Method for producing cultured meat, and cultured meat

Abstract

The present disclosure relates to a method for producing cultured meat, the method comprising: a washing step for bringing bovine tissue into contact with a washing liquid containing peracetic acid; a recovery step for recovering stem cells from the washed bovine tissue; a preparation step for preparing bio-ink containing the recovered stem cells and fragmented extracellular matrix components; a printing step for printing the bio-ink to form a structure containing the stem cells; and a culturing step for subjecting the stem cells in the structure to differentiation induction culturing to form a tissue body.

Description

Method for producing cultured meat and cultured meat

[0001] The present invention relates to a method for producing cultured meat and cultured meat.

[0002] As the demand for sustainable food supply increases and the utilization and development of various protein sources are underway, cell-based foods, particularly cultured meat produced from cells, have recently attracted attention. Various technical means for producing cultured meat from cells have been proposed so far (Non-Patent Documents 1 to 2).

[0003] Dong-Hee Kang et al., “Engineered whole cut meat-like tissue by the assembly of cell fibers using tendon-gel integrated bioprinting” Nature Communications volume 12, Article number: 5059 (2021)Fiona Louis et al., “Mimicking Wagyu beef fat in cultured meat: Progress in edible bovine adipose tissue production with controllable fatty acid composition” Materials Today Bio, Volume 21, August 2023, 100720

[0004] An object of the present invention is to provide a new method for producing cultured meat. Another object of the present invention is to provide cultured meat obtained by the production method.

[0005] In other words, the present invention relates to, for example, the following inventions: [1] A method for producing cultured meat, comprising a washing step of contacting bovine tissue with a washing solution containing peracetic acid; a recovery step of recovering stem cells from the washed bovine tissue; a preparation step of preparing a bioink containing the recovered stem cells and fragmented extracellular matrix components; a printing step of printing the bioink to form a structure containing the stem cells; and a culture step of differentiating and inducing the stem cells in the structure to form a tissue. [2] The method according to [1], wherein the stem cells are at least one selected from the group consisting of satellite cells and adipose-derived stem cells. [3] The method according to [1] or [2], wherein the recovery step comprises a step of refining the bovine tissue; a step of enzymatically treating the refined bovine tissue with trypsin to disperse it into cell clumps containing the stem cells; a step of culturing the cell clumps in a culture medium for stem cells; a step of cryopreserving the cultured stem cells; and a step of activating and culturing the stem cells after cryopreservation. [4] The method according to any one of [1] to [3], wherein the fragmented extracellular matrix component is a defibration extracellular matrix component. [5] The method according to any one of [1] to [4], wherein the fragmented extracellular matrix component is a fragmented collagen component. [6] The method according to any one of [1] to [5], wherein the fragmented extracellular matrix component is a defibration collagen component. [7] The method according to any one of [1] to [6], wherein the bioink further comprises fibrinogen. [8] The method according to any one of [1] to [7], wherein the structure in the printing step is formed by solidifying the bioink discharged from the nozzles of a printer device having a plurality of nozzles. [9] The method according to any one of [1] to [8], wherein the differentiation induction culture is carried out in the presence of fetal bovine serum (FBS).

[10] The method according to any one of [1] to [9], wherein the stem cells include adipose-derived stem cells, and the differentiation induction culture is carried out in the presence of oleic acid.

[11] The method according to any one of [1] to

[10] , wherein the tissue is fibrous.

[12] The method according to any one of [1] to

[11] , wherein the bovine tissue is bovine tissue taken from the sirloin area.

[13] Cultured meat obtained by any of the methods described in [1] to

[12] .

[14] Cultured meat containing docosahexaenoic acid and γ-nonalactone.

[0006] According to the present invention, a novel method for producing cultured meat can be provided. Furthermore, according to the present invention, cultured meat obtained by this production method can be provided.

[0007] The results of taste sensor evaluations of three varieties of ribeye meat are shown, with (A) showing the results when a 5-fold diluted hot water extract was used and (B) showing the results when a 100-fold diluted hot water extract was used. Muscle fibers prepared by the method according to this embodiment are shown, with (A) showing muscle fibers solidified with gelatin, (B) showing the state after the gelatin has been dissolved and the fibers have been removed, and (C) showing the fibers after the water has been removed. (A) shows the taste sensor evaluation results of the muscle fibers, and (B) shows the weight per muscle fiber. Photographs of the ribeye meat used in the sensory evaluation and the parts of the ribeye meat used in the sensory evaluation are shown. A graph showing the measurement results of the water content of cultured meat prepared by the method according to this embodiment is shown. The amino acid analysis results of lean meat are shown, with (A) showing the analysis results of alanine (sweetness), (B) showing glutamic acid (umami), and (C) showing leucine (bitterness). The results of amino acid analysis of cultured lean meat are shown, with (A) showing the analysis results for alanine (sweetness), (B) showing the analysis results for glutamic acid (umami), and (C) showing the analysis results for leucine (bitterness). The results of analysis of substances that enhance the sweetness response are shown, with (A) showing the amino acids and nucleosides detected in livestock muscle tissue, (B) showing the muscle fibers, and (C) showing the amino acids and nucleosides detected in Wagyu cultured meat and Holstein cultured meat. The results of fatty acid analysis of Wagyu fat and Wagyu cultured meat fat are shown, with (A) showing the fatty acid composition, and (B) and (C) showing the fatty acid amount. The results of fatty acid analysis of Wagyu cultured meat fat are shown. The results of γ-nonalactone analysis are shown, with (A) showing the results for three types of livestock meat, (B) showing the results for cultured meat muscle fibers, and (C) showing the results for cultured meat fat fibers. The method and results of mechanical analysis of livestock meat are shown, with (A) being a diagram explaining hardness and elasticity, (B) showing the results for hardness, and (C) showing the results for elasticity. The methods and results of the mechanical analysis of cultured lean meat are shown, with (A) being a diagram showing the measurement method and (B) showing the results of the elastic modulus. Heat maps of three types of meat are shown. Principal component analysis results of three types of meat are shown. Gene expression volcano plots are shown, with (A) showing the results for crossbred cattle and Wagyu, (B) showing the results for Holstein and crossbred cattle, and (C) showing the results for Holstein and Wagyu. Heat maps of muscle fibers of three types of cultured meat are shown. Principal component analysis results of muscle fibers of three types of cultured meat are shown. Gene expression volcano plots are shown, with (A) showing the results for muscle fibers of crossbred cattle and Wagyu, (B) showing the results for muscle fibers of Holstein and crossbred cattle, and (C) showing the results for Holstein and Wagyu.This shows a protein heatmap analysis of stem cells (satellite cells). It also shows a protein expression analysis of cultured meat. The results of the protein expression analysis of stem cells (satellite cells) are shown. The results of the protein expression analysis of cultured meat are shown. Finally, there is a graph showing the gene expression evaluation results of cultured meat.

[0008] The embodiments for carrying out the present invention will be described in detail below. However, the present invention is not limited to the following embodiments.

[0009] [Method for producing cultured meat] The method for producing cultured meat according to this embodiment includes a washing step of contacting bovine tissue with a washing solution containing peracetic acid, a recovery step of recovering stem cells from the washed bovine tissue, a preparation step of preparing a bioink containing the recovered stem cells and fragmented extracellular matrix components, a printing step of printing the bioink to form a structure containing stem cells, and a culture step of differentiating and inducing the stem cells in the structure to form a tissue.

[0010] Cultured meat, also known as cellular meat or processed cellular meat products, is a cellular food obtained by culturing animal cells in a culture medium.

[0011] <Washing Process> In the washing process, bovine tissue is brought into contact with a washing solution containing peracetic acid.

[0012] The origin of the bovine tissue is not particularly limited, but examples include Japanese Black and Holstein cattle. The bovine tissue may also be of crossbreed origin. Examples of crossbreeds include crosses of Japanese Black and Holstein cattle.

[0013] Examples of bovine tissue parts include the sirloin and cheek meat. The sirloin is a part of the thigh meat, located between the outer thigh and the rump. Because the sirloin is tender, using it makes cell collection easier, further suppresses contamination, and increases the recovery rate of target cells such as fat cells.

[0014] The washing solution contains peracetic acid. This washing solution allows for the recovery of stem cells while maintaining their ability to self-replicate and their pluripotency.

[0015] The washing solution may be an aqueous solution containing peracetic acid and an aqueous medium. The aqueous medium is not particularly limited, but examples include water, physiological saline such as phosphate-buffered saline (PBS), etc.

[0016] The concentration of peracetic acid in the washing solution may be, for example, 0.10 w / v% or more, 0.12 w / v% or more, 0.14 w / v% or more, 0.16 w / v% or more, or 0.17 w / v% or more, relative to the total volume of the washing solution. The concentration of peracetic acid in the washing solution may be 0.19 w / v% or less, relative to the total volume of the washing solution, and may be 0.18 w / v% or less, as this will make the resulting tissue more suitable for food applications. The concentration of peracetic acid in the washing solution may be 0.10 w / v% to 0.19 w / v%, 0.12 w / v% to 0.19 w / v%, 0.14 w / v% to 0.19 w / v%, 0.16 w / v% to 0.19 w / v%, or 0.17 w / v% to 0.19 w / v%, relative to the total amount of washing solution, and may also be 0.10 w / v% to 0.18 w / v%, 0.12 w / v% to 0.18 w / v%, 0.14 w / v% to 0.18 w / v%, 0.16 w / v% to 0.18 w / v%, or 0.17 w / v% to 0.18 w / v%, relative to the total amount of washing solution.

[0017] The cleaning solution can also be prepared, for example, by diluting a commercially available concentrate (e.g., Persan® (Envirotec, containing 15 v / v% peracetic acid, 5 v / v% hydrogen peroxide, and 45 v / v% acetic acid)) in an aqueous medium.

[0018] The washing solution may further contain other ingredients that are suitable for food use.

[0019] The washing solution may be brought into contact with at least the surface of the bovine tissue, i.e., the entire tissue surface, or it may be brought into contact with the interior of the tissue in addition to the entire surface. The washing step may also be a step of bringing at least the surface of the bovine tissue into contact with the washing solution to remove bacteria from the surface of the bovine tissue. "Removing bacteria" means sterilizing bacteria attached to the surface of the collected bovine tissue, inhibiting the growth of bacteria attached to the surface of the bovine tissue, or physically removing bacteria from the surface of the bovine tissue. By bringing at least the surface of the bovine tissue into contact with the washing solution, stem cells can be recovered without contamination by bacteria.

[0020] The method of bringing the bovine tissue into contact with the cleaning solution is not particularly limited as long as at least the surface of the bovine tissue comes into contact with the cleaning solution. Examples include immersing the bovine tissue in the cleaning solution, or spraying or applying the cleaning solution to the surface of the bovine tissue.

[0021] In the washing process, the time for which at least the surface of the bovine tissue is in contact with the washing solution is not particularly limited, and may be, for example, 1 minute or more, 3 minutes or more, or 5 minutes or more, or 20 minutes or less, 15 minutes or less, or 10 minutes or less. In the washing process, the time for which at least the surface of the bovine tissue is in contact with the washing solution may be, for example, 1 minute to 20 minutes, 3 minutes to 20 minutes, or 5 minutes to 20 minutes, or 1 minute to 15 minutes, 3 minutes to 15 minutes, or 5 minutes to 15 minutes, or 1 minute to 10 minutes, 3 minutes to 10 minutes, or 5 minutes to 10 minutes.

[0022] The amount of washing solution used per tissue may be 6 mL or more, 8 mL or more, 10 mL or more, 12 mL or more, 14 mL or more, 16 mL or more, or 18 mL or more, and may be 40 mL or less, 35 mL or less, 30 mL or less, 25 mL or less, or 22 mL or less. The amount of washing solution used per tissue may be 6 mL to 40 mL, 8 mL to 40 mL, 10 mL to 40 mL, 12 mL to 40 mL, 14 mL to 40 mL, 16 mL to 40 mL, or 18 mL to 40 mL, and may be 6 mL to 35 mL, 8 mL to 35 mL, 10 mL to 35 mL, 12 mL to 35 mL, 14 mL to 35 mL, 16 mL to 35 mL, or 18 mL to 35 mL, and may be 6 mL to 30 mL, 8 mL to 30 mL, 10 mL to 30 mL, 12 It may be mL to 30 mL, 14 mL to 30 mL, 16 mL to 30 mL, or 18 mL to 30 mL, and may be 6 mL to 25 mL, 8 mL to 25 mL, 10 mL to 25 mL, 12 mL to 25 mL, 14 mL to 25 mL, 16 mL to 25 mL, or 18 mL to 25 mL, and may be 6 mL to 22 mL, 8 mL to 22 mL, 10 mL to 22 mL, 12 mL to 22 mL, 14 mL to 22 mL, 16 mL to 22 mL, or 18 mL to 22 mL.

[0023] <Recovery Process> In the recovery process, stem cells are recovered from the washed bovine tissue.

[0024] Stem cells recovered from bovine tissue include satellite cells (SCs) and adipose-derived stem cells (ADSCs).

[0025] The recovery process may include subdividing the washed bovine tissue. Subdividing the bovine tissue can be done using sterilized cutting instruments (e.g., sterilized scissors, tweezers). The washed bovine tissue may be shredded to a size of approximately 3 mm. Including subdividing the washed bovine tissue makes it easier to suppress the mixing of tissues with different hardness, such as tendons, in the bovine tissue. By suppressing the inclusion of heterogeneous tissue, the cell proliferation rate becomes more uniform, and as a result, the uniformity of the cells is further improved.

[0026] The recovery process may include dispersing the tissue into multiple cell clumps. A cell clump refers to a cell aggregate consisting of one cell or several cells (for example, about 2 to 10 cells) adhering to each other. Enzymatic methods can be used to disperse the tissue into multiple cell clumps.

[0027] As an enzymatic approach, the method of adding trypsin is preferred because it makes the product even more suitable for food applications.

[0028] The temperature during enzyme treatment can be set appropriately according to the optimal temperature of the enzyme, but for example, it may be 30°C to 40°C, and 37°C is particularly preferred. The duration of the enzyme treatment may be, for example, 10 minutes to 1 hour, or 20 minutes to 40 minutes. The enzyme treatment may be performed on tissue placed in the wells using a well plate. The rotation speed during enzyme treatment may be, for example, 300 rpm to 500 rpm, 350 rpm to 450 rpm, or 390 rpm to 410 rpm.

[0029] Enzyme inactivation can be achieved by adding a liquid culture medium containing fetal bovine serum (FBS). Enzyme inactivation can be achieved, for example, by mixing I-MEM (Integri Culture Tokyo, Japan) containing FBS at a concentration of 10 v / v% to 25 v / v% (e.g., 20 v / v%).

[0030] After dispersing the cells into multiple cell clumps, a procedure to purify the target cells from the multiple cell clumps (a procedure to select the target cells) may be performed. Purification of the target cells can be performed, for example, by selecting them using the difference in adhesion between the target cells and other cells. If the target cells (stem cells) are satellite cells, purification can be performed by separating impurities from the reaction solution after enzyme inactivation and then performing pre-plating. Separation of impurities can be done using centrifugation and a cell strainer, etc. Pre-plating can be performed by seeding the cell suspension obtained after separating impurities into an uncoated culture dish, culturing the cells in the culture medium for 2 to 3 days, and then collecting the cells that did not adhere (on the culture medium side). Pre-plating may be repeated, for example, two or more times.

[0031] The recovery process may include culturing stem cells under conditions that promote stem cell proliferation. The culture conditions can be set according to the type of cell. Stem cell culture may be performed in a stem cell culture medium. "Stem cell culture medium" means a medium in which stem cells can proliferate. There are no particular restrictions on the stem cell culture medium as long as it is a medium in which stem cells can proliferate, but examples include I-MEM, Dulbecco's Modified Eagle medium (DMEM), Iscove's Modified Dulbecco's medium (IMDM), etc. The medium may be a serum-added medium or a serum-free medium. The medium may be a mixed medium obtained by mixing two types of mediums. Furthermore, it is preferable that the stem cell culture medium is a medium in which cells other than stem cells do not proliferate easily.

[0032] For example, the culture temperature may be 20°C to 40°C or 30°C to 37°C. The pH of the culture medium may be 6 to 8 or 7.2 to 7.4. The culture time may be 1 to 2 days or 3 to 5 days. Subculturing may also be performed after a certain culture period. Subculturing refers to the operation of removing the culture medium from the cell culture system and transferring the cells to a new culture medium. When removing the culture medium from the culture system, an additional operation may be performed to detach cells adhering to the culture substrate from the cell substrate. The number of subculturings may be one or more, two or more, three or more, or four or more.

[0033] Stem cell culture is preferably carried out on a collagen-coated dish coated with type I collagen. The type I collagen may be manufactured by Nippi.

[0034] The recovery process may include cryopreserving the stem cells. Cryopreservation can be carried out in accordance with conventional methods.

[0035] Stem cells can be cryopreserved in a cryopreservation solution containing a cryoprotectant. To make the cryopreservation solution more suitable for food applications, it is preferable that it does not contain organic solvents such as dimethyl sulfoxide (DMSO). An example of such a cryopreservation solution is Banbankar® DMSO-free, manufactured by Nippon Genetics Co., Ltd.

[0036] The dormancy and culture of stem cells after cryopreservation may be carried out under conditions suitable for the type of stem cell. The culture medium used during dormancy and culture of stem cells after cryopreservation is not particularly limited as long as it allows stem cells to proliferate, but examples include I-MEM, Dulbecco's Modified Eagle medium (DMEM), Iscove's Modified Dulbecco's medium (IMDM), etc. The culture medium may be a serum-added medium or a serum-free medium. The culture medium may be a mixed medium of two types of media. The cell culture conditions should be conditions that allow stem cells to proliferate. The culture medium used during dormancy and culture of stem cells after cryopreservation is preferably I-MEM containing FBS, as this is more suitable for food applications.

[0037] As the cell-exfoliating enzyme used during culture, trypsin is preferred because the resulting tissue is suitable for food applications.

[0038] The recovery process may be carried out by a method that specifically includes the steps of: subdividing the bovine tissue; enzymatically treating the subdivided bovine tissue with trypsin to disperse it into cell clumps containing stem cells; culturing the cell clumps in a stem cell culture medium; cryopreserving the cultured stem cells; and activating and culturing the stem cells after cryopreservation.

[0039] If the stem cells are satellite cells, the selection of satellite cells can be performed using a method combining pipetting and centrifugation. From the perspective of further reducing processing time, the selection of satellite cells does not necessarily need to be performed using a cell sorter.

[0040] <Preparation Step> In the preparation step, a bioink containing the recovered stem cells and fragmented extracellular matrix components is prepared. The bioink contains an aqueous medium, which may include, for example, the liquid culture medium described above. The preparation step may include filling equipment capable of containing the bioink (for example, culture plates such as 96-well plates and syringes) before carrying out the printing step described later.

[0041] In this specification, "bio-ink" refers to an ink composition capable of forming structures by bioprinting. Specifically, bio-ink is an ink composition containing biocompatible materials that is liquid at the time of ejection from a printer and solidifies after ejection from the printer due to stimulation or the passage of time. The structure formed by the bio-ink may be fibrous.

[0042] The number of stem cells in the bio-ink can be appropriately set depending on the type of stem cells, etc. For example, the number of stem cells in the bio-ink can be set to, for example, 1 × 10⁻⁶ based on the total amount of bio-ink. ７ cells / mL or higher, 10 × 10 ７ It may be less than or equal to cells / mL.

[0043] (Fragmented Extracellular Matrix Component) The "fragmented extracellular matrix component" in this specification can be obtained by fragmenting an extracellular matrix component. The fragmented extracellular matrix component may be dispersed in the bioink.

[0044] The extracellular matrix component is an aggregate of extracellular matrix molecules formed by a plurality of extracellular matrix molecules. The extracellular matrix molecule may be a substance present outside cells in a multicellular organism. As the extracellular matrix molecule, any substance can be used as long as it does not adversely affect cell growth and the formation of cell aggregates. Examples of extracellular matrix molecules include, but are not limited to, collagen, laminin, fibronectin, vitronectin, elastin, tenascin, entactin, fibrillin, and proteoglycan. As the extracellular matrix component, these extracellular matrix molecules may be used alone or in combination of two or more.

[0045] The extracellular matrix molecule may be a modified form or variant of the above-described extracellular matrix molecule, or may be a polypeptide such as a chemically synthesized peptide. The extracellular matrix molecule may have a repeat of a sequence represented by Gly-X-Y characteristic of collagen. Here, Gly represents a glycine residue, and X and Y each independently represent an arbitrary amino acid residue. The plurality of Gly-X-Y may be the same or different. By having a repeat of the sequence represented by Gly-X-Y, the restriction on the arrangement of the molecular chain is reduced. In an extracellular matrix molecule having a repeat of the sequence represented by Gly-X-Y, the proportion of the sequence represented by Gly-X-Y may be 80% or more, preferably 95% or more, of the total amino acid sequence. Also, the extracellular matrix molecule may be a polypeptide having an RGD sequence. The RGD sequence refers to a sequence represented by Arg-Gly-Asp (arginine residue-glycine residue-aspartic acid residue). Examples of extracellular matrix molecules containing the sequence represented by Gly-X-Y and the RGD sequence include collagen, fibronectin, vitronectin, laminin, cadherin, etc.

[0046] Examples of collagen include fibrous collagen and non-fibrous collagen. Fibrous collagen refers to collagen that is the main component of collagen fibers, and specifically includes type I collagen, type II collagen, type III collagen, and the like. Examples of non-fibrous collagen include type IV collagen.

[0047] Examples of proteoglycans include chondroitin sulfate proteoglycan, heparan sulfate proteoglycan, keratan sulfate proteoglycan, and dermatan sulfate proteoglycan, but are not limited thereto.

[0048] The extracellular matrix component may contain at least one selected from the group consisting of collagen, laminin, and fibronectin, and preferably contains collagen. Collagen is preferably fibrous collagen, more preferably type I collagen. Fibrous collagen may be commercially available collagen, and specific examples thereof include type I collagen derived from porcine skin manufactured by Nippon Ham Foods Co., Ltd.

[0049] The extracellular matrix component may be an extracellular matrix component derived from an animal. Examples of animal species from which the extracellular matrix component is derived include, but are not limited to, humans, pigs, cows, and the like. The extracellular matrix component may be a component derived from one type of animal, or may be used in combination with components derived from multiple types of animals.

[0050] In this specification, "fragmentation" means reducing the size of aggregates of extracellular matrix molecules. Fragmentation may be carried out under conditions that cleave bonds within extracellular matrix molecules, or under conditions that do not cleave bonds within extracellular matrix molecules. Unlike enzymatic treatment, the molecular structure of extracellular matrix fragmented by the application of physical force usually does not change from that before fragmentation (the molecular structure is maintained). Fragmented extracellular matrix components may include defibrated extracellular matrix components (defibrated extracellular matrix components), which are components obtained by defibrating the above-mentioned extracellular matrix components by the application of physical force. Defibration is carried out under conditions that do not cleave bonds within extracellular matrix molecules.

[0051] There are no particular limitations on the method for fragmenting extracellular matrix components. For example, extracellular matrix components may be defibrated by applying physical force, such as using an ultrasonic homogenizer, agitator homogenizer, or high-pressure homogenizer. When using an agitator homogenizer, the extracellular matrix components may be homogenized directly or in an aqueous medium such as physiological saline. Furthermore, by adjusting the homogenization time and number of repetitions, it is possible to obtain defibrated extracellular matrix components of millimeter or nanometer size. Defibrated extracellular matrix components can also be obtained by repeated freeze-thaw cycles.

[0052] The fragmented extracellular matrix component may contain at least a portion of the defibrated extracellular matrix component. The fragmented extracellular matrix component may consist solely of the defibrated extracellular matrix component. In other words, the fragmented extracellular matrix component may be the defibrated extracellular matrix component. The defibrated extracellular matrix component preferably contains the defibrated collagen component. The defibrated collagen component preferably maintains the triple helix structure derived from collagen. The defibrated collagen component may be a component that completely or partially maintains the triple helix structure derived from collagen.

[0053] Examples of the shape of fragmented extracellular matrix components include fibrous structures. Fibrous structures refer to structures composed of thread-like fragmented extracellular matrix components, or structures composed of thread-like fragmented extracellular matrix components cross-linked between molecules. At least a portion of the fragmented extracellular matrix components may be fibrous. Fibrous extracellular matrix components include thin thread-like structures (fibrillaries) formed by the aggregation of multiple thread-like extracellular matrix molecules, thread-like structures formed by further aggregation of fibrillaries, and defibrillated versions of these thread-like structures. In fibrous extracellular matrix components, the RGD sequence is preserved without disruption.

[0054] The average length of the fragmented extracellular matrix components may be between 100 nm and 400 μm, or between 100 nm and 200 μm. In one embodiment, the average length of the fragmented extracellular matrix components may be between 5 μm and 400 μm, between 10 μm and 400 μm, between 22 μm and 400 μm, or between 100 μm and 400 μm. In another embodiment, the average length of the fragmented extracellular matrix components may be 100 μm or less, 50 μm or less, 30 μm or less, 15 μm or less, 10 μm or less, 1 μm or less, or 100 nm or more. The average length of most of the fragmented extracellular matrix components may fall within the above numerical range. Specifically, the average length of 95% of the fragmented extracellular matrix components may fall within the above numerical range. The fragmented extracellular matrix component may be a fragmented collagen component having an average length within the above range, or a defibrillated collagen component having an average length within the above range.

[0055] The average diameter of fragmented extracellular matrix components may be, for example, 20 nm to 30 μm, or 20 nm to 10 μm. Fragmented extracellular matrix components with an average diameter of nano-order (1000 nm or less) are also called nanofibers (NF). The average diameter of nanofibers may be, for example, 20 nm to 1000 nm, 20 nm to 500 nm, 20 nm to 200 nm, 20 nm to 150 nm, 40 nm to 130 nm, or 20 nm to 100 nm. Fragmented extracellular matrix components with an average diameter of micro-order (greater than 1000 nm) are also called microfibers (MF). The average diameter of the microfibers may be, for example, greater than 1 μm and 30 μm or less, greater than 1 μm and 20 μm or less, greater than 1 μm and 10 μm or less, greater than 1.5 μm and 8.5 μm or less, or greater than 2 μm and 8.5 μm or less. The fragmented extracellular matrix component may be a fragmented collagen component with an average diameter within the above range, or a defibrated collagen component with an average diameter within the above range. In this embodiment, the shape of the nanofibers and microfibers does not have to be fibrous, as long as they are the fragmented extracellular matrix described above.

[0056] The average length and average diameter of fragmented extracellular matrix components can be determined by measuring individual fragmented extracellular matrix components using an optical microscope and performing image analysis. In this specification, "average length" refers to the average value of the length in the longitudinal direction of the measured sample, and "average diameter" refers to the average value of the length in the direction perpendicular to the longitudinal direction of the measured sample.

[0057] The particle size of the fragmented extracellular matrix component may be less than 40 μm. Fragmented extracellular matrix component with a particle size of less than 40 μm is a fragmented extracellular matrix component that passes through a filter with a pore size of 40 μm. When the particle size of the fragmented extracellular matrix component is less than 40 μm, aggregation of components in the bioink becomes less likely, and the ejection by the 3D printer becomes smoother.

[0058] At least a portion of the fragmented extracellular matrix components may be crosslinked intermolecularly or intramolecularly. The fragmented extracellular matrix components may be crosslinked within the molecules constituting the fragmented extracellular matrix components, or crosslinked between the molecules constituting the fragmented extracellular matrix components.

[0059] Fragmented extracellular matrix components, in which at least a portion are crosslinked intermolecularly or intramolecularly, can be produced, for example, by a method including a step of crosslinking the fragmented extracellular matrix components (crosslinking step). Fragmented extracellular matrix components can include, for example, fragmented and crosslinked extracellular matrix components. Fragmented and crosslinked extracellular matrix components can be produced, for example, by a method comprising, in this order, a step of fragmenting an extracellular matrix component and a step of crosslinking the fragmented extracellular matrix component, or by a method comprising, in this order, a step of crosslinking an extracellular matrix component and a step of fragmenting the crosslinked extracellular matrix component.

[0060] Methods for crosslinking include, for example, physical crosslinking by applying heat, ultraviolet light, or radiation, and chemical crosslinking by using crosslinking agents or enzymatic reactions, but the method is not particularly limited. The crosslinking may be via covalent bonds.

[0061] When the fragmented extracellular matrix component includes fragmented collagen component, crosslinks may be formed between collagen molecules (triple helix structure) or between collagen fibrils formed by collagen molecules.

[0062] Fragmented extracellular matrix components can be crosslinked, for example, by using a crosslinking agent. The crosslinking agent may be, for example, a crosslinking agent capable of crosslinking carboxyl groups and amino groups, or a crosslinking agent capable of crosslinking amino groups with each other. The crosslinking agent may be at least one selected from the group consisting of aldehyde-based crosslinking agents, carbodiimide-based crosslinking agents, epoxide-based crosslinking agents, and imidazole-based crosslinking agents, for example, from the viewpoint of economy, safety, and ease of handling. Examples of crosslinking agents include water-soluble carbodiimides such as glutaraldehyde, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide sulfonate.

[0063] The content of fragmented extracellular matrix components is not particularly limited, but may be 1.0 mg / mL or more, 3.0 mg / mL or more, 5.0 mg / mL or more, 10.0 mg / mL or more, 12.0 mg / mL or more, 15.0 mg / mL or more, or 18.0 mg / mL or more, based on the total volume of the bioink, and may be 45.0 mg / mL or less, 40.0 mg / mL or less, 35.0 mg / mL or less, 30.0 mg / mL or less, or 25.0 mg / mL or less. The content of fragmented extracellular matrix components may be 5 mg / mL to 45 mg / mL, 5 mg / mL to 40 mg / mL, 5 mg / mL to 35 mg / mL, 5 mg / mL to 30 mg / mL, 5 mg / mL to 25 mg / mL, 10 mg / mL to 45 mg / mL, 10 mg / mL to 40 mg / mL, 10 mg / mL to 35 mg / mL, 10 mg / mL to 30 mg / mL, 10 mg / mL to 25 mg / mL, 15 mg / mL to 45 mg / mL, 15 mg / mL to 40 mg / mL, 15 mg / mL to 35 mg / mL, 15 mg / mL to 30 mg / mL, or 15 mg / mL to 25 mg / mL.

[0064] Fragmented extracellular matrix components can be obtained by a method comprising a fragmentation step of fragmenting extracellular matrix components in an aqueous medium to obtain a liquid containing the fragmented extracellular matrix components, and a drying step of freeze-drying the liquid containing the fragmented extracellular matrix components.

[0065] The method for fragmenting extracellular matrix components is as described above. In the drying step, the liquid component is removed from the liquid containing the fragmented extracellular matrix components obtained in the fragmentation step by freeze-drying. The drying step removes the liquid component, and a solid material containing the dried fragmented extracellular matrix components is obtained. It should be noted that the removal of the liquid component does not mean that no liquid component is attached to the solid material containing the dried fragmented extracellular matrix components at all, but rather that the amount of liquid component attached is reduced to a level that can be reasonably achieved by the general drying method described above.

[0066] The freeze-drying time may be 1 day or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, or 7 days or more. There is no particular upper limit on the freeze-drying time. For example, the freeze-drying time may be 10 days or less, 9 days or less, or 8 days or less. The freeze-drying time may be, for example, 1 to 10 days, 2 to 10 days, 3 to 10 days, 4 to 10 days, 5 to 10 days, 6 to 10 days, or 7 to 10 days, 1 to 9 days, 2 to 9 days, 3 to 9 days, 4 to 9 days, 5 to 9 days, 6 to 9 days, or 7 to 9 days, 1 to 8 days, 2 to 8 days, 3 to 8 days, 4 to 8 days, 5 to 8 days, 6 to 8 days, or 7 to 8 days.

[0067] The bioink may further contain fibrinogen. Fibrinogen is a substance that forms a fibrin gel through a reaction with thrombin. The fibrinogen content in the bioink may be 5 mg / mL or more, 10 mg / mL or more, or 15 mg / mL or more, and 30 mg / mL or less, or 25 mg / mL or less, based on the total amount of the bioink. The fibrinogen content in the bioink may be 5 mg / mL to 30 mg / mL, 10 mg / mL to 30 mg / mL, or 15 mg / mL to 30 mg / mL, based on the total amount of the bioink, and may be 5 mg / mL to 25 mg / mL, 10 mg / mL to 25 mg / mL, or 15 mg / mL to 25 mg / mL, based on the total amount of the bioink.

[0068] <Printing Process> In the printing process, bio-ink is printed (bioprint) to form structures containing stem cells.

[0069] The printing process can be carried out using a three-dimensional bioprinting method. The three-dimensional bioprinting method may be automated, semi-automated, computer-assisted, or manual. A dispenser with nozzles, such as a multi-nozzle dispenser, may be used to dispense a large amount of bio-ink containing stem cells at once. The conditions for bioprinting (e.g., temperature, print pressure, nozzle diameter, etc.) can be appropriately set according to the 3D printing device, the shape of the structure, and its application.

[0070] In the printing process, it is preferable to use a 3D printing device equipped with multiple nozzles in order to improve the production speed of cultured meat.

[0071] The number of nozzles in the 3D printing device may be 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or 11 or more, and may be 13 or less, for example, 12.

[0072] The structure can be formed by extruding bio-ink from the nozzle of a 3D printing device into a supporting bath containing gel, and then solidifying the bio-ink.

[0073] Examples of materials that make up the gel in the supporting bath include gellan gum and gelatin.

[0074] If the bioink contains fibrinogen, the supporting bath may further contain thrombin in addition to the gel. The thrombin content may be 1 U / mL or more, 5 U / mL or more, or 8 U / mL or more, and 15 U / mL or less, or 12 U / mL or less, relative to the total volume of liquid constituting the supporting bath. The thrombin content may be 1 U / mL to 15 U / mL, 5 U / mL to 15 U / mL, or 8 U / mL to 15 U / mL, and may be 1 U / mL to 12 U / mL, 5 U / mL to 12 U / mL, or 8 U / mL to 12 U / mL, relative to the total volume of liquid constituting the supporting bath.

[0075] To solidify the printed structure, it may be incubated in a supporting bath. The incubation temperature may be between 10°C and 40°C. The incubation time may be 0.5 hours or more, or 1.0 hour or more, and 2 hours or less, or 1.5 hours or less. The incubation time may be 0.5 hours to 2 hours, or 1.0 hour to 2 hours, or 0.5 hours to 1.5 hours, or 1.0 hour to 1.5 hours.

[0076] After the structure is formed, the materials constituting the supporting bath may be removed. The method for removing the materials is appropriately selected depending on the type of material, etc. Methods for removing the materials include removal by pipetting, and removal by, for example, immersion in an aqueous medium heated to 35-40°C and incubation for 1-3 hours.

[0077] <Culture Process> In the culture process, stem cells in the structure are differentiated and cultured to form a tissue. The stem cells in the structure may be cultured in the liquid medium described above before the culture process if necessary, before undergoing differentiation and culture.

[0078] Stem cell differentiation induction culture can be carried out by conventional methods. The conditions for differentiation induction culture are selected according to the type of stem cells, etc. The culture medium can be selected according to the type of cells to be cultured. Examples of culture media include I-MEM, Eagle's MEM medium, DMEM, Modified Eagle medium (MEM), Minimum Essential medium, RPMI, and GlutaMax medium. For food applications, the culture medium is preferably I-MEM, which consists only of food components. The culture medium may be a mixed medium obtained by mixing two types of media. The culture medium may be a serum-added medium or a serum-free medium. The temperature for differentiation induction culture may be 20°C to 40°C or 30°C to 37°C. The pH of the culture medium may be 6 to 8 or 7.2 to 7.4. The differentiation induction culture time may be 24 hours or more and 336 hours or less, 72 hours or more and 336 hours or less, 96 hours or more and 384 hours or less, or 96 hours or more and 288 hours or less.

[0079] If the stem cells are satellite cells, differentiation induction culture can be performed in DMEM containing FBS.

[0080] If the stem cells are adipose-derived stem cells, differentiation induction culture may be performed by culturing the adipose-derived stem cells in a culture medium containing oleic acid, as this provides more suitable conditions for food applications. It is preferable that the culture medium does not contain any fatty acids other than oleic acid, as this makes it more suitable for food applications.

[0081] As for oleic acid, it is preferable to use purified oleic acid. The oleic acid content in the culture medium may be, for example, 200 μM or more, 300 μM or more, 400 μM or more, or 450 μM or more, based on the total amount of the culture medium, and may be 700 μM or less, 650 μM or less, 600 μM or less, or 550 μM or less. The oleic acid content in the culture medium may be 200 μM to 700 μM, 300 μM to 700 μM, 400 μM to 700 μM, or 450 μM to 700 μM based on the total amount of the culture medium; it may be 200 μM to 650 μM, 300 μM to 650 μM, 400 μM to 650 μM, or 450 μM to 650 μM; it may be 200 μM to 600 μM, 300 μM to 600 μM, 400 μM to 600 μM, or 450 μM to 600 μM; it may be 200 μM to 550 μM, 300 μM to 550 μM, 400 μM to 550 μM, or 450 μM to 550 μM.

[0082] The culture vessel (support) used for culturing stem cells is not particularly limited and may be, for example, a well insert, a low-adhesion plate, or a plate with a bottom shape such as U-shaped or V-shaped. The cells may be cultured while attached to the support, or without being attached to the support, or they may be separated from the support during culture. When culturing the cells without being attached to the support, or when separating them from the support during culture, it is preferable to use a plate with a bottom shape such as U-shaped or V-shaped that inhibits cell adhesion to the support, or a low-adhesion plate.

[0083] By using satellite cells as stem cells and differentiating and culturing structures formed into a fibrous structure by bioprinting, muscle fibers can be obtained as tissue. By using adipose-derived stem cells as stem cells and differentiating and culturing structures formed into a fibrous structure by bioprinting, adipose fibers can be obtained as tissue.

[0084] [Cultured Meat] One embodiment of cultured meat is obtained by the method for producing cultured meat described above. The cultured meat may be formed by bonding together a plurality of structures that have been formed into fibers by bioprinting using the method described above. The structures can be bonded together, for example, using an adhesive such as gelatin.

[0085] Cultured meat may contain at least one of the group consisting of muscle fibers and adipose fibers, and may contain both muscle fibers and adipose fibers. When cultured meat contains both muscle fibers and adipose fibers, the ratio of the number of adipose fibers used to the number of muscle fibers used (number of fibers) (adipose fibers / muscle fibers) is not particularly limited, but may be, for example, 1 / 2 to 2 / 1.

[0086] According to the method of this embodiment described above, cultured meat containing fatty acids such as docosahexaenoic acid can be produced. Therefore, as another embodiment of the present invention, cultured meat containing docosahexaenoic acid is provided.

[0087] The cultured meat may further contain γ-nonalactone and may generate γ-nonalactone upon heating. Heating conditions include using 20 mg of cultured meat as a heating sample and heating the sample at 200°C for 30 minutes. Docosahexaenoic acid, γ-nonalactone, and its precursors may be cell-derived.

[0088] Cultured meat may further contain one or more fatty acids other than docosahexaenoic acid (C22:6n3). Specific examples of fatty acids include saturated fatty acids and unsaturated fatty acids. Specific examples of unsaturated fatty acids include monounsaturated fatty acids and polyunsaturated fatty acids.

[0089] Examples of saturated fatty acids include myristic acid (C14:0), pentadecanoic acid (C15:0), palmitic acid (C16:0), margaric acid (C17:0), stearic acid (C18:0), and arachidic acid (C20:0).

[0090] Examples of monounsaturated fatty acids include myristoleic acid (C14:1), palmitoleic acid (C16:1), elaidic acid (C18:1n9t), oleic acid (C18:1n9c), gandrelic acid (C20:1n9), and erucic acid (C22:1n9).

[0091] Examples of polyunsaturated fatty acids include linoleic acid (C18:2n6c), alpha-linolenic acid (C18:3n3), dihomo-γ-linolenic acid (C20:3n6), and arachidonic acid (C20:4n6).

[0092] Cultured meat may further contain at least one fatty acid selected from the group consisting of myristic acid (C14:0), pentadecanoic acid (C15:0), palmitic acid (C16:0), margaric acid (C17:0), stearic acid (C18:0), arachidic acid (C20:0), myristoleic acid (C14:1), palmitoleic acid (C16:1), elaidic acid (C18:1n9t), oleic acid (C18:1n9c), gandrelic acid (C20:1n9), erucic acid (C22:1n9), linoleic acid (C18:2n6c), alpha-linolenic acid (C18:3n3), dihomo-γ-linolenic acid (C20:3n6), and arachidonic acid (C20:4n6).

[0093] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0094] [Production of Cultured Meat] <Origin and Source of Cattle> Source (Beef Acquisition Route) and Source (Part of the Beef) In this example, beef obtained from Sankyo Meat Co., Ltd. (Kyushu) and IH Meat Packer Co., Ltd. (Tohoku) was used. For Japanese Black, crossbred (Japanese Black x Holstein), and Holstein beef, bovine tissue was collected from the sirloin area of ​​the carcass immediately after slaughter, so that cell collection could be performed the day after slaughter. The bovine tissue was transported at chilled temperatures. Number of times bovine tissue was provided (May 2023 - January 2024) (Up to 6 heads per provision, approximately 30g per head) Total 56 times (301 heads) Japanese Black: 37 times (213 heads) Crossbred: 6 times (32 heads) Holstein: 13 times (56 heads)

[0095] <Recovery of bovine satellite cells (bSCs) and bovine adipose-derived stem cells (bADSCs)> Bovine satellite cells (bSCs) and bovine adipose-derived stem cells (bADSCs) were recovered in the same manner as described in Non-Patent Literature 1 (Nature Communications volume 12, Article number: 5059 (2021)) and Non-Patent Literature 2 (Materials Today Bio, Volume 21, August 2023, 100720), except that the sterilization, meat subdivision, enzyme treatment, enzyme inactivation treatment, and serum were performed under the conditions shown below.

[0096] (Sterilization) Bovine tissue collected from the sirloin area was washed with a washing solution for 5 minutes. The washing solution was prepared by adding 214 μL of peracetic acid (15% [v / v]) to 20 mL of PBS. The concentration of peracetic acid in the washing solution was 0.18% [w / v] (calculation formula: 15% [v / v] × 0.214 / 20 × 1.12 (specific gravity)). Approximately 20 mL of the washing solution was used per tissue. After processing, the surface of the meat was discolored, so it was trimmed and the inner portion was collected.

[0097] (Meat subdivision process) Muscle tissue was extracted from sterilized bovine tissue and finely chopped with sterilized tweezers and scissors to create bovine tissue fragments of approximately 3 mm.

[0098] (Enzymatic treatment of bSC and bADSC) The bovine tissue samples described above were transferred to a well plate at a rate of approximately 2-3 g per well, and treated with trypsin at a concentration of 3 mL (4,000 U / mL) per well. The enzymatic treatment was carried out by shaking the plate for 30 minutes at 400 rpm and 37°C.

[0099] (Enzyme inactivation treatment) I-MEM containing 20% ​​fetal bovine serum (FBS) was added to the enzyme-treated wells to stop the trypsin reaction.

[0100] (Serum) As serum, Gibco Fetal Bovine Serum (FBS), qualified, Brazil, product number: 10270106 was used.

[0101] <Cell purification and cryopreservation> Except for the following conditions for bSC purification, coating of dishes used for subculturing, and cryopreservation, and the absence of a cell sorting step for satellite cells, cell purification and cryopreservation were carried out in the same manner as described in Non-Patent Document 1.

[0102] (bSC purification and coating of dishes used for subculturing) After inactivation, impurities were removed from the trypsin reaction solution by centrifugation and cell straining, and the pre-plating procedure was repeated twice. Pre-plating aims to increase the purity of bSCs by seeding the cell suspension into an uncoated culture dish and collecting the cells that did not adhere (on the culture medium side) after 2-3 days. After repeating pre-plating twice, the cells were seeded into a collagen-coated dish and cultured. The collagen-coated dish was made using type I collagen from Nippi.

[0103] (Cryopreservation) For cell cryopreservation, we used Banbankar (registered trademark) DMSO-free, manufactured by Japan Genetics Co., Ltd.

[0104] <Post-freezing dormancy and extended culture> Post-freezing dormancy and extended culture were performed in the same manner as described in Non-Patent Document 1, except that the conditions for bSC dormancy and culture, bSC selection, bADSC dormancy and culture, and cell exfoliation enzymes during culture were set as shown below.

[0105] (bSC dormancy and culture) bSC dormancy and culture were performed by adding 5 ml of I-MEM containing 20% ​​FBS, bFGF, and p38i, along with cells, to a 15 ml tube and centrifugation (1000 rpm, 3 minutes, room temperature).

[0106] (bSC selection) bSCs were separated by pipetting and centrifugation. They were detached with trypsin, and their adhesion to the collagen coating was also utilized.

[0107] (bADSC dormancy and culture) bADSC dormancy and culture were performed using I-MEM containing 10% FBS.

[0108] (Cell exfoliation enzyme during culture) Trypsin (20,000 U / mL) was used to collect the cells.

[0109] <Bio-ink preparation> (Collagen microfiber preparation method) The collagen microfibers (CMF) used in this example are prepared in the same manner as the CMF preparation method described in the non-patent document mentioned above. In this example, the freeze-drying period was changed to one week from the conditions described in the non-patent document.

[0110] (Bio-ink composition) The bio-ink was prepared in the same manner as in Non-Patent Document 1, except that the composition was changed to include stem cells (bSC and bADSC), CMF, and fibrinogen (except that Matrigel was not used).

[0111] <Printing> Bio-ink printing was performed in the same manner as in Non-Patent Document 1, except that multiple bio-prints were performed simultaneously using a three-dimensional printer equipped with 12 nozzles.

[0112] <Differentiation Induction> Differentiation induction of bSCs was carried out in the same manner as in Non-Patent Document 1, except that DMEM containing 2% FBS was used as the bSC differentiation induction medium.

[0113] The differentiation induction of bADSCs was carried out in the same manner as in Non-Patent Document 1, except that DMEM containing 10% FBS and 500 μM purified oleic acid was used as the bADSC differentiation induction medium (FBS was used, and only purified oleic acid was used as the fatty acid for differentiation culture).

[0114] Hereinafter, artificial tissue constructed using bSC will be referred to as muscle fiber (or muscle fiber), and artificial tissue constructed using bADSC will be referred to as adipose fiber (or adipose fiber).

[0115] [Evaluation of Cultured Meat] 1. Taste evaluation using a taste sensor <Sample preparation, measurement, and analysis> [Sample preparation] Based on the example of beef using intelligent sensor technology, the sample was prepared using the following method. (1) Finely chop beef or cellular beef. (2) Place (1) and pure water in a beaker (5 to 200 times weight dilution) and cover with plastic wrap. (3) Heat the beaker from (2) in a water bath over high heat until boiling, then reduce heat to low and heat for 1 hour. (4) Allow the beaker to return to room temperature and add the amount of water that has evaporated. After cooling the beaker thoroughly in ice water, centrifuge at 3,000 rpm at 1°C for 10 minutes to remove fat. (5) Filter the contents of the beaker through a stoma filter and measure this as the stock solution. (6) For umami, prepare a dilution series and measure.

[0116] [Analytical equipment] Insent Co., Ltd. Taste recognition device TS-5000Z

[0117] [Analysis Items] (Initial Taste) Sourness, bitterness, off-flavors, astringency, stimulating taste, umami, saltiness (Aftertaste) Bitterness, astringency, umami, richness The initial taste is the taste felt the moment it enters the mouth. The aftertaste is the persistent taste that remains after swallowing.

[0118] [Analysis] (i) Taste items with a negative difference from the reference solution (30 mM KCl, 0.3 mM tartaric acid: equivalent to human saliva) were excluded from evaluation (no taste). However, since the reference solution contains sourness and saltiness, sourness values ​​of "-13" and saltiness values ​​of "-6" or lower were excluded from evaluation. (ii) For umami, the intensity of umami was plotted against the logarithm of the dilution ratio, and relative comparisons between samples were made at dilutions within the range where linearity was possible.

[0119] The values ​​for each taste item are shown as relative values. The limit of concentration difference (taste difference) that humans can distinguish is "1.0". Sensor output values: ▲ < ● < ○ < ◎ The sensor output values ​​indicate that ◎ is the highest intensity and ▲ is the lowest intensity.

[0120] Table 1 shows the results of taste evaluation using a taste sensor. Taste items, sensors, and taste characteristics are described at URL: https: / / www.insent.co.jp / taste-sensor / .

[0121]

[0122] <Selection of "flavorful" taste items in beef, and calculation of required amount of cellular beef sample (preliminary study)> In the case of meat, hot water extraction is usually performed with a weight dilution of 2.5 to 5 times (using 30g to 100g of meat), but it is currently difficult to produce cellular beef on the order of tens of grams. Therefore, with the aim of reducing the required amount of sample as much as possible, we investigated the behavior of the sensor when the dilution ratio during hot water extraction was increased.

[0123] The following was found as a result. The values ​​in Table 2 are those when the reference solution is set to "0". ・In beef thigh meat, the items that could be evaluated (had taste) using a taste sensor were bitterness / off-flavors, umami, and umami / richness (however, umami / richness at 100x and 200x dilutions were not evaluated). ・As the dilution level increased, the intensity of umami / richness (aftertaste) decreased, while the intensity of bitterness / off-flavors increased (Table 2). Since bitterness / off-flavors (initial taste) are related to richness and complexity, this change in value was considered reasonable. ・Umami could also be measured in hot water extracts diluted 100x and 200x.

[0124]

[0125] Based on the above, we decided to prepare approximately 1 g of cellular beef from each breed and extract it with hot water at a weight dilution of 100 to 150 times (120 mL to 150 mL of the test solution is required).

[0126] <Evaluation of three varieties of ribeye meat from the same cut (between the 9th and 10th ribs)> Lean meat was minced, extracted with hot water, and fat was removed by cooling and centrifugation before being measured with a taste sensor. (N=3 for each variety). The results are shown in Figures 1(A) and (B). A concentration difference that can be distinguished by humans is 1.0. The dashed lines in Figures 1(A) and (B) indicate the concentration difference that can be distinguished by humans.

[0127] While there were slight differences in bitterness and off-flavors in Holstein cattle between 5x dilution hot water extraction (standard measurement) and 100x dilution hot water extraction (higher dilution ratio for cultured meat), no significant differences were observed between breeds in either case. It is thought that the taste sensor cannot distinguish differences in meat quality between breeds because it removes fat before measurement.

[0128] <Preparation of muscle fibers derived from each of the three varieties> Six lots of muscle fibers derived from each variety were prepared, two lots each, as shown in Table 3. Figure 2(A) is a photograph of 96 fibers after 3D printing, differentiation and culture, and removal after solidification with gelatin. Figure 2(B) is a photograph of the fibers after the gelatin has been dissolved and they have been removed. Figure 2(C) is a photograph of the fibers after the water has been removed, they have been weighed, and they are being frozen for storage.

[0129]

[0130] <Evaluation of the fabricated muscle fibers> The muscle fibers were finely cut, extracted with hot water, cooled, centrifuged, and then measured with a taste sensor. The results are shown in Figures 3(A) and (B).

[0131] The muscle fibers exhibited a savory flavor and richness comparable to that of ribeye. However, the savory flavor was slightly lower, while bitterness and bitter off-flavors tended to be higher. The bitterness and off-flavors of the muscle fibers showed significant variability between lots, with higher values ​​tending to be observed in smaller average weights. No differences were detected based on the origin of the muscle fibers.

[0132] <Sensory Evaluation of Three Varieties of Ribeye Meat> For the sensory evaluation, three varieties of ribeye meat were used, which had been castrated, frozen five days after slaughter, and thawed at 4°C for two days. The loin core was cut into pieces 5 cm wide and approximately 8 cm thick (indicated as a in Figure 4), and used as the sample before cooking. The cooked sample was divided into two, and one piece from each was provided to the sensory evaluators. The sensory evaluation of the three varieties of ribeye meat was conducted by 14 sensory evaluators (5 males, 9 females). The results of the sensory evaluation are shown in the table below. For the evaluation items of "aroma of the meat," "juiciness of the meat," "tenderness of the meat," "perception of juiciness," and "overall evaluation," sensory evaluators were asked to select one option from the choices shown in each table. For the evaluation items of "taste when chewed" and "reason for overall evaluation," multiple answers were allowed from the choices shown in each table.

[0133]

[0134] The sensory evaluation revealed differences and characteristics between breeds. Japanese Black cattle received a high number of responses for items that appeared to be related to marbling. Of the 14 evaluators, three rated Japanese Black cattle as somewhat undesirable and Holstein cattle as desirable.

[0135] In sensory evaluation, clear differences were observed in the texture, aroma, and taste of the three varieties' meat. However, taste sensors revealed that there was no discernible difference in the taste of the lean meat after removing the fat.

[0136] <Evaluation of muscle fibers from each of the three varieties> No differences were observed in the taste sensor depending on the variety from which the muscle fibers originated. It showed a similar level of umami and richness as ribeye meat, but the umami was low, the bitterness was slightly high, and the bitter and off-flavors were high.

[0137] <Nutritional Analysis of Meat> In accordance with the provisions of the Food Labeling Act, the protein, lipids, carbohydrates, salt equivalent, moisture content, and ash content were measured per 100g each of lean meat and fat meat. The results are shown in the table below.

[0138] Wagyu lean beef contained 26% fat (compared to 8% for Holstein) and 57% water (compared to 70% for Holstein).

[0139] <Moisture Content of Livestock Meat and Cultured Meat> Livestock meat (Real meat) and cultured meat (Cultured meat) were freeze-dried for one day and their weight was measured. The moisture content was calculated using the following formula: Moisture content (%) = (Weight before freeze-drying [mg] - Weight after freeze-drying [mg]) / Weight before freeze-drying [mg] × 100

[0140] Figures 5(A) and (B) show the results of moisture content measurement. The cultured meat contained more than 95% water.

[0141] <Amino Acid Analysis of Lean Meat> Figures 6(A) to (C) show the results of amino acid analysis of lean meat. (A) shows the analysis results for alanine (sweetness), (B) shows the analysis results for glutamic acid (umami), and (C) shows the analysis results for leucine (bitterness). Wagyu beef contained 2.0 times more alanine (sweetness), 1.03 times more glutamic acid (umami), and 1.07 times more leucine (bitterness) than Holstein beef.

[0142] <Amino Acid Analysis of Cultured Meat> Figures 7(A) to (C) show the results of amino acid analysis of cultured meat. (A) shows the analysis results for alanine (sweetness), (B) shows the analysis results for glutamic acid (umami), and (C) shows the analysis results for leucine (bitterness). The analytical value of Holstein cultured meat (Value of H's CM) was set to 1. Wagyu beef contained 0.2 times the amount of alanine, 0.2 times the amount of glutamic acid, and 1.5 times the amount of leucine compared to Holstein. It was revealed that cultured meat has a lower amino acid content compared to livestock meat, but this is expected to improve due to a reduction in water content.

[0143] <Analysis of substances that enhance sweetness response> Figure 8 shows the analysis results of substances that enhance sweetness response, with (A) showing livestock muscle tissue, (B) showing muscle fibers, and (C) showing amino acids and nucleosides detected in Wagyu cultured meat and Holstein cultured meat. It was revealed that substances that enhance sweetness response are characteristically abundant in Wagyu cultured meat. It is expected that achieving an increase in amino acid content in cultured meat will result in sweeter Wagyu beef.

[0144] <Analysis of Fatty Acids> Figure 9 shows the results of fatty acid analysis of Wagyu beef fat and Wagyu cultured meat fat, where (A) shows the fatty acid composition and (B) and (C) show the fatty acid content. Wagyu beef fat contained 44% oleic acid (Holstein: 36.6%, Crossbred cattle: 37.9%), and Wagyu cultured meat fat contained 34% oleic acid (Holstein: 31.1%, Crossbred cattle: 36.5%). Wagyu beef fat contained 1.2 times more oleic acid than Holstein, and Wagyu cultured meat fat contained 1.2 times more oleic acid than Holstein. From these results, it can be seen that cultured meat fat was able to achieve the same fatty acid composition as Wagyu beef fat.

[0145] <Analysis of Saturated Fatty Acids (SFA), Monounsaturated Fatty Acids (MUFA), and Polyunsaturated Fatty Acids (PUFA)> The table below shows the analysis results of saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), and polyunsaturated fatty acids (PUFA). As shown in the table below, it was confirmed that cultured meat contains DHA. Figure 10 shows the analysis results of fatty acid components (C14:0-C22:6n3) of ADSC-derived cultured meat.

[0146]

[0147] <Analysis of γ-nonalactone> Minced meat was prepared by mincing three types of livestock meat. Approximately 20 mg of the minced meat was taken and heated at 200°C for 30 minutes. Cultured meat was also prepared and heated at 200°C for 30 minutes at a rate of approximately 20 mg per container. The aroma components containing γ-nonalactone were analyzed in the minced meat and cultured meat before and after heating. The extraction temperature was 25°C, the extraction time was 50 minutes, and the desorption time was 2 minutes.

[0148] Figure 11 shows the results of γ-nonalactone analysis, with (A) showing the results for three types of livestock meat, (B) showing the results for cultured meat muscle fibers, and (C) showing the results for cultured meat fat fibers.

[0149] When heated, Wagyu beef produced six times more gamma-nonalactone, a sweet-smelling compound, than Holstein beef, and 1.5 times more than crossbred beef. The production of gamma-nonalactone was also confirmed in cultured meat. In addition, 58 other aromatic compounds were analyzed from the cultured meat.

[0150] <Mechanical Analysis of Livestock Meat and Cultured Meat Lean Meat> Figure 12 shows the method and results of the mechanical analysis of livestock meat, with (A) illustrating the hardness and elasticity, (B) showing the results for hardness, and (C) showing the results for elasticity. Wagyu beef showed 0.5 times the hardness and 1.6 times the elasticity of Holstein beef.

[0151] Figure 13 shows the method and results of the mechanical analysis of cultured lean meat, where (A) is a diagram showing the measurement method and (B) shows the results of the elastic modulus analysis. Wagyu beef showed an elastic modulus 0.7 times that of Holstein beef.

[0152] Cultured Wagyu beef was able to replicate 70% of the elastic modulus of Wagyu beef, and cultured Holstein beef was able to replicate 100% of the elastic modulus of Holstein beef.

[0153] <Heatmaps and Principal Component Analysis of Three Types of Meat> Figure 14 shows the heatmaps of the three types of meat. Genes with the lowest gene expression values ​​(FPKM values) in the bottom 25% were excluded. Figure 15 shows the results of the principal component analysis of the three types of meat. It was found that Wagyu, crossbred cattle, and Holstein meat each have different gene expression patterns.

[0154] <Analysis of differences in gene expression among three types of livestock> Figure 16 shows the gene expression volcano plot, with (A) showing the results for crossbred cattle and Wagyu, (B) showing the results for Holstein and crossbred cattle, and (C) showing the results for Holstein and Wagyu. It was found that the difference in gene expression between Wagyu and Holstein was greater than that between crossbred cattle.

[0155] <Heatmap and Principal Component Analysis of Three Types of Cultured Meat Muscle Fibers> Figure 17 shows the heatmap of the three types of cultured meat muscle fibers. Those with the lowest gene expression values ​​(FPKM values) in the bottom 25% were excluded. Figure 18 shows the results of the principal component analysis of the three types of cultured meat muscle fibers. It was found that Wagyu, crossbred cattle, and Holstein each have different gene expression patterns.

[0156] <Analysis of differences in gene expression in three types of cultured meat muscle fibers> Figure 19 shows the gene expression volcano plot, with (A) showing the results for crossbred cattle and Wagyu beef muscle fibers, (B) showing the results for Holstein and crossbred cattle muscle fibers, and (C) showing the results for Holstein and Wagyu beef. It was found that Wagyu beef showed a large difference in expression compared to both crossbred cattle and Holstein beef. The results showed a large difference in expression compared to livestock meat.

[0157] <Protein Heatmap Analysis of Stem Cells (Satellite Cells) and Protein Expression Analysis of Cultured Meat> Figure 20 shows the protein heatmap analysis of stem cells (satellite cells). Figure 21 shows the protein expression analysis of cultured meat. In stem cells, Wagyu and crossbred cattle were closely related, while in cultured meat, Wagyu differed from Holstein and crossbred cattle. It was confirmed that differentiation induction altered the protein expression of cultured Wagyu beef.

[0158] <Protein Expression Analysis of Stem Cells (Satellite Cells) and Cultured Meat> Figure 22 shows the results of protein expression analysis of stem cells (satellite cells). The expression trends of metabolic proteins were generally the same among the three species, with slightly higher expression of glycolytic proteins in Holstein.

[0159] Figure 23 shows the results of protein expression analysis of cultured meat. In cultured meat, Cori cycle expression was decreased and citric acid cycle expression was increased compared to cells.

[0160] <Evaluation of Gene Expression in Cultured Meat> Figure 24 is a graph showing the results of the gene expression evaluation of cultured meat. It was confirmed that cultured meat expressed 46 more genes than actual meat.

[0161] <Production of Cellular Beef> We achieved an efficient bioprinting process that allows us to bioprint 576 tissue fibers (equivalent to 6 print containers) in one hour, which is necessary for the target cellular beef (2 cm long x 2 cm wide x 1 cm high). The perfusion culture incubator can accommodate 6 print containers for culture after bioprinting, and differentiation induction can be carried out for one week without human intervention. The final operational results using these devices are as follows: ・Number of cellular muscle tissue fibers and differentiation-inducing tissue fiber prints using bSCs: 9 print containers (864 fibers) ・Number of cellular adipose tissue fibers and differentiation-inducing tissue fiber prints using bADSCs: 3 print containers (288 fibers)

[0162] Regarding tissue fiber retrieval, we established a method in which gelatin is sealed in a print container to fix the fibers, and then a jig is used to bundle the tissue fibers in an aligned state into groups of eight.

Claims

1. A method for producing cultured meat, comprising: a washing step of contacting bovine tissue with a washing solution containing peracetic acid; a recovery step of recovering stem cells from the washed bovine tissue; a preparation step of preparing a bioink containing the recovered stem cells and fragmented extracellular matrix components; a printing step of printing the bioink to form a structure containing the stem cells; and a culture step of differentiating and inducing the stem cells in the structure to form a tissue.

2. The method according to claim 1, wherein the stem cells include at least one selected from the group consisting of satellite cells and adipose-derived stem cells.

3. The method according to claim 1 or 2, wherein the recovery step includes a step of subdividing the bovine tissue, an enzymatic treatment of the subdivided bovine tissue with trypsin to disperse it into cell clumps containing the stem cells, a step of culturing the cell clumps in a culture medium for stem cells, a step of cryopreserving the cultured stem cells, and a step of activating and culturing the stem cells after cryopreservation.

4. The method according to claim 1 or 2, wherein the fragmented extracellular matrix component is a defibrated extracellular matrix component.

5. The method according to claim 1 or 2, wherein the fragmented extracellular matrix component is a fragmented collagen component.

6. The method according to claim 1 or 2, wherein the fragmented extracellular matrix component is a defibrillated collagen component.

7. The method according to claim 1 or 2, wherein the bioink further comprises fibrinogen.

8. The method according to claim 1 or 2, wherein the structure in the printing step is formed by solidifying the bio-ink discharged from the nozzles of a printer device having a plurality of nozzles.

9. The method according to claim 1 or 2, wherein the differentiation induction culture is carried out in the presence of fetal bovine serum (FBS).

10. The method according to claim 1 or 2, wherein the stem cells include adipose-derived stem cells, and the differentiation induction culture is carried out in the presence of oleic acid.

11. The method according to claim 1 or 2, wherein the tissue is fibrous.

12. The method according to claim 1 or 2, wherein the bovine tissue is bovine tissue taken from the sirloin area.

13. Cultured meat obtained by the method described in claim 1 or 2.

14. Cultured meat containing docosahexaenoic acid and gamma-nonalactone.

15. Cultured meat according to claim 14, further comprising at least one fatty acid selected from the group consisting of myristic acid (C14:0), pentadecanoic acid (C15:0), palmitic acid (C16:0), margaric acid (C17:0), stearic acid (C18:0), arachidic acid (C20:0), myristoleic acid (C14:1), palmitoleic acid (C16:1), elaidic acid (C18:1n9t), oleic acid (C18:1n9c), gandrelic acid (C20:1n9), erucic acid (C22:1n9), linoleic acid (C18:2n6c), α-linolenic acid (C18:3n3), dihomo-γ-linolenic acid (C20:3n6), and arachidonic acid (C20:4n6).

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