Medium additive for culturing cultured meat, cell culture medium for culturing cultured meat comprising same, and method for culturing cultured meat using same

Abstract

The present invention relates to an invention utilizing a microalgae extract as a medium additive for culturing cultured meat. More specifically, the present invention uses an extract of chlorella, which is a kind of microalgae, as a medium additive component, thereby making it possible to effectively induce cross-differentiation from muscle stem cells into adipocytes and provide cultured meat having improved flavor by inducing cells in which muscle fat is present at the same time. In addition, the use of the medium additive of the present invention replaces fetal bovine serum, which is a culture medium component for cultured meat, or is used in combination with fetal bovine serum, thereby enabling a reduction in the use of expensive fetal bovine serum and enhancing the culture effect of cultured meat, thus providing cultured meat having excellent cost effectiveness.

Description

Cultured meat culture medium additive, cultured meat cell culture medium containing the same, and culture method of cultured meat using the same

[0001] The present invention relates to a medium additive containing an extract of chlorella, a type of microalgae, a cell culture medium for cultured meat containing the same, and a method for culturing cultured meat using the same.

[0002] Cultured meat refers to edible meat obtained through cell engineering technology by culturing cells from living animals in a laboratory without going through the process of raising livestock. It is also called in vitro meat or lab-grown meat because it is grown in a test tube, artificial meat because it is synthesized using human stem cells rather than natural ones, clean meat because it is produced in clean production facilities rather than traditional livestock facilities, and bio-artificial muscles (BAMs) because the muscle fibers that make up cultured meat are cultivated.

[0003] Cultured meat is manufactured by maturing cells until muscle tissue is formed from stem cells to replicate the texture of actual meat, which takes a long time and makes the production cost of cultured meat very high, making commercialization still a high barrier to reality.

[0004] In particular, a large amount of culture medium is used in the process of mass proliferation of cells, and the culture medium for manufacturing cultured meat currently contains expensive fetal bovine serum (FBS) containing various growth factors, cytokines, hormones, and proteins essential for cell growth. FBS accounts for a very large portion of the total production cost of cultured meat, and is acting as a major barrier to the commercialization and popularization of cultured meat.

[0005] Furthermore, fetal bovine serum, a substance primarily used as an additive in basal media for cell growth, is collected from the blood of live calves, making stable production difficult and raising ethical concerns about the loss of young lives. Furthermore, because the slaughter rate of cattle is fixed, and thus the production volume, the unit price is also very high, making it unsuitable for use in cultured meat.

[0006] Accordingly, there is a need for research and product development on low-cost cultured meat culture media by replacing or using FBS in the culture media used for cultured meat culture.

[0007] The present invention has been completed by finding that chlorella extract has excellent differentiation ability from muscle cells to adipocytes or adipocyte-like cells, and that cultured meat culture can be controlled by controlling the amount of this extract used. That is, the present invention provides a cultured meat culture medium additive using chlorella extract, a cultured meat culture medium, and a cultured meat culture method using the same.

[0008] Accordingly, the technical problems to be solved by the present invention are not limited to the technical problems mentioned above, and other technical problems not mentioned can be clearly understood by a person having ordinary skill in the technical field pertaining to the present invention from the description below.

[0009] The present invention, which aims to solve the above problem, relates to a medium additive for cultured meat cultivation, which includes a chlorella extract.

[0010] As a preferred embodiment of the present invention, the chlorella extract is an extract obtained by performing an extraction process on chlorella, which includes at least one selected from among Chlorella vulgaris, Chlorella pyrenidosa, Chlorella regularis, Chlorella saccharophila, Chlorella sorokiniana, and Chlorella capsulata.

[0011] As a preferred embodiment of the present invention, the extract may be a hot water extract.

[0012] In a preferred embodiment of the present invention, the hot water extract may be prepared by performing a process including: a first step of preparing a chlorella suspension; a second step of sonicating the suspension of the first step to prepare a chlorella cell lysate; a third step of performing a reflux extraction process on the chlorella cell lysate to obtain a hot water extract; a fourth step of treating the hot water extract with a base, performing a centrifugation process to obtain a supernatant, and then neutralizing the supernatant to obtain a chlorella extract; and a fifth step of drying the chlorella extract to obtain a powdered chlorella extract.

[0013] Another object of the present invention is to provide a cell culture medium for cultured meat culturing comprising the medium additive described above.

[0014] As a preferred embodiment of the present invention, the cell culture medium for culturing cultured meat of the present invention may be a serum-free medium including a basal medium and the medium additive; or a serum medium including a basal medium, fetal bovine serum (FBS), and the medium additive.

[0015] In a preferred embodiment of the present invention, the basal medium of the serum-free medium or serum medium is DMEM (Dulbecco's Modified Eagle's Medium), GenGro medium, DMEM / F-12 medium, Leibovitz's L-15 medium, IMDM (Iscove's Modified Dulbecco's Medium), McCoy's 5A medium, M199 medium, MEM (Minimum Essential Medium Eagle), α-MEM (Minimum Essential Medium Eagle, Alpha), Ham's F-10 medium (Ham's F-10 Nutrient Mixture), Ham's F-12 medium (Ham's F-12 Nutrient Mixture), RPMI1640 medium, BME (Basal Media Eagle), CMRL1066 medium, Fischer's Medium, Weymouth It may include one or more selected from MB752 / 1 medium (Waymouth MB752 / 1 Medium), MCDB medium, and WME (Williams' Medium E).

[0016] As a preferred embodiment of the present invention, the basal medium may further include at least one selected from insulin, transferrin, sodium selenite, FGF-2 (Fibroblast growth factor), EGF (Epidermal Growth Factor), TGFβ3 (Transforming growth factor), APS (l-ascorbate-2-phosphate trisodium salt), PDGF-BB (Platelet Derived Growth Factor), and HGF (Hepatocyte Growth Factor, recombinant).

[0017] As a preferred embodiment of the present invention, the medium additive may include, in addition to the chlorella extract, at least one selected from a phosphodiesterase inhibitor, insulin, a synthetic glucocorticoid thiazolidinedione, lecithin, an antibiotic, glutamine, an amino acid, and vitamins.

[0018] As a preferred embodiment of the present invention, the cell culture medium for culturing cultured meat of the present invention may further include fatty acids as other additives.

[0019] As a preferred embodiment of the present invention, the medium additive of the cell culture medium for cultured meat of the present invention may include 0.001 to 0.500 g / L of chlorella extract per 1 L of the cell culture medium.

[0020] As a preferred embodiment of the present invention, the cell culture medium for cultured meat of the present invention may further include at least one selected from 11.2 to 112.0 g / L of phosphodiesterase inhibitor, 0.01 to 1.00 g / L of insulin, 4.0 to 40.0 g / L of synthetic glucocorticoid, 3.6 to 36.0 g / L of thiazolidinedione, 0.012 to 0.120 g / L of lecithin, antibiotic, 1.5 to 15.0 g / L of glutamine, 0.1 to 2.0 g / L of amino acid, and 0.05 to 5.00 g / L of vitamin per 1 L of the cell culture medium.

[0021] As a preferred embodiment of the present invention, the cell culture medium for cultured meat of the present invention may further include 5.7 to 57.0 g / L of fatty acids per 1 L of the cell culture medium.

[0022] As a preferred embodiment of the present invention, the culture target of the cell culture medium for cultured meat of the present invention may be muscle stem cells.

[0023] As a preferred embodiment of the present invention, the muscle stem cells may be animal muscle stem cells.

[0024] Another object of the present invention is to provide a method for culturing cultured meat using the cell culture medium described above, which performs a cell culture process for cross-differentiating animal muscle stem cells into adipocytes.

[0025] As a preferred embodiment of the present invention, the cultured meat culture can be performed in an incubator at 37 to 41°C and 5.0 to 10.0% CO₂ atmosphere.

[0026] As a preferred embodiment of the present invention, the cultured meat culturing method of the present invention can satisfy the following equation 1 for increasing cross-differentiation efficiency.

[0027] [Equation 1]

[0028] 2.0% ≤ (BA) / B×100% ≤ 15.0%

[0029] In Equation 1, A and B represent the cross-differentiation rates of muscle stem cells into adipocytes when culturing muscle stem cells in a cell culture medium. A is the cross-differentiation rate into adipocytes cultured using a cell culture medium that does not contain chlorella extract, and B is the cross-differentiation rate into adipocytes cultured using a cell culture medium that contains chlorella extract.

[0030] The present invention relates to a cultured meat culture medium additive and a cell culture medium for cultured meat using the same, wherein by using an extract of chlorella, a type of microalgae, as a medium additive component, cross-differentiation from muscle stem cells into adipocytes can be effectively induced, and cells in which intramuscular fat exists simultaneously can be induced, thereby providing cultured meat with improved flavor. In addition, by using the medium additive of the present invention, it is possible to replace fetal bovine serum, a cultured meat culture medium component, or to use it in combination with fetal bovine serum, thereby reducing the use of expensive fetal bovine serum and increasing the culture effect of cultured meat, thereby providing cultured meat with excellent cost-effectiveness.

[0031] The effects of the present invention are not limited to those mentioned above, and effects not mentioned will be clearly understood by those skilled in the art from the following description.

[0032] Figures 1a and 1b show the results of a cell experiment to evaluate cell viability according to treatment with chlorella extract.

[0033] Figure 2 shows the results of measuring the efficiency of cross-differentiation of chicken muscle stem cells into adipocytes according to the composition of the cell culture medium performed in Experimental Example 2.

[0034] Figure 3 is a DIC (Digital image correlation) measurement image of cross-differentiation of muscle stem cells performed in Experimental Example 2.

[0035] Figure 4 shows the results of measuring the efficiency of cross-differentiation of C2C12 myoblasts into adipocytes according to the composition of the cell culture medium performed in Experimental Example 3.

[0036] Figure 5 is a DIC (Digital image correlation) measurement image of cross-differentiated C2C12 myogenic cells performed in Experimental Example 3.

[0037] The numerical ranges described in the present invention include all values ​​within the range of start and end points, and for example, the range of “20°C to 100°C” means that the values ​​of ‘20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, 95°C, 100°C’ as well as any value between integers that fall within the scope of the described range, such as ‘20°C to 40°C, 50°C to 70°C, 80°C to 100°C’. Additionally, the range of “10 minutes to 100 minutes” is meant to include all integers including, for example, “10 minutes to 20 minutes,” “20 minutes to 30 minutes,” “30 minutes to 40 minutes,” “40 minutes to 50 minutes,” “60 minutes to 70 minutes,” “70 minutes to 80 minutes,” “80 minutes to 90 minutes,” and “90 minutes to 100 minutes,” as well as any integers between 20 minutes to 40 minutes, 80 minutes to 100 minutes, which fall within the ranges described above.

[0038] In the present invention, the terms “medium,” “culture medium,” “culture medium,” “medium composition,” “culture composition,” and “culture medium composition” refer to a culture solution containing nutrients capable of supporting the growth and survival of stem cells under in vitro culture conditions, and are not distinguished in this specification and may be used interchangeably.

[0039] In the present invention, "muscle stem cells" include precursor cells such as quiescent satellite cells and activated satellite cells called myoblasts. Muscle stem cells can differentiate into muscle cells, and in particular, muscle stem cells derived from animals such as livestock can be used in actual cultured meat production, making them highly useful in agrobiological applications.

[0040] In the present invention, “cultivation of muscle stem cells” means cell proliferation of muscle stem cells while maintaining their differentiation ability, which is a stem cell ability.

[0041] The present invention is described in more detail below.

[0042] The cultured meat culture medium additive of the present invention includes a chlorella extract.

[0043] The method for producing the above chlorella extract comprises the steps of: 1) producing a chlorella suspension; 2) ultrasonically treating the chlorella suspension of step 1 to produce a chlorella cell lysate; 3) performing a reflux extraction process on the chlorella cell lysate to obtain a hot water extract; 4) treating the hot water extract with a base, then performing a centrifugation process to obtain a supernatant, and then neutralizing the supernatant to obtain a chlorella extract; and 5) drying the chlorella extract to obtain a powdered chlorella extract.

[0044] The chlorella suspension of step 1 can be prepared by mixing and stirring chlorella powder and water in a weight ratio of 1:5 to 20, preferably 1:8 to 15, and more preferably 1:9 to 12.

[0045] The chlorella constituting the chlorella powder of the first step may include at least one selected from among Chlorella vulgaris, Chlorella pyrenidosa, Chlorella regularis, Chlorella saccharophila, Chlorella sorokiniana, and Chlorella capsulata, preferably at least one selected from among Chlorella vulgaris, Chlorella pyrenidosa, and Chlorella capsulata, and more preferably at least one selected from among Chlorella vulgaris and Chlorella capsulata.

[0046] Next, the second-stage ultrasonic treatment can be performed under conditions of a temperature of 0 to 20°C and an intensity of 10 to 50 kHz, and preferably, can be performed for 30 minutes to 2 hours under conditions of a temperature of 0 to 10°C and an intensity of 15 to 30 kHz. At this time, if the ultrasonic intensity exceeds 50 kHz, there may be a problem that the effective ingredient is decomposed and reduced due to the strong intensity, and if the ultrasonic treatment temperature exceeds 40°C, there may be a problem that the effective ingredient is denatured due to the high temperature and the content is reduced, so it is advantageous to perform the ultrasonic treatment under the above conditions.

[0047] Next, the 3-step reflux extraction process is to prepare a mixture by mixing chlorella ultrasonically treated in step 2 with water in a weight ratio of 1:1 to 100, preferably 1:5 to 50, and then performing reflux extraction on the mixture using a reflux extractor at 25 to 100°C for 10 minutes to 3 hours, preferably at 85 to 95°C for 10 minutes to 1 hour, thereby obtaining a hot water extract.

[0048] Next, the fourth step is the base treatment process. The reason for mixing the base into the hot water extract is to separate the available active ingredients from the cell wall.

[0049] The above base treatment can be performed by mixing the hot water extract and water, adding a base, and then heat treating at 80 to 105°C for 20 to 50 minutes.

[0050] At this time, the base may be at least one selected from sodium hydroxide and potassium hydroxide. At this time, the concentration of the base is suitably 0.100 to 2.000 N, preferably 0.100 to 1.000 N. If the concentration of the base is less than 0.100 N, the available effective ingredient may not be separated well from the cell wall, and if it exceeds 2.000 N, there may be a problem of the effective ingredient being destroyed.

[0051] And, after the base treatment is completed, it is centrifuged, the supernatant is taken, and then the supernatant is subjected to a neutralization process.

[0052] And, the neutralization process is performed by neutralizing the pH of the supernatant to pH 7.0±0.3, preferably 7.0±0.1, and the neutralization treatment can use an acid such as hydrochloric acid.

[0053] The cell culture medium for cultured meat of the present invention uses the chlorella extract prepared by the method described above as a medium additive, thereby reducing the amount of fetal bovine serum used in the cell culture medium or replacing it, thereby lowering the unit cost of cultured meat production.

[0054] The cell culture medium for cultured meat of the present invention may include a serum-free medium including a basal medium and the medium additives; or a serum medium including a basal medium, fetal bovine serum (FBS), and the medium additives.

[0055] In addition, the above-mentioned medium additive includes a chlorella extract, and may further include other additives in addition to the chlorella extract.

[0056] Among the cell culture medium components, the basal medium can be arbitrarily selected from among commonly used media suitable for stem cell culture in the relevant field. Furthermore, the culture conditions can also be arbitrarily selected from among appropriate conditions used in the relevant field. That is, the medium and culture conditions can be selected based on the type of cell to be cultured. The medium used for culture is a cell culture minimum medium (CCMM), which can generally include carbon sources, nitrogen sources, and trace elements.

[0057] As a preferred example of the basal medium, the basal medium is DMEM (Dulbecco's Modified Eagle's Medium), GenGro medium, DMEM / F-12 medium, Leibovitz's L-15 medium, IMDM (Iscove's Modified Dulbecco's Medium), McCoy's 5A medium, M199 medium, MEM (Minimum Essential Medium Eagle), α-MEM (Minimum Essential Medium Eagle, Alpha), Ham's F-10 medium (Ham's F-10 Nutrient Mixture), Ham's F-12 medium (Ham's F-12 Nutrient Mixture), RPMI1640 medium, BME (Basal Media Eagle), CMRL1066 medium, Fischer's Medium, Waymouth MB752 / 1 medium (Waymouth It may include one or more selected from MB752 / 1 Medium), MCDB Veggie and WME (Williams' Medium E).

[0058] In addition to the basal medium, the above serum-free medium may further include at least one selected from insulin, transferrin, sodium selenite, FGF-2 (Fibroblast growth factor), EGF (Epidermal Growth Factor), TGFβ3 (Transforming growth factor), APS (l-ascorbate-2-phosphate trisodium salt), PDGF-BB (Platelet Derived Growth Factor), and HGF (Hepatocyte Growth Factor, recombinant).

[0059] In addition, the amount of basal medium in the serum-free medium or serum medium is appropriately used at 500 to 1,000 g / L, preferably 530 to 972 g / L, and more preferably 600 to 900 g / L per 1 L of cell culture medium. If the amount of serum-free medium used is less than 500 g / L, there may be a problem of insufficient effect, and if it exceeds 60 g / L, there may be a problem of cell death.

[0060] In addition, the amount of fetal bovine serum used in the serum medium is appropriately 1 to 200 g / L per 1 L of cell culture medium, preferably 50 to 150 g / L.

[0061] And, the medium additive of the serum-free medium or serum medium may include chlorella extract at 0.001 to 0.500 g / L, preferably 0.005 to 0.200 g / L, and more preferably 0.010 to 0.080 g / L per 1 L of cell culture medium. If the concentration of the chlorella extract is less than 0.001 g / L, the cross-differentiation enhancing effect due to its use is insignificant, and even if it is used in excess of 0.500 mg / L, the cross-differentiation enhancing effect due to its use is insignificant or there is no further enhancing effect, and there may be a cytotoxicity problem.

[0062] In addition, the medium additive may further include, in addition to the chlorella extract, one or more additives selected from among a phosphodiesterase inhibitor, insulin, synthetic glucocorticoid thiazolidinedione, lecithin, antibiotics, glutamine, amino acids, and vitamins.

[0063] Among other additive components, the phosphodiesterase inhibitor may include at least one selected from IBMX (3-Isobutyl-1-0methylxanthine), sildenafil, tadalafil, vardenafil, avanafil, roflumilast, apremilast, ibudilast, cilostazol, milrinone, theophylline, aminophylline, and methylxanthine.

[0064] Among other additive components, the synthetic glucocorticoid may include at least one selected from dexamethasone, prednisone, methylprednisolone, hydrocortisone, and cortisol.

[0065] Among other additive components, the thiazolidinedione may include at least one selected from rosiglitazone, pioglitazone, lobeglitazone, troglitazone, ciglitazone, darglitazone, englitazone, netoglitazone, and rivoglitazone.

[0066] Among other additive ingredients, the antibiotic may include at least one selected from soy lecithin, sunflower lecithin, coconut lecithin, egg yolk lecithin, quinoa lecithin, and rice bran lecithin.

[0067] In addition, the other additives may further include a fatty acid, and the fatty acid may further include at least one selected from canola oil, olive oil, avocado oil, peanut oil, and high-oleic sunflower oil, which contain oleic acid.

[0068]

[0069] As a preferred embodiment of the medium additive of the cell culture medium for cultured meat culture of the present invention, in which chlorella extract and other additives are used, when the other additives include a phosphodiesterase inhibitor, insulin, synthetic glucocorticoid, fatty acid, thiazolidinedione, lecithin, antibiotic, glutamine, amino acid and vitamin, the appropriate contents of these components in the cell culture medium are as follows.

[0070] In addition, when using a phosphodiesterase inhibitor as another additive and IBMX as the phosphodiesterase inhibitor, it is advantageous in terms of cross-differentiation to use IBMX at 11.2 to 112.0 g / L per 1 L of cell culture medium, preferably 15.0 to 60.0 g / L.

[0071] In addition, when using insulin as another additive, it is advantageous in terms of cross-differentiation to use it at 0.01 to 1.00 g / L per 1 L of cell culture medium, preferably 0.20 to 0.90 g / L.

[0072] In addition, as other additives, when using synthetic glucocorticoids and dexamethasone as the synthetic glucocorticoid, it is advantageous in terms of cross-differentiation to use dexamethasone at 4 to 40 g / L per 1 L of cell culture medium, preferably 5.0 to 30.0 g / L.

[0073] In addition, when using thiazolidinedione as other additives and rosiglicazone as the thiazolidinedione, it is advantageous in terms of cross-differentiation to use rosiglicazone at 3.6 to 36 g / L, preferably 6.0 to 30.0 g / L per 1 L of cell culture medium.

[0074] In addition, when using lecithin as another additive, it is advantageous in terms of cross-differentiation to use it at 0.012 to 0.120 g / L per 1 L of cell culture medium, preferably 0.030 to 0.100 g / L.

[0075] In addition, when using antibiotics as other additives, it is advantageous in terms of stably culturing cells to use them at 0.1 to 5.0 volume%, preferably 0.3 to 1.2 volume%, per 1 L of cell culture medium. In addition, penicillin, streptomycin, and / or gentamicin can be used as the antibiotics.

[0076] In addition, when using glutamine as another additive, it is advantageous in terms of cross-differentiation to use it at 1.5 to 15.0 g / L per 1 L of cell culture medium, preferably 2.5 to 12.0 g / L.

[0077] In addition, when using amino acids as other additives, it is advantageous in terms of cross-differentiation to use them at 0.1 to 2.0 g / L per 1 L of cell culture medium, preferably 0.3 to 1.6 g / L.

[0078] In addition, when using vitamins as other additives, it is advantageous in terms of cross-differentiation to use them at 0.05 to 5.00 g / L per 1 L of cell culture medium, preferably 0.20 to 3.50 g / L.

[0079] In addition, as other additives, when using more fatty acids and canola oil containing oleic acid as a fatty acid, it is advantageous in terms of cross-differentiation to use it at 5.7 to 57 g / L per 1 L of cell culture medium, preferably 6.5 to 45.0 g / L.

[0080] The target cells to be cultured in the cell culture medium for culturing the cultured meat of the present invention include muscle stem cells, mesenchymal stem cells, adipose stem cells, fibroblasts, embryonic stem cells, induced pluripotent stem cells, vascular stem cells and / or animal cells, and preferably, muscle stem cells, mesenchymal stem cells, adipose stem cells, fibroblasts, embryonic stem cells, induced pluripotent stem cells, vascular stem cells derived from pigs, cows or chickens, and more preferably, muscle stem cells derived from chickens.

[0081] As a preferred embodiment of the method for culturing cultured meat using the cell culture medium for culturing cultured meat of the present invention, a cell culture process for cross-differentiating muscle stem cells into adipocytes can be performed.

[0082] To be more specific, the above cell culture process uses the cell culture medium for cultured meat culture described above for culture. In addition, the cultured meat culture conditions are advantageous in terms of cross-differentiation when culture is performed in an incubator at 37 to 41°C and 5.0 to 10.0% CO₂ atmosphere, preferably in an incubator at 38 to 40°C and 5.0 to 7.0% CO₂ atmosphere.

[0083] The method for culturing cultured meat using a cell culture medium containing the medium additive of the present invention exhibits excellent cross-differentiation efficiency of muscle cells or muscle stem cells into adipocytes. The cross-differentiation efficiency increase rate of the culture medium of the present invention can satisfy the following equation 1.

[0084] [Equation 1]

[0085] 2.0% ≤ (BA) / B×100% ≤ 15.0%, preferably 2.5% ≤ (BA) / B×100% ≤ 12.5%, more preferably 4.5% ≤ (BA) / B×100% ≤ 12.0%,

[0086] In Equation 1, A and B represent the cross-differentiation rate of muscle stem cells into adipocytes when culturing muscle stem cells in a cell culture medium, A is the cross-differentiation rate of muscle stem cells into adipocytes when cultured using a cell culture medium not containing chlorella extract, and B is the cross-differentiation rate of muscle stem cells into adipocytes when cultured using a cell culture medium containing chlorella extract.

[0087] Using the cell culture medium for cultured meat culture described above, high-quality cultured meat can be provided at a high cost-effectiveness.

[0088] In the present invention, the cultured meat may contain muscle cells and intramuscular fat at the same time.

[0089] Furthermore, the present invention can provide a food composition including the cultured meat described above.

[0090]

[0091] The present invention is described in detail through preferred embodiments, and the following examples are illustrative of the present invention, but the present application is not limited to the following examples.

[0092] [Example]

[0093] Example 1: Preparation of chlorella extract (culture medium additive)

[0094] As chlorella, Chlorella vulgaris (target chlorella powder) was prepared.

[0095] Next, the chlorella powder and distilled water were mixed in a weight ratio of 1:10 to prepare a suspension, which was then placed in an ultrasonic processor, and ultrasonic treatment was performed for 1 hour under the conditions of an output of 700 kW, a temperature of 4°C, and an ultrasonic intensity of 20 kHz to obtain a chlorella cell lysate with the cell walls broken.

[0096] Next, the chlorella cell lysate was subjected to reflux extraction using a reflux extractor at 95°C for 30 minutes to obtain a hot water extract.

[0097] Next, a mixture was prepared by mixing the hot water extract and the same amount of distilled water used in preparing the suspension, and then a base was added to the mixture so that the concentration of 0.25 N sodium hydroxide became 0.125 N, and then the mixture was placed in an autoclave and heat-treated at 100°C for 30 minutes to perform base treatment.

[0098] Next, the base-treated mixture was cooled, centrifuged at 3900 rpm for 20 minutes, and only the supernatant was collected.

[0099] Next, the supernatant was neutralized to pH 7.0±0.1 using hydrochloric acid to obtain a chlorella extract.

[0100] Next, the chlorella extract was freeze-dried to obtain a powdered chlorella extract.

[0101]

[0102] Manufacturing Example 1: Manufacturing of cell culture medium for cultured meat cultivation

[0103] The chlorella extract prepared in Example 1 was used as an additive to a culture medium to prepare a cell culture medium for cultured meat cross-differentiation with the following composition.

[0104] The above-mentioned cell culture medium for cultured meat cross-differentiation comprises, as a base medium, 900 g / L of DMEM (Dulbecco's Modified Eagle's Medium High glucose), 0.01 g / L of insulin, 100 g / L of FBS, the chlorella extract of Example 1 as a medium additive, and as other additives of the medium additive, 11.2 g / L of IBMX (3-Isobutyl-1-0methylxanthine), a phosphodiesterase inhibitor, 0.1 g / L of insulin, 4.0 g / L of dexamethasone, a synthetic glucocorticoid, and 3.6 g / L of rosiglicazone, a thiazolidinedione.

[0105] At this time, the medium additive (chlorella extract of Example 1) was added at 0.001 g / L, 0.01 g / L, 0.03 g / L, 0.05 g / L, 0.07 g / L, and 0.1 g / L, respectively, to prepare a separate culture medium.

[0106]

[0107] Comparative Manufacturing Example 1

[0108] A cell culture medium having the same composition as in Manufacturing Example 1 was prepared, but instead of the chlorella extract of Example 1, chlorella powder (containing 1 wt% of total chlorophyll) from Company D was used as a medium additive at 0.001 g / L, 0.01 g / L, 0.03 g / L, 0.05 g / L, 0.07 g / L, and 0.1 g / L.

[0109]

[0110] Comparative Manufacturing Example 2

[0111] A cell culture medium having the same composition as in Manufacturing Example 1 was prepared, but instead of the chlorella extract of Example 1, commercially available chlorella powder (Shaanxi Ruiwo Phytochem Growth Factor Chlorella Extract CGF OD1200) was used as a medium additive at 0.001 g / L, 0.01 g / L, 0.03 g / L, 0.05 g / L, 0.07 g / L, and 0.1 g / L.

[0112]

[0113] Comparative Manufacturing Example 3 (Positive Control Group)

[0114] A cell culture medium having the same composition as in Manufacturing Example 1 was prepared, but a cell culture medium without using a medium additive (chlorella extract of Example 1) was prepared as a control.

[0115] Classification (g / L) Negative control Comparative manufacturing example 3 (positive control) Manufacturing example 1, Comparative manufacturing example 1~2 Base medium DMEM 900 875.48 875.38 Insulin 00.01 0.01 Fetal bovine serum (FBS) 100 100 100 Medium additive Chlorella extract 000.001 ~ 0.100 IBMX 011.2 11.2 Dexamethasone 04.0 4.0 Rosiglicazone 03.6 3.6 Lecithin 00.01 20.012

[0116]

[0117] Experimental Example 1: Cytotoxicity Test of Cell Culture Medium

[0118] The cytotoxicity (or cell viability) of the cell culture medium containing the chlorella extracts prepared in Manufacturing Example 1 and Comparative Manufacturing Examples 1 to 2 against chicken muscle stem cells was measured using a CCK8 assay, and the results are shown in Fig. 1a (Manufacturing Example 1), Fig. 1b (Comparative Manufacturing Example 1), and Fig. 1c (Comparative Manufacturing Example 2).

[0119] Looking at Figures 1a to 1c, it was confirmed that not only Manufacturing Example 1 but also Comparative Manufacturing Examples 1 and 2 exhibited cytotoxicity when the concentration of chlorella extract in the cell culture medium was 30 mg / L (0.03 g / L) or higher. In addition, it was confirmed that cell viability decreased in a concentration-dependent manner at concentrations of 100 mg / L (0.1 g / L) or higher. Therefore, the cross-differentiation rate measurement experiment was conducted at a concentration of 10 mg / L (0.01 g / L), which is a level that does not cause toxicity to cells.

[0120]

[0121] Experimental Example 2: Experiment to measure the cross-differentiation rate of chicken muscle stem cells into adipocytes.

[0122] An experiment was conducted to measure the cross-differentiation rate into adipocytes by culturing chicken muscle stem cells using a cell culture medium for cultured meat containing 10 mg / L (0.01 g / L) of the chlorella extract prepared in Manufacturing Example 1 and Comparative Manufacturing Examples 1 to 2 and the cell culture medium of Comparative Manufacturing Examples 1 to 2, respectively.

[0123] The above chicken muscle stem cells were obtained from cells extracted from the chest and legs of chickens hatched on the 17th, 18th, and 19th days using fertilized eggs cultured in a sterile laboratory.

[0124] The experimental method was to remove the medium from the adipocytes cross-differentiated from chicken muscle stem cells, add DPBS to wash the cells, mix thoroughly, and then remove the medium.

[0125] Next, 4% formalin solution was added at room temperature and left for at least 1 hour. Next, after removing formalin, wash three times with distilled water, add 60% by volume isopropanol, leave for 5 minutes, remove isopropanol, and dry completely. Add Oil Red O working solution and leave for 10 minutes at room temperature. Remove Oil Red O solution and immediately add triple-distilled water. After confirming that the cells were stained by observing under a microscope, photographs were taken. Discard the water and dry the flask. Add 100% by volume isopropanol and shake gently for 10 minutes to completely dissolve Oil Red O. The absorbance was measured at 490 nm using a microplate reader, and the absorbance at 630 nm was measured again to correct the absorbance at 490 nm.

[0126] And, the results are shown in Table 3 and Figure 2 below, and Figure 3 shows the DIC (Digital image correlation) measurement images of the cross-differentiated muscle stem cells of the positive control group and Manufacturing Example 1.

[0127] Additionally, the cross-differentiation increase rate in Table 2 represents the cross-differentiation increase rate compared to the cross-differentiation rate of the positive control group.

[0128] The negative control group and positive control group in Table 2 were tested using a medium having the composition in Table 2.

[0129] Negative control group Positive control group Manufacturing example 1 Comparative manufacturing example 1 Comparative manufacturing example 2 Cross-differentiation rate of muscle stem cells into adipocytes -24% 29% 26% 24% Cross-differentiation increase rate --5% 1% 0%

[0130] Looking at Table 2 and Figure 2 above, it can be confirmed that the cell culture medium of Manufacturing Example 1 has the highest cross-differentiation rate, and Comparative Manufacturing Example 2 did not have a cross-differentiation enhancement effect despite containing chlorella extract. In addition, it can be confirmed that Manufacturing Example 1 has a cross-differentiation enhancement effect that is 5 times higher than that of the cell culture medium of Comparative Manufacturing Example 1.

[0131]

[0132] Experimental Example 3: Experiment to measure the cross-differentiation rate of C2C12 myoblasts

[0133] An experiment was performed to measure the cross-differentiation rate into adipocytes by culturing C2C12 myoblasts in a cell culture medium for cultured meat containing 10 mg / L (0.01 g / L) of the chlorella extract prepared in Manufacturing Example 1. The experimental method and conditions were the same as in Experimental Example 2, and the results are shown in Table 3 and Fig. 4 below. Fig. 5 shows DIC measurement images of the cross-differentiation of C2C12 myoblasts of the positive control group and Manufacturing Example 1.

[0134] And, the positive control group in Table 3 below is an experiment conducted using a medium having the composition in Table 2 above.

[0135] Comparative manufacturing example 3 (positive control group) Manufacturing example 1 Cross-differentiation rate into adipocytes 28% 57% Cross-differentiation increase rate -29%

[0136] Through the above examples and experimental examples, it was confirmed that the chlorella extract prepared by the method presented in the present invention has excellent cell proliferation and culture effects as an additive to a cultured meat culture medium, and that by using it as a cultured meat culture medium, the cross-differentiation rate of muscle cells or muscle stem cells into adipocytes is very excellent.

Claims

1. A culture medium additive for cultured meat, containing chlorella extract.

2. In paragraph 1, the chlorella extract, A cultured meat culture medium additive, comprising an extract obtained by performing an extraction process of chlorella comprising at least one selected from Chlorella vulgaris, Chlorella pyrenidosa, Chlorella regularis, Chlorella saccharophila, Chlorella sorokiniana, and Chlorella capsulata.

3. A culture medium additive for cultured meat, wherein the extract in the second paragraph is a hot water extract.

4. In the second paragraph, the hot water extract is Step 1: Preparing a chlorella suspension; Step 2: preparing chlorella cell disruption solution by sonicating the suspension from step 1; Step 3: performing a reflux extraction process on the above chlorella cell disruption solution to obtain a hot water extract; Step 4: treating the above hot water extract with a base, performing a centrifugation process to obtain a supernatant, and then neutralizing the supernatant to obtain a chlorella extract; and A cultured meat culture medium additive manufactured by performing a process including the step of drying the above chlorella extract to obtain a powdered chlorella extract.

5. A cell culture medium for cultured meat, comprising the medium additive of clause 1.

6. In paragraph 5, a serum-free medium comprising a base medium and the medium additive; or A serum medium comprising a basal medium, fetal bovine serum (FBS) and the medium additives; a cell culture medium for culturing cultured meat.

7. In the 6th antibody, the base medium of the serum-free medium or serum medium is, DMEM (Dulbecco's Modified Eagle's Medium), GenGro medium, DMEM / F-12 medium, Leibovitz's L-15 medium, IMDM (Iscove's Modified Dulbecco's Medium), McCoy's 5A medium, M199 medium, MEM (Minimum Essential Medium Eagle), α-MEM (Minimum Essential Medium Eagle, Alpha), Ham's F-10 medium, Ham's F-12 medium, RPMI1640 medium, BME (Basal Media Eagle), CMRL1066 medium, Fischer's Medium, Waymouth MB752 / 1 Medium, MCDB medium, and A cell culture medium for cultured meat, comprising at least one selected from WME (Williams' Medium E).

8. In the 6th paragraph, the basal medium further comprises at least one selected from insulin, transferrin, sodium selenite, FGF-2 (Fibroblast growth factor), EGF (Epidermal Growth Factor), TGFβ3 (Transforming growth factor), APS (l-ascorbate-2-phosphate trisodium salt), PDGF-BB (Platelet Derived Growth Factor), and HGF (Hepatocyte Growth Factor, recombinant).

9. In paragraph 6, the badge additive, in addition to the chlorella extract, A cell culture medium for culturing cultured meat, further comprising at least one selected from a phosphodiesterase inhibitor, insulin, a synthetic glucocorticoid thiazolidinedione, lecithin, an antibiotic, glutamine, an amino acid, and vitamins.

10. A cell culture medium for cultured meat, wherein the medium additive comprises 0.001 to 0.500 g / L of chlorella extract per 1 L of cell culture medium.

11. A cell culture medium for culturing cultured meat, comprising 11.2 to 112.0 g / L of a phosphodiesterase inhibitor, 0.01 to 1.00 g / L of insulin, 4.0 to 40.0 g / L of a synthetic glucorticoid, 3.6 to 36.0 g / L of a thiazolidinedione, 0.012 to 0.120 g / L of lecithin, 1.5 to 15.0 g / L of an antibiotic, 0.1 to 2.0 g / L of an amino acid, and 0.05 to 5.00 g / L of vitamins per 1 L of the cell culture medium in claim 10.

12. A cell culture medium for cultured meat, wherein the culture target of the cell culture medium is muscle stem cells, according to any one of claims 5 to 11.

13. A cell culture medium for cultured meat, wherein the muscle stem cells in clause 12 are animal muscle stem cells.

14. A method for culturing cultured meat using the cell culture medium of any one of claims 5 to 11, A method for culturing cultured meat, which comprises performing a cell culture process to cross-differentiate animal muscle stem cells into adipocytes.

15. In paragraph 14, the cultured meat culture is, A method for culturing cultured meat, which involves culturing in an incubator at a temperature of 37 to 41°C and an atmosphere of 5.0 to 10.0% CO₂.

16. A method for culturing cultured meat in clause 14, wherein the cross-differentiation efficiency increase rate satisfies the following equation 1. [Equation 1] 2.0% ≤ (BA) / B×100% ≤ 15.0% In Equation 1, A and B represent the cross-differentiation rates of muscle stem cells into adipocytes when culturing muscle stem cells in a cell culture medium, A is the cross-differentiation rate into adipocytes cultured using a cell culture medium that does not contain chlorella extract, and B is the cross-differentiation rate into adipocytes cultured using a cell culture medium containing chlorella extract.

Images