Additive for culturing cell-cultured food comprising placenta extract, medium composition for culturing cell-cultured food comprising same, and method for producing cell-cultured food using same

Abstract

The present invention relates to: an additive for culturing a cell-cultured food containing a placenta extract; a medium composition for culturing a cell-cultured food, comprising same; and a method for producing a cell-cultured food using same.

Description

Additive for culturing cell culture food containing placental extract, medium composition for culturing cell culture food containing the same, and method for producing cell culture food using the same

[0001] The present invention relates to an additive for culturing cell culture food containing a placental extract, a medium composition for culturing cell culture food containing the same, and a method for producing cell culture food using the same.

[0002] Since Singapore became the first country in the world to commercially approve cultured meat products in 2020, interest in cell-cultured foods has been growing worldwide. Unlike traditional meat derived directly from animals, cell-cultured foods are artificial meat produced by culturing cells. This process requires an appropriate culture medium that allows cells to grow and differentiate. Conventional culture mediums typically contain reagent-grade chemicals and animal-derived ingredients like fetal bovine serum (FBS).

[0003] The world's first cultured meat product, developed by the American startup Eat Just, is a chicken nugget-shaped product made from cultured chicken cells. The culture medium used to produce the meat also contained fetal bovine serum (FBS).

[0004] FBS, containing cell growth factors, is recognized as the most important ingredient in cell media for the production of cultured meat products. However, as an animal-derived ingredient, it is very expensive, difficult to standardize due to variations, and suffers from high costs and variable quality, necessitating the search for alternatives.

[0005] Currently, several companies are conducting research on ingredients to replace FBS, but there is still a need for the development of a cost-effective and stable quality culture medium for commercial production of cell cultured foods.

[0006] [Prior Art Literature]

[0007] [Patent Document] U.S. Patent Publication No. 2022-0073870

[0008] The purpose of the present invention is to provide an additive optimized for the production of cell-cultured foods and a food medium composition containing the additive. In particular, the present invention aims to provide an additive and medium composition for culturing cell-cultured foods that can replace or reduce the amount of existing fetal bovine serum (FBS), while maintaining the production efficiency of cell-cultured foods and, further, maintaining equivalence with products produced in existing media without causing genetic mutations in the final product.

[0009] Another object of the present invention is to provide an efficient and economical method for producing cell culture food using the above additive and medium composition.

[0010]

[0011] Other purposes and advantages of this application will be further clarified by the detailed description below, taken in conjunction with the appended claims and drawings. Any details not described herein are readily apparent and inferable to those skilled in the technical field of this application or similar technical fields, and therefore, their description is omitted.

[0012] Hereinafter, embodiments of the present invention will be described in detail with reference to the attached drawings so that those skilled in the art can easily practice the present invention. The present invention may be implemented in various different forms and is not limited to the embodiments described herein. In the drawings, parts irrelevant to the description are omitted to clearly explain the present invention, and the same reference numerals are used throughout the specification for identical or similar components. In addition, detailed descriptions of widely known and publicly known technologies are omitted.

[0013] Throughout the specification, whenever a part is said to "include" a component, this does not mean that it excludes other components, but rather that it may include other components, unless otherwise stated.

[0014] The term "about" as used herein means within 10%, preferably within 5%, and more preferably within 1% of a given value or range.

[0015] In one embodiment, the present invention relates to an additive for culturing cell culture food comprising a placental extract.

[0016] The term "cell-cultured food" as used herein refers to a food produced using cells cultured outside of a living organism, such as in a laboratory, rather than through traditional farming or livestock breeding methods, and is understood to encompass all types of food commonly referred to as "cultured meat." Initial cells are collected from living animals, such as chickens, pigs, cows, sheep, and shrimp. The collected cells are then propagated through initial culture, produced through ocean culture, and processed into various food forms. In one specific example, the cell-cultured food may be cultured chicken meat cultured from cells derived from a Gallus gallus cell line.

[0017] Accordingly, the "additive for culturing cell culture food" of the present invention may refer to an additive used for cell culture in the process of manufacturing a cell culture food product, and more specifically, may be an additive for cell culture.

[0018] In one specific example, the additive according to the present invention is suitable for culturing cells used in cell culture foods, wherein the cells may be at least one type of muscle cells (myoblasts) or fat cells (adipocytes). In addition, connective tissue cells (fibroblasts) and the like may be additionally used.

[0019] Muscle cells are a key component of cell-cultured foods, particularly cultured meat, and are responsible for providing meat texture and protein. Muscle cells are primarily derived from satellite cells or progenitor cells, which differentiate into muscle fibers through the culture process.

[0020] Fat cells are often responsible for the flavor and tenderness of meat. The presence of fat can enhance the taste and texture, making it more meat-like. Fat cells are derived from adipocyte progenitor cells and, under appropriate culture conditions, accumulate fat.

[0021] In one embodiment, the additive according to the present invention comprises a placental extract.

[0022] The term "placenta" used herein refers to the structure that mediates the exchange of substances necessary for the survival and growth of the fetus between the fetus and the mother. The placenta is known to contain essential amino acids, melatonin, nucleic acid components such as RNA and DNA, the antioxidant enzyme SOD (super oxide dismutase), hyaluronic acid, antioxidants, cytokines, insulin-like growth factor, epidermal growth factor (EGFS), and senescent cell activating factors (SCAFS). However, for reasons such as safety, purity, and consistency, the placenta is not used as is, but rather is manufactured into an extract through hydrolysis, hot water extraction, and enzyme (proteolytic enzyme) treatment, and then used as a raw material for foods, etc.

[0023] In the present invention, the placenta may preferably be that of a mammal, for example, a placenta of a human, pig, horse, sheep, cow, etc. may be used. However, the use of the human placenta in industries other than pharmaceuticals is regulated and has ethical issues, so the placenta of an animal other than a human is more preferably used.

[0024] In one specific embodiment, the placental extract may be a placental extract from at least one selected from the group consisting of porcine placenta, horse placenta, sheep placenta, and bovine placenta. More preferably, the placental extract of the present invention may be at least one of porcine placenta extract and horse placenta extract. The placental extract may be obtained without limitation by a method commonly known in the art, such as acid, base hydrolysis, hot water extraction, enzyme (proteolytic enzyme) treatment, salting out, solvent precipitation, ultracentrifugal filtration, dialysis, gel filtration chromatography, etc.

[0025] The placental extract of the present invention contains various essential amino acids, growth factors, etc. necessary for cell growth, and thus can be used as a substitute for or in conjunction with fetal bovine serum (FBS), which is commonly used in the cultivation and production of cell culture products. When the placental extract of the present invention is used together with FBS, the content of FBS conventionally used can be reduced.

[0026] In another embodiment, the additive of the present invention may further include, in addition to the placental extract, at least one selected from the group consisting of whey protein, yeast extract, Chlorella A or B, Spirulina, and egg yolk.

[0027] In one embodiment, the present invention provides a medium composition for culturing a cell culture food product, comprising the above-described cell culture food product culturing additive.

[0028] In one specific embodiment, the additive of the present invention may be included at a concentration of 10 to 10,000 ug / ml, preferably 50 to 8,000 ug / ml, more preferably 100 to 5,000 ug / ml, and especially 1,000 ug / ml.

[0029] In one specific embodiment, the composition of the present invention may include a serum-free medium. The serum-free medium may be any medium known in the art to be commonly used in cell culture for the production of cell culture foods, and may include, without limitation, Essential 8™ Medium, Pro293™ CDM, ExpiCHO™ SFM, StemPro™-34 SFM, Neurobasal™ Medium, VitaCyte™, Fungi™ SFM, BalanCD® CHO Growth A, Ham's medium, NCTC 109, NCTC 135, Waymouth's MB 752 / 1, Waymouth's MB 705 / 1, Brich & Pirt media, Higuchi media, MCDB media, etc.

[0030] In another specific embodiment, the food medium composition of the present invention comprises various amino acids, vitamins, minerals, etc., and is formulated at specific concentrations to optimize cell proliferation and differentiation. For example, the food medium composition of the present invention may include the following ingredients:

[0031] - Amino acids: L-Arginine, L-Asparagine, L-Aspartic Acid, L-Glutamic Acid, L-Histidine, L-Isoleucine, L-Leucine, L-Methionine, L-Phenylalanine, L-Threonine, L-Tryptophan, L-Valine, L-Tyrosine, L-Cystine, Glycine, L-Alanine, L-Lysine HCl, L-Proline, L-Serine, L-Glutamine

[0032] - Vitamins: Riboflavin, Folic acid, Choline chloride, D-Calcium Pantothenate, i-Inositol, Nicotinic acid, Pyridoxine HCl, Thiamine HCl

[0033] - Minerals: Iron(II) gluconate, Calcium chloride, Sodium bicarbonate, Potassium chloride, Sodium chloride, Magnesium sulfate, Monosodium phosphate

[0034] - Carbohydrate: D-Glucose monohydrate

[0035] Each component of the above composition may be blended at a specific concentration. Preferably, the concentration of each component is as follows:

[0036] - L-Arginine: 47 ~ 213 mg / L

[0037] - L-Asparagine: 19 to 86 mg / L

[0038] - L-Aspartic acid: 21 ~ 95 mg / L

[0039] - L-Glutamic acid: 78 ~ 352 mg / L

[0040] - L-Histidine: 17 to 77 mg / L

[0041] - L-Isoleucine: 42 ~ 189 mg / L

[0042] - L-Leucine: 45 ~ 204 mg / L

[0043] - L-Methionine: 14 ~ 63 mg / L

[0044] - L-Phenylalanine: 30 ~ 136 mg / L

[0045] - L-Threonine: 38 ~ 171 mg / L

[0046] - L-Tryptophan: 6 ~ 29 mg / L

[0047] - L-Valine: 37 ~ 168 mg / L

[0048] - L-Tyrosine: 42 ~ 188 mg / L

[0049] - L-Cystine: 25 ~ 113 mg / L

[0050] - Glycine: 12 ~ 54 mg / L

[0051] - L-Alanine: 20 ~ 89 mg / L

[0052] - L-Lysine HCl: 58 ~ 263 mg / L

[0053] - L-Proline: 18 ~ 82 mg / L

[0054] - L-Serine: 32 ~ 145 mg / L

[0055] - Riboflavin: 0.2 ~ 0.7 mg / L

[0056] - Folic acid: 1.6 ~ 7.2 mg / L

[0057] - Choline chloride: 1.6 ~ 7.2 mg / L

[0058] - D-Calcium pantothenate: 1.6 ~ 7.2 mg / L

[0059] - i-Inositol: 2.8 ~ 12.6 mg / L

[0060] - Nicotinamide: 1.6 ~ 7.2 mg / L

[0061] - Pyridoxine HCl: 1.6 ~ 7.2 mg / L

[0062] - Thiamine HCl: 1.6 ~ 7.2 mg / L

[0063] - Iron(II) gluconate: 0.1 ~ 0.4 mg / L

[0064] - L-Glutamine: 234 ~ 1051 mg / L

[0065] - D-Glucose monohydrate: 1800 ~ 8100 mg / L

[0066] - Magnesium sulfate: 39 ~ 176 mg / L

[0067] - Monosodium phosphate: 50 ~ 225 mg / L

[0068] - Potassium chloride: 160 ~ 720 mg / L

[0069] - Sodium chloride: 2560 ~ 11520 mg / L

[0070] - Calcium chloride: 80 ~ 360 mg / L

[0071] - Sodium bicarbonate: 7400 ~ 33300 mg / L

[0072] In one specific embodiment, the present invention provides a medium composition comprising the above-described component. The medium composition can be usefully used for culturing cell culture foods even without a placental extract.

[0073] In another specific embodiment, the composition of the present invention may further comprise growth factors, antibiotics, and fetal bovine serum (FBS).

[0074] The growth factors that can be used herein are those known in the art to be commonly used in cell culture for the production of cell cultured foods, and can be used without limitation, and can include, for example, FGF (Fibroblast Growth Factor), EGF (Epidermal Growth Factor), IGF (Insulin-like Growth Factor), TGF-β (Transforming Growth Factor-beta), HGF (Hepatocyte Growth Factor), PDGF (Platelet-Derived Growth Factor), VEGF (Vascular Endothelial Growth Factor), SCF (Stem Cell Factor), NGF (Nerve Growth Factor), BMP (Bone Morphogenetic Protein), etc. Additionally, sub-classes of each of these growth factors may also be used, for example, in the case of FGF, it should be understood that they include FGF-1 (Acidic FGF), FGF-2 (Basic FGF), FGF-3, FGF-4, FGF-5, FGF-6, FGF-7 (Keratinocyte Growth Factor), FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, FGF-15, FGF-16, FGF-17, FGF-18, FGF-19, FGF-20, FGF-21, FGF-22, FGF-23, etc. In one specific example, the composition of the present invention may include FGF, particularly FGF-2, as a growth factor.

[0075] The growth factor may be included at a concentration of 1 to 100 ng / ml, specifically, as follows:

[0076] FGF-2 (Fibroblast Growth Factor-2): 1-100 ng / mL

[0077] EGF (Epidermal Growth Factor): 10-50 ng / mL

[0078] IGF (Insulin-like Growth Factor): 10-100 ng / mL

[0079] TGF-β (Transforming Growth Factor-beta): 1-10 ng / mL

[0080] HGF (Hepatocyte Growth Factor): 10-50 ng / mL

[0081] PDGF (Platelet-Derived Growth Factor): 10-50 ng / mL

[0082] VEGF (Vascular Endothelial Growth Factor): 5-50 ng / mL

[0083] SCF (Stem Cell Factor): 10-100 ng / mL

[0084] NGF (Nerve Growth Factor): 10-50 ng / mL

[0085] BMP (Bone Morphogenetic Protein): 10-100 ng / mL

[0086] Growth factors that can be used herein are those known in the art to be commonly used in cell culture for the production of cell cultured foods, and may be used without limitation, and may include, for example, penicillin, streptomycin, gentamicin, kanamycin, amphotericin B, neomycin, tetracycline, ciprofloxacin, vancomycin, erythromycin, etc. In one specific example, the composition of the present invention may include at least one of penicillin and streptomycin, and in particular, a combination of penicillin / streptomycin may be used.

[0087] The above antibiotic may be included in a concentration of 1 to 100 μg / ml, specifically, as follows:

[0088] Penicillin: 100 IU / mL

[0089] Streptomycin: 100 μg / mL

[0090] Gentamicin: 10-50 μg / mL

[0091] Kanamycin: 100 μg / mL

[0092] Amphotericin B: 0.25-2.5 μg / mL

[0093] Neomycin: 50 μg / mL

[0094] Tetracycline: 1-10 μg / mL

[0095] Ciprofloxacin: 1-10 μg / mL

[0096] Vancomycin: 10-50 μg / mL

[0097] Erythromycin: 50-100 μg / mL

[0098] The composition of the present invention may contain 1 to 15 wt% of fetal bovine serum (FBS) based on the total volume of the composition. In one specific example, the fetal bovine serum may contain 1 wt%, 3 wt%, 5 wt%, 10 wt%, or 15 wt%.

[0099] At this time, the additive of the present invention may be included in an amount of 0.005 to 1 wt%, preferably 0.01 to 0.5 wt%, and in specific examples, 0.01, 0.05, 0.1, 0.5 wt%, based on the total volume of the composition.

[0100] When the placental extract of the present invention is used together with FBS as an additive, the content of FBS used conventionally can be reduced, and for example, when 15 wt% of FBS and 0.1 wt% of placental extract are used, a cell proliferation rate at a level similar to that of a case containing 20 wt% of FBS can be obtained.

[0101] In one embodiment, the present invention provides a method for producing a cell cultured food product, comprising the step of inoculating cells with the medium composition for culturing the cell cultured food product. The cells may be proliferating cells or differentiated cells, and may be, for example, cells derived from a Gallus gallus cell line.

[0102] In one specific example, the method for producing a cell culture food according to the present invention comprises the following steps.

[0103] Cells are inoculated into the food medium composition of the present invention. In one specific example, the cell inoculation amount is 1x10 4 cells / mL to 5x10 6 It can be in the range of cells / mL.

[0104] The above-mentioned inoculated cells are cultured at 37°C and 5% CO2. More preferably, the culture conditions are maintained at pH 7.2 to 7.4.

[0105] Regularly replace the culture medium during the culture process to optimize cell growth. Ideally, replace the medium every two to three days.

[0106] After a certain period of culture, the cells are harvested and processed into food. The culture period is preferably 7 to 14 days.

[0107] When using a cell culture food culture additive containing a placental extract and a cell culture food culture medium composition containing the same, compared to existing commercially available media, cell proliferation and differentiation can be improved while reducing the amount of FBS used, and a protein expression pattern at an equivalent level can be shown without showing significant genetic mutations.

[0108] More specifically, the food medium composition of the present invention has the following effects: even when the content of FBS is reduced, it exhibits a cell proliferation rate almost equivalent to that of a case where a high content of FBS is used, the analysis of the region where DNA mutations occur does not show significant genetic mutations (gene stability), and in the protein expression pattern, it exhibits an equivalent protein expression pattern between the food medium and the reagent medium (protein expression pattern similarity).

[0109] Accordingly, the additive and medium composition of the present invention can greatly contribute to improving the quality of cell cultured food and reducing production costs, and can be used as a culture medium optimized for manufacturing cell cultured food.

[0110] Figure 1 is a graph showing the results of screening for cell proliferation rates of food medium additive candidates.

[0111] Figure 2 is a graph showing the effect of cell proliferation rate in a culture medium containing a placenta food additive.

[0112] Figure 3 is a graph showing the effect of cell proliferation rate in a culture medium containing a placenta food additive.

[0113] Figure 4 shows the results of Reference sequencing according to Experimental Example 3.1.1, showing the total number of reads and mean depth in food media and reagent media. P<0.01; ns, non-significant.

[0114] Figure 5 shows the results of Reference sequencing according to Experimental Example 3.1.1, showing Whole genome reads homology. P<0.01; ns, non-significant.

[0115] Figure 6 shows the number of DNA mutations as a result of searching for base sequence SNPs and InDels according to Experimental Example 3.1.2. SNP; single nucleotide polymorphisms, P<0.01; ns, non-significant

[0116] Figure 7 shows the results of analyzing the region where DNA mutations occurred according to Experimental Example 3.1.3.

[0117] Figures 8 to 10 show the results of analyzing the degree of similarity grouping based on transcript expression levels according to Experimental Example 3.2.1. Figure 8 is a tree of hierarchical clustering for each sample, Figure 9 is a heatmap of hierarchical clustering analysis that groups samples and genes with similar expression levels for significant data for each sample, and Figure 10 shows the results of principal component analysis of food media and reagent media for proliferating and differentiating cells.

[0118] Figure 11 shows the results of comparing protein production patterns of proliferating cells according to the medium according to Experimental Example 3.3.1. Specific spots for each medium are indicated.

[0119] Figure 12 shows the results of the protein production pattern ratio of differentiated cells according to the medium according to Experimental Example 3.3.2. Light green indicates a spot specific to the reagent medium.

[0120] Hereinafter, preferred examples are presented to aid in understanding the present invention. However, the following examples are provided solely to facilitate a better understanding of the present invention, and the scope of the present invention is not limited by the following examples.

[0121]

[0122] Manufacturing Example 1. Manufacturing of food medium

[0123]

[0124] (1) Stock solution manufacturing

[0125]

[0126] First, the solvents described in Table 1 were prepared. 1.2 M HCl was prepared by diluting 37% HCl (12 M) 10-fold in purified water. 2.0 M HCl was prepared by diluting 37% HCl (12 M) 6-fold in purified water. 1.0 M NaOH was prepared by dissolving 2 g of sodium hydroxide in 50 mL of purified water. 0.1 M NaOH was prepared by diluting 1.0 M NaOH 10-fold in purified water.

[0127] Next, the raw materials were accurately weighed using an electronic balance according to the information in Table 1.

[0128] Stock solutions were prepared by adding each solvent to the weighed raw materials and completely dissolving them. The raw materials were imported from overseas manufacturers or purchased through domestic distributors.

[0129]

[0130] Stock solution Raw material number Raw material name Concentration (mg / mL) Solvent 1L-Arginine1001.2M HCl2L-Asparagine1001.2M HCl3L-Aspartic Acid1001.2M HCl4L-Glutamic Acid1001.2M HCl5L-Histidine1001.2M HCl6L-Isoleucine1001.2M HCl7L-Leucine1001.2M HCl8L-Methionine1001.2M HCl9L-Phenylalanine1001.2M HCl10L-Threonine1001.2M HCl11L-Tryptophan1001.2M HCl12L-Valine1001.2M HCl13L-Tyrosine1002.0M HCl 14 L-Cystine 100 2.0M HCl 15 Glycine 100 Purified water 16 L-Alanine 100 Purified water 17 L-Lysine HCl 100 Purified water 18 L-Proline 100 Purified water 19 L-Serine 100 Purified water 20 Riboflavine 100.1M NaOH 21 Folic acid 10 1.0M NaOH 22 Choline chloride 10 Purified water 23 D-Calcium Pantothenate 10 Purified water 24 i-Inositol 10 Purified water 25 Nicotinic acid 10 Purified water 26 Pyridoxine HCl 10 Purified water 27 Thiamine HCl 10 Purified water 28 Iron(II) gluconate 1 Purified water

[0131] (2) Mixing of raw materials

[0132]

[0133] Half the volume of the medium to be prepared was added to the beaker with purified water and stirred at 400 rpm. The stock solution prepared in item 1 above was added to the purified water at the concentration of the medium to be prepared (Table 2). L-Glutamine, D-Glucose monohydrate, Magnesium sulfate, Monosodium phosphate, Potassium chloride, Sodium chloride, Calcium chloride, and Sodium bicarbonate were weighed using an electronic balance at the concentration of the medium to be prepared and added additionally. The mixture was stirred at 400 rpm for 30 minutes and visually checked for complete dissolution.

[0134]

[0135] Medium raw material concentration Raw material concentration (mg / L) L-Arginine47 ~ 213L-Asparagine19 ~ 86L-Aspartic acid21 ~ 95L-Glutamic acid78 ~ 352L-Histidine17 ~ 77L-Isoleucine42 ~ 189L-leucine45 ~ 204L-Methionine14 ~ 63L-Phenylalanine30 ~ 136L-Threonine38 ~ 171L-Tryptophan6 ~ 29L-Valine37 ~ 168L-Tyrosine42 ~ 188L-Cystine25 ~ 113Glycine12 ~ 54L-Alanine20 ~ 89L-Lysine HCl58 ~ 263L-Proline18 ~ 82L-Serine32 ~ 145Riboflavin0.2 ~ 0.7Folic acid1.6 ~ 7.2Choline chloride1.6 ~ 7.2D-Calcium pantothenate1.6 ~ 7.2i-Inositol2.8 ~ 12.6Nicotinamide1.6 ~ 7.2Pyridoxine HCl1.6 ~ 7.2Thiamine HCl1.6 ~ 7.2Iron(II) gluconate0.1 ~ 0.4L-Glutamine234 ~ 1051D-Glucose monohydrate1800 ~ 8100Magnesium sulfate39 ~ 176Monosodium phosphate50 ~ 225Potassium chloride160 ~ 720Sodium chloride2560 ~ 11520Calcium chloride80 ~ 360Sodium bicarbonate296 ~ 1332

[0136] (3) pH titration

[0137]

[0138] The final pH was titrated to 7.2–7.4 with 1 M HCl or 1 M NaOH using a pH meter.

[0139]

[0140] (4) Sterile filtration

[0141]

[0142] Sterile filtration was performed using a 0.2 μm filter.

[0143]

[0144] Manufacturing Example 2. Selection of Materials for Food Additives

[0145]

[0146] Raw materials known to be rich in nutrients and growth factors were selected as candidates for food additives, and their cell proliferation rates were evaluated as follows to determine the final additives to be used. The candidates used were whey protein, yeast extract, chlorella (Chlorella A or B), spirulina, egg yolk, and placenta (Porcine placenta or Horse placenta).

[0147]

[0148] Evaluation of cell proliferation rate in culture medium containing food additives

[0149]

[0150] Chicken embryo-derived muscle stem cells were cultured in collagen-coated dishes (37°C, 5% CO2) and seeded in 24-well cell culture plates. After 24 hours, cell attachment to the plates was confirmed, and the medium was replaced with a culture medium containing food medium additives and cultured for 1 to 2 days. To the basal medium containing the food ingredients listed in Table 2, 1 to 15% FBS, 100 to 5,000 μg / mL food medium additive candidates, 5 ng / mL FGF-2 (Fibroblast Growth Factor), and 1% penicillin / streptomycin were added. After 1 to 2 days, the cells were treated with the cell viability assay kit (WST) solution and incubated in an incubator for 2 to 4 hours. After that, the absorbance was measured at a wavelength of 450 nm. The cell proliferation rate of the control group containing 20% ​​FBS was set at 100%, and the cell proliferation rates of the experimental groups were compared. Subsequently, a medium food additive with superior cell proliferation rate was selected, and an FBS replacement test was conducted using the same method as above.

[0151] The experimental results for this are as follows.

[0152]

[0153] (1) Screening of cell proliferation rate of food medium additives

[0154]

[0155] To utilize food additives in cell proliferation media, screening evaluations were conducted on various raw materials, including whey protein, yeast extract, chlorella (Chlorella A or B), spirulina, egg yolk, and placenta (Porcine placenta or Horse placenta). As shown in Figure 1, the cell proliferation rate in the Porcine placenta and Horse placenta groups was significantly increased compared to the control group (p<0.05).

[0156] Therefore, among the candidate materials for food additives, pig placenta and horse placenta were selected and FBS replacement tests were conducted as follows.

[0157]

[0158] (2) Effect of cell proliferation rate in culture medium containing pig placenta food additives

[0159]

[0160] Experiments were conducted under two conditions depending on the cell seeding amount and placental processing time, and the results are shown in Figure 2.

[0161] Condition 1: 16,000 cells / well, treated for 1 day (Fig. 2a).

[0162] Condition 2: 11,000 cells / well, treated for 2 days (Fig. 2b).

[0163] When the amount of FBS added was reduced to 15%, 10%, 5%, 3%, 1%, and 0%, the cell proliferation rate was confirmed to drop to 90%, 86%, 75%, 55%, 43%, and 42%. At this time, when the pig placenta was mixed and treated by concentration, the cell proliferation rate increased in most treatment groups. However, there was no experimental group that showed a cell proliferation rate higher than the 20% FBS treatment group (Fig. 2a).

[0164] When the amount of FBS added was reduced to 15%, 10%, 5%, 3%, 1%, and 0%, the cell proliferation rates were 99%, 99%, 80%, 69%, 49%, and 48%. At this time, when the placenta was mixed and treated by concentration, the cell proliferation rate increased only in the 15% FBS mixed treatment group, and it showed a higher cell proliferation rate than the 20% FBS treatment group, but there was no statistically significant difference (p<0.05) (Fig. 2b).

[0165]

[0166] (3) Effect of cell proliferation rate in culture medium containing placental food additives

[0167]

[0168] As in item (2), experiments were conducted under two conditions depending on the cell seeding amount and placental processing time, and the results are shown in Fig. 3.

[0169] When the amount of FBS added was reduced to 15%, 10%, 5%, 3%, 1%, and 0%, the cell proliferation rate was confirmed to drop to 90%, 86%, 75%, 55%, 43%, and 42%. At this time, when the placenta was mixed and treated according to concentration, the cell proliferation rate increased in most treatment groups. However, there was no experimental group that showed a cell proliferation rate higher than the 20% FBS treatment group (Fig. 3a).

[0170] When the amount of FBS added was reduced to 15%, 10%, 5%, 3%, 1%, and 0%, the cell proliferation rates were 99%, 99%, 80%, 69%, 49%, and 48%. At this time, when the placenta was mixed and treated according to concentration, the cell proliferation rate increased in the 15% FBS mixed treatment group, and showed a higher cell proliferation rate than the 20% FBS treatment group, but there was no statistically significant difference (p<0.05) (Fig. 3b).

[0171]

[0172] Experimental example: Evaluation of the effects of food media and reagent media on the genome, transcriptome, and proteome in the growth of proliferative and differentiated cells of Gallus gallus cell lines.

[0173]

[0174] 1. Experimental objectives

[0175]

[0176] The aim of this study was to evaluate the effects of food media and reagent media on the genome, transcriptome, and proteome of the Gallus gallus cell line during proliferation and differentiation.

[0177]

[0178] 2. Research materials and contents

[0179] 2.1. Research materials

[0180]

[0181] Proliferating and differentiated cells of the Gallus gallus cell line were used as materials, and the experiment was conducted twice with each sample.

[0182] The food media used were food ingredients listed in Table 2, 20% FBS, 5 ng / mL FGF-2, 1% penicillin / streptomycin, porcine placenta, and horse placenta, and the reagent media were Ham's F10 or DMEM, 20% FBS, 5 ng / mL FGF-2, 1% penicillin / streptomycin, porcine placenta, and horse placenta. FBS and Ham's F10 were purchased from Gibco, FGF-2 was purchased from Miltenyi, penicillin / streptomycin was purchased from Sigma, and DMEM was purchased from Hyclon. Porcine placenta and horse placenta were supplied by Neocrema Co., Ltd.

[0183]

[0184] Distinctive use media sample name Repeated proliferation cell food medium CIC-FCIC-F1 CIC-F2 Reagent medium CIC-RCIC-R1 CIC-R2 Differentiating cell food medium CAC-FCAC-F1 CAC-F2 Reagent medium CAC-RCAC-R1 CAC-R2

[0185] Food ingredients (medium) corresponding to the composition of food medium 2 78.8% FBS 20% FGF-25 ng / mL Porcine placenta 0.1% Horse placenta 0.1% Penicillin / streptomycin 1%

[0186] Composition of reagent medium: Ham's F10 or DMEM 79% FBS 20% FGF-25 ng / mL penicillin / streptomycin 1%

[0187] 2.2. Research contents and methods

[0188] 2.2.1. Genome Analysis

[0189]

[0190] DNA libraries were isolated and purified using the TruSeq Nano DNA High Throughput Library Prep Kit (Illumina). The extracted products were quantified according to the qPCR Quantification Protocol Guide. After verification using the Agilent Technologies 4200 TapeStation D1000 screentape (Agilent technologies), paired-end sequencing (2 x 150 bp) was performed at Macrogen using NovaSeq (Illumina). After sequencing, FastQC (v. 0.11.8) was performed to check read quality, and Trimmomatic (v. 0.38) was used to remove adapter sequences and low-quality reads. The filtered data from each sample were mapped to Gallus gallus (GenBank assembly: GCF_016699485.2) using BWA (v. 0.7.17) and the mem algorithm. After mapping, duplicate reads were removed using Sambaba (v. 0.6.8), and genome coverage, mapped reads, and ratios were calculated using SAMtools (v. 1.9) and BCFtools (v. 1.6). Using the mapped reads, we identified candidates for SNPs and short indels with a phred score of 30 or higher. SnpEff (v. 4.3t) was used to identify variants in these candidates.

[0191]

[0192] 2.2.2. Transcriptome Analysis

[0193]

[0194] For RNA library construction, total RNA concentration was quantified using Quant-IT RiboGreen (Invitrogen). 1 μg of total RNA was obtained from each sample using the Illumina TruSeq Standed mRNA Sample Prep Kit (Illumina). To analyze mRNA, the RNA was purified using an mRNA purification kit. For short-read sequencing, the fragments were randomly fragmented. The fragmented RNA fragments were then copied into first-strand cDNA using SuperScript II reverse transcriptase (Invitrogen) and random primers. The final cDNA was then synthesized using DNA Polymerase I, RNase H, and dUHP. Different adapters were attached to both ends of the cDNA fragments and ligation was performed. The library was quantified using the KAPA Library Quantification kits for the Illumina Sequencing platform according to the qPCR Quantification Protocol Guide (KAPA BIOSYSTEMS). The final verification was performed using TapeStation D1000 ScreenTape (Agilent Technologies). Afterwards, the library was paired-end sequenced (2X100 bp) at Macrogen using Illumina NovaSeq (Illumina). The raw reads obtained through sequencing were analyzed for quality control to remove artifacts. The preprocessed reads were mapped to Gallus gallus (GenBank assembly: GCF_016699485.2) using HISAT2 (v.2.2.1). After mapping, transcript assembly was performed using StringTie (v.2.2.0) based on the aligned reads.

[0195]

[0196] 2.2.3. Proteome Analysis

[0197]

[0198] Proteins were extracted from the samples and measured using the Bradford method (Bio-Rad). For 2-DE analysis, pH 3-10 IPG strips (GE-Healthcare Life Sciences) were rehydrated in a buffer containing 7 M urea, 2 M thiourea, 2.5% DTT, and 4% CHAPS. Protein lysates were separated on 14% sodium dodecyl SDS-PAGE using IPGphor III (GE-Healthcare Life Sciences). After separation, the gels were fixed and stained with Colloidal Coomassie Blue G-250 solution (BioRad). After destaining, images were acquired using an Image scanner (BioRad). Image analysis was performed using ImageMasterRM 2D Plantinum software (Amersham Biosciences), and more than 200 landmarks were identified for protein spot comparison. Among the selected landmarks, spots with a target ratio of 2 or higher were analyzed using the method of Bahk et al. (2004), the gel was extracted using trypsin. The extracted solution was concentrated and desalted using a reversed-phase column as described in Gobom et al. (1999). The analytical instrument used was a nano ACQUITY UPLC equipped with an LTQ-orbitrap-mass spectrometer (Thermo Electron) and LC-MS / MS. The mobile phases used were a 0.1% formic acid solution and an acetonitrile solution supplemented with 0.1% formic acid. The analytical column used was a BEH C18 1.7 μm, 100 μm 100 mm column (Waters). SEQUEST software (Thermo Quest) was used to acquire protein information, and the generated peak list was analyzed using the MASCOT program (Matrix Science).

[0199]

[0200] 3. Research Results

[0201] 3.1. Genome Analysis: DNA CNV (copy number variation) analysis (equivalence assessment through observation of chromosomal abnormalities)

[0202]

[0203] The purpose of this study was to examine differences between samples by observing chromosomal abnormalities in proliferating and differentiated cells using reagent media and food media.

[0204]

[0205] 3.1.1. Reference sequencing

[0206]

[0207] To compare proliferating and differentiated cells using food media and reagent media based on Gallus gallus (GenBank assembly: GCA_016699485.1), two replicates of resequencing were performed. Mapped sites were identified at over 99% accuracy, and mapped reads were also similar at over 99% accuracy, demonstrating no statistically significant differences (Table 6, Fig. 4).

[0208]

[0209] Mapped data statsSampleMapped Sites(>=1x)Total ReadsMapped ReadsMapped BasesMean DepthVariantCIC-F11,047,157,051666,020,812662,482,17292,617,820,49987. 939,042,657CIC-F21,047,250,608627,534,424624,027,47184,703,276,86280.41 9,172,172CIC-R11,047,186,512707,849,370704,150,53497,557,655,91792.629 ,042,754CIC-R21,047,284,186632,786,906629,314,39085,376,604,44681.059,2 07,489CAC-F11,047,432,327682,385,888678,733,49994,973,469,36290.169,24 2,250CAC-F21,047,318,941702,567,056698,838,45794,124,212,24089.369,230, 405CAC-R11,047,180,292685,391,306681,890,02094,830,616,39990.039,307,2 67CAC-R21,047,346,448731,147,022727,199,30197,492,034,76492.569,244,771

[0210] Based on the number of reads excluding variant values ​​in the mapped reads shown in Table 6, the homology with the chromosome of Gallus gallus (GenBank assembly: GCA_016699485.1) was analyzed. As a result, the proliferating cells showed 60.2% ± 2.6 homology in the food medium and 62.4% ± 5.0 in the reagent medium, and the differentiating cells showed 64.5% ± 1.4 homology in the food medium and 66.0% ± 3.0 in the reagent medium, and there was no significant difference between the media (Fig. 5).

[0211]

[0212] 3.1.2. Search for base sequence SNPs and InDels

[0213]

[0214] Although base sequence mutations are a general phenomenon that occurs when the genome is replicated, the mutation rate can vary depending on environmental factors, so we attempted to identify base sequence mutations according to the medium. Regarding single nucleotide polymorphisms (SNPs), 8,385,816 ± 84,220 SNPs were identified in the growth medium (reagent medium) with a minimum of 8,254,562 and a maximum of 8,469,898 differentiated cells (reagent medium). In addition, the number of insertions and deletions was confirmed to be 374,592 ± 7,024 and 425,813 ± 9,495, respectively, but we confirmed that there was no significant difference in each cell type depending on the medium (Table 7, Fig. 6). Although no significant difference was observed, the number of SNPs increased when the reagent medium was used.

[0215]

[0216] Number of DNA mutations Library name Number of SNPs Number of insertions Number of deletionsCIC-F18,256,497369,127417,033CIC-F28,381,625369,636420,911CIC-R18,254,562370,099418,093CIC-R28,414,671370,22042 2,598CAC-F18,441,090374,863426,297CAC-F28,428,460375,501426, 444CAC-R18,469,898390,232447,137CAC-R28,439,727377,057427,987

[0217] 3.1.3. Analysis of DNA mutation occurrence areas

[0218]

[0219] The genome can be divided into genes, regulatory elements that control genes (mostly located upstream of genes), and base sequences that are neither genes nor regulatory elements. Furthermore, eukaryotic genes are divided into exons and introns, which contain protein composition information. Exons are regions that code for amino acids, and introns are noncoding regions that exist between exons. Intron variants refer to mutations that occur in noncoding regions, intergenic regions refer to mutations in the region between genes, upstream variants refer to mutations that occur within 5 kb from the start of a gene transcript, and conversely, downstream variants refer to mutations that occur within 5 kb from the end of a gene transcript. 3 prime UTR variant, 5 prime UTR variant refers to mutations occurring in the untranslated region of the 3 prime end and 5 prime end of the transcript, synonymous variants are mutations that occur in the coding region but do not change the amino acid sequence, intragenic variants are mutations that occur in the noncoding region between two genes, missense variants are mutations that change one amino acid sequence, and lastly, 5 prime UTR premature start codon gain variant refers to a mutation in the start codon of the 5 prime end. Intragenic variant, missense variant, and 5 prime UTR premature start codon gain variant, where mutations mainly occurred, accounted for more than 88.5% of all samples, but it was analyzed that most of the locations where genomic mutations occurred were non-coding variants that do not affect protein expression or variants that affect non-coding genes. In addition, no significant difference was found in the media difference (Fig. 7).

[0220]

[0221] 3.2. Transcriptome Analysis: Comparison of Transcriptome Sequencing

[0222]

[0223] We aimed to compare and analyze the expression levels of transcribed RNAs from the genomes of proliferating and differentiated cells using reagent media and food media through transcriptome sequencing. For RNAs with significantly increased or decreased expression, the genes transcribing the corresponding RNAs were identified, and functional classification and gene annotation were performed based on gene pathway information.

[0224] The expression profile was extracted using the normalization values ​​FPKM (Fragments Per Kilobase of transcript per Million mapped reads) / RPKM (Reads Per Kilobase of transcript per Million mapped reads) and TPM (Transcripts Per Kilobase Million) obtained through transcript quantification, and the TPM values ​​were organized as the results based on the normalization values.

[0225]

[0226] 3.2.1. Similarity grouping based on transcript expression level

[0227]

[0228] Twice repeated analyses of expressed transcripts per sample revealed groupings into proliferating and differentiating cells, but no differentiation by medium. The values ​​for this grouping, normalized as described above, are hierarchically clustered to show the degree of similarity in expression levels (Figure 8).

[0229] The expression levels of significant data of each analyzed sample were grouped through hierarchical clustering analysis and displayed as a heatmap. As a result, it was confirmed that the cells were similar and that the medium did not have a significant effect (Fig. 9).

[0230] This was confirmed by principal component analysis, which showed that the same group was formed for each cell, and that similar expression patterns were confirmed without differences in the medium (Fig. 10).

[0231]

[0232] 3.3. Proteome Analysis: Comparison of Protein Expression Using 2D Gels

[0233]

[0234] We aimed to confirm the equivalence of protein expression by examining the translated protein patterns in proliferating and differentiated cells using reagent media and food media.

[0235]

[0236] 3.3.1. Comparison of protein patterns in proliferating cells

[0237]

[0238] The difference in protein expression was confirmed using 2D gel with proteins isolated from the sample.

[0239] The similarity between the media was confirmed to be 99.7% by comparing and analyzing the spots with a ratio of 2.0 or higher among the proteins of the proliferating cells obtained by loading the 2D gel. The number of proteins detected was 616 in the food medium and 615 in the reagent medium. In terms of expression levels between the media, 7 spots were confirmed to be visible only in the food medium and 6 spots were confirmed to be visible only in the reagent medium. Among these, the protein information was confirmed through LC-MS / MS analysis of spots with an intensity value of 0.2 or higher, and it was confirmed that they were proteins mainly expressed due to environmental influences (Table 8, Fig. 11).

[0240]

[0241] Media-specific proteins of proliferating cells Protein GenBank EC No. Protein name Note AAB86581.1 - Heat shock protein 10 Oligomerisation interface AAF73513.1 - Voltage-dependent anion channel Voltage-dependent anion channel of the outer mitochondrial membrane

[0242] 3.3.2. Comparison of protein patterns in differentiated cells

[0243]

[0244] The protein expression differences were confirmed using 2D gels for proteins isolated from the samples. The similarity between the media was confirmed to be 82.0% by comparing the spots with a ratio of 2.0 or higher among the proteins of the differentiated cells obtained by loading the 2D gels. The number of proteins detected was 217 in the food medium and 238 in the reagent medium. Regarding the expression levels between the media, 5 spots were identified that appeared only in the food medium and 26 spots were identified that appeared only in the reagent medium. Among these, the protein information was confirmed through LC-MS / MS analysis of spots with an intensity value of 0.2 or higher, and it was confirmed that they were proteins mainly expressed due to environmental influences (Table 9, Fig. 12).

[0245]

[0246] Reagent medium used Differentiated cell-specific protein Protein GenBank EC No. Protein name Note NP_001193425.14.1.2.13 Fructose-bisphosphate aldolase CB Rain-type aldolase CAH65352.1-Hypothetical protein NP_001041541.2-Vimentin Vimentin C-terminus P13648.1-Lamin-A3J0S_A-α-actin Nucleotide-Binding Domain of the sugar kinase 0703290 B-Tubulin alpha

[0247] 4. Results

[0248]

[0249] As a result of genome comparison analysis, when compared with Gallus gallus, which was used as a reference genome, proliferating cells and differentiating cells showed homology of approximately 60.2 ± 2.6% - 66.0 ± 3.0%, and no significant difference was observed between the media.

[0250] In the case of transcriptome analysis, when analyzed based on the amount of expressed transcripts, grouping into proliferating cells and differentiating cells was performed, but the difference between the media was not significant.

[0251] The similarity between proteins produced in proliferating cells and differentiated cells was 82.0% and 99.7%, respectively, but there was no significant difference between the media.

[0252]

[0253] The present invention,

[0254] Assignment Number: 2022M3A9I308237112

[0255] Ministry of Science and ICT

[0256] Research Management Specialist Organization: National Research Foundation of Korea

[0257] Research Project Name: Development of Technology to Secure and Enhance the Value of Natural Products

[0258] Research Project Name: (Joint Project 6) Development of Source Technology for Customized New Food Materials for Future Alternative Meat (Including Cultured Meat)

[0259] Host organization: Neocrema Co., Ltd. (Contribution ratio: 1 / 1)

[0260] Research period: January 1, 2024 - December 31, 2024

[0261] It is a technology developed through .

Claims

1. An additive for cell culture food containing placental extract.

2. An additive for cell culture food culture, wherein the additive in paragraph 1 is an additive for culturing muscle cells or fat cells.

3. An additive for culturing cell culture food, wherein the placental extract in paragraph 1 is a placental extract from at least one species selected from the group consisting of pig placenta, horse placenta, sheep placenta, and bovine placenta.

4. An additive for culturing cell culture food in the first paragraph, wherein the placental extract is at least one of a pig placental extract or a horse placental extract.

5. A medium composition for culturing cell culture food, comprising the cell culture food culturing additive of paragraph 1.

6. A medium composition for culturing cell culture food, wherein the additive is included in a concentration of 10 to 400 ㎍ / ㎖ in paragraph 5.

7. A medium composition for culturing cell culture food, wherein the composition further comprises growth factors, antibiotics, and fetal bovine serum (FBS) in the fifth paragraph.

8. A medium composition for culturing cell culture food, wherein the growth factor in paragraph 7 is FGF (Fibroblast Growth Factor).

9. A medium composition for culturing cell culture food, wherein the antibiotic in paragraph 7 is at least one selected from penicillin and streptomycin.

10. In the 7th paragraph, the medium composition for culturing the cell culture food is, 1 to 15 wt% of fetal bovine serum based on the total volume of the composition Growth factor 1-100 ng / mL, and Antibiotic 1 to 100 μg / ml, and A medium composition for culturing cell culture food, comprising:

11. A medium composition for culturing cell culture food in claim 10, wherein the growth factor is FGF-2 (Fibroblast Growth Factor) and the antibiotic is 1% penicillin / streptomycin.

12. A method for producing a cell cultured food product, comprising a step of inoculating cells with a medium composition for culturing a cell cultured food product according to any one of claims 5 to 11.

13. A method for producing a cell cultured food according to claim 12, wherein the cell is a cell derived from a Gallus gallus cell line.

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