Medium additive composition for culturing cultured meat, comprising laxogenin or 5-alpha hydroxy laxogenin

Abstract

The present invention relates to: a cultured meat medium additive and a cultured meat medium composition that can induce muscle growth by differentiating muscle stem cells into muscle cells and inhibiting the expression of myostatin, which inhibits muscle growth; and a method for preparing cultured meat by using same. Specifically, the cultured meat medium additive and the cultured meat medium composition comprise laxogenin and 5-alpha hydroxy laxogenin.

Description

A composition of a culture medium additive for cultured meat containing lacsogenin or 5 alpha hydroxy lacsogenin

[0001] The present invention relates to a medium additive composition capable of producing cultured meat, and a medium additive for cultured meat culture capable of differentiating muscle stem cells into muscle cells and inducing muscle growth by suppressing the expression of myostatin, which suppresses muscle growth.

[0002] Cultured meat is a lean meat produced using cell engineering technology, which involves extracting and culturing stem cells from living animals to produce meat without the need for livestock farming. While cultured meat is not yet commercially available, some research and experimental studies using in vitro meat technology are underway. Cultured meat is produced using tissue engineering techniques pioneered in the field of biomedicine. By producing edible resources in a laboratory without the need for livestock farming, it not only has the potential to overcome the limitations of meat production due to climate change, but it is also attracting attention as a potential food source for space, where livestock farming is unavailable. Consequently, various food tech companies are actively conducting research and development on cultured meat worldwide.

[0003] The methods developed to date for producing cultured meat involve extracting stem cells from living livestock, culturing them into mature muscle tissue in a culture medium, and ultimately manufacturing this into a food product. Typically, stem cells are isolated from the skeletal muscle of livestock, cultured in a cell culture dish, and processed into tissue through proliferation, differentiation, and maturation stages to enable the cells to form tissue. Stem cells that can be used for cultured meat are primarily satellite cells, which are undeveloped muscle cells found in skeletal muscle. However, numerous studies have reported successful differentiation into muscle tissue from embryonic stem cells, induced pluripotent stem cells (iPSCs), and adipose-derived stem cells. While no cell culture alone has yet been used to develop a method that mimics the flavor of conventional meat, the formation of cultured meat is currently evaluated based on whether the cells have differentiated to a similar degree to meat in terms of color, appearance, and texture. Whether stem cells, which are mainly used in cultured meat production, differentiate into muscle cells, which make up the majority of meat, and form tissue, and whether factors related to muscle differentiation are expressed, are receiving attention as challenges that must be addressed in producing high-quality cultured meat that can impart the unique characteristics of meat.

[0004] [Prior Art Literature]

[0005] [Patent Document]

[0006] Korean Patent Publication No. 10-2022-0029490 (Published on March 8, 2022)

[0007] The purpose of the present invention is to provide a cultured meat culture medium additive capable of inducing muscle growth by differentiating muscle stem cells into muscle cells and suppressing the expression of myostatin, which suppresses muscle growth.

[0008] The present invention provides a cultured meat culture medium additive comprising at least one selected from Laxogenin and 5 Alpha Hydroxy Laxogenin.

[0009] In addition, the present invention provides a cultured meat culture medium composition comprising at least one selected from Laxogenin and 5 Alpha Hydroxy Laxogenin as an effective ingredient.

[0010] In addition, the present invention provides a method for producing cultured meat, including a step of inducing muscle differentiation and muscle growth of cells derived from an animal other than a human using a cultured meat culture medium composition including at least one selected from Laxogenin and 5 Alpha Hydroxy Laxogenin.

[0011] According to the present invention, when Laxogenin and 5 Alpha Hydroxy Laxogenin, which are selected as substances that bind to Myostatin through in silico analysis, are added to a differentiation medium for culturing muscle stem cells derived from cows, pigs, and chickens and mouse myoblasts, the activity of creatine kinase specific for muscle differentiation increases, the expression level of muscle differentiation markers MYOD (myoblast determination protein 1), MYOG (Myogenin), or MYH (myosin heavy chain) increases, and the expression level of Myostatin, SMAD2 (Mothers against decapentaplegic homolog 2), SMAD3 (Mothers against decapentaplegic homolog 3), or ACVR2b (Activin receptor type-2B), which inhibit muscle growth, decreases, thereby allowing muscle stem cells and myoblasts to differentiate and grow into muscle cells. As confirmed, the composition comprising Laxogenin and 5 Alpha Hydroxy Laxogenin can be provided as a medium additive for cultured meat culture.

[0012] Figure 1 shows the results of in silico analysis of the binding free energy level of Laxogenin or 5 Alpha Hydroxy Laxogenin to Myostatin.

[0013] Figure 2a shows the results of analyzing the effect of lacsogenin (LXG) on the activity of creatine kinase in bovine muscle stem cells.

[0014] Figure 2b shows the results of analyzing the effect of lacsogenin (LXG) on the gene and protein expression of muscle differentiation markers and myostatin (MSTN) in bovine muscle stem cells.

[0015] Figure 2c shows the results of analyzing the effect of lacsogenin (LXG) on ROS levels in bovine muscle stem cells.

[0016] Figure 2d shows the results of analyzing the effect of 5 alpha hydroxy laxogenin (5HLXG) on the activity of creatine kinase in bovine muscle stem cells.

[0017] Figure 2e shows the results of analyzing the effects of 5alpha-hydroxy laxogenin (5HLXG) on the gene and protein expression of muscle differentiation markers and myostatin (MSTN) in bovine muscle stem cells.

[0018] Figure 2f shows the results of analyzing the effect of 5 alpha hydroxy laxogenin (5HLXG) on ROS levels in bovine muscle stem cells.

[0019] Figure 3a shows the results of analyzing the effect of lacsogenin (LXG) on the activity of creatine kinase in porcine muscle stem cells.

[0020] Figure 3b shows the results of analyzing the effect of lacsogenin (LXG) on the gene and protein expression of muscle differentiation markers and myostatin (MSTN) in porcine muscle stem cells.

[0021] Figure 3c shows the results of analyzing the effect of lacxogenin (LXG) on ROS levels in porcine muscle stem cells.

[0022] Figure 3d shows the results of analyzing the effect of 5 alpha hydroxy laxogenin (5HLXG) on the activity of creatine kinase in porcine muscle stem cells.

[0023] Figure 3e shows the results of analyzing the effects of 5 alpha hydroxy laxogenin (5HLXG) on the gene and protein expression of muscle differentiation markers and myostatin (MSTN) in porcine muscle stem cells.

[0024] Figure 3f shows the results of analyzing the effect of 5 alpha hydroxy laxogenin (5HLXG) on ROS levels in porcine muscle stem cells.

[0025] Figure 4a shows the results of analyzing the effect of lacsogenin (LXG) on the activity of creatine kinase in chicken muscle stem cells.

[0026] Figure 4b shows the results of analyzing the effect of lacsogenin (LXG) on the gene and protein expression of muscle differentiation markers and myostatin (MSTN) in chicken muscle stem cells.

[0027] Figure 4c shows the results of analyzing the effect of lacsogenin (LXG) on ROS levels in chicken muscle stem cells.

[0028] Figure 4d shows the results of analyzing the effect of 5 alpha hydroxy laxogenin (5HLXG) on the activity of creatine kinase in chicken muscle stem cells.

[0029] Figure 4e shows the results of analyzing the effects of 5-alpha-hydroxy laxogenin (5HLXG) on the gene and protein expression of muscle differentiation markers and myostatin (MSTN) in chicken muscle stem cells.

[0030] Figure 4f shows the results of analyzing the effect of 5 alpha hydroxy laxogenin (5HLXG) on ROS levels in chicken muscle stem cells.

[0031] Figure 5a shows the results of immunocytochemistry analysis of the effect of lacsogenin (LXG) on MYH protein expression during the differentiation process of myoblast cell lines (C2C12).

[0032] Figure 5b shows the results of analyzing the effect of lacsogenin (LXG) on the activity of creatine kinase during the differentiation process of a myoblast cell line (C2C12).

[0033] Figure 5c shows the results of immunocytochemistry analysis of the effect of 5-alpha hydroxy laxogenin (5HLXG) on MYH protein expression during the differentiation process of myoblast cell lines (C2C12).

[0034] Figure 5d shows the results of analyzing the effect of 5alpha-hydroxy laxogenin (5HLXG) on the activity of creatine kinase during the differentiation process of a myoblast cell line (C2C12).

[0035] Figure 5e shows the results of analyzing the effect of lacsogenin (LXG) on the gene and protein expression of muscle differentiation markers during the differentiation process of a myoblast cell line (C2C12).

[0036] Figure 5f shows the results of analyzing the effect of 5alpha-hydroxy laxogenin (5HLXG) on the gene and protein expression of muscle differentiation markers during the differentiation process of a myoblast cell line (C2C12).

[0037] Figure 6a shows the results of analyzing the effect of laxogenin (LXG) on the expression of myostatin (MSTN, Myostatin)-related genes and proteins during the differentiation process of a myoblast cell line (C2C12).

[0038] Figure 6b shows the results of analyzing the effect of lacsogenin (LXG) on ROS levels and expression of related genes during the differentiation process of myoblast cells (C2C12).

[0039] Figure 6c shows the results of analyzing the effect of 5alpha-hydroxy laxogenin (5HLXG) on the expression of myostatin (MSTN)-related genes and proteins during the differentiation process of a myoblast cell line (C2C12).

[0040] Figure 6d shows the results of analyzing the effect of 5 alpha hydroxy laxogenin (5HLXG) on ROS levels and expression of related genes during the differentiation process of myoblast cell lines (C2C12).

[0041] The terms used in this specification have been selected from widely used, current terms, taking into account the functions of the present invention. However, these terms may vary depending on the intentions of those skilled in the art, precedents, the emergence of new technologies, etc. Furthermore, in certain cases, terms may be arbitrarily selected by the applicant, and in such cases, their meanings will be described in detail in the relevant description of the invention. Therefore, the terms used in this invention should not be defined simply as names, but rather based on their inherent meanings and the overall content of the present invention.

[0042] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and shall not be interpreted in an idealized or overly formal sense unless explicitly defined herein.

[0043] Hereinafter, the present invention will be described in more detail.

[0044] The present invention provides a cultured meat culture medium additive comprising at least one selected from Laxogenin and 5 Alpha Hydroxy Laxogenin.

[0045] The above Laxogenin is a type of plant growth hormone found in plants and is a substance belonging to brassinosteroids, and has a structure represented by the following chemical formula 1.

[0046] [Chemical Formula 1]

[0047]

[0048] The above 5 Alpha Hydroxy Laxogenin is a derivative of the above lacsogenin and has a structure represented by the following chemical formula 2.

[0049] [Chemical Formula 2]

[0050]

[0051] The above Laxogenin and 5 Alpha Hydroxy Laxogenin can bind to the active site of Myostatin. Specifically, Laxogenin interacts with the amino acids Arg17, Ala34, Pro35, Arg37, Tyr38, Lys39, Asn41, Pro81, Ile82, Asn83, Met84, Leu85, Tyr95, and Val102 that constitute Myostatin, and 5 Alpha Hydroxy Laxogenin can interact with the amino acids Ala34, Pro35, Arg37, Tyr38, Lys39, Pro81, Ile82, Asn83, Met84, Leu85, Tyr95, and Val102 that constitute Myostatin.

[0052] The above cultured meat culture medium additive can be used to induce muscle differentiation and muscle growth of cells selected from the group consisting of embryonic stem cells, muscle stem cells, mesenchymal stem cells, induced pluripotent stem cells, myoblasts, and fibroblasts.

[0053] The above cells may be derived from chicken, cow, pig or mouse.

[0054] Muscle differentiation of the above cells means an increase in the activity of creatine kinase, an enzyme specifically expressed in muscles, or an increase in the gene and protein expression levels of muscle differentiation-related markers MYOD (myoblast determination protein 1), MYOG (Myogenin), or MYH (myosin heavy chain).

[0055] Inducing the above muscle growth means a decrease in the gene and protein expression levels of myostatin, a myokine that inhibits muscle growth, and its related markers, SMAD2 (Mothers against decapentaplegic homolog 2), SMAD3 (Mothers against decapentaplegic homolog 3), or ACVR2b (Activin receptor type-2B). In addition, inducing the above muscle growth means a decrease in the ROS (Reactive oxygen species) stress level.

[0056] In addition, the present invention provides a cultured meat culture medium composition comprising at least one selected from Laxogenin and 5 Alpha Hydroxy Laxogenin as an effective ingredient.

[0057] One selected from the above Laxogenin and 5 Alpha Hydroxy Laxogenin may be included in the cultured meat culture medium composition at a concentration of 0.1 nM to 1000 nM.

[0058] The above medium composition may include fetal bovine serum (FBS) and penicillin / streptomycin (P / S) based on DMEM (Dulbecco's Modified Eagle's Medium) medium, and the fetal bovine serum (FBS) may be included in an amount of 1% to 3% by weight and the penicillin / streptomycin (P / S) may be included in an amount of 0.5% to 1.5% by weight relative to 100% by weight of the medium composition.

[0059] In addition, the present invention provides a method for producing cultured meat, including a step of inducing muscle differentiation and muscle growth of cells derived from an animal other than a human using a cultured meat culture medium composition including at least one selected from Laxogenin and 5 Alpha Hydroxy Laxogenin.

[0060] Hereinafter, to aid understanding of the present invention, experimental examples and examples will be described in detail. However, the following experimental examples and examples are merely illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental examples and examples of the present invention are provided to more fully explain the present invention to those with average knowledge in the art.

[0061] <Experimental Example> Experimental Materials and Methods

[0062] The following experimental examples are intended to provide experimental examples commonly applied to each embodiment according to the present invention.

[0063] 1. Computer analysis (in silico)

[0064] The components of garlic were collected through a literature search, and their structures were collected from the PubChem (https: / pubchem.ncbi.nlm.nih.gov / ) compound database. To determine whether the collected components could bind to myostatin, Discovery Studio and Autodock molecular binding analysis programs were used.

[0065] 2. Isolation of muscle stem cells from cow, pig, and chicken muscles

[0066] To extract muscle stem cells, muscle tissues were collected from beef rump, pig foreleg, and chicken embryo leg, minced, placed in a 0.1% pronase enzyme solution, and stirred at 37°C for 2 hours. After the reaction, centrifugation was performed at 1,000 g for 3 minutes, the supernatant was removed, and the tissues were suspended in DMEM (Dulbecco's Modified Eagle's Medium) containing 1% penicillin / streptomycin (P / S) and 10% fetal bovine serum (FBS), and the dissociated cells were separated from the muscle tissue through a 100 μm filter. The separated cells were centrifuged at 1,000 g for 5 minutes, the supernatant was removed, and the cells were suspended in Ham's F10 medium containing 1% penicillin and streptomycin, 20% fetal bovine serum, and 5 ng / mL fibroblast growth factor 2 (FGF2). The cells were then transferred to cell culture dishes and cultured in a 5% CO2 environment at 37°C.

[0067] 3. Analysis of muscle differentiation rate according to treatment with lacsogenin and 5-alpha-hydroxy lacsogenin

[0068] When mouse myoblasts and bovine, porcine, and chicken muscle stem cells proliferated to more than 90% of the culture dish surface, the differentiation medium (DMEM containing 1% P / S and 2% FBS) was replaced, and lacsogenin or 5 alpha hydroxy lactogenin (5 alpha hydroxy lactogenin) was diluted to concentrations of 0.1 nM, 1 nM, 10 nM, 100 nM, and 1000 nM, respectively, and treated. After 4 days of differentiation culture, all media were removed and washed with physiological saline (phosphate buffer saline, PBS). After adding 200 μL of new physiological saline, the cells were harvested. The harvested cells were disrupted by sonication and the supernatant was separated by centrifugation. For the creatine kinase activity assay, a muscle-specific enzyme, the assay mixture (buffer 100 μL, substrate 100 μL, enzyme 1 μL) of the creatine enzyme activity assay kit was prepared, and 10 μL of the supernatant obtained through cell disruption was mixed with 100 μL of the assay mixture and reacted. The reacted sample was measured for absorbance at 340 nm using a spectrophotometer (microplate reader) at reaction times of 20 and 25 minutes, respectively. The measured creatine kinase activity was calculated using the following mathematical equation 1.

[0069] [Mathematical Formula 1]

[0070] Creatine kinase activity (%) = (25-minute measurement OD340nm - 20-minute measurement OD340nm) / (calibrator OD340nm - distilled water OD340nm) *600

[0071] 4. RNA extraction, cDNA synthesis, and gene expression confirmation (real-time RT-PCR)

[0072] RNA extraction: 1 mL of TRIzol reagent was added to cultured cells, and the cells were disrupted. 200 μL of chloroform was added to the disrupted sample, and centrifugation was performed at 12,000 rpm for 10 minutes. After centrifugation, the clear aqueous layer on top was transferred to a new tube, and 500 μL of isopropyl alcohol was added. After mixing thoroughly, centrifugation was performed at 12,000 rpm for 10 minutes. The supernatant except for the RNA precipitate was removed, and 1 mL of 70% ethanol was added and washed. All ethanol was removed, and the RNA precipitate was dissolved in distilled water containing DEPC (Diethyl pyrocarbonate).

[0073] cDNA was synthesized by reacting with reverse transcriptase.

[0074] Gene expression confirmation: Gene expression was confirmed using real-time polymerase chain reaction (PCR) analysis. The expression levels of muscle differentiation-related markers MYOD (myoblast determination protein 1), MYOG (myogenin), and MYH (myosin heavy chain) were analyzed. In addition, the expression levels of myostatin (MSTN) and related markers SMAD2 (Mothers against decapentaplegic homolog 2), SMAD3 (Mothers against decapentaplegic homolog 3), and ACVR2b (Activin receptor type-2B) were analyzed. The synthesized cDNA, primer sets, and SYBR green fluorescent material-containing reagents were mixed. The primers were designed using the primer design tool provided by the National Center for Biotechnology Information (NCBI) and are shown in Table 1 below. Real-time PCR analysis was performed using the 7500 real-time PCR from Applied Biosystems.

[0075] Gene Species End product (bp) Tm (℃) Forward primer Reverse primer GAPDH Mouse 15559 TGC TGG TGC TGA GTA TGT CGCAA GCA GTT GGT GGT ACA GGMYOD Mouse 21359 AGG AGC ACG CAC ACT TCT CTTCT CGA AGG CCT CAT TCA CTMYOG Mouse 18559 TCC AGT ACA TTG AGC GCC TACAA ATG ATC TCC TGG GTT GMYH Mouse 14159 CTG AAG CAG AGG CAA GTA GTCGA AAT GAG GAT GGG TGC TMSTN Mouse 16359 ACG CTA CCA CGG AAA CAA TCGGA GTC TTG ACG GGT CTG AGSMAD2 Mouse 20358 GAG CTC AAG GCA ATC GAA AACCT GCT GGG AAA TTT GTG TTSMAD3Mouse19759CTG GGC CTA CTG TCC AAT GTGGT GGG ATC TTG CAG ACA GTACVR2bMouse19758AAC TTC CAG AGA GAC GCC TTATC GTG GGC CTC ATC TTC TTGAPDHBeef20859GGG TCA TCT CTG CAC CTACA GTC TTC TGG GTG GCA GTMYODBeef22959GAT GAC CCG TGT TTC GAC TCTAG TCG TCT TGC GTT TGC ACMYOGBeef19759TGG GCG TGT AAG GTG TGT AATGC AGG CGC TCT ATG TAC TGMYHBeef23557GGA GAT GCG AGA TGA AAA GCCAT GTT GGC CAT TTC CTT CTGAPDHPig14760TCG GAG TGA ACG GAT TTG GCTGC CGT GGG TGG AAT CAT ACMYODPig19660GCA CTA CAG CGG TGA CTC AGCAC GAT GCT GGA CAG ACA GTMYOGPig16458CAG TGA ATG CAG TTC CCA CACCA CAT CCT CCA CTG TGATGMYHPig16258TTC CTT CCA AAC CGT CTC TGTTA CAC CTC AGC TGG TGC AGGAPDHChicken15759CAA CAT CAA ATG GGC AGA TGGAC ACC CCA TCA CAA ACA TGMYODChicken16059AGC TCT CGC AGG AGA AAC AGCTG GAG GCA GTA TGG GAC AMYOGChicken20559CGC CAT CAG TAC AAT CGA GATC GCT CAG GAG GTG ATC TMYHchicken12859CTG CCG ATG AAA AAG TGG CTAGC CTT GTC TGC AAC TTC T

[0076] 5. Western blot

[0077] All cultured cell media were removed and washed with saline. Then, a mixture containing RIPA (Radioimmunoprecipitation) buffer and 1% protease inhibitor was added to the cells and the cells were scraped. After centrifugation at 12,000 rpm for 10 minutes, the supernatant was collected and the protein concentration was measured using a spectrophotometer. 60 μg of the extracted protein was electrophoresed on a 10% polyacrylamide gel and transferred to a PVDF (Polyvinylidene fluoride) membrane. After blocking in TBS (Tris-buffered saline) solution containing 3% skim milk and 1% surfactant for 1 hour, the primary antibody was added. After incubation for 16 hours at 4°C, the cells were washed three times for 10 minutes in TBS (Tris-buffered saline) buffer containing 1% surfactant. Then, it was reacted with the secondary antibody at room temperature for 1 hour. After washing three times for 10 minutes in TBS (tris-buffered saline) buffer containing 1% surfactant, a chemical developer (Super Signal West Pico Chemiluminescent Substrate) was added and developed.

[0078] 6. Reactive Oxygen Species (ROS) Analysis

[0079] To compare the production of reactive oxygen species during differentiation, myoblasts and muscle stem cells from bovine, porcine, and chicken were differentiated for 4 days after treatment with lacsogenin or 5α-hydroxy lacsogenin, respectively. The medium was removed, and 10 μM 2',7'-dichlorofluorescein (Sigma-Aldrich, St. Louis) was added, followed by incubation at 37°C for 2 hours. After washing twice with PBS, fluorescence was measured using a spectrophotometer (excitation 498 nm, emission 522 nm).

[0080] 7. Statistical analysis

[0081] The Tukey test was used to analyze differences between gene expression means. The software used was SAS version 9.0, and one-way analysis of variance was performed using PROC GLM. A value of p ≤ 0.05 indicates statistical significance.

[0082]

[0083] Example 1. Analysis of intermolecular binding of myostatin

[0084] In silico analysis revealed that among the garlic components, lacsogenin and 5 alpha hydroxy lacsogenin were most ideally predicted to bind to the active site of myostatin. The binding free energy level of lacsogenin and myostatin was confirmed to be -7.90 kcal / mol, and the binding free energy level of 5 alpha hydroxy lacsogenin and myostatin was confirmed to be -8.50 kcal / mol (Fig. 1). Laxogenin was found to interact with the amino acids Arg17, Ala34, Pro35, Arg37, Tyr38, Lys39, Asn41, Pro81, Ile82, Asn83, Met84, Leu85, Tyr95, and Val102 of myostatin. 5alpha-hydroxy laxogenin was found to interact with the amino acids Ala34, Pro35, Arg37, Tyr38, Lys39, Pro81, Ile82, Asn83, Met84, Leu85, Tyr95, and Val102 that constitute myostatin (Table 2).

[0085] IndexMSTN + LXGMSTN + 5HLXGBonding energy (kcal / mol) -7.90-8.50Bonding amino acidsArg17, Ala34, Pro35, Arg37, Tyr38, Lys39, Asn41, Pro81, lle82, Asn83, Met84, Leu85, Tyr95, Val102Ala34, Pro35, Arg37, Tyr38, Lys39, Pro81, lle82, Asn83, Met84, Leu85, Tyr95, Val102Hydrogen bondLYS39:HN - LIG0:03LYS39:HZ3-LIG0:03LYS39:HN - LIG0:04LYS39:HZ3-LIG0:04Hydrophobic bondLIGO - ILE82LIGO:C31 - ALA34LIGO:C31 - LEU85PRO35 - LIG0VAL 102 - LIG0LIGO - ILE82LIGO:C32 - ALA34LIGO:C32 - LEU85PRO35 - LIGOVAL 102 - LIGO

[0086] Additionally, the small intestinal absorption rates of laxogenin and 5-alpha-hydroxy laxogenin were predicted to be 97.12% and 96.78%, respectively, and negative results were also confirmed in the prediction models for cancer risk, hepatotoxicity, and skin irritation (Table 3).

[0087] Parameters LXG5HLXGMolecular formula C 27 H 42 O4C 27 H 42 O5 Molecular weight (g / mol) 430.6446.6 Absorption rate (human absorption rate) 97.12% 96.78% Toxicity Carcinogenicity (AMES toxicity) No No Hepatotoxicity (Hepatotoxicity) No No Skin irritation (Skin sensitisation) No No

[0088]

[0089] Example 2. Effects on the differentiation process of bovine muscle stem cells

[0090] Bovine muscle stem cells were cultured for differentiation for 4 days after adding lacsogenin or 5-alpha-hydroxy lacsogenin at concentrations of 0.1 nM, 1 nM, 10 nM, 100 nM, and 1000 nM, respectively.

[0091] When treated with lacsogenin (LXG), the activity of creatine kinase, a muscle-specific expression enzyme, increased by more than 10% from 10 nM (Fig. 2a), and after treatment with 10 nM, which showed the effect of increasing creatine kinase expression, the gene and protein expression of muscle differentiation-related markers MYOD (myoblast determination protein 1), MYOG (Myogenin), and MYH (myosin heavy chain) increased, whereas the gene and protein expression of myostatin (MSTN), which was predicted to bind to lacsogenin, decreased (Fig. 2b). In addition, it was confirmed that the level of ROS generated during muscle development was reduced by about 10% or more when treated with 10 nM lacsogenin (Fig. 2c).

[0092] When treated with 5 alpha-hydroxy lactogenein (5HLXG), creatine kinase activity increased by about 15% or more from 1 nM (Fig. 2d), and when treated with 10 nM, which showed an effect of increasing creatine kinase expression, the expression of muscle-related markers was examined, and the gene and protein expression of MYOD (myoblast determination protein 1), MYOG (Myogenin), and MYH (myosin heavy chain) were found to increase (Fig. 2e). The expression of myostatin MSTN (Myostatin), which was predicted to bind to 5 alpha-hydroxy lactogenein, was found to decrease in both gene and protein levels. In addition, it was confirmed that the level of ROS generated during muscle development was reduced by about 10% or more when treated with 10 nM 5 alpha-hydroxy lactogenein (Fig. 2f).

[0093]

[0094] Example 3. Effects on the differentiation process of pig muscle stem cells

[0095] Porcine muscle stem cells were cultured for differentiation for 4 days after adding lacsogenin or 5-alpha-hydroxy lacsogenin at concentrations of 0.1 nM, 1 nM, 10 nM, 100 nM, and 1000 nM, respectively.

[0096] When treated with lacxogenin (LXG), the activity of creatine kinase, a muscle-specific enzyme, increased by approximately 18% starting from 10 nM (Fig. 3a). The gene and protein expression of muscle differentiation-related markers, MYOD (myoblast determination protein 1), MYOG (myogenin), and MYH (myosin heavy chain), increased, whereas the gene and protein expression of myostatin (MSTN, Myostatin) decreased (Fig. 3b). In addition, the level of ROS generated during muscle development was found to decrease to approximately 7% (Fig. 3c).

[0097] When treated with 5alpha-hydroxy laxogenin (5HLXG), the activity of creatine kinase was found to increase the most by approximately 18% at 10 nM (Fig. 3d). The gene and protein expression of muscle differentiation-related markers, MYOD (myoblast determination protein 1), MYOG (myogenin), and MYH (myosin heavy chain), increased, whereas the gene and protein expression of myostatin (MSTN, Myostatin) decreased (Fig. 3e). In addition, the level of ROS generated during muscle development was found to decrease to approximately 9% (Fig. 3f).

[0098]

[0099] Example 4. Effects on the differentiation process of chicken muscle stem cells

[0100] Chicken muscle stem cells were cultured for differentiation for 4 days after adding lacsogenin or 5-alpha-hydroxy lacsogenin at concentrations of 0.1 nM, 1 nM, 10 nM, 100 nM, and 1000 nM, respectively.

[0101] When treated with lacsogenin (LXG), the activity of creatine kinase increased by approximately 9% from 0.1 nM (Fig. 4a). The gene and protein expression of muscle differentiation-related markers, MYOD (myoblast determination protein 1), MYOG (myogenin), and MYH (myosin heavy chain), increased, whereas the gene and protein expression of myostatin (MSTN, Myostatin) decreased (Fig. 4b). In addition, the level of ROS generated during muscle development was found to decrease to approximately 35% when treated with 10 nM lacsogenin (Fig. 4c).

[0102] When treated with 5alpha-hydroxy laxogenin (5HLXG), the expression of ROS increased from 0.1 nM to a maximum of 22% at a concentration of 1000 nM (Fig. 4d). The gene and protein expression of muscle differentiation-related markers, MYOD (myoblast determination protein 1), MYOG (myogenin), and MYH (myosin heavy chain), increased, whereas the gene and protein expression of myostatin (MSTN) decreased (Fig. 4e). In addition, the level of ROS generated during muscle development was found to decrease to approximately 8% when treated with 10 nM of 5alpha-hydroxy laxogenin (Fig. 4f).

[0103]

[0104] Example 5. Effects on the differentiation process of myoblast cells

[0105] C2C12 cells were treated with lacsogenin or 5α-hydroxy lacsogenin at a concentration of 10 nM during differentiation and cultured for 4 days. After culture, myosin heavy chain, which mainly constitutes muscle, was stained by immunocytofluorescence and observed under a fluorescence microscope. When treated with lacsogenin or 5α-hydroxy lacsogenin, not only was the formation of distinct myotubes common, but a thickening effect was also observed (Figs. 5a and 5c). And when the two substances were treated at concentrations of 0.1 nM, 1 nM, 10 nM, 100 nM, and 1000 nM, respectively, and the activity of creatine kinase was analyzed, in the case of lacsogenin, a 19% increase was confirmed at 100 nM, and in the case of 5alpha-hydroxy lacsogenin, a synergistic effect of about 10% was confirmed from 0.1 nM to 100 nM (Fig. 5b and Fig. 5d). And after treating the two substances at a concentration of 10 nM each, the gene expression patterns of muscle development factors that change in the early (2 days), middle (4 days), and late (6 days) stages of differentiation were confirmed. At this time, when lacsogenin or 5alpha-hydroxy lacsogenin was treated at all stages, muscle development-related genes (MYOD, MYOG, MYH) increased, and protein expression also increased in the same pattern (Fig. 5e and Fig. 5f).

[0106]

[0107] Example 6. Myostatin inhibitory effect

[0108] In silico analysis, lacsogenin and 5α-hydroxy lacsogenin were confirmed to have myostatin potential. Therefore, their effects on myostatin-related gene and protein expression were examined. When lacsogenin or 5α-hydroxy lacsogenin was treated at a concentration of 10 nM, myostatin gene and protein expression were decreased. In addition, SMAD2 and 3 transcription factors, which are increased in cells affected by myostatin, were also reduced, and protein analysis showed that phosphorylated SMAD2, the active form of SMAD2, was reduced. In addition, it was effective in reducing ACVR2b receptors, which are present on the cell membrane and directly bind to myostatin to transmit cellular signals (Figures 6a and 6c).

[0109]

[0110] Example 7. ROS reduction effect

[0111] Differentiated myoblasts treated with 10 nM each of lacsogenin and 5-alpha-hydroxy-lacsogenin showed a decrease in ROS compared to the control group. Gene expression of the transcription factor NRF2, which is expressed when ROS is lowered, increased, and gene expression of SOD2, an enzyme that actually removes ROS, also increased (Fig. 6b and 6d).

[0112]

[0113] While specific aspects of the present invention have been described in detail above, it should be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. In other words, the substantial scope of the present invention is defined by the appended claims and their equivalents.

[0114] Numerical ranges are inclusive of the values ​​defined in the ranges above. Any maximum numerical limit given throughout this specification includes any lower numerical limit, as if that lower numerical limit were explicitly stated. Any minimum numerical limit given throughout this specification includes any higher numerical limit, as if that higher numerical limit were explicitly stated. Any numerical limit given throughout this specification will include any better numerical range within that broader numerical range, as if that narrower numerical limit were explicitly stated.

Claims

1. A culture medium additive for cultured meat, comprising at least one selected from among Laxogenin and 5 Alpha Hydroxy Laxogenin.

2. A cultured meat culture medium additive according to claim 1, characterized in that the lacsogenin and 5 alpha hydroxy lacsogenin bind to myostatin.

3. In the first paragraph, the cultured meat culture medium additive is characterized in that it induces muscle differentiation and muscle growth of cells selected from the group consisting of embryonic stem cells, muscle stem cells, mesenchymal stem cells, induced pluripotent stem cells, myoblasts, and fibroblasts.

4. A cultured meat culture medium additive according to claim 3, characterized in that the cells are derived from chicken, cow or pig.

5. In the third paragraph, a cultured meat culture medium additive characterized by increased activity of creatine kinase or increased gene and protein expression levels of MYOD (myoblast determination protein 1), MYOG (Myogenin), or MYH (myosin heavy chain) in the muscle differentiation of the cells.

6. In the third paragraph, a cultured meat culture medium additive characterized in that the muscle growth inducing step is caused by a decrease in the gene and protein expression levels of Myostatin, SMAD2 (Mothers against decapentaplegic homolog 2), SMAD3 (Mothers against decapentaplegic homolog 3), or ACVR2b (Activin receptor type-2B).

7. A cultured meat culture medium additive according to claim 3, characterized in that the muscle growth inducing agent is characterized by a decrease in the ROS (Reactive oxygen species) stress level.

8. A cultured meat culture medium composition comprising at least one selected from among Laxogenin and 5 Alpha Hydroxy Laxogenin as an active ingredient.

9. A cultured meat culture medium composition according to claim 8, characterized in that one selected from among Laxogenin and 5 Alpha Hydroxy Laxogenin is included in the cultured meat culture medium composition at a concentration of 0.1 nM to 1000 nM.

10. A culture medium composition for cultured meat, characterized in that the medium composition in claim 8 is based on a DMEM (Dulbecco's Modified Eagle's Medium) medium and includes fetal bovine serum (FBS) and penicillin / streptomycin (P / S).

11. A cultured meat culture medium composition according to claim 10, characterized in that the fetal bovine serum (FBS) is contained in an amount of 1 to 3 wt% and the penicillin / streptomycin (P / S) is contained in an amount of 0.5 to 1.5 wt%, based on 100 wt% of the medium composition.

12. A method for producing cultured meat, comprising the step of inducing muscle differentiation and muscle growth of cells derived from an animal other than a human using a cultured meat culture medium composition containing at least one selected from among Laxogenin and 5 Alpha Hydroxy Laxogenin.

13. A method for producing cultured meat, characterized in that the cells in paragraph 12 are derived from chicken, cow, pig or mouse.

Images