KR20220040425A - Porous cell culture support for producing cultured meat, method for producing cultured meat using the same, and special processed food containing cultured meat prepared accordingly Special processed food comprising cultured meat - Google Patents

Abstract

The present invention relates to a porous cell culture support for producing cultured meat, a method for producing cultured meat using the same, and a special processed food containing cultured meat, wherein the porous cell culture support for producing cultured meat has a structure including a first cross-linked network having a protein and a second cross-linked network having a polysaccharide. The present invention is to provide cultured meat close to actual meat by using a cell culture support having a large surface area and many dense pores through cross-linking, and to provide a convenient special processed food including the cultured meat.

Description

Porous cell culture support for producing cultured meat, method for producing cultured meat using the same, and special processed food containing cultured meat {Porous cell culture support for producing cultured meat, method for producing cultured meat using the same, and special processed food containing cultured meat prepared accordingly Special processed food comprising cultured meat}

The present invention relates to a porous cell culture support for producing cultured meat, a method for producing cultured meat using the same, and a special processed food comprising cultured meat prepared according to the method for producing cultured meat.

Outer space is a space in a state of zero gravity with almost no gravity, and astronauts easily experience physiological problems such as loss of appetite, bone density and muscle loss. Because the supply of nutrients essential for astronauts in outer space is directly related to the problem of survival. Research on the development of space food has been continued, mainly in the United States and Russia.

Space food is food made for astronauts to consume in outer space, such as spaceships, space stations, and space bases built on planets such as the moon and Mars. Since space food has to be light so that spacecraft can be launched, most of the space food developed so far is in a dried state with moisture removed. In addition, even if the various microorganisms present in food are beneficial to our body, they can change to a threatening existence in outer space, so space food must be stored in a completely sterile state. In order to satisfy these conditions, high-pressure sterilization, freeze-drying, and irradiation techniques have been introduced and used in space food.

In particular, processed meat products need to be consumed as a source of essential amino acids for the human body, so steak is being used as a space food in a processed form. However, because there is a risk of food safety problems due to the growth of food-borne pathogens, in order to be used in space food, a strict level of processing technology for controlling microorganisms is required, and special treatment to suppress the growth of microorganisms is required.

On the other hand, 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.

Cultured meat provides the advantage of being able to consume healthy animal protein by preventing the abuse of antibiotics or growth promoters that occur in the process of raising and slaughtering livestock. In addition, cultured meat can be used in food for various purposes in that it can be designed to contain beneficial nutrients in the manufacturing stage.

The present invention has been devised from research for various applications of cultured meat, and aims to secure microbiological stability for long-term safe storage of food in the extreme environment described above.

Another object of the present invention is to provide a cultured meat having a high cell content by providing a cell culture support having a large surface area and a large number of dense pores, which is advantageous for cell adhesion and culture.

The porous cell culture support for producing cultured meat according to the present invention exhibits a structure in which a first cross-linked network including a protein and a second cross-linked network including a polysaccharide are finely phase-separated from each other, so that proliferation and differentiation of cells can be effectively performed through dense pores An object of the present invention is to provide cultured meat exhibiting excellent chewiness and hardness, and special processed food containing the same.

Specially processed food containing cultured meat according to the present invention provides high-quality animal protein without fear of deterioration due to microbial propagation even after long-term storage.

In addition, the special processed food containing cultured meat according to the present invention is processed in various forms such as flakes or powder, and aims to provide rich flavor even after restoration.

In order to solve the above problems, the present invention includes a first cross-linked network comprising a protein and a second cross-linked network comprising a polysaccharide, wherein the first cross-linked network and the second cross-linked network are microphase-separated structures from each other. It provides a porous cell culture support for producing cultured meat comprising a porous polymer support.

In one aspect of the present invention, the polysaccharide may include a hydroxyl group, a carboxyl group, or an amine group in the structural unit.

In one aspect of the present invention, the protein and polysaccharide may be included in a weight ratio of 2:8 to 8:2.

In one aspect of the present invention, the protein may be collagen, gelatin, or fibrinogen.

In one aspect of the present invention, the polysaccharide may be hyaluronic acid, agar, chitosan, alginate, cellulose, or starch.

In addition, the present invention may provide a method for producing cultured meat comprising the step of culturing cells using the above-described cell culture support for producing cultured meat.

In one aspect of the present invention, the cell culture support for preparing cultured meat comprises: a) preparing a support solution by mixing an aqueous protein solution and a polysaccharide aqueous solution; b) crosslinking the protein in the support solution; c) crosslinking the polysaccharide in the support solution; And d) lyophilizing the support solution; may be prepared according to a manufacturing method comprising a.

In one aspect of the present invention, the aqueous protein solution and the polysaccharide aqueous solution may be included in an amount of 2 to 20 wt%.

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

In addition, the present invention can provide a special processed food containing the cultured meat.

In one aspect of the present invention, the special processed food may be space food, combat food, medical food or leisure food.

According to the present invention, a cultured meat having a high cell content can be prepared by providing a cell culture support in the form of a large surface area and dense pores, which is advantageous for cell adhesion and culture.

The porous cell culture support for producing cultured meat according to the present invention exhibits a structure in which a first cross-linked network including a protein and a second cross-linked network including a polysaccharide are finely phase-separated from each other, so that proliferation and differentiation of cells can be effectively performed through dense pores can be done In this case, the tissue can be formed to be close to the muscle tissue in the living body, thereby exhibiting excellent chewability and hardness.

In addition, the present invention can provide a high-quality animal protein that is microbiologically stable and does not deteriorate by being processed in a state that can be safely stored for a long time in an extreme environment.

In addition, the present invention can provide a special processed food that shortens the restoration time and maintains the umami and flavor provided by animal protein and meat even after restoration.

1 shows an SEM image of a gelatin/alginate support prepared according to Example 1 of the present invention.
Figure 2 shows an image of the gelatin / chitosan support prepared according to Example 2 of the present invention.
3 is a microscopic image showing the proliferation of C2C12 cells attached to the gelatin/chitosan support prepared according to Example 2 of the present invention.
4 is a cultured meat model prepared using a support according to an embodiment of the present invention, and shows pictures of before and after cooking in the form of breadcrumbs.

Hereinafter, a porous cell culture support for producing cultured meat according to the present invention, a method for producing cultured meat using the same, cultured meat prepared according to the method for producing cultured meat, and special processed food including 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.

Throughout this specification, the term "combination(s) of these" included in the expression of the Markush form means one or more mixtures or combinations selected from the group consisting of the components described in the expression of the Markush form, It means to include one or more selected from the group consisting of the above components.

Throughout this specification, reference to “A and/or B” means “A or B, or A and B”.

Hereinafter, the porous cell culture support for producing cultured meat according to the present invention will be described in detail. The porous cell culture support for producing cultured meat according to the present invention includes a first cross-linked network comprising a protein and a second cross-linked network comprising a polysaccharide, wherein the first cross-linked network and the second cross-linked network are microphase-separated structures from each other It may include a porous polymer support.

The porous polymer support has a large surface area through cross-linking, and is characterized in that it has a form with many dense pores.

As a specific example, the porous polymer support may include a vegetable protein. When the porous polymer support contains a plant protein, a coating treatment may be additionally applied to secure a large surface area to contain a large number of factors such as hormones, cytokines, growth factors, or inhibitors secreted from cells. The coating may be to form a natural polymer film on the plant protein support. In one embodiment of the present invention, the vegetable protein support may be plasma-treated, negative charges are introduced, and then a natural polymer coating layer may be deposited. The natural polymer is preferably a biocompatible porous polymer.

Or in one embodiment of the present invention, the protein may be collagen, gelatin, or fibrinogen. In this case, the protein may be cross-linked to form a first cross-linked network.

The porous polymer support may include a second cross-linked network in which polysaccharides are cross-linked. The polysaccharide may include a hydroxyl group, a carboxyl group, or an amine group in the structural unit, specifically, for example, hyaluronic acid, agar, chitosan, alginate, cellulose, or starch, but is not limited thereto.

The protein and polysaccharide may be included in a weight ratio of 2:8 to 8:2 or 3:5 to 5:3 by weight.

The present invention also provides a method for producing cultured meat comprising the step of culturing cells using the cell culture support for producing cultured meat.

The cell culture support for producing cultured meat comprises: a) preparing a support solution by mixing an aqueous protein solution and an aqueous polysaccharide solution; b) crosslinking the protein in the support solution; c) crosslinking the polysaccharide in the support solution; and d) freeze-drying the support solution.

Specifically, the protein and polysaccharide may be included in an amount of 1 to 50 wt%, or 2 to 20 wt%, based on the total weight of each aqueous solution.

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

In addition, the present invention can provide a special processed food containing the cultured meat.

Here, the special processed food may be space food, combat food, medical food, or food for leisure, but is not necessarily limited thereto.

Specifically, cultured meat is stem cells, for example, mesenchymal stem cells (MSCs), induced pluripotent stem cells (induced pluripotent stem cells: iPSCs), satellite cells (Satellite cells), adipose-derived adult stem cells ( Adipose-derived stem cells (ASCs) or embryonic stem cells are cultured to proliferate and differentiate. More specifically, the stem cells may be derived from mammals other than humans, birds, reptiles, fish, crustaceans or molluscs.

Stem cells 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, epidermal growth factor (EGF), insulin-like growth factor (IGF-1), platelet-derived growth factor (PDGF), transforming growth factor Beta (Transforming growth factor-beta: TGF-β), vascular endothelial growth factor (VEGF), leukemia inhibitory factor (LIF), or fibroblast growth factor (basic fibroblast growth factor: bFGF) ) and so on. The composition of the culture medium is not limited to the above-described composition because it can be adjusted as needed.

The culture form may be a conventional two-dimensional or three-dimensional culture known in the art. However, in order to create an environment similar to an actual living body due to the interaction between cells and cells, a three-dimensional culture may be preferable. Specific examples of three-dimensional culture include 3D porous scaffolds, scaffold-free platforms using cells themselves or cell sheet technology, a method of arranging cells in a microchip, a method using a hydrogel, a living organism There is a method using a reactor.

In one embodiment of the present invention, three-dimensional culture using a scaffold may be preferable. As a three-dimensional scaffold for cell proliferation, a scaffold provides a biological environment for cells to facilitate cell adhesion, proliferation and differentiation by mimicking the various roles of the extracellular matrix of a living body in a given environment. It may be desirable for the scaffold to be porous so that cells and cultures can be immobilized.

Specifically, the scaffold may be any one selected from the group consisting of alginate, gelatin, agar, starch, collagen, fibrinogen, chitosan, cellulose, and combinations of two or more thereof, but if it is an edible biocompatible polymer, it is not particularly limited can be used without

Alternatively, the scaffold may be 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 any one selected from the group consisting of (hydroxyapatite) and a combination of two or more thereof, and may be used without limitation as long as it can be used in conventional cell culture. The strength of the scaffold may be 0.1 to 200 kPa depending on the type of cell.

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. The formation of muscle tissue takes a long time, and a processing process of mixing a colorant and/or fat may be added to realize the appearance and texture of actual meat.

In a preferred embodiment of the present invention, the cultured meat may be an aggregate of muscle cells in which no muscle tissue is formed. Specifically, the muscle cell aggregate is a stage before muscle tissue is formed, and may be in the form of a cell cluster, cell aggregation, or organoid, and may be in the form of agglomerated muscle fibers, but a tissue form in which muscle fibers are connected to each other by connective tissue may not be the structure of

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.

In one embodiment of the present invention, the specially processed food may be space food, combat food, medical food or leisure food. However, it is not particularly limited thereto, and may include convenience food such as meal replacement food, emergency food in case of disaster or emergency, and portable food for outdoor use such as mountain climbing or fishing.

Specially processed food may require long-term storage in extreme environments, and may be in an environment without water, fire, or cooking utensils, so it should be restored and easy to eat. It is also necessary to have a high-nutrition diet.

These specially processed foods may be subjected to radiation treatment, gas treatment, or high-temperature and high-pressure treatment to ensure aseptic conditions. In addition, processes such as freeze-drying and vacuum packaging may be added for long-term storage.

The specially processed food containing cultured meat according to the present invention may be in the form of freeze-drying the cultured meat itself and vacuum-packing it in powder form, or freeze-drying the cultured meat-added food and vacuum-packing it.

In one embodiment of the present invention, the special processed food may further include a process of rapid freezing and/or irradiation with radiation. Through this process, it is possible to form a sterile state by completely killing microorganisms, and long-term storage is possible.

Hereinafter, the special processed food containing the 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 gelatin/alginate porous support

12wt% edible fish gelatin aqueous solution and 8wt% edible alginate aqueous solution were prepared, respectively, and the two solutions were mixed in a 1:1 volume ratio to prepare a support solution. Subsequently, genipin at a concentration of 0.03 mM was added to the support solution. The solvent used to prepare the genipin solution is 70% ethanol. After stirring the genipine-added support solution for 30 minutes, it was poured onto a flat plastic bottom and hardened for 12 hours. In the meantime, a 5 wt% CaCl ₂ aqueous solution was prepared as a secondary crosslinking solution. The hardened and molded support was immersed in the prepared CaCl ₂ aqueous solution for 1 hour, and then washed with tertiary distilled water. The gelatin/alginate support produced through two rounds of crosslinking was frozen in a deep freezer for 24 hours, followed by freeze-drying.

As a result, an SEM image of the prepared gelatin/alginate support is shown in FIG. 1 .

Example 2. Preparation of porous gelatin/chitosan support

15 wt% edible fish gelatin solution (solvent: tertiary distilled water) and 2 wt% edible chitosan solution (tertiary distilled water with acetic acid added 2 v/v%) were prepared and prepared, respectively. After mixing the prepared two solutions in a volume ratio of 1:1, the double network hydrogel was induced to form through stirring for 12 hours, and then gelled at room temperature for 1 hour. Thereafter, the gelatin/chitosan gel was immersed in 1 wt% microbial transglutaminase (mTG) aqueous solution to cross-link the gelatin network for 3 hours. After washing the gel with 1X PBS, it was immersed in 20 wt% phytic acid aqueous solution to perform cross-linking of the chitosan network for 3 hours. The cross-linked gel was washed with 1X PBS and lyophilized.

As a result, the SEM image of the prepared gelatin/chitosan support is shown in FIG. 2 .

Experimental Example 1. Change in porosity of porous support through crosslinking

In Example 1, gelatin and alginate were not crosslinked (bare) and the porosity change of the porous support according to the crosslinking time was observed, and the results are shown in Table 1 below.

[Table 1]

As a result of analysis of the porosity of non-crosslinked gelatin/alginate support (Bare) and gelatin/alginate support crosslinked for 6 hours, 16 hours, and 48 hours, it was confirmed that the total porosity increased from 62.84% to 77.4% as the crosslinking time increased. .

If high porosity is given, the surface area within the support is maximized, and it can contain high content of cytokines or growth factors secreted from cells, so that the unique taste of livestock meat can be imitated, and cell adhesion and activity are maximized. As a result, it is expected that it will be possible to produce high-quality cultured meat.

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 (11)

A porous cell for producing cultured meat comprising a first cross-linked network comprising a protein and a second cross-linked network comprising a polysaccharide, wherein the first cross-linked network and the second cross-linked network have a porous polymer support having a microphase-separated structure from each other; culture support. The method of claim 1,
The polysaccharide comprises a hydroxyl group, a carboxyl group or an amine group in the structural unit, a cell culture support for producing cultured meat. The method of claim 1,
The protein and polysaccharide are contained in a weight ratio of 2:8 to 8:2, a cell culture support for producing cultured meat. The method of claim 1,
The protein is collagen, gelatin, or fibrinogen, a cell culture support for producing cultured meat. The method of claim 1,
The polysaccharide is hyaluronic acid, agar, chitosan, alginate, cellulose, or starch, a cell culture support for producing cultured meat. A method for producing cultured meat comprising; culturing cells using the cell culture support for producing cultured meat according to claim 1 . 7. The method of claim 6,
The cell culture support for producing cultured meat comprises: a) preparing a support solution by mixing an aqueous protein solution and an aqueous polysaccharide solution; b) crosslinking the protein in the support solution; c) crosslinking the polysaccharide in the support solution; And d) lyophilizing the support solution; will be prepared according to a manufacturing method comprising a, cultured meat manufacturing method. 8. The method of claim 7,
The method for producing cultured meat, wherein the protein aqueous solution and the polysaccharide aqueous solution are contained in an amount of 2 to 20 wt%. A cultured meat prepared according to the method according to any one of claims 6 to 8. A specially processed food containing the cultured meat according to claim 9. 11. The method of claim 10,
The special processed food is a special processed food that is space food, combat food, medical food or leisure food.

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