KR102669272B1 - Scaffold for manufacturing cultured meat and manufacturing method thereof - Google Patents

Abstract

The present invention relates to a scaffold for producing cultured meat containing jellyfish collagen extract and a method for producing the same. The scaffold containing jellyfish collagen extract produced by the production method of the present invention has excellent cell adhesion and cell growth, and is suitable for use in meat. It can be usefully used as a scaffold for cultured meat production by being able to reproduce the texture of.

Description

Scaffold for manufacturing cultured meat and manufacturing method thereof}

The present invention relates to a scaffold for producing cultured meat containing jellyfish collagen extract and a method for producing the same.

Due to the recent increase in sea temperature, the population of jellyfish has rapidly increased, causing problems such as deteriorating the quality of fisheries resources and polluting the ocean. However, the jellyfish has a large amount of collagen, so if jellyfish are used as a source of collagen extraction, disturbance in the balance of the marine ecosystem can be resolved and collagen production costs can be reduced.

Collagen is a major component of the extracellular matrix (ECM) and is a fibrous polymer protein distributed in various tissues such as skin, bones, cartilage, blood vessel walls, teeth, and muscles. It is used in various fields such as food, medicine, cosmetics, and cell culture, and is one of the important substances that can promote interaction with cells and, in particular, provides a microenvironment for cell attachment and growth.

Collagen is generally supplied from cow, pig, and chicken tissue, but it is difficult to secure the supply of raw materials or product stability for such animal collagen due to the increase in the incidence of animal diseases. There is also a method of producing it by extracting it from human corpses or placental tissue, but there are problems such as low productivity, high processing costs, and unethicality.

Meanwhile, cultured meat refers to new meat obtained by collecting cells from living animals and proliferating them using cell engineering technology, without slaughtering the animals. To produce cultured meat, cells, culture media, incubators, and scaffolds are required. Among these, the scaffold is an important element in the development of cultured meat because it affects the shape, texture, and flavor of cultured meat.

As interest in sustainable, eco-friendly materials grows, research is continuing on the development of cultured meat scaffolds made using various seaweed and plant-derived materials rather than animal materials. Representative examples of cultured meat scaffolds include alginate, agar, and gellan gum.

Alginate is a natural polymer extracted from brown algae. Alginate hydrogel can be prepared quickly and easily by adding cations such as calcium ions to sodium alginate. In addition, it has excellent biocompatibility and is used in various fields such as tissue engineering and cell therapy. Agar is a polymer composed of agarose and agaropectin. Agar hydrates at high temperatures and forms an elastic gel with strong coagulation at room temperature. In addition, it is a low-calorie, high-fiber food material and is also used as a fat and protein substitute. Gellan gum is a water-soluble dietary fiber produced through carbohydrate fermentation using a microorganism called Pseudomonas elodea, isolated from aquatic plants of the genus Elodea ( Elodea sp.). It is a tasteless, odorless substance and is used as a thickening stabilizer. A gel can be formed in small amounts.

Alginate, agar, and gellan gum can all be consumed, and the process of dissolving and molding the materials is simple, making it easy to create three-dimensional structures. However, scaffolds derived from seaweed and plant materials have very low cell adhesion and have limitations in cell growth.

Against this background, the inventors of the present invention conducted intensive research to develop a scaffold for producing cultured meat capable of excellent cell adhesion and growth. As a result, the scaffold coated with jellyfish collagen extract prepared by the production method of the present invention contained hydroxyproline ( The present invention was completed by confirming that the hydroxyproline) content was high, cell adhesion and cell growth were excellent, and the texture of meat could be reproduced.

KR 10-0760989 B1

The purpose of the present invention is to provide a composition for producing a cultured meat scaffold containing jellyfish collagen extract, a natural polymer gelling agent, and a gelation promoter.

Another object of the present invention is to provide a cultured meat scaffold prepared from the composition.

Another object of the present invention is to provide a method for manufacturing the cultured meat scaffold.

Another object of the present invention is to provide cultured meat produced by culturing cells derived from non-human animals on the cultured meat scaffold prepared by the above production method.

The advantages and features of the present invention and methods for achieving them will become clear by referring to the embodiments described in detail below along with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below and may be implemented in various different forms. The present embodiments are merely provided to ensure that the disclosure of the present invention is complete and to provide a general understanding of the technical field to which the present invention pertains. It is provided to fully inform the skilled person of the scope of the present invention, and the present invention is only defined by the scope of the claims.

The terminology used herein is for describing embodiments and is not intended to limit the invention. As used herein, singular forms also include plural forms, unless specifically stated otherwise in the context. As used in the specification, “comprises” and/or “comprising” does not exclude the presence or addition of one or more other elements in addition to the mentioned elements. Like reference numerals refer to like elements throughout the specification, and “and/or” includes each and every combination of one or more of the referenced elements. Although “first”, “second”, etc. are used to describe various components, these components are of course not limited by these terms. These terms are merely used to distinguish one component from another. Therefore, it goes without saying that the first component mentioned below may also be a second component within the technical spirit of the present invention.

Unless otherwise defined, all terms (including technical and scientific terms) used in this specification may be used with meanings commonly understood by those skilled in the art to which the present invention pertains. Additionally, terms defined in commonly used dictionaries are not to be interpreted ideally or excessively unless clearly specifically defined.

The present invention provides a composition for producing a cultured meat scaffold comprising jellyfish collagen extract, a natural polymer gelling agent, and a gelling accelerator.

Collagen of the present invention is a major component of the extracellular matrix and is distributed in skin, bone, and cartilage proteins, and its structure is a fibrous polymer protein with a triple helix structure. Collagen is a functional material that is widely used industrially in food, medicine, cosmetics, and cell culture. Most collagen derived from land animals is extracted from the cartilage, bones, and leather of land animals, and existing collagen derived from land animals has the same risk of metastasis to the human body as mad cow disease. To solve this problem, the need to develop collagen using aquatic organisms is emerging.

The jellyfish of the present invention is effective in controlling fatigue, skin elasticity, and blood circulation, as well as treating arthritis, high blood pressure, bronchitis, asthma, and hay fever, and has high potential for use in high-protein diet foods, cosmetics, and medicines. However, the appearance of a large number of jellyfish due to global warming causes ecosystem disturbance, and the large number of jellyfish is difficult to deal with.

The jellyfish collagen extract of the present invention is in demand as an alternative collagen source because it does not have the problems of collagen derived from land animals. Collagen extracted from the jellyfish has an amino acid content different from existing collagen, and can be used to manufacture cultured meat scaffolds by providing physical properties suitable for substitute meat.

In the present invention, the term jellyfish collagen extract may be used interchangeably with collagen extracted from jellyfish.

The natural polymer gelling agent of the present invention may include, but is not limited to, any one selected from the group consisting of alginate, agar, and gellangum.

The alginate is a sticky mucilage component extracted from brown algae such as seaweed and kelp. It does not dissolve in water but swells and is a colloid, that is, a complex polysaccharide in which fine particles are aggregated or sprayed in a gas or liquid. It is used for various purposes such as a thickener, emulsion stabilizer, and gelling agent, and can be applied to various fields such as food, industry, and medicine. In its natural state, it exists in combination with sodium, calcium, potassium, etc., and when included in cosmetics, it has excellent adsorption properties, is effective in removing toxins, and has excellent moisturizing properties. When a hydrogel is produced by adding a multivalent metal such as Ca ²⁺ and crosslinking alginate, the tissue of an actual organism can be simulated.

The agar is also called agar, and is extracted from the seaweed Agar agar. It is a freeze-dried material made from agar agar. Its main component is galactose, and it is a transparent substance with physical properties similar to gelatin. It can be dissolved in water and used for food or as an industrial material. It is a transparent and soft substance that dissolves well in water. It is also used to prepare solid media for fixing and culturing microorganisms, plant tissues, and animal cells. It is a low-calorie, high-dietary fiber material that has high potential as a fat and protein substitute.

The gellan gum is a water-soluble dietary fiber produced by carbohydrate fermentation using a microorganism called Pseudomonas elodea, isolated from water plants of the genus Elodea ( Elodea sp.), and is a tasteless, odorless substance, and is used as a thickening stabilizer. And a gel can be formed in a small amount.

The gelation accelerator of the present invention may be a Group 1 or Group 2 metal salt. The gelation accelerator may be one or more metal salts selected from the group consisting of potassium chloride, calcium chloride, magnesium chloride, and calcium lactate, but is not limited thereto.

With respect to a total of 100 parts by weight of the composition of the present invention, the jellyfish collagen extract may include 0.01 to 2 parts by weight, the natural polymer gelling agent may include 1 to 5 parts by weight, and the gelation accelerator may include 0.5 to 3 parts by weight. Not limited.

If the jellyfish collagen extract is less than 0.01 parts by weight based on 100 parts by weight of the total composition, the strength of the hydrogel itself is very weak, the amount of cells that can be cultured is limited, and the jellyfish collagen extract coating on the hydrogel may not be achieved well. . Additionally, if the amount exceeds 2 parts by weight, the jellyfish collagen extract may interfere with crosslinking of the hydrogel raw material.

If the natural polymer gelling agent is used in an amount of less than 1 part by weight relative to the total 100 parts by weight of the composition, it is difficult to maintain the hydrogel form due to a lack of components necessary for crosslinking, and if it is more than 5 parts by weight, a very hard hydrogel is formed, and the hydrogel As the size of the pores decreases, the amount of water absorbed decreases, making cell culture difficult.

The jellyfish collagen extract of the present invention may contain 100 parts by weight of all amino acids, 10 to 25 parts by weight of glycine, 4 to 11 parts by weight of hydroxyproline, and 4 to 11 parts by weight of proline. , more specifically, 20 to 25 parts by weight of glycine, 7 to 11 parts by weight of hydroxyproline, and 7 to 11 parts by weight of proline, but is not limited thereto.

Hydroxyproline is hydroxyproline and is abundant in collagen. It accounts for approximately 4% of all amino acids found in animal or plant tissue and plays an important role in the stability of collagen. Hydroxyproline, an amino acid that makes up collagen, is uniquely contained in large amounts only in collagen, so it can be used as an indicator to quantify collagen.

In a specific example of the present invention, it was confirmed that hydroxyproline, a quantitative index component of collagen, was contained more than twice as much as the previously known jellyfish collagen extract. A high content of hydroxyproline residues can increase the thermal stability of collagen, and indicate that the purity of jellyfish collagen itself extracted through the extraction method of the present invention is high.

Cultured meat of the present invention is also called clean meat, cell-based meat, or cultivated meat, and means suitable or edible for human or non-human animals to eat.

Additionally, the present invention provides a cultured meat scaffold prepared from the composition.

The cultured meat scaffold of the present invention may include jellyfish collagen extract and a natural polymer gelling agent. The cultured meat scaffold is a scaffold for producing cultured meat that is similar to the structure of meat and has cell adhesion ability.

The terms of the present invention, scaffold for producing cultured meat, may be used interchangeably with cultured meat scaffold.

The cultured meat scaffold of the present invention can increase cell attachment and proliferation. In a specific embodiment of the present invention, the cultured meat scaffold coated with the jellyfish collagen extract of the present invention has better cell adhesion and cell adhesion of muscle stem cells than a scaffold not coated with the jellyfish collagen extract or a scaffold coated with the fish collagen extract. Growth was excellent, and many cells were observed to attach and grow in an evenly distributed state.

Additionally, the present invention provides a method for manufacturing the cultured meat scaffold.

The method for producing a cultured meat scaffold of the present invention includes the steps of i) preparing jellyfish collagen extract; ii) preparing a scaffold based on a natural polymer gelling agent; iii) solution neutralizing the jellyfish collagen extract; iv) immersing the natural polymer gelling agent-based scaffold prepared in step ii) into a solution of the jellyfish collagen extract neutralized in step iii); and v) freeze-drying.

The step i) of preparing the jellyfish collagen extract includes the steps of i-i) washing the jellyfish; i-ii) pulverizing and ultrasonically crushing the washed jellyfish; i-iii) centrifugation step; i-iv) dialysis; i-v) adding sodium chloride; and i-vi) dialysis.

In step i-i), salts and impurities can be removed by placing the jellyfish in distilled water and immersing it at 1 to 7°C, and more specifically, immersing it at 4°C, but is not limited thereto.

In step i-ii), the ultrasonic disruption may be performed for 20 to 40 minutes, and more specifically, may be performed for 30 minutes, but is not limited thereto.

In step i-iii), the centrifugation may be performed at 8,000 to 12,000 rpm and 1 to 7°C for 20 to 40 minutes, and more specifically, may be performed at 10,000 rpm and 4°C for 30 minutes, but is limited thereto. It doesn't work.

In step i-iv), the dialysis may be performed at 1 to 7° C. using a phosphate buffer solution of pH 7 to pH 7.5, and more specifically, using a phosphate buffer solution of pH 7.2, 4 It may be performed at, but is not limited to, ℃. The phosphate buffer solution may be prepared by mixing disodium hydrogen phosphate (Na ₂ HPO ₄ ) and potassium dihydrogen phosphate (KH ₂ PO ₄ ).

In steps i-v), the sodium chloride may be added at a concentration of 0.5 to 1.5 M, and more specifically, the sodium chloride may be added at a concentration of 0.9 M, but is not limited thereto.

In step i-vi), the dialysis may be performed at 1 to 7° C. using distilled water after dissolving in 0.1 to 1 M acetic acid.

The step ii) of preparing a scaffold based on a natural polymer gelling agent includes ii-i) dissolving the natural polymer gelling agent in distilled water at room temperature; ii-ii) freeze-drying; ii-iii) immersing in calcium chloride solution; ii-iv) washing with distilled water; and ii-v) freeze-drying.

The natural polymer gelling agent may include, but is not limited to, any one selected from the group consisting of alginate, agar, and gellangum.

The freeze-drying may be performed for 40 to 60 hours after freezing at -70 to -90°C for 20 to 30 hours, or more specifically, freezing at -80°C for 24 hours and then freeze-drying for 48 hours. It is not limited to this.

In step iii), the solution may be neutralized with a sodium hydroxide solution to a pH in the range of 7.0 to 7.2, but is not limited thereto.

In step iv), the immersion may be performed at 1 to 7°C for 10 to 20 hours, and more specifically, may be performed at 4°C for 16 hours, but is not limited thereto.

Additionally, the present invention provides cultured meat produced by culturing cells derived from non-human animals on a cultured meat scaffold prepared by the above production method.

The non-human animal-derived cells may be one or more selected from the group consisting of muscle stem cells, muscle cells, and progenitors thereof.

The muscle stem cells may be satellite cells or myoblasts, for example, but are not limited thereto.

The muscle cells may be myocytes or myotubes, for example, but are not limited thereto.

The progenitor cells refer to cells capable of generating cells differentiated into multiple lineages, such as myoblasts, fibroblasts, adipocytes, stromal cells, pericytes, smooth muscle cells, and endothelial cells. Progenitor cells differ from stem cells in that they do not have extensive self-replication capabilities.

The non-human animal-derived cells include fat cells (fat cells or adipocytes) and/or their progenitor cells, stromal cells (connective tissue) and/or their progenitor cells, or endothelial cells (blood vessels) and/or their progenitor cells. It may include, but is not limited to this.

Non-human animals of the present invention may be selected from the group consisting of cattle, sheep, pigs, poultry, crustaceans, and fish, but are not limited thereto.

The present invention provides a scaffold for producing cultured meat containing jellyfish collagen extract and a production method thereof, and the scaffold containing jellyfish collagen extract prepared by the production method of the present invention has a high hydroxyproline content, cell adhesion and cell adhesion. It has excellent growth and can reproduce the texture of meat, making it useful as a scaffold for producing cultured meat.

Figure 1 is a diagram showing the process of producing jellyfish collagen extract.
Figure 2 is a diagram showing the results of analyzing the amino acid composition of jellyfish collagen extract.
Figure 3 is a diagram showing the cell adhesion and growth effects of jellyfish collagen extract.
Figure 4 is a diagram showing a method of manufacturing a scaffold coated with jellyfish collagen extract.
Figure 5 shows an alginate, agar or gellan gum scaffold coated with jellyfish collagen extract.
Figure 6 is a diagram showing the chemical structure analysis of a scaffold coated with jellyfish collagen extract.
Figure 7 is a diagram showing the surface analysis of a scaffold coated with jellyfish collagen extract.
Figure 8 is a diagram showing cell attachment and growth by coating with fish collagen extract or jellyfish collagen extract on an alginate-based scaffold.
Figure 9 is a diagram showing cell attachment and growth by coating with fish collagen extract or jellyfish collagen extract on an agar-based support.
Figure 10 is a diagram showing cell attachment and growth by coating with fish collagen extract or jellyfish collagen extract on a gellan gum-based scaffold.
Figure 11 is a diagram showing the texture analysis of a scaffold coated with jellyfish collagen extract.

Hereinafter, the contents of the present invention will be described in more detail through the following examples and experimental examples. However, the scope of the present invention is not limited to the following examples and experimental examples, and includes modifications of the technical idea equivalent thereto.

Example 1. Preparation of jellyfish collagen extract

To extract collagen from jellyfish, the following manufacturing method was performed. First, the jellyfish is placed in distilled water and immersed at 4°C to remove salts and impurities in the jellyfish. Afterwards, it was placed in a sodium hydroxide (NaOH) solution and immersed at 4°C. Jellyfish immersed in sodium hydroxide solution were washed with flowing distilled water and then pulverized using a grinder with a 0.1 to 1 M acetic acid solution.

The structure of the jellyfish was extracted by crushing the pulverized solution using an ultrasonicator for 30 minutes, and only the supernatant was separated by centrifugation at 10,000 rpm and 4°C for 30 minutes. The material precipitated after centrifugation was mixed with acetic acid solution and subjected to ultrasonic disruption and centrifugation once again under the same conditions.

The separated supernatant was desalted through dialysis at 4°C using a phosphate buffer solution of pH 7.2. The phosphate buffer solution is prepared by mixing disodium hydrogen phosphate (Na ₂ HPO ₄ ) and potassium dihydrogen phosphate (KH ₂ PO ₄ ). After completing dialysis, the solution inside the dialysis membrane was centrifuged at 10,000 rpm and 4°C for 30 minutes to obtain precipitated material.

The precipitated material was dissolved in 0.1 to 1 M acetic acid, and then sodium chloride (NaCl) was added to 0.9 M and stirred at 4°C to separate only collagen. Afterwards, the precipitated material was obtained by centrifugation at 10,000 rpm, 4°C for 30 minutes, which was dissolved in 0.1 to 1 M acetic acid, and then dialyzed at 4°C using distilled water to obtain collagen extracted from jellyfish. (Figure 1).

Example 2. Analysis of amino acid composition of jellyfish collagen extract

Hydroxyproline, an amino acid that makes up collagen, is uniquely contained in large amounts only in collagen, so it can be used as an indicator to quantify collagen. Through the method of Example 1, the amino acid composition of collagen extract extracted from jellyfish was analyzed (FIG. 2).

In the case of the jellyfish collagen extract prepared in Example 1, it was confirmed that it had the amino acid composition and content of glycine (23.45%), hydroxyproline (8.16%), and proline (8.55%). did. Generally, collagen extracts from jellyfish contain a hydroxyproline content of 4.3 to 5.4%, but it was confirmed that the jellyfish collagen extract extracted using the preparation method of Example 1 was extracted at a high content of 8.16%.

Through amino acid composition analysis, it was confirmed that the jellyfish collagen extract contains a high amount of collagen, and the jellyfish collagen extract solution was used in the experiment by diluting jellyfish collagen, which contains 8.16% of the total amino acid hydroxyproline. .

Example 3. Analysis of cell adhesion effect of jellyfish collagen extract

In order to confirm whether collagen extracted from jellyfish through the method of Example 1 is effective in adhesion to muscle cells, 0.1 to 1 part by weight of jellyfish collagen extract solution was dissolved in sodium hydroxide solution at pH 7.0 to 7.2 based on 100 parts by weight of the total composition. It was neutralized to the range, placed in a petri dish, and stored at 37°C for 3 hours. Afterwards, the solution was removed and left at room temperature for more than 15 hours to be coated.

Muscle stem cells were seeded in the Petri dish and cultured for 7 days, and cell growth was confirmed using an optical microscope. The muscle stem cells are non-human derived muscle stem cells and were directly isolated and used from animal muscle tissues such as cows, chickens, or pigs through primary cell culture.

In the case of Petri dishes coated with jellyfish collagen extract, higher cell attachment and proliferation were confirmed than those of Petri dishes not coated with jellyfish collagen extract. On the 7th day of cell culture, the Petri dish not coated with jellyfish collagen extract was 13.31%, and the Petri dish coated with jellyfish collagen extract was 38.08%, a significant difference of more than two times (Figure 3).

This shows that the coating of jellyfish collagen extract has an excellent effect on cell attachment and growth.

Preparation Example 1. Preparation of scaffold coated with jellyfish collagen extract

To prepare an alginate scaffold, 2 parts by weight of alginate was dissolved in distilled water at room temperature based on a total of 100 parts by weight of the composition. This was placed in a frame, frozen at -80°C for 24 hours, and then freeze-dried for 48 hours. For cross-linking of alginate, after primary freeze-drying, the composition was immersed in a solution of 1 part by weight of calcium chloride (CaCl ₂ ) for a total of 100 parts by weight, and washed with distilled water for 2 hours to remove unreacted calcium chloride. Scaffolds were manufactured by freezing at 80°C for 24 hours and then freeze-drying for 48 hours.

In the case of the agar scaffold, 2 parts by weight of agar was dissolved in distilled water at 80°C for a total of 100 parts by weight of the composition. Through this, it was manufactured by freezing at -80°C for 24 hours and then freeze-drying for 48 hours.

In the case of the gellan gum scaffold, 2 parts by weight of gellan gum was dissolved in distilled water at 80°C for a total of 100 parts by weight of the composition. Afterwards, the manufacturing process was performed in the same manner as the alginate scaffold manufacturing process.

In order to coat the prepared alginate, agar, and gellan gum scaffolds with the jellyfish collagen extract, the jellyfish collagen extract extracted in Example 1 was neutralized with a sodium hydroxide (NaOH) solution to a pH range of 7.0 to 7.2. Thereafter, the prepared alginate, agar, and gellan gum scaffolds were placed in 0.1 to 1 part by weight of jellyfish collagen extract solution based on a total of 100 parts by weight of the composition, and immersed at 4° C. for 16 hours. Then, scaffolds coated with jellyfish collagen extract were prepared by freezing at -80°C for 24 hours and then freeze-drying for 48 hours (Figures 4 and 5).

Experimental Example 1. Chemical structure analysis of scaffold coated with jellyfish collagen extract

Chemical structural analysis was performed to confirm whether the scaffold prepared by the method of Preparation Example 1 was coated with jellyfish collagen extract. After freeze-drying the jellyfish collagen extract and the prepared scaffold, the observed peaks were compared using FT-IR (Fourier Transform Infrared Spectroscopy).

It was confirmed that a new peak, which was not visible in the scaffold not coated with jellyfish collagen extract, appeared at a location around 1630 cm ^-1 and 1550 cm ^-1 in the scaffold coated with jellyfish collagen extract. This was also observed in the FT-IR results of jellyfish collagen extract (Figure 6).

The observed peak is the peak position of the amide band that can be observed in collagen, and may appear due to the C=O functional group and C-N and N-H functional groups in the peptide, respectively.

This indicates that the jellyfish collagen extract is properly coated on the scaffold.

Experimental Example 2. Surface analysis of scaffolds coated with jellyfish collagen extract

In order to confirm the difference in surface structure of the scaffold depending on the presence or absence of jellyfish collagen extract coating, the scaffold prepared in Preparation Example 1 was coated with platinum at 20 mA and then observed through SEM (Scanning Electron Microscopy).

The surfaces of scaffolds made of alginate, agar, or gellan gum were confirmed to have almost no pores and were smooth.

On the other hand, in the case of the scaffold coated with jellyfish collagen extract, collagen was confirmed to be attached to the surface, unlike the scaffold not coated with jellyfish collagen extract (Figure 7).

This indicates that the jellyfish collagen extract is coated on the scaffold of alginate, agar, and gellan gum.

Experimental Example 3. Muscle stem cell culture on scaffold coated with jellyfish collagen extract

To determine whether the scaffold coated with jellyfish collagen extract was more suitable for the attachment and growth of muscle cells than the scaffold not coated with jellyfish collagen extract, the following experiment was performed.

First, muscle stem cells were seeded on the scaffold, and the muscle stem cells were cultured on the scaffold for 7 days. To confirm attachment and growth on the scaffold, LIVE&DEAD ASSAY (Thermofisher, L3223) was used. The LIVE&DEAD ASSAY reagent has the characteristic of staining living cells green and dead cells red. Using this, attachment and growth of muscle stem cells were confirmed through fluorescent staining photos at 1, 4, and 7 days after culture.

As a result of observing each cell image through LIVE&DEAD ASSAY, in the case of a scaffold made of alginate, the number of cells tends to decrease over time, but in the case of a scaffold coated with jellyfish collagen extract, the cells adhere evenly throughout. It was confirmed that it is growing. It was confirmed that the scaffold made of agar and the scaffold made of gellan gum did not adhere well to cells. In addition, it was confirmed that scaffolds coated with fish collagen extract did not show good cell attachment and growth. On the other hand, on the scaffold coated with jellyfish collagen extract, the cells were evenly dispersed and attached, and many cells were observed to extend and grow (Figures 8 to 10).

Through this, excellent cell adhesion and proliferation effects were observed by coating jellyfish collagen extract on scaffolds made of alginate, agar, or gellan gum, which do not adhere well to cells. Scaffolds derived from seaweed and plants have the problem of low adhesion to animal cells, but the scaffold coated with the jellyfish collagen extract of the present invention can improve this problem, suggesting that it is suitable as a scaffold for cultured meat.

Experimental Example 4. Texture analysis of scaffolds coated with jellyfish collagen extract

In order to analyze the texture of the scaffold coated with the jellyfish collagen extract of the present invention, the physical properties of the prepared scaffold were observed.

As a result, the hardness, gumminess, chewiness, springiness, cohesiveness, and resilience of the scaffold coated with jellyfish collagen extract were comparable to those of alginate, agar, or gellan gum scaffolds. It was confirmed that the results were similar to (FIG. 11).

Through this, it was observed that the jellyfish collagen extract of the present invention did not cause changes in the physical properties of the scaffold. Additives for increasing cell adhesion change the physical properties of the scaffold, making it difficult to reproduce the texture of meat. However, the scaffold coated with the jellyfish collagen extract of the present invention can improve the above problems and are excellent at reproducing the texture of meat. Indicates that it can be used.

Although the embodiments of the present invention have been described above, the present invention is not limited to the above embodiments and can be manufactured in various different forms, and those skilled in the art will understand the technical idea of the present invention. It will be understood that it can be implemented in other specific forms without changing the essential features. Therefore, the embodiments described above should be understood in all respects as illustrative and not restrictive.

Claims (15)

A composition for manufacturing a cultured meat scaffold comprising a jellyfish collagen extract, a natural polymer gelling agent, and a gelation accelerator, wherein the jellyfish collagen extract is prepared through the steps of i) washing the jellyfish; ii) crushing and ultrasonically crushing the washed jellyfish; iii) separating the supernatant by centrifugation; iv) dialyzing the separated supernatant; v) adding sodium chloride to the dialyzed solution; and vi) dialysis, wherein the jellyfish collagen extract contains 7 to 11 parts by weight of hydroxyproline based on 100 parts by weight of total amino acids of the collagen extract. Composition for manufacturing. According to paragraph 1,
A composition for producing a cultured meat scaffold, wherein the natural polymer gelling agent includes any one selected from the group consisting of alginate, agar, and gellangum. According to paragraph 1,
A composition for producing a cultured meat scaffold, wherein the gelation accelerator is one or more metal salts selected from the group consisting of potassium chloride, calcium chloride, magnesium chloride, and calcium lactate. A cultured meat scaffold manufactured from the composition of any one of claims 1 to 3. According to clause 4,
A cultured meat scaffold, characterized in that the scaffold increases non-human animal derived cell attachment and proliferation. Method for producing the cultured meat scaffold of claim 4 comprising the following steps:
i) preparing jellyfish collagen extract;
ii) preparing a scaffold based on a natural polymer gelling agent;
iii) solution neutralizing the jellyfish collagen extract;
iv) immersing the natural polymer gelling agent-based scaffold prepared in step ii) into a solution of the jellyfish collagen extract neutralized in step iii); and
v) Freeze drying. According to clause 6,
The step i) of preparing the jellyfish collagen extract includes the steps of ii) washing the jellyfish; i-ii) pulverizing and ultrasonically crushing the washed jellyfish; i-iii) centrifugation to separate the supernatant; i-iv) dialyzing the separated supernatant; iv) adding sodium chloride to the dialyzed solution; and i-vi) dialysis. In clause 7,
In step i-iv), the dialysis is performed at 1 to 7 ° C using a phosphate buffer solution of pH 7 to pH 7.5. In clause 7,
In step iv), the sodium chloride is added at a concentration of 0.5 to 1.5 M. In clause 7,
In step i-vi), the dialysis is carried out at 1 to 7° C. using distilled water after dissolving in 0.1 to 1 M acetic acid. According to clause 6,
In step ii), the natural polymer gelling agent is characterized in that it includes any one selected from the group consisting of alginate, agar, and gellangum. According to clause 6,
The step ii) of preparing a scaffold based on a natural polymer gelling agent includes ii-i) dissolving the natural polymer gelling agent in distilled water at room temperature; ii-ii) freeze-drying; ii-iii) immersing in a gelation accelerator solution; ii-iv) washing with distilled water; and ii-v) freeze-drying. Cultured meat produced by culturing cells derived from non-human animals on a cultured meat scaffold prepared by the production method of claim 6. According to clause 13,
Cultured meat, characterized in that the non-human animal-derived cells are one or more selected from the group consisting of muscle stem cells, muscle cells, and progenitors thereof. According to clause 13,
Cultured meat, characterized in that the non-human animal is selected from the group consisting of cattle, sheep, pigs, poultry, crustaceans and fish.