation gene expression and muscle protein synthesis protein expression in a cell experiment. [Figure 3] 1 shows the results of analyzing body composition and body fat mass using DEXA in animal experiments. [Figure 4] 1 shows the analysis results of muscle weight and muscle function measurements in animal experiments. [Figure 5] 1 is a diagram showing the results of histological analysis in an animal experiment. [Figure 6] 1 shows the results of analysis of muscle atrophy inhibition and muscle protein synthesis signaling protein expression in animal experiments. [Figure 7] 1 is a diagram showing the results of an analysis of intestinal microorganisms in animal experiments. [Figure 8]1 is a diagram showing the participation status and analysis groups of human subject trials. [Figure 9] 10 is a diagram showing changes in left hamstring muscle strength. [Figure 10] 1 is a graph showing the average change in hamstring strength. [Figure 11] 1 is a graph showing the change in muscle mass (ASM / Height2, g / m2). [Figure 12] This is a graph showing the change in muscle mass (ASM / Height2, g / m2) for subjects in their 50s. [Figure 13] 10 is a graph showing the change in SPPB-total score (points). [Figure 14] 10 is a graph showing the amount of change in SPPB-walking speed (sec). [Figure 15] 1 is a graph showing the change in SPPB-chair standing (sec). [Figure 16] 1 is a graph showing the change in CRP (mg / L). [Figure 17] 1 is a diagram showing the analysis results of fecal flora α-Diversity (Shannon Index) (PP Set) in a human application test. [Figure 18] 1 is a diagram showing the analysis results of fecal flora β-Diversity (Bray-Curtis dissimilarity) (PP Set) in a human application test. [Figure 19] 1 is a graph showing the results of analysis of test group-specific fecal flora abundance changes (MaAsLin2) after 12 weeks of intake in a human application test. DETAILED DESCRIPTION OF THE INVENTION

[0013] The present invention provides a pharmaceutical composition for preventing or treating muscle diseases, which contains as an active ingredient a fermentation-cultured heat-treated composite of Lactiplantibacillus plantarum KM2 strain, which is deposited under the accession number KCTC 14637BP, a concentrate thereof, a dried product thereof, a fermentation metabolite thereof, or a mixture thereof.

[0014] Preferably, the pharmaceutical composition increases beneficial Veillonella spp. and decreases harmful Shigella spp. in changing the intestinal community composition. More preferably, the Veillonella spp. is one or more selected from the group consisting of Veillonella sp. S12025-13, Veillonella dispar, Veillonella nakazawae, and Veillonella atypica, and the Shigella spp. is one or more selected from the group consisting of Shigella sonnei, Shigella boydii, Shigella dysenteriae, and Shigella flexneri, but is not limited thereto.

[0015] Preferably, the muscle disease is, but is not limited to, muscular atrophy, sarcopenia, atony, muscular dystrophy, myasthenia gravis, or amyotrophic lateral sclerosis.

[0016] Preferably, the pharmaceutical composition can strengthen or improve the strength of the hamstring muscles, which are responsible for flexion among thigh muscles, but is not limited thereto.

[0017] Preferably, the pharmaceutical composition can increase muscle mass in people in their 50s, who are prone to significant muscle mass loss, but is not limited thereto.

[0018] The pharmaceutical compositions of the present invention can be prepared using pharmaceutically suitable and physiologically acceptable excipients in addition to the active ingredient. Examples of such excipients include solubilizers, such as excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, glidants, or flavoring agents. The pharmaceutical compositions of the present invention can be formulated as pharmaceutical compositions containing one or more pharmaceutically acceptable carriers in addition to the active ingredient for administration. For compositions formulated as liquid solutions, acceptable pharmaceutical carriers are sterile and biocompatible, and include saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and mixtures of one or more of these components. Other common additives, such as antioxidants, buffers, and bacteriostats, can be added as needed. Furthermore, the compositions can be formulated into injectable forms, such as aqueous solutions, suspensions, and emulsions, as well as pills, capsules, granules, or tablets, by adding diluents, dispersants, surfactants, binders, and lubricants.

[0019] The pharmaceutical compositions of the present invention may be in the form of granules, powders, coated tablets, tablets, capsules, suppositories, syrups, juices, suspensions, emulsions, infusions, or injectable solutions, as well as sustained-release formulations of the active compound. The pharmaceutical compositions of the present invention may be administered in a conventional manner via intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, intranasal, inhalation, topical, rectal, oral, intraocular, or intradermal routes. The effective amount of the active ingredient of the pharmaceutical composition of the present invention refers to the amount required for the prevention or treatment of a disease. Therefore, the effective amount may be adjusted depending on various factors, including the type of disease, the severity of the disease, the type and content of the active ingredient and other ingredients contained in the composition, the type of dosage form, the patient's age, weight, general health, sex, and diet, the administration time, administration route, the excretion rate of the composition, the duration of treatment, and concomitant medications.

[0020] The present invention also provides a functional health food composition for preventing or improving muscle diseases, which contains as an active ingredient a fermented, cultured, and heat-treated complex of Lactiplantibacillus plantarum KM2 strain, which is deposited under KCTC 14637BP, a concentrate thereof, a dried product thereof, a fermented metabolite thereof, or a mixture thereof.

[0021] Preferably, the functional health food composition increases beneficial Veillonella spp. and reduces harmful Shigella spp. in changing the intestinal community composition, and more preferably, the Veillonella spp. is at least one selected from the group consisting of Veillonella sp. S12025-13, Veillonella dispar, Veillonella nakazawae, and Veillonella atypica, and the Shigella spp. is at least one selected from the group consisting of Shigella sonnei, Shigella boydii, Shigella dysenteriae, and Shigella flexneri, but is not limited thereto.

[0022] Preferably, the muscle disease is, but is not limited to, muscle atrophy, sarcopenia, hypotonia, muscle dystrophy, myasthenia gravis, or amyotrophic lateral sclerosis.

[0023] The present invention also provides a health functional food composition for improving muscle strength or increasing muscle mass, which contains as an active ingredient a fermented, cultured, and heat-treated composite of Lactiplantibacillus plantarum KM2 strain, which is designated as KCTC 14637BP, a concentrate thereof, a dried product thereof, a fermented metabolite thereof, or a mixture thereof. Preferably, the health functional food composition can strengthen or improve the strength of the hamstring muscles, which are responsible for flexion among thigh muscles, but is not limited thereto.

[0024] Preferably, the health functional food composition can increase muscle mass in people in their 50s, who are prone to significant muscle mass loss, but is not limited thereto.

[0025] The health functional food composition of the present invention is provided in the form of powder, granules, tablets, capsules, syrup or drink, and the health functional food composition is used together with other foods or food additives in addition to the active ingredient, and is used appropriately in a conventional manner. The amount of the active ingredient mixed is determined appropriately depending on the purpose of use, such as prevention, health or therapeutic treatment.

[0026] The effective dose of the active ingredient contained in the health functional food composition can be used in accordance with the effective dose of the pharmaceutical composition, but in the case of long-term intake for the purpose of health and hygiene or health regulation, it is within the above range, and it is certain that the active ingredient can be used in an amount greater than the above range because there is no problem in terms of safety.

[0027] The types of the health foods are not particularly limited, and examples include meats, sausages, bread, chocolate, candies, snacks, sweets, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, drinking water, tea, energy drinks, alcoholic beverages, and vitamin complexes.

[0028] The present invention will be described in detail below with reference to examples to aid in understanding the present invention. However, the following examples are merely illustrative of the content of the present invention, and the scope of the present invention is not limited to the following examples. The examples of the present invention are provided to more completely explain the present invention to those skilled in the art. <Experimental Example>

[0029] The following experimental examples are provided to provide examples that are commonly applicable to each embodiment of the present invention.

[0030] 1. Preparation of Lactiplastid Bacillus plantarum KM2 Fermentation Culture Heat Treatment Composite

[0031] Lactipranthibacillus plantarum KM2 was inoculated (2%, v / v) into MRS medium and cultured at 30°C for 12 hours, followed by heat treatment at 90°C for 1 hour. The strain-free supernatant of L. plantarum (CFS-L. plantarum) was collected using a centrifugal separator and filtered through a 0.2 μm membrane. CFS-L. plantarum was freeze-dried with the addition of 7% (w / v) soybean flour, and the centrifuged cells were dried and converted into CFS powder. The heat-treated microorganisms were then lyophilized. 9 cells / g to prepare a Lactiplantibacillus plantarum KM2 fermented culture heat-treated composite (KLP-KM2).

[0032] 2. Cell Experiments

[0033] (1) C2C12 cell culture and viability measurement

[0034] Mouse C2C12 myoblast cells (ATCC, Manassas, VA, USA) were subcultured in DMEM containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C in a 5% CO2 incubator. C2C12 myoblast cells were cultured in a 96-well plate at 5 × 10 3 After dispensing at 100 cells / well, the cells were treated with KLP-KM2 (200, 400, 800 μg / mL) and cultured for 48 hours, after which cell viability was analyzed using an MTT assay.

[0035] (2) C2C12 cell differentiation and muscle atrophy model formation

[0036] To differentiate C2C12 myoblasts into myotubes, 8 × 10 cells were cultured in a 6-well plate. 5After distributing cells / well, when the cells reached 100% fullness, they were replaced with DMEM medium containing 2% horse serum and 1% penicillin-streptomycin and allowed to differentiate for 6 days. As shown in the figure, C2C12 cells differentiated into myotubes were treated with dexamethasone (DEX: 100 μM), a known inducer of muscle atrophy, to induce muscle atrophy. After incubation with DEX for 24 or 48 hours, analysis was performed.

[0037] (3) Jenner-Giema staining of C2C12 cell morphological changes

[0038] Morphological changes in muscle cells due to muscle atrophy were assessed by measuring myotube diameter using Jenner-Giemsa staining. Differentiated C2C12 cells were stained with Jenner-Giemsa solution, and the fusion index (%) and myotube width were measured using a DM IL LED microscope (Leica Microsystems, Germany).

[0039] (4) Analysis of muscle atrophy improvement using C2C12 myotubes

[0040] Because root hair cells (C2C12) increase the expression of MyoD and myogenin as they differentiate into myotubes, we analyzed the increase in myogenesis-related factor expression following sample treatment using real-time quantitative PCR (q-RT PCR). Because DEX treatment induces the expression of proteolytic factors FoxO3a, MuRF1, and Atrogin-1, we analyzed the suppression of proteolytic factor expression following sample addition using q-RT PCR and Western blotting. Because muscle atrophy is mediated by decreased activation of the mTOR / PI3K / Akt pathway, we analyzed the increase in the expression of protein synthesis factors (mTOR, PI3K, and Akt) following sample treatment using real-time quantitative PCR (q-RT PCR).

[0041] 3. Animal Experiments

[0042] (1) Preparation and design of animal experiments

[0043] Experimental animals were 7-week-old male C57BL / 6J mice purchased from Orient Bio. The animal room environment was controlled at 21±1°C, 40-60% relative humidity, and a 12-hour light-dark cycle. Food and water were available ad libitum. This study was approved by the Kookmin University Animal Experiment Ethics Committee (KMU-2023-05) and conducted in accordance with the Standard Operating Guidelines. After an adaptation period, the experimental groups were divided into five groups: normal control (NOR), negative control (dexamethasone; DEX), low-dose sample treatment group (KLP-KM2 900 mg / kg; KBL), high-dose sample treatment group (KLP-KM2 1800 mg / kg; KBH), and positive control (oxymetholone 50 mg / kg; OXM), each consisting of eight mice. To create the muscle atrophy model, dexamethasone (5 mg / kg) was administered intraperitoneally to the DEX, KLP-KM2, and OXM groups daily between 10:00 and 11:00 AM for 8 weeks. KLP-KM2 and OXM were administered orally for 8 weeks, while the NOR and DEX groups were administered saline orally for the same period. Food intake and body weight were measured every 2 weeks.

[0044] (2) Analysis of gymnastics

[0045] Before sacrificing the experimental animals, the physical fitness of the mice was measured using dual energy X-ray absorptiometry (DEXA; Medikors, Seongnam, Korea) at 7 weeks, and changes in muscle mass relative to body weight were analyzed.

[0046] (3) Serum biochemical analysis

[0047] After sacrifice, blood was collected from the heart and immediately centrifuged (3,000 × g, 10 min, 4°C) to separate plasma. Creatine phosphokinase (CPK), lactate dehydrogenase (LDH), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and blood urea nitrogen (BUN) were measured using a chemical analyzer (Fuji DRI-CHEM 3500i, Fuji Photo Film, Ltd., Tokyo, Japan).

[0048] (4) Grip strength measurement

[0049] Grip strength was measured after muscle atrophy induction using a grip strength meter (DBI, Co., Eumsung, Korea). A stainless steel T-bar was attached to the cage, and the mouse was allowed to grasp the T-bar with both forepaws. The tail was pulled at a constant speed, and grip strength was measured five times per mouse. The average value excluding the highest and lowest values was recorded.

[0050] (5) Evaluation of athletic ability

[0051] Mouse motility was measured using a treadmill, and recorded videos were analyzed using an EthoVision video tracking system (Noldus Information Technology, Wageningen, Netherlands). After 2 days of training, mice were set to run for 10 minutes at a speed of 10 m / min with a 10° incline, and the speed was increased by 2 m / min every 3 minutes until a maximum speed of 30 m / min was reached.

[0052] (6)Histological analysis

[0053] The gastrocnemius muscles were fixed in 4% formaldehyde, then paraffin blocks were prepared and subjected to H&E staining. The cross-sectional area of muscle tissue was measured using KFBIO Slide Manager (KFBIO, Ningbo, China), and the cross-sectional area of muscle fibers (μm 2 ) were expressed as an average of 100 cross-sectional areas of representative images measured at 20x magnification using ImageJ software (Version 1.8.0, National Institutes of Health, USA).

[0054] (7) Analysis of muscle breakdown and synthesis-related protein expression

[0055] Muscle cells and tissues were homogenized in RIPA (radioimmunoprecipitation assay) buffer containing 1% protease inhibitor and 1% kinase inhibitor using a bullet blender (Next Advance, Troy, NY, USA) before use. The homogenized tissue was incubated at 4°C for 50 minutes and then centrifuged at 4°C and 15,000 x g for 15 minutes to obtain the supernatant. Equal amounts of protein were separated by 12% SDS-PAGE and transferred to polyvinylidene fluoride membranes (Bio-Rad, Hercules, CA, USA). After blocking with 5% bovine serum albumin in Tris-buffered saline with Tween 20 (TBST, 0.1%), the cells were incubated overnight at 4°C with antibodies to AKT (Cat. 4691S), p-AKT (Cat. 4060S), mTOR (Cat. 2986S), p-mTOR (Cat. 5536S), p-FoxO3a (Cat. 9466S), and β-actin (Cat. 4970S, Cell Signaling Technology).

[0056] After incubation with horseradish peroxidase-conjugated secondary antibodies for 1 hour at room temperature, sections were washed four times with TBST buffer. Protein bands were detected using enhanced chemiluminescence detection kits (BioRad, Hercules, CA, USA). Band intensities were normalized for β-actin protein and quantified using Image Lab software 5.1 (BioRad).

[0057] (8) Analysis of muscle breakdown and synthesis-related gene expression

[0058] RNA was extracted from muscle cells using NucleoZol Reagent (Macherey-Nagel, Düren, Germany), and from gastrocnemius muscle tissue using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). cDNA was synthesized using the High-Capacity RNA-to-cDNA Kit (Applied Biosystems) and analyzed using TaqMan. (R) qRT-PCR was performed using the StepOne Plus RT-PCR system (Applied Biosystems) with Gene Expression Master Mix (Applied Biosystems). Quantitative results were expressed relative to a reference mRNA (GAPDH) using the 2-ΔΔCT method (Livak & Schmittgen, 2001). Primers used for analysis were as follows: GAPDH (Mm99999915_g1), FoxO3a (Mm01185722_m1), atrogin-1 (Mm00499523_m1), and MuRF1 (Mm01185221_m1).

[0059] (9) Analysis of intestinal microorganisms

[0060] To confirm the effect of the samples on the intestinal microbiota of the animal model, the animals were sacrificed, the cecum was removed, and DNA was extracted and analyzed using Shotgun metagenome sequencing. The extracted DNA was subjected to adapter ligation using the Truseq Nano DNA prep kit, followed by eight cycles of PCR to create libraries. The library size was confirmed using a Tapestation 4200 instrument, and libraries of 450-650 bp in length were sequenced using a Nextseq 2000 instrument to generate data. Raw reads were subjected to a process to remove low-quality reads and adapters using Trimmomatic v0.39 (AM Bolger, M Lohse, B Usadel, 2014). Trimmed reads were then aligned to the PhiX reference genome (NC_001422.1) using BWA v0.7.17-r1188 (Li and Heng, 2013) and then purified using SAMtools v1.15.1 (Li, Heng, et al., 2009). Taxonomy was performed using Kraken2 v2.1.2 (Wood et al., 2019), and species richness was estimated using Bracken v2.55 (Lu, Jennifer, et al., 2017). Alpha diversity was measured using the Shannon index via the rtk v0.2.6.1 R package (Saary, Paul, et al., 2017), and beta diversity analysis was performed using the phyloseq v1.34.0 R package (McMurdie & Holmes, 2013) to calculate weighted and unweighted UniFrac distances.

[0061] (10) Statistical analysis

[0062] Data are expressed as mean ± standard error, and statistical analysis of the experimental results was performed using SPSS Statistics V.28 (SPSS Inc., USA). First, one-way ANOVA was used to determine whether there was a difference between treatment groups. If a significant difference was found (P < 0.05), Duncan's multiple comparison test was used to verify the significance between treatment groups.

[0063] 4. Human application test

[0064] (1)Design

[0065] The human trial was designed as a 12-week, randomized, double-blind, placebo-controlled, parallel study. Subjects who voluntarily signed the human trial consent form participated in the trial. Demographic surveys, medical and medication history, non-drug treatment history surveys, lifestyle surveys, physical examinations, vital signs (blood pressure, pulse), anthropometric measurements (height, weight, body mass index (BMI)), clinical pathology tests, pregnancy tests (only for women of childbearing age), electrocardiograms, skeletal muscle mass (in-body), grip strength, and blood indicator tests were conducted. Subjects who met the inclusion / exclusion criteria were randomly assigned to the study. Subjects assigned to the test or control group consumed the test or control food for a total of 12 weeks. The allocation ratio for each group was 1:1.

[0066] (2) Selection of subjects

[0067] Subjects were selected from those aged 50 to 85 years, with a grip strength of less than 36 kg for men or 22 kg for women, and with reduced muscle strength, with skeletal muscle mass measured by Inbody less than 100% of the standard range, who did not meet the exclusion criteria.

[0068] (3) Food for human application tests

[0069] The Lactiplantibacillus plantarum KM2 fermented culture heat-treated composite (hereafter referred to as KLP-KM2) and the control food were manufactured by Kokumin Bio Co., Ltd. The subjects took 9g of the compound in the form of small pellets twice a day, two packets per day, with plenty of water for 12 weeks.

[0070] (4) Random assignment

[0071] The human application test was conducted in parallel by randomly assigning subjects to either the test group or the control group, and the required number of subjects for the human application test was 80 in total, with 40 subjects per group, taking into account the dropout rate (25%).

[0072] At Visit 2 (Randomization visit, Week 0), all human study subjects who met the inclusion / exclusion criteria were assigned to each group using a blocked random allocation code. To ensure balanced random allocation between the intake groups, the ratio of subjects in each group was kept at 1:1.

[0073] The random assignment table was created using SAS (R) The permutation of random numbers (A, B) generated by the randomization program of the SAS system is applied sequentially from human application test subject number 1. (R) The human application test client attached the human application test food label according to the random allocation table when packaging the food for human application testing, and then supplied it to the human application testing institution before the start of the human application testing.

[0074] (5) Measurement of thigh muscle strength

[0075] Thigh muscle strength was measured at visits 2, 3, and 4 using an isokinetic test device (Con-Trex) at an angular velocity of 60° / sec. The subject sat in the dynamometer chair with their torso fixed to the backrest. The knee joint axis of the leg being measured was aligned with the axis of the dynamometer, and the distal thigh, ankle joint, and upper leg were firmly fixed using resistance pads. The range of motion of the joints was determined to be the maximum possible range.

[0076] Measurements were taken five times on each side, and this was repeated three times. The maximum quadriceps strength measured each time was recorded.

[0077] When measuring lower limb muscle strength using isokinetic evaluation equipment (Con-Trex), the independent contractions of the thigh muscle extensors (quadriceps) and flexors (hamstrings) are primarily involved. Therefore, in the human subject application test study, the efficacy evaluation items were collected by dividing them into thigh muscle extensors (quadriceps strength) and flexors (hamstrings strength).

[0078] (6) Muscle mass (ASM / Height) 2 ) measurement

[0079] Baumgartner first proposed diagnostic criteria for sarcopenia in 1998, presenting the results of the New Mexico Elder Health Survey (NMEHS). In this study, Baumgartner defined appendicular skeletal muscle mass (ASM) as the sum of muscle mass measured by DEXA, minus bone mineral mass, and divided it by the square of height, similar to body mass index (BMI), to correct for the increase in appendicular muscle mass with increasing height. Appendicular fat mass was measured using DEXA at visits 2, 3, and 4, and ASM was calculated by subtracting bone mineral mass from appendicular fat mass, then corrected by the square of height.

[0080] (7) Short Physical Performance Battery (SPPB)

[0081] The Simple Physical Performance Test (SPPB), administered on visits 2, 3, and 4, was first used by the US National Institute of Aging (NIA) in a study to establish an epidemiological study of the elderly and has been used to easily assess the physical function of the elderly. The performance test to evaluate lower limb function consisted of three items: upright balance test, walking speed, and five repeated chair rises. A score of 0 was given for failure to perform each task, and a score of 1 to 4 was given depending on performance. Successful completion of each task resulted in a total of 12 points.

[0082] -Standing balance: The balance test consisted of sub-items: side-by-side stance, semi-tandem stance, and tandem stance. Each stance was tested in turn, and an experienced examiner demonstrated it to the subject before evaluating them. If the subject was able to maintain the side-by-side stance for more than 10 seconds, they were given 1 point. If they were unable to maintain the side-by-side stance, the next test, the walking speed test, was conducted. If the side-by-side stance was given 1 point, the subject was then tested in semi-tandem stance and given 1 point if they were able to maintain it for more than 10 seconds, and then the tandem stance was performed. In the tandem stance, if the subject maintained it for more than 3 seconds, they were given 1 point, and if they maintained it for more than 10 seconds, they were given 2 points. The maximum score for the balance test was 4 points.

[0083] -Gait speed test: Walking speed was evaluated based on the time it took to walk 4 meters. Participants were instructed to walk at their normal speed, and if they were unable to do so, they received a score of 0, if it took more than 8.7 seconds, they received a score of 1, if it took 6.21 to 8.7 seconds, they received a score of 2, if it took 4.82 to 6.20 seconds, they received a score of 3, and if it took less than 4.82 seconds, they received a score of 4. The test was conducted twice in total, and the fastest time was used as the standard. If participants were able to walk using walking aids such as a cane, the test was conducted in the same way.

[0084] Repeated chair stands: Chair stands were assessed based on the time it took to repeatedly stand up and sit down from a chair five times while folding arms across the chest. Failure to complete the test within 60 seconds was scored as 0; 16.7 seconds or longer was scored as 1; 13.7-16.6 seconds was scored as 2; 11.2-13.6 seconds was scored as 3; and 11.1 seconds or less was scored as 4. If the subject used their hands or arms to stand up, or if there was concern about the subject's safety, such as a fall, the examiner could discontinue the test and score 0. This test is intended to assess lower-body muscle strength, which is required for many tasks, such as climbing stairs, walking, standing up from a chair or bathroom, and getting out of a car. Increasing this motor ability reduces the likelihood and frequency of falls.

[0085] (8) CRP

[0086] CRP (C-Reactive Protein) increases when skeletal muscle mass decreases due to sarcopenia, and it has been reported that high CRP levels are directly related to functional decline in muscles.

[0087] Blood samples were collected and tested at visits 1 and 4.

[0088] (9) Analysis of intestinal microorganisms

[0089] To confirm the effects of the sample on the intestinal microbiota, feces were collected after sample administration, and DNA was extracted and analyzed using Shotgun metagenome sequencing. The extracted DNA was subjected to adapter ligation using the Truseq Nano DNA prep kit, followed by eight cycles of PCR to create libraries. The library size was confirmed using a Tapestation 4200 instrument, and libraries of 450-650 bp in length were sequenced using a Nextseq 2000 instrument to generate data. Raw reads were subjected to a process to remove low-quality reads and adapters using Trimmomatic v0.39 (AM Bolger, M Lohse, B Usadel, 2014). To remove PhiX sequences added during the sequencing process, trimmed reads were aligned to the PhiX reference genome (NC_001422.1) using BWA v0.7.17-r1188 (Li and Heng, 2013) and then purified using SAMtools v1.15.1 (Li, Heng, et al., 2009). Taxonomy was performed using Kraken2 v2.1.2 (Wood et al., 2019), and species richness was estimated using Bracken v2.55 (Lu, Jennifer, et al., 2017). Alpha diversity was measured using the Shannon index via the rtk v0.2.6.1 R package (Saary, Paul, et al., 2017), and beta diversity analysis was performed using the phyloseq v1.34.0 R package (McMurdie & Holmes, 2013) to calculate weighted and unweighted UniFrac distances.

[0090] (10) Abnormal cases

[0091] For abnormal cases, a safety set analysis was conducted as the main analysis, and the analysis included 40 test subjects and 40 control subjects who had consumed the human test food at least once after being randomly assigned to the human test. The type and incidence of abnormal cases, and the relationship between the severity of symptoms and the human test food were evaluated.

[0092] (11) Statistical analysis method

[0093] Statistical analysis was performed using SAS (R) (Version 9.4, SAS Institute, Cary, North Carolina, USA) was used for the analysis.

[0094] Efficacy data, demographic and nutritional analysis data, and safety data were subjected to two-sided statistical tests with a significance level of 0.05. p-values for all analyses were presented to four decimal points, and p-values <0.05 for all analyses were considered significant. Numerical values with decimal points, such as means, standard deviations, and percentages, were presented to two decimal points.

[0095] Comparisons between groups were performed using a normality test (Kolmogorov-Smirnov) with a p-value of 0.05. When normality was satisfied in both the test and control groups, a two-sample t-test was used for comparison analysis. When normality was not satisfied in either group, a Wilcoxon rank sum test was used for comparison analysis. Comparisons within groups were performed using a normality test. When normality was satisfied, a paired t-test was used for comparison analysis. When normality was not satisfied, a Wilcoxon signed rank test was used for comparison analysis. ANCOVA was performed using the baseline of each variable as a covariate.

[0096] Example 1: Cell experiment

[0097] 1. Cytotoxicity and Myotube Diameter Analysis

[0098] Analysis of the cytotoxicity of KLP-KM2 samples in muscle cells revealed no significant change in cell viability up to 800 μg / mL. Treatment with 200-800 μg / mL samples significantly increased myotube diameter, which had been reduced by DEX, compared to the control group, with a 33% increase at the highest concentration of 800 μg / mL (Figure 1).

[0099] 2. Analysis of Muscle Proteolysis Gene Expression and Muscle Protein Synthesis Protein Expression

[0100] Analysis of the effects of KLP-KM2 on muscle protein breakdown and synthesis factors in muscle cells showed that the sample significantly reduced gene expression of muscle protein breakdown factors FoxO3a, Atrogin-1, and MuRF1 in a concentration-dependent manner, with reductions of 36%, 33%, and 30%, respectively, at the highest concentration of 800 μg / mL. Protein expression of muscle protein synthesis factors Akt, mTOR, and FoxO3a also increased significantly in a concentration-dependent manner. These results also confirmed that KLP-KM2 has the effect of ameliorating muscle loss at the cellular level by suppressing muscle protein breakdown genes and increasing muscle protein synthesis (Figure 2).

[0101] Cellular experiments showed that KLP-KM2 (KB) significantly increased the myotube diameter, which had decreased in muscle cells in which muscle atrophy was induced by dexamethasone, and promoted muscle protein synthesis and inhibited muscle protein degradation.

[0102] Example 2: Animal experiment

[0103] 1. Analysis of body composition and body fat mass using DEXA

[0104] To evaluate the efficacy of DEX in preventing muscle loss, we conducted a dexamethasone-induced animal efficacy study and measured body weight and food intake. All DEX-treated groups had significantly lower body weights than the NOR group, but there was no significant difference in food intake between groups. Using DEXA to measure changes in physical activity, the DEX group showed a significant decrease in muscle mass relative to total body weight compared to the NOR group, while the KLP-KM2 (KB900, KB1800) and OXM groups showed a significant increase relative to the DEX group. Hindlimb and forelimb muscles were significantly reduced in all DEX-treated groups, but there were no significant differences between the DEX groups (Figure 3).

[0105] 2. Analysis of Muscle Weight and Muscle Function Measurements

[0106] Immediately after the animals were sacrificed, the weights of the gastrocnemius and quadriceps muscles of the mice were measured. The results showed that the weights of the gastrocnemius and quadriceps muscles in the KLP-KM2 group were significantly increased in a concentration-dependent manner compared to the DEX group.

[0107] To evaluate whether administration of the sample improved muscle function loss in a DEX-induced animal model, we evaluated the muscle strength and running performance of mice using a grip strength meter and a treadmill. The KLP-KM2 group maintained significantly higher muscle strength than the DEX group. During treadmill exercise, the DEX group significantly decreased running distance and speed compared to the NOR group over the same time period, while the KLP-KM2 group significantly increased running distance and speed compared to the DEX group, maintaining similar exercise performance to the NOR group, demonstrating the muscle function improvement effect of KLP-KM2 (Figure 4).

[0108] 3.Histological analysis

[0109] Because muscle fiber atrophy is an important indicator of muscle damage, we examined the cross-sectional area of gastrocnemius muscles after H&E staining to assess muscle structural changes following administration. The DEX group exhibited decreased muscle fiber size and increased interfiber spacing compared to the NOR group, while the KLP-KM2 group mitigated muscle tissue damage compared to the DEX group. To quantify morphological changes in muscle fibers, we measured the CSA of muscle fibers. The DEX group showed a significant decrease in CSA compared to the NOR group, while the KLP-KM2 group significantly recovered CSA. Consequently, KLP-KM2 administration maintained muscle cross-sectional area at a similar level to that before muscle atrophy (Figure 5).

[0110] 4. Serum biochemical analysis

[0111] ALT and AST are biomarkers commonly used to assess hepatotoxicity, and DEX administration increased ALT and AST. To assess the in vivo toxicity of KLP-KM2, serum levels of ALT, AST, and BUN were analyzed. AST was significantly increased in the DEX group compared with the NOR group, and ALT levels also tended to increase. However, there was no significant difference in the KLP-KM2 group compared with the DEX group. BUN is a biomarker commonly used to assess nephrotoxicity, and measurements showed no significant increase in BUN levels in the KLP-KM2 group compared with the NOR group. These results suggest that KLP-KM2 administration did not induce hepatotoxicity or nephrotoxicity in mice and provided partial protection against DEX-induced liver and kidney damage (Table 1).

[0112] [Table 1]

[0113] 5. Muscle atrophy suppression and muscle protein synthesis signaling protein expression

[0114] Analysis of the effects of KLP-KM2 on muscle protein breakdown and synthesis in gastrocnemius muscle tissue showed that the expression of protein breakdown-related factors Atrogin-1 and MuRF1 was significantly reduced in the KLP-KM2 group compared to the DEX group, while the expression of protein synthesis-related factors Akt, mTOR, and FoxO3a was significantly increased in the KLP-KM2 group compared to the DEX group. This indicates that KLP-KM2 intake effectively modulates DEX-induced muscle loss (Figure 6).

[0115] 6. Analysis of intestinal microorganisms

[0116] The Firmicutes to Bacteroidetes F / B ratio has been proposed as an important indicator of gut microbial health, and similar to findings reported in patients with chronic liver disease with low muscle mass, the F / B ratio was significantly decreased in the DEX group and significantly increased by KLP-KM2 intake.

[0117] Sudologranulum regulates abnormal inflammatory and immune conditions and is a butyrate-producing microorganism that may positively affect muscle mass through butyrate and its metabolites. At the genus level, the relative abundance of Sudologranulum was significantly decreased in the DEX group and significantly increased by KLP-KM2 administration.

[0118] The genus Alistipes is a microorganism present in the gastrointestinal tract of healthy humans and is considered an important indicator of gut microbial community imbalance when depleted. At the genus level, Alistipes was significantly decreased in the DEX group and significantly increased by KLP-KM2 administration.

[0119] Although it has been reported that the abundance of Akkermansia increased in elderly chronic patients experiencing sarcopenia, the proportion of Akkermansia at the genus level was significantly decreased in the KLP-KM2 and OXM groups compared to the DEX group.

[0120] Porphyromonas gingivalis is known to be involved in elevated blood glucose levels in patients with type 2 diabetes, which may lead to sarcopenia by impairing insulin sensitivity. Intake of KLP-KM2 reduced Porphyromonas gingivalis at the species level, which was similar to that of the NOR group.

[0121] Faecalibacterium prausnitzii, a microorganism known to produce SCFAs, is known to increase gastrocnemius muscle mass and increase the expression of the mitochondrial respiratory chain protein ATP5A when administered to rats fed a high-fat diet. At the species level, the relative abundance of Faecalibacterium prausnitzii was highest in rats fed KLP-KM2 (Figure 7).

[0122] Animal experiments showed that administration of KLP-KM2 (KB) in an animal model in which muscle atrophy was induced with dexamethasone significantly increased muscle weight (gastrocnemius and quadriceps) and body fat mass, maintained significantly higher muscle strength, and maintained the same level of exercise capacity as the normal control, demonstrating improved muscle function. Histological analysis showed that muscle cross-sectional area showed the same level of muscle atrophy as the normal control. Regarding the gut microbiome, administration of KLP-KM2 increased microbial diversity, increasing the abundance of microorganisms that increase SCFA production and decreasing muscle atrophy-related microorganisms.

[0123] In conclusion, in a C57BL / 6J rodent model in which muscle atrophy was induced by DEX treatment, administration of KLP-KM2 preparations increased body fat mass, muscle weight, grip strength, running speed, muscle tissue cross-sectional area, and muscle protein synthesis factors, and positively regulated muscle atrophy-related biomarkers through a decrease in muscle protein breakdown-related factors. Overall, KLP-KM2 intake is considered to be a postbiotic formulation that is effective in ameliorating muscle loss by regulating gut microbiota.

[0124] <Example 3> Human application test

[0125] 1. Selection of study subjects to be included in the analysis

[0126] Figure 8 summarizes the participation status of human trial subjects and the analysis groups from the start to the end of the human trial.

[0127] 2. Demographic information and other pre-intake characteristics

[0128] All pre-intake characteristics, including demographic information, of the human study participants were compared between the intake groups to identify factors that may have contributed to the differences.

[0129] An investigation into the demographic information and characteristics of the subjects in the human application test before intake revealed that, in terms of gender, the test group included 4 men (12.50%) and 28 women (87.50%), while the control group included 4 men (11.76%) and 30 women (88.24%), with no statistically significant difference between the intake groups.

[0130] The mean age of the test group was 58.97±6.48 years, while the mean age of the control group was 60.00±6.42 years, and no statistically significant difference was observed between the intake groups.

[0131] Additionally, no statistically significant differences were observed between the intake groups in height, weight, or BMI, allowing for the assumption of comparability between the groups (Table 2).

[0132] [Table 2]

[0133] 3.Evaluation of thigh muscle strength effectiveness

[0134] In a human subject application test study, after 12 weeks of intake, a significant increase was confirmed in the average strength of the hamstring muscles located behind the femur, with a statistically significant increase in left hamstring strength in the test group compared to the control group. After intake of the Lactiplantibacillus plantarum KM2 fermented, cultured, and heat-treated complex, right hamstring strength increased, but the right leg, which is used more, did not show a significant change in muscle strength, and a statistically significant increase in muscle strength was confirmed in the left leg, which is used less (Figures 9 and 10).

[0135] 4. Evaluation of muscle mass, simple physical performance assessment, CRP and gut microbiota efficacy

[0136] Analysis of the change in muscle mass after 6 weeks of intake showed that the test group had a muscle mass of 57.66±130.31g / m 2 increased (p=0.0178), while the control group was 0.78±138.26g / m 2After 12 weeks of intake, the test group had a mean blood cholesterol level of 17.01±137.13g / m 2 increased (p=0.4882), and the control group was 30.17±118.23g / m 2 decreased (p=0.1463), showing a positive trend.

[0137] Further analysis of muscle mass by specifying the age of the subjects as those in their 50s, when physical function begins to decline rapidly, showed that after 12 weeks of intake, the test group had a muscle mass of 60.63±119.44g / m 2 The difference was statistically significant, with an increase of 31.89±124.55g / m² in the test group and a decrease of 31.89±124.55g / m² in the control group. This means that the limb muscle mass of the test group increased significantly compared to the control group in their 50s (Figures 11 and 12).

[0138] In the case of the SPPB, analysis of the change in the total SPPB score showed that after 6 weeks of intake, the test group increased by 0.03±0.31 points (p=1.0000), while the control group decreased by 0.03±0.17 points (p=1.0000), but no statistically significant difference was observed between the intake groups. After 12 weeks of intake, the test group increased by 0.06±0.25 points (p=0.5000), while the control group showed no change.

[0139] Analysis of the change in SPPB walking speed showed that after 6 weeks of intake, the test group increased by 0.03±0.18 seconds (p=1.0000), while the control group decreased by 0.03±0.17 seconds (p=1.0000), but no statistically significant difference was observed between the intake groups. After 12 weeks of intake, the test group increased by 0.03±0.18 seconds (p=1.0000), while the control group showed no change.

[0140] Analysis of the change in SPPB chair stand time showed that after 6 weeks of intake, the test group increased by 0.03±0.18 seconds (p=1.0000), while the control group showed no change. After 12 weeks of intake, the test group increased by 0.03±0.18 seconds (p=1.0000), while the control group showed no change.

[0141] Because most subjects received scores close to perfect at baseline, it was difficult to confirm statistically significant changes between the test and control groups after 6 and 12 weeks of intake. However, after 12 weeks of intake, there was no change in the control group, while there was an increase in the test group. This change may be confirmed through further research using SPPB (Figures 13 to 15).

[0142] In the analysis of changes in CRP, measurements were taken at baseline and 12 weeks, and subjects with a CRP result of <0.2 at 12 weeks were excluded from the analysis. After 12 weeks of intake, the test group had a decrease of 0.13±2.22 mg / L (p=0.0554), while the control group had an increase of 0.43±3.09 mg / L (p=0.8516), confirming a significant increase in the test group compared to the control group (Figure 16).

[0143] The diversity of the microbial flora composition in the feces was analyzed using α-diversity (Shannon Index). At baseline, the test group and the control group showed the same diversity, but after 12 weeks of intake, a statistically significant level of higher diversity was confirmed in the test group compared to the control group (Figure 17).

[0144] Analysis of the structural differences in the microbial flora in the feces using β-diversity (Bray-Curtis dissimilarity) showed that at baseline, the test group and the control group had almost the same structure, but after 12 weeks of intake, the intestinal microbial flora structures of the control group and the test group were statistically significantly separated (Figure 18).

[0145] An additional analysis of fecal microbiota after 12 weeks of supplementation using a general linear model-based microbiome multivariable association linear model (MaAsLin2) revealed statistically significant positive and negative correlations in the test group after 12 weeks of supplementation (p<0.05). Among the positively correlated strains, a significant increase in the genus Veillonella was observed. Among Veillonella strains, Veillonella atypica has been reported to be a bacterium that utilizes lactic acid. Among the negatively correlated strains, a significant decrease in the genus Shigella was observed. Shigella has been reported to induce diarrhea and intestinal mucosal damage, inhibiting nutrient absorption and disrupting the supply of essential nutrients for muscle recovery and growth. Furthermore, a systemic inflammatory response caused by infection can promote muscle protein breakdown and exacerbate muscle damage (Figure 19).

[0146] In conclusion, the human subject trial confirmed a statistically significant increase in average hamstring muscle strength, located behind the femur, after 12 weeks of supplementation. The left hamstring muscle strength was significantly greater in the test group than in the control group. Fecal microbiota analysis confirmed that 12 weeks of supplementation with the Lactiplantibacillus plantarum KM2 fermented, cultured, and heat-treated complex increased the diversity of intestinal microorganisms in the test group compared to the control group, and significantly altered the composition of the intestinal community between the test and control groups. Furthermore, beneficial Veillonella spp. increased, including Veillonella atypica, which is known to utilize lactic acid. Decreased harmful Shigella spp. was also confirmed. Shigella has been reported to induce diarrhea and intestinal mucosal damage, inhibiting nutrient absorption and disrupting the supply of essential nutrients for muscle recovery and growth. In addition, a systemic inflammatory response caused by infection can accelerate the breakdown of muscle proteins and exacerbate muscle damage. Furthermore, we further analyzed muscle mass by limiting the age of subjects in the human application test to their 50s, an age at which physical function begins to decline rapidly. After 12 weeks of supplementation, muscle mass increased in the test group and decreased in the control group, demonstrating a statistically significant difference. This means that limb muscle mass in the test group increased significantly compared to the control group in people in their 50s, confirming the effectiveness of the Lactiplantibacillus plantarum KM2 fermented culture heat-treated complex in improving muscle strength and increasing muscle mass.

[0147] 5. Abnormal Cases

[0148] In the test group, there were 22 abnormal cases among a total of 13 (32.50%) human test subjects, and in the control group, there were 15 abnormal cases among a total of 13 (32.50%) human test subjects, and no statistically significant difference was observed between the intake groups.

[0149] In a survey of the severity of abnormalities that occurred during the subject application test period, there were 22 mild cases in the test group and 15 mild cases in the control group.

[0150] In terms of the relationship with the food for human application tests, the test group had 4 cases where it was "possibly related" and 18 cases where it was "clearly considered unrelated," while the control group had 2 cases where it was "not considered related" and 13 cases where it was "clearly considered unrelated," as judged by the testers.

[0151] One serious abnormality (urinary tract infection) occurred in one (2.50%) human application test subject in the test group, and no subjects dropped out due to abnormalities (Table 3).

[0152] [Table 3]

[0153] Four abnormal cases occurred in the test group, and a possible link to the human test food could not be ruled out; all were digestive system disorders. The abnormal cases were one case of "thin stool" and three cases of "indigestion." All of the subjects continued and completed the study. One abnormal case of "indigestion" was cured, while one abnormal case of "thin stool" and two abnormal cases of "indigestion" could not be followed up after the subjects' last visit, so it was not possible to confirm whether the abnormal cases had disappeared (Table 4).

[0154] [Table 4]

[0155] Therefore, the safety analysis and statistical analysis of abnormal cases in 80 human application test subjects showed no correlation between the test group and abnormal reactions under any conditions.

[0156] Although the present invention has been described in detail above, it is obvious to those skilled in the art that the specific details are merely preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the true scope of the present invention is defined by the claims and their equivalents.

[0157] [Accession number]

[0158] Depository institution: Korea Center for Biological Resources (KCTC) Accession number: KCTC14637BP Date of acceptance: 2021.07.14

Claims

1. A pharmaceutical composition for preventing or treating muscle diseases, comprising as an active ingredient a fermentation-cultured heat-treated complex of Lactiplantibacillus plantarum KM2 strain, which is deposited under KCTC 14637BP, a concentrate thereof, a dried product thereof, a fermentation metabolite thereof, or a mixture thereof.

2. The pharmaceutical composition for preventing or treating muscle diseases according to claim 1, wherein the pharmaceutical composition increases beneficial bacteria, Veillonella spp., and decreases harmful bacteria, Shigella spp., in changing the intestinal community composition.

3. 3. The pharmaceutical composition for preventing or treating a muscle disease according to claim 2, wherein the Veillonella spp. is at least one selected from the group consisting of Veillonella sp. S12025-13, Veillonella dispar, Veillonella nakazawae, and Veillonella typica, and the Shigella spp. is at least one selected from the group consisting of Shigella sonnei, Shigella boydii, Shigella dysenteriae, and Shigella flexneri.

4. 2. The pharmaceutical composition for preventing or treating muscle diseases according to claim 1, wherein the muscle disease is muscle atrophy, sarcopenia, hypotonia, muscle dystrophy, myasthenia gravis, or amyotrophic lateral sclerosis.

5. 10. The pharmaceutical composition for preventing or treating muscle diseases according to claim 1, wherein the pharmaceutical composition strengthens or improves the strength of hamstring muscles, which are responsible for flexion force among thigh muscles.

6. The pharmaceutical composition for preventing or treating muscle diseases according to claim 1, wherein the pharmaceutical composition increases muscle mass in people in their 50s, who are prone to significant muscle mass loss.

7. A functional health food composition for preventing or improving muscle diseases, comprising as an active ingredient a fermented, cultured, and heat-treated complex of Lactiplantibacillus plantarum KM2 strain, which is deposited under KCTC 14637BP, a concentrate thereof, a dried product thereof, a fermented metabolite thereof, or a mixture thereof.

8. The functional health food composition for preventing or improving muscle diseases according to claim 7, characterized in that the composition increases beneficial bacteria, Veillonella spp., and reduces harmful bacteria, Shigella spp., in changing the intestinal community composition.

9. The health functional food composition for preventing or improving muscle diseases according to claim 7, characterized in that the muscle disease is muscle atrophy, sarcopenia, hypotonia, muscle dystrophy, myasthenia gravis, or amyotrophic lateral sclerosis.

10. A functional health food composition for improving muscle strength or increasing muscle mass, comprising as an active ingredient a fermented, cultured, and heat-treated complex of Lactiplantibacillus plantarum KM2 strain, which is deposited under KCTC 14637BP, a concentrate thereof, a dried product thereof, a fermented metabolite thereof, or a mixture thereof.

11. The functional health food composition according to claim 7 or 10, characterized in that the functional health food composition strengthens or improves the strength of the hamstring muscles, which are responsible for flexion force among the thigh muscles.

12. The functional health food composition according to claim 7 or 10, wherein the pharmaceutical composition increases muscle mass in people in their 50s, who are prone to significant muscle mass loss.

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