Method for promoting differentiation of animal cell

Abstract

The problem addressed is to provide a method for promoting differentiation of animal cells and a technique related thereto. The problem is solved by culturing animal cells in the presence of a taste substance.

Description

Method for promoting differentiation of animal cells

[0001] The present invention relates to a method for promoting differentiation of animal cells and related techniques.

[0002] Culture media containing serum, such as fetal bovine serum, are widely used for culturing animal cells. However, from the viewpoints of safety and economy, the development of serum-free culture media is desired. For example, serum-free culture media in which serum is replaced with alternative components, such as growth factors, have been developed.

[0003] It is also known that intracellular calcium ions are involved in muscle differentiation (Non-Patent Document 1).

[0004] It is also known that tastants increase the intracellular calcium ion concentration in taste cells through binding to taste receptors on the cells (Non-Patent Document 2).

[0005] Sansrity Sinha et al., Ca2+ as a coordinator of skeletal muscle differentiation, fusion and contraction. FEBS J. 2022 Nov;289(21):6531-6542. Raise Ahmad and Julie E Dalziel, G Protein-Coupled Receptors in Taste Physiology and Pharmacology. Front Pharmacol. 2020 Nov 30;11:587664.

[0006] An objective of the present invention is to provide a method for promoting differentiation of animal cells and techniques related thereto.

[0007] The present inventors have discovered that differentiation of animal cells can be promoted in the absence of serum by using taste substances, and have completed the present invention.

[0008] That is, the present invention can be exemplified as follows: [1] A composition for promoting differentiation of animal cells, containing a taste substance. [2] The composition (specifically described in [1]) which is a medium or a medium additive. [3] The composition (specifically described in [1] or [2]) which is a medium that is a basal medium, a feed medium, or a perfusion medium. [4] The composition (specifically described in [1] or [2]) which is a medium that is a sweet substance, a salty substance, a sour substance, a bitter substance, an umami substance, a fatty substance, a rich substance, a succinic acid receptor agonist, and a GABA B The composition (specifically, the composition according to any one of [1] to [3]) is at least one component selected from the group consisting of receptor agonists. [5] The sweet substance is at least one component selected from the group consisting of sugars, sugar alcohols, high-intensity sweeteners, and amino acids; the salty substance is at least one inorganic salt; the sour substance is at least one component selected from the group consisting of organic acids, inorganic acids, and amino acids; the bitter substance is at least one component selected from the group consisting of amino acids, polyphenols, alkaloids, terpenoids, inorganic salts, diphenidol, and salicin; the umami substance is at least one component selected from the group consisting of amino acids, nucleic acids, organic acids, amide compounds, and high-potency umami substances; the fatty substance is at least one component selected from the group consisting of fatty acids and fatty acid receptor agonists; the kokumi substance is at least one component selected from the group consisting of γ-glutamyl peptides and calcium-sensing receptor agonists; the succinate receptor agonist is succinic acid; and the GABA B The composition (specifically, the composition described in [4]), wherein the receptor agonist is GABA and / or SKF97541. [6] The composition (specifically, the composition described in [4]), wherein the tastant is a sweet taste receptor agonist, a salty taste receptor agonist, a sour taste receptor agonist, a bitter taste receptor agonist, an umami taste receptor agonist, a fatty acid receptor agonist, a calcium-sensing receptor agonist, a succinate receptor agonist, and GABA. B[7] The composition (specifically described in any one of [1] to [5]) wherein the tastant is at least one component selected from the group consisting of receptor agonists. [7] The composition (specifically described in any one of [6]) wherein the sweet receptor is T1R2 / 3, the salty receptor is ENaC, TMC4, or TMC6, the sour receptor is GPR91, PKD2L1, PKD1L3, or Otop1, the bitter receptor is T2R, the umami receptor is T1R1 / 3, the fatty acid receptor is GPR120, and the succinic acid receptor is GPR91. [8] The composition (specifically described in any one of [1] to [7]) wherein the tastant is at least one component selected from the group consisting of ATF100, sucralose, cinacalcet, TUG-891, difenidol, salicin, SKF97541, and succinic acid. [9] The composition (specifically, according to any one of [1] to [8]) wherein the animal is a chicken and the tastant is salicin.

[10] The composition (specifically, according to any one of [1] to [9]) wherein the tastant is a tastant other than sugar, amino acid, and calcium.

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

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

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

[11] ) wherein the animal is a meat animal.

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

[12] ) wherein the animal is a cow or a chicken.

[14] The composition (specifically, according to any one of [1] to

[13] ) wherein the differentiation is muscle differentiation.

[15] The composition (specifically, according to any one of [1] to

[14] ) wherein the cells are satellite cells or myoblasts.

[16] The composition (specifically, the composition described in any one of [1] to

[15] ) that is substantially free of serum.

[17] The composition (specifically, the composition described in any one of [1] to

[16] ) that is substantially free of animal-derived albumin.

[18] A method for producing cultured meat, comprising the steps of culturing animal cells in the presence of a taste substance and differentiating the animal cells.

[19] A method for promoting differentiation of animal cells, comprising the step of culturing animal cells in the presence of a tastant, and differentiating the animal cells.

[20] The tastant is a sweet substance, a salty substance, a sour substance, a bitter substance, an umami substance, a fatty substance, a substance that imparts kokumi, a succinic acid receptor agonist, or GABA. B The method (specifically, the method described in

[18] or

[19] ) above, wherein the component is at least one component selected from the group consisting of receptor agonists.

[21] The sweet substance is at least one component selected from the group consisting of sugars, sugar alcohols, high-intensity sweeteners, and amino acids; the salty substance is at least one inorganic salt; the sour substance is at least one component selected from the group consisting of organic acids, inorganic acids, and amino acids; the bitter substance is at least one component selected from the group consisting of amino acids, polyphenols, alkaloids, terpenoids, inorganic salts, difenidol, and salicin; the umami substance is at least one component selected from the group consisting of amino acids, nucleic acids, organic acids, amide compounds, and high-potency umami substances; the fatty substance is at least one component selected from the group consisting of fatty acids and fatty acid receptor agonists; the kokumi-imparting substance is at least one component selected from the group consisting of γ-glutamyl peptides and calcium-sensing receptor agonists; the succinic acid receptor agonist is succinic acid; and the GABA B

[22] The method (specifically, the method described in

[20] ) above, wherein the receptor agonist is GABA and / or SKF97541.

[23] The method (specifically, the method described in

[20] ) above, wherein the tastant is a sweet taste receptor agonist, a salty taste receptor agonist, a sour taste receptor agonist, a bitter taste receptor agonist, an umami taste receptor agonist, a fatty acid receptor agonist, a calcium-sensing receptor agonist, a succinate receptor agonist, and a GABA receptor agonist. BThe method (specifically, the method described in any one of

[18] to

[21] ) wherein the sweet taste receptor is T1R2 / 3, the salty taste receptor is ENaC, TMC4, or TMC6, the sour taste receptor is GPR91, PKD2L1, PKD1L3, or Otop1, the bitter taste receptor is T2R, the umami taste receptor is T1R1 / 3, the fatty acid receptor is GPR120, and the succinate receptor is GPR91.

[24] The method (specifically, described in any one of

[18] to

[23] ) wherein the tastant is at least one component selected from the group consisting of ATF100, sucralose, cinacalcet, TUG-891, difenidol, salicin, SKF97541, and succinic acid.

[25] The method (specifically, described in any one of

[18] to

[24] ) wherein the animal is a chicken and the tastant is salicin.

[26] The method (specifically, described in any one of

[18] to

[25] ) wherein the tastant is a tastant other than sugars, amino acids, and calcium.

[27] The method (specifically, described in any one of

[18] to

[26] ) wherein the concentration of the tastant in the medium during the culture is 0.05 nM to 500 mM.

[28] The method (specifically, the method described in

[20] ) has one, two, three, four, five, six, seven, eight, or nine properties selected from the following (1) to (8): (1) the concentration of the sweet substance in the medium during the culture is 100 nM to 2 mM; (2) the concentration of the salty substance in the medium during the culture is 0.05 nM to 500 mM; (3) the concentration of the sour substance in the medium during the culture is 10 nM to 100 μM; (4) the concentration of the bitter substance in the medium during the culture is 10 nM to 200 μM or 5 μM to 100 mM; (5) the concentration of the umami substance in the medium during the culture is 0.1 nM to 2 μM; (6) the concentration of the fatty substance in the medium during the culture is 10 nM to 100 μM; (7) The concentration of the kokumi-imparting substance in the culture medium during the culture is 0.1 nM to 2 μM; (8) The concentration of the succinate receptor agonist in the culture medium during the culture is 10 nM to 100 μM; (9) The GABA in the culture medium during the culture. BThe concentration of the receptor agonist is 10 nM to 100 μM.

[29] The method (specifically, the method described in

[22] ) has one, two, three, four, five, six, seven, eight, or nine properties selected from the following (1) to (8): (1) the concentration of the sweet taste receptor agonist in the culture medium during the culture is 100 nM to 2 mM; (2) the concentration of the salty taste receptor agonist in the culture medium during the culture is 0.05 nM to 500 mM; (3) the concentration of the sour taste receptor agonist in the culture medium during the culture is 10 nM to 100 μM; (4) the concentration of the bitter taste receptor agonist in the culture medium during the culture is 10 nM to 200 μM or 5 μM to 100 mM; (5) the concentration of the umami taste receptor agonist in the culture medium during the culture is 0.1 nM to 2 μM; (6) the concentration of the fatty acid receptor agonist in the culture medium during the culture is 10 (7) the concentration of the calcium-sensing receptor agonist in the culture medium during the culture is 0.1 nM to 2 μM; (8) the concentration of the succinate receptor agonist in the culture medium during the culture is 10 nM to 100 μM; (9) the concentration of the GABA receptor agonist in the culture medium during the culture is 0.1 nM to 2 μM; BThe concentration of the receptor agonist is 10 nM to 100 μM.

[30] The method (specifically described in any of

[18] to

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

[31] The method (specifically described in any of

[18] to

[30] ) wherein the animal is a meat-producing animal.

[32] The method (specifically described in any of

[18] to

[31] ) wherein the animal is a cow or chicken.

[33] The method (specifically described in any of

[18] to

[32] ) wherein the differentiation is muscle differentiation.

[34] The method (specifically described in any of

[18] to

[33] ) wherein the cells are satellite cells or myoblasts.

[35] The method (specifically described in any of

[18] to

[34] ) wherein the culture is performed in a medium substantially free of serum.

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

[18] to

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

[0009] 1 shows the differentiation of bovine muscle stem cells in serum-free medium in the presence of various taste substances, and 2 shows the differentiation of chicken muscle stem cells in serum-free medium in the presence of various taste substances.

[0010] The present invention will be described in detail below.

[0011] <1> Active ingredient In the present invention, a taste substance is used as an active ingredient.

[0012] Tasting substances are also called "active ingredients."

[0013] The active ingredient can be used for culturing animal cells. Specifically, animal cells can be cultured in the presence of the active ingredient. More specifically, animal cells can be cultured in the presence of the active ingredient to differentiate the animal cells.

[0014] The use of an active ingredient, specifically, culturing animal cells in the presence of the active ingredient, may promote differentiation of animal cells, i.e., an effect of promoting differentiation of animal cells may be obtained. This effect is also referred to as a "differentiation-promoting effect." In other words, the active ingredient may have the function of promoting differentiation of animal cells. This function is also referred to as a "differentiation-promoting function." In other words, culturing animal cells in the presence of the active ingredient may promote differentiation of animal cells compared to culturing animal cells in the absence of the active ingredient. Examples of culturing animal cells in the absence of an active ingredient include 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 differentiation-promoting effect may be obtained, for example, when animal cells are cultured in the substantial absence of serum (i.e., when animal cells are cultured in a medium substantially free of serum). The differentiation-promoting effect may be obtained, for example, when animal cells are cultured in the substantial absence of animal-derived albumin (i.e., when animal cells are cultured in a medium substantially free of animal-derived albumin). The differentiation-promoting effect may be obtained, for example, by culturing animal cells in the substantial absence of serum and animal-derived albumin (i.e., by culturing animal cells in a medium substantially free of serum and animal-derived albumin). Examples of differentiation include muscle differentiation. Examples of differentiation promotion include an increase in the differentiation rate and an increase in the proportion of differentiated cells. The differentiation-promoting effect can be confirmed by measuring and comparing the differentiation of animal cells (e.g., the differentiation rate and the proportion of differentiated cells) in the presence and absence of the active ingredient. The differentiation of animal cells can be measured, for example, using the expression of proteins that serve as indicators of differentiation. For example, in muscle differentiation, Sprouty1, Pax7, MyoD, Myogenin, and Myosin heavy chain (MHC) can be expressed in sequence as differentiation progresses (Manuel Schmidt et al. Adult stem cells at work: regenerating skeletal muscle. Cell Mol Life Sci. 2019 Jul;76(13):2559-2570.). Furthermore, differentiation of animal cells, for example, in the case of muscle differentiation, can be measured using the Fusion Index as an indicator.

[0015] In one aspect, a product may be produced by culturing animal cells. Examples of the product include cultured meat. When a product is produced by culturing animal cells, the use of an active ingredient may improve product production. That is, culturing animal cells in the presence of an active ingredient may improve product production compared to culturing animal cells in the absence of the active ingredient. For example, the use of an active ingredient may promote differentiation of animal cells, thereby improving product production. That is, one aspect of the differentiation-promoting effect may be the effect of improving product production. The effect of improving product production may be obtained, for example, when animal cells are cultured in the substantial absence of serum (i.e., when animal cells are cultured in a medium that is substantially free of serum). The effect of improving product production may be obtained, for example, when animal cells are cultured in the substantial absence of animal-derived albumin (i.e., when animal cells are cultured in a medium that is substantially free of animal-derived albumin). The effect of improving product production may be obtained, for example, when animal cells are cultured under conditions substantially free of serum and animal-derived albumin (i.e., when animal cells are cultured in a medium substantially free of serum and animal-derived albumin). Examples of improved production include improved production amount and improved production rate. When a product is produced by culturing animal cells, a product may be produced that has been imparted or enhanced with a flavor corresponding to a flavor component.

[0016] The active ingredient may be one kind of tasting substance or a combination of two or more kinds of tasting substances. There is no particular limitation on the combination of tasting substances selected as the active ingredient.

[0017] "Tastant" may refer to a substance involved in taste. Examples of tastant substances include substances that exhibit a taste and substances that impart or enhance a taste. "Tastant substance" may refer to a substance that exhibits a taste by itself. "Substance that imparts or enhances a taste" may refer to a substance that imparts a taste to a subject or enhances the taste of a subject, regardless of whether it exhibits a taste by itself. "Imparting a taste to a subject" and "enhancing the taste of a subject" may both mean increasing the taste of a subject. "Imparting a taste to a subject" may mean, for example, imparting a taste to a subject that does not exhibit a taste. "Enhancing the taste of a subject" may mean, for example, enhancing the taste of a subject that exhibits a taste. The type of taste is not particularly limited. Examples of taste include sweet taste, salty taste, sour taste, bitter taste, umami (umami), fatty taste, and kokumi (kokumi). Substances that exhibit a taste include substances that exhibit a sweet taste (also called "sweet substances"), substances that exhibit a salty taste (also called "salty substances"), substances that exhibit a sour taste (also called "sour substances"), substances that exhibit a bitter taste (also called "bitter substances"), substances that exhibit an umami taste (also called "umami substances"), and substances that exhibit a fatty taste (also called "fatty substances"). Substances that impart or enhance a taste include substances that impart or enhance kokumi (also called "kokumi substances").

[0018] Sweet substances include sugars, sugar alcohols, high-intensity sweeteners, and amino acids. Sweet sugars include monosaccharides such as glucose, fructose, galactose, and isomerized sugar; and disaccharides or higher oligosaccharides such as sucrose, lactose, maltose, trehalose, palatinose, lactofructose oligosaccharides, maltooligosaccharides, isomaltooligosaccharides, and fructooligosaccharides. Sweet sugar alcohols include erythritol, xylitol, sorbitol, mannitol, maltitol, and lactitol. High-intensity sweeteners (all of which have a sweet taste) include aspartame (APM), acesulfame K (Ace-K), advantame, sucralose, neotame, and saccharin. Sweet amino acids include glycine, alanine, threonine, serine, glutamine, proline, and asparagine.

[0019] Sweet substances also include components that activate sweet receptors (also called "sweet receptor agonists"). Sweet receptors include T1R2 / 3. "T1R2 / 3" may refer to a heterodimer of T1R2 and T1R3. For example, any of the sweet substances listed above may be sweet receptor agonists. Specifically, sucralose may be a T1R2 / 3 agonist.

[0020] Examples of salty substances include inorganic salts, such as chlorides, sodium chloride, potassium chloride, and ammonium chloride.

[0021] Salty substances also include components that activate salty taste receptors (also called "salty taste receptor agonists"). Examples of salty taste receptors include ENaC (epithelial sodium channel), TMC4 (transmembrane channel-like 4), and TMC6 (transmembrane channel-like 6). For example, any of the salty substances listed above can be salty taste receptor agonists.

[0022] Sour substances include organic acids, inorganic acids, and amino acids. Organic acids that exhibit a sour taste include acetic acid, lactic acid, malic acid, citric acid, tartaric acid, gluconic acid, succinic acid, and adipic acid. Inorganic acids that exhibit a sour taste include phosphoric acid. Amino acids that exhibit a sour taste include glutamic acid and aspartic acid.

[0023] Sour taste substances also include components that activate sour taste receptors (also called "sour taste receptor agonists"). Examples of sour taste receptors include GPR91, PKD2L1, PKD1L3, and Otopetrin 1 (OTOP-1). For example, any of the sour taste substances listed above can be sour taste receptor agonists. Specifically, succinic acid can be a GPR91 agonist.

[0024] Bitter substances include amino acids, polyphenols, alkaloids, terpenoids, and inorganic salts. Bitter amino acids include branched-chain amino acids such as valine, leucine, and isoleucine; aromatic amino acids such as phenylalanine, tryptophan, and tyrosine; and other amino acids such as methionine, cysteine, lysine, arginine, histidine, and ornithine. Bitter polyphenols include catechin, naringin, and chlorogenic acid. Bitter alkaloids include caffeine, theobromine, and nicotine. Bitter terpenoids include humulone, cucurbitacin, momordicine, limonin, and bile acids. Bitter inorganic salts include potassium salts such as potassium chloride and magnesium salts such as magnesium chloride. Bitter substances also include other organic compounds such as diphenidol and salicin.

[0025] Bitter substances also include components that activate bitter taste receptors (also called "bitter taste receptor agonists"). Bitter taste receptors include T2Rs. T2Rs include hT2R1, hT2R2, hT2R3, hT2R5, hT2R7, hT2R8, hT2R9, hT2R10, hT2R13, hT2R14, hT2R16, hT2R38, hT2R39, hT2R40, hT2R41, hT2R43, hT2R44, hT2R45, hT2R46, hT2R47, Human T2Rs such as hT2R48, hT2R49, hT2R50, and hT2R60, mT2R102, mT2R103, mT2R104, mT2R105, mT2R106, mT2R108, mT2R109, mT2R110, mT2R113, mT2R115, mT2R116, mT2R118, mT2R119, and mT2R120. Mouse T2Rs such as R120, mT2R121, mT2R122, mT2R123, mT2R125, mT2R126, mT2R130, mT2R134, mT2R135, mT2R137, mT2R138, mT2R139, mT2R140, mT2R143, and mT2R144, as well as bT2R3, bT2R4, and bT2R5. , bT2R8, bT2R10, bT2R12, bT2R16, bT2R31, bT2R38, bT2R39, bT2R40, bT2R41, bT2R46, bT2R55, bT2R60, bT2R65, bT2R109, and other bovine T2Rs, and chicken T2Rs such as ggT2R1, ggT2R2, and ggT2R7. For example, any of the bitter substances exemplified above can be bitter receptor agonists. Specifically, for example, difenidol can be an agonist of hT2R1, hT2R4, hT2R7, hT2R10, hT2R13, hT2R14, hT2R16, hT2R38, hT2R39, hT2R40, hT2R43, hT2R44, hT2R46, hT2R47, hT2R49, and mT2R108. Also specifically, for example, salicin can be an agonist of hT2R16 and mT2R126.

[0026] Examples of umami substances include amino acids, nucleic acids, organic acids, amide compounds, and high-potency umami substances. Examples of amino acids that impart umami include glutamic acid and aspartic acid. Examples of nucleic acids that impart umami include inosinic acid, guanylic acid, and xanthylic acid. Examples of organic acids that impart umami include succinic acid. Examples of amide compounds that impart umami include the amide compounds described in WO2014 / 208751 (specifically, compounds having two amide bonds). Examples of amide compounds described in WO2014 / 208751 include ATF100 (N-(R)-1-(2-(pyridin-2-yl)ethylcarbamoyl)-2-(benzyloxy)ethyl-4-methoxy-3-methylbenzamide). A "high-potency umami substance" may refer to a substance with a taste potency 50 times or more that of monosodium glutamate (MSG) (WO 2013 / 133404). For example, a substance whose minimum concentration (threshold) at which umami can be recognized in a human taste test is 0.00024% (w / w) or less is a high-potency umami substance. Also, for example, a substance whose concentration at which umami is recognized in a human taste test is 0.01% (w / w) or less is a high-potency umami substance.High-potency umami substances (all of which exhibit umami taste) include amides described in WO2005 / 015158A2, WO2005 / 041684A2, and US2006-0045953A1; compounds comprising linked organic moieties described in WO2006 / 084184A2; compounds comprising linked heteroaryl moieties described in WO2006 / 084186A2; isosorbide derivatives described in US2009-0220662A1; alkyldienamides as described in US 2004-0202619A1 and US 2004-0202760A1; saturated and unsaturated N-alkamides as described in US 2006-0057268A1 and US 2006-0068071A1; N-alkamides; oxalamides described in WO2011 / 095533A1; and compounds described in WO2011 / 020908A1, WO2011 / 004016A1, WO2010 / 094679A1, WO2007 / 027095A1, WO2008 / 046895A1, US2009-0110796A1, WO2007 / 124152A2, US2009-0311401A1, EP2168442A2, WO2006 / 009425, and EP1989944A1.

[0027] Umami substances also include components that activate umami receptors (also called "umami receptor agonists"). Examples of umami receptors include T1R1 / 3. "T1R1 / 3" may refer to a heterodimer of T1R1 and T1R3. For example, all of the umami substances listed above can be umami receptor agonists. Specifically, ATF100 can be a T1R1 / 3 agonist.

[0028] Fatty substances include fatty acids, and fatty acids that impart a fatty taste include C8 to C24 fatty acids. Fatty acids that impart a fatty taste (specifically, C8-C24 fatty acids) include caprylic acid (8:0), capric acid (10:0), lauric acid (12:0), myristic acid (14:0), palmitic acid (16:0), stearic acid (18:0), arachidic acid (20:0), behenic acid (22:0), lignoceric acid (24:0), palmitoleic acid (16:1), oleic acid (18:1), linoleic acid (18:2), linolenic acid (18:3), eicosenoic acid (20:1), erucic acid (22:1), arachidonic acid (20:4), eicosapentaenoic acid (EPA; 20:5), and docosahexaenoic acid (DHA; 22:6) (numbers in parentheses indicate the number of carbon atoms and double bonds).

[0029] Fatty acid substances also include components that activate fatty acid receptors (also referred to as "fatty acid receptor agonists"). Fatty acid receptors include GPR40, GPR41, GPR43, GPR83, and GPR120. For example, any of the fatty acid substances listed above may be fatty acid receptor agonists. Fatty acid receptor agonists, such as GPR120 agonists, include 4-[(4-Fluoro-4'-methyl[1,1'-biphenyl]-2-yl)methoxy]-benzenepropanoic acid, 2-[3-fluoro-(pyridin-3-yloxy)phenyl]-1,2-benzisothiazol-3(2H)-one-1,1-dioxide, and TUG-891 (3-[4-[[5-fluoro-2-(4-methylphenyl)phenyl]methoxy]phenyl]propanoic acid).

[0030] Examples of kokumi substances include γ-glutamyl peptides. γ-glutamyl peptides include γ-glutamyl tripeptides represented by the general formula: γ-Glu-X-Gly (X represents an amino acid or an amino acid derivative) and γ-glutamyl dipeptides represented by the general formula: γ-Glu-Y (Y represents an amino acid or an amino acid derivative). In the above general formula, "γ-" means that X or Y is bound via the carboxyl group at the γ-position of glutamic acid. As the γ-glutamyl peptide, one type of γ-glutamyl peptide may be used, or two or more types of γ-glutamyl peptides may be used in combination.

[0031] Specific examples of amino acids selected as X and Y include neutral amino acids such as Gly, Ala, Val, Leu, Ile, Ser, Thr, Cys, Met, Asn, Gln, Pro, and Hyp, acidic amino acids such as Asp and Glu, basic amino acids such as Lys, Arg, and His, aromatic amino acids such as Phe, Tyr, and Trp, and other amino acids such as Orn, Sar, Cit, Nva, Nle, Abu, Tau, Hyp, t-Leu, Cl, Aib, Pen, and Hse.

[0032] In the present invention, the abbreviations for amino acid residues refer to the following amino acids. (1) Gly: glycine (2) Ala: alanine (3) Val: valine (4) Leu: leucine (5) Ile: isoleucine (6) Met: methionine (7) Phe: phenylalanine (8) Tyr: tyrosine (9) Trp: tryptophan (10) His: histidine (11) Lys: lysine (12) Arg: arginine (13) Ser: serine (14) Thr: threonine (15) Asp: aspartic acid (16) Glu: glutamic acid (17) Asn: asparagine (18) Gln: glutamine (19) Cys: cysteine ​​(20) Pro: proline (21) Orn: ornithine (22) Sar: sarcosine (23) Cit: Citrulline (24) Nva: Norvaline (25) Nle: Norleucine (26) Abu: α-aminobutyric acid (27) Tau: Taurine (28) Hyp: Hydroxyproline (29) t-Leu: Tert-leucine (30) Cl: Cycloleucine (31) Aib: α-aminoisobutyric acid (also known as 2-methylalanine) (32) Pen: Penicillamine (33) Hse: Homoserine

[0033] The amino acid derivative refers to various derivatives of the amino acids described above. Examples of the amino acid derivative include special amino acids, unnatural amino acids, amino alcohols, and amino acids in which one or more functional groups such as a terminal carbonyl group, a terminal amino group, and a cysteine ​​thiol group are substituted with various substituents. Specific examples of the substituent include an alkyl group, an acyl group, a hydroxyl group, an amino group, an alkylamino group, a nitro group, a sulfonyl group, and various protecting groups. Specific examples of the amino acid derivative include Arg(NO ２ ): N-γ-nitroarginine, Cys(SNO): S-nitrocysteine, Cys(S-Me): S-methylcysteine, Cys(S-allyl): S-allylcysteine, Val-NH ２: valinamide, Val-ol: valinol (also known as 2-amino-3-methyl-1-butanol), Met(O): methionine sulfoxide, and Cys(S-Me)(O): S-methylcysteine ​​sulfoxide.

[0034] Specific examples of γ-glutamyl peptides include γ-Glu-Val-Gly, γ-Glu-Cys-Gly, γ-Glu-Abu-Gly, γ-Glu-Nva-Gly, γ-Glu-Abu, and γ-Glu-Nva.

[0035] Unless otherwise specified, the amino acids and amino acid derivatives constituting the γ-glutamyl peptide are all in the L-configuration.

[0036] As kokumi substance, the component (also referred to as " CaSR agonist ") that activates calcium-sensing receptor (CaSR) can also be mentioned.For example, all of the kokumi substance exemplified above can be CaSR agonist.In addition, as CaSR agonist, can be mentioned cation such as calcium, gadolinium, etc.; basic peptide such as polyarginine, polylysine, etc.; polyamine such as putrescine, spermine, spermidine, etc.; protein such as protamine, etc.; amino acid such as phenylalanine, etc.; cinacalcet, aroma component such as methional, isovaleraldehyde, etc.

[0037] Tasting substances also include components that activate succinic acid receptors (also referred to as "succinic acid receptor agonists"). Examples of succinic acid receptors include GPR91. Examples of succinic acid receptor agonists, such as GPR91 agonists, include succinic acid. Succinic acid can be, for example, a sour taste substance. That is, succinic acid receptors, such as GPR91, can also be, for example, sour taste receptors.

[0038] GABA is a taste-producing substance. B Components that activate receptors (GABA B GABA receptor agonists are also included. B Receptor agonists include GABA (γ-aminobutyric acid) and SKF97541 (3-aminopropyl(methyl)phosphinic acid).B Receptor agonists can function, for example, to enhance the taste of other tastants or objects containing them. GABA, for example, can enhance the salty taste.

[0039] Taste substances include compounds that activate taste receptors (also called "taste receptor agonists"). Taste receptors include sweet taste receptors, salty taste receptors, sour taste receptors, bitter taste receptors, umami taste receptors, fatty acid receptors, CaSRs, succinic acid receptors, and GABA receptors. B Receptors include sweet taste receptors, salty taste receptors, sour taste receptors, bitter taste receptors, umami taste receptors, fatty acid receptors, CaSRs, succinate receptors, and GABA receptors. B The receptors and their agonists are described above.

[0040] Whether a substance activates a receptor can be confirmed, for example, by contacting the receptor with the substance and measuring receptor activation due to contact with the substance. Contact between the receptor and the substance and measurement of receptor activation due to this can be performed, for example, with reference to a method for screening taste substances using receptor response as an indicator (e.g., JP 2018-014999 A and WO 2011 / 081186 A). The receptor may be used by being supported on a cell, such as an animal cell. When using a receptor supported on a cell, "receptor activation" may refer to activation of the cell supporting the receptor. Receptor-supporting cells can be obtained, for example, by introducing a gene encoding the receptor into the cell and expressing it. In the case of a receptor that functions by forming a heterocomplex, receptor-supporting cells can be obtained, for example, by introducing genes encoding each of the subunits constituting the heterocomplex into the cell and co-expressing them. Receptor activation can be measured, for example, using an increase in cell membrane current or an increase in intracellular calcium ion concentration as an indicator (see, for example, JP 2018-014999 A and WO 2011 / 081186 A). That is, for example, if the cell membrane current or intracellular calcium ion concentration increases under conditions where the receptor is in contact with a substance compared to conditions where the receptor is not in contact with the substance, it can be determined that the substance has activated the receptor. Examples of methods for measuring cell membrane current include patch clamping and voltage clamping. Examples of methods for measuring intracellular calcium ion concentration include calcium imaging using a calcium indicator.

[0041] The amino acids selected as taste substances may be in the D-form, the L-form, or a combination thereof, as long as the object of the present invention can be achieved (for example, a differentiation-promoting effect can be obtained). The ratio of D- to L-forms in the combination is not particularly limited. The ratio of D- or L-forms in the combination may be, for example, 20 to 80%, 30 to 70%, 40 to 60%, or 45 to 55% in molar ratio. The amino acids selected as taste substances may particularly be in the L-form. Note that when a D- or L-form amino acid is selected, it is sufficient to use the D- or L-form of the amino acid as an active ingredient, and this does not preclude the use of the L- or D-form of the amino acid in combination.

[0042] Taste substances include, in particular, sweet substances, salty substances, sour substances, bitter substances, umami substances, fatty substances, rich substances, succinic acid receptor agonists, GABA B Receptor agonists are included.

[0043] Taste substances include, in particular, sweet substances, sour substances, bitter substances, umami substances, fatty substances, rich substances, succinic acid receptor agonists, GABA B Receptor agonists are included.

[0044] Taste substances include, in particular, sweet taste receptor agonists, salty taste receptor agonists, sour taste receptor agonists, bitter taste receptor agonists, umami taste receptor agonists, fatty acid receptor agonists, CaSR agonists, succinic acid receptor agonists, and GABA receptor agonists. B Receptor agonists are included.

[0045] Taste substances include, in particular, sweet taste receptor agonists, sour taste receptor agonists, bitter taste receptor agonists, umami taste receptor agonists, fatty acid receptor agonists, CaSR agonists, succinic acid receptor agonists, GABA receptor agonists, B Receptor agonists are included.

[0046] Taste substances include, inter alia, ATF100, sucralose, cinacalcet, TUG-891, diphenidol, salicin, SKF97541, and succinic acid.

[0047] In one embodiment, examples of tastants include tastants other than ATF100, sucralose, cinacalcet, TUG-891, diphenidol, SKF97541, and succinic acid.For example, when the animal cells are avian cells such as chicken, examples of tastants include tastants other than ATF100, sucralose, cinacalcet, TUG-891, diphenidol, SKF97541, and succinic acid.Tastants other than ATF100, sucralose, cinacalcet, TUG-891, diphenidol, SKF97541, and succinic acid include those exemplified above except ATF100, sucralose, cinacalcet, TUG-891, diphenidol, SKF97541, and succinic acid. Taste substances other than ATF100, sucralose, cinacalcet, TUG-891, difenidol, SKF97541, and succinic acid include, in particular, sweet substances, salty substances, sour substances, bitter substances, umami substances, fatty substances, rich substances, succinic acid receptor agonists, and GABA B Among receptor agonists, those other than ATF100, sucralose, cinacalcet, TUG-891, diphenidol, SKF97541, and succinic acid are included. Taste substances other than ATF100, sucralose, cinacalcet, TUG-891, diphenidol, SKF97541, and succinic acid are more particularly sweet substances, sour substances, bitter substances, umami substances, fatty substances, rich substances, succinic acid receptor agonists, and GABA B Among receptor agonists, those other than ATF100, sucralose, cinacalcet, TUG-891, difenidol, SKF97541, and succinic acid are included. Taste substances other than ATF100, sucralose, cinacalcet, TUG-891, difenidol, SKF97541, and succinic acid include, in particular, sweet taste receptor agonists, salty taste receptor agonists, sour taste receptor agonists, bitter taste receptor agonists, umami taste receptor agonists, fatty acid receptor agonists, CaSR agonists, succinic acid receptor agonists, and GABA receptor agonists. BAmong receptor agonists, those other than ATF100, sucralose, cinacalcet, TUG-891, difenidol, SKF97541, and succinic acid are included. Taste substances other than ATF100, sucralose, cinacalcet, TUG-891, difenidol, SKF97541, and succinic acid are more particularly sweet taste receptor agonists, sour taste receptor agonists, bitter taste receptor agonists, umami taste receptor agonists, fatty acid receptor agonists, CaSR agonists, succinic acid receptor agonists, and GABA receptor agonists. B Among receptor agonists, those other than ATF100, sucralose, cinacalcet, TUG-891, diphenidol, SKF97541, and succinic acid are included. Taste substances other than ATF100, sucralose, cinacalcet, TUG-891, diphenidol, SKF97541, and succinic acid include, in particular, salicin.

[0048] In one embodiment, the tastants particularly include tastants other than sugars, amino acids, organic acids, and calcium. In other words, in one embodiment, the present invention may exclude cases in which the active ingredient is one or more tastants selected from the group consisting of sugars, amino acids, organic acids, and calcium. Tastants other than sugars, amino acids, organic acids, and calcium include those exemplified above other than sugars, amino acids, organic acids, and calcium. Tastants other than sugars, amino acids, organic acids, and calcium particularly include sweet substances, salty substances, sour substances, bitter substances, umami substances, fatty substances, rich substances, succinic acid receptor agonists, and GABA. B Among receptor agonists, those other than sugars, amino acids, organic acids, and calcium are included. Taste substances other than sugars, amino acids, organic acids, and calcium include, more particularly, sweet substances, sour substances, bitter substances, umami substances, fatty substances, kokumi substances, succinic acid receptor agonists, and GABA receptor agonists. BAmong receptor agonists, those other than sugars, amino acids, organic acids, and calcium are included. Taste substances other than sugars, amino acids, organic acids, and calcium include, in particular, sweet taste receptor agonists, salty taste receptor agonists, sour taste receptor agonists, bitter taste receptor agonists, umami taste receptor agonists, fatty acid receptor agonists, CaSR agonists, succinate receptor agonists, and GABA receptor agonists. B Among receptor agonists, those other than sugars, amino acids, organic acids, and calcium are included. Taste substances other than sugars, amino acids, organic acids, and calcium include, more particularly, sweet taste receptor agonists, sour taste receptor agonists, bitter taste receptor agonists, umami taste receptor agonists, fatty acid receptor agonists, CaSR agonists, succinate receptor agonists, and GABA receptor agonists. B Among the receptor agonists are sugars, amino acids, organic acids, and those other than calcium.

[0049] In one embodiment, the tastants particularly include tastants other than glucose, amino acids, organic acids, and calcium. In other words, in one embodiment, the present invention may exclude cases in which the active ingredient is one or more tastants selected from the group consisting of glucose, amino acids, organic acids, and calcium. Tastants other than glucose, amino acids, organic acids, and calcium include those exemplified above other than glucose, amino acids, organic acids, and calcium. Tastants other than glucose, amino acids, organic acids, and calcium particularly include sweet substances, salty substances, sour substances, bitter substances, umami substances, fatty substances, rich substances, succinic acid receptor agonists, and GABA. B Among receptor agonists, those other than glucose, amino acids, organic acids, and calcium are included. Taste substances other than glucose, amino acids, organic acids, and calcium include, more particularly, sweet substances, sour substances, bitter substances, umami substances, fatty substances, kokumi substances, succinic acid receptor agonists, and GABA receptor agonists. BAmong receptor agonists, those other than glucose, amino acids, organic acids, and calcium are included. Taste substances other than glucose, amino acids, organic acids, and calcium include, in particular, sweet taste receptor agonists, salty taste receptor agonists, sour taste receptor agonists, bitter taste receptor agonists, umami taste receptor agonists, fatty acid receptor agonists, CaSR agonists, succinate receptor agonists, and GABA receptor agonists. B Among receptor agonists, those other than glucose, amino acids, organic acids, and calcium are included. More particularly, taste substances other than glucose, amino acids, organic acids, and calcium include sweet taste receptor agonists, sour taste receptor agonists, bitter taste receptor agonists, umami taste receptor agonists, fatty acid receptor agonists, CaSR agonists, succinate receptor agonists, and GABA receptor agonists. B Among the receptor agonists are those other than glucose, amino acids, organic acids, and calcium.

[0050] Furthermore, the medium components described below may include those that fall under the category of taste substances. In one embodiment, taste substances particularly include taste substances other than the medium components described below. In other words, in one embodiment, the present invention may exclude cases in which the active ingredient is composed of the medium components described below. Taste substances other than the medium components described below include those exemplified above other than the medium components described below. Taste substances other than the medium components described below particularly include sweet substances, salty substances, sour substances, bitter substances, umami substances, fatty substances, rich substances, succinic acid receptor agonists, and GABA B Among the receptor agonists, those other than the medium components described below can be mentioned. The taste substances other than the medium components described below can more particularly be sweet substances, sour substances, bitter substances, umami substances, fatty substances, rich substances, succinic acid receptor agonists, and GABA receptor agonists. BAmong the receptor agonists, those other than the medium components described below can be mentioned. Examples of taste substances other than the medium components described below include sweet taste receptor agonists, salty taste receptor agonists, sour taste receptor agonists, bitter taste receptor agonists, umami taste receptor agonists, fatty acid receptor agonists, CaSR agonists, succinate receptor agonists, and GABA receptor agonists. B Among the receptor agonists, those other than the medium components described below can be mentioned. More particularly, the taste substances other than the medium components described below include sweet taste receptor agonists, sour taste receptor agonists, bitter taste receptor agonists, umami taste receptor agonists, fatty acid receptor agonists, CaSR agonists, succinate receptor agonists, and GABA receptor agonists. B Examples of receptor agonists include those other than the medium components described below.

[0051] When an active ingredient can form a salt, the active ingredient may be used in its free form, its salt, or a combination thereof. That is, unless otherwise specified, the term "active ingredient" may refer to the active ingredient in its free form, its salt, or a combination thereof. "Free form" means a form in which no salt is formed. Also, when an active ingredient can form a hydrate, the active ingredient may be used in its non-hydrate form, its hydrate, or a combination thereof. That is, the term "active ingredient" (e.g., "active ingredient in its free form" or "salt of the active ingredient") may include both the non-hydrate and the hydrate unless otherwise specified. The active ingredient may be in any form, such as an ion, when used.

[0052] The salt is not particularly limited as long as it is acceptable for the purpose of the present invention. For example, when producing a product for consumption by culturing animal cells, an orally ingestible salt may be selected. For example, salts of acidic groups such as carboxyl groups include ammonium salts, salts with alkali metals such as sodium and potassium, salts with alkaline earth metals such as calcium and magnesium, aluminum salts, zinc salts, salts with organic amines such as triethylamine, ethanolamine, morpholine, pyrrolidine, piperidine, piperazine, and dicyclohexylamine, and salts with basic amino acids such as arginine and lysine. Specific examples of salts of basic groups such as amino groups include salts with inorganic acids such as hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, and hydrobromic acid; salts with organic carboxylic acids such as acetic acid, citric acid, benzoic acid, maleic acid, fumaric acid, tartaric acid, succinic acid, tannic acid, butyric acid, hybenzic acid, pamoic acid, enanthic acid, decanoic acid, teoclic acid, salicylic acid, lactic acid, oxalic acid, mandelic acid, malic acid, methylmalonic acid, and adipic acid; and salts with organic sulfonic acids such as methanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid. For example, examples of salts of succinic acid include sodium salts. One type of salt may be used, or two or more types of salts may be used in combination.

[0053] The active ingredient may be a commercially available product or may be obtained by appropriate manufacturing. The method for manufacturing the active ingredient is not particularly limited. The active ingredient can be manufactured, for example, by chemical synthesis, enzymatic reaction, fermentation, extraction, or a combination thereof. The active ingredient may be purified to a desired degree or may not be purified. That is, the active ingredient may be a purified product, or a material containing the active ingredient. For example, the active ingredient may be a material containing the active ingredient at a content of 1% (w / w) or more, 5% (w / w) or more, 10% (w / w) or more, 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.

[0054] When a material containing an active ingredient is used, the amount of the active ingredient (e.g., content (concentration) or amount used) is calculated based on the amount of the active ingredient itself in the material. When the active ingredient forms a salt or hydrate, the amount of the active ingredient (e.g., content (concentration) or amount used) is calculated based on the mass of the salt or hydrate converted to the mass of an equimolar unhydrated free form.

[0055] <2> Composition of the Present Invention The composition of the present invention is a composition containing an active ingredient (i.e., a taste substance).

[0056] The composition of the present invention can be used, for example, for culturing animal cells. That is, the composition of the present invention may be a composition for animal cell culture. The composition of the present invention can be used, for example, for culturing animal cells in the manner described in the method of the present invention described below.

[0057] 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 medium, feed medium, and perfusion medium. "Basal medium" may refer to the medium used at the start of culture. Basal medium is also called "initial medium." "Fed-batch medium" may refer to the medium supplied to a culture system after the start of culture in fed-batch culture. "Perfusion medium" may refer to the medium supplied to a culture system after the start of culture in continuous culture (this is not limited to perfusion culture).

[0058] The composition of the present invention (specifically, a composition for animal cell culture) may be, for example, a medium additive. The term "medium additive" may refer to a composition that is added to a medium before use. Examples of media to which the medium additive is added include basal medium, feed medium, and perfusion medium.

[0059] The use of the composition of the present invention may promote differentiation of animal cells, i.e., an effect of promoting differentiation of animal cells (differentiation-promoting effect) may be obtained. For example, a differentiation-promoting effect may be obtained by culturing animal cells in a medium containing the composition of the present invention. Furthermore, for example, a differentiation-promoting effect may be obtained by culturing animal cells in a medium containing the composition of the present invention, which is a medium additive. In other words, the composition of the present invention may have a function of promoting differentiation of animal cells (differentiation-promoting function). Thus, the composition of the present invention may be used to promote differentiation of animal cells. That is, the composition of the present invention may be, for example, a composition for promoting differentiation of animal cells.

[0060] In one aspect, a product (e.g., cultured meat) may be produced by culturing animal cells. When a product is produced by culturing animal cells, the use of the composition of the present invention may improve production of the product. For example, the use of the composition of the present invention may promote differentiation of animal cells, thereby improving production of the product. In other words, one aspect of the composition of the present invention (specifically, a composition for animal cell culture) may be a composition for improving production of a product.

[0061] The composition of the present invention may consist of an active ingredient, or may contain ingredients other than the active ingredient. Ingredients other than the active ingredient are also referred to as "additional ingredients." That is, the active ingredient may be used as is or in combination with additional ingredients in the composition of the present invention. The additional ingredients may be a single ingredient, or two or more ingredients may be used in combination.

[0062] That is, the present invention provides a method for producing the composition of the present invention, which comprises using an active ingredient. The manner of use of the active ingredient in the production of the composition of the present invention is not particularly limited, as long as a composition containing the active ingredient is obtained. Use of the active ingredient in the production of the composition of the present invention includes blending the active ingredient into the composition. "Bundling an active ingredient into a composition" may mean containing the active ingredient in the composition. "Bundling an active ingredient into a composition" may specifically mean combining the active ingredient with an additional ingredient.

[0063] The additional components are not particularly limited as long as they can achieve the object of the present invention (e.g., a differentiation-promoting effect can be obtained). The additional components can be appropriately selected depending on various conditions, such as the type of animal cell and the mode of use of the composition of the present invention. Examples of the additional components include medium components. Medium components will be described later. For example, at least when the composition of the present invention is a medium, the composition of the present invention may contain medium components.

[0064] The composition of the present invention may be formulated as appropriate, for example. When formulating, additives may be used as appropriate. That is, the additional components include additives used in formulation. Examples of additives include excipients, binders, disintegrants, lubricants, stabilizers, flavoring agents, diluents, and surfactants. The additives can be selected as appropriate depending on various conditions, such as the shape of the composition of the present invention.

[0065] The form of the composition of the present invention is not particularly limited, and the composition of the present invention may be in any form, such as powder, flakes, tablets, paste, or liquid.

[0066] The content and content ratio of each component (i.e., active component and optional additional component) in the composition of the present invention are not particularly limited as long as the object of the present invention can be achieved (e.g., a differentiation-promoting effect can be obtained). That is, for example, the content of the active component in the composition of the present invention may be an effective amount (e.g., an amount that can achieve a differentiation-promoting effect). The content and content ratio of each component in the composition of the present invention can be appropriately set depending on various conditions, such as the type of animal cell and the mode of use of the composition of the present invention.

[0067] The content of the active ingredient in the composition of the present invention is more than 0% (w / w) and not more than 100% (w / w). The content of the active ingredient in the composition of the present invention may be, for example, 1 ppb (w / w) or more, 10 ppb (w / w) or more, 100 ppb (w / w) or more, 1 ppm (w / w) or more, 10 ppm (w / w) or more, 100 ppm (w / w) or more, 1000 ppm (w / w) or more, 1% (w / w) or more, 2% (w / w) or more, 5% (w / w) or more, or 10% (w / w) or more, and may be 100% (w / w) or less, less than 100% (w / w), 99.9% (w / w) or less, 90% (w / w) or less, 50% (w / w) or less, 20% (w / w) or less, 10% (w / w) or less, 5% (w / w) or less, 2% (w / w) or less, 1% (w / w) or less, 1000 ppm (w / w) or less, 100 The content of the active ingredient in the composition of the present invention may be, for example, 1 ppb (w / w) to 1 ppm (w / w), 1 ppm (w / w) to 10 ppm (w / w), 10 ppm (w / w) to 100 ppm (w / w), 100 ppm (w / w) to 1000 ppm (w / w), 1000 ppm (w / w) to 1% (w / w), 1% (w / w) to 10% (w / w), or 10% (w / w) to 20% (w / w). The content of the active ingredient in the composition of the present invention may be, for example, 1 ppb (w / w) to 10% (w / w), 1 ppb (w / w) to 1% (w / w), or 1 ppb (w / w) to 1000 ppm (w / w). When the composition of the present invention contains two or more active ingredients, the contents of those two or more active ingredients in the composition of the present invention may be set independently or in total within the range of the content of the active ingredients in the composition of the present invention exemplified above (with the proviso that the total content of those two or more active ingredients in the composition of the present invention is 100% (w / w) or less).In addition, when the composition of the present invention contains two or more active ingredients, the "content of the active ingredients in the composition of the present invention" means the total content of those two or more active ingredients in the composition of the present invention, unless otherwise specified.

[0068] In one embodiment, the composition of the present invention may be substantially free of serum. "The composition of the present invention is substantially free of serum" 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 a 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 serum).

[0069] In one embodiment, the composition of the present invention may be substantially free of animal-derived albumin. "The composition of the present invention is substantially free of 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 a 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).

[0070] The content of each component (i.e., the active component and any additional component) in the composition of the present invention can be set, for example, so as to obtain the concentration of each component in the medium in the method of the present invention described below.

[0071] When the composition of the present invention contains two or more components, the components may be mixed together and contained in the composition of the present invention, or may be contained separately or in any combination. For example, the composition of the present invention may be provided as a set consisting of a package of an active ingredient and a package of an additional ingredient. In such a case, the components contained in the set can be used together as appropriate when used.

[0072] <3> Method of the Present Invention The method of the present invention is a method for culturing animal cells, which comprises a step of culturing animal cells in the presence of an active ingredient (i.e., a taste substance). This step is also referred to as a "culturing step." Specifically, the culturing step may be a step of culturing animal cells in the presence of the active ingredient to differentiate the animal cells.

[0073] The use of an active ingredient, specifically, culturing animal cells in the presence of the active ingredient, may promote differentiation of animal cells, i.e., an effect of promoting differentiation of animal cells (differentiation-promoting effect) may be obtained. That is, one aspect of the method of the present invention (specifically, the method for culturing animal cells) may be, for example, a method for promoting differentiation of animal cells.

[0074] The purpose of culturing animal cells is not particularly limited.

[0075] In one aspect, a product (e.g., cultured meat) may be produced by culturing animal cells. That is, if animal cells are capable of forming cultured meat through culture, then cultured meat can be produced by culturing those cells. Cultured meat may be composed primarily of muscle tissue. Thus, "animal cells are capable of forming cultured meat through culture" may mean, for example, that animal cells are capable of differentiating through culture to form muscle tissue, and specifically, that animal cells are capable of differentiating into myotube cells through culture to form muscle tissue. That is, one aspect of the method of the present invention (specifically, the method for culturing animal cells) may be a method for producing a product, including a step of culturing animal cells in the presence of an active ingredient. Furthermore, the culturing 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. When a product is produced by culturing animal cells, the use of an active ingredient may improve product production. For example, the use of an active ingredient may promote differentiation of the animal cells, thereby improving product production. That is, one embodiment of the method of the present invention (specifically, the method for culturing animal cells) may be a method for improving the production of a product.

[0076] "Animal cells" refers to cells of an animal. There are no particular limitations on the animal cells, as long as they have the ability to differentiate. Animal cells can be selected appropriately depending on 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 limitations on the animal cells, as long as they can form cultured meat through culture. "Animal" may refer to organisms classified under the 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. Birds include chickens, turkeys, ducks, geese, guinea fowl, quail, and ostriches. Fish include eels, tuna, grouper, sea bream, salmon, cod, and pufferfish. Aquatic organisms include aquatic mammals and fish, as well as crustaceans such as shrimp and crab, shellfish such as scallops and oysters, and other aquatic organisms such as squid and octopus. Aquatic organisms particularly include fish and crustaceans. Animals suitable for producing cultured meat include animals raised for meat. Meat animals include those animals listed above whose meat can be used for food. Meat animals (particularly mammals) particularly include cattle, pigs, sheep, goats, and rabbits. Meat animals (particularly mammals) particularly include cattle and pigs. Meat animals (particularly mammals) particularly include cattle. Examples of meat-producing animals (particularly birds) include chickens, turkeys, ducks, geese, guinea fowl, quails, and ostriches. Examples of meat-producing animals (particularly birds) include chickens, more particularly. Examples of meat-producing animals (particularly aquatic organisms) include the fish and crustaceans exemplified above. Examples of meat-producing animals (particularly aquatic organisms) include eels, tuna, and shrimp, more particularly. The tissues or cells from which animal cells are derived are not particularly limited.Examples of tissues or cells from which animal cells are derived include 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. Examples of tissues or cells from which animal cells are derived include muscle. Specific examples of animal cells include somatic cells, stem cells, and progenitor cells. Somatic cells include myoblasts and fibroblasts. Examples of stem cells include adult stem cells such as hematopoietic stem cells, satellite cells (muscle stem cells), neural stem cells, mesenchymal stem cells, mammary stem cells, olfactory mucosa stem cells, neural crest stem cells, hepatic stem cells, pancreatic stem cells, germ stem cells, intestinal stem cells, and hair follicle stem cells; pluripotent stem cells such as embryonic stem cells (ES cells), embryonic tumor cells, embryonic germ 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). Animal cells suitable for producing cultured meat include stem cells such as satellite cells (muscle stem cells), mesenchymal stem cells, embryonic stem cells (ES cells), and induced pluripotent stem cells (iPS cells), as well as myoblasts. Animal cells particularly suitable for producing cultured meat include satellite cells (muscle stem cells) and myoblasts. Myoblasts may be classified as stem cells. Animal cells may, for example, express taste receptors that correspond to tastants (i.e., taste receptors that are activated by tastants).

[0077] "Animal cells satisfy a certain condition" means that the animal cells must satisfy the condition for at least a portion of the culture period, and does not require the animal cells to satisfy the condition for the entire culture period. In other words, "animal cells satisfy a certain condition" is not limited to cases where animal cells satisfy the condition at the start of culture (i.e., culture is initiated using animal cells that satisfy the condition), but may also include cases where animal cells satisfy the condition after the start of culture. For example, "animal cells are myoblasts" is not limited to cases where culture is initiated using animal cells that are myoblasts, but may also include cases where animal cells differentiate into myoblasts after the start of culture.

[0078] The animal cells are cultured in the presence of an active ingredient. "The animal cells are cultured in the presence of an active ingredient" may mean, for example, that the animal cells are cultured in a medium containing the active ingredient. "The animal cells are cultured in the presence of an active ingredient" may mean, for example, that the active ingredient is supplied to the culture system during the culture of the animal cells.

[0079] The active ingredient may be used in the form of, for example, the composition of the present invention for culturing animal cells. The composition of the present invention may be used, for example, as a culture medium for culturing animal cells. That is, for example, when 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). That is, "culturing animal cells in a culture medium containing an active ingredient" may also include culturing 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 culturing animal cells. That is, for example, when 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 supplemented with the composition of the present invention. That is, "culturing animal cells in a culture medium containing an active ingredient" may also include culturing animal cells in a culture medium supplemented with the composition of the present invention, which is a culture medium additive. Furthermore, for example, the composition of the present invention or a culture medium supplemented with the composition of the present invention may be supplied to a culture system during animal cell culture. That is, "supplying an active ingredient to a culture system during animal cell culture" may also include supplying the composition of the present invention or a culture medium supplemented with the composition of the present invention to a culture system during animal cell culture.

[0080] The medium composition and culture conditions are not particularly limited as long as the objectives of the present invention can be achieved (e.g., a differentiation-promoting effect can be obtained), except that the animal cells are cultured in the presence of an active ingredient and the animal cells differentiate. The medium composition and culture conditions can be appropriately set depending on various conditions, such as the type of animal cells and the purpose of the animal cell culture. For example, when producing cultured meat by culturing animal cells, the medium composition and culture conditions are not particularly limited as long as cultured meat can be formed by culturing the animal cells. Except for the presence of an active ingredient, the animal cells can be cultured using, for example, a conventional medium and conditions used for culturing animal cells (specifically, differentiation of animal cells), either as is or with appropriate modifications. When producing cultured meat by culturing animal cells, except for the presence of an active ingredient, the animal cells can be cultured using, for example, a conventional medium and conditions used for the formation of muscle tissue by differentiation of animal cells (e.g., the production of cultured meat), either as is or with appropriate modifications.

[0081] Culture can be carried out using, for example, a liquid medium. Culture can be carried out by batch culture, fed-batch culture, continuous culture, or a combination thereof. Continuous culture includes perfusion culture and chemostat culture. The medium at the start of culture is also called the "initial medium" or "basal medium." The medium supplied to a culture system (e.g., initial medium) in fed-batch culture is also called the "feed medium." The medium supplied to a culture system (e.g., initial medium) in continuous culture (not limited to perfusion culture) is also called the "perfusion medium." Supplying a feed medium or perfusion medium to a culture system in fed-batch or continuous culture is also simply called "medium supply." Medium supply may be carried out throughout the entire culture period or only for a portion of the culture period. Medium supply may be carried out continuously or intermittently. During culture (particularly 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 for a portion of the culture period. The culture medium may be withdrawn continuously or intermittently. The culture medium may be withdrawn and the medium may be supplied simultaneously, or not. The culture vessel may be coated with a cell adhesion molecule such as fibronectin. Culturing may be carried out three-dimensionally, for example, using a scaffold. When producing cultured meat by culturing animal cells, culturing may be carried out three-dimensionally, for example, using a scaffold that matches the shape of the cultured meat to be formed.

[0082] The media used for culture, for example, the basal medium, the feed medium, and the perfusion medium, can be selected independently.

[0083] The medium used for the culture may be a commercially available medium or an appropriately prepared medium.

[0084] The medium used for the culture includes a medium containing components essential for culturing animal cells (for example, a carbon source, a nitrogen source, inorganic salts, etc.).

[0085] Specific examples of media used for culture include Dulbecco's Modified Eagle's Medium (DMEM), Ham's Nutrient Mixture F12, DMEM / F12, 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.

[0086] Furthermore, specific examples of media used for culture (for example, media particularly used for culturing stem cells (particularly pluripotent stem cells)) include STEMPRO (registered trademark) hESC SFM medium (Life Technologies), mTeSR1 medium (STEMCELL Technologies), TeSR2 medium (STEMCELL Technologies), TeSR-E8 medium (STEMCELL Technologies), Essential 8 medium (Life Technologies), HEScGRO (trademark) Serum-Free Medium for hES cells (Millipore), PluriSTEM (trademark) Human ES / iPS Medium (EMD Millipore), NutriStem (registered trademark) hESC XF medium (Biological Industries Israel Beit-Haemek), NutriStem (trademark) XF / FF Culture Medium (Stemgent), AF NutriStem (registered trademark) hESC XF medium (Biological Industries Israel Examples of suitable medium include hESF9 medium, hESF-FX medium, CDM medium, DEF-CS 500 Xeno-Free 3D Spheroid Culture Medium (Cellartis), and StemFlex medium (Thermo Fisher Scientific).

[0087] Other commercially available media include animal cell culture media such as CELLiST Basal Media BASAL3, BASAL4P, and 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-CELL™302 (Sigma-Aldrich), IS CHO-CD (Irvine Scientific), and IS CHO-CDXP (Irvine Scientific).

[0088] The media exemplified above may be used for culture by adding, for example, an active ingredient.

[0089] Furthermore, the medium used for culture may be, for example, the composition of the present invention (specifically, the composition of the present invention that is a medium). That is, when the composition of the present invention is a medium, the composition of the present invention may be used for culture as is, or may be appropriately prepared as a liquid medium of a desired composition. For example, the composition of the present invention may be diluted with an aqueous medium such as water or an aqueous buffer solution to prepare a liquid medium and use it for culture.

[0090] Furthermore, the medium used for culture may be, for example, a medium supplemented with the composition of the present invention (specifically, the composition of the present invention as a medium additive). The medium supplemented with the composition of the present invention may be a commercially available medium or an appropriately prepared medium.

[0091] The medium may contain various medium components. Examples of medium components include carbon sources, amino acid sources, peptides, proteins, vitamins, fatty acids, lipids, organic acids, nucleic acids, amines, antioxidants, inorganic components, pH buffers, growth factors, cytokines, hormones, cell adhesion factors, extracellular matrix components, serum, lecithin, pea-derived materials, yeast extract, antibiotics, and gene expression inducers. Any of these medium components may be essential or effective for, for example, the survival or proliferation of animal cells. Any of these medium components may be pre-contained in the medium, such as those exemplified above, or may be added to the medium, such as those exemplified above.

[0092] Examples of carbon sources include sugars such as glucose, fructose, sucrose, and maltose.

[0093] The amino acid source may include amino acids. In addition, both peptides and proteins may be examples of amino acid sources. The amino acids may 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. The amino acids may be, for example, L-form.

[0094] Examples of peptides include dipeptides and tripeptides. Specific examples of peptides include glycylglycylglycine and soybean peptide. The descriptions of amino acids also apply mutatis mutandis to the amino acids that make up peptides.

[0095] Examples of proteins include albumin and transferrin.

[0096] 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 precursors thereof.

[0097] Fatty acids include oleic acid, arachidonic acid, and linoleic acid.

[0098] Lipids include cholesterol.

[0099] Examples of organic acids include pyruvic acid (such as sodium pyruvate).

[0100] Nucleic acids include hypoxanthine and thymidine.

[0101] The amines include polyamines such as putrescine.

[0102] Antioxidants include lipoic acid.

[0103] Examples of 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).Specific examples of inorganic components include inorganic salts such as sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, and sodium dihydrogen phosphate.

[0104] Examples of pH buffers include sodium bicarbonate, phosphate, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and N-[tris(hydroxymethyl)methyl]glycine (Tricine).

[0105] Examples of growth factors include fibroblast growth factor (FGF), 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). Examples of FGF include basic fibroblast growth factor (bFGF). Examples of growth factors include bFGF in particular. That is, at least bFGF may be used as a medium component. That is, the medium may contain at least bFGF. Examples of bFGF include bovine basic fibroblast growth factor (bbFGF). "Bovine basic fibroblast growth factor (bbFGF)" may refer to bovine-derived bFGF. The term "bovine" in reference to bbFGF may refer to organisms of the genus Bos. Bos organisms include Bos taurus. bbFGF is not limited to bFGF found in organisms of the genus Bos, and may be a modified form thereof.

[0106] Cytokines include interleukins.

[0107] Hormones include dexamethasone, hydrocortisone, estradiol, progesterone, glucagon, and insulin.

[0108] Examples of cell adhesion factors or extracellular matrix components include Type I collagen, Type II collagen, fibronectin, laminin, poly-L-lysine, and poly-D-lysine.

[0109] Antibiotics include amphotericin B, kanamycin, gentamicin, streptomycin, and penicillin.

[0110] "Pea-derived material" means a material derived from peas. 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 proteins.

[0111] When a medium component corresponding to a taste substance is used for cultivation, the medium component may or may not be selected as an active ingredient.

[0112] In one embodiment, the medium may be substantially free of serum. For example, the medium may be substantially free of serum throughout the entire culture period. "The medium is substantially free of serum" may mean that the serum content in the 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 a case where the serum content in the medium is 0 (zero) (i.e., the medium does not contain serum).

[0113] In one aspect, the medium may be substantially free of animal-derived albumin. Examples of animals from which albumin is derived include the animals exemplified for animal cells. The medium may be substantially free of animal-derived albumin, for example, throughout the entire culture period. "The medium is substantially free of animal-derived albumin" may mean that the content of animal-derived albumin in the 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 a case where the content of animal-derived albumin in the medium is 0 (zero) (i.e., the medium does not contain animal-derived albumin).

[0114] Various components, such as active ingredients, may be contained in the initial medium, feed medium, perfusion medium, or a combination thereof. That is, various components, such as active ingredients, may be supplied to the medium alone or in any combination during the culture process. These components may be supplied once, multiple times, or continuously. The compositions (e.g., the types and / or concentrations of the components) of the initial medium, feed medium, and perfusion medium may or may not be the same. That is, the types of components contained in the initial medium may or may not be the same as the types of components contained in the feed medium or perfusion medium. Furthermore, the concentrations of the components contained in the initial medium may or may not be the same as the concentrations of the components contained in the feed medium or perfusion medium. For example, when a feed medium is used for perfusion culture, the compositions of the initial medium and the feed medium may be the same. Furthermore, two or more feed media or perfusion media with different compositions (e.g., the types and / or concentrations of the components contained) may be used. For example, when the feed medium or perfusion medium is supplied intermittently multiple times, the composition of the feed medium or perfusion medium may or may not be the same for each supply. In addition, various components such as active ingredients may be supplied to the medium in a form not contained in the feed medium or perfusion medium, such as a powder.

[0115] The seeding amount of animal cells at the start of culture is, for example, 1 × 10 in terms of viable cell number. 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, but not more than 1 x 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 x 10 3The seeding amount of animal cells at the start of culture may be, for example, 1 × 10 cells / mL or less in terms of 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 x 10 7 ~1×10 8 The seeding amount of animal cells at the start of culture may be, for example, 1 × 10 cells / mL in terms of the number of viable cells. 2 ~1×10 8 cells / mL, 1 × 10 3 ~1×10 7 cells / mL, or 1 x 10 3 ~1×10 6 The number of viable cells may be measured using, for example, a viable cell autoanalyzer, Vi-CELL TM Measurement can be performed using XR (Beckman Coulter).

[0116] The culture may be carried out under, for example, 5 to 15% CO ２ CO etc. ２The cultivation may be carried out under a pH-containing atmosphere. The pH of the medium may be, for example, near neutral. "Near neutral" may mean, for example, pH 6 to 8, pH 6.5 to 7.5, or pH 6.8 to 7.2. The pH of the medium can be adjusted as needed during cultivation. The pH of the 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 cultivation temperature may be, for example, 30 to 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 compatible combination thereof. Specific examples of the culture period may be, for example, 1 to 60 days, 3 to 25 days, or 5 to 20 days. Culture may be continued, for example, until the product is produced to a desired level.

[0117] The amount (e.g., concentration in the medium) or ratio of amounts used (e.g., concentration ratio in the medium) of each component (e.g., medium component such as an active ingredient) used in the method of the present invention is not particularly limited, as long as the object of the present invention can be achieved (e.g., a differentiation-promoting effect can be obtained). That is, for example, the amount of an active ingredient used in the method of the present invention may be an effective amount (e.g., an amount that can achieve a differentiation-promoting effect). Specifically, for example, the concentration of the active ingredient in the medium may be an effective concentration (e.g., a concentration that can achieve a differentiation-promoting effect). The amount and ratio of amounts used of each component in the method of the present invention can be appropriately determined depending on various conditions, such as the type of animal cell, the purpose of culturing the animal cell, and the type of component.

[0118] The concentration of the active ingredient in the medium may be, for example, 0.05 nM or more, 0.1 nM or more, 0.2 nM or more, 0.5 nM or more, 1 nM or more, 2 nM or more, 5 nM or more, 10 nM or more, 20 nM or more, 50 nM or more, 100 nM or more, 200 nM or more, 500 nM or more, 1 μM or more, 2 μM or more, 5 μM or more, 10 μM or more, 20 μM or more, 50 μM or more, 100 μM or more, 200 μM or more, 500 μM or more, 1 mM or more, 2 mM or more, 5 mM or more, 10 mM or more, 20 mM or more, 50 mM or more, 100 mM or more, or 200 mM or more, or 500 mM or less, 200 mM or less, 100 mM or less, 50 mM or less, 20 mM or less, 10 mM or less, 5 mM or less, 2 mM or less, 1 mM or less, 500 μM or less, 200 The concentration may be 0.1 μM or less, 100 μM or less, 50 μM or less, 20 μM or less, 10 μM or less, 5 μM or less, 2 μM or less, 1 μM or less, 500 nM or less, 200 nM or less, 100 nM or less, 50 nM or less, 20 nM or less, 10 nM or less, 5 nM or less, 2 nM or less, 1 nM or less, 0.5 nM or less, 0.2 nM or less, or 0.1 nM or less, or any compatible combination thereof. Specifically, the concentration of the active ingredient in the medium is, for example, 0.05 nM to 0.1 nM, 0.1 nM to 0.2 nM, 0.2 nM to 0.5 nM, 0.5 nM to 1 nM, 1 nM to 2 nM, 2 nM to 5 nM, 5 nM to 10 nM, 10 nM to 20 nM, 20 nM to 50 nM, 50 nM to 100 nM. nM, 100 nM to 200 nM, 200 nM to 500 nM, 500 nM to 1 μM, 1 μM to 2 μM, 2 μM to 5 μM, 5 μM to 10 μM, 10 μM to 20 μM, 20 μM to 50 μM, 50 μM to 100 μM, 100 μM to 200 μM, 200 μM to 500 μM, 500 μM to 1 mM, 1 mM to 2 mM, 2 mM to 5 mM, 5 mM to 10 mM, 10 mM to 20 mM, 20 mM to 50 mM, 50 mM to 100 mM, 100 mM to 200 mM, or 200 mM to 500 mM.The concentration of the active ingredient in the medium may be, for example, 0.05 nM to 500 mM, 0.2 nM to 100 mM, or 1 μM to 20 mM. When the medium contains two or more active ingredients, the concentrations of those two or more active ingredients in the medium may be set independently or in total within the range of the active ingredient concentrations in the medium exemplified above. When the medium contains two or more active ingredients, the "concentration of the active ingredients in the medium" refers to the total concentration of those two or more active ingredients in the medium, unless otherwise specified.

[0119] The concentration of an umami substance such as ATF100 or an umami receptor agonist, or a kokumi substance such as cinacalcet or a CaSR agonist in the medium may be, for example, within the range of the concentration of the active ingredient in the medium exemplified above. The concentration of the umami substance or umami receptor agonist such as ATF100, or the kokumi substance or CaSR agonist such as cinacalcet in the culture medium may be, for example, 0.1 nM or more, 0.2 nM or more, 0.5 nM or more, 1 nM or more, 2 nM or more, 5 nM or more, 10 nM or more, 20 nM or more, 50 nM or more, 100 nM or more, 200 nM or more, 500 nM or more, or 1 μM or more, or 2 μM or less, 1 μM or less, 500 nM or less, 200 nM or less, 100 nM or less, 50 nM or less, 20 nM or less, 10 nM or less, 5 nM or less, 2 nM or less, 1 nM or less, 0.5 nM or less, or 0.2 nM or less, or any compatible combination thereof. The concentration of the umami substance or umami receptor agonist such as ATF100, or the kokumi substance or CaSR agonist such as cinacalcet in the medium may be, specifically, for example, 0.1 nM to 0.2 nM, 0.2 nM to 0.5 nM, 0.5 nM to 1 nM, 1 nM to 2 nM, 2 nM to 5 nM, 5 nM to 10 nM, 10 nM to 20 nM, 20 nM to 50 nM, 50 nM to 100 nM, 100 nM to 200 nM, 200 nM to 500 nM, 500 nM to 1 μM, or 1 μM to 2 μM. The concentration of the umami substance or umami receptor agonist such as ATF100, or the kokumi substance or CaSR agonist such as cinacalcet in the medium may be, specifically, for example, 0.1 nM to 2 μM, 0.5 nM to 500 nM, or 2 nM to 100 nM.

[0120] The concentration of a sweet substance such as sucralose or a sweet taste receptor agonist in the medium may be, for example, within the range of the concentrations of the active ingredients in the medium exemplified above. The concentration of a sweet substance such as sucralose or a sweet taste receptor agonist in the medium may be, for example, 100 nM or more, 200 nM or more, 500 nM or more, 1 μM or more, 2 μM or more, 5 μM or more, 10 μM or more, 20 μM or more, 50 μM or more, 100 μM or more, 200 μM or more, 500 μM or more, or 1 mM or more, or 2 mM or less, 1 mM or less, 500 μM or less, 200 μM or less, 100 μM or less, 50 μM or less, 20 μM or less, 10 μM or less, 5 μM or less, 2 μM or less, 1 μM or less, 500 nM or less, or 200 nM or less, or a compatible combination thereof. The concentration of a sweet substance such as sucralose or a sweet taste receptor agonist in the medium may be, for example, 100 nM to 200 nM, 200 nM to 500 nM, 500 nM to 1 μM, 1 μM to 2 μM, 2 μM to 5 μM, 5 μM to 10 μM, 10 μM to 20 μM, 20 μM to 50 μM, 50 μM to 100 μM, 100 μM to 200 μM, 200 μM to 500 μM, 500 μM to 1 mM, or 1 mM to 2 mM. The concentration of a sweet substance such as sucralose or a sweet taste receptor agonist in the medium may be, for example, 100 nM to 2 mM, 500 nM to 500 μM, or 2 μM to 100 μM.

[0121] Fatty substances such as TUG-891 or fatty acid receptor agonists, GABA receptor agonists such as SKF97541 in the culture medium B The concentrations of the sour taste substance or sour taste receptor agonist, such as succinic acid, or the succinic acid receptor agonist, may be within the range of the concentrations of the active ingredients in the medium exemplified above. BThe concentration of the receptor agonist, sour taste substance such as succinic acid or sour taste receptor agonist, or succinic acid receptor agonist such as succinic acid may be, for example, 10 nM or more, 20 nM or more, 50 nM or more, 100 nM or more, 200 nM or more, 500 nM or more, 1 μM or more, 2 μM or more, 5 μM or more, 10 μM or more, 20 μM or more, or 50 μM or more, or 100 μM or less, 50 μM or less, 20 μM or less, 10 μM or less, 5 μM or less, 2 μM or less, 1 μM or less, 500 nM or less, 200 nM or less, 100 nM or less, 50 nM or less, or 20 nM or less, or a compatible combination thereof. B The concentration of a receptor agonist, a sour taste substance such as succinic acid or a sour taste receptor agonist, or a succinic acid receptor agonist such as succinic acid may be, for example, 10 nM to 20 nM, 20 nM to 50 nM, 50 nM to 100 nM, 100 nM to 200 nM, 200 nM to 500 nM, 500 nM to 1 μM, 1 μM to 2 μM, 2 μM to 5 μM, 5 μM to 10 μM, 10 μM to 20 μM, 20 μM to 50 μM, or 50 μM to 100 μM. B The concentration of the receptor agonist, the sour taste substance such as succinic acid or the sour taste receptor agonist, or the succinic acid receptor agonist such as succinic acid may be, for example, 10 nM to 100 μM, 50 nM to 20 μM, or 200 nM to 5 μM.

[0122] The concentration of the bitter substance such as difenidol or the bitter taste receptor agonist (particularly a T2R108 agonist) in the medium may be, for example, within the range of the concentration of the active ingredient in the medium exemplified above. The concentration of the bitter substance such as difenidol or bitter taste receptor agonist (particularly a T2R108 agonist) in the culture medium may be, for example, 10 nM or more, 20 nM or more, 50 nM or more, 100 nM or more, 200 nM or more, 500 nM or more, 1 μM or more, 2 μM or more, 5 μM or more, 10 μM or more, 20 μM or more, 50 μM or more, or 100 μM or more, or 200 μM or less, 100 μM or less, 50 μM or less, 20 μM or less, 10 μM or less, 5 μM or less, 2 μM or less, 1 μM or less, 500 nM or less, 200 nM or less, 100 nM or less, 50 nM or less, or 20 nM or less, or a compatible combination thereof. The concentration of the bitter substance such as difenidol or the bitter taste receptor agonist (particularly a T2R108 agonist) in the medium may be, specifically, for example, 10 nM to 20 nM, 20 nM to 50 nM, 50 nM to 100 nM, 100 nM to 200 nM, 200 nM to 500 nM, 500 nM to 1 μM, 1 μM to 2 μM, 2 μM to 5 μM, 5 μM to 10 μM, 10 μM to 20 μM, 20 μM to 50 μM, 50 μM to 100 μM, or 100 μM to 200 μM. The concentration of the bitter substance such as difenidol or bitter taste receptor agonist (particularly a T2R108 agonist) in the medium may be, specifically, for example, 10 nM to 200 μM, 50 nM to 50 μM, or 200 nM to 10 μM.

[0123] The concentration of a bitter substance such as salicin or a bitter receptor agonist (particularly a T2R126 agonist) in the medium may be, for example, within the range of the concentrations of the active ingredients in the medium exemplified above. The concentration of a bitter substance such as salicin or a bitter receptor agonist (particularly a T2R126 agonist) in the medium may be, for example, 5 μM or more, 10 μM or more, 20 μM or more, 50 μM or more, 100 μM or more, 200 μM or more, 500 μM or more, 1 mM or more, 2 mM or more, 5 mM or more, 10 mM or more, 20 mM or more, or 50 mM or more, or 100 mM or less, 50 mM or less, 20 mM or less, 10 mM or less, 5 mM or less, 2 mM or less, 1 mM or less, 500 μM or less, 200 μM or less, 100 μM or less, 50 μM or less, 20 μM or less, or 10 μM or less, or a compatible combination thereof. The concentration of a bitter substance such as salicin or a bitter taste receptor agonist (particularly a T2R126 agonist) in the medium may be, for example, 5 μM to 10 μM, 10 μM to 20 μM, 20 μM to 50 μM, 50 μM to 100 μM, 100 μM to 200 μM, 200 μM to 500 μM, 500 μM to 1 mM, 1 mM to 2 mM, 2 mM to 5 mM, 5 mM to 10 mM, 10 mM to 20 mM, 20 mM to 50 mM, or 50 mM to 100 mM. The concentration of a bitter substance such as salicin or a bitter taste receptor agonist (particularly a T2R126 agonist) in the medium may be, for example, 5 μM to 100 mM, 20 μM to 20 mM, or 100 μM to 5 mM.

[0124] The concentration of the salty substance or salty taste receptor agonist in the medium may be, for example, within the range of the concentrations of the active ingredients in the medium exemplified above.

[0125] Each of the various components, such as the active ingredient, may be contained in the medium throughout the entire culture period, or only during a portion of the culture period. In other words, "culture is carried out in a medium containing a certain component" means that the component is contained in the medium during at least a portion of the culture period, and does not necessarily have to be contained in the medium throughout the entire culture period. Each of the various components, such as the active ingredient, may be contained in the medium at the start of culture, or may be supplied to the medium after the start of culture. Furthermore, each of the various components, such as the active ingredient, may be contained in the medium at the start of culture, and further supplied to the medium after the start of culture (e.g., after the active ingredient has been consumed).

[0126] Each of the various components, such as the active ingredient, may be contained in the medium at the concentrations exemplified above throughout the entire culture period, or only during a portion of the culture period. That is, "culture is carried out in a medium containing a certain component at a certain concentration," "a certain component is contained in the medium at a certain concentration during culture," or "the concentration of a certain component in the medium during culture is a certain concentration" means that the concentration of the component in the medium is within the range for at least a portion of the culture period, but does not necessarily need to be within the range for the entire culture period. Each of the various components, such as the active ingredient, may be contained in the medium at the concentrations exemplified above, for example, at the start of culture, or may be supplied to the medium after the start of culture to achieve the concentrations exemplified above. Furthermore, each of the various components, such as the active ingredient, may be contained in the medium at the concentrations exemplified above at the start of culture, and then further supplied to the medium after the start of culture (e.g., after the component has been consumed) to achieve the concentrations exemplified above.

[0127] The length of the "partial culture period" is not particularly limited as long as the object of the present invention can be achieved (e.g., a differentiation-promoting effect can be obtained). The length of the "partial culture period" can be appropriately set depending on various conditions, such as the type of animal cell and the length of the culture period. The "partial culture 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. Furthermore, the "partial culture 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 after the animal cells have differentiated into myoblasts, muscle cells, or myotubes. Furthermore, a "partial 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.

[0128] Furthermore, the concentrations of various components, such as active ingredients, in the culture medium may be set to the concentrations exemplified above, for example, as average values ​​over a specific period during culture. That is, "culture is performed in a medium containing a certain component at a certain concentration," "a certain component is contained in the medium at a certain concentration during culture," or "the concentration of a certain component in the medium during culture is a certain concentration" may mean that the average concentration of the component in the medium over a specific period during culture falls within the range of concentrations. The "average concentration of a certain component in the medium over a specific period during culture" is not particularly limited as long as it allows for understanding fluctuations in the concentration of the component over a specific period during culture. For example, it may mean the average concentration of the component in the medium measured every 60 minutes, every 30 minutes, every 20 minutes, or every 10 minutes over a specific period during culture. The "specific period during culture" may refer to the entire culture period or a portion of the culture period. The "partial culture period" is as described above.

[0129] Each of the various components, such as the active ingredient, may be supplied to the medium throughout the entire culture period, or may be supplied to the medium only during a portion of the culture period. The "part of the culture period" is as described above. Each of the various components, such as the active ingredient, may be supplied to the medium continuously or intermittently, for example. Each of the various components, such as the active ingredient, may be supplied to the medium every day, or every few days, for example.

[0130] The concentrations of various components, such as active ingredients, in the feed medium or perfusion medium may be within the range of the concentrations of the components in the media exemplified above, or may be, for example, 1-fold or more, 1.1-fold or more, 1.3-fold or more, 1.5-fold or more, 2-fold or more, 3-fold or more, 5-fold or more, 7-fold or more, 10-fold or more, 15-fold or more, or 20-fold or more, or 100-fold or less, 70-fold or less, 50-fold or less, 30-fold or less, 20-fold or less, 15-fold or less, 10-fold or less, 7-fold or less, 5-fold or less, 3-fold or less, 2-fold or less, or any combination thereof that is consistent therewith. The concentrations of various components such as active ingredients in the feed medium or perfusion medium may be, for example, 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 concentrations of the components in the above-exemplified media. The concentrations of various components such as active ingredients in the feed medium or perfusion medium may be, for example, 1 to 100 times, 2 to 50 times, or 5 to 20 times the concentrations of the components in the above-exemplified media.

[0131] The concentrations of any of the components, including the active ingredient, can be measured by known methods used to detect or identify compounds, such as HPLC, UPLC, LC / MS, GC / MS, and NMR.

[0132] Animal cells can be cultured as described above. In one embodiment, a product (e.g., cultured meat) may be obtained by culturing animal cells as described above.

[0133] <4> Use of Active Ingredient The present invention also discloses the use of the active ingredient in the above-exemplified applications. That is, the present invention discloses, for example, the use of the active ingredient for culturing animal cells, the use of the active ingredient for promoting differentiation of animal cells, and the use of the active ingredient in the production of the composition of the present invention, such as a composition for animal cell culture.

[0134] The present invention also discloses active ingredients for use in the above-mentioned applications, i.e., active ingredients for use in culturing animal cells, active ingredients for use in promoting differentiation of animal cells, and active ingredients for use in producing a composition for animal cell culture.

[0135] The present invention will now be described in more detail with reference to the following non-limiting examples.

[0136] (1) Evaluation of the activity of taste substances on muscle differentiation using bovine and chicken muscle stem cells (1-1) Preparation of bovine and chicken muscle stem cells Muscle tissue was collected from beef shanks and CD56-positive cells were isolated using a magnetically activated cell sorting system (MACS) to prepare bovine muscle stem cells. Separately, muscle stem cells were isolated from chicken breast meat.

[0137] (1-2) Evaluation of Taste Substance Activity Using Bovine Muscle Stem Cells. Bovine muscle stem cells obtained in (1-1) were suspended in serum-containing medium. The serum-containing medium used was Advanced DMEM (Thermo Fisher Scientific, Cat No. 12491015) supplemented with 20% FBS (Thermo Fisher Scientific, Cat No. 10270106), 10% horse serum (Thermo Fisher Scientific, Cat No. 16050-130), and 1% GlutaMAX™ Supplement (Thermo Fisher Scientific, Cat No. 35050061). The cell suspension was seeded at a cell density of 8,000 cells onto a 96-well plate coated with 1% Matrigel (Corning, Cat No. 356234). The following day, the medium was replaced with serum- or serum-free differentiation medium. Serum-containing differentiation medium was DMEM / F12 (Thermo Fisher Scientific, Cat No. 08460-95) supplemented with 2% FBS (Thermo Fisher Scientific, Cat No. 26140-079), while serum-free differentiation medium was DMEM / F12 supplemented with 1% NEAA (Thermo Fisher Scientific, Cat No. 11140050), 1 mM Glutamine (Ajinomoto Healthy Supply Co., Ltd.), 73 mg / mL 0.2% selenium yeast (Medience), 2.5 g / L pea protein (Roquette, Product Name: NUTRALYS S85F), 30 ng / mL IGF-1 (Laurus Bio, Cat No. Rc LR3-IGF-1), and 10 μg / mL transferrin (Laurus Bio, Cat No. RTRF-P).Furthermore, to evaluate taste substances, the serum-free differentiation medium was supplemented with ATF100 (N-(R)-1-(2-(pyridin-2-yl)ethylcarbamoyl)-2-(benzyloxy)ethyl-4-methoxy-3-methylbenzamide; AJINOMOTO), sucralose (SIGMA, Cat No. 69293-100G), cinacalcet (SIGMA, Cat No. SML2012), TUG-891 (SIGMA, Cat No. SML1914), diphenidol (MedchemExpress, Cat No. HY-A0082), salicin (SIGMA, Cat No. S0625-25G), SKF97541 (Tocris Bioscience, Cat No. 556-88751), or sodium succinic acid (SIGMA, Cat No. Table 1 lists the group numbers and additives.

[0138] On day 2, myosin heavy chain (MyHC) expression was measured by immunostaining. Cells were fixed with 4% paraformaldehyde (Nacalai Tesque, Cat. No. 09154-85) for 15 minutes. After washing twice with Dulbecco's phosphate-buffered saline (Ca, Mg-free) (Nacalai Tesque, Cat. No. 14249-24), they were immersed in Dulbecco's phosphate-buffered saline (Ca, Mg-free) containing 0.1% Triton X-100 (Sigma, Cat. No. T9284) for 10 minutes. The blocking buffer was then replaced with Dulbecco's phosphate-buffered saline (Ca, Mg-free) supplemented with 5% horse serum and 0.1% Triton X-100 for 1 hour. The plate was then replaced with blocking buffer containing 0.2% Anti-Myosin Heavy Chain Antibody, clone A4.1025 (Merck, Cat No. 05-716-I-25UL) and incubated overnight at 4°C. The next day, the plate was washed three times with Dulbecco's phosphate-buffered saline (Ca- and Mg-free) and then replaced with blocking buffer containing 0.1% Goat anti-Mouse IgG2a Antibody, Alexa Fluor 555 (Thermo Fisher Scientific, Cat No. A21137) and 0.1% Cellstain Hoechst 33342 solution (Dojindo Laboratories, Cat No. H342) and incubated at room temperature for 1 hour. The plate was then washed three times with Dulbecco's phosphate-buffered saline (Ca- and Mg-free) and then replaced with Dulbecco's phosphate-buffered saline (Ca- and Mg-free). MyHC-positive cells were detected using a fluorescence microscope ECLIPSE Ti2-E (Nikon), and the fusion index (= number of MyHC-positive nuclei / total number of nuclei × 100 (%)) was calculated.

[0139] The results are shown in Figure 1. Both tastants promoted bovine muscle differentiation. It is known that intracellular calcium ions are involved in muscle differentiation (Sansrity Sinha et al., Ca2+ as a coordinator of skeletal muscle differentiation, fusion, and contraction. FEBS J. 2022 Nov;289(21):6531-6542). Tastants are also known to increase intracellular calcium ion concentrations in taste cells via binding to taste receptors on the cells (Raise Ahmad and Julie E Dalziel, G Protein-Coupled Receptors in Taste Physiology and Pharmacology. Front Pharmacol. 2020 Nov 30;11:587664). In other words, in this example, it is believed that intracellular calcium ion concentrations increased in muscle stem cells via binding of tastants to taste receptors on muscle stem cells, thereby promoting muscle differentiation of muscle stem cells. Therefore, it is believed that the use of taste substances can promote differentiation, such as muscle differentiation, in cells that express the corresponding taste receptors.

[0140]

[0141] (1-3) Evaluation of Taste Substance Activity Using Chicken Muscle Stem Cells The chicken muscle stem cells obtained in (1-1) were suspended in serum medium. The serum medium used was DMEM (Nacalai Tesque, Cat. No. 08456-65) supplemented with 10% chicken serum (Thermo Fisher Scientific, Cat. No. 16110-082) and 5% horse serum. The cell suspension was diluted to 10 μg / cm 2Cells were seeded at a density of 60,000 cells onto a gelatin (Sigma, Cat No. G1393-20ML)-coated 96-well plate and the following day, the medium was replaced with serum or serum-free differentiation medium. The serum-free medium was DMEM / F12 supplemented with 2% FBS. The serum-free medium was DMEM / F12 supplemented with 1% NEAA (Thermo Fisher Scientific, Cat No. 11140050), 1 mM Glutamine (Ajinomoto Healthy Supply Co., Ltd.), 73 mg / mL 0.2% selenium yeast (Medience), 30 ng / mL IGF-1 (Laurus Bio, Cat No. Rc LR3-IGF-1), and 10 μg / mL Transferrin (Laurus Bio, Cat No. RTRF-P). Furthermore, to evaluate taste substances, ATF100, Sucralose, Cinacalcet, TUG-891, Diphenidol, Salicin, SKF97541, or Sodium succinic acid was added to the serum-free differentiation medium. Table 1 summarizes the group numbers and additives.

[0142] Then, on the second day, the expression of myosin heavy chain (MyHC) was measured by the same method as in (1-2).

[0143] The results are shown in Figure 2. Only salicin promoted muscle differentiation in chickens.

[0144] According to the present invention, differentiation of animal cells can be promoted.

Claims

1. A composition for promoting differentiation of animal cells, comprising a taste substance.

2. The composition of claim 1 which is a culture medium or a culture medium additive.

3. The composition of claim 2, wherein the medium is a basal medium, a feed medium, or a perfusion medium.

4. The taste substances are sweet substances, salty substances, sour substances, bitter substances, umami substances, fat substances, rich substances, succinic acid receptor agonists, and GABA B The composition according to claim 1 or 2, wherein the at least one component is selected from the group consisting of receptor agonists.

5. The sweet substance is at least one component selected from the group consisting of sugars, sugar alcohols, high-intensity sweeteners, and amino acids, the salty substance is at least one inorganic salt, the sour substance is at least one component selected from the group consisting of organic acids, inorganic acids, and amino acids, the bitter substance is at least one component selected from the group consisting of amino acids, polyphenols, alkaloids, terpenoids, inorganic salts, diphenidol, and salicin, the umami substance is at least one component selected from the group consisting of amino acids, nucleic acids, organic acids, amide compounds, and high-potency umami substances, the fatty substance is at least one component selected from the group consisting of fatty acids and fatty acid receptor agonists, the full-bodied substance is at least one component selected from the group consisting of γ-glutamyl peptides and calcium-sensing receptor agonists, the succinic acid receptor agonist is succinic acid, and the GABA B The composition of claim 4, wherein the receptor agonist is GABA and / or SKF97541.

6. The tasting substance is a sweet taste receptor agonist, a salty taste receptor agonist, a sour taste receptor agonist, a bitter taste receptor agonist, an umami taste receptor agonist, a fatty acid receptor agonist, a calcium-sensing receptor agonist, a succinate receptor agonist, and a GABA receptor agonist. B The composition according to claim 1 or 2, wherein the at least one component is selected from the group consisting of receptor agonists.

7. The composition of claim 6, wherein the sweet receptor is T1R2 / 3, the salty receptor is ENaC, TMC4, or TMC6, the sour receptor is GPR91, PKD2L1, PKD1L3, or Otop1, the bitter receptor is T2R, the umami receptor is T1R1 / 3, the fatty acid receptor is GPR120, and the succinate receptor is GPR91.

8. The composition according to claim 1 or 2, wherein the tasting substance is at least one component selected from the group consisting of ATF100, sucralose, cinacalcet, TUG-891, diphenidol, salicin, SKF97541, and succinic acid.

9. The composition of claim 1 or 2, wherein the animal is a chicken and the tastant is salicin.

10. The composition according to claim 1 or 2, wherein the tastant is a tastant other than sugar, amino acid, and calcium.

11. The composition of claim 1 or 2, wherein the animal is a mammal, a bird, a fish, or a crustacean.

12. The composition according to claim 1 or 2, wherein the animal is a meat animal.

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

14. The composition according to claim 1 or 2, wherein the differentiation is muscle differentiation.

15. The composition of claim 1 or 2, wherein the cells are satellite cells or myoblasts.

16. The composition of claim 1 or 2, which is substantially free of serum.

17. The composition of claim 1 or 2, which is substantially free of animal-derived albumin.

18. A method for producing cultured meat, comprising the steps of culturing animal cells in the presence of a tastant and causing differentiation of the animal cells.

19. A method for promoting differentiation of animal cells, comprising the step of culturing animal cells in the presence of a taste substance to differentiate the animal cells.

20. The taste substances are sweet substances, salty substances, sour substances, bitter substances, umami substances, fatty substances, kokumi-imparting substances, succinic acid receptor agonists, and GABA receptor agonists. B 20. The method of claim 18 or 19, wherein the at least one component is selected from the group consisting of receptor agonists.

21. The sweet substance is at least one component selected from the group consisting of sugars, sugar alcohols, high-intensity sweeteners, and amino acids; the salty substance is at least one inorganic salt; the sour substance is at least one component selected from the group consisting of organic acids, inorganic acids, and amino acids; the bitter substance is at least one component selected from the group consisting of amino acids, polyphenols, alkaloids, terpenoids, inorganic salts, diphenidol, and salicin; the umami substance is at least one component selected from the group consisting of amino acids, nucleic acids, organic acids, amide compounds, and high-potency umami substances; the fatty substance is at least one component selected from the group consisting of fatty acids and fatty acid receptor agonists; the kokumi-imparting substance is at least one component selected from the group consisting of γ-glutamyl peptides and calcium-sensing receptor agonists; the succinic acid receptor agonist is succinic acid; and the GABA B 21. The method of claim 20, wherein the receptor agonist is GABA and / or SKF97541.

22. The tasting substance is a sweet taste receptor agonist, a salty taste receptor agonist, a sour taste receptor agonist, a bitter taste receptor agonist, an umami taste receptor agonist, a fatty acid receptor agonist, a calcium-sensing receptor agonist, a succinate receptor agonist, and a GABA receptor agonist. B 20. The method of claim 18 or 19, wherein the at least one component is selected from the group consisting of receptor agonists.

23. The method of claim 22, wherein the sweet receptor is T1R2 / 3, the salty receptor is ENaC, TMC4, or TMC6, the sour receptor is GPR91, PKD2L1, PKD1L3, or Otop1, the bitter receptor is T2R, the umami receptor is T1R1 / 3, the fatty acid receptor is GPR120, and the succinate receptor is GPR91.

24. The method of claim 18 or 19, wherein the tastant is at least one component selected from the group consisting of ATF100, sucralose, cinacalcet, TUG-891, diphenidol, salicin, SKF97541, and succinic acid.

25. The method of claim 18 or 19, wherein the animal is a chicken and the tastant is salicin.

26. The method of claim 18 or 19, wherein the tastant is a tastant other than sugar, amino acid, and calcium.

27. The method according to claim 18 or 19, wherein the concentration of the tasting substance in the medium during the culture is 0.05 nM to 500 mM.

28. The method according to claim 20, having one, two, three, four, five, six, seven, eight, or nine properties selected from the following (1) to (8): (1) the concentration of the sweet substance in the medium during the culture is 100 nM to 2 mM; (2) the concentration of the salty substance in the medium during the culture is 0.05 nM to 500 mM; (3) the concentration of the sour substance in the medium during the culture is 10 nM to 100 μM; (4) the concentration of the bitter substance in the medium during the culture is 10 nM to 200 μM or 5 μM to 100 mM; (5) the concentration of the umami substance in the medium during the culture is 0.1 nM to 2 μM; (6) the concentration of the fatty substance in the medium during the culture is 10 nM to 100 μM; (7) The concentration of the kokumi-imparting substance in the medium during the culture is 0.1 nM to 2 μM; (8) The concentration of the succinate receptor agonist in the medium during the culture is 10 nM to 100 μM; (9) The concentration of the GABA receptor agonist in the medium during the culture is 10 nM to 100 μM. B The concentrations of receptor agonists range from 10 nM to 100 μM.

29. The method according to claim 22, having one, two, three, four, five, six, seven, eight, or nine properties selected from the following (1) to (8): (1) the concentration of the sweet taste receptor agonist in the culture medium during the culture is 100 nM to 2 mM; (2) the concentration of the salty taste receptor agonist in the culture medium during the culture is 0.05 nM to 500 mM; (3) the concentration of the sour taste receptor agonist in the culture medium during the culture is 10 nM to 100 μM; (4) the concentration of the bitter taste receptor agonist in the culture medium during the culture is 10 nM to 200 μM or 5 μM to 100 mM; (5) the concentration of the umami taste receptor agonist in the culture medium during the culture is 0.1 nM to 2 μM; (6) the concentration of the fatty acid receptor agonist in the culture medium during the culture is 10 nM to 100 (7) the concentration of the calcium-sensing receptor agonist in the culture medium during the culture is 0.1 nM to 2 μM; (8) the concentration of the succinate receptor agonist in the culture medium during the culture is 10 nM to 100 μM; (9) the concentration of the GABA receptor agonist in the culture medium during the culture is 0.5 nM to 1 μM; B The concentrations of receptor agonists range from 10 nM to 100 μM.

30. The method of claim 18 or 19, wherein the animal is a mammal, a bird, a fish, or a crustacean.

31. The method of claim 18 or 19, wherein the animal is a meat animal.

32. The method of claim 18 or 19, wherein the animal is a cow or a chicken.

33. The method of claim 18 or 19, wherein the differentiation is muscle differentiation.

34. The method of claim 18 or 19, wherein the cells are satellite cells or myoblasts.

35. The method of claim 18 or 19, wherein the culturing is carried out in a medium that is substantially free of serum.

36. The method according to claim 18 or 19, wherein the culturing is carried out in a medium substantially free of animal-derived albumin.

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