Activator, medium, culture method, and method for producing cultured meat

Abstract

To provide an activator that can activate stem cells, myoblasts, myotube cells, muscle cells, preadipocytes, adipocytes, hepatoblasts, hepatocytes, osteoprogenitor cells, preosteoblasts, osteoblasts, bone cells, vascular cells, and blood cells; to provide a useful activator in the production of cultured meat; to provide a culture medium; to provide a culture method; and to provide a method for producing cultured meat.SOLUTION: The solution is an activator containing a plant-derived polyamine-containing extract as an active ingredient. The activator can be at least one activator selected from the group consisting of stem cells, myoblasts, myotube cells, muscle cells, preadipocytes, adipocytes, hepatoblasts, hepatocytes, osteoprogenitor cells, preosteoblasts, osteoblasts, bone cells, vascular cells, and blood cells. The activator can be used to produce cultured meat. The activator can be used as a medium additive.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] The present invention relates to an activator, a culture medium, a culture method, and a method for producing cultured meat. [Background technology]

[0002] Cultured meat has been attracting attention as a technology to solve the food crisis caused by artificial population increase, or as a technology to avoid or reduce greenhouse gas emissions from livestock (especially greenhouse gas emissions from cow burps) (see Patent Documents 1 to 4). For example, cultured minced meat and cultured steak meat are known as cultured meat. Cultured minced meat is typically an aggregate of muscle cells oriented in different directions. On the other hand, cultured steak meat typically contains muscle tissue. In other words, cultured steak meat has a structure closer to real meat than cultured minced meat. Although different from cultured meat, cultured foie gras, for example, cultured foie gras produced through the cultivation of liver-derived cells, is also known (see Patent Documents 5 and 6).

[0003] Incidentally, Patent Document 7 describes that normal human skin fibroblasts and human hair papilla cells could be activated by culturing them in a medium to which a polyamine-containing extract extracted from soybean germ was added. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2021 / 132478 [Patent Document 2] JP 2020-141573 A [Patent Document 3] Patent No. 7201972 [Patent Document 4] Special Publication No. 2023-512686 [Patent Document 5] Special Publication No. 2020-523015 [Patent Document 6] International Publication No. 2020 / 096004 [Patent Document 7] Patent No. 5018171 Summary of the Invention [Problem to be solved by the invention]

[0005] Although Patent Document 7 describes that a polyamine-containing extract extracted from soybean germ can activate normal human skin fibroblasts and human hair papilla cells, it is unclear whether it can activate other cells. Moreover, since neither normal human skin fibroblasts nor human hair papilla cells are used in the production of cultured meat, it is also unclear whether the extract is useful for the production of cultured meat.

[0006] An object of the present invention is to provide an activating agent capable of activating at least one selected from the group consisting of stem cells, myoblasts, myotube cells, muscle cells, preadipocytes, adipocytes, hepatoblasts, hepatocytes, osteoprogenitor cells, preosteoblasts, osteoblasts, bone cells, vascular cells, and blood cells.

[0007] The present invention also aims to provide an activator that is useful in producing cultured meat.

[0008] The present invention also aims to provide a culture medium, a culture method, and a method for producing cultured meat. [Means for solving the problem]

[0009] As a result of conducting research into the cell activation effect of plant-derived polyamine-containing extracts, the present inventors have found that plant-derived polyamine-containing extracts can improve the dehydrogenase activity of adipose-derived mesenchymal stem cells, which are known to have the ability to differentiate into myoblasts, adipocytes, etc. In addition, the present inventors have also found that plant-derived polyamine-containing extracts can improve the dehydrogenase activity of pre-osteoblasts.

[0010] The present invention, which has been completed based on these findings, is as follows. [1] Contains plant-derived polyamine-containing extracts as active ingredients. At least one activating agent selected from the group consisting of stem cells, myoblasts, myotube cells, muscle cells, fat precursor cells, adipocytes, hepatoblasts, hepatocytes, osteoprogenitor cells, pre-osteoblasts, osteoblasts, bone cells, vascular cells, and blood cells. [2] The activator according to [1], wherein the plant-derived polyamine-containing extract is extracted from at least one material selected from the group consisting of soybean seeds, soybean germs, soybean embryos, soybean sprouts, wheat seeds, wheat germs, wheat embryos, wheat sprouts, soy milk, and okara. [3] The activator according to [1] or [2], wherein the plant-derived polyamine-containing extract is extracted from at least one of soybean germ and wheat germ. [4] The activating agent according to any one of [1] to [3], wherein the plant-derived polyamine-containing extract is obtained by a method including a step of extracting with an aqueous solution having a pH of 6.0 or less. [5] The activating agent according to any one of [1] to [4], which is for addition to a culture medium. [6] A medium comprising the activator according to any one of [1] to [5]. [7] A culture method comprising culturing at least one type of cell selected from the group described above in the medium according to [6]. [8] A method for producing cultured meat, comprising culturing at least one type of cell selected from the group consisting of stem cells, myoblasts, myotube cells, muscle cells, preadipocytes, adipocytes, vascular cells, and blood cells in the medium described in [6]. [9] Contains plant-derived polyamine-containing extracts as active ingredients. An enhancer used to produce cultured meat.

[10] A medium containing the activator described in [9]. [Brief description of the drawings]

[0011] [Figure 1] FIG. 1 is a graph showing dehydrogenase activity measured in Example 1 using MC3T3-E1 cells derived from mouse calvaria as an activation rate. [Diagram 2] FIG. 1 is a graph showing dehydrogenase activity measured in Example 2 using human adipose-derived mesenchymal stem cells (hADSCs) as an activation rate. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] Hereinafter, an embodiment of the present invention will be described.

[0013] <1. Activator> The activator of the present embodiment includes a plant-derived polyamine-containing extract. This allows the activator of the present embodiment to improve the metabolic activity of cells, for example, dehydrogenase activity. Therefore, the activator of the present embodiment can improve the metabolic activity of cells when producing cultured products such as cultured meat and cultured foie gras. Therefore, the activator of the present embodiment is useful for producing cultured products such as cultured meat and cultured foie gras. Moreover, the activator of the present embodiment is excellent in safety in the sense that the plant-derived polyamine-containing extract contained in the activator of the present embodiment is derived from plants. In other words, high safety can be expected. Therefore, the activator of the present embodiment is particularly useful for producing edible cultured products (for example, edible cultured meat and edible cultured foie gras).

[0014] As used herein, "activation" refers to an improvement in some metabolic activity of cells (e.g., stem cells, myoblasts, myotubes, muscle cells, preadipocytes, adipocytes, hepatoblasts, hepatocytes, osteoprogenitor cells, preosteoblasts, osteoblasts, osteocytes, vascular cells, blood cells, etc.). "Some metabolic activity" may be, for example, dehydrogenase activity. This will be explained. Dehydrogenase is involved in oxidation-reduction reactions that are important for cells to obtain energy, for example, oxidation-reduction reactions that constitute respiration. According to this, it can be considered that the energy obtaining ability of cells is reflected in dehydrogenase activity. Therefore, "some metabolic activity" may be dehydrogenase activity.

[0015] <1.1. Plant-derived polyamine-containing extracts> The plant-derived polyamine-containing extract is a plant extract containing polyamines. The plant-derived polyamine-containing extract can be extracted from plants and / or processed plant products.

[0016] Examples of plants include dicotyledonous plants, monocotyledonous plants, herbaceous plants, woody plants, cucurbitaceous plants, solanaceous plants, gramineous plants, cruciferous plants, legumes, mallows, Asteraceae, Chenopodiaceae, and legumes. Among these, gramineous plants and legumes are preferred.

[0017] Examples of grasses include wheat, barley, rice, corn, sorghum, sugarcane, turfgrass, oats, rye, and foxtail millet. Among them, wheat is preferred because it has a large net food supply per person per year, that is, the annual consumption per person is high, and it is also available at a low cost.

[0018] Examples of legumes include soybeans, alfalfa, adzuki beans, kidney beans, and cowpeas. Among them, soybeans are preferred because they have a large net food supply per person per year, that is, they have a large annual consumption per person, and are also available at low cost.

[0019] Examples of plants other than grasses and legumes include sweet potato, tomato, cucumber, pumpkin, melon, watermelon, tobacco, Arabidopsis, bell pepper, eggplant, taro, spinach, carrot, strawberry, potato, rapeseed, eucalyptus, poplar, kenaf, eucommia, sugar beet, cassava, sago palm, chenopodium, lily, orchid, carnation, rose, chrysanthemum, petunia, torenia, snapdragon, cyclamen, gypsophila, geranium, sunflower, cotton, enoki mushroom, shimeji mushroom, matsutake mushroom, shiitake mushroom, mushrooms, ginseng, agaricus, turmeric, ginseng, citrus fruits, banana, and kiwi.

[0020] Examples of plant parts from which an extract (i.e., a plant-derived polyamine-containing extract) can be obtained include flowers, buds, ovaries, fruits, leaves, cotyledons, stems, shoots, roots, seeds, dry seeds, germs, and germs. Among these, fruits, leaves, stems, shoots, seeds, dry seeds, germs, and germs are preferred, seeds, dry seeds, germs, and germs are more preferred, and germs (e.g., soybean germ, wheat germ, rice germ, barley germ, corn germ, milo germ, and sunflower germ) are even more preferred. Of course, an extract may be obtained from the entire body of a plant.

[0021] As the plant, soybean is preferable as mentioned above. That is, the plant-derived polyamine-containing extract is preferably a soybean-derived polyamine-containing extract. The soybean-derived polyamine-containing extract may be obtained from unprocessed soybeans (e.g., soybean germ) or from a product obtained by adding some processing to soybeans, i.e., a processed product. Examples of the processed product include soybean extract (e.g., soybean germ extract, soybean germ extract), fermented as necessary, natto, soy milk, and okara.

[0022] As mentioned above, wheat is also preferred as a plant. That is, the plant-derived polyamine-containing extract is preferably a wheat-derived polyamine-containing extract. The wheat-derived polyamine-containing extract may be obtained from unprocessed wheat (e.g., wheat germ) or from a product obtained by adding some processing to wheat, i.e., a processed product. Examples of processed products include wheat extract (e.g., wheat germ extract, wheat germ extract) and wheat flour that are fermented as necessary.

[0023] In addition, even if the plant is derived from a source other than soybean or wheat, the plant-derived polyamine-containing extract may be obtained from a processed product, such as green tea, black tea, oolong tea, fruit juice, etc.

[0024] The plant-derived polyamine-containing extract is preferably one extracted from at least one material selected from the group consisting of soybean seeds, soybean germs, soybean embryos, soybean sprouts, wheat seeds, wheat germs, wheat embryos, wheat sprouts, soy milk, and okara, because the raw materials are inexpensive, available in large quantities, and rich in polyamines. Of these, the plant-derived polyamine-containing extract is more preferably one extracted from at least one of soybean germs and wheat germs, and even more preferably one extracted from soybean germs.

[0025] The plant-derived polyamine-containing extract can be extracted with an aqueous solution having a pH of 6.0 or less (hereinafter sometimes referred to as an "acidic aqueous solution"). By extracting with an acidic aqueous solution, the recovery rate of polyamines can be increased compared to extraction with an organic solvent such as ethanol or methanol. Moreover, by extracting with an acidic aqueous solution, the residual organic solvent used for extraction can be avoided compared to extraction with an organic solvent. Therefore, the safety of the plant-derived polyamine-containing extract can be increased. Moreover, by having a pH of 6.0 or less, the recovery rate of polyamines can be further increased and the stability of polyamines in the acidic aqueous solution can be improved.

[0026] An example of the extraction procedure is to crush the plant and / or the processed plant product as necessary, and then extract the plant-derived polyamine-containing extract with an acidic aqueous solution. A more specific example of the extraction procedure is to add an acidic aqueous solution to the plant and / or the processed plant product, further add a polyphenol adsorbent as necessary, crush the plant and / or the processed plant product as necessary, and then extract the plant-derived polyamine-containing extract in the acidic aqueous solution.

[0027] The pH of the acidic aqueous solution is preferably 5.0 or less, more preferably 4.0 or less, more preferably 3.0 or less, and even more preferably 2.0 or less. On the other hand, the pH of the acidic aqueous solution can be, for example, 1.0 or more.

[0028] Examples of the acidic aqueous solution include an aqueous solution of a mineral acid dissolved in water, an aqueous solution of an organic acid dissolved in water, an aqueous solution of both a mineral acid and an organic acid dissolved in water, and acidic water. Examples of the acidic aqueous solution include an aqueous solution of a mineral acid such as hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, or perchloric acid dissolved in water, and an aqueous solution of an organic acid such as formic acid, acetic acid, propionic acid, butyric acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, malic acid, lactic acid, tartaric acid, citric acid, benzoic acid, trichloroacetic acid, or sulfosalicylic acid dissolved in water. Among them, 0.01N to 6N hydrochloric acid, sulfuric acid, nitric acid, acetic acid, phosphoric acid, trichloroacetic acid, sulfosalicylic acid, citric acid, or lactic acid, or 0.1% to 10% perchloric acid are preferable, and 0.0625N to 1N hydrochloric acid and 0.25% to 5% perchloric acid are more preferable.

[0029] For example, a mixer, blender, homogenizer, mortar, ultrasonic crusher, etc. can be used to crush the plant and / or the processed plant product. By crushing the plant and / or the processed plant product, the plant and / or the processed plant product can be made into, for example, a slurry, fine particles, granules, or powder, and then extraction can be performed. The operation of crushing the plant and / or the processed plant product may be performed in an acidic aqueous solution. That is, the plant and / or the processed plant product may be crushed after immersing it in an acidic aqueous solution.

[0030] The extraction time is preferably 10 minutes or more, more preferably 20 minutes or more, and even more preferably 30 minutes or more. Meanwhile, the extraction time may be 48 hours or less, 24 hours or less, 12 hours or less, 6 hours or less, or 3 hours or less. The extraction temperature, i.e., the temperature of the acidic aqueous solution during extraction, may be, for example, 10°C or more, 15°C or more, or 20°C or more. Meanwhile, the extraction temperature may be 40°C or less, 35°C or less, or 30°C or less. Stirring may be performed during extraction.

[0031] The extraction may be performed in the presence of a polyphenol adsorbent. By performing the extraction in the presence of a polyphenol adsorbent, polyphenols can be removed. Examples of polyphenol adsorbents include PVPP (polyvinyl polypyrrolidone), PVP (polyvinyl pyrrolidone), and PEG (polyethylene glycol). Among them, PVPP is preferable. An example of a commercially available polyphenol adsorbent is Polyclar (registered trademark) from ISP Co., Ltd. The polyphenol adsorbent can be added to the acidic aqueous solution to be, for example, 0.1% (w / v) to 30% (w / v), preferably 0.5% (w / v) to 20% (w / v), and more preferably 1% (w / v) to 10% (w / v). In the following, "% (w / v)" may be referred to as "w / v%".

[0032] After extraction, the extract is recovered by centrifugation and / or filtration, if necessary.

[0033] The pH of the extract is adjusted as necessary. In order to adjust the pH of the extract, a basic aqueous solution such as an aqueous sodium hydroxide solution or an aqueous sodium carbonate solution can be added. The pH of the extract after the pH adjustment is preferably neutral or close to neutral, and may be, for example, 6.0 or more, 6.2 or more, 6.5 or more, or 6.7 or more. The pH of the extract after the pH adjustment may be, for example, 8.0 or less, 7.8 or less, 7.5 or less, or 7.3 or less.

[0034] The extract is dried, if necessary. By such a procedure, a plant-derived polyamine-containing extract can be obtained.

[0035] The plant-derived polyamine-containing extract contains polyamines. Examples of polyamines include putrescine, spermidine, and spermine. The plant-derived polyamine-containing extract can contain one or more polyamines. The plant-derived polyamine-containing extract preferably contains putrescine, spermidine, and spermine. Of course, the plant-derived polyamine-containing extract may contain other polyamines. In addition, when the plant-derived polyamine-containing extract is a soybean germ extract, it is known to contain putrescine, spermidine, and spermine (see Japanese Patent No. 5018171). When the plant-derived polyamine-containing extract is a wheat germ extract, it is known to contain putrescine, spermidine, and spermine (see Japanese Patent No. 5018171).

[0036] The plant-derived polyamine-containing extract may contain a fraction of less than 1 kDa. The "fraction of less than 1 kDa" refers to a component that passes through an ultrafiltration membrane with a cut-off of 1 kDa, i.e., an ultrafiltration membrane with a nominal molecular weight limit (NMWO) of 1 kDa. An example of a product having an ultrafiltration membrane with a cut-off of 1 kDa is an ultrafiltration tube with a cut-off of 1 kDa (Microsep Advance with 1K Omega, MCP001C41, Pall).

[0037] Substances that may be contained in the fraction less than 1 kDa include polyamines (eg, putrescine, spermidine, spermine) and water-soluble substances other than polyamines.

[0038] The fraction less than 1 kDa may have an effect of improving the metabolic activity (e.g., dehydrogenase activity) of cells, i.e., the fraction less than 1 kDa may contain an active ingredient.

[0039] The active ingredient may not lose its effect of improving the metabolic activity (e.g., dehydrogenase activity) of cells upon heat treatment at 121° C. for 15 minutes. In other words, the active ingredient may not be inactivated upon heat treatment at 121° C. for 15 minutes.

[0040] The active ingredient may be insoluble in ethanol.

[0041] The content of the plant-derived polyamine-containing extract in the activator of this embodiment can be appropriately set depending on the properties of the activator (for example, liquid, powder, etc.). The content of the plant-derived polyamine-containing extract may be 1% by mass or more, 3% by mass or more, 50% by mass or more, 80% by mass or more, 90% by mass or more, 95% by mass or more, 97% by mass or more, 99% by mass or more, or 100% by mass.

[0042] The enhancer of the present embodiment may further contain one or more other components, such as an excipient, a carrier, a diluent, and the like.

[0043] The activator of the present embodiment may be in liquid form or powder form.

[0044] <1.2.Application> The activator of the present embodiment can be suitably used to produce cultured products such as cultured meat and cultured foie gras. Cultured meat is meat produced by a method including a step of culturing animal cells. In this specification, cultured meat refers to a cultured product that includes at least one of muscle fibers or myotube cells. Cultured meat may or may not faithfully have the original structure of meat. It is more preferable that cultured meat includes muscle fiber bundles. Cultured meat may be, for example, minced or filleted (which may also be called steak-shaped). Cultured meat may be edible or inedible (for example, for drug screening or medical research). Meanwhile, in this specification, cultured foie gras refers to a cultured product that includes hepatocytes. Cultured foie gras does not necessarily have to faithfully have the original structure of the liver. Cultured foie gras is preferably in the form of a paste.

[0045] Examples of cell cultures used in producing cultured products such as cultured meat and cultured foie gras include two-dimensional cell culture and three-dimensional cell culture. Examples of three-dimensional cell cultures include the culture of one or more tissues, or a three-dimensional cell population before one or more tissues are completed. Examples of tissues include muscle tissue, fat tissue, and nerve tissue. The activator of this embodiment may be used in two-dimensional cell culture, three-dimensional cell culture, or both. In addition, a bio 3D printer, a scaffold, etc. may be used when producing a cultured product.

[0046] Examples of animals for which animal cells are produced include vertebrates and invertebrates. Among these, vertebrates are preferred, since the meat of vertebrates generally tends to be more commercially valuable than that of invertebrates.

[0047] Examples of vertebrates include cyclostomes, fish, amphibians, reptiles, birds, and mammals, with fish, birds, and mammals being preferred, given that the meat of fish, birds, and mammals generally tends to be more commercially valuable than that of other animals.

[0048] Examples of fish include eels such as Japanese eel, giant mottled eel, European eel, and American eel, tuna such as bluefin tuna, southern bluefin tuna, yellowfin tuna, and albacore, salmon such as chum salmon, sockeye salmon, coho salmon, pink salmon, cherry salmon, Biwa salmon, and chinook salmon, and, given their potential commercial value, pufferfish such as tiger puffer, red puffer, and spotted puffer, and grouper.

[0049] Birds include Anatidae, such as geese, mallards, ducks, and ducklings, and Phasianidae, such as chickens and pheasants. Given the potential commercial value of cultured foie gras, Anatidae are preferred, with geese being more preferred.

[0050] Examples of mammals include Monotremes, Marsupialia, Insectivora, Chiroptera, Primates (e.g., Tarsiers, humans), Rodentia, Rodents (e.g., mice such as Mus musculus and Chinese caddisfly), Cetacea, Proboscidea, Carnivora, Perissodactyla (e.g., horses, donkeys), and Artiodactyla (e.g., cattle, pigs, and sheep). Among these, in light of the potential commercial value of cultured meat, especially cultured meat for consumption, Artiodactyla is preferred, with cattle, pigs, and sheep being more preferred, and cattle being even more preferred. Meanwhile, in light of the utility value of cultured meat in drug screening and medical research, primates are preferred, and humans are more preferred.

[0051] Invertebrates include, for example, arthropods and mollusks. An example of an arthropod is crustaceans. Crustaceans are preferred in view of their potential commercial value. Crustaceans include crabs such as red king crab, snow crab, and hairy crab, lobsters, and shrimps such as white crab and tiger prawn.

[0052] The activator of the present embodiment can be suitably used as an activator for stem cells, myoblasts, myotube cells, muscle cells, preadipocytes, adipocytes, hepatoblasts, hepatocytes, osteoprogenitor cells, preosteoblasts, osteoblasts, bone cells, vascular cells, blood cells, and the like. In particular, when the activator of the present embodiment is used to produce cultured meat, it is preferable to use it as at least one activator selected from the group consisting of stem cells, myoblasts, myotube cells, muscle cells, preadipocytes, and adipocytes. In other words, it is preferable that the activator of the present embodiment is used to activate at least one selected from this group. In particular, it is preferable to use it as at least one activator selected from the group consisting of stem cells, myoblasts, myotube cells, and muscle cells.

[0053] Examples of stem cells include pluripotent stem cells and tissue stem cells. Examples of pluripotent stem cells include embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), and embryonic germ stem cells (EG cells). Examples of tissue stem cells include neural stem cells, mesenchymal stem cells (MSCs), skeletal muscle stem cells, adipose stem cells, vascular endothelial progenitor cells, and vascular pericytes.

[0054] Tissue stem cells may be, for example, cells extracted from an animal, or cells (which may be cell lines) that have been cultured (for example, primary culture or subculture) after being extracted from an animal. Tissue stem cells may also be cells produced by differentiation induction from pluripotent stem cells such as iPS cells and ES cells.

[0055] Mesenchymal stem cells (MSCs) can have the ability to differentiate into, for example, myoblasts and adipocytes (i.e., differentiation potential). Mesenchymal stem cells may also have the ability to differentiate into other cells, for example, osteoblasts, chondroblasts, and the like (i.e., differentiation potential).

[0056] When mesenchymal stem cells (MSCs) are cells extracted from an animal or cells cultured after extraction from an animal, the mesenchymal stem cells may be derived from, for example, adipose tissue, bone marrow, dental pulp, umbilical cord, placenta-amniotic membrane, or amniotic fluid. In addition, mesenchymal stem cells derived from adipose tissue are preferred in the sense that they are relatively easy to obtain.

[0057] An example of a skeletal muscle stem cell is a satellite cell. A satellite cell is a mononuclear cell present in a skeletal muscle. A satellite cell is responsible for the regeneration of a skeletal muscle. When a skeletal muscle is damaged, the satellite cell is activated, starts to proliferate, differentiates into a myoblast, and migrates to the damaged site while repeatedly proliferating, and fuses with a muscle fiber. Thus, a satellite cell has at least the ability to differentiate into a myoblast (i.e., differentiation ability).

[0058] Myoblasts have the ability to differentiate into muscle cells (i.e., differentiation potential). Myoblasts can fuse in a lined-up state (hereinafter, sometimes referred to as "cell fusion"). On the other hand, myotubes can be early muscle cells that have begun cell fusion, and therefore can be multinucleated cells. Muscle cells are muscle fibers. Muscle cells can be multinucleated.

[0059] Preosteoblasts are cells that typically arise during the differentiation of mesenchymal stem cells into osteoblasts. In this differentiation, mesenchymal stem cells typically differentiate into osteoprogenitor cells, preosteoblasts, and osteoblasts in this order. Mature osteoblasts can then transform into bone cells.

[0060] Vascular cells are any cells that make up blood vessels. Examples of vascular cells include vascular endothelial cells and vascular smooth muscle cells. Of these, vascular endothelial cells are preferred. Examples of blood cells include erythroblasts, red blood cells, myeloblasts, myelocytes, lymphoblasts, leukocytes, megakaryoblasts, megakaryocytes, and platelets.

[0061] The above-mentioned cells (i.e., stem cells, myoblasts, myotube cells, muscle cells, preadipocytes, adipocytes, hepatoblasts, hepatocytes, osteoprogenitor cells, preosteoblasts, osteoblasts, bone cells, vascular cells, blood cells, etc.) may be the cells themselves extracted from an animal, or they may be cells (which may be cell lines) that have been cultured (e.g., primary culture or subculture) after being extracted from an animal. The above-mentioned cells may also be cells produced by differentiation induction from ES cells, iPS cells, etc.

[0062] Among the above-mentioned cells (i.e., stem cells, myoblasts, myotube cells, muscle cells, preadipocytes, adipocytes, hepatoblasts, hepatocytes, osteoprogenitor cells, preosteoblasts, osteoblasts, osteocytes, vascular cells, blood cells, etc.), mesenchymal stem cells (MSCs) are preferred, and adipose tissue-derived mesenchymal stem cells are more preferred, in the sense that their activation has been demonstrated in the Examples described below.

[0063] The activating agent of the present embodiment can be added to a medium. That is, the activating agent of the present embodiment can be used as an activating agent for addition to a medium (specifically, a culture medium), that is, as a medium additive.

[0064] <2. Culture medium> The medium of this embodiment contains the above-mentioned activating agent. Therefore, the medium of this embodiment can be a medium to which the above-mentioned activating agent has been added.

[0065] The medium of the present embodiment can be suitably used for producing cultured meat or cultured foie gras. The medium of the present embodiment can be suitably used for culturing the above-mentioned cells (i.e., stem cells, myoblasts, myotube cells, muscle cells, preadipocytes, adipocytes, hepatoblasts, hepatocytes, osteoprogenitor cells, preosteoblasts, osteoblasts, bone cells, vascular cells, blood cells, etc.).

[0066] Examples of media to which the activating agent of this embodiment is added include Alpha-MEM, D-MEM (for example, Poweredby10 (Glycotechnica Inc.)), Plusoid-M (Glycotechnica Inc.), G031101 (Glycotechnica Inc.), M061101 (Glycotechnica Inc.), etc. Serum, antibiotics, cell growth factors, etc. may be added to the medium as necessary. An example of serum is fetal calf serum (FCS). The medium of this embodiment may not contain serum, for example fetal calf serum. In other words, the medium of this embodiment may be a serum-free medium.

[0067] The polyamine concentration in the medium (specifically, the concentration of polyamine derived from the plant-derived polyamine-containing extract) can be appropriately set. The polyamine concentration in the medium may be, for example, 0.1 μM or more, 0.5 μM or more, 1.0 μM or more, 5.0 μM or more, or 10 μM or more. The polyamine concentration in the medium may be 5000 μM or less, 3000 μM or less, 1000 μM or less, 500 μM or less, 200 μM or less, 100 μM or less, or 50 μM or less.

[0068] <3.Culture method> The culture method of this embodiment includes culturing the above-mentioned cells (i.e., stem cells, myoblasts, myotube cells, muscle cells, preadipocytes, adipocytes, hepatoblasts, hepatocytes, osteoprogenitor cells, preosteoblasts, osteoblasts, bone cells, vascular cells, blood cells, etc.) in the above-mentioned medium. Examples of the method for culturing the above-mentioned cells in the above-mentioned medium include a method of seeding the above-mentioned cells in the above-mentioned medium and incubating them at 37°C in a 5% CO2 atmosphere, and a method of replacing the medium with the above-mentioned medium and incubating them at 37°C in a 5% CO2 atmosphere. Note that when culturing the above-mentioned cells, cells other than these may be included in the cells to be cultured.

[0069] <4. Manufacturing method of cultured meat> The method for producing cultured meat in this embodiment includes culturing a group consisting of stem cells, myoblasts, myotubes, muscle cells, preadipocytes, adipocytes, vascular cells, and blood cells in the above-mentioned medium. Methods for culturing these cells in the above-mentioned medium include, for example, a method of seeding these cells in the above-mentioned medium and incubating them at 37°C in a 5% CO2 atmosphere, and a method of replacing the medium with the above-mentioned medium and incubating them at 37°C in a 5% CO2 atmosphere. Note that when culturing these cells, cells other than these may be included among the cells being cultured. EXAMPLES

[0070] The present invention will be described in more detail below with reference to examples. However, the present invention is not limited to the following examples as long as it does not depart from the gist of the present invention.

[0071] <Preparation of soybean germ extract> The soybean germ extract was obtained by the method of Example 3 described in the Detailed Description of the Invention section of Japanese Patent No. 5018171. Specifically, 500 mL of 5% aqueous perchloric acid solution was added to 100 g of soybean germ (manufactured by For You Co., Ltd.) and left at room temperature for 1 hour. Then, 16 g of polychlor VT (manufactured by ISP Co., Ltd.), a polyphenol adsorbent, was added, and the soybean germ was thoroughly crushed with a blender mixer, and left at room temperature for 30 minutes to extract under acidic conditions. The crushed material was centrifuged at 2°C and 22,000×g for 20 minutes to collect a liquid fraction, which was neutralized with a 30% sodium hydroxide solution to make it neutral. The neutralized liquid fraction was desalted using an electrodialysis device (Acilyzer, manufactured by Astom Co., Ltd.) and then freeze-dried. The powder obtained in this manner, i.e., the powder of soybean germ extract, was used for various evaluations. The amounts of putrescine, spermidine, and spermine in the soybean germ extract were also determined. To determine these amounts, diluted internal standard solution (1,7-diaminoheptane, internal standard amount = 1200 nmol) and 500 mL of 5% aqueous perchloric acid solution were added to 100 g of soybean germ (manufactured by For You Co., Ltd.) and left at room temperature for 1 hour. Then, 16 g of polychlor VT (manufactured by ISP Co., Ltd.), a polyphenol adsorbent, was added, and the soybean germ was thoroughly crushed using a blender mixer, and left at room temperature for 30 minutes to extract under acidic conditions. The crushed material was centrifuged at 2°C and 22,000 x g for 20 minutes to collect the liquid fraction, which was then neutralized with 30% sodium hydroxide solution to make it neutral. The amounts of putrescine, spermidine, and spermine were determined using the neutralized liquid fraction according to the method described in the Detailed Description of the Invention section of Japanese Patent No. 5018171. The soybean germ extract contained 23.5 mg of putrescine, 24.0 mg of spermidine, and 9.6 mg of spermine for a total of 57.1 mg.

[0072] Example 1: Activation effect of soybean germ extract on MC3T3-E1 <1.1. Cells and reagents used> Cells: Mouse calvarial osteoblasts MC3T3-E1 (10 cm dish, 60% confluency) Culture medium: MEM Alpha basic (1x) (gibco) (unless otherwise specified, containing 10% FCS and 1% Penicillin-Streptmycin (hereinafter referred to as "PS")) Cell activation measurement reagent: Viable cell count measurement reagent SF (Nacalai Tesque, Inc.)

[0073] <1.2. Preculture> The MC3T3-E1 culture supernatant was aspirated off and the cells were washed twice with 5 mL of PBS, after which 2 mL of trypsin-EDTA solution was added and incubated at 37°C for 3 to 5 minutes. After adding an equal volume of medium, the cells were combined into a 50 mL centrifuge tube and collected by centrifugation. After removing the supernatant, the cells were suspended in an appropriate volume of medium containing 1% FCS and 1% PS, and the cells were counted. Based on the cell count results, 4 × 10 4 Prepare a cell suspension of 1 × 10 cells / mL and seed 250 μL of the suspension into a 48-well plate (1 × 10 4 The cells were incubated overnight in a CO2 incubator (37°C, 5.0%).

[0074] <1.3. Sample addition and incubation> Soybean germ extract was added to the cell culture medium prepared in 1.2 at final concentrations of 0.01 w / v%, 0.05 w / v%, 0.1 w / v%, and 0.2 w / v% (n=3 for each condition), and cultured for 3 days in a CO2 incubator (37°C, 5.0%). The control was a condition in which no soybean germ extract was added. The polyamine concentrations in the medium to which soybean germ extract was added at final concentrations of 0.01 w / v%, 0.05 w / v%, 0.1 w / v%, and 0.2 w / v% were 1.4 μM, 7.0 μM, 14 μM, and 28 μM.

[0075] <1.4. Measurement of dehydrogenase activity> After 3 days of incubation, the plate was removed and observed under a microscope. 250 μL of medium containing 1% FCS and 1% PS was added to each of the 3 blank wells (Blank). 20 μL of the viable cell count measurement reagent SF was added to each well, and after incubation for 2 hours in a CO2 incubator (37°C, 5.0%), the absorbance at 450 nm and 600 nm was measured.

[0076] <1.5. Data organization - Calculation of activation rate> The cell activation effect, i.e., the activation rate, was calculated by the following formula. Activation rate=(As-Ab) / (Ac-Ab)×100 In this formula, Ab, Ac, and As represent the following: Ab: Absorbance difference of blank (i.e. Abs450-Abs600) Ac: Absorbance difference of control As: absorbance difference between various samples Here, "absorbance difference" means the difference between the absorbance at a wavelength of 450 nm and the absorbance at a wavelength of 600 nm (ie, absorbance at a wavelength of 450 nm - absorbance at a wavelength of 600 nm). The activation rate of the control is set to 100%, and the activation rate when soybean germ extract was added is shown in FIG.

[0077] <1.6.Comments> As shown in FIG. 1, the soybean germ extract was able to improve the dehydrogenase activity of preosteoblastic cells, MC3T3-E1.

[0078] Example 2: Activation effect of soybean germ extract on hADSCs <2.1. Cells and reagents used> Cells: Human adipose-derived mesenchymal stem cells (hADSC) (passage number: P13, 10 cm dish, 70% confluency) Culture medium: MEM Alpha basic (1x) (gibco) (unless otherwise noted, containing 10% FCS and 1% PS) Cell activation measurement reagent: Viable cell count measurement reagent SF (Nacalai Tesque, Inc.)

[0079] <2.2. Preculture> The culture supernatant of hADSCs was aspirated off and the cells were washed twice with 5 mL of PBS, after which 2 mL of trypsin-EDTA solution was added to detach the cells from the bottom of the dish. After adding an equal volume of medium, the cells were combined into a 50 mL centrifuge tube and collected by centrifugation. After removing the supernatant, the cells were suspended in an appropriate volume of medium containing 1% FCS and 1% PS, and the cells were counted. Based on the cell counting results, 4 × 10 4 Prepare a cell suspension of 1 × 10 cells / mL and seed 250 μL of the suspension into a 48-well plate (1 × 10 4 The cells were incubated overnight in a CO2 incubator (37°C, 5.0%).

[0080] <2.3. Sample addition and incubation> The plates precultured in 2.2 were removed from the CO2 incubator, and soybean germ extract was added to final concentrations of 0.01 w / v%, 0.05 w / v%, 0.1 w / v%, and 0.2 w / v% (n=3 for each condition), and cultured in a CO2 incubator (37°C, 5.0%) for 7 days. The condition without soybean germ extract was used as the control.

[0081] <2.4. Measurement of dehydrogenase activity> After 7 days of incubation, the plate was removed and observed under a microscope. 250 μL of medium containing 1% FCS and 1% PS was added to each of the 3 blank wells (Blank). 20 μL of the viable cell count measurement reagent SF was added to each well, and after incubation for 3 hours in a CO2 incubator (37°C, 5.0%), the absorbance at 450 nm and 600 nm was measured.

[0082] <2.5. Data organization - Calculation of activation rate> The cell activation effect, i.e., the activation rate, was calculated by the method described in Example 1 (i.e., the method described in 1.5.). The activation rate of the control was set to 100%, and the activation rate when soybean germ extract was added is shown in Figure 2.

[0083] <2.6.Comments> As shown in FIG. 2, the soybean germ extract was able to improve the dehydrogenase activity of human adipose-derived mesenchymal stem cells (hADSCs). [Industrial Applicability]

[0084] The present invention has industrial applicability because it can provide an activator, a culture medium, a culture method, and a method for producing cultured meat.

Claims

1. Contains plant-derived polyamine-containing extracts as active ingredients. At least one activating agent selected from the group consisting of stem cells, myoblasts, myotube cells, muscle cells, fat precursor cells, adipocytes, hepatoblasts, hepatocytes, osteoprogenitor cells, pre-osteoblasts, osteoblasts, bone cells, vascular cells, and blood cells.

2. 2. The activator according to claim 1, wherein the plant-derived polyamine-containing extract is extracted from at least one material selected from the group consisting of soybean seeds, soybean germs, soybean embryos, soybean sprouts, wheat seeds, wheat germs, wheat embryos, wheat sprouts, soy milk, and soybean lees.

3. The activator according to claim 1 , wherein the plant-derived polyamine-containing extract is extracted from at least one of soybean germ and wheat germ.

4. The activating agent according to claim 1 , wherein the plant-derived polyamine-containing extract is obtained by a method comprising a step of extracting with an aqueous solution having a pH of 6.0 or less.

5. The activating agent according to claim 1 , which is used for adding to a culture medium.

6. A medium comprising the activating agent according to any one of claims 1 to 5.

7. A culture method comprising culturing at least one type of cell selected from the group in the medium according to claim 6.

8. A method for producing cultured meat, comprising culturing at least one type of cell selected from the group consisting of stem cells, myoblasts, myotubes, muscle cells, preadipocytes, adipocytes, vascular cells, and blood cells in the medium according to claim 6.

9. Contains plant-derived polyamine-containing extracts as active ingredients. An enhancer used to produce cultured meat.

10. A medium comprising the activating agent according to claim 9.

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