KR102570450B1 - Medium additive for culturing cell cultured meat comprising Lycii fructus extract, medium composition for culturing cell cultured meat comprising the same, and method for producing cell cultured meat using the same - Google Patents

Abstract

The present invention relates to a medium additive for cultured meat culture containing a Lycii fructus extract, a medium composition for cultured meat culture containing the same, and a method for producing cultured meat using the same, which is conventionally high in unit price and collected from live calves. It is possible to improve the production efficiency of cultured meat while reducing the amount of fetal bovine serum (FBS) used by replacing or using it in combination with fetal bovine serum (FBS), which has ethical problems to be done.

Description

Medium additive for culturing cultured meat comprising goji berry extract, medium composition for culturing cultured meat comprising the same, and method for manufacturing cultured meat using the same same, and method for producing cell cultured meat using the same}

The present invention relates to a medium additive for cultured meat culture containing goji berry extract, a medium composition for cultured meat culture containing the same, and a method for manufacturing cultured meat using the same, and more particularly, to a cultured meat medium containing a cell growth factor It relates to an additive that can be used to replace or reinforce fetal bovine serum (FBS), a medium composition containing the same, and a method for producing cultured meat using the same.

The eating habits of modern people are gradually changing to a form in which meat occupies an increasing proportion. Since ethical and environmental problems related to meat production can be exacerbated with the increase in meat consumption, cell cultured meat is attracting attention as an alternative to solving meat shortage and environmental pollution problems.

Cell cultured meat is called by various names such as cultured meat, in vitro meat, lab grown meat, cell-based meat, and cultured alternative protein. Cells are collected from live livestock and grown in an incubator in a laboratory to make meat. By obtaining edible meat with only cell proliferation without slaughtering, it is suitable for the current meat production system in terms of animal welfare, hygiene, and environment for meat production. greatly advantageous compared to

Today, the livestock industry consumes a huge portion of land and natural resources used by mankind and emits a huge amount of greenhouse gases. In addition, it has a great impact on antibiotic resistance and causes problems such as water shortage, climate change, and resource depletion, so experts predict that cell cultured meat will become popular within 10 years. Already, the Netherlands, Norway, and the United States are preparing for massive research support and commercialization of cell cultured meat.

In Korea, domestic universities and companies related to cell cultured meat are conducting research on cell proliferation and differentiation, and large companies' investments and startups are starting to emerge, planning to launch the first cultured meat in 2022 or 2023. Worldwide, since 2016, research and development of cultured meat has been actively carried out by various food tech companies, and cell-based cultured meat is being developed in the United States, Israel, the Netherlands, Canada, Japan, Singapore, Spain, China, the United Kingdom, and Turkey. are developing Professor Mark Post of the Netherlands held the world's first cultured meat tasting event in London in August 2013. Recently, Mark Post's research team established a company called Mosa meat to market commercially available cultured meat patties within the next five years. It is being developed for the release of cultured meat. Memphis Meats held the first cultured meatball tasting event in December 2015, Aleph Farms cultivated cow cells to make artificial beef for steaks in 2017, and Shiok Meats cultivated cells using shrimp and crab meat to produce cell-cultured seafood. release is in progress.

In cultured meat, muscle satellite cells, which are stem cells, are mainly used. In addition, cells such as embryonic stem cells, induced pluripotent stem cells, and adipose-derived adult stem cells have sufficient differentiation capacity, but there is no case of successfully differentiating muscle tissue. On the other hand, muscle satellite cells play a regenerative role when skeletal muscle is injured and are known to be most suitable for producing cultured meat because they differentiate only into muscle tissue. However, in order for muscle satellite cells to develop into muscle fibers, sufficient cell proliferation is achieved, and culture conditions must be changed in order for myoblasts to differentiate into myotube cells. The medium used in the proliferation and differentiation of these muscle satellite cells is one of the most important factors in culture because it determines nutrients, growth factors, hormones, pH control, and osmotic pressure during cell culture. As mentioned above, since nutrients required for each stage of cell injection, proliferation, differentiation, and maturation are different, the composition of the culture medium should also be different.

The most important substance in the cell medium is fetal bovine serum (FBS) containing cell growth factors. These serums are very expensive, difficult to standardize according to mutations, and may contain unwanted components in cells. Since FBS is collected from the fetus of an unborn cow, it cannot be free from problems arising from breeding and slaughter, and ethical problems may arise. This problem does not fit the basic purpose of cultured meat as a substitute for live animals, and the use of inedible FBS also has limitations in its use as food. In addition, in the production of cultured meat, FBS accounts for about 80% of the total production cost. If muscle satellite cells have been previously used for medical research purposes, a more efficient and economical process is needed to industrially use them for the purpose of producing cultured meat. It is required.

Currently, fetal bovine serum replacement products are on the market, but research on completely replacing FBS in the culture of muscle cells for cell culture meat is very insufficient. Therefore, it is required to develop a cell culture technology that can replace serum in order to release industrially suitable cell cultured meat.

Korean Patent Publication No. 10-2017-0120431

An object of the present invention is to reduce the amount of fetal bovine serum used by substituting or combining fetal bovine serum (FBS), which is necessary for the medium used for culturing cultured meat, but is expensive and has ethical problems collected from fetal bovine fetuses, It is to provide a medium additive for cultured meat culture that can also increase the production efficiency of cultured meat, a medium for cultured meat culture containing the same, and a method for manufacturing cultured meat using the same.

According to one aspect of the invention,

A medium additive for culturing cultured meat containing a Lycii fructus extract is provided.

The medium additive for culturing the cultured meat may be a medium additive for growth of muscle satellite cells (satellite cell).

The extraction solvent of the goji berry extract may be water, alcohol having 1 to 4 carbon atoms, or a mixture thereof.

The goji berry extract may be used instead of or in parallel with fetal bovine serum (FBS).

According to another aspect of the present invention,

There is provided a medium composition for cultured meat culture comprising a medium additive for cultured meat culture containing the goji berry extract.

The medium for culturing the cultured meat may be a medium for growth of muscle satellite cells (satellite cell).

The medium composition for cultured meat culture is based on 100 parts by weight of serum-free medium, 1 to 30 parts by weight of fetal bovine serum (FBS); 0.5 to 1.5 parts by weight of an antibiotic/antibacterial agent; may include.

The goji berry extract may be included in a concentration of 10 to 100 μg/ml in the medium for culturing the cultured meat.

The serum-free medium may be any one selected from Ham's medium, NCTC 109, NCTC 135, Waymouth's MB752/1, Waymouth's MB705/1, Brich & Pirt media, Higuchi media, and MCDB media.

The antibiotic/antibacterial agent is at least one antifungal agent selected from amphotericin-B clotrimazole, miconazole, ketoconazole, and nystatin; and

One or more antibiotics selected from penicillin, streptomycin and tetracycline; may include.

According to another aspect of the present invention,

Proliferating muscle satellite cells using a medium composition for culturing cultured meat containing the goji berry extract; a method for producing cultured meat is provided.

The goji berry extract may be used to reduce the amount of fetal bovine serum (FBS) by replacing the amount of fetal bovine serum (FBS) corresponding to the content thereof, or may be additionally used together with the fetal bovine serum (FBS).

When the goji berry extract is used to replace fetal bovine serum (FBS), it can be cultured for 6 to 10 days.

The medium additive for cultured meat culture of the present invention contains Lycii fructus extract used as an herbal medicine as an active ingredient, which is not only harmless to the human body, but also has problems in cost and ethical aspects in the production process. Fetal bovine serum (FBS) can be replaced or used in combination to reduce the amount of fetal bovine serum (FBS) and improve the production efficiency of cultured meat.

1 is an analysis result of the growth rate of muscle satellite cells according to the mixed concentration of goji berry extract and FBS in Experimental Example 1.
Figure 2 is the result of analysis of the growth rate of muscle satellite cells according to the culture period after the addition of goji berry extract of Experimental Example 2.

Since the present invention can apply various transformations and have various embodiments, specific embodiments will be illustrated in the drawings and described in detail in the detailed description. However, it should be understood that this is not intended to limit the present invention to specific embodiments, and includes all transformations, equivalents, and substitutes included in the spirit and scope of the present invention. In describing the present invention, if it is determined that a detailed description of related known technologies may obscure the gist of the present invention, the detailed description will be omitted.

Hereinafter, the medium additive for cultured meat culture of the present invention will be described.

The medium additive for culturing cultured meat of the present invention contains Lycii fructus extract as an active ingredient. Here, goji ja is all using the fruit part.

The culture medium additive for cultured meat is characterized in that it is a medium additive for growth of muscle satellite cells (satellite cell).

Goji ja (Lycii fructus) has thorns similar to barn trees (Ku: 枸) and stems similar to willow trees (Ki: 杞), so it is called 枸杞 (Gugi) by combining the two letters. It is almost odorless, astringent, slightly sweet and cold, weak blood, yellow face, white hair, and is effective for insomnia or short thirst. As for pharmacological action, non-specific immunity enhancement action, hematopoiesis action, cholesterol lowering action, anti-fatty liver action, blood pressure lowering, blood sugar lowering, growth promotion, anticancer action, etc. have been reported. The rind is red or dark red. The outer surface is wrinkled and the inside contains yellowish white seeds. Seeds are flat oval. Goji's nickname is Cheomchaeja (甛菜子), Seogugi (西枸杞), Gojija (苟杞子), Goji (枸杞), Gugye (枸), Gugeuk (枸棘), Gogi (苦杞), Cheonjeong (天精). , Phalanx (地骨), Jibo (地輔), Jiseon (地仙), Memorandum (却暑), Yangyu (羊乳), Cactus (仙人杖), and Seowang Mojang (西王母杖).

The extraction solvent of the goji berry extract may be water, alcohol having 1 to 4 carbon atoms, or a mixture thereof.

The extraction solvent is not particularly limited, but 80 to 95% ethanol aqueous solution or hot water extract is preferred.

In the present specification, the term 'extract' used while referring to the Lycii fructus extract includes not only the crude extract obtained by treating the extraction solvent, but also the processed product of the wolfberry extract. For example, goji berry extract may be prepared in a powder state by additional processes such as distillation under reduced pressure and freeze drying or spray drying.

Also, when extracting Goji (Lycii fructus) can be extracted as a dried fruit, or can be extracted by crushing the dried fruit.

The goji berry extract may be used instead of or in parallel with fetal bovine serum (FBS). When used in parallel, it may be used in addition to the same amount of fetal bovine serum (FBS) as conventionally used, or may be used as a partial substitute to reduce the amount of fetal bovine serum (FBS) used.

The present invention provides a medium composition for cultured meat culture comprising a medium additive for cultured meat culture containing the goji berry extract.

The medium for culturing the cultured meat is characterized in that it is a medium for growth of muscle satellite cells (satellite cell).

The medium composition for cultured meat culture is based on 100 parts by weight of serum-free medium, 1 to 30 parts by weight of fetal bovine serum (FBS); It is preferable to include; 0.5 to 1.5 parts by weight of an antibiotic/antibacterial agent.

The fetal bovine serum (FBS) may include more preferably 2 to 27 parts by weight, more preferably 10 to 25 parts by weight. The scope of the present invention is not limited thereto, and the concentration may be adjusted according to the concentration or culture conditions of goji berry extract.

The goji berry extract may be included in the cultured meat culture medium composition at a concentration of 10 to 100 μg/ml, more preferably 30 to 80 μg/ml, and even more preferably 40 to 60 μg/ml. .

The serum-free medium may be any one selected from Ham's medium, NCTC 109, NCTC 135, Waymouth's MB752/1, Waymouth's MB705/1, Brich & Pirt media, Higuchi media, and MCDB media.

The antibiotic/antibacterial agent is at least one antifungal agent selected from amphotericin-B clotrimazole, miconazole, ketoconazole, and nystatin; and at least one antibiotic selected from penicillin, streptomycin and tetracycline.

In addition, the present invention provides a method for producing cultured meat.

The method for producing cultured meat of the present invention is characterized by comprising the step of proliferating muscle satellite cells using the medium composition for culturing cultured meat containing the above-described goji berry extract.

If the wolfberry extract is a wolfberry extract, the wolfberry extract replaces the fetal bovine serum (FBS) corresponding to its content to reduce the amount of fetal bovine serum (FBS), or together with the fetal bovine serum (FBS) can be used additionally.

When the goji berry extract is used to replace fetal bovine serum (FBS), it is preferably cultured for 6 to 10 days, more preferably 7 to 9 days.

Hereinafter, preferred embodiments are presented to aid understanding of the present invention, but the following examples are merely illustrative of the present invention, and various changes and modifications are possible within the scope and spirit of the present invention. It is obvious to those skilled in the art, It goes without saying that these variations and modifications fall within the scope of the appended claims.

[Example]

Preparation Example 1: Preparation of Lycii fructus extract

Goji ja (Lycii fructus) used in this study was purchased from the Korea Research Institute of Bioscience and Biotechnology, Goji berry Extract Bank (Daejeon). 59 g of dried and powdered goji berry in the shade was added to 1 L of ethyl alcohol 95% (GR grade) and 30 cycles (40 KHz, 1,500 W, ultrasonication for 15 minutes -120 minutes, standing per cycle), followed by filtration (Qualitative Filter No. 100, HYUNDAI MICRO CO., LTD) and drying under reduced pressure to obtain 9.7 g of goji berry extract. The obtained Goji ethanol extract was dissolved in a dimethyl sulfoxide (DMSO) solvent at a concentration of 100 mg/ml to prepare a stock solution and used in the experiment.

Example 1: Culture of muscle satellite cells using goji berry extract additives

, 5% CO₂ 인큐베이터 환경에서 배양되었다. Muscle satellite cells isolated from Korean cattle were provided by Chungbuk National University. Cells are BioCoat?? (Corning, Kennebunk, ME, USA) in a T-75 flask coated with 37

, and cultured in a 5% CO ₂ incubator environment.

The culture medium used for Maintain was 20% heat-inactivated fetal bovine serum (Gibco, Gaithersburg, MD, USA) and 1% Hams F-10 Nutrient Mix (Gibco, Grand Island, NY, USA). A mixture of penicillin/streptomycin/amphotericin-B (Lonza, Walkersville, MD, USA) was used. Subculture was performed when the cells reached 80% confluency in the plate.

A 96-well plate was coated with collagen (Thermo Fisher Scientific, A1048301) dissolved in 20 mM acetic acid (biossesang, Gyeonggi, Korea). The coating method was performed based on the collagen type 1 manual.

Cells were seeded at 2,000 cells/well with 20% FBS culture medium. After 24 hours, the concentration of FBS was mixed with Hams F-10 Nutrient Mix (Gibco, Grand Island, NY, USA) at 2%, 5%, 10%, and 20%, respectively, and the goji berry extract of Preparation Example 1 was set to a final concentration of 50 μg/ml. The media was replaced every 2 days, and after the addition of goji berry extract, 4, 6, and 8 days later, it was measured using Cell Counting Kit-8 (Enzo Life Sciences Inc, Burlington, Ontario, Canada).

Comparative Example 1: Muscle satellite cell culture using a medium containing only FBS

Muscle satellite cells were cultured under the same conditions as in Example 1, except that the medium containing FBS alone was used without mixing with the goji berry extract.

[Experimental Example]

In the following experimental example, the abbreviation of the culture group "number d / number / capital letter" means "cultivation period (days) / FBS concentration (%) / C: control (no addition of plant extract), L: goji berry". For example, "6d2C" means cultured in 2% FBS medium conditions for 6 days, and "6d10L" means cultured in 10% FBS + goji berry extract supplemented medium conditions for 6 days, which are summarized in Table 1 below.

In the following experimental example, the growth rate of the corresponding culture groups was measured after 4 days, 6 days, and 8 days, respectively, using Cell Counting Kit-8 (Enzo Life Sciences Inc, Burlington, Ontario, Canada). Specifically, it was performed by observing the absorbance at 3 hours after CCK-8 treatment according to the manual. The measured value was normalized based on the OD value of the group cultured for 6 days (6d20C) in the medium of the goji berry extract-free group containing 20% FBS.

abbreviation culture conditions 4d10C 4-day culture 10% FBS control 4d10L Goji berry extract added to 4-day cultured 10% FBS 4d20C 4-day culture 20% FBS control 4d20L Goji berry extract added to 20% FBS cultured for 4 days 6d2C 6-day culture 2% FBS control 6d2L Goji berry extract added to 2% FBS for 6 days culture 6d5C 6-day culture 5% FBS control 6d5L Goji berry extract added to 5% FBS cultured for 6 days 6d10C 6-day culture 10% FBS control 6d10L Goji berry extract added to 6-day cultured 10% FBS 6d20C 6 days culture 20% FBS control 6d20L Goji berry extract added to 6-day cultured 20% FBS 8d10C 8-day culture 10% FBS control 8d10L Goji berry extract added to 10% FBS cultured for 8 days 8d20C 8-day culture 20% FBS control 8d20L Goji berry extract added to 20% FBS cultured for 8 days

Experimental Example 1: Growth rate analysis of muscle satellite cells according to the concentration of goji berry extract and FBS mixture

Experiments were conducted to confirm the proliferation rate of cells for each concentration of FBS used with the goji berry extract of Preparation Example 1. Conventional muscle satellite cell culture method was cultured by adding 20% FBS to Hams F-10 Nutrient Mix, but in order to replace FBS or reduce its amount, the goji berry extract of Preparation Example 1 was additionally added along with FBS Muscle satellite cells were cultured.

The growth rate of the cultured group according to Example 1 and Comparative Example 1, in which the FBS composition ratio of the cell culture medium was changed to 2%, 5%, 10%, and 20%, and 50 μg/ml of Goji berry extract of Preparation Example 1 was added, was compared. The results are shown in Figure 1.

According to this, 6d10C using only FBS without adding goji berry extract showed a growth rate of 0.5 compared to 6d20C, but 6d10L reduced the FBS content to 0.62 level and the growth rate was significantly improved in the group added with goji berry extract. (p=0.0228). In addition, when comparing 6d20C and 6d20L, it was confirmed that 6d20L containing the same 20% FBS but with the addition of goji berry extract significantly increased the proliferation rate to 1.33 (p<0.0001).

Experimental Example 2: Analysis of the growth rate of muscle satellite cells according to the culture period after adding goji berry extract

In order to confirm whether the goji berry extract of Preparation Example 1 can expect a shortening of the culture period or an increase in yield, the FBS composition ratio of the culture medium was 10% and 20%, respectively, and the culture period was divided into 4 days, 6 days, and 8 days, muscle satellite cells The proliferation rate was measured and the results are shown in FIG. 2 .

According to this, when 10% FBS was added, the proliferation rate tended to increase when the goji berry extract of Preparation Example 1 was added. 6d10C and 6d10L were 0.5 and 0.62 compared to 6d20C, confirming a significant increase according to the addition of goji berry (p=0.228). When cultured for 8 days by increasing the culture period, 8d10C was 0.87 compared to 6d20C and 8d10L was 0.98.

When 20% FBS was added, the growth rate by day was checked by dividing the group with the addition of the goji berry extract of Preparation Example 1 and the group without addition. Proliferation rate increased (p=0.0492). When cultured for 6 days, the growth rate of 6d20L, which is a group with the addition of goji berry extract, increased to 1.33 compared to 6d20C (p<0.0001), and when cultured for 8 days, 8d20C and 8d20L increased significantly to 1.19 and 1.63, respectively (p=0.0001). 0.0007).

In particular, when the goji berry extract of Preparation Example 1 was added to 20% FBS, the cell proliferation ability significantly increased on all culture days, and even when 10% FBS was added, the goji berry extract of Preparation Example 1 was added on all culture days. When added, the growth rate tended to increase.

That is, Goji berry extract can increase the proliferation of muscle satellite cells, and in particular, the proliferation rate of 8d10C was still lower than that of 6d20C, but the proliferation rate of 8d10L increased to a level that was not significantly different from 6d20C. Therefore, by mixing goji berry extract, FBS can be substituted, the cost of cultured meat can be reduced, and disadvantages such as ethical problems or difficulties in standardization can be expected due to the use of FBS. In addition, even if Goji is additionally added while maintaining the FBS composition ratio at the conventional level, the cell proliferation ability is greatly increased on all culture days, so not only the increase in the output of cultured meat but also the effect of reducing the overall production cost can be expected. In addition, since the addition of goji berry extract is an extract of goji berry, it is easy to obtain raw materials and mass-produce, and it is expected to be of great help in changing consumers' awareness of cultured meat.

Although the embodiments of the present invention have been described above, those skilled in the art can add, change, delete, or add components within the scope not departing from the spirit of the present invention described in the claims. The present invention can be variously modified and changed by the like, and this will also be said to be included within the scope of the present invention.

Claims (13)

A medium additive for culturing cultured meat containing a Lycii fructus extract. According to claim 1,
The medium additive for cultured meat culture is a medium additive for cultured meat culture, characterized in that the medium additive for growth of muscle satellite cells (satellite cell). According to claim 1,
Cultured meat culture medium additive, characterized in that the extraction solvent of the goji berry extract is water, alcohol having 1 to 4 carbon atoms, or a mixture thereof. According to claim 1,
The goji berry extract is a medium additive for cultured meat culture, characterized in that used instead of or in parallel with fetal bovine serum (FBS). A medium composition for cultured meat culture comprising a medium additive for cultured meat culture containing the goji berry extract of claim 1. According to claim 5,
The cultured meat culture medium composition is a medium composition for cultured meat culture, characterized in that for the growth of muscle satellite cells (satellite cell). According to claim 6,
The medium composition for cultured meat culture is based on 100 parts by weight of serum-free medium, 1 to 30 parts by weight of fetal bovine serum (FBS); 0.5 to 1.5 parts by weight of antibiotics and antifungal agents; Cultured meat culture medium composition comprising a. According to claim 7,
The goji berry extract is a medium composition for cultured meat culture, characterized in that contained in the cultured meat culture medium composition at a concentration of 10 to 100 μg / ml. According to claim 7,
The serum-free medium is cultured meat culture medium composition, characterized in that any one selected from Ham's medium, NCTC 109, NCTC 135, Waymouth's MB752 / 1, Waymouth's MB705 / 1, Brich & Pirt media, Higuchi media and MCDB media. According to claim 7,
The antibiotic and antifungal agent is at least one antibiotic and antifungal agent selected from amphotericin-B clotrimazole, miconazole, ketoconazole and nystatin; and
At least one antibiotic selected from penicillin, streptomycin and tetracycline; Cultured meat culture medium composition comprising a. Proliferating muscle satellite cells using any one cultured medium composition selected from claims 5 to 10; A method for producing cultured meat comprising: According to claim 11,
The goji berry extract replaces the fetal bovine serum (FBS) corresponding to its content to reduce the amount of fetal bovine serum (FBS), or cultured meat characterized in that additionally used together with the fetal bovine serum (FBS) Manufacturing method of. According to claim 12,
Method for producing cultured meat, characterized in that incubated for 6 to 10 days when the goji berry extract is used to replace fetal bovine serum (FBS).