KR20220040424A - Method for manufacturing Cultured meat, cultured meat prepared therefrom, and food composition comprising the saME - Google Patents

Abstract

The present invention relates to a producing method of a cultured meat, the cultured meat produced therefrom, and a food composition comprising the cultured meat. Specifically, by producing the cultured meat using cell sheet technology introducing a porous polymer support, the present invention has an effect of having an advantage of providing a food texture similar to that of real meat by forming a high-density muscular tissue, allowing a high-quality animal protein to be easily consumed by being treated and used for various types of food groups, and reducing a negative perception of cultured meat by a consumer.

Description

Method for manufacturing cultured meat, cultured meat prepared therefrom, and food composition comprising the saME

The present invention relates to a method for producing cultured meat, to a cultured meat prepared therefrom, and to a food composition comprising the cultured meat.

According to a report by the Food and Agriculture Organization of the United Nations (FAO), the world population is projected to increase from 7.6 billion in 2018 to 9.5 billion in 2050. However, due to global climate abnormalities such as global warming, crop yields are expected to decrease, while the demand for feed grains increases, increases production costs and reduces production area, so livestock products are expected to become expensive food sources.

Animal-derived foods such as livestock products are expensive in terms of energy and production, but have contributed directly to human nutrition as they are the best source of high-quality protein and micronutrients essential for normal human growth and health. In particular, essential amino acids are not synthesized in the body or are synthesized in very small amounts, so they must be consumed with food. There is a limit to the amount of protein required to supply essential amino acids through traditional livestock production methods.

Recently, alternative meat has been attracting attention as a way to solve the problem of meat shortage in the future. Substitute meat is a concept that encompasses both plant-based meat and cultured meat, which are mainly made of protein extracted from soybeans, wheat, and mold. This is being marketed. However, since plant meat does not contain animal protein, there is a limit in that it is not easy to realize the taste of actual meat.

Cultured meat refers to edible meat obtained through cell propagation using cell engineering technology by culturing live animal cells in a laboratory without going through the process of raising livestock. In vitro meat or lab-grown meat in the sense of growing in vitro; artificial meat in the sense of being synthesized using human stem cells rather than natural; They are also called bio-artificial muscles (BAMs) in the sense of culturing the muscle fibers that make up the muscles.

Currently, cultured meat is produced by collecting tissue from live animals, separating stem cells, culturing and growing them into muscle cells, and then going through stages such as muscle fiber coloring and fat mixing. At this time, in order to implement a texture similar to that of meat, research is being actively conducted to prepare cultured meat that exhibits a texture similar to that of actual muscle tissue.

As a substitute for meat, it is one of the important tasks to realize a texture similar to actual meat, but cultured meat can be freed from the abuse of antibiotics and the use of growth promoters resulting from the factory-style group breeding method, and cell engineering As meat is produced without raising animals using technology, it is expected to be in the spotlight as an eco-friendly alternative technology field that reduces environmental pollution. is in need.

The present invention has been devised to solve the above technical problem, and an object of the present invention is to provide high-quality animal protein through a food composition comprising cultured meat according to the present invention.

Another object of the present invention is to provide a method for producing cultured meat exhibiting a texture similar to actual livestock meat or other meat substitutes with a high cell content by introducing a micro-sized porous polymer support into a cell sheet.

In addition, according to the present invention, by utilizing the cell sheet technology in which the porous polymer support is introduced, gas or nutrients are effectively delivered to the attached cells, and cells are stably attached to proliferate and proliferate by providing cell adhesion based on the extracellular matrix component It aims to provide a differentiated environment.

In order to solve the above technical problem, the present invention comprises the steps of introducing a porous polymer support to a substrate for cell culture; seeding cells usable for preparing cultured meat on the porous polymer support; culturing the seeded cells to prepare a single-layered cell sheet; immobilizing the cell sheet to a substrate; and stacking a plurality of the cell sheets.

In one aspect of the present invention, the porous polymer may be selected from the group consisting of alginate, gelatin, agar, starch, collagen, fibrinogen, chitosan, cellulose, and combinations of two or more thereof.

In one aspect of the present invention, the porous polymer support comprises the steps of emulsifying the porous polymer; and a drying step; may be a fine support obtained through.

In one aspect of the present invention, the porous polymer support may be cross-linked.

In one aspect of the present invention, the single-layered cell sheet may include a cross-linked porous polymer micro-support.

In one aspect of the present invention, the plurality of cell sheets may be stacked according to an electrostatic interaction between the cell sheet and the cell sheet, hydrogen bonding, covalent bonding, ionic bonding, or a combination thereof.

In addition, the present invention can provide a cultured meat prepared according to the above-described manufacturing method.

In addition, the present invention can provide a food composition comprising the cultured meat.

In one aspect of the present invention, the food may be at least one selected from the group consisting of snacks, dumplings, fried foods, fried foods, soy sauce, seasonings, powder mixes, breads, processed canned foods, beverages, dried seaweeds, and processed noodles. .

The present invention can reduce the production cost of cultured meat through various applications by introducing an edible polymer material in the development of cell-based cultured meat.

In addition, by introducing a micro-sized porous polymer platform and utilizing it as a gas and nutrient carrier or a three-dimensional cell adhesion substrate, it is possible to provide improved texture and high-quality cultured meat through a high-density cell sheet.

The present invention can provide high-quality animal protein by including cultured meat as a substitute for meat in general foods containing meat. In addition, by adding various types of cultured meat to general foods that do not contain meat, it is possible to conveniently provide the umami of animal protein and meat.

1 shows a microscope image of a cell sheet prepared according to Comparative Example 1 and Example 1 of the present invention. Bare sheet means existing cell sheet without gelatin support, and GMS sheet means cell sheet with gelatin support.
2 is a graph showing the results of analyzing the DNA content of the cell sheets prepared according to Comparative Example 1 and Example 1 of the present invention.
3 is a graph showing the results of analyzing the glycosaminoglycan (GAG) content of the cell sheets prepared according to Comparative Example 1 and Example 1 of the present invention.
4 is a graph showing changes in the storage modulus of the cell sheets of Comparative Examples 1 and 1 according to whether or not the gelatin micro-support is contained in Experimental Example 2 of the present invention.
5 is a graph showing changes in the storage modulus of cultured meat, roasted cultured meat, chicken breast and TVP (Textured vesitable protein) by introduction of a gelatin micro support according to Experimental Example 2 of the present invention.
6 is a photograph showing the state before and after cooking of a cultured meat model cultured on a gelatin support prepared according to Example 1 of the present invention.
7 is a photograph showing the form of minced meat, which is one of the cultured meat models cultured on a gelatin support, prepared according to Example 1 of the present invention, and shows an example of being baked and cooked.

Hereinafter, the method for producing cultured meat according to the present invention, the cultured meat prepared according to the method, and a food composition comprising the cultured meat will be described in detail.

At this time, unless otherwise defined, all technical and scientific terms have the meanings commonly understood by those of ordinary skill in the art to which this invention belongs, and the terms used in the description of the present invention are only specific examples is intended to effectively describe, and is not intended to limit the present invention.

In addition, in the following description, descriptions of well-known effects and configurations that may unnecessarily obscure the gist of the present invention will be omitted. In the following specification, the units used without special mention are based on weight, and for example, the unit of % or ratio means weight % or weight ratio.

In addition, in describing the components of the present invention, terms such as first, second, A, B (a), (b), etc. may be used. These terms are only for distinguishing the elements from other elements, and the essence, order, or order of the elements are not limited by the terms.

Also, the singular form used in the specification of the present invention may be intended to include the plural form as well, unless the context specifically dictates otherwise.

Hereinafter, the method for producing cultured meat according to the present invention will be described in detail.

The method for producing cultured meat according to the present invention is cost-effective and can provide high-quality cultured meat with a high cell content by introducing a micro-sized porous polymer platform. The cell culture form for the production of cultured meat may be a conventional two-dimensional or three-dimensional culture known in the art, but three-dimensional culture is advantageous in order to create an environment similar to an actual living body due to the interaction between cells and cells. .

Specifically, the present invention relates to a method for producing cultured meat using a cell sheet technology, comprising the steps of introducing a porous polymer support into a substrate for cell culture; seeding cells usable for preparing cultured meat on the porous polymer support; culturing the seeded cells to prepare a single-layered cell sheet; immobilizing the cell sheet to a substrate; and stacking a plurality of the cell sheets.

The cell sheet may serve as a three-dimensional support for mass proliferation by injecting cells usable for the production of cultured meat. That is, by mimicking the various roles of the extracellular matrix of a living body in a given environment, a biological environment is provided to cells so that cell adhesion, proliferation, and differentiation can be made well. In this case, the cell sheet into which the micro-sized porous polymer is introduced can allow the cells and the culture medium to be easily fixed.

Specifically, the porous polymer may be, for example, a natural polymer with excellent biocompatibility, and may be selected from the group consisting of alginate, gelatin, agar, starch, collagen, fibrinogen, chitosan, cellulose, and combinations of two or more thereof. In particular, since collagen and gelatin have excellent physiological activity and biocompatibility, a gelatin support or a collagen support may be implemented according to a preferred embodiment of the present invention.

Alternatively, the porous polymer is polydimethylsiloxane, polyethylene glycol, polystyrene polylactic acid, polypropylene, poly(lactic acid-co-glycolic acid), polyglycolic acid, polyvinyl alcohol, polyethylene oxide, polystyrene, poly(ethylene-co-vinyl alcohol) ), polycaprolactone, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), polyacrylonitrile, polyurethane, polyethylene terephthalate, polyacrylic acid, silk-like polymer, hydroxyapatite It may be selected from the group consisting of (hydroxyapatite) and a combination of two or more thereof, and is not particularly limited as long as it can be used in conventional cell culture. However, the strength of the porous polymer may be 0.1 to 200 kPa depending on the type of cell.

The cell sheet may be manufactured as a cell sheet having a higher density than a conventional cell sheet by introducing a porous polymer support. The porous polymer support may be a fine support obtained by emulsifying the porous polymer and then drying. The emulsification of the porous polymer may be performed by a method known in the art, but in a non-limiting embodiment of the present invention, the emulsion may be formed by sequentially dropping the porous polymer into oil.

In addition, the porous polymer support may be further cross-linked. When the polymer support is cross-linked, it exhibits a rough structure with multiple pores, which increases the surface area, so that during cell culture for cultured meat production, polymer loading and release behavior of cell growth factors, etc. can occur actively.

In addition, the growth factors inside the porous polymer support can be incorporated and immobilized through crosslinking. Specifically, a functional group of a polymer that forms a cross-linkage is immobilized on a support by electrostatic interaction or hydrogen bonding with a functional group such as a growth factor.

In this case, the porous polymer may be introduced by treating the substrate for cell culture with 0.1 to 20 wt%, 0.5 to 15 wt%, or 1 to 10 wt% of a porous polymer solution in order to maintain a uniform shape of the support and secure mechanical rigidity. there is. In addition, the porous polymer has a high surface area, and since nutrients required for the production of cultured meat can be loaded in the pores, as a nutrient carrier, cell proliferation and differentiation can be further improved.

The cell sheet introduced with the porous polymer support according to the present invention can effectively induce interactions with cells, such as cell adhesion or proliferation, making it possible to produce high-quality cultured meat from high-density cell sheets. That is, the cell sheet can enjoy the effects of high surface area and excellent porous structure due to the cross-linking by including the cross-linked porous polymer micro-support. It is known that the cell content is 10% or less when using a support for conventional cultured meat production. When cultured meat is prepared by using the micro-sized porous natural polymer platform according to the present invention, a high cell content can be exhibited. Specifically, it can show a synergistic effect of at least 25 to 40% compared to the conventional cell sheet.

In addition, the cell sheet may be coated with a polymer of an extracellular matrix component. A single-layered cell sheet is coated with an ECM-related polymer material, making it possible to easily transmit continuous physical and chemical stimuli between multi-layered cell sheets.

In addition, in the present invention, by stacking a plurality of the cell sheets, it is possible to create a three-dimensional cellular environment in a living body and induce the formation of a tissue similar to a living tissue. A plurality of cell sheets may be stably stacked according to an electrostatic interaction between the cell sheet and the cell sheet, hydrogen bonding, covalent bonding, ionic bonding, or a combination thereof. The number of cell sheets to be laminated is not limited, but may be preferably 2 to 100, 5 to 80, or 10 to 60.

Cells that can be used for the production of cultured meat are myoblast, myocyte, fibroblast, adipocyte, myosatellite cell, and mesenchymal stem cells isolated from livestock. cells: MSCs), induced pluripotent stem cells (iPSCs), satellite cells, adipose-derived stem cells (ASCs), or embryonic stem cells (ESCs) ) can be Hereinafter, in the present specification, cells usable for producing cultured meat are referred to as 'stem cells, etc.'.

The stem cells and the like may be derived from mammals other than humans, birds, reptiles, fish, crustaceans or molluscs. Stem cells, etc. are isolated from the tissue after collecting the tissue from a living animal. As the method of extracting stem cells, a known stem cell extraction method may be applied. Thereafter, the extracted stem cells are cultured, and the cultured cells are mixed with a culture medium containing growth factors and nutrients, and the culture medium contains a number of growth factors.

Specifically, for example, growth factors include epidermal growth factor (EGF), insulin like growth factor (IGF-1), platelet-derived growth factor (PDGF), Transforming growth factor-beta (TGF-β), vascular endothelial growth factor (VEGF), leukemia inhibitory factor (LIF), or basic fibroblast growth factor factor: bFGF) and the like.

When the cultured cells are mixed with a culture medium and subjected to proliferation and differentiation steps, they grow from myoblasts to muscle cells, and further, muscle tissue is formed. It is then common to process the muscle tissue into food using food technology. In order to realize the appearance and texture of the actual meat, a processing process of mixing a colorant and/or fat may be added.

The present invention provides a food composition comprising the cultured meat prepared according to the above-described manufacturing method.

In one embodiment of the present invention, the food is any one or more selected from the group consisting of snacks, dumplings, fried foods, fried foods, soy sauce, seasonings, powder mixes, breads, beverages, processed canned foods, dried seaweeds, and processed noodles. can The form added to food may be pulverized into various particle sizes depending on the purpose of use in food. It can be added to food by grinding uniformly or non-uniformly within the range of 1 μm to 10 cm.

In one embodiment of the present invention, the composition may further include a fat and/or a colorant. The fat may be included in a method of co-culture in the process of muscle cell proliferation by being injected into fat cells together during the production of cultured meat, or may be included in a method of adding fat in a liquid form. In this case, you can replace the saturated fatty acids in meat with beneficial fats, which is good for your health. Specifically, the fat includes vegetable oils such as soybean oil, corn oil, canola oil, rice bran oil, sesame oil, extracted sesame oil, perilla oil, extracted perilla oil, safflower oil, sunflower oil, cottonseed oil, peanut oil, olive oil, palm oil, palm oil, red pepper seed oil, etc.; Animal fats and oils such as edible tallow, edible lard, raw tallow, raw lard, and fish oil, and processed edible oils and fats such as mixed edible oil, flavored oil, processed oil, shortening, margarine, imitation cheese, and vegetable cream can be used.

Coloring agent refers to a compound that gives color to food. To reproduce the red meat color of beef or pork, artificial colorants, natural colorants, and natural extracts (eg, beet root extract, pomegranate fruit extract, cherry) extract, carrot extract, red cabbage extract, red seaweed extract), modified natural extract, natural juice (eg, beet root juice, pomegranate juice, cherry juice, carrot juice, red cabbage juice, red seaweed juice), Modified Natural Juice, FD&C (Food Drug & Cosmetics) Red No. 3 (erythrosine), FD&C Green No. 3 (fast green FCF), FD&C Red No. 40 (allura red AC), FD&C Yellow No. 5 (tartazine), FD&C Yellow No. 6 (sunset yellow FCF), FD&C Blue No. 1 (brilliant blue FCF), FD&C Blue No. 2 (indigotine) , titanium oxide, annatto, anthocyanin, betanin, beta-APE 8 carotene, beta-carotene, black currant, burnt sugar, canthaxanthin, caramel, carmine/carminic acid , cochineal extract, curcumin, lutein, carotenoids, monascin, paprika, riboflavin, saffron, turmeric, and combinations thereof may be used, but are not particularly limited thereto. Additionally, a coloring agent such as nitrite and ascorbic acid, erythobric acid, or a salt thereof that promotes the color development of the nitrite may be further added as a color development aid.

In addition, antioxidants, emulsifier salts, etc. for stabilizing the protein may be added to prevent rancidity of fat, color change, or separation of fat. The antioxidants, emulsifier salts, etc. can be used without limitation as long as they are widely used in the art.

Hereinafter, the method for producing cultured meat according to the present invention will be described in more detail through examples. However, the following examples are only a reference for describing the present invention in detail, and the present invention is not limited thereto, and may be implemented in various forms.

Example 1. Preparation of cell sheet introduced with gelatin micro support

1.875 g of fish gelatin was added to 30 ml of tertiary distilled water to prepare a gelatin solution. The gelatin solution was sequentially dropped into olive oil to form a gelatin emulsion. A gelatin emulsion was obtained through a filter process, washed with acetone, and dried in an oven at 50° C. for 12 hours to prepare a gelatin micro support. The gelatin micro support was placed in 1w t% microbial transglutaminase (mTG) crosslinking solution and crosslinked for 6 hours. The cross-linked micro-support was washed with 1XPBS and dried completely. C2C12 cells (ATCC, Manassas, VA, USA), which are mouse-derived myoblasts, were seeded on a culture dish with gelatin micro support introduced, and cultured at 37 ° C., 5% CO ₂ condition using DMEM medium containing 10% FBS. and passaged at 80% saturation. The passaged C2C12 cells were aliquoted on a culture dish having a gelatin micro support introduced therein, and 20 ml of DMEM containing 5% horse serum was added. The culture was continued until the cells became confluent on the culture dish in which the gelatin micro support was introduced. At this time, the cross-linked micro-support was added to the cell suspension during myoblast seeding process, and put into a culture dish together with the cells and cultured. After 3 days, the culture medium was replaced, and after 2 days of incubation, all of the culture medium in the saturated culture dish was removed and the cell sheet was washed with 1X PBS, and then all of the PBS was also removed. A cell sheet in the form of a monolayer film was peeled off from the culture dish using a scraper to obtain it.

As shown in the microscopic image of FIG. 1 , it can be seen that the gelatin micro support in the cell sheet is evenly spread without tearing.

Comparative Example 1. Preparation of cell sheet without gelatin micro support introduced

Except for the process of introducing the gelatin micro support, the same procedure as in Example 1 was performed to obtain a cell sheet in which the gelatin micro support was not introduced. In the case of Example 1, it took 5 days to obtain a cell sheet, whereas in Comparative Example 1, it took about 10 days to obtain a cell sheet.

Experimental Example 1. Analysis of cell content of cell sheets into which gelatin micro supports were introduced

1-1. DNA content analysis

DNA quantification was performed using the Quant-iTTM PicoGreen dsDNA assay kit (Invitrogen). The reagent solution was prepared by diluting the dsDNA reagent 200-fold with 1X TE buffer according to the manufacturer's instructions. Then, the prepared reagent solution was added to the provided standard and digested cell sheet solution, and incubated for 5 minutes at room temperature in a light-blocked condition. The fluorescence intensity (λ _ex = 480 nm, λ _em = 520 nm) of the sample was measured using photoluminescence (FP-8300, JASCO). DNA was quantified by plotting fluorescence emission intensity versus DNA concentration.

As a result, as shown in FIG. 2 , in Example 1 in which the gelatin micro support was present, the DNA content was higher, confirming that the cell content of the cell sheet was higher due to the gelatin micro support.

1-2. Analysis of glycosaminoglycan (GAG) content

GAG quantification was performed using a dimethylmethylene blue (DMMB, Sigma Aldrich) assay. Digested cell sheet solution was used for GAG analysis. 1.6 mg of DMMB was dissolved in 100 ml of aqueous solution containing 304 mg of glycine, 160 mg of sodium chloride and 9.5 ml of 0.1M acetic acid to prepare 100 ml of dye solution. 30 μl of the sample and 270 μl of the dye solution were placed in a 96-well plate, and absorbance was measured at 570 nm. GAG concentrations were calculated from standard curves prepared by serial dilutions (0, 1.25, 2.5, 5, 7.5 and 10 μg/ml) of chondroitin sulfate solution (Sigma Aldrich).

As a result, as shown in FIG. 3 , in the case of Example 1 in which the gelatin micro support was present, the GAG content was higher, confirming that the cell content of the cell sheet was higher due to the gelatin micro support.

1-3. Analysis of cell sheet characteristics according to the introduction of gelatin micro support

Looking at the results in Table 1 below, when the gelatin micro support is present, the cell sheet culture period and the used culture medium cost are less than those of the conventional cell sheet, but it can be confirmed that the cell content is higher through DNA and GAG quantitative analysis. .

[Table 1]

Experimental Example 2. Changes in physical properties of cultured meat by introduction of gelatin micro support

2-1. Analysis of storage modulus of cell sheet according to the presence or absence of gelatin micro support

Two types of samples were prepared. The first is a model in which a conventional cell sheet is stacked in 10 layers, and the second is a model in which each cell sheet including a gelatin micro support is stacked in 10 layers. In order to evaluate the texture characteristics of the cultured meat model, rheometer measurements were performed. The rheological properties were measured at room temperature using a rheometer (MCR 302, Anton Paar, Austria) using an accessory equipped with an 8 mm diameter parallel plate. The measurement method was measured by the strain-sweep method, and the measurement was carried out within the strain range from 0.01% to 10% under the condition of 10 rad/s.

As shown in FIG. 4 , as a result of shear storage modulus analysis according to Example 1 and Comparative Example 1, it was confirmed that the physical properties of the cell sheet of Example 1 in which the gelatin micro support was introduced were high.

2-2. Analysis of the physical properties of cultured meat according to the presence or absence of gelatin micro support

In order to evaluate the texture characteristics of the 10-layer cell sheet cultured meat model containing the gelatin micro support, the above-described rheometer measurement was performed, and the measurement method was the same as that described in Experimental Example 2-2. The analyzed samples were chicken breast, vegetable protein support and a cultured meat model before roasting and a roasted cultured meat model.

As shown in FIG. 5 , as a result of analyzing the shear storage modulus of commercially available vegetable substitute meat (TVP) and chicken breast, it can be confirmed that the properties of the baked GMS sheet were the highest.

Although the preferred embodiment of the present invention has been described above, the present invention is not limited thereto, and various modifications can be made within the scope of the claims, the description of the invention, and the accompanying drawings, and this is also It goes without saying that it falls within the scope of the invention.

Claims (10)

introducing a porous polymer support to a substrate for cell culture;
seeding cells usable for preparing cultured meat on the porous polymer support;
culturing the seeded cells to prepare a single-layered cell sheet;
immobilizing the cell sheet to a substrate; and
A method for producing cultured meat comprising; stacking a plurality of the cell sheets. The method of claim 1,
The porous polymer is selected from the group consisting of alginate, gelatin, agar, starch, collagen, fibrinogen, chitosan, cellulose, and combinations of two or more thereof, cultured meat production method. The method of claim 1,
The porous polymer support comprises the steps of emulsifying the porous polymer; and a drying step; a method for producing cultured meat, which is a fine support obtained through. The method of claim 1,
Wherein the porous polymer support is cross-linked, cultured meat production method. The method of claim 1,
The single-layered cell sheet comprises a cross-linked porous polymer micro-support, cultured meat production method. The method of claim 1,
The method for producing cultured meat, wherein the single-layered cell sheet is coated with a polymer of an extracellular matrix component. The method of claim 1,
The method for producing cultured meat, wherein the plurality of cell sheets are stacked according to an electrostatic interaction between the cell sheet and the cell sheet, hydrogen bonding, covalent bonding, ionic bonding, or a combination thereof. A cultured meat prepared according to the method of any one of claims 1 to 7. A food composition comprising cultured meat prepared according to the method of any one of claims 1 to 7. 10. The method of claim 9,
The food is at least one food composition selected from the group consisting of snacks, dumplings, fried foods, fried foods, soy sauce, seasonings, powder mixes, breads, processed canned foods, beverages, dried seaweeds, and processed noodles.

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