Method for producing cultured meat using FBS(fetal bovine serum) substitute derived from slaughter by-products

Abstract

The present invention relates to a method for producing cultured meat using a fetal bovine serum (FBS) substitute derived from slaughterhouse by-products. More specifically, the invention relates to a method for producing a cultured meat culture medium additive (FBS substitute) from slaughterhouse by-products, a cultured meat culture medium additive produced by said method, a cultured meat culture medium composition containing said additive, and a method for producing cultured meat using said additive. By using a cultured meat culture medium additive (FBS substitute) produced by the method unique to the present invention, it is possible to completely replace or use in combination with fetal bovine serum (FBS), which conventionally has a high unit cost and raises ethical issues as it must be extracted from live calves, thereby reducing the amount of FBS used and improving the production efficiency of cultured meat. Furthermore, there are advantages such as increasing the value of slaughterhouse by-products—which are inevitably generated during the slaughter of livestock for meat production—by enhancing their usability, and significantly increasing economic effects such as reduced disposal costs and increased income.

Description

Technology Field

[0001] The present invention relates to a method for producing cultured meat using a fetal bovine serum (FBS) substitute derived from slaughterhouse by-products, and more specifically, to a method for producing a cultured meat culture medium additive (FBS substitute) from slaughterhouse by-products, a cultured meat culture medium additive produced by the said method, a cultured meat culture medium composition containing said additive, and a method for producing cultured meat using said additive. Background Technology

[0003] According to a report by the Food and Agriculture Organization of the United Nations (FAO), the world population is projected to increase to 9.5 billion by 2050. However, due to abnormal global climate conditions such as global warming, crop yields are expected to decrease, while the demand for feed grains is rising, leading to increased production costs and a reduction in production area. Consequently, livestock products are expected to become expensive food items as a food resource.

[0004] Although animal-derived foods such as livestock products are expensive in terms of energy input and production, they have made a direct contribution to ensuring human nutrition because they are the best source of high-quality protein and micronutrients essential for normal human growth and health. In particular, essential amino acids are nutrients that must be obtained through food because they are not synthesized in the body or, even if synthesized, are in very small quantities. Given the prediction that the demand for animal-derived foods will reach 550 million tons by 2050—double the current amount—there are limitations to meeting the protein requirements for supplying essential amino acids through traditional livestock production methods.

[0005] Recently, 'cultured meat' has been attracting attention as a solution to address future meat shortages, serving as an alternative to the method of producing meat by directly raising livestock. Cultured meat refers to edible meat obtained by extracting cells from living animals and multiplying them using cell engineering technology; it can be considered a field of cellular agriculture that produces meat without the process of raising livestock. In Korean, it is referred to as cultured meat, alternative meat, or artificial meat. In English, it is called 'in vitro meat' (meaning grown in a test tube), 'artificial meat' (meaning synthesized by humans using stem cells rather than being natural), 'clean meat' (meaning produced in clean facilities rather than traditional livestock farms), and 'lab-grown meat' (since some prototypes are created in laboratories).

[0006] Cultured meat primarily utilizes muscle satellite cells, which are a type of stem cell. While other cells such as embryonic stem cells, induced pluripotent stem cells, and adipose-derived adult stem cells possess sufficient differentiation potential, there have been no successful cases of them differentiating into muscle tissue. In contrast, muscle satellite cells are known to be the most suitable for cultured meat production because they perform a regenerative role in the event of skeletal muscle injury and differentiate exclusively into muscle tissue.

[0007] The most important substance in the cell medium used for the proliferation and differentiation processes of such cultured meat is fetal bovine serum (FBS). FBS can be obtained from blood fractions collected from unborn fetuses when pregnant cows are inevitably slaughtered. Compared to general serum, FBS contains fewer antibodies and a higher concentration of growth factors, making it effective for cell culture. Furthermore, because it is obtained from fetuses prior to birth, it contains only a minimal amount of antibodies, which reduces the likelihood of triggering immune responses in various cells. In addition, while the demand for FBS used in biotechnological research, such as vaccine and pharmaceutical development, is expected to increase, obtaining this essential FBS requires the slaughter of pregnant cows and the utilization of their fetuses, which presents the disadvantages of unstable supply and high costs.

[0008] To address the disadvantages of FBS, various studies have been conducted using FBS-free media, but serum-free media have the disadvantage of significantly slowing down cell division or cell growth rates. In addition, domestic technologies for replacing FBS include the development of FBS-substitute serum-free biomaterials based on marine organisms, such as Korean registered patent 10-1950245 (Patent Document 1), but the feasibility of their actual public use remains unknown.

[0009] Therefore, there is a need to develop an alternative substance that can solve the aforementioned problems caused by existing FBS and is effective for cell culture. Prior art literature

[0011] Korean Registered Patent 10-1950245 The problem to be solved

[0012] The present invention aims to solve the problems of the prior art described above. While researching a method to produce a high-quality FBS substitute suitable for cell culture using slaughterhouse by-products from discarded animals such as cattle, pigs, or chickens, the inventors confirmed that by using an FBS substitute (additive to cultured meat media) manufactured by the method unique to the present invention, it is possible to completely replace or use in combination with Fetal Bovine Serum (FBS), which has a high unit cost and raises ethical issues due to the need to extract it from live calves, thereby reducing the amount of FBS used while improving the production efficiency of cultured meat. Based on this, the present invention was completed.

[0013] Accordingly, the objective of the present invention is,

[0014] (i) A step of obtaining serum or plasma from blood separated from animals other than humans as a byproduct at the time of slaughter;

[0015] (ii) a step of decolorizing the serum or plasma obtained in step (i) above; and

[0016] (iii) a step of filtering the decolorized serum or plasma obtained in step (ii) above;

[0017] The present invention provides a method for manufacturing a culture medium additive (FBS substitute) for cultured meat from slaughter by-products, comprising:

[0018] Another objective of the present invention is to provide a culture medium additive (FBS substitute) for cultured meat, prepared by the manufacturing method of the present invention.

[0019] Another objective of the present invention is to provide a culture medium composition for cultured meat comprising the culture medium additive (FBS substitute) of the present invention.

[0020] Another objective of the present invention is to provide a method for producing cultured meat comprising the step of proliferating muscle satellite cells using the cultured meat culture medium composition of the present invention.

[0021] However, the technical problems that the present invention aims to solve are not limited to those mentioned above, and other unmentioned problems will be clearly understood by those skilled in the art to which the present invention belongs from the description below. means of solving the problem

[0023] In order to achieve the above objectives, the present invention

[0024] (i) A step of obtaining serum or plasma from blood separated from animals other than humans as a byproduct at the time of slaughter;

[0025] (ii) a step of decolorizing the serum or plasma obtained in step (i) above; and

[0026] (iii) a step of filtering the decolorized serum or plasma obtained in step (ii) above;

[0027] A method for manufacturing a culture medium additive (FBS substitute) for cultured meat from slaughter by-products is provided, comprising:

[0028] In one embodiment of the present invention, the animal may be characterized as being one or more selected from the group consisting of cattle, chickens, and pigs, but is not limited thereto.

[0029] In one embodiment of the present invention, the cattle may be characterized as being 30 to 48 months old, the chickens as being 2 to 8 weeks old, and the pigs as being 180 days old, but are not limited thereto.

[0030] In one embodiment of the present invention, the decolorization of step (ii) may be characterized by being performed by mixing kaolin into the serum or plasma obtained in step (i), but is not limited thereto.

[0031] In one embodiment of the present invention, the decolorization of step (ii) may be characterized by being performed at a temperature of 50 to 70 ℃, but is not limited thereto.

[0032] In one embodiment of the present invention, the filtration process of step (iii) may be characterized by filtering through pores with a diameter of 0.05 to 0.3 μm, but is not limited thereto.

[0033] In one embodiment of the present invention, the filtration process of step (iii) may additionally include a pre-filtration process with pores of diameter 2 μm to 25 μm, but is not limited thereto.

[0034] In one embodiment of the present invention, the filtration of step (iii) may be characterized as being membrane filter filtration, but is not limited thereto.

[0035] In one embodiment of the present invention, the filtration may additionally be performed using a method selected from the group consisting of filter paper, syringe filters, and combinations thereof, in addition to membrane filter filtration, but is not limited thereto.

[0036] In one embodiment of the present invention, the method for producing a culture medium additive (FBS substitute) for cultured meat from slaughter by-products of the present invention may additionally include a sterilization process after the filtration process in step (iii), but is not limited thereto.

[0037] In one embodiment of the present invention, the sterilization may be performed at a temperature of 50 to 70 ℃, but is not limited thereto.

[0038] To achieve another objective of the present invention, the present invention provides a culture medium additive (FBS substitute) prepared by the manufacturing method of the present invention (a method for preparing a culture medium additive (FBS substitute) from slaughter by-products).

[0039] In one embodiment of the present invention, the culture medium additive for cultured meat of the present invention may be characterized as a culture medium additive for the proliferation of muscle satellite cells, but is not limited thereto.

[0040] In one embodiment of the present invention, the culture medium additive for cultured meat of the present invention may be characterized by being used to replace fetal bovine serum (FBS) or in parallel (combined) with fetal bovine serum (FBS), but is not limited thereto.

[0041] To achieve another objective of the present invention, the present invention provides a culture medium composition for cultured meat comprising a culture medium additive (FBS substitute) of the present invention.

[0042] In one embodiment of the present invention, the culture medium composition for cultured meat of the present invention may be characterized as being for the proliferation of muscle satellite cells, but is not limited thereto.

[0043] In one embodiment of the present invention, the culture medium composition for cultured meat of the present invention may be characterized by comprising, but is not limited to, 1 to 20 parts by weight of the culture medium additive (FBS substitute) of the present invention and 0.5 to 1.5 parts by weight of an antibiotic / antimicrobial agent, based on 100 parts by weight of a serum-free medium.

[0044] In one embodiment of the present invention, the serum-free medium may be characterized as being selected from Ham's 10 medium, Ham's 12 medium, DMEM, MEM, MCDB-201 medium and RPMI 1640 medium, but is not limited thereto.

[0045] In one embodiment of the present invention, the antibiotic / antimicrobial agent may comprise one or more antibiotics selected from penicillin and streptomycin; and one or more antifungal agents selected from amphotericin-B, clotrimazole, miconazole, ketoconazole and nitatin, but is not limited thereto.

[0046] In one embodiment of the present invention, the culture medium composition for cultured meat of the present invention may additionally include fetal bovine serum (FBS), but is not limited thereto. In one embodiment, the culture medium composition for cultured meat of the present invention may be characterized by comprising, with respect to 100 parts by weight of serum-free medium, 10 to 20 parts by weight of the culture medium additive (FBS substitute) of the present invention; 0 to 20 parts by weight of fetal bovine serum (FBS); and 0.5 to 1.5 parts by weight of an antibiotic / antimicrobial agent, but is not limited thereto.

[0047] To achieve another objective of the present invention, the present invention,

[0048] The present invention provides a method for producing cultured meat comprising the step of proliferating muscle satellite cells using the cultured meat culture medium composition of the present invention. Effects of the invention

[0050] By using a cultured meat culture medium additive (FBS substitute) manufactured by the method unique to the present invention, it is possible to completely replace or use in combination Fetal Bovine Serum (FBS), which is conventionally expensive and raises ethical issues due to the need to extract it from live calves, thereby reducing the amount of FBS used while improving the production efficiency of cultured meat. Furthermore, there are advantages such as increasing the value of slaughterhouse by-products—which are inevitably generated during the slaughter of livestock for meat production—by enhancing their usability, thereby providing economic benefits like reduced disposal costs and increased income, as well as significant environmental protection benefits.

[0051] Specifically, to address the problems associated with the conventional use of fetal bovine serum (FBS), the inventors of the present invention propose the utilization of blood generated during the slaughter of livestock for meat production as a substitute for existing FBS. By utilizing an FBS substitute (culture medium additive) produced by the unique method of the present invention using blood obtained as a byproduct during livestock slaughter, the issues of supply instability and high prices of existing FBS products can be resolved. Furthermore, by utilizing discarded slaughter byproducts, unnecessary disposal costs can be reduced and profits generated, while also protecting the environment. Ultimately, these effects can serve as a foundation for stably supplying additives capable of promoting growth in various cell cultures, as well as for cell culture for cultured meat production, at a low cost. Brief explanation of the drawing

[0053] Figure 1 is a schematic diagram briefly illustrating the process of producing an FBS substitute using livestock blood in the present invention. Figure 2 is a figure illustrating a series of processes in which a normal animal slaughter situation is reenacted by sacrificing an animal (chicken) using CO2 in a laboratory, blood is obtained from the slaughtered livestock, and serum or plasma is separated and obtained from it. Figure 3 is a figure showing the decolorization process of serum or plasma. Figure 4 is a figure showing a series of filtration and sterilization processes to remove impurities from a serum or plasma solution after decolorization. Figure 5 is a diagram showing a series of processes for separating satellite cells from muscle tissue of an animal (livestock). Figure 6 shows the results of confirming the presence or absence of cytotoxicity following the use of the FBS substitutes of the present invention (each bovine plasma (BP), bovine serum (BS), chicken plasma (CP), and chicken serum (CS) produced by the method of Example 1) on bovine cells, and no cytotoxicity causing significant cell death was confirmed. Figure 7 shows the results of confirming the presence or absence of cytotoxicity following the use of the FBS substitutes of the present invention (each bovine plasma (BP), bovine serum (BS), chicken plasma (CP), and chicken serum (CS) produced by the method of Example 1) on chicken satellite cells, and no cytotoxicity causing significant cell death was confirmed. FIG. 8 shows the degree of growth and proliferation of myoblasts cultured based on bovine cells by partially applying the FBS substitutes of the present invention (each bovine plasma (BP), bovine serum (BS), chicken plasma (CP), and chicken serum (CS) produced by the method of Example 1) to the cell culture medium (basically, Ham's F-10 medium supplemented with 20% (v / v) FBS and 1% (v / v) P / S / A (CTL, control) was used as the cell growth and proliferation medium, and eight treatment groups were conducted in which each of the FBS substitutes of the present invention replaced 25% (v / v) (5% (v / v) of the total medium composition) or 50% (v / v) (10% (v / v) of the total medium composition) of the total FBS amount). Figure 9 shows the degree of differentiation of myoblasts cultured based on bovine cells by partially applying the FBS substitutes of the present invention (each bovine plasma (BP), bovine serum (BS), chicken plasma (CP), and chicken serum (CS) produced by the method of Example 1) to the cell culture medium (basically, Ham's F-10 medium supplemented with 20% (v / v) FBS and 1% (v / v) P / S / A (CTL, control) was used as the cell growth and proliferation medium, and eight treatment groups were conducted in which each of the FBS substitutes of the present invention replaced 25% (v / v) (5% (v / v) of the total medium composition) or 50% (v / v) (10% (v / v) of the total medium composition) of the total FBS amount). FIG. 10 shows the degree of growth and proliferation of myoblasts cultured based on chicken satellite cells by partially applying the FBS substitutes of the present invention (each bovine plasma (BP), bovine serum (BS), chicken plasma (CP), and chicken serum (CS) produced by the method of Example 1) to the cell culture medium (basically, a medium (CTL, control) supplemented with 20% (v / v) FBS and 1% (v / v) P / S / A to Ham's F-10 medium was used as the cell growth and proliferation medium, and eight treatment groups were conducted in which each of the FBS substitutes of the present invention replaced 25% (v / v) (5% (v / v) of the total medium composition) or 50% (v / v) (10% (v / v) of the total medium composition) of the total FBS amount). FIG. 11 shows the degree of differentiation of myoblasts cultured based on chicken satellite cells by partially applying the FBS substitutes of the present invention (each bovine plasma (BP), bovine serum (BS), chicken plasma (CP), and chicken serum (CS) produced by the method of Example 1) to the cell culture medium (basically, Ham's F-10 medium supplemented with 20% (v / v) FBS and 1% (v / v) P / S / A (CTL, control) was used as the cell growth and proliferation medium, and eight treatment groups were conducted in which each of the FBS substitutes of the present invention replaced 25% (v / v) (5% (v / v) of the total medium composition) or 50% (v / v) (10% (v / v) of the total medium composition) of the total FBS amount). FIG. 12 is a graph showing the growth and proliferation of myoblasts cultured in bovine cells, in which the existing FBS in the cell culture medium was completely replaced with the FBS substitutes of the present invention (bovine serum (BS), porcine serum (PS), and chicken serum (CS), respectively produced by the method of Example 1), and added at concentrations of 20% (v / v), 30% (v / v), or 40% (v / v) in the total cell culture medium (basically, Ham's F-10 medium supplemented with 20% (v / v) FBS and 1% (v / v) P / S / A (CTL, control))). FIG. 13 is a graph showing the growth and proliferation of myoblasts cultured based on porcine cells, in which the existing FBS in the cell culture medium was completely replaced with the FBS substitutes of the present invention (bovine serum (BS), porcine serum (PS), and chicken serum (CS), respectively produced by the method of Example 1), and added at concentrations of 20% (v / v), 30% (v / v), or 40% (v / v) in the total cell culture medium (basically, Ham's F-10 medium supplemented with 20% (v / v) FBS and 1% (v / v) P / S / A (CTL, control))). FIG. 14 is a graph showing the growth and proliferation of myoblasts cultured in chicken cells, in which the existing FBS in the cell culture medium was completely replaced with the FBS substitutes of the present invention (bovine serum (BS), porcine serum (PS), and chicken serum (CS), respectively produced by the method of Example 1), and added at concentrations of 20% (v / v), 30% (v / v), or 40% (v / v) in the total cell culture medium (basically, Ham's F-10 medium supplemented with 20% (v / v) FBS and 1% (v / v) P / S / A (CTL, control))). Specific details for implementing the invention

[0054] The present invention relates to a method for producing an FBS substitute to replace the expensive conventional FBS (fetal bovine serum), which is essential for cell growth, by utilizing slaughterhouse by-products generated during the slaughter of livestock. Conventional FBS is produced from blood collected from stillborn bovine fetuses within the mother's womb. This production method not only struggles to meet the increasing demand for FBS driven by active research in cultured meat but also raises ethical issues regarding animals. Therefore, the inventors focused on creating an FBS substitute effective for cultured meat growth by utilizing blood generated during the slaughter of ordinary livestock, rather than fetuses within the mother's womb. The inventors devised a method to obtain blood generated during the slaughter of ordinary livestock in the form of serum and plasma and convert it into an FBS substitute. Furthermore, by obtaining satellite cells necessary for cultured meat production from the muscles of livestock of various species and conducting cell culture using the FBS substitute of the present invention, the inventors verified the effectiveness of the FBS substitute compared to conventional FBS. In this process, the individual-specific effects of FBS substitutes derived from cattle, pigs, and chickens were confirmed, and it was verified that methods utilizing plasma as well as serum are feasible. Additionally, it was confirmed that the FBS substitute of the present invention is effective for the formation and growth (proliferation) of myoblasts for cultured meat production from satellite cells.

[0056] Specifically, the entire process for producing the FBS substitute of the present invention is broadly composed of the supply of by-products (supplementation of serum or plasma from blood), a decolorization process, a filtration process, and a sterilization process. The produced FBS substitute was applied to satellite cell cultures from bovine, porcine, and chicken to verify its effectiveness by confirming the myoblast growth rate, cytotoxicity, and degree of cell differentiation. The above process is illustrated in FIG. 1, and the detailed process is described through the examples in this specification.

[0058] Accordingly, the present invention

[0059] (i) A step of obtaining serum or plasma from blood separated from animals other than humans as a byproduct at the time of slaughter;

[0060] (ii) a step of decolorizing the serum or plasma obtained in step (i) above; and

[0061] (iii) a step of filtering the decolorized serum or plasma obtained in step (ii) above;

[0062] A method for manufacturing a culture medium additive (FBS substitute) for cultured meat from slaughter by-products is provided, comprising:

[0063] In one embodiment of the present invention, the method for preparing a culture medium additive for cultured meat from slaughter by-products of the present invention will be understood by those skilled in the art as a method for obtaining an FBS substitute from animals other than humans, comprising steps (i) to (iii).

[0064] In one embodiment of the present invention, the animal is not particularly limited in type as long as it is a mammal other than a human, but in a preferred embodiment, it may be a type of livestock. In a specific embodiment, the livestock may be characterized as being one or more selected from the group consisting of cattle, pigs, and chickens, but not limited thereto.

[0065] In step (i) above, the blood (serum and plasma) is obtained at the time of slaughter of the animal (livestock), and the slaughter age of each animal is well known in the industry.

[0066] In a specific embodiment of the present invention, when the slaughtered animal is a cow, preferably the cow may be 30 to 48 months old, and more preferably the cow may be 33 to 46 months old.

[0067] In a specific embodiment of the present invention, when the slaughtered animal is a chicken, preferably the chicken may be 2 to 8 weeks old, and more preferably the chicken may be 2 to 6 weeks old.

[0068] In a specific embodiment of the present invention, when the slaughtered animal is a pig, the pig may preferably be 180 days old. In Korea, the slaughter age of pigs is fixed at 180 days old.

[0069] In one embodiment of the present invention, preferably, in step (i), the blood (whole blood) separated from animals other than humans as a byproduct at slaughter may be treated with an anticoagulant. The type of anticoagulant is not particularly limited as long as it is known in the art, and may be, for example, one or more selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), ethyleneglycoltetraacetic acid (EGTA), citrate, citrate salt diethylenetriaminepentaacetic acid (DTPA), 1,2-diaminocyclohexanetetraacetic acid (DCTA), heparin, warfarin, and oxalate.

[0070] In the present invention, in step (i), serum or plasma is separated and obtained from blood (whole blood) separated from animals other than humans as a byproduct during slaughter. Methods for separating serum or plasma from whole blood are well known in the art, and the method is not particularly limited in the present invention. For example, methods using centrifugation, methods using gels for separating serum or plasma, and commercially available products for this purpose are known in the art.

[0072] In one embodiment of the present invention, the decolorization of step (ii) may be characterized by being performed by mixing kaolin into the serum or plasma obtained in step (i).

[0073] In one embodiment of the present invention, the decolorization of step (ii) may be characterized by being performed preferably at a temperature of 50 to 70 ℃, and more preferably at 55 to 65 ℃.

[0074] In one embodiment of the present invention, the decolorization of step (ii) may be performed by appropriately selecting and changing conditions such as time and / or stirring according to the amount of blood or plasma and / or the decolorization efficiency desired by the person skilled in the art.

[0076] In one embodiment of the present invention, the filtration process of step (iii) is preferably characterized by filtration through pores with a diameter of 0.05 to 0.3 μm. Although not limited thereto, for example, the size of the pores may be 0.05 μm, 0.1 μm, 0.15 μm, 0.2 μm, 0.25 μm, or 0.3 μm in diameter, and the present invention includes all ranges with two values ​​selected from these as boundaries (upper and lower limits). At this time, the filtration process may additionally include a pre-filtration process through pores with a diameter of 2 μm to 25 μm, but is not limited thereto.

[0077] In the present invention, the type of filtration is not particularly limited as long as it is a filtration means known in the art. In a preferred embodiment of the present invention, the filtration may be characterized as membrane filter filtration, and additional filtration may be performed using a method selected from the group consisting of filter paper, syringe filters, and combinations thereof.

[0079] In one embodiment of the present invention, the method for producing a culture medium additive (FBS substitute) for cultured meat from slaughter by-products of the present invention may additionally include a sterilization process after the filtration process in step (iii), but is not limited thereto.

[0080] In a specific embodiment of the present invention, the sterilization method is not particularly limited as long as it uses a method for sterilizing blood, serum, or plasma known in the art. Although not limited thereto, for example, sterilization methods such as low-temperature sterilization or radiation irradiation may be used so as not to destroy the active ingredients in the culture medium additive (FBS substitute) of the present invention for cultured meat. In one embodiment of the present invention, the sterilization may be performed at a temperature of 50 to 70°C, and conditions such as time may be appropriately selected and changed by a person skilled in the art according to the amount of blood or plasma and / or the sterilization efficiency desired by the person skilled in the art.

[0082] In addition, the present invention provides a culture medium additive for cultured meat (an FBS substitute of the present invention) prepared by the manufacturing method of the present invention (a method for preparing a culture medium additive for cultured meat (an FBS substitute) from slaughter by-products).

[0083] In one embodiment of the present invention, the culture medium additive for cultured meat of the present invention may be characterized as being a culture medium additive for the proliferation of muscle satellite cells.

[0084] In one embodiment of the present invention, the culture medium additive for cultured meat of the present invention may be characterized by being used to replace fetal bovine serum (FBS) or in parallel with fetal bovine serum (FBS). Specifically, the culture medium additive for cultured meat of the present invention may be provided as a substitute for fetal bovine serum (FBS), or provided as a preparation prepared by additionally adding the culture medium additive for cultured meat of the present invention to Fetal Bovine Serum (FBS).

[0085] In the example of parallel (combined use) with the above fetal bovine serum (FBS), the cultured meat culture medium additive of the present invention, when the amount (amount, weight, volume, etc.) of fetal bovine serum (FBS) conventionally added to the cell culture medium is considered as 100%, is 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% may be used in combination. In this case, the % notation may mean '%(v / v)' and is also understood as a volume portion (volume ratio).

[0087] In addition, the present invention provides a culture medium composition for cultured meat comprising the culture medium additive (FBS substitute) of the present invention.

[0088] In one embodiment of the present invention, the culture medium composition for cultured meat of the present invention may be characterized as being for the proliferation of muscle satellite cells.

[0089] In one embodiment of the present invention, the culture medium composition for cultured meat of the present invention may be characterized by comprising, with respect to 100 parts by weight of serum-free medium, 1 to 20 parts by weight of the culture medium additive (FBS substitute) of the present invention; and 0.5 to 1.5 parts by weight of an antibiotic / antimicrobial agent.

[0090] In one embodiment of the present invention, the culture medium composition for cultured meat of the present invention may be characterized by comprising, with respect to 100 volume parts (volume ratio) of serum-free medium, 1 to 20 volume parts of the cultured meat culture medium additive (FBS substitute) of the present invention; and 0.5 to 1.5 volume parts of an antibiotic / antimicrobial agent.

[0092] In one embodiment of the present invention, the culture medium composition for cultured meat of the present invention does not contain FBS, and the culture medium additive of the present invention can be used in a form that completely replaces FBS instead of FBS.

[0093] In a preferred specific embodiment of the present invention, when the cultured meat culture medium additive (FBS substitute) of the present invention is produced from cattle, it may be characterized by being included in the medium composition in an amount of 20 to 40 parts by weight (volume) (more preferably 20 to 30 parts by weight (volume)) per 100 parts by weight (volume) of serum-free medium. In addition, when the cultured meat culture medium additive (FBS substitute) of the present invention is produced from cattle, it may be preferable to use it when culturing muscle satellite cells derived from cattle or chickens.

[0094] In addition, when the cultured meat culture medium additive (FBS substitute) of the present invention is produced from chicken, it may be characterized by being included in the medium composition in an amount of 20 to 40 parts by weight (volume) (more preferably 30 to 40 parts by weight (volume)) per 100 parts by weight (volume) of serum-free medium. Furthermore, when the cultured meat culture medium additive (FBS substitute) of the present invention is produced from chicken, it may be preferable to use it when culturing muscle satellite cells derived from pigs or chickens.

[0095] In addition, when the cultured meat culture medium additive (FBS substitute) of the present invention is produced from pigs, it may be characterized by being included in the medium composition in an amount of 20 to 40 parts by weight (volume) per 100 parts by weight (volume) of serum-free medium. Furthermore, when the cultured meat culture medium additive (FBS substitute) of the present invention is produced from pigs, it may be preferable to use it when culturing muscle satellite cells derived from cattle, pigs, or chickens.

[0096] In one embodiment of the present invention, the serum-free medium is not particularly limited in type as long as it is known in the art for use in cell culture, and, for example, one or more selected from Ham's (F)10 medium, Ham's (F)12 medium, DMEM (Dulbecco Modified Eagle Medium), MEM (Minimum Essential Medium), MCDB-201 medium, and RPMI 1640 medium may be used.

[0097] In one embodiment of the present invention, the antibiotic / antimicrobial agent may comprise, but is not limited to, one or more antibiotics selected from penicillin and streptomycin, provided that such agent is known in the art to be used in cell culture; and one or more antifungal agents selected from amphotericin-B, clotrimazole, miconazole, ketoconazole and nitstatin.

[0099] In one embodiment of the present invention, the culture medium composition for cultured meat of the present invention may additionally include fetal bovine serum (FBS), but is not limited thereto. That is, in one embodiment of the present invention, the culture medium composition for cultured meat of the present invention may be used in a form in which the culture medium additive of the present invention (the FBS substitute of the present invention) is used in combination with fetal bovine serum (FBS).

[0100] In one embodiment, the culture medium composition for cultured meat of the present invention may be characterized by comprising, but is not limited to, 10 to 20 parts by weight (volume) of the cultured meat culture medium additive (FBS substitute) of the present invention, with respect to 100 parts by weight (volume) of serum-free medium; 0 to 20 parts by weight (volume) of fetal bovine serum (FBS); and 0.5 to 1.5 parts by weight (volume) of an antibiotic / antimicrobial agent.

[0102] Furthermore, the present invention provides a method for producing cultured meat comprising the step of proliferating (culturing) muscle satellite cells using the cultured meat culture medium composition of the present invention. In a specific embodiment, the present invention provides a method for producing cultured meat comprising the step of culturing muscle satellite cells in a culture vessel containing the cultured meat culture medium composition of the present invention. In a preferred embodiment of the present invention, the muscle satellite cells may be cultured in a culture vessel (e.g., plate, dish, etc.) coated with Matrigel.

[0103] In the present invention, the muscle satellite cells may be obtained according to methods known in the art, without any particular limitation on the type of animal from which they are obtained or the age of the animal. In one embodiment of the present invention, the muscle satellite cells may be obtained at the time of slaughter in step (i) of the method for preparing a culture medium additive for cultured meat from slaughter by-products of the present invention.

[0105] Hereinafter, preferred embodiments and experimental examples are presented to aid in understanding the present invention. However, the following embodiments and experimental examples are provided merely to facilitate a better understanding of the present invention, and the scope of the present invention is not limited by the following embodiments and experimental examples.

[0107] Example 1. Obtaining the FBS (fetal bovine serum) substitute of the present invention from slaughterhouse by-products

[0108] (Process 1) After obtaining blood from animal slaughter by-products, obtain serum and plasma

[0109] Blood was obtained by slaughtering livestock. Specifically, beef blood was obtained by slaughtering cows aged 45 months, chicken blood was obtained by slaughtering laying hens (young chickens) aged about 6 weeks, and pig blood was obtained by slaughtering sows aged 180 days.

[0110] When cardiac blood collection was possible, blood was obtained using a tube treated with EDTA (anticoagulant) or coagulant gel. When cardiac blood collection was not possible, blood was obtained using a bottle containing a solution of dissolved Na-EDTA.

[0111] The obtained blood was centrifuged at 3,000 rpm for 10 to 20 minutes to obtain the supernatant (serum obtained).

[0112] Anticoagulant-treated blood was centrifuged at 3,000 rpm for 10 to 20 minutes to obtain the supernatant (plasma obtained). The obtained blood in serum / plasma form (meaning serum or plasma) was frozen and stored at -10 to -20 ℃.

[0113] Figure 2 briefly illustrates the processes of Process 1 (i.e., a series of collecting blood from a slaughtered animal and obtaining serum / plasma from it).

[0115] (Process 2) Serum / plasma Decolorization

[0116] Serum / plasma (50ml tube) was mixed with 1X PBS (phosphate buffer saline) at a ratio of 1:5 to 10, and then homogenized using a sonicator. Subsequently, 3 to 5% (v / v) kaolin was added to the mixed solution, and the mixture was reacted for 30 minutes in a water bath at 55 to 65 ℃ while shaking.

[0117] Figure 3 briefly shows the processes of Process 2 above.

[0119] (Process 3) Filtration and Sterilization

[0120] To remove kaolin and impurities, filtration was performed using filter paper of various sizes (with pore sizes ranging from a minimum of 2 μm to 25 μm) as a preliminary filtration step. Additionally, filtration using a 0.2 μm syringe filter may be performed, and after filtration using a 0.1 μm membrane filter as a final filtration step, the solution was divided into 15 / 50 ml tubes.

[0121] The divided tubes were sealed with Parafilm and sterilized by reacting in a 65°C constant temperature water bath with shaking for 30 minutes to 1 hour.

[0122] The produced FBS substitute was stored in tube form by freezing at -10 to -20 ℃.

[0123] Figure 4 briefly shows the processes of process 3 above.

[0125] Example 2. Separation of satellite cells from animal muscle

[0126] As a step to obtain satellite cells used in the production of cultured meat, animal muscle tissue was collected and the muscle tissue was minced (finely chopped) in a 1X PBS solution mixed with antibiotics (1–3% (v / v) penicillin / streptomycin) to remove impurities such as blood, fat, and connective tissue. Then, an enzyme solution mixed with collagenase D (11088858001, Sigma Aldrich, USA) and dipase II (4942074800, Roche, Swiss) was added to the tissue transferred to a tube, and the reaction was carried out at 37°C for 30 minutes to 1 hour to decompose the protein. Once the reaction was complete, a 1X PBS solution was added to stop the enzymatic reaction, and the undigested (undecomposed) tissue was filtered out and the cells were obtained by sequentially filtering using strainers of 100㎛, 70㎛, and 40㎛, respectively. The filtered cell solution was centrifuged to remove the supernatant, and a cell pellet was obtained. The pellet was dispersed in medium (Ham's F-10 (11550043, Gibco, USA)) and incubated for 1 to 2 hours. After incubation, only the supernatant was transferred to a Matrigel-coated dish, to which growth factor (bFGF, 354060, Corning, USA) and medium were added for culture. The cell culture medium used was a Ham's F-10 (11550043, Gibco, USA) base supplemented with 20% (v / v) FBS (35-015-CV, Corning, USA) and 1% (v / v) P / S / A (03-033-1B, Biological Industries, Israel). Figure 5 briefly illustrates the series of processes for isolating satellite cells from animal muscle tissue.

[0128] Example 3. Production of cultured meat using the FBS (fetal bovine serum) substitute of the present invention

[0129] 3-1. Confirmation of the cytotoxicity of the FBS substitute of the present invention

[0130] To confirm the cytotoxicity of the FBS substitute of the present invention produced in Example 1 above, an experiment was conducted using the MTT assay method. Thiazolyl Blue Tetrazolium Bromide (sigma M 2128) was used to prepare the MTT reagent for the analysis. For the analysis, 1 x 10⁶ of each satellite cell obtained in Example 2 above were placed in a 96-well plate coated with Matri gel. 4 Cells were added one by one and cultured in FBS substitute media prepared at different concentrations.

[0131] The experiment was conducted with a CTL (control) using a standard medium composition [Ham's F-10 + 20% (v / v) FBS + 1% (v / v) P / S / A], and eight treatment groups in which 25% (v / v) (5% (v / v) of the total medium composition) or 50% (v / v) (10% (v / v) of the total medium composition) of the total FBS amount was replaced with bovine plasma (BP), bovine serum (BS), chicken plasma (CP), and chicken serum (CS) produced by the method of Example 1.

[0132] After 2–3 days, when the cells had grown sufficiently, the supernatant was removed. Under dark conditions, MTT reagent prepared at 5 mg / ml (in 1X PBS) was diluted with the medium (media, Ham's F-10) at a ratio of 1:9 and added. The plates were wrapped in foil and incubated at 37°C in a 5% CO2 incubator for 4 hours. Subsequently, the added supernatant was removed, DMSO was added, and the mixture was incubated again under dark conditions for 10–15 minutes; afterward, the absorbance was measured at 540 nm using an ELISA reader.

[0133] The results of cytotoxicity experiments following the use of the FBS substitute of the present invention (each bovine plasma (BP), bovine serum (BS), chicken plasma (CP), and chicken serum (CS) produced by the method of Example 1) in all treatment groups are shown in Figures 6 and 7. In both experiments using bovine cells and chicken cells, no cytotoxicity causing significant cell death was confirmed when the FBS substitute was added. Since cell growth similar to or slightly higher than that of CTLs was confirmed in most treatment groups, it was determined that the FBS substitute prepared by the method of Example 1 of the present invention can be used for cell culture.

[0135] 3-2. Confirmation of the myoblast growth-promoting effect using the FBS substitute of the present invention Partial use of the FBS substitute of the present invention as a substitute for conventional FBS in the growth (proliferation) medium

[0136] After each cell (Bovine cell and Chicken cell) grew in Example 3-1 above, the medium was replaced to induce differentiation, and the degree of differentiation according to cell growth was confirmed.

[0137] As the cell growth and proliferation medium, Ham's F-10 medium supplemented with 20% (v / v) FBS and 1% (v / v) P / S / A (CTL, control) was basically used. As experimental groups, eight treatments were conducted in which the respective bovine plasma (BP), bovine serum (BS), chicken plasma (CP), and chicken serum (CS) produced by the method of Example 1 were replaced with 25% (v / v) (5% (v / v) of the total medium composition) or 50% (v / v) (10% (v / v) of the total medium composition) of the total FBS amount, and the cells were cultured for 3 to 7 days according to cell growth rate and measured.

[0138] Cell differentiation medium was prepared by adding 2% (v / v) horse serum and 1% (v / v) P / S / A to DMEM medium, and measurements were taken after culturing for 3 to 5 days according to cell differentiation.

[0140] Figures 8 and 9 show the degree of cell growth, proliferation, and differentiation resulting from cell culture. As can be seen in Figures 8 and 9, it was confirmed that the CS-treated myoblasts cultured on bovine cells proliferated the most compared to the control group (CTL). When comparing plasma (BP and CP) and serum (BS and CS), greater cell proliferation was observed in the serum for both bovine blood and chicken blood, and it was confirmed that greater cell proliferation occurred in the treatment group using chicken blood compared to bovine blood. Among them, the cell proliferation rate of BP was the lowest, while the proliferation levels of the other treatment groups were similar. Accordingly, when the proliferated cells were differentiated, it was confirmed that although the cell density was relatively low, the cells differentiated in a form similar to the control group in all alternative treatment groups.

[0141] In addition, as can be seen in Figures 10 and 11, in the case of myoblasts cultured based on chicken cells, the CS treatment group exhibited the highest cell proliferation, and a greater amount of cell growth was observed compared to bovine cells. In particular, it was confirmed that more cell growth occurred than in the control group (CTL) in a medium supplemented with 20% FBS (v / v). Accordingly, it was confirmed that the degree of cell differentiation in the group of chicken cells treated with CS was similar to that of CTLs, and other treatment groups also differentiated without cellular toxicity in a form similar to the control group, although the density was relatively lower. Furthermore, as in the previous experiment, it was confirmed that cell growth was more dominant in serum (BS and CS) than in plasma (BP and CP), and in chicken blood than in bovine blood.

[0142] Taken together, these results indicate that using chicken blood (CP and CS) can significantly increase the growth and proliferation of chicken cells compared to FBS in the control group (CTL).

[0143] It is believed that this trend was not clearly observed in bovine cells because blood obtained from livestock at the age of slaughter was utilized. As evidence for this, the control group in the bovine cell experiment using a medium supplemented with bovine fetal serum was confirmed to exhibit the most superior cell growth and proliferation.

[0145] 3-3. Confirmation of Myoblast Growth-Promoting Effect and Cytotoxicity Using the FBS Substitute of the Present Invention Use of the FBS substitute of the present invention as a complete (100%) replacement for existing FBS in the growth (proliferation) medium

[0146] The effects on myoblast differentiation and proliferation (growth) were confirmed using each FBS substitute (bovine serum (BS), porcine serum (PS), and chicken serum (CS)) produced by the method of Example 1 from the blood of three representative livestock species (cattle, pig, and chicken). Each satellite cell (cattle, pig, and chicken) obtained in Example 2 was used, and the cells were cultured in the same manner as in Example 3-2, except that the FBS in the cell growth and proliferation medium was completely replaced (100% replacement). The MTT assay was performed using the same measurement method as described in Example 3-1.

[0148] As can be seen in Figure 12, it was found that BS and PS could sufficiently replace FBS in bovine cells when added at an amount equal to FBS, specifically 20% (v / v). On the other hand, CS showed a cell viability of approximately 70% when replaced up to 30% (v / v), while adding more than that resulted in negative outcomes.

[0149] As can be seen in Figure 13, when BS or CS was added at 20% (v / v) in Porcine cells, cell survival was nearly similar to that of FBS. Addition beyond this level was not effective in increasing cell viability relative to the amount added. In the case of PS, adding 30% (v / v) showed an effect approximately 1.4 times higher than FBS.

[0150] As can be seen in Figure 14, in chicken cells, all treatment groups showed cell viability similar to or slightly higher than that of FBS when 20% (v / v) was added. In the case of BS and CS, cell viability could be slightly increased when the amount added was increased up to 30% (v / v).

[0152] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will understand that other specific forms can be easily modified without altering the technical spirit or essential features of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims

Claim 1 A method for producing a culture medium additive for muscle satellite cell cultured meat from slaughter by-products, comprising: (i) a step of obtaining serum or plasma from blood separated from an animal as a by-product at slaughter; (ii) a step of decolorizing the serum or plasma obtained in step (i); and (iii) a step of filtering the decolorized serum or plasma obtained in step (ii); for producing muscle satellite cell cultured meat by proliferating and differentiating muscle satellite cells. Claim 2 A method according to claim 1, characterized in that the animal is one or more selected from the group consisting of cattle, pigs, and chickens. Claim 3 A method according to paragraph 2, characterized in that the cattle are 30 to 48 months old, the chickens are 2 to 8 weeks old, and the pigs are 180 days old. Claim 4 A method according to claim 1, characterized in that the decolorization is performed by mixing kaolin into the serum or plasma obtained in step (i). Claim 5 A method according to claim 1, characterized in that the decolorization is performed at a temperature of 50 to 70 ℃. Claim 6 A method according to claim 1, wherein the filtration is characterized by filtering through pores with a diameter of 0.05 to 0.3 μm. Claim 7 In claim 6, the filtration method further comprises a process of pre-filtering with pores of a diameter of 2 μm to 25 μm. Claim 8 A method according to claim 6, characterized in that the filtration is membrane filter filtration. Claim 9 In claim 8, the filtration is further performed by a method selected from the group consisting of filter paper, syringe filters, and combinations thereof. Claim 10 A method according to claim 1, characterized in that the method further includes a sterilization process after the filtration process in step (iii). Claim 11 A method according to claim 10, characterized in that the sterilization is performed at a temperature of 50 to 70 ℃. Claim 12 A culture medium additive for muscle satellite cell cultured meat, for producing muscle satellite cell cultured meat by proliferating and differentiating muscle satellite cells manufactured by the method of claim 1. Claim 13 delete Claim 14 In claim 12, the cultured meat culture medium additive is characterized by being used to replace fetal bovine serum (FBS) or in combination with fetal bovine serum (FBS). Claim 15 A culture medium composition for muscle satellite cells to produce muscle satellite cell cultured meat by proliferating and differentiating muscle satellite cells, comprising the cultured meat culture medium additive of claim 12. Claim 16 delete Claim 17 In claim 15, the culture medium composition for cultured meat is characterized by comprising, with respect to 100 parts by weight of serum-free medium, 1 to 20 parts by weight of the culture medium additive for cultured meat of claim 12; and 0.5 to 1.5 parts by weight of an antibiotic / antimicrobial agent. Claim 18 A culture medium composition for cultured meat according to claim 17, characterized in that the serum-free medium is selected from any one of Ham's 10 medium, Ham's 12 medium, DMEM, MEM, MCDB-201 medium, and RPMI 1640 medium. Claim 19 A culture medium composition for cultured meat according to claim 17, characterized in that the antibiotic / antimicrobial agent comprises one or more antibiotics selected from penicillin and streptomycin; and one or more antifungal agents selected from amphotericin-B, clotrimazole, miconazole, ketoconazole and nitatin. Claim 20 In claim 15, the culture medium composition for cultured meat comprises, additionally, fetal bovine serum (FBS). Claim 21 In claim 20, the culture medium composition for cultured meat is characterized by comprising, with respect to 100 parts by weight of serum-free medium, 10 to 20 parts by weight of the culture medium additive of claim 12; 0 to 20 parts by weight of fetal bovine serum (FBS); and 0.5 to 1.5 parts by weight of an antibiotic / antimicrobial agent. Claim 22 A method for producing muscle satellite cell cultured meat, comprising the step of proliferating and differentiating muscle satellite cells using the muscle satellite cell cultured meat culture medium composition of claim 15.

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