Methods for culturing animal cells

Abstract

This invention provides methods for culturing animal cells and related technologies. [Solution] Animal cells are cultured in the presence of lecithin.

Description

[Technical Field]

[0001] This invention relates to a method for culturing animal cells and related technologies. [Background technology]

[0002] Culture media containing serum, such as fetal bovine serum, are widely used for culturing animal cells. However, from the perspectives of safety and cost-effectiveness, the development of serum-free media is desired. For example, serum-free media in which serum is replaced with alternative components such as growth factors have been developed. [Overview of the Initiative] [Problems that the invention aims to solve]

[0003] The object of this invention is to provide a method for culturing animal cells and related technologies. [Means for solving the problem]

[0004] The inventors of this invention have discovered that animal cells can be cultured in the absence of serum by utilizing lecithin, and have completed this invention.

[0005] In other words, the present invention can be illustrated as follows. [1] A composition for animal cell culture containing lecithin. [2] The composition described above (specifically, the composition described in [1]) is a composition for improving the growth of animal cells. [3] A culture medium or culture medium additive, as described above (specifically, as described in [1] or [2]). [4] The composition (specifically as described in [3]) wherein the culture medium is a basal medium, a fed-batch medium, or a perfusion medium. [5] The composition (specifically, as described in any of [1] to [4]) wherein the lecithin is soy lecithin. [6] Furthermore, a composition containing a material derived from broad bean, said (specifically, the composition according to any one of [1] to [5]). [7] The composition according to the above (specifically, the composition according to [6]), wherein the material derived from broad bean is a broad bean fermented product and / or broad bean protein. [8] The composition according to the above (specifically, the composition according to [7]), wherein the broad bean fermented product is broad bean soy sauce. [9] The composition according to the above (specifically, the composition according to any one of [1] to [8]), wherein the animal is a mammal, bird, fish, or crustacean.

[10] The composition according to the above (specifically, the composition according to any one of [1] to [9]), wherein the animal is a carnivorous animal.

[11] The composition according to the above (specifically, the composition according to any one of [1] to

[10] ), wherein the animal is a cow.

[12] The composition according to the above (specifically, the composition according to any one of [1] to

[11] ), which substantially does not contain serum.

[13] The composition according to the above (specifically, the composition according to any one of [1] to

[12] ), which substantially does not contain animal-derived albumin.

[14] A method for producing a product, comprising: A method including a step of culturing animal cells in the presence of lecithin.

[15] The method according to the above (specifically, the method according to

[14] ), wherein the product is cultured meat.

[16] A method for culturing animal cells, comprising: A method including a step of culturing animal cells in the presence of lecithin.

[17] A method for improving the growth of animal cells, comprising: A method including a step of culturing animal cells in the presence of lecithin.

[18] The method according to the above (specifically, the method according to any one of

[14] to

[17] ), wherein the lecithin is soybean lecithin.

[19] The concentration of lecithin in the culture medium during the culture is 0.1 to 100 μg / mL. Physically, the method described in any of

[14] to

[18] .

[20] The method described above (specifically, any of

[14] to

[19] ), wherein the culture is carried out in the presence of a pea-derived material. [twenty one] The method (specifically as described in

[20] ) wherein the pea-derived material is a pea ferment and / or pea protein. [twenty two] The method (specifically as described in

[21] ), wherein the fermented pea product is pea soy sauce. [twenty three] The composition (specifically as described in

[21] or

[22] ) wherein the concentration of the pea ferment in the culture medium during the culture is 0.1 to 100 mg / mL. [twenty four] The composition (specifically, one of

[21] to

[23] ) wherein the concentration of the pea protein in the culture medium during the culture is 0.05 to 50 mg / mL. [twenty five] The method described above (specifically, any of

[14] to

[24] ), wherein the animal is a mammal, a bird, a fish, or a crustacean.

[26] The method described above (specifically, any of

[14] to

[25] ), wherein the animal is an animal raised for meat.

[27] The method described above (specifically, any of

[14] to

[26] ), wherein the animal is a cattle.

[28] The method (specifically, any of

[14] to

[27] ) wherein the culture is carried out in a culture medium that is substantially free of serum.

[29] The method (specifically, as described in any of

[14] to

[28] ) wherein the culture is carried out in a medium that is substantially free of animal-derived albumin. [Effects of the Invention]

[0006] According to the present invention, animal cells can be cultured. [Brief explanation of the drawing]

[0007] [Figure 1] A diagram showing the proliferation of bovine muscle stem cells in serum-free medium in the presence of soy lecithin. [Figure 2] A figure showing the proliferation rate of bovine muscle stem cells in serum-free medium in the presence of soy lecithin. [Figure 3] A diagram showing the proliferation of bovine muscle stem cells in serum-free medium in the presence of a combination of pea soy sauce, pea protein, and soy lecithin. [Figure 4] A figure showing the proliferation rate of bovine muscle stem cells in serum-free medium in the presence of pea soy sauce and a combination of pea protein and soy lecithin. [Modes for carrying out the invention]

[0008] <1> Active ingredients <1-1> Active ingredients In this invention, lecithin is used as an active ingredient.

[0009] Lecithin is also called the "active ingredient."

[0010] The active ingredient can be used in the culture of animal cells. Specifically, animal cells can be cultured in the presence of the active ingredient.

[0011] The use of the active ingredient, specifically by culturing animal cells in the presence of the active ingredient, may improve the growth of animal cells; that is, an effect of improving the growth of animal cells may be obtained. This effect is also called the "growth-enhancing effect." In other words, the active ingredient may have a function that improves the growth of animal cells. This function is also called the "growth-enhancing function." That is, by culturing animal cells in the presence of the active ingredient, the growth of animal cells may be improved compared to culturing animal cells in the absence of the active ingredient. An example of culturing animal cells in the absence of the active ingredient is culturing animal cells under the same conditions as culturing animal cells in the presence of the active ingredient, except that the active ingredient is not used. The growth-enhancing effect may be obtained, for example, when culturing animal cells under conditions in which serum is substantially absent (i.e., culturing animal cells in a medium that substantially does not contain serum). The growth-enhancing effect may be obtained, for example, when culturing animal cells under conditions in which animal-derived albumin is substantially absent (i.e., culturing animal cells in a medium that substantially does not contain animal-derived albumin). The growth-enhancing effect may be obtained, for example, by culturing animal cells under conditions where serum and animal-derived albumin are substantially absent (i.e., culturing animal cells in a medium that substantially does not contain serum and animal-derived albumin). An improvement in growth is characterized by an increase in proliferation rate. The growth-enhancing effect can be confirmed by measuring and comparing the growth (e.g., proliferation rate) of animal cells in the presence and absence of the active ingredient.

[0012] In one embodiment, the product may be produced by culturing animal cells. Examples of products include cultured meat and the target substance. The target substance is not particularly limited as long as it can be produced by animal cells. Examples of target substances include proteins and viruses. Examples of products include: Cultured meat is a particular example. When a product is produced by culturing animal cells, the production of the product may be improved by using an active ingredient. That is, by culturing animal cells in the presence of an active ingredient, the production of the product may be improved compared to culturing animal cells in the absence of the active ingredient. For example, the production of the product may be improved by improving the growth of animal cells through the use of an active ingredient. That is, one aspect of the growth-improving effect may be an effect of improving the production of the product. The effect of improving the production of the product may be obtained, for example, when culturing animal cells under conditions in which serum is substantially absent (i.e., culturing animal cells in a medium that substantially does not contain serum). The effect of improving the production of the product may be obtained, for example, when culturing animal cells under conditions in which animal-derived albumin is substantially absent (i.e., culturing animal cells in a medium that substantially does not contain animal-derived albumin). The effect of improving the production of the product may be obtained, for example, when culturing animal cells under conditions in which serum and animal-derived albumin are substantially absent (i.e., culturing animal cells in a medium that substantially does not contain serum and animal-derived albumin). Improvements in production include increasing production volume and increasing production speed.

[0013] "Lecithin" may refer collectively to materials containing phospholipids. Lecithin may contain phospholipids as its main component. The phospholipid content in lecithin may be, for example, 50% (w / w). The above may be 70% (w / w) or more, 90% (w / w) or more, or 95% (w / w) or more. The type of lecithin is not particularly limited as long as it has the desired function (e.g., growth-enhancing function). Examples of lecithin include plant lecithin, egg yolk lecithin, and their derivatives. Examples of plant lecithin include soybean lecithin, rapeseed lecithin, and sunflower lecithin. Examples of derivatives include fractional lecithin, enzyme-treated lecithin, and enzymatically hydrolyzed lecithin. Soybean lecithin is particularly noteworthy as a type of lecithin. One type of lecithin may be used, or two or more types of lecithin may be used in combination.

[0014] Commercially available lecithin may be used, or lecithin may be manufactured and obtained as appropriate. The method of producing lecithin is not particularly limited. Lecithin can be produced, for example, by separation from a raw material containing lecithin. Specifically, soy lecithin can be produced, for example, by separation from soybean oil.

[0015] <1-2> Other ingredients The active ingredient may be used in combination with other ingredients, for example. Other ingredients include pea-derived materials and yeast extract. That is, the active ingredient may be used in combination with pea-derived materials and / or yeast extract, for example. Other ingredients include pea-derived materials in particular. That is, the active ingredient may be used in combination with, for example, at least pea-derived materials. Other ingredients (e.g., pea-derived materials and yeast extract) may have, for example, growth-enhancing functions. The description of the growth-enhancing function of the active ingredient can be applied mutatis mutandis to the growth-enhancing function of the other ingredients.

[0016] <1-2-1> Pea-derived material "Pea-derived ingredients" refers to ingredients derived from peas.

[0017] There are no particular restrictions on the type of pea-derived material. For example, a pea-derived material with a desired function (e.g., growth-enhancing function) may be selected. Examples of pea-derived materials include processed pea products. Examples of pea-derived materials (e.g., processed pea products) include fermented pea products and pea protein. A single pea-derived material may be used, or a combination of two or more materials may be used.

[0018] The type of pea is not particularly limited. As for the pea, for example, one may be selected that yields a pea-derived material having a desired function (e.g., growth-enhancing function). Examples of peas include garden peas and snow peas. Examples of garden peas include mature peas such as green peas, red peas, and yellow peas, and immature peas such as green peas. As for peas, garden peas are particularly noteworthy. As for peas, mature peas are particularly noteworthy. One type of pea may be used, or two or more types of peas may be used in combination. That is, the pea-derived material may be derived from one type of pea, or from two or more types of peas. That is, the pea-derived material may be derived from one type of pea, or from two or more types of peas in combination.

[0019] <1-2-1-1> Fermented peas "Pea ferment" refers to the product obtained by fermenting peas with microorganisms. Here, "fermentation" may mean that the product is used as a culture medium component for cultivation. In other words, "fermenting peas with microorganisms" may mean cultivating microorganisms in a culture medium containing peas. Specifically, "pea ferment" may refer to the product obtained by cultivating microorganisms in a culture medium containing peas. As pea ferment, one type of ferment may be used, or two or more types of ferment may be used in combination.

[0020] The type of pea from which the pea ferment is derived is not particularly limited, as long as it is possible to produce the pea ferment. The peas are as described above. The pea ferment may be derived from one type of pea, or from two or more types of peas. That is, the pea ferment may be made from a ferment derived from one type of pea, or from a combination of ferment derived from two or more types of peas.

[0021] An example of a fermented pea product is pea soy sauce. "Pea soy sauce" may mean a soy sauce-like seasoning manufactured using peas as a raw material. "Soy sauce-like seasoning" may mean a liquid seasoning or a processed product thereof that can be used in the same way as "soy sauce" according to the Japanese Agricultural Standards. Examples of processed products include dried products (e.g., dried powder). An example of pea soy sauce is one manufactured by the method described in Japanese Patent Publication No. 2014-110771.

[0022] The following is an example of a method for producing fermented peas. In other words, the fermented peas may be produced by this method.

[0023] Fermented pea products can be produced by fermenting peas with microorganisms (specifically, by cultivating microorganisms in a culture medium containing peas).

[0024] In other words, first, microorganisms may be cultured in a culture medium containing peas. That is, the method for producing pea fermentation may include a step of culturing microorganisms in a culture medium containing peas. This step is also called the "culturing step." Specifically, the culturing step may be a step of culturing microorganisms in a culture medium containing peas to obtain a culture. This culture is also called the "fermentation product."

[0025] The type of microorganism used is not particularly limited, as long as it can produce pea ferment. Examples of microorganisms include bacteria and fungi.

[0026] Lactobacillus is one example of a bacterium that can be found in this context.

[0027] Examples of lactic acid bacteria include Tetragenococcus species such as Tetragenococcus halophilus, and Bifidobacterium longum. Bifidobacterium breve, Bifidobacterium Bifidobacterium bifidum, Bifidobacterium adolescent Bifidobacterium bacteria such as Bifidobacterium adolescentis, Bifidobacterium angulatum, Bifidobacterium dentium, Bifidobacterium pseudocatenulatum, Bifidobacterium animalis, Bifidobacterium pseudolongum, Bifidobacterium thermophilum, Lactobacillus mali, Lactobacillus hilgardii, Lactobacillus Brevis, Lactobacillus delbrueckii, Lactobacillus ca Examples of Lactobacillus bacteria include Lactobacillus casei, Lactobacillus paracasei, Lactobacillus gasseri, and Lactobacillus acidophilus. Among lactic acid bacteria, Tetragenococcus bacteria are particularly noteworthy. More specifically, Tetragenococcus halophilus is a prime example. The lactic acid bacteria may be, for example, salt-tolerant lactic acid bacteria. "Salt-tolerant lactic acid bacteria" may mean, for example, lactic acid bacteria that can grow in a culture medium containing 8% (w / w) sodium chloride. For example, Tetragenococcus halophilus may be an example of a salt-tolerant lactic acid bacterium. As for the lactic acid bacteria, for example, lactic acid bacteria commonly used in soy sauce production may be selected.

[0028] Examples of fungi include yeasts and filamentous fungi.

[0029] The yeast may be a budding yeast or a fission yeast. The yeast may be a haploid yeast or a diploid or more polyploid yeast. Examples of yeast include Zygosaccharomyces genus yeasts such as Zygosaccharomyces rouxii and Zygosaccharomyces sapae, and Saccharomyces Examples include yeasts of the genus Saccharomyces such as Saccharomyces cerevisiae, yeasts of the genus Pichia (also called the genus Wickerhamomyces) such as Pichia farinosa, Pichia ciferrii, Pichia sydowiorum, and Pichia pastoris, yeasts of the genus Candida such as Candida versatilis, Candida etschelsii, and Candida utilis, yeasts of the genus Hansenula such as Hansenula polymorpha, and yeasts of the genus Schizosaccharomyces such as Schizosaccharomyces pombe. Notable yeast species include Zygosaccharomyces rouxii, Zygosaccharomyces sapae, Pichia farinosa, Candida versatilis, Candida etschelsii, Saccharomyces cerevisiae, and Schizosaccharomyces pombe. Furthermore, yeasts of the genus Zygosaccharomyces are particularly noteworthy. Even more specifically, Zygosaccharomyces rouxii is a notable example. An example of Zygosaccharomyces rouxii is Zygosaccharomyces rouxii NBRC 1130 strain (ATCC 56077).Examples of Saccharomyces cerevisiae include strains NBRC 10217 (ATCC 18824), BY4742 (ATCC 201389), BY4743 (ATCC 201390), and S288C (ATCC 26108). An example of Schizosaccharomyces pombe is strain NBRC 1628. The yeast may be, for example, a salt-tolerant yeast. "Salt-tolerant yeast" may mean, for example, a yeast that can grow in a medium containing 8% (w / w) sodium chloride. For example, Zygosaccharomyces rouxii, Zygosaccharomyces sapae, Pichia farinosa, Candida versatilis, and Candida etschelsii are all examples of salt-tolerant yeasts. For the yeast, you can choose, for example, a yeast commonly used in soy sauce production.

[0030] Examples of filamentous fungi include Aspergillus species such as Aspergillus oryzae, Aspergillus sojae, Aspergillus niger, Aspergillus tamarii, Aspergillus kawachii, Aspergillus awamori, and Aspergillus saitoi. In particular, Aspergillus oryzae and Aspergillus sojae are noteworthy filamentous fungi. For example, filamentous fungi commonly used in soy sauce production (specifically, koji production) may be selected.

[0031] These strains can be obtained, for example, from the American Type Culture Collection (ATCC, Address: 10801 University Boulevard Manassas, VA 20110, United States of America). Each strain is assigned a registration number, which can be used to obtain them (see http: / / www.atcc.org / ). The registration numbers for each strain are listed in the American Type Culture Collection catalog. These strains can also be obtained, for example, from the NBRC (NITE Biological Resource Center). Furthermore, these strains can be obtained, for example, from the depositary institutions where each strain is deposited.

[0032] As for the microorganisms, one type of microorganism may be used, or two or more types of microorganisms may be used in combination. For example, filamentous fungi, yeast, and lactic acid bacteria may be used in combination. When two or more types of microorganisms are used in combination, they may be cultured at the same time or not. For example, filamentous fungi may be cultured first, and then yeast and lactic acid bacteria may be cultured. Alternatively, a certain microorganism A may be cultured first, and then When culturing a certain microorganism B, the "peas" in the cultivation of microorganism B are microorganism A This can mean peas after cultivation (i.e., peas after fermentation by microorganism A).

[0033] The culture conditions are not particularly limited, as long as they allow for the production of pea ferment. The culture can be carried out under ordinary conditions used for culturing microorganisms, for example, except that a culture medium containing peas is used. The culture may be carried out, for example, by solid culture (i.e., in a solid medium) or by liquid culture (i.e., in a liquid medium). The culture may be carried out particularly by liquid culture (i.e., in a liquid medium).

[0034] The culture medium used is not particularly limited, as long as it contains peas and can produce pea fermentation products. In addition to peas, the culture medium may contain, as needed, a carbon source, nitrogen source, phosphorus source, sulfur source, and other components selected from various organic and inorganic sources. The culture medium may also consist of peas themselves; that is, microorganisms may be directly inoculated onto peas for cultivation. The types and concentrations of culture medium components can be appropriately determined by those skilled in the art. For specific culture medium compositions, for example, the usual culture medium compositions used for culturing microorganisms can be referenced. The culture medium may contain one type of pea, or a combination of two or more types of peas.

[0035] The form in which peas are used in cultivation is not particularly limited, as long as a fermented pea product can be produced. Peas may be used in cultivation after being separated from the pods, for example, or they may be used in cultivation without being separated from the pods. As for the peas, fresh peas (specifically, fresh edible parts) may be used in cultivation as is, or after being processed as appropriate. Processing methods include cutting, crushing, straining, juicing, fractionation, dilution, concentration, drying, and heating. These processing methods may be carried out individually or in combination as appropriate.

[0036] Sugars can be cited as a carbon source. Specifically, sugars include glucose and fructose. Examples of carbon sources include sucrose, galactose, xylose, arabinose, sucrose, lactose, cellobiose, molasses, starch hydrolysates, and biomass hydrolysates. As a carbon source, one carbon source may be used, or two or more carbon sources may be used in combination.

[0037] Examples of nitrogen sources include ammonium salts such as ammonium sulfate, ammonium chloride, and ammonium phosphate; organic nitrogen sources such as peptone, casein peptone, soy peptone, potato peptone, wheat peptone, casamino acids, yeast extract, malt extract, meat extract, and corn steep liquor; ammonia; and urea. One nitrogen source may be used, or two or more nitrogen sources may be used in combination.

[0038] Examples of phosphate sources include phosphates such as potassium dihydrogen phosphate and dipotassium hydrogen phosphate, and phosphate polymers such as pyrophosphate. A single phosphate source may be used, or two or more phosphate sources may be used in combination.

[0039] Specific examples of sulfur sources include inorganic sulfur compounds such as sulfates, thiosulfates, and sulfites, and sulfur-containing amino acids such as cysteine, cystine, and glutathione. A single sulfur source may be used, or a combination of two or more sulfur sources may be used.

[0040] Other various organic and inorganic components include, specifically, inorganic salts such as sodium chloride and potassium chloride; trace metals such as iron, manganese, magnesium, and calcium; and vitamins such as vitamin B1, vitamin B2, vitamin B6, nicotinic acid, nicotinamide, and vitamin B12. Examples of organic components include methyl compounds; amino acids; nucleic acids; peptones containing these, casamino acids, yeast extract, malt extract, meat extract, and corn steep liquor. Other organic and inorganic components may be used individually, or in combination of two or more components.

[0041] The culture medium may contain, for example, at least sodium chloride. That is, the culture medium may contain, for example, peas and sodium chloride.

[0042] Culturing may be carried out, for example, under aerobic conditions. Under aerobic conditions, the dissolved oxygen concentration of the culture medium may be controlled to, for example, 5-50%, preferably 10-20%, as a percentage of the saturated dissolved oxygen concentration being 100%. Culturing under aerobic conditions may be carried out by, for example, static culture, aeration culture, shaking culture, stirring culture, or a combination thereof. The culture temperature may be, for example, 25-45°C, 25-35°C, preferably 27-33°C, more preferably 28-32°C. The culture period may be, for example, 10 hours to 6 months, 10 hours to 2 months, 10 hours to 1 month, 10 hours to 200 hours, preferably 15 hours to 120 hours. The pH of the culture medium may be, for example, pH 3-10, preferably pH 3-8. The pH of the culture medium may be adjusted as needed during cultivation. To adjust the pH, an inorganic or organic acidic or alkaline substance, such as ammonia gas, can be used. Cultivation may be carried out by batch culture, fed-batch culture, continuous culture, or a combination thereof. Cultivation may also be carried out in two stages: pre-culture and main culture. For example, pre-culture may be carried out on a solid medium such as agar, and main culture may be carried out on a liquid medium. Note that "the medium contains a certain component" means that, if cultivation is carried out in two stages, the medium contains that component at least during the main culture. That is, for example, peas are contained in the medium at least during the main culture. Cultivation may be continued until a culture with a desired function (e.g., growth-enhancing function) is obtained.

[0043] Furthermore, the culture can be carried out, for example, in the manner described in Japanese Patent Application Publication No. 2014-110771. That is, for example First, peas that have undergone appropriate pretreatment can be inoculated with filamentous fungi and cultured to produce pea koji. The culture period for producing pea koji is, for example, 25-45 This can be done at ℃ and 85-95% humidity for 12-240 hours. Next, the pea koji is mixed with sodium chloride. A fermented product can be produced by mixing it with an aqueous solution of um, inoculating it with yeast and lactic acid bacteria, and culturing it (specifically, maturation). Maturation may be carried out for 3 to 6 months at 15 to 35°C with appropriate stirring. Alternatively, maturation may be carried out for 1 to 2 months at 35 to 55°C with appropriate stirring.

[0044] The content of peas and other culture medium components such as sodium chloride in the culture medium is not particularly limited, as long as it is sufficient to produce a pea ferment.

[0045] The pea content in the culture medium can be, for example, 0.5% (w / w) or more, 1% (w / w) or more, 2% (w / w) or more, 3% (w / w) or more, 4% (w / w) or more, 5% (w / w) or more, 7% (w / w) or more, 10% (w / w) or more, 15% (w / w) or more, 20% (w / w) or more, 25% (w / w) or more, 30% (w / w) or more, 40% (w / w) or more, or 50% (w / w). It may be 70%(w / w) or more, and may be 100%(w / w) or less, 70%(w / w) or less, 50%(w / w) or less, 40%(w / w) or less, 30%(w / w) or less, 25%(w / w) or less, 20%(w / w) or less, 15%(w / w) or less, 10%(w / w) or less, 7%(w / w) or less, 5%(w / w) or less, 4%(w / w) or less, 3%(w / w) or less, 2%(w / w) or less, or 1%(w / w) or less, and any non-contradictory combination thereof is also acceptable. The pea content in the culture medium may be, specifically, 0.5-1% (w / w), 1-2% (w / w), 2-3% (w / w), 3-4% (w / w), 4-5% (w / w), 5-7% (w / w), 7-10% (w / w), 10-15% (w / w), 15-20% (w / w), 20-25% (w / w), 25-30% (w / w), 30-40% (w / w), 40-50% (w / w), 50-70% (w / w), or 70-100% (w / w). The pea content in the culture medium may be, specifically, 0.5-100% (w / w), 0.5-50% (w / w), 1-30% (w / w), or 2-10% (w / w).

[0046] The sodium chloride content in the culture medium may be, for example, 0% (w / w) or more, 0.1% (w / w) or more, 0.2% (w / w) or more, 0.5% (w / w) or more, 1% (w / w) or more, 2% (w / w) or more, 3% (w / w) or more, 4% (w / w) or more, 5% (w / w) or more, 6% (w / w) or more, 7% (w / w) or more, 8% (w / w) or more, 9% (w / w) or more, 10% (w / w) or more, 12% (w / w) or more, or 15% (w / w) or more. It may be 0%(w / w) or less, 15%(w / w) or less, 12%(w / w) or less, 10%(w / w) or less, 9%(w / w) or less, 8%(w / w) or less, 7%(w / w) or less, 6%(w / w) or less, 5%(w / w) or less, 4%(w / w) or less, 3%(w / w) or less, 2%(w / w) or less, 1%(w / w) or less, 0.5%(w / w) or less, 0.2%(w / w) or less, or 0.1%(w / w) or less, or any non-contradictory combination thereof. The sodium chloride content in the culture medium may be, specifically, 0-0.1% (w / w), 0.1-0.2% (w / w), 0.2-0.5% (w / w), 0.5-1% (w / w), 1-2% (w / w), 2-3% (w / w), 3-4% (w / w), 4-5% (w / w), 5-6% (w / w), 6-7% (w / w), 7-8% (w / w), 8-9% (w / w), 9-10% (w / w), 10-12% (w / w), 12-15% (w / w), or 15-20% (w / w). The sodium chloride content in the culture medium may be, for example, 0-20% (w / w), 0.1-20% (w / w), 1-20% (w / w), 5-20% (w / w), 5-15% (w / w), or 5-12% (w / w). Note that "0% (w / w) or more" for the sodium chloride content in the culture medium includes both cases where the culture medium does not contain sodium chloride (i.e., 0% (w / w)) and cases where the culture medium contains sodium chloride (i.e., greater than 0% (w / w)). Therefore, for example, "the sodium chloride content in the culture medium is 0-20% (w / w)" means that the culture medium does not contain sodium chloride, or that the culture medium contains sodium chloride at a content of 20% (w / w) or less (specifically, a content greater than 0% (w / w) and 20% (w / w) or less).

[0047] The culture medium components, such as peas and sodium chloride, may be present in the medium for the entire duration of cultivation, or for only a portion of the cultivation period. In other words, "cultivation is carried out in a medium containing a certain component" means that the component is present in the medium for at least a portion of the cultivation period, and it is not necessary for the component to be present in the medium for the entire duration of cultivation. The culture medium components, such as peas and sodium chloride, may be present in the medium at the start of cultivation, for example, or they may be supplied to the medium after the start of cultivation. Furthermore, the culture medium components, such as peas and sodium chloride, may be present in the medium at the start of cultivation, and may be supplied to the medium again after the start of cultivation (for example, after the component has been consumed).

[0048] The culture medium components, such as peas and sodium chloride, may be included in the culture medium at the concentrations exemplified above for the entire duration of cultivation, or they may be included in the culture medium at the concentrations exemplified above for only a portion of the cultivation period. In other words, "cultivation is carried out in a culture medium containing a certain component at a certain concentration" means that the concentration of the component in the culture medium is within the range of that concentration for at least a portion of the cultivation period, and it is not necessary for the concentration of the component in the culture medium to be within the range of that concentration for the entire duration of cultivation. The culture medium components, such as peas and sodium chloride, may be included in the culture medium at the concentrations exemplified above at the start of cultivation, or they may be supplied to the culture medium after the start of cultivation to reach the concentrations exemplified above. Furthermore, the culture medium components, such as peas and sodium chloride, may be included in the culture medium at the concentrations exemplified above at the start of cultivation, and they may be further supplied to the culture medium after the start of cultivation (for example, after the consumption of the component) to reach the concentrations exemplified above.

[0049] The length of "part of the culture period" is not particularly limited, as long as it is possible to produce pea ferment. The length of "part of the culture period" can be set appropriately depending on various conditions such as the type of culture medium components, the type of microorganism, and the length of the culture period. "Part of the period" may be, for example, 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, 95% or more, 97% or more, or 99% or more of the total culture period. Alternatively, "part of the period" may be, for example, 10 hours or more, 20 hours or more, 40 hours or more, 60 hours or more, 80 hours or more, 100 hours or more, 120 hours or more, or 150 hours or more. Note that "total culture period" means the entire period of the main culture if the culture is divided into seed culture and main culture.

[0050] By culturing microorganisms in this manner, a culture (i.e., pea fermentation product) is obtained.

[0051] The cultured material may be used as is, or after being processed as appropriate, as a fermented pea product. That is, the method for producing the fermented pea product may include a step of processing the cultured material. For example, the cultured material may be used as is, or after being processed as appropriate, as a fermented pea product in the production of the composition of the present invention. Alternatively, for example, the cultured material may be used as is, or after being processed as appropriate, as a fermented pea product in the method of the present invention. The processing is not particularly limited as long as it can achieve the objective of the present invention (for example, an effect of improving growth is obtained). Examples of processing include fractionation, dilution, concentration, drying, heating, and sterilization. For example, in the case of fractionation, it is sufficient to recover the fraction having the desired function (for example, a function of improving growth). Specifically, the fraction recovered by fractionation may be a fraction containing a component having the desired function (for example, a function of improving growth). The shape of the fermented pea product is not particularly limited. The fermented pea product may be in any shape, such as powder, flakes, paste, or liquid. That is, the cultured material may be processed into a desired shape. In other words, the method for producing pea fermentation may include a step of processing the pea fermentation into a desired shape. For example, the culture may be dried and powdered and used as pea fermentation. The method for producing pea fermentation may include, for example, a step of drying and powdering the pea fermentation. Examples of drying methods include freeze-drying, spray-drying, and drum-drying. Additives such as excipients may be added to the pea fermentation.

[0052] <1-2-1-2> Pea Protein "Pea protein" refers to protein derived from peas. "Pea protein" is not limited to full-length pea protein, but may also include its fragments (i.e., partial proteins). Examples of fragments of full-length pea protein include hydrolysates of full-length pea protein. In other words, pea protein may or may not be hydrolyzed. In one embodiment, pea protein does not need to be hydrolyzed. Pea protein may be a single protein, or a combination of two or more proteins may be used.

[0053] The type of pea from which the pea protein is derived is not particularly limited. As for the pea from which the pea protein is derived, for example, a pea containing pea protein having a desired function (e.g., growth-enhancing function) may be selected. The pea is as described above. The pea protein may be derived from one type of pea, or from two or more types of peas. That is, as the pea protein, a protein derived from one type of pea may be used, or a combination of proteins derived from two or more types of peas may be used.

[0054] As for pea protein, commercially available products may be used, or products may be manufactured and obtained as appropriate. The method for producing pea protein is not particularly limited. Pea protein can be produced, for example, by extraction from peas. Pea protein may or may not be purified to a desired degree. That is, as for pea protein, a purified product may be used, or a material containing pea protein may be used. An example of a material containing pea protein is a crude extract of pea protein (i.e., protein crudely extracted from peas). An example of a material containing pea protein is crushed peas (e.g., pea powder). Crushed peas (e.g., pea powder) can be produced by crushing peas. Peas may be crushed after being separated from the pods, for example, or crushed without being separated from the pods. The pea protein content in materials containing pea protein can be, for example, 1% (w / w) or more, 5% (w / w) or more, or 10% (w / w). The percentages may be 30% (w / w) or more, 50% (w / w) or more, 70% (w / w) or more, 90% (w / w) or more, or 95% (w / w) or more.

[0055] Furthermore, the amount of pea protein (e.g., content (concentration) or amount used) shall be calculated based on the amount of pea protein itself in the material when using a material containing pea protein. The pea protein content in the object (e.g., material or composition) can be measured by known protein quantification methods such as the Kjeldahl method.

[0056] <1-2-2> Yeast Extract "Yeast extract" may refer to an extract of yeast. A single yeast extract may be used, or a combination of two or more yeast extracts may be used.

[0057] The type of yeast used for yeast extract is not particularly limited. For example, a yeast that yields a yeast extract with a desired function (e.g., growth-enhancing function) may be selected. Examples of yeasts used for yeast extract include those exemplified in the method for producing fermented pea products. Particularly noteworthy examples of yeasts used for yeast extract include Saccharomyces yeasts such as Saccharomyces cerevisiae, Schizosaccharomyces yeasts such as Schizosaccharomyces pombe, and Candida yeasts such as Candida utilis. Even more specifically, Saccharomyces yeasts and Candida yeasts are used as examples of yeasts used for yeast extract. A single type of yeast may be used for yeast extract, or a combination of two or more types of yeast may be used.

[0058] As for the yeast extract, commercially available products may be used, or products that are manufactured and obtained as appropriate may be used. The method of producing the yeast extract is not particularly limited. Yeast extract can be produced from yeast cells by known methods, for example. Methods for preparing yeast extract include autolysis and enzymes. Methods include decomposition, acid decomposition, alkaline decomposition, physical crushing, and freeze-thaw methods.

[0059] The form of the yeast extract is not particularly limited. The yeast extract may be in any form, such as powder, flakes, paste, or liquid.

[0060] <2> Composition of the present invention The composition of the present invention is a composition containing an active ingredient (i.e., lecithin).

[0061] The compositions of the present invention can be used, for example, in the culture of animal cells. That is, the compositions of the present invention may be compositions for animal cell culture. The compositions of the present invention can be used, for example, in the manner described in the method of the present invention described later.

[0062] The composition of the present invention (specifically, a composition for animal cell culture) may be, for example, a culture medium. Examples of culture media include basal media, feed medium, and perfusion medium. Examples include: "basal medium" may mean the medium used at the start of culture. The basal medium is also called the "initial medium". "Fed-batch medium" may mean the medium supplied to the culture system after the start of culture in fed-batch culture. "Perfusion medium" may mean the medium supplied to the culture system after the start of culture in continuous culture (this is not limited to perfusion culture).

[0063] The composition of the present invention (specifically, a composition for animal cell culture) may be, for example, a culture medium additive. "Culture medium additive" may mean a composition used by adding it to a culture medium. Examples of culture media to which the culture medium additive is added include basal media, fed-batch media, and perfusion media.

[0064] The use of the composition of the present invention may improve the growth of animal cells, that is, it may provide an effect of improving the growth of animal cells (growth-enhancing effect). For example, a growth-enhancing effect may be obtained by culturing animal cells in a culture medium containing the composition of the present invention. Alternatively, a growth-enhancing effect may be obtained by culturing animal cells in a culture medium to which the composition of the present invention, which is a culture medium additive, has been added. In other words, the composition of the present invention may have a function of improving the growth of animal cells (growth-enhancing function). Therefore, the composition of the present invention may be used to improve the growth of animal cells. That is, the composition of the present invention may, for example, be a composition for improving the growth of animal cells.

[0065] In one embodiment, the product (e.g., cultured meat or the target substance) may be produced by culturing animal cells. When the product is produced by culturing animal cells, the production of the product may be improved by using the composition of the present invention. For example, the production of the product may be improved by improving the growth of animal cells by using the composition of the present invention. That is, one embodiment of the composition of the present invention (specifically, the composition for animal cell culture) may be a composition for improving the production of the product.

[0066] The composition of the present invention may consist of an active ingredient, or it may contain other components. Components other than the active ingredient are also referred to as "additional components." That is, the active ingredient may be used as is, or in combination with additional components, as part of the composition of the present invention. As additional components, one component may be used, or two or more components may be used in combination.

[0067] The additional components are not particularly limited as long as they can achieve the objectives of the present invention (for example, by providing growth-enhancing effects). The additional components can be appropriately selected depending on various conditions, such as the type of animal cell and the manner in which the composition of the present invention is used. Examples of additional components include culture medium components. Culture medium components will be described later. For example, at least when the composition of the present invention is a culture medium, the composition of the present invention may contain culture medium components. Specific examples of additional components include pea-derived materials and yeast extract. That is, the composition of the present invention may contain, for example, pea-derived materials. The composition may contain a material and / or yeast extract. The composition of the present invention may, for example, contain at least a pea-derived material.

[0068] The composition of the present invention may be formulated as appropriate, for example. Additives may be used as appropriate in the formulation process. That is, additional components include additives used in the formulation. Examples of additives include excipients, binders, disintegrants, lubricants, stabilizers, flavoring and deodorizing agents, diluents, and surfactants. Additives can be appropriately selected, for example, depending on various conditions such as the shape of the composition of the present invention.

[0069] The form of the composition of the present invention is not particularly limited. The composition of the present invention may be in any form, such as powder, flakes, tablets, paste, or liquid.

[0070] The content and content ratio of each component (i.e., the active ingredient and optionally additional components) in the composition of the present invention are not particularly limited, as long as the objective of the present invention can be achieved (for example, a growth-enhancing effect can be obtained). The content and content ratio of each component in the composition of the present invention can be appropriately set according to various conditions such as the type of animal cell and the manner in which the composition of the present invention is used.

[0071] The content of the active ingredient in the composition of the present invention is greater than 0% (w / w) and less than or equal to 100% (w / w). For example, the content of the active ingredient in the composition of the present invention is 0.1 μg / g or more, and 0.2 μg / g or less. The amount may be μg / g or more, 0.5 μg / g or more, 1 μg / g or more, 2 μg / g or more, 5 μg / g or more, 10 μg / g or more, 20 μg / g or more, 50 μg / g or more, 100 μg / g or more, 1 mg / g or more, 10 mg / g or more, or 100 mg / g or more, and may also be 1000 mg / g or less, 100 mg / g or less, 10 mg / g or less, 1 mg / g or less, 100 μg / g or less, 50 μg / g or less, 20 μg / g or less, 10 μg / g or less, 5 μg / g or less, 2 μg / g or less, 1 μg / g or less, 0.5 μg / g or less, or 0.2 μg / g or less, and any non-contradictory combination thereof is also acceptable. The content of the active ingredient in the composition of the present invention may specifically be, for example, 0.1 μg / g to 0.2 μg / g, 0.2 μg / g to 0.5 μg / g, 0.5 μg / g to 1 μg / g, 1 μg / g to 2 μg / g, 2 μg / g to 5 μg / g, 5 μg / g to 10 μg / g, 10 μg / g to 20 μg / g, 20 μg / g to 50 μg / g, 50 μg / g to 100 μg / g, 100 μg / g to 1 mg / g, 1 mg / g to 10 mg / g, 10 mg / g to 100 mg / g, or 100 mg / g to 1000 mg / g. The content of the active ingredient in the composition of the present invention may specifically be, for example, 0.1 μg / g to 1000 mg / g, 0.2 μg / g to 1000 mg / g, 0.5 μg / g to 1000 mg / g, or 1 μg / g to 1000 mg / g.

[0072] The content of additional components in the composition of the present invention is less than 100% (w / w).

[0073] If the composition of the present invention contains fermented peas, the content of fermented peas in the composition of the present invention may be, for example, 0.1 mg / g or more, 0.2 mg / g or more, 0.5 mg / g or more, 1 mg / g or more, 2 mg / g or more, 5 mg / g or more, 10 mg / g or more, 20 mg / g or more, 50 mg / g or more, 100 mg / g or more, 200 mg / g or more, 500 mg / g or more, 1000 mg / g or more, 2000 mg / g or more, 5000 mg / g or more, 10000 mg / g or more, 2000 mg / g or more, or 50000 mg / g or more, and may also be 100000 mg / g or less, 50000 mg / g or less, 20000 mg / g or less, 10000 mg / g or less, 5000 mg / g or less, 2000 mg / g or less, 10000 mg / g or less, 2000 mg / g or less, 1000 mg / g or less, 500 The amount may be less than or equal to mg / g, less than or equal to 200 mg / g, less than or equal to 100 mg / g, less than or equal to 50 mg / g, less than or equal to 20 mg / g, less than or equal to 10 mg / g, less than or equal to 5 mg / g, less than or equal to 2 mg / g, less than or equal to 1 mg / g, less than or equal to 0.5 mg / g, or less than or equal to 0.2 mg / g, or any non-contradictory combination thereof. The content of fermented peas in the composition of the present invention may specifically be, for example, 0.1-0.2 mg / g, 0.2-0.5 mg / g, 0.5-1 mg / g, 1-2 mg / g, 2-5 mg / g, 5-10 mg / g, 10-20 mg / g, 20-50 mg / g, 50-100 mg / g, 100-200 mg / g, 200-500 mg / g, 500-1000 mg / g, 1000-2000 mg / g, 2000-5000 mg / g, 5000-10000 mg / g, 10000-20000 mg / g, 20000-50000 mg / g, or 50000-100000 mg / g. The content of fermented peas in the composition of the present invention may specifically be, for example, 0.1 to 100,000 mg / g, 0.2 to 20,000 mg / g, 0.5 to 5,000 mg / g, or 1 to 1,000 mg / g. Unless otherwise specified, "content of fermented peas" means the content of fermented peas converted to the content of the original culture, the content of fermented peas converted to the content of the original culture supernatant, or the content of fermented peas provided and distributed as a product.In other words, for example, "the content of fermented peas is 0.1 mg / g or more" means that, unless otherwise specified, at least one of the following is sufficient: the content of fermented peas converted to the content of the original culture, the content of fermented peas converted to the content of the original culture supernatant, and the content of fermented peas provided and distributed as a product. "Original culture" means a culture that has not undergone any concentration changes such as concentration or dilution after cultivation, and specifically, it may mean the culture at the end of cultivation. The original culture is also called the "original fermented product." "Original culture supernatant" means the supernatant of the culture that has not undergone any concentration changes such as concentration or dilution other than solid-liquid separation after cultivation, and specifically, it may mean the supernatant obtained by solid-liquid separation of the culture at the end of cultivation. Examples of fermented peas provided and distributed as a product include commercially available pea soy sauce. Note that a content exceeding 100% (w / w) (i.e., 1000 mg / g) means that the culture has been concentrated and included in the composition of the present invention. In other words, for example, a content of 2000 mg / g means that the culture is concentrated twice after cultivation and included in the composition of the present invention.

[0074] If the composition of the present invention contains pea protein, the content of pea protein in the composition of the present invention may be, for example, 0.05 mg / g or more, 0.1 mg / g or more, 0.2 mg / g or more, 0.5 mg / g or more, 1 mg / g or more, 2 mg / g or more, 5 mg / g or more, 10 mg / g or more, 20 mg / g or more, 50 mg / g or more, 100 mg / g or more, 200 mg / g or more, 500 mg / g or more, 700 mg / g or more, or 900 mg / g or more, and may be less than 1000 mg / g, 900 mg / g or less, 700 mg / g or less, 500 mg / g or less, 200 mg / g or less, 100 mg / g or less, 50 mg / g or less, 20 mg / g or less, 10 mg / g or less, 5 mg / g or less, 2 mg / g or less, 1 mg / g or less, 0.5 mg / g or less, 0.2 mg / g or less, or 0.1 The pea protein content in the composition of the present invention may be, for example, 0.05-0.1 mg / g, 0.1-0.2 mg / g, 0.2-0.5 mg / g, 0.5-1 mg / g, 1-2 mg / g, 2-5 mg / g, 5-10 mg / g, 10-20 mg / g, 20-50 mg / g, 50-100 mg / g, 100-200 mg / g, 200-500 mg / g, 500-700 mg / g, 700-900 mg / g, or 900 mg / g or more and less than 1000 mg / g. The pea protein content in the composition of the present invention may specifically be, for example, 0.05 mg / g or more and less than 1000 mg / g, 0.1 mg / g or more and less than 1000 mg / g, 0.2 mg / g or more and less than 1000 mg / g, or 0.5 mg / g or more and less than 1000 mg / g.

[0075] If the composition of the present invention contains yeast extract, the yeast extract content in the composition of the present invention may be, for example, 0.01 mg / g or more, 0.02 mg / g or more, 0.05 mg / g or more, 0.1 mg / g or more, 0.2 mg / g or more, 0.5 mg / g or more, 1 mg / g or more, 2 mg / g or more, 5 mg / g or more, 10 mg / g or more, or 20 mg / g. Above, 50 mg / g or more, 100 mg / g or more, 200 mg / g or more, 500 mg / g or more, 700 mg / g or more, It may be 900 mg / g or more, but may also be less than 1000 mg / g, 900 mg / g or less, 700 mg / g or less, 500 mg / g or less, 200 mg / g or less, 100 mg / g or less, 50 mg / g or less, 20 mg / g or less, 10 mg / g or less, 5 mg / g or less, 2 mg / g or less, 1 mg / g or less, 0.5 mg / g or less, 0.2 mg / g or less, 0.1 mg / g or less, 0.05 mg / g or less, or 0.02 mg / g or less, and any non-contradictory combination thereof is also acceptable. The yeast extract content in the composition of the present invention may specifically be, for example, 0.01-0.02 mg / g, 0.02-0.05 mg / g, 0.05-0.1 mg / g, 0.1-0.2 mg / g, 0.2-0.5 mg / g, 0.5-1 mg / g, 1-2 mg / g, 2-5 mg / g, 5-10 mg / g, 10-20 mg / g, 20-50 mg / g, 50-100 mg / g, 100-200 mg / g, 200-500 mg / g, 500-700 mg / g, 700-900 mg / g, or 900 mg / g or more and less than 1000 mg / g. The yeast extract content in the composition of the present invention may specifically be, for example, 0.01 mg / g or more and less than 1000 mg / g, 0.02 mg / g or more and less than 1000 mg / g, 0.05 mg / g or more and less than 1000 mg / g, or 0.1 mg / g or more and less than 1000 mg / g. "Yeast extract content" means the yeast extract content calculated based on the dry weight of the yeast extract.

[0076] In one embodiment, the composition of the present invention does not need to contain substantially any serum. "Substantially no serum in the composition of the present invention" may mean that the serum content in the composition of the present invention is 1% (w / w) or less, 0.1% (w / w) or less, 0.01% (w / w) or less, or 0.001% (w / w) or less, and may also include the case where the serum content in the composition of the present invention is 0 (zero) (i.e., the composition of the present invention does not contain any serum).

[0077] In one embodiment, the composition of the present invention does not need to substantially contain animal-derived albumin. "Substantially containing animal-derived albumin" may mean that the content of animal-derived albumin in the composition of the present invention is 1% (w / w) or less, 0.1% (w / w) or less, 0.01% (w / w) or less, or 0.001% (w / w) or less, and may also include the case where the content of animal-derived albumin in the composition of the present invention is 0 (zero) (i.e., the composition of the present invention does not contain animal-derived albumin).

[0078] The content of each component (i.e., the active ingredient and optionally additional components) in the composition of the present invention can be set, for example, to obtain the concentration of each component in the culture medium in the method of the present invention described later.

[0079] If the composition of the present invention contains two or more components, these components may be mixed together and contained in the composition of the present invention, or they may be contained separately, or separately in any combination. For example, the composition of the present invention may be provided as a set of a package of an active ingredient and a package of additional components. In such a case, the components contained in the set can be used in combination as appropriate at the time of use.

[0080] <3> Method of the present invention The present invention relates to a method for culturing animal cells, comprising the step of culturing animal cells in the presence of an active ingredient (i.e., lecithin). This step is also referred to as the "animal cell culture step."

[0081] By utilizing the active ingredient, specifically by culturing animal cells in the presence of the active ingredient, the growth of animal cells may be improved; that is, an effect of improving the growth of animal cells (growth-enhancing effect) may be obtained. In other words, one aspect of the method of the present invention (specifically, a method for culturing animal cells) may be, for example, a method for improving the growth of animal cells.

[0082] The purpose of culturing animal cells is not particularly limited.

[0083] In one embodiment, a product (e.g., cultured meat or the target substance) may be produced by culturing animal cells. That is, if animal cells can form cultured meat through culture, cultured meat can be produced by culturing the same cells. The cultured meat may be mainly composed of muscle tissue. Therefore, "animal cells can form cultured meat through culture" may mean, for example, that animal cells can differentiate through culture and form muscle tissue, and more specifically, that animal cells can differentiate into myotubes through culture and form muscle tissue. Furthermore, if animal cells have the ability to produce the target substance, the target substance can be produced by culturing the same cells. That is, one embodiment of the method of the present invention (specifically, a method for culturing animal cells) may be a method for producing a product that includes a step of culturing animal cells in the presence of an active ingredient. Furthermore, the animal cell culture step may specifically be a step of culturing animal cells in the presence of an active ingredient to produce a product. In the method for producing cultured meat, the cultured animal cells may form cultured meat. In the method for producing the target substance, the cultured animal cells may produce the target substance. When producing a product by culturing animal cells, the production of the product may be improved by utilizing an active ingredient. For example, the production of the product may be improved by improving the growth of animal cells through the use of an active ingredient. In other words, one aspect of the method of the present invention (specifically, a method for culturing animal cells) may be a method for improving the production of a product.

[0084] "Animal cells" means cells of animals. There are no particular restrictions on animal cells. Animal cells can be appropriately selected according to various conditions, such as the purpose of culturing the animal cells. For example, when producing cultured meat by culturing animal cells, there are no particular restrictions on animal cells as long as they can form cultured meat through cultivation. "Animals" may mean organisms classified in the animal kingdom (Animalia). Examples of animals include vertebrates and aquatic organisms. Examples of vertebrates include mammals, birds, reptiles, amphibians, and fish. Examples of vertebrates in particular include mammals, birds, and fish. Examples of vertebrates in particular include mammals. Examples of mammals include primates such as humans, monkeys, and chimpanzees; rodents such as hamsters, mice, rats, and guinea pigs; other terrestrial mammals such as cows, pigs, sheep, goats, rabbits, horses, deer, water buffalo, reindeer, donkeys, camels, dogs, and cats; and aquatic mammals such as whales, dolphins, reindeer, and sea lions. Examples of birds include chickens, turkeys, ducks, geese, guinea fowl, quail, and ostriches. Examples of fish include eels, tuna, grouper, sea bream, salmon, cod, and pufferfish. Examples of aquatic organisms include aquatic mammals and fish, as well as crustaceans such as shrimp and crabs, shellfish such as scallops and oysters, and other aquatic organisms such as squid and octopus. Examples of aquatic organisms include fish and crustaceans in particular. Examples of animals suitable for the production of cultured meat include meat-producing animals. Examples of meat-producing animals include those whose meat can be eaten from the animals listed above. Examples of meat-producing animals (especially mammals) include cattle, pigs, sheep, goats, and rabbits in particular. Examples of meat-producing animals (especially mammals) include cattle and pigs in particular. Examples of meat-producing animals (especially mammals) include cattle in particular. Examples of animals used for meat (especially birds) include chickens, turkeys, ducks, geese, guinea fowl, quail, and ostriches. Chickens are even more specifically mentioned as an example of animals used for meat (especially birds). Aquatic animals used for meat include the fish and crustaceans mentioned above. Eels, tuna, and shrimp are even more specifically mentioned as an example of animals used for meat (especially aquatic animals). There are no particular limitations on the tissues or cells from which animal cells are derived.Tissues or cells derived from animal cells include the ovaries, kidneys, adrenal glands, tongue epithelium, olfactory epithelium, pineal gland, thyroid gland, melanocytes, skin, spleen, liver, lungs, pancreas, uterus, stomach, colon, small intestine, large intestine, bladder, prostate, testes, thymus, muscle, connective tissue, bone, cartilage, vascular tissue, blood (including umbilical cord blood), bone marrow, heart, eyes, brain, and nervous tissue. Animal cells may be differentiated or undifferentiated. Specifically, animal cells include germ cells, somatic cells, stem cells, and progenitor cells. Germ cells include sperm and eggs. Somatic cells include myoblasts, fibroblasts, bone marrow cells, B lymphocytes, T lymphocytes, neutrophils, erythrocytes, platelets, macrophages, monocytes, osteocytes, pericytes, dendritic cells, adipocytes, mesenchymal cells, epithelial cells, epidermal cells (e.g., keratinocytes, corneocytes, etc.), endothelial cells, vascular endothelial cells, hepatocytes, chondrocytes, cumulus cells, nerve cells, glial cells, oligodendrocytes, microglia, astrocytes, cardiac cells, esophageal cells, muscle cells (e.g., smooth muscle cells, skeletal muscle cells), pancreatic beta cells, melanocytes, and mononuclear cells. Examples of stem cells include adult stem cells such as hematopoietic stem cells, satellite cells, neural stem cells, mesenchymal stem cells, mammary gland stem cells, olfactory mucosa stem cells, neural corona stem cells, hepatic stem cells, pancreatic stem cells, muscle stem cells, germline stem cells, intestinal stem cells, and hair follicle stem cells; pluripotent stem cells such as embryonic stem cells (ES cells), embryonic tumor cells, embryonic germline stem cells, and induced pluripotent stem cells (iPS cells); and cancer stem cells. Examples of progenitor cells include satellite cells, pancreatic progenitor cells, vascular progenitor cells, vascular endothelial progenitor cells, and hematopoietic progenitor cells (such as CD34-positive cells derived from umbilical cord blood). Suitable animal cells for the production of cultured meat include stem cells such as mesenchymal stem cells, embryonic stem cells (ES cells), and induced pluripotent stem cells (iPS cells), as well as myoblasts.

[0085] The culture of animal cells is carried out in the presence of the active ingredient. "The culture of animal cells is carried out in the presence of the active ingredient" may mean, for example, culturing animal cells in a culture medium containing the active ingredient. "The culture of animal cells is carried out in the presence of the active ingredient" may mean, for example, culturing animal cells in a culture medium containing the active ingredient. This may also mean that the active ingredients are supplied to the culture system during cell culture.

[0086] The active ingredient may be used, for example, in the form of the composition of the present invention for the culture of animal cells. The composition of the present invention may be used, for example, as a culture medium for the culture of animal cells. That is, for example, if the composition of the present invention is a culture medium, animal cells may be cultured in the composition of the present invention (i.e., the culture medium). In other words, "cultivating animal cells in a culture medium containing the active ingredient" may also include cultivating animal cells in the composition of the present invention, which is a culture medium. Furthermore, the composition of the present invention may be used, for example, as a culture medium additive for the culture of animal cells. That is, for example, if the composition of the present invention is a culture medium additive, the composition of the present invention (i.e., the culture medium additive) may be added to the culture medium, and animal cells may be cultured in the culture medium to which the composition of the present invention has been added. In other words, "cultivating animal cells in a culture medium containing the active ingredient" may also include cultivating animal cells in a culture medium to which the composition of the present invention, which is a culture medium additive, has been added. Furthermore, for example, the composition of the present invention or a culture medium to which it has been added may be supplied to the culture system during the culture of animal cells. That is, "the active ingredient is supplied to the culture system during the culture of animal cells" may also include supplying the composition of the present invention or a culture medium to which it has been added to the culture system during the culture of animal cells.

[0087] The culture medium composition and culture conditions are not particularly limited, except that the animal cell culture is carried out in the presence of the active ingredient, as long as the purpose of the animal cell culture is achieved. The culture medium composition and culture conditions can be appropriately set according to various conditions, such as the type of animal cell and the purpose of the animal cell culture. For example, when producing cultured meat by culturing animal cells, the culture medium composition and culture conditions are not particularly limited, as long as cultured meat can be formed by culturing animal cells. Animal cell culture can be carried out using, for example, the usual culture medium and conditions used for animal cell culture, either as they are or with appropriate modifications, except that the culture is carried out in the presence of the active ingredient. When producing cultured meat by culturing animal cells, animal cell culture can be carried out using, for example, the usual culture medium and conditions used for the formation of muscle tissue by differentiation of animal cells (e.g., production of cultured meat), either as they are or with appropriate modifications, except that the culture is carried out in the presence of the active ingredient.

[0088] Culture can be carried out, for example, using a liquid medium. Culture can be carried out by batch culture, fed-batch culture, continuous culture, or a combination thereof. Examples of continuous culture include perfusion culture and chemostat culture. The medium used at the start of culture is also called the "initial medium" or "basal medium." In fed-batch culture, the medium supplied to the culture system (e.g., initial medium) is also called the "feed medium." In continuous culture (which is not limited to perfusion culture), the medium supplied to the culture system (e.g., initial medium) is also called the "perfusion medium." The act of supplying fed-batch medium or perfusion medium to the culture system in fed-batch culture or continuous culture is simply called "medium supply." Medium supply may be carried out throughout the entire period of culture, or only during a part of the period of culture. Medium supply may also be carried out continuously or intermittently. During culture (especially continuous culture such as perfusion culture), the culture medium may be withdrawn. The culture medium may be withdrawn throughout the entire culture period or only during a portion of the culture period. The culture medium may be withdrawn continuously or intermittently. The culture medium may be withdrawn and the culture medium supplied simultaneously or separately. The culture vessel may be coated with a cell adhesion molecule such as fibronectin. Culture may be carried out three-dimensionally, for example, using scaffolding material. When producing cultured meat by culturing animal cells, culture may be carried out three-dimensionally, for example, using scaffolding material that matches the shape of the cultured meat to be formed.

[0089] The culture medium used for cultivation can be selected independently, for example, from basal medium, fed-batch medium, and perfusion medium.

[0090] The culture medium used may be a commercially available medium or a medium prepared as appropriate.

[0091] Culture media used for culturing include media containing components essential for the culture of animal cells (e.g., carbon sources, nitrogen sources, inorganic salts, etc.).

[0092] Examples of culture media used include Dulbecco's Modified Eagle's Medium (DMEM), Ham's Nutrient Mixture F12, DMEM / F12 medium, McCoy's 5A medium, Minimum Essential Medium (MEM), Eagle's Minimum Essential Medium (EMEM), alpha Modified Eagle's Minimum Essential Medium (αMEM), Roswell Park Memorial Institute (RPMI) 1640 medium, Iscove's Modified Dulbecco's Medium (IMDM), MCDB131 medium, William's Medium E, and Fischer's Medium.

[0093] Furthermore, the culture medium used for culturing (for example, a medium used for culturing stem cells (especially pluripotent stem cells)) may include, specifically, STEMPRO® hESC SFM medium (Life Technologies), mTeSR1 medium (STEMCELL Technologies), TeSR2 medium (STEMCELL Technologies), TeSR-E8 medium (STEMCELL Technologies), Essential 8 medium (Life Technologies), HEScGRO® Serum-Free Medium for hES cells (Millipore), PluriSTEM® Human ES / iPS Medium (EMD Millipore), and NutriStem® hESC XF medium. (Biological Industries Israel Beit-Haemek), NutriStem (trademark) XF / FF Culture Examples include Medium (Stemgent), AF NutriStem® hESC XF medium (Biological Industries Israel Beit-Haemek), S-medium (DS Pharma Biomedical Co., Ltd.), StemFit® AK03N medium (Ajinomoto Co., Inc.), hESF9 medium, hESF-FX medium, CDM medium, DEF-CS 500 Xeno-Free 3D Spheroid Culture Medium (Cellartis), StemFlex medium (Thermo Fisher Scientific), etc.

[0094] Other commercially available culture media include D-MEM (Dulbecco's Modified Eagle Medium), CELLiST Basal Media BASAL3, BASAL4P, BASAL10 (Ajinomoto Co., Inc.), Opti-MEM (Thermo Fisher Scientific), RPMI 1640 (Thermo Fisher Scientific), CD293 (Thermo Fisher Scientific), CHO-S-SFMII (Thermo Fisher Scientific), CHO-SF (Sigma-Aldrich), EX-CELL CD CHO (Sigma-Aldrich), EX-CELLTM302 (Sigma-Aldrich), IS CHO-CD (Irvine Scientific), IS CHO-CDXP (Irvine Scientific), and other animal cell culture media.

[0095] The culture medium described above may be used for cultivation after adding an active ingredient, for example.

[0096] Furthermore, the culture medium used for cultivation may be, for example, the composition of the present invention (specifically, the composition of the present invention which is a culture medium). That is, when the composition of the present invention is a culture medium, the composition of the present invention may be used as is, or as appropriate, to prepare a liquid culture medium of a desired composition and used for cultivation. For example, the composition of the present invention may be diluted with an aqueous medium such as water or an aqueous buffer to prepare a liquid culture medium and used for cultivation.

[0097] Furthermore, the culture medium used for cultivation may be, for example, a medium to which the composition of the present invention (specifically, the composition of the present invention as a culture medium additive) has been added. The culture medium to which the composition of the present invention has been added may be a commercially available culture medium or a culture medium prepared as appropriate.

[0098] The culture medium may contain various culture medium components. These components may include a carbon source, an amino acid source, Examples of culture medium components include peptides, proteins, vitamins, fatty acids, lipids, inorganic components, pH buffers, growth factors, cytokines, hormones, cell adhesion factors, extracellular matrix components, serum, pea-derived materials, yeast extract, antibiotics, and gene expression inducers. Any of these culture medium components may be essential or effective, for example, for the survival or proliferation of animal cells. Any of these culture medium components may be pre-contained in the culture medium as exemplified above, or may be added to the culture medium as exemplified above.

[0099] Examples of carbon sources include sugars such as glucose, fructose, sucrose, and maltose.

[0100] Amino acids can be considered sources of amino acids. Peptides and proteins can also be examples of amino acid sources. Examples of amino acids include glycine, alanine, valine, leucine, isoleucine, cysteine, methionine, phenylalanine, tyrosine, tryptophan, histidine, lysine, arginine, serine, threonine, aspartic acid, glutamic acid, asparagine, glutamine, proline, and ornithine. Amino acids may be in their L-form, for example.

[0101] Examples of peptides include dipeptides and tripeptides. Specifically, examples of peptides include glycylglycylglycine and soy peptides. The descriptions of amino acids can also be applied to the amino acids that make up peptides.

[0102] Examples of proteins include albumin and transferrin.

[0103] Examples of vitamins include vitamin A, vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B7, vitamin B9, vitamin B12, vitamin C, vitamin D, vitamin E, and vitamin K and their precursors.

[0104] Examples of fatty acids include oleic acid, arachidonic acid, and linoleic acid.

[0105] Cholesterol is an example of a lipid.

[0106] Inorganic components include sodium, potassium, calcium, magnesium, phosphorus, and various trace elements (e.g., Co, Cu, F, Fe, Mn, Mo, Ni, Se, Si, Ni, Bi, V, and Zn). Specifically, inorganic salts such as sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, and sodium dihydrogen phosphate are examples of inorganic components.

[0107] Examples of pH buffering agents include sodium bicarbonate, phosphates, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and N-[tris(hydroxymethyl)methyl]glycine (Tricine).

[0108] Growth factors include fibroblast growth factor (FGF) and hepatocytes. Examples of growth factors include hepatocyte growth factor (HGF), epidermal growth factor (EGF), transforming growth factor (TGF)-α, transforming growth factor (TGF)-β, vascular endothelial growth factor (VEGF), activin A, and insulin-like growth factor-1 (IGF-1). Basic fibroblast growth factor (bFGF) is an example of FGF. In particular, bFGF is a notable growth factor. That is, at least bFGF may be used as a component of the culture medium. That is, the culture medium may contain at least bFGF. Bovine basic fibroblast growth factor (bbFGF) is an example of bFGF. "Bovine basic fibroblast growth factor (bbFGF)" may mean bovine-derived bFGF. The term "bovine" in relation to bbFGF may refer to organisms of the genus Bos. An example of a Bos organism is Bos taurus. bbFGF is not limited to bFGF found in Bos organisms, but may also be a modified version thereof.

[0109] Interleukins are an example of cytokines.

[0110] Examples of hormones include dexamethasone, hydrocortisone, estradiol, progesterone, glucagon, and insulin.

[0111] Examples of cell adhesion factors or extracellular matrix components include Type I collagen and Type II collagen. Examples include collagen, fibronectin, laminin, poly-L-lysine, and poly-D-lysine.

[0112] Examples of antibiotics include amphotericin B, kanamycin, gentamicin, streptomycin, and penicillin.

[0113] In one embodiment, the culture medium does not need to contain substantially any serum. For example, the culture medium does not need to contain substantially any serum throughout the entire period of culture. "Substantially serum-free" means that the serum content in the culture medium is 1% (w / w) or less, 0.1% (w / w) or less, 0.01% (w / w) or less, or 0.001% (w / w) or less, and may also include the case where the serum content in the culture medium is 0 (zero) (i.e., the culture medium does not contain any serum).

[0114] In one embodiment, the culture medium does not need to substantially contain animal-derived albumin. Examples of animals from which albumin is derived include those exemplified for animal cells. The culture medium does not need to substantially contain animal-derived albumin, for example, throughout the entire culture period. "The culture medium does not substantially contain animal-derived albumin" may mean that the content of animal-derived albumin in the culture medium is 1% (w / w) or less, 0.1% (w / w) or less, 0.01% (w / w) or less, or 0.001% (w / w) or less, and may also include the case where the content of animal-derived albumin in the culture medium is 0 (zero) (i.e., the culture medium does not contain animal-derived albumin).

[0115] The various components such as the active ingredient may all be contained in the initial medium, the fed-batch medium, the perfusion medium, or a combination thereof. That is, during the culturing process, the various components such as the active ingredient may be supplied to the medium individually or in any combination. These components may all be supplied once or multiple times, or may be supplied continuously. The compositions (for example, the types and / or concentrations of the contained components) of the initial medium, the fed-batch medium, and the perfusion medium may be the same or different. That is, the types of components contained in the initial medium may be the same as or different from the types of components contained in the fed-batch medium or the perfusion medium. Also, the concentration of each component contained in the initial medium may be the same as or different from the concentration of each component contained in the fed-batch medium or the perfusion medium. For example, when the fed-batch medium is used for perfusion culture, the compositions of the initial medium and the fed-batch medium may be the same. Also, two or more fed-batch media or perfusion media having different compositions (for example, the types and / or concentrations of the contained components) may be used. For example, when the supply of the fed-batch medium or the perfusion medium is intermittently performed multiple times, the compositions of the fed-batch medium or the perfusion medium may be the same or different each time. Also, the various components such as the active ingredient may all be supplied to the medium in a form not contained in the fed-batch medium or the perfusion medium, such as powder etc.

[0116] The seeding amount of animal cells at the start of culture is, for example, in terms of the number of viable cells, 1×10 2 cells / mL or more, 1×10 3 cells / mL or more, 1×10 4 cells / mL or more, 1×10 5 cells / mL or more, 1×10 6 cells / mL or more, or 1×10 7 cells / mL or more, and may be 1×10 8 cells / mL or less, 1×10 7 cells / mL or less, 1×10 6 cells / mL or less, 1×10 5 cells / mL or less, 1×10 4 cells / mL or less, or 1×103 The number of cells / mL may be less than or equal to a certain number, and any non-inconsistent combination thereof may also be acceptable. Specifically, the seeding rate of animal cells at the start of culture should be, for example, 1 × 10⁻⁶ when converted to the number of viable cells. 2 ~1 × 10 3 cells / mL, 1 × 10 3 ~1 × 10 4 cells / mL, 1 × 10 4 ~1 × 10 5 cells / mL, 1 × 10 5 ~1 × 10 6 cells / mL, 1 × 10 6 ~1 × 10 7 cells / mL, or 1 × 10⁶ 7 ~1 × 10 8 It may also be cells / mL. Specifically, the seeding rate of animal cells at the start of culture should be, for example, 1 × 10⁻⁶ when converted to the number of viable cells. 2 ~1 × 10 8 cells / mL, 1 × 10 3 ~1 × 10 7 cells / mL, or 1 × 10⁶ 3 ~1 × 10 6 The number of live cells may be expressed as cells / mL. For example, the number of live and dead cells can be measured using a Vi-CELL autoanalyzer. TM It can be measured using XR (Beckman Coulter).

[0117] The culture may be carried out in a CO2-containing atmosphere, such as 5-15% CO2. The pH of the culture medium may be, for example, near neutral. "Near neutral" may mean, for example, pH 6-8, pH 6.5-7.5, or pH 6.8-7.2. The pH of the culture medium can be adjusted as needed during the culture. The pH of the culture medium can be adjusted using various alkaline or acidic substances such as ammonia gas, ammonia water, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, sodium hydroxide, potassium hydroxide, calcium hydroxide, and magnesium hydroxide. The culture temperature may be, for example, 30-38°C. The culture period may be, for example, 0.5 days or more, 1 day or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, 7 days or more, 8 days or more, 9 days or more, 10 days or more, 12 days or more, 15 days or more, or 20 days or more, or 60 days or less, 50 days or less, 40 days or less, 30 days or less, 25 days or less, 20 days or less, 15 days or less, 12 days or less, 10 days or less, 9 days or less, 8 days or less, or 7 days or less, or any non-contradictory combination thereof. Specifically, the culture period may be, for example, 1 to 60 days, 3 to 25 days, or 5 to 20 days. Cultivation may be continued, for example, until the product is produced to a desired extent.

[0118] The concentration of the active ingredient in the culture medium may be, for example, 0.1 μg / mL or higher, 0.2 μg / mL or higher, 0.5 μg / mL or higher, 1 μg / mL or higher, 2 μg / mL or higher, 5 μg / mL or higher, 7 μg / mL or higher, 10 μg / mL or higher, 15 μg / mL or higher, 20 μg / mL or higher, 30 μg / mL or higher, or 50 μg / mL or higher, or 100 μg / mL or lower, 50 μg / mL or lower, 30 μg / mL or lower, 20 μg / mL or lower, 15 μg / mL or lower, 10 μg / mL or lower, 7 μg / mL or lower, 5 μg / mL or lower, 2 μg / mL or lower, 1 μg / mL or lower, 0.5 μg / mL or lower, or 0.2 μg / mL or lower, or any non-contradictory combination thereof. The concentration of the active ingredient in the culture medium may be, specifically, for example, 0.1-0.2 μg / mL, 0.2-0.5 μg / mL, 0.5-1 μg / mL, 1-2 μg / mL, 2-5 μg / mL, 5-7 μg / mL, 7-10 μg / mL, 10-15 μg / mL, 15-20 μg / mL, 20-30 μg / mL, 30-50 μg / mL, or 50-100 μg / mL. The concentration of the active ingredient in the culture medium may be, specifically, for example, 0.1-100 μg / mL, 0.2-50 μg / mL, 0.5-30 μg / mL, or 1-15 μg / mL.

[0119] The culture of animal cells may be carried out in the presence of pea-derived material and / or yeast extract. The culture of animal cells may, in particular, be carried out in the presence of at least pea-derived material. "The culture of animal cells is carried out in the presence of a certain component (e.g., pea-derived material or yeast extract)" may mean, for example, that the animal cells are cultured in a medium containing the component. "The culture of animal cells is carried out in the presence of a certain component (e.g., pea-derived material or yeast extract)" may mean, for example, that the component is supplied to the culture system during the culture of animal cells.

[0120] If the culture medium contains pea ferment, the concentration of pea ferment in the culture medium may be, for example, 0.1 mg / mL or more, 0.2 mg / mL or more, 0.5 mg / mL or more, 1 mg / mL or more, 2 mg / mL or more, 5 mg / mL or more, 10 mg / mL or more, 20 mg / mL or more, or 50 mg / mL or more, or 100 mg / mL or less, 50 mg / mL or less, 20 mg / mL or less, 10 mg / mL or less, 5 mg / mL or less, 2 mg / mL or less, 1 mg / mL or less, 0.5 mg / mL or less, or 0.2 mg / mL or less, or any non-contradictory combination thereof. The concentration of pea ferment in the culture medium may specifically be, for example, 0.1-0.2 mg / mL, 0.2-0.5 mg / mL, 0.5-1 mg / mL, 1-2 mg / mL, 2-5 mg / mL, 5-10 mg / mL, 10-20 mg / mL, 20-50 mg / mL, or 50-100 mg / mL. The concentration of pea ferment in the culture medium may specifically be, for example, 0.1-100 mg / mL, 0.2-50 mg / mL, 0.5-20 mg / mL, or 1-10 mg / mL. Unless otherwise specified, "concentration of pea ferment" means the concentration of pea ferment converted to the concentration of the original culture, the concentration of pea ferment converted to the concentration of the original culture supernatant, or the concentration of pea ferment provided and distributed as a product. In other words, for example, "the concentration of pea ferment is 0.1 mg / mL or higher" means that, unless otherwise specified, at least one of the following must be 0.1 mg / mL or higher: the concentration of pea ferment converted to the concentration of the original culture, the concentration of pea ferment converted to the concentration of the original culture supernatant, and the concentration of pea ferment provided and distributed as a product. For example, if a culture that has been concentrated twice after cultivation is added to the culture medium to a concentration of 0.1 mg / mL, the concentration of pea ferment in the culture medium will be 0.2 mg / mL when converted to the concentration of the original culture.

[0121] If the culture medium contains pea protein, the concentration of pea protein in the medium may be, for example, 0.05 mg / mL or more, 0.1 mg / mL or more, 0.2 mg / mL or more, 0.5 mg / mL or more, 1 mg / mL or more, 2 mg / mL or more, 5 mg / mL or more, 7 mg / mL or more, 10 mg / mL or more, 15 mg / mL or more, 20 mg / mL or more, or 30 mg / mL or more, or 50 mg / mL or less, 30 mg / mL or less, 20 mg / mL or less, 15 mg / mL or less, 10 mg / mL or less, 7 mg / mL or less, 5 mg / mL or less, 2 mg / mL or less, 1 mg / mL or less, 0.5 mg / mL or less, 0.2 mg / mL or less, or 0.1 mg / mL or less, or any non-contradictory combination thereof. The concentration of pea protein in the culture medium may be, specifically, for example, 0.05-0.05 mg / mL, 0.1-0.1 mg / mL, 0.2-0.2 mg / mL, 0.5-0.5 mg / mL, 1-1 mg / mL, 2-2 mg / mL, 5-5 mg / mL, 7-7 mg / mL, 10-10 mg / mL, 15-15 mg / mL, 20-20 mg / mL, or 30-50 mg / mL. The concentration of pea protein in the culture medium may be, specifically, for example, 0.05-50 mg / mL, 0.1-30 mg / mL, 0.2-20 mg / mL, or 0.5-15 mg / mL.

[0122] If the culture medium contains yeast extract, the concentration of yeast extract in the culture medium may be, for example, 0.01 mg / mL or more, 0.02 mg / mL or more, 0.05 mg / mL or more, 0.1 mg / mL or more, 0.2 mg / mL or more, 0.5 mg / mL or more, 1 mg / mL or more, 2 mg / mL or more, or 5 mg / mL or more, or 10 mg / mL or less, 5 mg / mL or less, 2 mg / mL or less, 1 mg / mL or less, 0.5 mg / mL or less, 0.2 mg / mL or less, 0.1 mg / mL or less, 0.05 mg / mL or less, or 0.02 mg / mL or less, or any non-contradictory combination thereof. The concentration of yeast extract in the culture medium may specifically be, for example, 0.01-0.02 mg / mL, 0.02-0.05 mg / mL, 0.05-0.1 mg / mL, 0.1-0.2 mg / mL, 0.2-0.5 mg / mL, 0.5-1 mg / mL, 1-2 mg / mL, 2-5 mg / mL, or 5-10 mg / mL. The concentration of yeast extract in the culture medium may specifically be, for example, 0.01-10 mg / mL, 0.02-5 mg / mL, 0.05-2 mg / mL, or 0.1-1 mg / mL. "Yeast extract concentration" means the concentration of yeast extract calculated based on the dry weight of the yeast extract.

[0123] The active ingredients and other components may be present in the culture medium for the entire duration of the culture, or for only a portion of the culture duration. In other words, "the culture is carried out in a culture medium containing a certain component" means that the component is present in the culture medium for at least a portion of the culture duration, or that the component is present in the culture medium for the entire duration of the culture. This is not required. The active ingredients and other components may, for example, be contained in the culture medium at the start of cultivation, or they may be supplied to the culture medium after cultivation has begun. Furthermore, the active ingredients and other components may, for example, be contained in the culture medium at the start of cultivation, and then be supplied to the culture medium again after cultivation has begun (for example, after the active ingredients have been consumed).

[0124] The active ingredients and other components may, for example, be contained in the culture medium at the concentrations exemplified above for the entire duration of the culture, or they may be contained in the culture medium at the concentrations exemplified above for only a portion of the culture duration. In other words, "the culture is carried out in a culture medium containing a certain component at a certain concentration," "a certain component is contained in the culture medium at a certain concentration during the culture," or "the concentration of a certain component in the culture medium during the culture is at a certain concentration" means that it is sufficient for the concentration of the component in the culture medium to be within that range for at least a portion of the culture duration, and it is not necessary for the concentration of the component in the culture medium to be within that range for the entire duration of the culture. The active ingredients and other components may, for example, be contained in the culture medium at the concentrations exemplified above at the start of the culture, or they may be supplied to the culture medium after the start of the culture to reach the concentrations exemplified above. Furthermore, the active ingredients and other components may, for example, be contained in the culture medium at the concentrations exemplified above at the start of the culture, and then be supplied to the culture medium again after the start of the culture (for example, after the consumption of the component) to reach the concentrations exemplified above.

[0125] The length of "a portion of the culture period" is not particularly limited, as long as the objective of the present invention can be achieved (for example, an improvement in growth can be obtained). The length of "a portion of the culture period" can be appropriately set according to various conditions such as the type of animal cells and the length of the culture period. "A portion of the period" may be, for example, a period of 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, 95% or more, 97% or more, or 99% or more of the total culture period. Alternatively, "a portion of the period" may be, for example, a period of 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, 95% or more, 97% or more, or 99% or more of the total culture period after the animal cells have differentiated into myotubes. Furthermore, “a certain period” may be, for example, a period of 0.5 days or more, 1 day or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, 7 days or more, 8 days or more, 9 days or more, 10 days or more, 12 days or more, or 15 days or more.

[0126] Furthermore, the concentrations of various components, such as active ingredients, in the culture medium may all be set to the concentrations exemplified above, for example, as average values ​​over a specific period during cultivation. That is, "cultivation is carried out in a culture medium containing a certain component at a certain concentration," "a certain component is contained in the culture medium at a certain concentration," or "the concentration of a certain component in the culture medium at a certain concentration" may mean that the average value of the concentration of that component in the culture medium over a specific period during cultivation is within that range. "Average value of the concentration of a certain component in the culture medium over a specific period during cultivation" is not particularly limited as long as it allows for the capture of fluctuations in the concentration of that component during a specific period during cultivation, but for example, it may mean the average value of the concentration of that component in the culture medium measured every 60 minutes, every 30 minutes, every 20 minutes, or every 10 minutes over a specific period during cultivation. "A specific period during cultivation" can refer to the entire period of cultivation or a part of the period of cultivation. "A part of the period of cultivation" is as described above.

[0127] The active ingredients and other components may be supplied to the culture medium throughout the entire culture period, or only for a portion of the culture period. The "portion of the culture period" is as described above. The active ingredients and other components may be supplied to the culture medium continuously, or intermittently. The active ingredients and other components may be supplied to the culture medium daily, or every few days.

[0128] The concentrations of various components, such as active ingredients, in the fed-batch medium or perfusion medium may all be within the range of the concentrations of those components in the medium as exemplified above. The concentrations of the active ingredients and other components may be, for example, 1 or more, 1.1 or more, 1.3 or more, 1.5 or more, 2 or more, 3 or more, 5 or more, 7 or more, 10 or more, 15 or more, or 20 or more times the concentration of the component in the culture medium as exemplified above, or 100 or less, 70 or less, 50 or less, 30 or less, 20 or less, 15 or less, 10 or less, 7 or less, 5 or less, 3 or less, or 2 or less, and any non-contradictory combination thereof is also acceptable. The concentrations of various components, such as active ingredients, in the fed-batch or perfusion medium may, specifically, be 1 to 2 times, 1.1 to 2 times, 1.3 to 2 times, 1.5 to 2 times, 2 to 3 times, 3 to 5 times, 5 to 7 times, 7 to 10 times, 10 to 15 times, 15 to 20 times, 20 to 30 times, 20 to 50 times, 20 to 70 times, or 20 to 100 times the concentration of the component in the medium exemplified above. The concentrations of various components, such as active ingredients, in the fed-batch or perfusion medium may, specifically, be 1 to 100 times, 2 to 50 times, or 5 to 20 times the concentration of the component in the medium exemplified above.

[0129] The concentrations of active ingredients and other components can all be measured by known methods used for the detection or identification of compounds, for example. Such methods include HPLC, UPLC, LC / MS, GC / MS, and NMR.

[0130] Animal cells can be cultured in the manner described above. In one embodiment, a product (e.g., cultured meat or a target substance) may be obtained by culturing animal cells in the manner described above. If a target substance is produced, it may accumulate in the culture, for example, in the culture medium, on the cell surface, inside the cell, or a combination thereof. The target substance may be recovered from the culture as appropriate. That is, the method for producing the target substance may further include a step of recovering the target substance.

[0131] <4> Use of active ingredients Furthermore, the present invention discloses the use of the active ingredients in the applications exemplified above. Specifically, the present invention discloses, for example, the use of the active ingredients for culturing animal cells, the use of the active ingredients for improving the growth of animal cells, and the use of the active ingredients in the manufacture of compositions of the present invention, such as compositions for animal cell culture.

[0132] Furthermore, the present invention discloses active ingredients for use in the applications exemplified above. Specifically, the present invention discloses, for example, active ingredients for use in the culture of animal cells, active ingredients for use in improving the growth of animal cells, and active ingredients for use in the production of compositions for animal cell culture. [Examples]

[0133] The present invention will be described in more detail below with reference to non-limiting embodiments.

[0134] (1) Evaluation of soy lecithin activity using bovine muscle stem cells in serum-free medium (1-1) Preparation of bovine muscle stem cells Muscle tissue was collected from beef shank meat intended for consumption, and CD56-positive cells were isolated using a magnetically activated cell separation system (MACS) to create bovine muscle stem cells.

[0135] (1-2) Evaluation of soy lecithin activity using bovine muscle stem cells in serum-free medium The bovine muscle stem cells obtained in (1-1) were suspended in serum medium. The serum medium used was Advanced DMEM (Thermo Fisher Scientific, Cat No. 12491015) with 20% FBS (Thermo Fisher Scientific, Cat No. 10270106), 10% horse serum (Thermo Fisher Scientific, Cat No. 16050-130), and 1% GlutaMAX. TM A cell suspension containing Supplement (Thermo Fisher Scientific, Cat No. 35050061) was used. 25300 cells / 2 mL / cm³ were added to a 6-well plate (BD FALCON, Cat#353046) coated with fibronectin (Corning, Cat#F2006). 2 The cells were seeded to achieve the specified cell density, and the culture medium was changed every other day. On the fourth day of culture, the cells were subcultured in serum-free medium or serum-free medium containing soy lecithin, and the culture medium was changed every other day. StemFit was used as the serum-free medium. R Basic03 (Ajinomoto) contains 5 ng / mL HGF (Peprotech, Cat#294HGN005), 20 ng / mL EGF (Sigma, Cat#E9644), 10 ng / mL bFGF (Peprotech, Cat#AF-450-62), and Bacto TM Yeast extract (Thermo Fisher Scientific, Cat No. 212750) was added to the culture medium. For serum-free medium containing soy lecithin, serum-free medium with 10 mg / L soy lecithin (Nacalai Tesque, Cat#20335-52), 10 mg / L soy lecithin (Tsuji Oil Co., Ltd., product name: SLP-White), or 10 mg / L α-glycerophosphatidylcholine (αGPC) (Bioactives Japan, product name: GlycerophosphatidylCholine) added was used. Table 1 summarizes the group numbers and culture media. Cell counting and subculturing were performed every 4 days, and the growth rate of two consecutive subculturings from day 4 to day 12 was calculated.

[0136] The results are shown in Figures 1-2. 10 mg / L soy lecithin (Nacalai Tesque) and 10 mg / L soy lecithin (Tsuji Oil Co.) were found to promote cell proliferation in bovine muscle stem cells, but αGPC... No cell proliferation-promoting effect was observed in bovine muscle stem cells.

[0137] [Table 1]

[0138] (1-3) Activity evaluation of pea soy sauce and combinations of pea protein and soy lecithin The bovine muscle stem cells obtained in (1-1) were suspended in serum medium. The serum medium is as described in (1-2). The cell suspension was diluted to 1 μg / cm³. 2 4200 cells / 2 mL / cm³ in a 6-well plate coated with fibronectin. 2 The cells were seeded to achieve the specified cell density, and the culture medium was changed every other day. On the fourth day of culture, the cells were subcultured in serum-free medium or serum-free medium containing pea-derived material, and the culture medium was changed every other day. StemFit was used as the serum-free medium. R Basic03 (Ajinomoto) was used, supplemented with 10 ng / mL bbFGF, 20 ng / mL EGF, and 5 ng / mL HGF. As a serum-free medium containing pea-derived substances, serum-free medium was used, supplemented with 0.33% pea soy sauce (San-J, product name: No Soy Gluten Free Tamari), 2.5 g / L pea protein (Roquette, product name: NUTRALYS S85F), 2.5 g / L pea protein (HENGYUAN BIOTECHNOLOGY, product name: HYPP-A85%), 0.33% pea soy sauce and 2.5 g / L pea protein, or 0.33% pea soy sauce, 2.5 g / L pea protein, and soy lecithin (Tsuji Oil Co., Ltd., product name: SLP-White). Table 2 summarizes the group numbers and media. Cell counting and subculturing were performed every four days, and the growth rate of two consecutive subculturings from day 4 to day 12 was calculated.

[0139] The results are shown in Figures 3-4. Combining pea soy sauce and pea protein significantly promoted cell proliferation. Furthermore, combining pea soy sauce, pea protein, and soy lecithin further promoted cell proliferation.

[0140] [Table 2]

Claims

1. A composition for animal cell culture containing lecithin.

2. The composition according to claim 1, which is a composition for improving the growth of animal cells.

3. The composition according to claim 1 or 2, which is a culture medium or a culture medium additive.

4. The composition according to claim 3, wherein the culture medium is a basal medium, a fed-batch medium, or a perfusion medium.

5. The composition according to claim 1 or 2, wherein the lecithin is soy lecithin.

6. Furthermore, the composition according to claim 1 or 2, further containing a pea-derived material.

7. The composition according to claim 6, wherein the pea-derived material is a fermented pea product and / or pea protein.

8. The composition according to claim 7, wherein the fermented pea product is pea soy sauce.

9. The composition according to claim 1 or 2, wherein the animal is a mammal, a bird, a fish, or a crustacean.

10. The composition according to claim 1 or 2, wherein the animal is an animal used for meat.

11. The composition according to claim 1 or 2, wherein the animal is a cow.

12. The composition according to claim 1 or 2, which is substantially free of serum.

13. The composition according to claim 1 or 2, which substantially does not contain animal-derived albumin.

14. A method for producing the product, A method comprising the step of culturing animal cells in the presence of lecithin.

15. The method according to claim 14, wherein the product is cultured meat.

16. A method for culturing animal cells, A method comprising the step of culturing animal cells in the presence of lecithin.

17. A method for improving the growth of animal cells, A method comprising the step of culturing animal cells in the presence of lecithin.

18. The method according to any one of claims 14 to 17, wherein the lecithin is soy lecithin.

19. Claim 1, wherein the concentration of lecithin in the culture medium during the culture is 0.1 to 100 μg / mL The method described in any one of items 4 to 17.

20. The method according to any one of claims 14 to 17, wherein the cultivation is carried out in the presence of a pea-derived material.

21. The method according to claim 20, wherein the pea-derived material is a fermented pea product and / or pea protein.

22. The method according to claim 21, wherein the fermented pea product is pea soy sauce.

23. The composition according to claim 21, wherein the concentration of the fermented pea product in the culture medium during cultivation is 0.1 to 100 mg / mL.

24. The composition according to claim 21, wherein the concentration of the pea protein in the culture medium during the culture is 0.05 to 50 mg / mL.

25. The method according to any one of claims 14 to 17, wherein the animal is a mammal, a bird, a fish, or a crustacean.

26. The method according to any one of claims 14 to 17, wherein the animal is an animal intended for meat consumption.

27. The method according to any one of claims 14 to 17, wherein the animal is a cow.

28. The method according to any one of claims 14 to 17, wherein the culture is carried out in a culture medium that is substantially free of serum.

29. The method according to any one of claims 14 to 17, wherein the culture is carried out in a culture medium that is substantially free of animal-derived albumin.

Images