Cell proliferation-promoting agent

Abstract

Provided is a cell proliferation-promoting agent comprising a plant-based material, wherein the material is easy to procure and prepare and exhibits excellent safety.　The present invention provides a cell proliferation-promoting agent containing a germ or a germ extract as an active ingredient. Cells of which the proliferation has been promoted by using a cell proliferation-promoting agent according to the present invention can be used for the production of a cell-based food.

Description

Cell proliferation promoter

[0001] The present invention relates to a cell proliferation promoter and a culture medium using the same, as well as a method for preparing cells for the production of cellular foods and a method for producing cellular foods using the same.

[0002] In recent years, the demand for cell-based foods has been increasing. The culture media used to cultivate cells for these foods require low cost, easy mass procurement, stable growth performance, and safety. There is a need for strategies to promote cell proliferation while meeting these various requirements.

[0003] Biotechnol Prog. 2000 Sep Oct; 16(5):688-9 Journal of Biological Standardization Vol5, 4 1977J Agric Food Chem. 2004 Jun 30;52(13) Nutr Cancer 2010;62(8):1007-16. doi: 10.1080 / 01635581.2010.492090. J Agric Food Chem. 2015 Mar 4;63(8):2264-76. doi: 10.1021 / jf506361r. Atherosclerosis. 2006 Jun; 186(2):260-6. doi: 10.1016 / j. atherosclerosis. 2005.07.027.

[0004] International Publication No. 2022 / 171696, Japanese Patent Publication No. 2013-247927, Japanese Patent Publication No. 2010-273683, Japanese Patent Publication No. 2013-014627, Japanese Patent Publication No. 2024-127424, Japanese Patent Publication No. 2003-339395, Japanese Patent Publication No. 2002-003393, Japanese Patent Publication No. 2020-533986, Japanese Patent Publication No. Hei 08-070859

[0005] The present invention aims to provide a cell proliferation promoter that uses plant-derived materials that are easy to procure and prepare and have high safety.

[0006] As a result of diligent research, the inventors have discovered that the above problems can be solved by culturing cells using grain germ or grain germ extract, and have completed the following invention.

[0007] The present invention provides the following: (1) A cell proliferation promoter comprising grain germ or grain germ extract as an active ingredient, wherein the cells are edible cells. (2) The cell proliferation promoter according to (1), wherein the grain germ is the germ of a grain or rice seed. (3) The cell proliferation promoter according to (1) or (2), which is heat-resistant. (4) The cell proliferation promoter according to any one of (1) to (3), wherein the edible cells are cells selected from the group consisting of fibroblasts, muscle cells, and adipocytes. (5) The cell proliferation promoter according to any one of (1) to (4), wherein the grain germ or grain germ extract contains a non-proteinogenic or non-peptide substance with a molecular weight of 3 kDa to 30 kDa as a cell proliferation promoting component. (6) The cell proliferation promoter according to any one of (1) to (5), for use in serum-free culture media. (7) A culture medium comprising a cell proliferation promoter as described in any one of (1) to (6). (8) A method for preparing cells for the production of cellular food, comprising the step of culturing the cells in the culture medium described in (7). (9) A method for producing cellular food, comprising the step of culturing cells in the culture medium described in (7), and the step of producing cellular food using the cells cultured in the first step.

[0008] The present invention makes it possible to provide a cell proliferation promoter using plant-based materials that are easy to prepare and highly safe. Furthermore, cells whose proliferation has been promoted using the cell proliferation promoter of the present invention can be used in the production of cell-based foods.

[0009] Figure 1 shows the cell number or cell density before proliferation (at seeding: white bar on the far left), after proliferation in serum-free medium (-: gray bar in the center), and after proliferation in the positive control (10% FBS: black bar on the far right) using various animal cells in Example 1. Figure 1A shows the number of viable cells of chicken fibroblasts before and after adherent culture, Figure 1B shows the cell density (number of viable cells per unit of medium) of the same cells before and after suspension culture, Figure 1C shows the total number of cells of porcine fibroblasts, Figure 1D shows bovine myoblasts, and Figure 1E shows the total number of cells of bovine adipose-derived stem cells before and after adherent culture. Figure 2A shows the cell proliferation promoting activity of various plant-derived components using chicken fibroblasts in Example 2-1, as a percentage (%) relative to the positive control. Results are shown for serum-free medium (-: white bar on the far left), groups using plant-derived components at each concentration (bar in the center), and the positive control (FBS: black bar on the far right). Figure 2B shows the cell proliferation-promoting activity using porcine fibroblasts in Example 2, Section 2-2, as a percentage (%) relative to the positive control. Results are shown for serum-free medium (-: white bar on the far left), the group using chlorella powder (chlorella: bar with a diagonal line in the middle), and the positive control (FBS: black bar on the far right). Figure 2C shows the cell proliferation-promoting activity using porcine fibroblasts in Example 2, Section 2-3, as a percentage (%) relative to the positive control. Results are shown for serum-free medium (-: white bar on the far left), the group using wheat germ powder (wheat germ: bar with a diagonal line in the middle), and the positive control (FBS: black bar on the far right). Figures 3A, C, D, and E show the results for Example 3, and show the results for serum-free medium (-: white bar on the far left (A, D) or second from the left (C, E)), the group using wheat germ extract at each concentration (% (w / v)) (bar with a diagonal line in the middle), and the positive control (FBS: black bar on the far right). Figure 3E also shows the results before proliferation (at seeding: the gray bar on the far left). Figure 3A shows the cell proliferation-promoting activity of chicken fibroblasts in adherent culture as a percentage (%) of the positive control. Figure 3B is a line graph showing the cell density (number of viable cells per unit of medium) of chicken fibroblasts in suspension culture using 0.1% (w / v) wheat germ extract over time, compared to serum-free medium (-) and the positive control (10% FBS). Figure 3C shows the total number of cells in adherent culture of porcine fibroblasts.Figure 3D shows the cell proliferation-promoting activity in bovine myoblasts as a percentage (%) relative to the positive control. Figure 3E shows the total cell number in adherent culture of bovine adipose-derived stem cells. Figure 4 is a photograph showing the results after culturing chicken fibroblasts in Example 4 using serum-free medium (upper left), 0.025% (w / v) wheat germ extract (upper right), 0.05% (w / v) wheat germ extract (lower left), and the positive control of FBS (FBS: lower right). Figure 5 shows the proliferation-promoting activity when using wheat germ extracts after various heat treatments in Example 5 as a percentage (%) relative to the 4°C extract. Results are shown for serum-free medium (-: white bar on the far left), groups using various heat-treated extracts (shaded bar in the center), and the positive control of the 4°C extract (control: black bar on the far right). Figure 6 shows the growth-promoting activity of wheat germ extracts using various solvents in Example 6, with the left axis representing the percentage relative to the water extract. The results are shown for serum-free medium (-: white bar on the far left), groups using various solvent extracts (diagonal bar in the center), water extract control (control: black bar second from the right), and FBS control (FBS: black bar on the far right). The right vertical axis and circles indicate protein concentration. Figure 7 shows the growth-promoting activity of each molecular weight fraction of wheat germ extract in Example 7, as a percentage relative to the control of 70% ethanol extract of wheat germ. The results are shown for serum-free medium (-: white bar on the far left), groups using each fraction (diagonal bar in the center), and the control of 70% ethanol extract (control: black bar on the far right). Figure 8 shows the growth-promoting activity of wheat germ extracts extracted under various pH conditions in Example 8, as a percentage relative to the positive control of the water extract. The results are shown for serum-free medium (-: white bar on the far left), wheat germ extract groups at various pH levels (center bar with a diagonal line), wheat germ extract groups with NaCl (NaCl: second bar from the right with a horizontal line), and the positive control with water extract (control: black bar on the far right).

[0010] In recent years, the demand for cellular foods has been increasing. However, as mentioned above, the culture media used to cultivate the cells that serve as raw materials for these foods must meet requirements such as low cost, ease of mass procurement, stable growth performance, and safety. However, in general, in basic research, serum such as fetal bovine serum (FBS) is often used to promote cell proliferation. Although serum has a high cell culture promoting effect, it does not adequately meet these requirements as a culture medium for cells usable in cellular foods, and furthermore, there are issues in terms of livestock species selectivity, environmental impact, and animal ethics. Therefore, there is a need for measures to promote cell proliferation that can serve as an alternative to serum while meeting these various requirements.

[0011] For example, the following research is being conducted: 1) Use of chemically defined media 2) Use of culture products 3) Use of food components such as food processing residues and food powders

[0012] However, the above measures have the following problems: 1) The cost is high because it is for pharmaceutical and basic research purposes, and there are safety concerns because it may contain compounds that have no history of being consumed as food. 2) The use of cultured products requires large-scale culture facilities, which presents challenges in terms of time, effort, equipment, and cost. 3) The use of food components such as food processing residues and food powders requires complex operations, which is impractical.

[0013] Thus, there is a need for plant-derived components that are easy to procure and prepare, have high safety, and promote cell proliferation while solving the various problems mentioned above. As such plant-derived components, the use of grain bran (Patent Documents 1 and 2) and proteins or peptides derived from wheat or beans (Non-Patent Documents 1 to 3, Patent Document 3) has been reported. However, the plant-derived components described in these documents require proteins or peptides, which are inactivated by heat, and therefore have the disadvantage of not being heat-sterilizable. Furthermore, algal hydrolysates have also been reported to function as cell proliferation promoters, but they require complex operations and present challenges in terms of time, effort, and cost. In addition, enzymatic hydrolysates have the problem of lot-to-lot differences in each hydrolysis.

[0014] Patent Document 4 discloses that wheat germ extract has a heparin-binding epidermal growth factor-like growth factor (HB-EGF) production-promoting effect and promotes the proliferation of epidermal keratinocytes. Patent Document 5 discloses the use of wheat germ as an ATP production promoter and experimental results using serum-containing culture media. On the other hand, there are also reports (Non-Patent Documents 4-6) that consuming whole grains, including wheat, has an inhibitory effect on cancers, including colon cancer, suggesting an effect opposite to cell proliferation promotion, and the effect on cell proliferation has been a subject of debate.

[0015] One aspect of the present invention relates to a cell proliferation promoter containing grain germ or grain germ extract as an active ingredient. Cell proliferation promotion can be expressed by an index related to cell proliferation, such as an increase in cell number or concentration, when using the cell proliferation promoter of the present invention compared to when the cell proliferation promoter of the present invention is not used (control). In one embodiment, cell proliferation promotion is an increase in cell number or concentration when using the cell proliferation promoter of the present invention compared to when the cell proliferation promoter of the present invention is not used (control), with a statistically significant difference (e.g., a t-test) at a significance level of 5%. In another embodiment, cell proliferation promotion is an increase in cell number or concentration when using the cell proliferation promoter of the present invention compared to when the cell proliferation promoter of the present invention is not used (control), for example, by 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 100% or more, or more. Cell number or concentration can be measured using any known technique, such as flow cytometry, cell counters, absorbance, DNA quantification, density quantification, concentration quantification, or image analysis. However, cell proliferation may be measured using other indicators or measurement methods, but is not limited to those mentioned above.

[0016] As factors contributing to cell proliferation, various factors can be considered, such as increased DNA synthesis, elevated metabolic activity, cytoskeleton remodeling, increased proteins and cell membrane components, activation of signal transduction pathways, changes in cell membrane potential, changes in gene expression, and promoted ATP production. However, the cell proliferation of the present invention of the present application is not limited to any specific factor. In one embodiment of the present invention of the present application, the promotion of cell proliferation is not caused by the promotion of ATP production, and cell proliferation promotion caused by promotion of ATP production is excluded.

[0017] The starting cells whose proliferation is promoted using the cell proliferation promoter of the present invention are not limited, and refer to any cells including, for example, those from animals such as mammals, birds, fish, crustaceans, reptiles and amphibians, and microorganisms such as yeast, lactic acid bacteria and algae, and can be appropriately selected depending on the application. Examples include non-human mammals including mammals such as pigs, cattle, horses, sheep, deer and wild boars; birds such as chickens, turkeys, ducks, pheasants, pigeons and quails; fish such as tuna, sea bream, yellowtail, Pacific saury and sardines; crustaceans such as crabs and shrimps; and mollusks such as squids and octopuses. When cells of the aforementioned animals such as mammals, birds or fish are used for edible purposes, raw materials can be obtained without the need to breed, capture or slaughter these animals, which is preferable from the viewpoints of saving time, labor, food and cost, reducing equipment and the number of processes, securing resources, and ethical considerations.

[0018] Methods for culturing cells are not limited, and examples thereof include adhesion culture, suspension culture, tank culture, three-dimensional culture, and dynamic culture. Furthermore, use of the cell proliferation promoter of the present invention or a medium containing the cell proliferation promoter has the advantage that suspension culture of cells can be performed.

[0019] When animal cells are used, although not limited thereto, cells obtained from animals such as fibroblasts, muscle cells, adipocytes, hepatocytes, egg cells, nerve cells, immune cells, blood cells, epithelial cells, and endothelial cells may be used as starting materials or cells derived therefrom, and if necessary, cells obtained by subjecting these cells to treatment such as genetic recombination, stem cells, pluripotent stem cells, iPS cells, or the like may be used. In one embodiment, the starting cells are cells excluding keratinocytes, epithelial cells, and / or endothelial cells.

[0020] Cells obtained as a result of culturing using the cell proliferation promoter of the present invention may be cells used for any applications such as food, medical treatment, and cosmetics, and are not limited. However, for example, the cell proliferation promoter can be preferably used for edible purposes in order to satisfy requirements such as large-scale procurement, cost, stable proliferation performance, and safety. In addition, the obtained cells may be any cells, including but not limited to fibroblasts, muscle cells, adipocytes, hepatocytes, egg cells, nerve cells, immune cells, blood cells, epithelial cells, and endothelial cells. From the perspective of being preferably usable for edible purposes, in one embodiment, cells obtained as a result of culturing using the cell proliferation promoter of the present invention are cells selected from the group consisting of fibroblasts, muscle cells, and adipocytes. In another embodiment, cells obtained as a result of culturing using the cell proliferation promoter of the present invention are cells excluding keratinocytes, epithelial cells, and / or endothelial cells.

[0021] Examples of starting cells for obtaining muscle cells include mesenchymal stem cells, muscle satellite cells, myoblasts, and muscle cells obtained from animal skeletal muscle, cardiac muscle, or smooth muscle, or cells derived therefrom. Examples of muscle cells obtained as a result of culturing using the cell proliferation promoter of the present invention include mature or immature muscle cells, myotube cells, smooth muscle cells, and cardiomyocytes. Examples of starting cells for obtaining adipocytes include mesenchymal stem cells, preadipocytes, dedifferentiated adipocytes (DFA, adipose-derived stem cells), and adipocytes obtained from animal adipose tissue or cells derived therefrom. Examples of adipocytes obtained as a result of culturing using the cell proliferation promoter of the present invention include mature or immature adipocytes, mature white adipocytes, mature brown adipocytes, and mature beige adipocytes. Examples of starting cells for obtaining fibroblasts include mesenchymal stem cells, and fibroblasts obtained from the dermal layer of animal skin, or connective tissues such as the lung, heart, aortic adventitia, and uterus, or cells derived therefrom. Examples of fibroblasts obtained as a result of culturing using the cell proliferation promoter of the present invention include mature or immature fibroblasts and myofibroblasts.

[0022] The content of grain germ or grain germ extract in the cell proliferation promoter of the present invention is not limited as long as it exhibits a cell proliferation-promoting effect, but examples include 1 to 100% by weight, 5 to 90% by weight, 10 to 80% by weight, 20 to 30% by weight, 30 to 40% by weight, 40 to 50% by weight, 50 to 60% by weight, 70 to 80% by weight, 90 to 100% by weight, etc. The lower limit is not limited, but examples include about 1.0% by weight or more, about 5.0% by weight or more, about 10.0% by weight or more, about 15.0% by weight or more, about 20.0% by weight or more, about 25.0% by weight or more, about 30.0% by weight or more, etc. While there is no upper limit, examples include approximately 50.0% by weight or less, approximately 60.0% by weight or less, approximately 70.0% by weight or less, approximately 80.0% by weight or less, approximately 90.0% by weight or less, approximately 95.0% by weight or less, and 100.0% by weight.

[0023] The grains used in the cell proliferation promoter of the present invention include seeds of grasses (wheat, barley, oats, rye, rye wheat, oats, Job's tears, rice, millet, barnyard millet, foxtail millet, and sorghum, etc.), seeds of leguminous crops (soybeans, black beans, adzuki beans, chickpeas, lentils, broad beans, etc.), and pseudocereals (buckwheat, amaranth, quinoa, etc.). In one embodiment, the grains are seeds of grasses, wheat, and rice. As demonstrated in Example 4, which is detailed below, the germ of grains such as wheat can also be used to promote cell proliferation, so the germ of grains itself may be used in the cell proliferation promoter. However, from the viewpoint of usability and ease of handling, it is sometimes preferable to use an extract. As an example, the grain germ extract can be wheat germ extract or rice (brown rice) germ extract.

[0024] The structure of grain seeds can be divided into three parts: the endosperm, the germ, and the outer shell (including bran and bran). The germ is located at the tip of the embryo and is the starting point for seed germination, growing into the stem and leaves after germination. It contains nutrients such as B vitamins, vitamin E, minerals, proteins, and lipids. The method for recovering the germ is not limited and can be obtained by any known method. For example, grain seeds such as wheat can be crushed using a crushing means such as a roller machine, and the germ can be separated and recovered by sieving, taking advantage of the differences in specific gravity and particle size of the endosperm, germ, and bran after crushing. For example, a method such as that described in Patent Document 6 may be used. Alternatively, in the case of rice germ, germ rice can be obtained by using a friction milling method, which removes only the bran layer on the surface by rubbing the brown rice, or an impact milling method, which removes the bran layer by applying an appropriate impact to the brown rice, and then the germ portion obtained by removing the endosperm can be used. Alternatively, grain germ is readily available as a commercial product.

[0025] The germ used in the grain germ extract may be used in the form of a whole grain with the outer layer removed, along with the endosperm. However, in one embodiment, only the germ portion, with the endosperm removed, is used. This embodiment is preferable because it allows for effective utilization of the cell proliferation-promoting components contained in the grain germ, as described later. Any method can be used to remove the endosperm, such as sieving or enzymatic treatment as described above.

[0026] Grain germ can be used fresh or dried, but from the viewpoint of preservation and ease of extraction, it can also be used as dried material, dried powder, raw material powder, or juice. The form to be used can be appropriately selected depending on the grain germ raw material, and sterilization or other treatments may be applied as needed.

[0027] The method for extracting a cereal germ extract is arbitrary, and can be performed by, for example, solvent extraction. In the case of solvent extraction, optionally dried cereal germ, for example, a germ portion from which an endosperm portion has been removed, is further optionally shredded or pulverized, followed by extraction with an aqueous extraction solvent, water such as cold water, warm water, or hot water having a boiling point or a temperature lower than the boiling point, an organic solvent such as ethanol, methanol, ether, 1,3-butylene glycol, ethyl acetate, hexane, chloroform, a hydrous organic solvent which is a water-containing form of such an organic solvent, an alkaline solution such as NaOH, KOH, NH ３ ・H ２ O, Na ２ CO ３ , NaHCO ３ , Ca(OH) ２ , an acidic solution such as HCl, CH ３ COOH, C ６ H ８ O ７ , H ２ CO ３ , extraction is performed by appropriately selecting a preferable polar or nonpolar solvent such as those listed above according to the properties of the raw material, the use of the composition, or the like, and using the solvent at normal temperature or under heating.

[0028] Examples of the temperature of the extraction solvent include, but are not limited to, 1 to 130°C, 2 to 120°C, 3 to 110°C, 4 to 100°C, 0 to 10°C, 1 to 5°C, 10 to 20°C, 20 to 30°C, 30 to 40°C, 40 to 50°C, 50°C to 60°C, 60 to 70°C, 70°C to 80°C, 80°C to 90°C, 90°C to 100°C, 90°C to 110°C, 100°C to 110°C, 110°C to 120°C, and the like. Although the lower limit is not limited, examples include more than about 0°C, about 0°C or higher, about 4°C or higher, about 10°C or higher, about 20°C or higher, about 30°C or higher, about 40°C or higher, about 50°C or higher, and the like. Although the upper limit is not limited, examples include about 140°C or lower, about 130°C or lower, about 120°C or lower, about 110°C or lower, about 100°C or lower, and the like.

[0029] When using an aqueous organic solvent as an extraction solvent, examples of aqueous organic solvents include aqueous ethanol, aqueous methanol, aqueous ether, aqueous 1,3-butylene glycol, and other aqueous lower alcohols (e.g., C1 to C4). In this case, the water content may be, for example, 0 to 10 v / v%, 10 to 40 v / v%, 20 to 30 v / v%, 30 to 40 v / v%, 30 to 50 v / v%, 60 to 70 v / v%, 70 to 80 v / v%, 50 to 80 v / v%, 80 to 99.5 v / v%, etc.

[0030] The pH of the extraction solvent is not limited and can be adjusted as appropriate. Examples include 3-11, 3-10, 3-9, 3-7, 3-5, 7-13, 7-11, 7-9, 9-13, 9-11, etc. The lower limit is not limited, but examples include greater than approximately 1, greater than approximately 1, greater than approximately 2, greater than approximately 2, greater than approximately 3, greater than approximately 4, greater than approximately 5, etc. The upper limit is not limited, but examples include less than approximately 13, less than approximately 13, less than approximately 12, less than approximately 12, less than approximately 11, less than approximately 9, less than approximately 7, less than approximately 5, etc.

[0031] However, the extraction method is not limited to solvent extraction and may be carried out by commonly known methods in the art. The extraction method and form of the extract used in the present invention are arbitrary as long as they do not impair the effects of the present invention. The form of the extract may be the extract itself, or it may be diluted or concentrated as appropriate by commonly known methods. Furthermore, it may be a powder or lump solid obtained by drying the extract, or it may be a juice that has been diluted or concentrated as appropriate by commonly known methods. If necessary, the extract may be fermented or treated with enzymes such as protease or pectinase, or dextrin or gum arabic may be added to powder it.

[0032] The grain germ or grain germ extract used in the cell proliferation promoter of the present invention contains or consists of a cell proliferation-promoting component. In this specification, a cell proliferation-promoting component refers to a component that contributes to cell proliferation. In one embodiment, the cell proliferation-promoting component is contained in a fraction of grain germ or grain germ extract within a specific range of molecular weight, such as wheat germ. Examples of such specific ranges of molecular weight include 1 kDa to 100 kDa, 3 kDa to 100 kDa, 3 kDa to 50 kDa, etc., but preferably within the range of 3 kDa to 30 kDa, for example, within the range of 3 kDa to 10 kDa, 10 kDa to 20 kDa, 20 kDa to 30 kDa, and 10 kDa to 20 kDa. The lower limit is not limited, but examples include approximately 1 kDa or more, approximately 3 kDa or more, greater than approximately 3 kDa, approximately 5 kDa or more, approximately 10 kDa or more, greater than approximately 10 kDa, approximately 15 kDa or more, approximately 20 kDa or more, approximately 25 kDa or more, etc. The upper limit is not limited, but examples include less than approximately 30 kDa, approximately 30 kDa or less, approximately 40 kDa or less, approximately 50 kDa or less, approximately 60 kDa or less, approximately 70 kDa or less, approximately 80 kDa or less, approximately 90 kDa or less, approximately 100 kDa or less, etc.

[0033] In one embodiment, the cell proliferation-promoting component includes one or more non-proteinaceous or non-peptideous substances, or consists of one or more such non-proteinaceous or non-peptideous substances.

[0034] In the cell proliferation promoter of the present invention, non-proteinoid or non-peptide-like substances refer to substances other than polypeptides or proteins in which amino acids are linked in a chain, contained in grain germ or grain germ extract. In this specification, non-proteinoid or non-peptide-like substances are heat-resistant. In this specification, heat resistance means that the substance maintains its cell proliferation-promoting activity even when exposed to heating conditions such as, for example, about 1 to 2 months at about 40 to 50°C, about 24 to 72 hours at about 50 to 60°C, about 4 to 10 hours at about 60 to 70°C, about 1 to 4 hours at about 70 to 80°C, about 30 minutes to 1 hour at about 80 to 90°C, about 20 to 30 minutes at about 90 to 100°C, about 10 to 20 minutes at about 110 to 120°C, or about 5 to 10 minutes at about 130 to 135°C, or equivalent heating conditions. Furthermore, the temperature and time in the above heating conditions are not limited to the above range, as long as the heating is sufficient to inactivate the protein or peptide. Maintaining cell proliferation-promoting activity means that even when exposed to the above heating conditions, the cell proliferation-promoting activity is maintained at, for example, about 50% or more, about 55% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 80% or more, about 85% or more, and about 90% or more compared to when not exposed to the above heating conditions. Proteins and peptides lose or lose their cell proliferation-promoting activity because their structure breaks down and they denature under the above heating conditions, but the non-proteinaceous or non-peptideous substances of the present invention have the advantage that their cell proliferation-promoting activity is maintained even under such heating conditions. In one embodiment, one or more non-proteinaceous or non-peptideous substances are included in the molecular weight fractions within the specific ranges described above.

[0035] In one embodiment, the cell proliferation promoter is heat-resistant. While serum, proteinaceous or peptide substances, and other components commonly used as cell proliferation promoters often lose their cell proliferation-promoting activity upon heating, the heat resistance of the cell proliferation promoter of the present invention is advantageous because it allows the cell proliferation promoter or the culture medium containing it to be heat-sterilized before use in cell culture. In one embodiment, the cell proliferation promoter contains or consists of one or more non-proteinaceous or non-peptide substances with a molecular weight of 3 kDa to 30 kDa, for example, 3 kDa to 10 kDa, 10 kDa to 20 kDa, 20 kDa to 30 kDa, or 10 kDa to 20 kDa, such as wheat germ or grain germ extract, and is heat-resistant.

[0036] One aspect of the present invention relates to a method for producing a cell proliferation promoter. The method may include the steps of providing grain germ or grain germ extract as a material for the cell proliferation promoter, and / or extracting grain germ. The method for obtaining or preparing grain germ or grain germ extract in the step of providing grain germ or grain germ extract, and the method for extracting grain germ, are as described above.

[0037] One aspect of the present invention relates to a culture medium for cell culture containing a cell proliferation promoter. The culture medium may contain any components such as sugars, organic acids, vitamins, proteins such as amino acids and peptides, lipids, buffers, and nutrients such as minerals and inorganic salts, but it is preferable from the viewpoint of ease of procurement, safety, environmental impact, animal ethics, and cost to not contain serum or to contain a small amount of serum. An example of such a culture medium is a serum-free medium in which the cell proliferation promoter of the present invention is contained in the basic medium, and the liquid component is obtained by removing blood cells (red blood cells, white blood cells, platelets) and coagulation factors (fibrinogen, etc.) from blood, and does not contain serum containing growth factors, hormones, globulins, etc. In this specification, serum-free media refers to media that do not contain serum itself as described above, and may contain additional nutrients such as albumin (BSA, bovine serum albumin), whey, protein, lipids, transferrin, and selenium, as well as hormones such as insulin and growth factors such as bFGF (basic fibroblast growth factor), depending on the type and properties of the cells. Note that BSA is albumin isolated and purified from serum, but does not contain other serum components such as growth factors, hormones, and globulins, and is different from serum itself. It is also possible to use cyclodextrin or microorganisms as a substitute for BSA, as described in Patent Documents 8 and 9. In this specification, basic media refers to media that contain the minimum nutrients and inorganic salts necessary for cell proliferation and survival, such as sugars, organic acids, vitamins, amino acids, peptides, proteins, lipids, buffers, and minerals, and is not limited to basic media.

[0038] The content of the cell proliferation promoter in the culture medium of the present invention is not limited as long as it exhibits a cell proliferation-promoting effect, but for example, the amount of grain germ extract per culture medium can be set in ranges such as 0.001 to 1.0% (w / v), 0.005 to 1.8% (w / v), preferably 0.01 to 0.4% (w / v), more preferably 0.025 to 0.4% (w / v), for example, 0.05 to 0.2% (w / v), 0.05 to 0.4% (w / v), 0.025 to 0.02% (w / v), 0.05 to 0.1% (w / v), 0.1 to 0.4% (w / v), and 0.2 to 0.4% (w / v). The lower limit is not limited, but examples include approximately 0.001% (w / v) or more, approximately 0.005% (w / v) or more, approximately 0.01% (w / v) or more, approximately 0.025% (w / v) or more, approximately 0.05% (w / v) or more, approximately 0.1% (w / v) or more, approximately 0.2% (w / v) or more, and approximately 0.4% (w / v) or more. The upper limit is not limited, but examples include approximately 1.0% (w / v) or less, approximately 0.8% (w / v) or less, approximately 0.7% (w / v) or less, approximately 0.6% (w / v) or less, approximately 0.5% (w / v) or less, approximately 0.4% (w / v) or less, approximately 0.2% (w / v), and approximately 0.1% (w / v) or less. The content of the cell proliferation promoter in the culture medium of the present invention may be set according to the type of cells used. For example, in the case of chicken fibroblasts, the amount of grain germ extract per culture medium can be set to 0.01-0.4% (w / v), 0.02-0.025% (w / v), etc. For example, in the case of pig fibroblasts, the amount of grain germ extract per culture medium can be set to 0.01-0.4% (w / v), 0.1-0.4% (w / v), 0.2-0.4% (w / v), etc. For example, in the case of bovine myoblasts, the amount of grain germ extract per culture medium can be set to 0.0001-1.0% (w / v), 0.025-0.8% (w / v), 0.025-0.4% (w / v), etc. For example, in the case of bovine adipose-derived stem cells, the amount of grain germ extract per culture medium can be set to 0.01-0.5% (w / v), 0.1-0.4% (w / v), 0.2-0.4% (w / v), etc.

[0039] Furthermore, one aspect of the present invention relates to a cell proliferation promoter of the present invention or a method for culturing cells using a culture medium containing the cell proliferation promoter. Another aspect of the present invention relates to a method for preparing cells for the production of cellular foods, and a method for producing cellular foods. These methods may include the step of providing cells cultured in a culture medium containing the cell proliferation promoter of the present invention, and / or the step of culturing cells in a culture medium containing the cell proliferation promoter of the present invention. These methods may also include culturing cells by suspension culture. The cell proliferation promoter or a culture medium containing the same, starting cells, cells obtained as a result of culturing using the cell proliferation promoter, methods for culturing cells, etc., are as described above.

[0040] A method for producing cellular food may include the step of producing cellular food using cells obtained as a result of the culture in the above step. In the step of producing cellular food, the cultured cells are collected and processed according to the desired flavor, texture, taste, and use, such as sterilization, cooking, addition of additives, packaging, freezing, refrigeration, etc. Any method of sterilization can be used, such as heat sterilization, filtration sterilization, drying sterilization, ultrasonic sterilization, etc. Any method of cooking can be used, such as adding seasonings and other ingredients, kneading, mixing, heating, seasoning, cutting, boiling, boiling and discarding, simmering, steaming, baking, roasting, smoking, fermentation, etc. Examples of additives include seasonings, fragrances, flavorings, colorings, preservatives, thickeners, pH adjusters, etc. Any method of packaging can be used, such as retort processing, pouch packaging, canning, bottle packaging, etc.

[0041] Cellular foods refer to foods made using cultured cells. Examples of cellular foods, though not limited to them, include ham, salami, sausages, cultured meat products such as cultured foie gras, cultured seafood, cultured eggs, cultured dairy products, and cultured fat products. Cellular foods are desirable from the standpoint of reducing time, effort, cost, equipment, and number of processes, as well as from the perspective of resource conservation and animal ethics, because they can be produced using methods that do not rely on conventional livestock farming.

[0042] Furthermore, one aspect of the present invention relates to grain germ such as wheat germ or grain germ extract for promoting the proliferation of cells such as edible cells, or to the use thereof. Another aspect of the present invention relates to a culture medium such as a serum-free medium containing grain germ such as wheat germ or grain germ extract for promoting the proliferation of cells such as edible cells, or to the use thereof.

[0043] In this specification, "approximately" means that the stated numerical values, concentrations, quantities, times, temperatures, activities, and other values ​​include errors and variations within the range that a person skilled in the art would understand to be acceptable for achieving the effects of the present invention. Typically, this range is within ±10%, more preferably within ±5%, of the stated value, but if the appropriate range of variation recognized by a person skilled in the art differs depending on the measurement item and operating conditions, "approximately" shall be interpreted within the applicable range.

[0044] The present invention will now be described in more detail with reference to examples. However, the present invention is not limited thereto.

[0045] Example 1: Confirmation of proliferation ability of each animal-derived cell under serum-free conditions The proliferation ability of each animal-derived cell under serum-free conditions was confirmed by the following method.

[0046] 1-1: Culture of chicken-derived fibroblast cells 1-1-1: Adherent culture Chicken fibroblast cell line (DF-1) cells purchased from ATCC were used. Serum-free medium (-) was prepared by adding 0.1% Lipid mixture (SIGMA-ALDRICH), 0.2% BSA (SIGMA-ALDRICH), and 10 μg / ml insulin (WAKO) to DMEM (ThermoFisher, 10569010). 10% FBS (fetal bovine serum) added to DMEM medium was used as a positive control. Cells were cultured in 6.25 × 10⁶ cells. ４ cells / cm ２ Sow the seeds at 37°C and CO2. ２ CO2 with a concentration set to 5% ２Adherent culture was performed by culturing in an incubator. After 3 days of culture, the number of viable cells obtained by enzyme treatment (ThermoFisher, 12605010) was counted, and the number of cells before and after proliferation was calculated and graphed (Figure 1A). Cell counting was performed by staining the cell suspension with trypan blue and then measuring the number of viable cells and total cells using TC20™ (manufactured by BIO RAD).

[0047] 1-1-2: For the suspension culture chicken fibroblast cell line (DF-1), cells purchased from ATCC were used. After enzyme treatment of the cells, they were seeded in a suspension culture spinner flask containing 10% FBS DMEM medium and acclimatized at 60 rpm until they increased stably. Subsequently, the cells were divided into 2 × 10⁶ cells. ５ Suspension culture was performed by seeding at cells / ml and culturing under the same conditions. After 7 days of culture, the number of viable cells was counted, and the cell density before and after proliferation was calculated and graphed (Figure 1B). All other materials and conditions were the same as in 1-1-1.

[0048] 1-2: Culture of Porcine Fibroblasts Porcine fibroblasts were collected from the skin tissue of slaughtered pigs using the following procedure: 1. After washing the tissue with ethanol and PBS, the dermis layer was peeled off and isolated in a clean bench. 2. The isolated tissue was finely chopped with scissors and placed in a culture dish containing DMEM with 10% FBS added, and then incubated at 37°C CO2. ２ The cells were cultured in an incubator for several days. 3. The migrating cells were collected and used for the following cultures in each medium.

[0049] For serum-free medium (-), DMEM medium supplemented with 0.1% lipid mixture, 0.2% BSA, and 10 μg / ml insulin was used. A positive control was prepared by adding 10% FBS to DMEM medium. Cells were measured in 1.25 × 10⁶ cells. ５ cells / cm ２ Sow the seeds at 37°C and CO2. ２ CO2 set to a concentration of 5% ２ The cells were cultured in an incubator. After 3 days of culture, the number of viable cells was counted, and the number of cells before and after proliferation was calculated and graphed (Figure 1C).

[0050] 1-3: Culture of Bovine Myoblasts Bovine myoblasts were obtained from the muscle tissue of slaughtered cattle, and cells were collected using the following procedure: 1. The collected muscle tissue was washed with ethanol and phosphate buffer (PBS), and then finely chopped with scissors in a clean bench. 2. The muscle tissue was digested by shaking culture in DMEM medium supplemented with 0.2% collagenase II (Worthingon) at 37°C for 1.5 hours. The reaction was stopped by adding 20% ​​FBS to the reaction solution after digestion. 3. The digested solution was centrifuged at 80 x g for 3 minutes, and the suspended tissue was removed with tweezers, and the supernatant was collected. 4. The supernatant obtained by centrifuging again at 80 x g for 3 minutes was passed through a nylon mesh (100 μm) for cell separation. 5. The filtrate was centrifuged at 1500 x g for 5 minutes, and the resulting precipitate was suspended in DMEM containing 20% ​​FBS. 6. The cell suspension was passed through a 100 μm nylon mesh, then again through a 40 μm nylon mesh, and the filtrate was centrifuged at 1500 × g for 5 minutes. 7. The precipitate was allowed to stand on ice for 5 minutes using erythrocyte lysate to remove hematopoietic cells. 8. After washing twice with phosphate buffer, the cells were pooled in DMEM medium containing 20% ​​FBS and 4 ng / ml human basic fibroblast growth factor (bFGF) and seeded into culture dishes. 9. The grown cells were harvested and used for the following cultures in each medium.

[0051] For serum-free medium (-), we used DMEM supplemented with 0.1% Lipid Mixture, 0.2% BSA, 1× ITS-G supplement (WAKO), and 5 ng / ml bFGF (WAKO). A positive control was prepared by adding 10% FBS and 5 ng / ml bFGF to DMEM medium. Bovine myoblasts were 6.25 × 10⁶ ４ cells / cm ２ Sow the seeds at 37°C and CO2. ２ CO2 set to a concentration of 5% ２ The cells were cultured in an incubator. After 3 days of culture, the number of viable cells was counted, and the number of cells before and after proliferation was calculated and graphed (Figure 1D).

[0052] 1-4: Collection of Bovine Adipose-Derived Stem Cells [Dedifferentiated Adipose-Derived Stem Cells (DFAT) Cells] Bovine adipose-derived stem cells were collected from subcutaneous adipose tissue near the buttocks using the following procedure: 1. The collected adipose tissue was washed with ethanol and phosphate buffer (PBS), and then finely chopped using scissors in a clean bench. 2. 0.25% collagenase I (Gibco), 5 mM CaCl ２ 1. The adipose tissue was digested by culturing in 20 mM HEPES (pH 8.0) with shaking at 37°C for 1 hour. The reaction was stopped by adding an equal volume of 10% FBS / DMEM to the reaction solution after digestion. 3. The cell digestate was passed through a stainless steel mesh and the filtrate was collected. 4. The filtrate was centrifuged at 186 × g for 10 minutes and the fraction containing the upper layer of adipose tissue was collected. 5. The adipose fraction was washed with 10% FBS / DMEM and then seeded into a flask. 6. The flask was filled with 10% FBS / DMEM, inverted with the adhesion surface facing upwards, and cultured in an incubator for 7 days until the adherent adipocytes dedifferentiated. 7. After 7 days, the medium was changed and the cells were grown until confluence. 8. The grown cells were collected and used for the following cultures in each medium.

[0053] For serum-free medium (-), DMEM medium supplemented with 0.1% lipid mixture, 0.2% BSA, and 10 μg / ml insulin was used. A positive control was prepared by adding 10% FBS to DMEM medium. Cells were measured in 5.0 × 10⁶ cells. ３ cells / cm ２ Sow the seeds at 37°C and CO2. ２ CO2 set to a concentration of 5% ２ The cells were cultured in an incubator. After 3 days of culture, the number of viable cells was counted, and the number of cells before and after proliferation was calculated and graphed (Figure 1E).

[0054] Investigation: As shown in Figures 1A to 1E, in all cases of animal cells, cell proliferation was not observed in serum-free medium (-), regardless of whether it was adherent culture or suspension culture, even when additional nutrients such as BSA, ITS-G supplement, protein, lipids, hormones such as insulin, or growth factors such as bFGF were added. From these results, it is clear that there is a high need for a substance that has an effective cell proliferation-promoting effect as a substitute for serum in cell proliferation.

[0055] Example 2: Search for plant-derived components that promote cell proliferation in serum-free media. Plant-derived components that have a high cell proliferation-promoting effect even in serum-free media were searched for using the following method.

[0056] 2-1: Screening of plant-derived components using chicken fibroblasts. Chicken fibroblasts DF-1, serum-free medium (-), and positive control were the same as in 1-1-1. Commercially available plant-derived components included wheat germ, rice germ, rice bran, wheat flour, rice flour, corn flour, okara (soy pulp), soybeans, chlorella, spirulina, black tea, and coffee. Each plant-derived component powder was added to water at a concentration of 10% (w / v) and voltexed for 3 minutes to perform water extraction. Subsequently, the mixture was centrifuged at 1500 rpm for 3 minutes at 4°C, and the supernatant was filtered through a 0.8 μm cellulose acetate filter to obtain the filtrate. The filtrate was used as an extract of each plant-derived component.

[0057] Extracts of various plant-derived components were added to serum-free medium (-) at a concentration of 10% (w / v) of the original solution, resulting in final concentrations of 0.01%, 0.02%, 0.05%, 0.1%, 0.2%, and 0.5% (w / v), respectively. Chicken fibroblast cells DF-1 were used in 6.25 × 10⁶ samples. ４ cells / cm ２ The seeds were then seeded in 96-well plates, and their growth-promoting activity against positive controls was evaluated three days later using Crystal Violet staining.

[0058] Crystal Violet staining was performed by washing cultured cells with PBS and then staining them with a 0.5% Crystal Violet / 20% methanol solution for 15 minutes. After washing with water and air drying, the absorbance (560 nm) of the dye eluted with 100% methanol was measured using a plate reader (Promega). The absorbance was calculated as the ratio (%) of the absorbance of the group using plant-derived components to the absorbance of the positive control and graphed (Figure 2A).

[0059] 2-2: Evaluation of Chlorella Proliferation Using Pig Fibroblasts Pig fibroblasts were collected in the same manner as in 1-2, and serum-free medium (-) and positive controls were the same as in 1-2. As a plant-derived component, Chlorella was used, which showed comparable proliferation to serum in chicken fibroblasts in 2-1. Chlorella powder was added to water at a concentration of 10% (w / v) and voltexed for 3 minutes to perform water extraction. Then, the mixture was centrifuged at 1500 rpm for 3 minutes at 4°C, and the supernatant was filtered through a 0.8 μm cellulose acetate filter to obtain the filtrate. The filtrate was used as the Chlorella extract.

[0060] Chlorella extract was added to serum-free medium (-) to a final concentration of 0.2% (w / v) in which significant proliferation was observed in chicken fibroblasts in 2-1. 6.25 × 10⁶ porcine fibroblasts were added. ４ cells / cm ２ The seeds were then seeded in 96-well plates, and the growth-promoting activity compared to the positive control was evaluated using Crystal Violet staining after 3 days. The percentage of absorbance of the Chlorella extract group relative to the absorbance of the positive control was calculated and graphed (Figure 2B).

[0061] 2-3: Evaluation of wheat germ proliferation using porcine fibroblasts. Pig fibroblasts were collected in the same manner as in 1-2, and serum-free medium (-) and positive controls were the same as in 1-2. As a plant-derived component, wheat germ was used, which showed comparable proliferation to serum in chicken fibroblasts in 2-1. Wheat germ powder was added to water at a concentration of 10% (w / v) and voltexed for 3 minutes to perform water extraction. The mixture was centrifuged at 1500 rpm for 3 minutes at 4°C, and the supernatant was filtered through a 0.8 μm cellulose acetate filter to obtain the filtrate. The filtrate was used as the wheat germ extract.

[0062] Wheat germ extract was used to significantly increase chicken fibroblasts in 2-1, and was added to serum-free medium (-) to achieve the optimal final concentration of 0.2% (w / v) for porcine fibroblasts. 3.13 × 10⁶ porcine fibroblasts ４ cells / cm ２ The seeds were then sown in 96-well plates, and the growth-promoting activity compared to the positive control was evaluated three days later using Crystal Violet staining. The percentage of absorbance of the wheat germ extract group relative to the absorbance of the positive control was calculated and graphed (Figure 2C).

[0063] Investigation: As shown in Figure 2A, the screening results revealed that wheat germ extract and chlorella extract showed high cell proliferation-promoting activity comparable to FBS in chicken fibroblasts. Brown rice germ extract, soybean extract, okara extract, and rice bran extract also showed proliferation-promoting activity.

[0064] When porcine fibroblasts were cultured using wheat germ extract and chlorella extract, which showed high proliferation-promoting activity in Figure 2A, the chlorella extract did not show very high proliferation-promoting activity as shown in Figure 2B. However, as shown in Figure 2C, wheat germ extract showed proliferation-promoting activity even in porcine fibroblasts, suggesting the high versatility of wheat germ extract.

[0065] Example 3: Investigation of the proliferation-promoting effect and effective concentration of wheat germ in the culture of various animal cells The proliferation-promoting effect and effective concentration of wheat germ were investigated by performing adherent and suspension cultures of chicken fibroblasts, as well as adherent cultures of porcine fibroblasts, bovine myoblasts, and bovine adipose-derived stem cells, using the following method.

[0066] 3-1: Exploration of the growth-promoting effect and effective concentration of wheat germ extract in adherent culture of chicken fibroblasts. Chicken fibroblasts DF-1, serum-free medium (-), and positive control were the same as in 1-1-1. Wheat germ extract was prepared as in 2-1 and added to serum-free medium (-) to final concentrations of 0.01, 0.025, 0.05, 0.1, 0.2, and 0.4% (w / v), respectively. Chicken fibroblasts DF-1 were cultured in 6.25 × 10⁶ units. ４ cells / cm ２ The seeds were then seeded in 96-well plates and cultured using adherent culture. After 3 days, the growth-promoting activity compared to the positive control was evaluated using Crystal Violet staining. Growth-promoting activity was calculated as the ratio (%) of the absorbance of the wheat germ extract group to the absorbance of the positive control and graphed (Figure 3A).

[0067] 3-2: Confirmation of the growth-promoting effect of wheat germ extract in suspension culture of chicken fibroblasts. Chicken fibroblasts DF-1, serum-free medium (-), and positive control were the same as in 1-1-1. Wheat germ extract was prepared in the same manner as in 2-1 and added to serum-free medium (-) to a final concentration of 0.1% (w / v), which is the optimal concentration shown in Figure 3A. Suspension chicken fibroblasts DF-1 were cultured in 2 × 10⁻⁶ units. ５ Cells / ml were seeded into 5 ml spinner flasks, and suspension culture (tank culture) was performed using the same method as in 1-1-2. The number of viable cells was counted after 3, 7, and 14 days of culture, and the number of viable cells before and after proliferation was calculated and graphed (Figure 3B).

[0068] 3-3: Exploration of the growth-promoting effect and effective concentration of wheat germ extract in adherent culture of porcine fibroblasts. Porcine fibroblasts were collected in the same manner as in 1-2, and serum-free medium (-) and positive controls were used in the same manner as in 1-1-1. Wheat germ food extract was prepared in the same manner as in 2-1 and added to serum-free medium (-) to final concentrations of 0.01, 0.025, 0.1, 0.2, and 0.4% (w / v), respectively. 1.25 × 10⁶ porcine fibroblasts ５ cells / cm ２ The cells were then seeded in a 96-well plate, and the total number of cells was counted after 3 days of culture. The total number of cells before and after proliferation was calculated and graphed (Figure 3C).

[0069] 3-4: Exploration of the growth-promoting effect and effective concentration of wheat germ extract in adherent culture of bovine myoblasts. Bovine myoblasts were collected in the same manner as in 1-3. The serum-free medium (-) was prepared by adding 0.2% BSA, 0.2% whey, 1× ITS-G, 1× p / s, 0.1% Lipid mixture, and 4 ng / ml bFGF to DMEM. The positive control was prepared by adding 20% ​​FBS and 4 ng / ml bFGF to DMEM. Wheat germ extract was prepared in the same manner as in 2-1 and added to the serum-free medium (-) to final concentrations of 0.001, 0.005, 0.01, 0.025, 0.05, 0.1, 0.2, 0.4, 0.8, and 1.0% (w / v), respectively. Bovine myoblasts were divided into 1.56 × 10⁶ cells. ４ cells / cm ２ The seeds were then seeded in collagen-coated culture dishes, and their growth-promoting activity against the positive control was evaluated using Crystal Violet staining after 3 days. Growth-promoting activity was calculated as the ratio (%) of the absorbance of the wheat germ extract group to the absorbance of the positive control, and graphed (Figure 3D).

[0070] 3-5: Exploration of the proliferation-promoting effect and effective concentration of wheat germ in adherent culture of bovine adipose-derived stem cells. Bovine adipose-derived stem cells were collected in the same manner as in 1-4, and serum-free medium (-) and positive control were used in the same manner as in 1-1-1. Wheat germ extract was prepared in the same manner as in 2-1 and added to serum-free medium (-) to final concentrations of 0.01, 0.025, 0.05, 0.1, 0.2, and 0.4% (w / v), respectively. Bovine adipose-derived stem cells were cultured in 6.25 × 10⁻⁶ cells. ４ cells / cm ２ The seeds were then sown in a culture dish, and the total number of cells was counted after 3 days of culture. The total number of cells before and after proliferation was calculated and graphed (Figure 3E).

[0071] Examination: As shown in Figures 3A to 3E, in all animal cell types, a concentration of wheat germ extract in the culture medium of 0.01–0.4% (w / v) showed good cell proliferation-promoting activity, and a concentration of 0.025–0.4% (w / v) showed an even better cell proliferation-promoting effect. Furthermore, as shown in Figure 3B, wheat germ extract can exert a cell proliferation-promoting effect equivalent to that of FBS even in suspension culture.

[0072] Example 4: Investigation of the cell proliferation-promoting effect and toxicity of wheat germ To determine whether the cell proliferation-promoting effect of wheat germ extract is due to the removal of toxic components and other components that hinder cell proliferation contained in wheat germ by water extraction, wheat germ powder was added to serum-free culture medium without going through the extraction process, and the cell proliferation-promoting effect was investigated. The experimental method is shown below.

[0073] Chicken fibroblasts DF-1, serum-free medium (-), and positive control were the same as in 1-1-1. Wheat germ powder was added to water at a concentration of 10% (w / v) and voltexed for 3 minutes to obtain a suspension. Without centrifugation or filtration, the suspensions were added to serum-free medium (-) to final concentrations of 0.025% and 0.05% (w / v), respectively. Chicken fibroblasts DF-1 were added in 6.25 × 10⁶ units. ４ cells / cm ２ The cells were then seeded into 96-well plates, and three days later, the cells and culture medium were photographed using a phase-contrast microscope (with a 4x objective lens) (Figure 4).

[0074] Review: In Examples 2 and 3, cell proliferation-promoting activity was confirmed using a water extract of wheat germ. However, in Example 4, qualitative evaluation results such as those shown in Figure 4, based on imaging, confirmed that wheat germ itself, not just the water extract, has cell proliferation-promoting activity, and that the fraction removed by water extraction does not contain toxic components. Therefore, it was found that wheat germ itself, rather than the water extract, can be used as a cell proliferation promoter.

[0075] Example 5: Examination of the heat resistance of wheat germ extract To investigate the cell proliferation-promoting components in wheat germ, a heat resistance test was conducted on wheat germ. The experimental method is as follows. Chicken fibroblast cells DF-1 and serum-free medium (-) were the same as in 1-1-1. The same wheat germ as in 2-1 was used, and extraction was performed for 3 minutes or 20 minutes in the same manner as in 2-1, except that hot water at 100°C was used (indicated as "100°C extraction" and "100°C, 20 min" in Figure 5, respectively). In addition, an extract obtained by exposing the same wheat germ extract as in 2-1 to heating conditions in an autoclave at 121°C for 15 minutes was also used (indicated as "autoclave" in Figure 5). For the positive control, an extract obtained by extracting wheat germ at 4°C was used in the same manner as in 2-1. Each extract thus obtained was added to serum-free medium (-) to a final concentration of 0.05% (w / v) in which significant proliferation was observed in Example 3 and Figure 3A. Chicken fibroblasts DF-1 6.25 × 10 ４ cells / cm ２ The seeds were then seeded in 96-well plates, and their growth-promoting activity against the positive control was evaluated three days later using Crystal Violet staining. Growth-promoting activity was calculated as the percentage of absorbance of each group using the post-heated extract relative to the absorbance of the positive control, and the results were graphed (Figure 5).

[0076] Investigation: As shown in Figure 5, even after heating, the wheat germ extract maintained approximately 90% or more of its cell proliferation-promoting activity compared to the control that was not exposed to heating conditions, indicating that the cell proliferation-promoting component of the wheat germ extract is heat-resistant. Therefore, it is suggested that the cell proliferation-promoting component of the wheat germ extract is not a protein or peptide substance.

[0077] Example 6: Cell proliferation promoting effect and protein concentration of various organic solvent extracts of wheat germ. Example 5 suggested that the cell proliferation promoting component is a non-protein or non-peptide substance. To clarify this and to investigate whether extraction is possible with solvents other than water, extraction was performed using various organic solvents, and the cell proliferation activity and protein concentration of these extracts were measured. The experimental method is as follows. Chicken fibroblast cells DF-1 and serum-free medium (-) were the same as in 1-1-1. For the water extract control, a water extract obtained by extracting wheat germ with water was used in the same manner as in 2-1. Wheat germ extracts were prepared in the same manner as in 2-1, except that 70 v / v% ethanol, ethanol, methanol, ethyl acetate, hexane, chloroform, and Milli-Q were used. After volatilizing each solvent in a rotary evaporator (90°C, 10 hPa), the solvents were replaced with the same amount of water and suspended, and these were used as various solvent extracts in the following experiments.

[0078] These various solvent extracts were added to serum-free medium (-) to a final concentration of 0.05% (w / v), as in Example 5. A 10% FBS DMEM was also used as a control (labeled "FBS" in Figure 6). Chicken fibroblast cells DF-1 were divided into 6.25 × 10⁶ cells. ４ cells / cm ２Seeds were seeded in 96-well plates, and the growth-promoting activity of each aqueous extract compared to the control was evaluated after 3 days using Crystal Violet staining. Growth-promoting activity was calculated as the ratio (%) of the absorbance of each solvent extract group to the absorbance of the wheat germ control, and graphed using the left vertical axis (bar graph portion of Figure 6). The protein concentrations of these various solvent extracts were quantified using the Pierce BCA Protein Assay Kit (manufactured by ThermoFisher). Quantification using this kit is based on the BCA method and was performed according to the manufacturer's specified method. Quantitative values ​​are indicated by circles using the right vertical axis (circles portion of Figure 6).

[0079] Study: As shown in Figure 6, cell proliferation activity was maintained at approximately 60% to 95% of the control (water extract) regardless of the solvent used. Therefore, it was found that cell proliferation-promoting components can be extracted not only with water but also with various polar or nonpolar solvents such as aqueous solvents, organic solvents, and aqueous organic solvents. Furthermore, this result suggests that the cell proliferation-promoting components are non-proteinogenic or non-peptide substances. For example, the proliferation activity of the ethyl acetate extract is equivalent to or higher than that of the FBS control, but the protein concentration is approximately 0 ng / ml = 63 pg / ml, which supports this finding.

[0080] Example 7: Molecular weight evaluation of cell proliferation-promoting components. To determine the approximate molecular weight of the cell proliferation-promoting components, molecular weight fractionation was performed by ultrafiltration. Ultrafiltration was performed using a 70% ethanol extract of wheat germ, which showed growth equivalent to that of water extraction in Example 6. The experimental method is shown below.

[0081] Chicken fibroblasts DF-1 and serum-free medium (-) were the same as in 1-1-1. A 70% ethanol extract of wheat germ was prepared in the same manner as in Example 6 and used as a positive control. Ultrafiltration of the 70% ethanol solution of wheat germ was performed using Amicon Ultra-15. Ultrafiltration was performed by centrifugation at 4000 g for 10-60 minutes at 4°C using an ultrafiltration unit in the following order: 100 kDa, 50 kDa, 30 kDa, 10 kDa, and 3 kD. The filtrate from each fraction was collected and added to serum-free medium (-) to the optimal final concentration of 0.1% (w / v) as shown in Example 3-1 and Figure 3A. Chicken fibroblasts DF-1 were added in 6.25 × 10⁶ units. ４ cells / cm ２ The seeds were then seeded in 96-well plates, and their growth-promoting activity against the positive control was evaluated three days later using Crystal Violet staining. Growth-promoting activity was calculated as the percentage of absorbance of each fraction used relative to the absorbance of the positive control, and the results were graphed (Figure 7).

[0082] Analysis: As shown in Figure 7, fractions with a molecular weight of 3 kDa or less did not show significantly higher cell proliferation-promoting activity compared to the negative control (serum-free medium (-)). However, a remarkable contribution to increased cell proliferation-promoting activity was observed in the 3 kDa to 10 kDa range (47.3%) and the 10 kDa to 30 kD range (52.7%). On the other hand, the increase in cell proliferation-promoting activity ceased above 30 kDa. These results suggest that the cell proliferation-promoting component is one or more substances with molecular weights ranging from 3 kDa to 10 kDa.

[0083] Example 8: Evaluation of optimal pH and salt concentration in extraction. To confirm the water extraction conditions for wheat germ, extraction was performed with various pH and salt solutions. The experimental method is as follows.

[0084] Chicken fibroblasts DF-1 and serum-free medium (-) were used as in 1-1-1. A wheat germ extract extracted in the same manner as in 2-1 was used as the positive control. Solutions at pH 1, 3, 5, 7, 9, 11, and 13 were prepared using HCl or NaOH as the extraction solution. A 150 mM salt solution was also prepared using NaCl. Wheat germ extract was prepared using each extraction solution in the same manner as in 2-1, and after centrifugation and filtration, the pH was adjusted to 7.4-7.8. The extracts obtained from each pH solution were added to serum-free medium (-) to a final concentration of 0.1% (w / v) as in Example 7. 6.25 × 10⁶ chicken fibroblasts DF-1 were added. ４ cells / cm ２ The seeds were then seeded in 96-well plates, and their growth-promoting activity against the positive control was evaluated three days later using Crystal Violet staining. Growth-promoting activity was calculated as the percentage of absorbance of each pH solution extract group relative to the absorbance of the positive control, and the results were graphed (Figure 8).

[0085] Investigation: As shown in Figure 8, when extracted within the pH range of 3 to 11, the growth-promoting activity was maintained at approximately 90% or more of that of the positive control. Furthermore, when extracted at pH 3 to 5, the growth-promoting activity was even higher than that of the positive control. Therefore, it is suggested that the preferred pH of the extraction solvent is 3 to 11, and more preferably 3 to 5. Moreover, even in salt solutions, the growth-promoting activity was maintained at approximately 80% or more of that of the positive control.

Claims

1. A cell proliferation promoter comprising grain germ or grain germ extract as an active ingredient, wherein the cells are edible cells.

2. The cell proliferation promoter according to claim 1, wherein the cereal germ is the germ of a grain or rice seed.

3. The cell proliferation promoter according to claim 1, which is heat resistant.

4. The cell proliferation promoter according to claim 1, wherein the edible cells are cells selected from the group consisting of fibroblasts, muscle cells, and adipocytes.

5. The cell proliferation promoter according to claim 1, wherein the grain germ or grain germ extract contains a non-protein or non-peptide substance with a molecular weight of 3 kDa to 30 kDa as a cell proliferation promoting component.

6. The cell proliferation promoter according to claim 1, for use in serum-free culture media.

7. A culture medium comprising the cell proliferation promoter according to any one of claims 1 to 6.

8. A method for preparing cells for the production of cellular food, comprising the step of culturing the cells in the culture medium described in claim 7.

9. A method for producing a cellular food, comprising the steps of culturing cells in the culture medium described in claim 7, and producing a cellular food using the cells cultured in the first step.

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