KR20230080363A - Support for culturing functional cells based on food materials and method for manufacturing cultured meat using the same - Google Patents

Abstract

The present invention relates to a scaffold for manufacturing cultured meat containing ingredients containing flour and soybean flour as main components and a method for manufacturing cultured meat using the same, without the need to separate and remove the scaffold after cell culture based on the material, It is possible to provide cultured meat that contains all of the nutrients and flavors contained in the ingredients.

Description

Support for culturing functional cells based on food materials and method for manufacturing cultured meat using the same}

The present invention relates to the development of a support for culturing functional cells based on food materials and a method for manufacturing cultured meat using the same.

Globally, meat production is not being met for population growth, and countermeasures are required. Meat consumption, which is needed to supply essential amino acids, is expected to increase by about 49.7% in 2050 compared to 2018. Traditional meat production methods have problems in that production costs are high, limited resources must be used, and carbon emissions can cause environmental pollution. In order to solve this problem, the development of cultured meat obtained by harvesting cells from living animals and then proliferating the cells by cell engineering technology has been conducted.

Support technology for producing cultured meat is a technology that separates myoblasts or satellite cells of animals and grows them in a bioreactor, and is an essential technology for the differentiation and proliferation of muscle cells. When manufacturing cultured meat, a support capable of supporting cells is required in order to realize the desired size, texture, and taste.

A support for cell culture must be sufficiently supplied with nutrients and oxygen to help cell growth, have a structure that can discharge waste products well, and must play a role of stably supporting cells to help them grow.

Recently, research has been focused on the development of various scaffolds, such as scaffolds grafted with 3D printing technology. In the case of the currently developed method for manufacturing cell scaffolds for cultured meat, there are many methods using 3D printing technology, so the manufacturing process is complicated and it is difficult to produce edible materials. Even if used, there is a problem that purification is required later. In addition, in the case of the prior art, there is an economic limitation in that the manufacturing cost is high, and there is a problem that cost is incurred even in the process of removing the support after preparing the cultured meat.

Therefore, in order to reduce production costs, it is necessary to develop various types of scaffolds and cultured meat using them through research related to self-organization technology.

In order to solve the above technical limitations, the present invention is to provide a support for cell culture having a porous structure using easily obtainable food material.

The present invention creates an environment that facilitates the supply of nutrients and oxygen to cells through a support for cell culture that includes a porous structure formed from oxygen plasma and freeze-drying, and coats the surface of the support with a biocompatible polymer so that the cells adhere to the support. Its purpose is to create an environment in which it can adhere well.

In addition, an object of the present invention is to provide cultured meat that has excellent nutrition, taste, and texture, including a food material-based support, and can be produced by a simple method and at a low cost.

In order to solve the above technical problem, the present invention can provide a scaffold composition for preparing cultured meat containing wheat flour and soybean flour.

In one embodiment of the present invention, the flour and soybean flour may be obtained from tortilla, dumpling skin, or tofu.

In addition, it is possible to provide a scaffold for producing cultured meat containing ingredients containing wheat flour and soybean flour as main components using the above composition.

In one embodiment of the present invention, the scaffold may further include a cell adhesive film.

In one embodiment of the present invention, the cell-adhesive film may include a biocompatible polymer containing an RGD sequence.

In one embodiment of the present invention, the surface of the scaffold may include an ionic functional group.

In one embodiment of the present invention, the ionic functional group may be introduced by plasma treatment.

In one embodiment of the present invention, the biocompatible polymer may be at least one selected from gelatin, collagen, and fibronectin.

In addition, the present invention comprises the steps of (S10) preparing a scaffold for producing cultured meat; (S20) filling the scaffold with a culture medium and inoculating animal cells; (S30) cell proliferation by incubating the culture solution inoculated with the animal cells; And (S40) organizing the proliferated cells in the culture medium; it is possible to provide a cultured meat manufacturing method comprising a.

In one embodiment of the present invention, prior to the step (S10), pre-treatment by freeze-drying the scaffold for preparing food-based cultured meat; may be further included.

In one embodiment of the present invention, the animal cells may include animal myoblasts and animal adipocytes.

A food material-based support for functional cell culture and a method for manufacturing cultured meat using the same according to the present invention use tortilla, dumpling skin, and tofu, which are ingredients composed of vegetable ingredients, wheat flour and soybean as main ingredients, as a support for cell culture, It can be stably attached to provide an environment in which it can grow well.

In addition, the present invention can provide an environment in which cells can adhere and grow better by imparting porosity through surface modification of the scaffold using oxygen plasma and freeze-drying technology and coating it with a biocompatible polymer.

In addition, the present invention is economical because it can reduce the cost of producing the support by using easily available materials to prepare the support for cell culture in a simple way, and even after manufacturing the cultured meat using ingredients, the support is not removed and the support is combined with the cultured meat. Ingestion can provide benefits.

1 is a photograph showing a food material-based functional cell culture support before undergoing a sterilization process prepared according to Example 1 of the present invention.
2 shows FE-SEM images before (a) and after (b) cell culture of a tortilla-based cell culture support according to an embodiment of the present invention.
Figure 3 shows FE-SEM images before (a) and after (b) cell culture of the dumpling skin-based cell culture support according to an embodiment of the present invention.
Figure 4 shows FE-SEM images before (a) and after (b) cell culture of the tofu-based cell culture scaffold according to an embodiment of the present invention.
5 is a photograph showing a state in which cells are cultured on a food material-based support for culturing functional cells prepared according to Example 1 of the present invention.
6 is a schematic diagram showing a process of manufacturing cultured meat using a food material-based support for culturing functional cells according to the present invention.
7 is a graph showing cell viability of functional cell culture scaffolds treated with oxygen plasma and/or lyophilization according to Experimental Example 1 according to the present invention.
8 shows an optical microscope image after attaching cells to a scaffold for culturing functional cells prepared according to Example 4 of the present invention.
9 is a graph showing the results of CCK-8 analysis after attaching cells to a support for culturing functional cells coated with a coating according to Experimental Example 1 of the present invention.
10 is a graph showing changes in physical properties after cooking a tofu support body cultured with cells and a tofu support body without cell culture according to Experimental Example 2 of the present invention.
11 is a graph showing flavor changes before (a) and after (b) cooking a tofu support body cultured with cells and a tofu support body without cell culture according to Experimental Example 2 of the present invention.

Hereinafter, with reference to the accompanying drawings and embodiments, a food material-based support for culturing functional cells and a method for manufacturing cultured meat using the same according to the present invention can be easily carried out by those skilled in the art to which the present invention belongs. Explain in detail.

At this time, unless there is another definition in the technical terms and scientific terms used, they have meanings commonly understood by those of ordinary skill in the art to which this invention belongs, and the gist of the present invention in the following description and accompanying drawings Descriptions of well-known functions and configurations that may be unnecessarily obscure are omitted.

The present invention may be embodied in many different forms and is not limited to the embodiments or figures set forth herein. In order to clearly describe the present invention in the drawings, parts not related to the description are omitted, and similar reference numerals are assigned to similar parts throughout the specification. The drawings are provided as examples so that the spirit of the present invention can be sufficiently conveyed to those skilled in the art to which the present invention belongs, and the present invention is not limited to the drawings presented and may be embodied in other forms. However, the drawings may be exaggerated to clarify the spirit of the present invention.

The singular form used herein may be intended to include the plural form as well, unless the context dictates otherwise.

In addition, the term "includes" in this specification is an open description having the same meaning as the expression "includes", "includes", "has" or "characterized by", and is not further listed. It does not exclude any element, material or process.

As used herein, the term "scaffold" may be used in the same sense as "support for cell culture".

The present invention relates to a scaffold for culturing functional cells based on food materials, and uses tortilla, dumpling skin, and tofu, which are food ingredients composed of wheat flour and soybean, which are vegetable ingredients, as a scaffold for cell culture, so that cells can stably attach and grow well. environment can be created.

Specifically, the present invention can provide a scaffold composition for producing cultured meat containing wheat flour and soybean flour, which can be obtained from tortillas, dumpling skins, or tofu.

Tofu made of 98.4% soybean flour and dumpling skin made of 95% wheat flour are vegetable-based ingredients that are edible and provide abundant nutrients for cell growth, so they can be used as a support for cell culture.

Soybean meal, the main component of tofu, is composed of globulin protein, which is a protein involved in cell recognition, binding, and adhesion processes. Wheat flour, the main component of dumpling skin, is composed of starch, which has a lot of OH groups on the surface, so it has excellent cell adhesion and can contribute to the growth and maturation of cells in vitro.

The present invention can significantly reduce the cost of manufacturing cultured meat in that it can be used as a support for cell culture using ingredients readily available on the market, and provides cultured meat with excellent nutrition, taste, and texture in a simple way. can do.

In addition, the present invention can provide a scaffold for producing cultured meat containing ingredients mainly composed of wheat flour and soybean flour.

As a specific example, commercially available tofu, dumpling skin, or tortilla can be cut into a predetermined size and used as a scaffold after sterilization.

In one embodiment, the scaffold may further include a cell adhesive film. Specifically, the cell-adhesive film may be a coating layer including a biocompatible polymer containing RGD sequences formed on the surface of the scaffold. The RGD sequence can bind to the cell surface through the integrin protein, which is a signal transduction receptor of the cell membrane of cultured cells, and can act on cell-substrate and cell-cell interactions. Specifically, the polymer may be gelatin, collagen, or fibronectin.

The film may have an average thickness of 100 to 2500 nm, 200 to 2000 nm, or 300 to 1000 nm, but the thickness may be appropriately adjusted as needed, so it is not limited thereto.

In one embodiment, the surface of the scaffold may include ionic functional groups. As a specific example, the ionic functional group may be introduced by plasma treatment. For example, by treating oxygen gas at a flow rate of 10 to 100 sccm, power of 100 to 300 W, and frequency of 10 to 20 kHz for about 1 to 3 minutes, chemically active sites are generated on the surface of grains according to the oxygen plasma reaction, thereby producing grain A functional group such as a hydroxy group, a carboxylic acid group, a carbonyl group or an oxygen radical may be introduced to the grain surface. Specifically, cell adhesion can be further improved by introducing a hydroxyl group suitable for functional surfaces for cell adhesion through strong electrostatic attraction with cells.

In addition, the present invention (S10) preparing a scaffold for producing cultured meat based on the above-described ingredients; (S20) filling the scaffold with a culture medium and inoculating animal cells; (S30) cell proliferation by incubating the culture solution inoculated with the animal cells; And (S40) organizing the proliferated cells in the culture medium; it is possible to provide a cultured meat manufacturing method comprising a.

In one embodiment, prior to the step (S10), pre-processing by freeze-drying the scaffold for preparing cultured meat based on food material; may be further included. The scaffold pretreated through freeze-drying can significantly increase porosity and cell binding activity. By proceeding at a temperature of -10 ° C or lower, 20 ° C or lower, or -40 ° C or lower, the water inside the scaffold is supercooled to generate nuclei, and then ice grows, where ice crystals were replaced by pores through the sublimation process. It is possible to impart porosity to the scaffold. In this case, cell adhesion is improved, and nutrients can be easily delivered from the food material-based scaffold during cell culture.

Animal cells include animal myoblasts and animal adipocytes, but are not limited as long as they can be used for producing cultured meat. It may include pluripotent stem cells (PSCs) and/or cells differentiated therefrom, non-embryonic stem cells (ESCs), or satellite cells.

(S30) after organizing the proliferated cells in the culture medium, if necessary (S40) applying a coloring agent to the tissue; may be further included.

Colorant refers to a compound that imparts color to food, artificial colorants, natural colorants, natural extracts (eg, beet root extract, pomegranate fruit extract, cherry) to reproduce the red meat color of beef or pork. 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 carotinal, 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 color developing agent such as nitrite and ascorbic acid, erythorbic acid or salts thereof, which promote color development of the nitrite, may be further added as a color developing aid.

In addition, the present invention may further include adding fat to the muscle tissue, if necessary. When fat cells are not co-cultured, separately cultured fat cells may be injected into muscle tissue, or when the muscle tissue is prepared into patties, liquid fat may be added and mixed. This is good because it can replace the saturated fatty acids contained in meat with beneficial fats. Since the taste of meat comes from the fat between the muscles, 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, and peanuts are used to realize the actual taste of meat. Vegetable oils such as oil, olive oil, palm oil, palm oil, and red pepper seed oil, edible beef tallow, edible lard, raw beef tallow, raw pork fat, animal fats and oils such as fish oil, mixed cooking oil, flavored oil, processed oil, shortening, margarine, imitation cheese, Edible oil and fat processed products such as vegetable cream can be used.

At this time, antioxidants, emulsifier salts, etc. for stabilizing proteins may be added to prevent rancidity of fat, change in color or separation of fat. The antioxidant, emulsifier salt, etc. can be used without limitation as long as they are widely used in the art.

The present invention is characterized by providing an environment in which cells can adhere and grow better by imparting porosity through surface modification of the scaffold using oxygen plasma and freeze-drying technology and coating it with a biocompatible polymer. The present invention can reduce the cost of scaffold production by preparing a scaffold for cell culture in a simple way using easily available materials. In addition, since the material is used, it is possible to provide the advantage of being ingested together with the cultured meat without removing the scaffold even after manufacturing the cultured meat.

The present invention will be described in more detail through the following examples. However, the following examples are only references for explaining the present invention in detail, and the present invention is not limited thereto, and may be implemented in various forms.

Example 1. Preparation of food material-based support for functional cell culture

Commercially-purchased food ingredients tortilla, dumpling skin, and tofu are cut into 3-5 cm × 3-5 cm in length to prepare a scaffold for culturing functional cells, and sterilized by soaking in an ethanol solution for 30 minutes before culturing cells on the scaffold. proceeded.

Example 2. Preparation of support for surface-modified functional cell culture (1) - Oxygen Plasma

After opening the oxygen gas cylinder so that the oxygen gas can be delivered to the oxygen plasma, the support for culturing functional cells prepared according to Example 1 is placed in a vacuum chamber. After stabilizing the gas gauge at 23-25 cc/min, oxygen plasma (flow rate: 20 sccm, power: 250 W, frequency: 20 KHz) is operated. When the oxygen plasma is operated, high power radio waves are applied to the vacuum chamber, and oxygen molecules are ionized due to low pressure to form plasma. Oxygen plasma activates the surface of the support to form a porous structure.

Example 3. Preparation of scaffold for surface-modified functional cell culture (2) - Freeze-drying

The scaffold for culturing functional cells prepared in Example 1 and the scaffold for culturing functional cells prepared in Example 2 and treated with oxygen plasma were put into deionized water to hydrate them, and then placed in liquid nitrogen to rapidly freeze them. Then, a support is inserted into the conical tube and the tube inlet is closed. The tube containing the support was placed in a lyophilizer and operated overnight at -40 °C. Water inside the scaffold is supercooled to undergo a nucleation process, and then ice is grown at a temperature near the melting point.

Example 4. Preparation of scaffold for surface-modified functional cell culture (3) - Fibronectin coating

A scaffold for culturing an orchid head-based functional cell is supported in a fibronectin solution having a concentration of fibronectin diluted to about 10 μg/mL, and the surface of the scaffold is coated with fibronectin. After covering the entire scaffold area, excess solution is removed, and air-dried for at least 45 minutes at room temperature to prepare a functional cell culture scaffold surface-modified with a fibronectin coating layer.

Experimental Example 1. Analysis of cell viability coefficient of support for surface-modified functional cell culture

Bovine myoblasts were inoculated into the tofu-based functional cell culture support prepared in Examples 1 to 3, and after proliferation for 3 to 7 days, Bio-RAD x-Mark ^TM spectrophotometer (Bio-Rad Laboratories, USA) ) was used to measure absorbance (Absorbance, 450 nm). The results are shown in FIG. 7 . In the case of the scaffold whose surface was modified by treatment with oxygen plasma or lyophilization, cell viability was remarkably increased.

Bovine myoblasts were inoculated on a functional cell culture scaffold coated with fibronectin prepared in Example 4, and after proliferation for 3 to 7 days, the cell proliferation rate was evaluated through the CCK-8 assay, and the results are shown in FIGS. 8 and 8 shown in 9. Figure 8 shows an optical microscope image, compared to the case of culturing in a cell culture dish, in the case of the scaffold according to Example 1 of the present invention, the cell density was higher, and the fibronectin-coated scaffold according to Example 4 showed a higher cell density. Higher cell attachment and proliferation were observed in the case of In the case of FIG. 9, the scaffold coated with fibronectin showed higher cell viability than the uncoated scaffold.

Experimental Example 2. Analysis of physical property changes before and after cooking of cultured meat

(Rheological property analysis)

The bovine myoblasts were inoculated into the follicular scaffold prepared in Example 1, and after proliferation for 3 to 7 days, it was replaced with a differentiation medium and then subjected to a tissue organization process for 5 days. Thereafter, the rheological properties were measured after cooking the follicular scaffolds cultured with cells and the scaffolds without cultured cells. The results are shown in FIG. 10 . When the tofu is roasted at a high temperature, the increase in the storage modulus of the tofu with cells is larger than that of the tofu without cells. It solidifies at , and the storage modulus increases.

Compared to the case without cells, the storage modulus (Storage modulus, G') tended to be higher in the presence of cells before and after cooking. In addition, when comparing before and after cooking, it can be seen that the after cooking is measured significantly higher than before cooking, resulting in significantly enhanced physical properties.

(flavor analysis)

Flavor analysis was performed by baking the tofu scaffolds cultured with cells and the tofu scaffolds without cell culture at 180 ° C. for 10 minutes. In the graph before cooking (a), the fat flavor of tofu with cell culture increased by 9.1% compared to tofu without cell culture. In addition, bitterness such as almonds decreased by 58% and soybean flavor, the main component of tofu, decreased by 39.3%. This indicates that the fat flavor, such as meat, increased due to the increase in cell content, and the intrinsic flavor of tofu decreased. In the graph after cooking (b), compared to tofu without cell culture, after cooking, fried tofu flavor increased by 57.4%, garlic flavor by 153.6%, oil flavor by 17.6%, and fatty flavor by 20.3%. This indicates that the cell content of tofu increases and the protein component increases to give a meat-like flavor. Soybean flavor, the main component of tofu, decreased by 17.2%, confirming that the unique flavor of tofu decreased.

What has been described above is only an example for carrying out the food material-based functional cell culture support and the cultured meat manufacturing method using the same according to the present invention, but is not limited thereto, and the gist of the present invention as claimed in the following claims Anyone skilled in the art without departing from it will say that the technical idea of the present invention exists to the extent that various changes can be made.

Claims (11)

A scaffold composition for preparing cultured meat containing flour and soybean flour. According to claim 1,
The flour and soybean flour are obtained from tortilla, dumpling skin, or tofu, scaffold composition for preparing cultured meat. A scaffold for manufacturing cultured meat containing ingredients mainly composed of wheat flour and soybean flour. According to claim 3,
The scaffold further comprises a cell adhesive film, a scaffold for producing cultured meat. According to claim 3,
The cell-adhesive film comprises a biocompatible polymer containing an RGD sequence, a scaffold for producing cultured meat. According to claim 3,
The surface of the scaffold comprises an ionic functional group, a scaffold for producing cultured meat. According to claim 3,
The ionic functional group is introduced by plasma treatment, a scaffold for producing cultured meat. According to claim 5,
The biocompatible polymer is at least one selected from gelatin, collagen, and fibronectin, scaffold for producing cultured meat. (S10) preparing a scaffold for preparing cultured meat according to any one of claims 3 to 8;
(S20) filling the scaffold with a culture medium and inoculating animal cells;
(S30) cell proliferation by incubating the culture solution inoculated with the animal cells; and
(S40) Organizing the proliferated cells in the culture medium; Cultured meat manufacturing method comprising. According to claim 9,
Prior to the step (S10), freeze-drying and pre-treating the scaffold for preparing food material-based cultured meat; further comprising a cultured meat manufacturing method. According to claim 9,
Wherein the animal cells include animal myoblasts and animal adipocytes.

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