Protein hydrolysate-based culture media and their preparation process

Abstract

This invention relates to a culture medium suitable for cell culture and a process for preparing the same. The culture medium can essentially be prepared by dissolving or mixing individual medium components in water or a suitable aqueous buffer. A key step in the preparation of the medium is a sterilization step. The culture medium according to the present invention may contain protein hydrolysates as an amino acid source. A favorable process for protein hydrolysis to shorter peptide chains and / or single amino acids is also provided by this invention.

Description

[Technical Field]

[0001] This invention relates to a protein hydrolysate-based culture medium suitable for cell culture and a process for preparing the same. The culture medium according to the present invention can be used, for example, for cell culture to prepare food containing cellular biomass for animal or human nutrition. [Background technology]

[0002] Mammalian cells are composed of various chemical compounds. The main component of cellular biomass is protein, which typically accounts for 60% to 80% of the cell's dry mass. Proteins are long polymers of amino acids. There are 20 amino acids that make up proteins, nine of which are essential in the diet of mammals. This means that they cannot be synthesized by the organism and must be supplied from the diet, or in the case of cultured cells, from the culture medium. Furthermore, some amino acids can only be synthesized from certain essential amino acids, or under certain conditions, their synthesis tends to be very slow, and therefore can be considered "semi-essential" or conditionally essential.

[0003] Cell line-specific spontaneous mutations or intentional modifications may cause cells to become unable to synthesize one or more amino acids, and therefore these amino acids must be provided in the culture medium (nutrient requirements). Such cells may acquire or regain the ability to synthesize specific amino acids through spontaneous mutations or intentional modifications. Even non-essential amino acids that the cells can synthesize may be important to the culture process, as the cells may also exhibit improved growth or metabolic characteristics when certain non-essential amino acids are provided. Cells can also synthesize non-essential amino acids using certain other nitrogen sources, particularly ammonia.

[0004] Generally, amino acids primarily function as building blocks for protein synthesis and are therefore consumed in the culture medium in proportion to the amino acid composition of cellular proteins. However, some amino acids can be used in energy metabolism, nucleic acid synthesis, and other similar processes, and may be consumed by cells at a higher rate than corresponding to their abundance in cellular proteins. To provide cells in culture with sufficient amino acids for protein synthesis, commonly used culture medium formulations contain individual amino acids in different concentration ratios. These amino acids are usually produced by fermentation processes using microorganisms engineered to produce specific amino acids. Some amino acids can also be synthesized chemically, but this is generally more expensive than microbial production. However, while microbial production works well for the needs of cell culture in research and therapeutic protein production applications, it is generally too expensive for the production of cultured meat.

[0005] Therefore, there is a need for culture media containing alternative and more economically advantageous amino acid sources. These culture media should be suitable for cell culture and economically viable. [Overview of the Initiative]

[0006] The drawbacks of conventional methods are solved by the present invention, which provides a culture medium suitable for cell culture and a process for preparing the same.

[0007] Culture media can be prepared by dissolving medium components in water or a suitable aqueous buffer. Complete media may be prepared outside the cell culture apparatus, for example, by forming them in a bioreactor and then introduced into the culture apparatus; or one or more component solutions may be prepared outside the culture apparatus and introduced separately into the culture apparatus; or individual components or mixtures of components may be introduced directly into the culture apparatus and dissolved; or a combination of the above methods may be used.

[0008] Sterilization of the culture medium is important to prevent contamination of the culture apparatus by unwanted microorganisms, for example, formed by a bioreactor. The culture medium may be sterilized after being introduced into the culture apparatus, or alternatively, the complete culture medium, the constituent solution of one or more components, the individual components or a mixture thereof may be sterilized before being introduced into the culture apparatus, or a combination of the aforementioned methods may be used.

[0009] The culture medium according to the present invention may contain a protein hydrolysate as an amino acid source. The protein hydrolysate may function as a source of all amino acids in the culture medium according to the present invention for the purpose of cell culture, or some amino acids may be supplied separately to the culture medium. Advantageous processes for protein hydrolysis into shorter peptide chains and / or single amino acids are also provided by the present invention.

Brief Description of the Drawings

[0010] [Figure 1] It is a diagram showing a culture system according to the present invention.

Modes for Carrying Out the Invention

[0011] Detailed Description of the Invention The drawbacks of the means according to the prior art are solved by the present invention which provides a culture medium suitable for cell culture and its preparation process.

[0012] The culture medium according to the present invention can be used for cell culture, for example, for the purpose of using cell biomass for animal or human nutrition. The culture medium according to the present invention can be used for the production of cultured meat.

[0013] The types of cells used in the culture process in the culture medium according to the present invention may include many types of non-human metazoan cells, such as embryonic stem cells (ESCs) and other cell types derived from blastocysts or other early embryos, muscle stem cells such as muscle satellite cells, mesenchymal stem cells or cells derived from bone marrow, adipose tissue, subcutaneous tissue or other tissues, or cells in which stem cell characteristics are later induced or established, such as induced pluripotent stem cells (iPSCs). Other cell types that can be used may include, for example, myoblasts, muscle cells, fibroblasts, fibroadipocytes, preadipocytes, adipocytes, epithelial cells, chondrocytes and tendon-derived cells, such as chondrocytes and chondrocytes, macrophages, keratinocytes, hepatocytes, testicular cells, Sertoli cells, or any other suitable cells.

[0014] Examples of cell lines used in the culture process in the culture medium according to the present invention include, for example, Chinese hamster ovary (CHO) cells, such as CHO-K1 or CHO-DG44, C2C12, Madin-Darby bovine kidney cells (MDBK), Madin-Darby canine kidney (MDCK) cells, UMNSAH / DF-1, or any other suitable cell line.

[0015] The cells used in the culture process in the culture medium according to the present invention may be any suitable non-human metazoan cells. The cells for culture may be non-human vertebrate cells. These cells may be, for example, cattle, pigs, fish (piscine), game animals (cervine), birds, rodents (cricetine, mouse), horses, or any other suitable cells. Cells for culture may be selected from at least one of the following animals, though not limited to: cattle (Bos taurus), chickens (Gallus domesticus), domestic pigs (Sus domesticus), house crickets (Acheta domesticus), snails (Helix pomatia), carp (Cyprinus carpio), horses (Equus ferus), edible crabs (Cancer pagurus), laughing frogs (Pelophylax ridibundus), common octopuses (Octopus vulgaris), European snapper (Sparus aurata), deer (Capreolus capreolus), edible sea urchins (Echinus esculentus), harbor seals (Phoca vitulina), European stag beetles (Lucanus cervus), and African elephants (Loxodonta). It may be derived from the African mouse (Mus musculus), the green sea turtle (Chelonia mydas), or any other suitable animal.

[0016] In one aspect of the present invention, the cultured cells may be bovine cells. Bovine cells may be selected from a group of stem cells, including embryonic stem cells and other cell types derived from blastocysts or other early embryos, muscle stem cells such as muscle satellite cells, mesenchymal stem cells or stem cells derived from bone marrow, adipose tissue, subcutaneous tissue or other tissues, or cells in which stem characteristics are subsequently induced or established, such as induced pluripotent stem cells. Other bovine cell types that may be used may be bovine myoblasts, muscle cells, fibroblasts, fibroadipogenic progenitor cells, preadipocytes, adipocytes, epithelial cells, cartilage and tendon-derived cells, such as chondrocytes and chondrocytes, macrophages, keratinocytes, hepatocytes, testicular cells, Sertoli cells, mesenchymal stem cells, muscle satellite cells, or combinations thereof.

[0017] In one aspect of the present invention, the cells used in the culture process in the culture medium according to the present invention may be at least one of the following forms: single cells; cell aggregates that can take the form of cell clumps (loosely linked aggregates), spheroids (dense, homogeneous aggregates), and / or organoids (dense, heterogeneous aggregates); cells attached to carriers such as microcarriers, macrocarriers, or microfragments; or any other suitable form of cells. The cells used in the culture process in the culture medium according to the present invention may be immortalized. Preparation of culture media - general explanation

[0018] Culture media can be prepared by dissolving individual media components in water or a suitable aqueous buffer. Components can be sterilized by a suitable sterilization method to remove fungi, bacteria, viruses, and other potential contaminants. Sterilization may be performed before or after dissolution of media components. Sterilization may be carried out by physical or chemical methods. Chemical methods may include treatment with ozone, chlorine dioxide, ethylene oxide, or any other suitable chemical compound. Physical methods may include treatment with moist or dry heat, ionizing radiation, or any other suitable physical effect. Furthermore, filtration can be used as a sterilization method for aqueous solutions of solid media components, aqueous solutions of liquid media components, or separate liquid media components. Advantageously, physical sterilization methods (including filtration) may be used to minimize the risk of the final product being contaminated with chemical disinfectant residues. Advantageously, several media components or their solutions may be mixed together before sterilization, thereby reducing the number of materials that need to be sterilized separately.

[0019] For filter sterilization of culture media or its components, the filter material may be polyethersulfone, cellulose acetate, ceramic, or any other suitable filter material. The maximum pore size of the filter may be in the range of 0.001 μm to 10 μm, or in the range of 0.01 μm to 2 μm, or in the range of 0.05 μm to 0.5 μm. Filtration may be carried out as dead-end filtration, tangential flow filtration, alternating flow filtration, or any other suitable filtration setting.

[0020] The culture medium may be stored as a final complete culture medium solution, or as a set or mixture of at least one solid component, or as a set of at least one concentrated solution of one or more components, or as a set of one or more liquids, or as a combination thereof.

[0021] The culture medium of the present invention can be prepared in a culture medium tank. The culture medium tank may include at least one of a mixing tank, a hydrolysis tank, a storage tank, a loading tank or a waste culture medium tank, or any other suitable device.

[0022] The culture medium components may be mixed in a mixing tank which may be made of stainless steel, glass, or any other suitable material. The mixing tank may include, for example, a stirring unit having a shaft with one or more impellers. The mixing tank may include a heating system. The temperature may be in the range of 10–40°C, or 15–38°C, or 18–35°C. The mixing tank may be connected to one or more storage tanks. The mixing tank may be connected to one or more culture devices formed, for example, by a bioreactor. The culture medium components may be mixed directly within the culture device.

[0023] The volume of the mixing tank is 500 mL to 100 m³. 3 The range, or 1L~10m 3 Within the range of 2L to 5m 3 Within the range of 500L to 3m 3 It could be within the range of.

[0024] The storage tanks can be made from stainless steel, glass, or any other suitable material. The volume of the storage tanks is 500 mL to 100 m³. 3 Within the range of 1L to 5m 3 Within the range of 2L~3m 3 Within the range of 500L~1m 3 It could be within the range of.

[0025] The culture medium components may be introduced into the mixing tank through a sterile filter, or sterilized before being placed in the mixing tank, or sterilized within the mixing tank.

[0026] The mixing tank may be equipped with different types of sensors, such as thermal sensors, pH probes, conductivity meters, or any other suitable sensors depending on the needs of the process.

[0027] The cell culture process in a culture medium according to the present invention can be carried out in a culture system. In one aspect of the present invention, the culture system 1 is as shown in Figure 1. The culture system 1 may include a seeding tank 2, a culture apparatus 3, a harvesting apparatus 4, a control unit 5, and sensors and analytical instruments 6, as shown in Figure 1. Optionally, the culture system 1 may further include an apparatus for preparing food (not shown in Figure 1).

[0028] The control unit can control and / or regulate all processes performed within the culture system. The control unit may operate using at least one printed circuit board (PCB) and / or microprocessor with software capable of controlling the culture apparatus regardless of the system's expansion and scale. The control unit may be connected to at least one central data storage. The culture system may include one or more sub-control units.

[0029] The culture medium according to the present invention may contain protein hydrolysates as an amino acid source.

[0030] The culture medium preparation process may further have the characteristics of a batch process, a continuous process, or a combination thereof, as described below. Culture medium preparation - batch process

[0031] In a batch process for preparing culture media, a complete culture medium can be prepared, introduced into a culture device, and then inoculated with cells.

[0032] As described above, the complete culture medium may be mixed in a mixing tank, or the culture medium components may be mixed directly in the culture apparatus.

[0033] Complete culture media can be sterilized separately within the mixing tank, within the culture apparatus, or during transfer from the mixing tank to the culture apparatus. Alternatively, a sterilized complete culture medium can be obtained by sterilizing individual culture medium components or mixtures thereof before adding them to the mixing tank or during transfer to the mixing tank. Culture medium preparation - continuous process

[0034] In a continuous process of culture medium preparation, the preparation of the culture medium can be carried out simultaneously with cell culture.

[0035] Individual culture medium components or mixtures of components may be introduced into the culture apparatus during cell culture. These components may be sterilized before or during transfer to the culture apparatus. They may be introduced into the culture apparatus in any form, for example, as an aqueous solution, thereby enabling simple filter sterilization and rapid mixing of all culture medium components within the culture apparatus.

[0036] The rate at which culture medium components are added may be fixed, or it may be adjusted to ensure optimal conditions for cell culture in the culture apparatus. These optimal conditions may include, but are not limited to, the pH range, osmotic pressure, shear protectant concentration, sugar concentration, and amino acid concentration as specified herein in the section entitled Culture Medium Composition.

[0037] Conditions within the culture apparatus can be determined by a pH probe, conductivity meter, osmometer, glucose prober, refractometer, UV-Vis spectrometer, Raman spectrometer, or any other suitable measurement method. Where appropriate, the concentration of a particular compound within the culture apparatus can also be approximated by a mass balance equation.

[0038] To achieve desired conditions within the culture apparatus, the flow rate of culture medium components can be adjusted using an appropriate control mechanism, such as a PID control loop.

[0039] In one aspect of the present invention, the continuous culture medium mixing process may include the following steps: - Introduction of concentrated basic medium into the culture apparatus at a specified flow rate: For example, if the basic medium is concentrated to X times the desired concentration in the final medium, the basic medium is introduced at a flow rate of 1 / X times the total discharge flow rate from the culture apparatus; - Introduction of a concentrated sugar solution into the culture apparatus, with the flow rate controlled so that the sugar concentration in the culture medium is maintained at a desired set point, e.g., 1 g / L; - Introduction of hydrolysate and amino acid solutions into the culture apparatus, with the flow rate controlled so that the concentration of amino acids in the culture medium is maintained at a desired setpoint; - Introduction of a concentrated NaCl solution into the culture apparatus, with the flow rate controlled so that the total osmotic pressure of the culture medium is maintained at a desired set point, e.g., 310 mOsm / kg; -Introduction of desalinated water into a culture device, wherein the flow rate is adjusted to be equal to the difference between the total output flow rate from the culture device and the sum of all other input flow rates, thereby maintaining a constant volume of liquid in the bioreactor. Preparation of hydrolysates - General explanation

[0040] Protein sources for hydrolysis can be selected from industrially scalable protein sources. Industrially scalable protein sources include terrestrial plants, green algae, red algae, brown algae or other phototrophic eukaryotes, phototrophic prokaryotes, such as cyanobacteria, or cultured heterotrophic prokaryotes or eukaryotes, such as bacteria or yeast. Organisms used as protein sources may be able to synthesize all amino acids from inorganic nitrogen sources such as ammonium ions, nitrate ions or molecular nitrogen. Hydrolysis can be carried out, for example, in a hydrolysis tank or in any other suitable apparatus.

[0041] Hydrolysis may be performed on protein isolates or concentrates from source organisms, or on the entire biomass of the source organism. Source organisms may be pre-treated mechanically or chemically to improve the rate and efficiency of the hydrolysis process. Sugars, fats, or other compounds may be removed from the biomass of the source organism to facilitate easier processing. Examples of suitable industrially scalable protein sources include soybeans, peas, rice, wheat, corn, broad beans, alfalfa, hemp, chickpeas, potatoes, pumpkins, rapeseed, red lentils, spirulina, chlorella, sunflowers, duckweed, mango beans, flax, or baker's yeast. The present invention is not limited to the exemplary protein sources listed.

[0042] Protein hydrolysates or multiple protein hydrolysates from the same or different source organisms can serve as a source of all essential amino acids in a culture medium for cell culture purposes, or some amino acids, such as methionine, which is found in very low concentrations in the most scalable protein sources, can be supplied separately. Other different individual amino acids can be supplied separately from sources different from the protein hydrolysates. Typically, methionine and some non-essential amino acids, such as asparagine or glutamic acid, are commercially available (in food grade) at prices suitable for use in industrial-scale cell culture. However, the majority of the essential amino acid content in the medium according to the present invention may come from hydrolysates. This approach may be more scalable and economically feasible than using individual free amino acids, as is commonly done in the biopharmaceutical industry or basic research.

[0043] The hydrolysis process involves breaking down the original protein molecule into shorter peptide chains and / or single amino acids. As used herein, the term “protein hydrolysate” is understood to mean a mixture of amino acids, peptides, and other molecules prepared from a suitable protein source by any suitable method, including hydrolysis by acid, base or enzyme, autolysis, or fermentation by suitable microorganisms capable of degrading proteins. The “protein hydrolysate” as relating to this disclosure may be, for example, an enzymatic hydrolysate of a plant protein, an extract or lysate of various yeasts (such as a whole yeast autolysate), or an acid hydrolysate of an alga.

[0044] Methods of protein hydrolysis include acid hydrolysis, base hydrolysis, enzymatic hydrolysis, or autolysis. Acid hydrolysis is usually performed at high temperatures, lowering the pH of the protein source to a very low level. The reaction can last for several hours or even several days. Unfortunately, acid hydrolysis results in the significant degradation of several amino acids, most notably tryptophan, which must then be supplied separately at a significant cost. Significant degradation of several amino acids also occurs during base hydrolysis, which typically exposes the protein source to very high temperatures and pH. Furthermore, the acid or base used in hydrolysis must be removed before the hydrolysate can be used in cell culture, adding further complexity. For example, when performing acid hydrolysis using hydrochloric acid, the acid can be removed by neutralization or evaporation. However, both processes are economically undesirable because i) the neutralization process results in undesirable high concentrations of salt that also need to be removed, and ii) evaporation is energy-intensive, and the resulting HCl vapor poses health and environmental hazards that need to be addressed. The autodegradation process relies on the activity of enzymes in the source organism that break down the protein source; therefore, this process is usually not very efficient and generally does not result in sufficient hydrolysis of the source protein. Furthermore, the protein can be broken down by fermentation by organisms such as Bacillus licheniformis or Aspergillus oryzae, which produce large amounts of proteolytic enzymes. However, in this approach, some of the amino acids derived from the source protein may be consumed by the organism used to break down the protein during the fermentation process. In addition, metabolic waste and other compounds from the fermenting organism may contaminate the resulting lysate and adversely affect its properties with respect to mammalian cell cultures.

[0045] The hydrolysates according to the present invention can be obtained by enzymatic hydrolysis of a suitable protein source. Industrially scalable protein sources are advantageous. In one aspect of the present invention, soy protein isolate can be used as a protein source for enzymatic hydrolysis. Advantageously, soy protein isolate has a favorable ratio of most amino acids for the purpose of mammalian cell culture, with the exception of methionine, which is present in relatively low concentrations. However, methionine can be added separately to the culture medium as described above.

[0046] Protein substrates for hydrolysis in a solvent may be subjected to an initial heat pretreatment to improve their solubility and sensitivity to hydrolysis. The temperature during the heat pretreatment may be in the range of 75–95°C, 80–92.5°C, or 85–90°C for 5–120 minutes, 15–60 minutes, or 30–45 minutes.

[0047] Enzymatic hydrolysis can utilize so-called proteases, which are enzymes that catalyze the breakdown of peptide bonds, to achieve protein hydrolysis under much milder conditions than acid or base hydrolysis, thus preserving all the amino acids of the original protein.

[0048] In one aspect of the present invention, the enzyme used for hydrolysis may be immobilized on a solid support. This approach sterically prevents the enzyme molecules from degrading each other and allows the enzyme to be separated from the reaction mixture after the reaction and reused. The solid support may be in the form of a solid carrier suspended in the reaction mixture, or a solid structure with a large surface area, such as a sponge or fibrous structure, through which the reaction mixture is perfused. The enzyme may also be added in a soluble (free) form. After hydrolysis is complete, the resulting hydrolysate is separated from the solid support on which the enzyme is immobilized simply by draining the reaction vessel (in the case of a large solid structure), or by removing the enzyme from the solid support by filtration or sedimentation (in the case of a suspension carrier). The reaction vessel may be formed, for example, by a hydrolysis tank. The filtration step may also remove any solid residues, such as cell wall residues, from the source protein. Free enzymes may be removed from the hydrolysate by ultrafiltration or inactivated at high temperatures when hydrolysis is complete. Ultrafiltration of the hydrolysate may further remove any larger peptide chains that were not digested by the enzyme. These peptide chains can be harmful to cells, and therefore their removal can be beneficial. The temperature increase used to inactivate the enzymes can also sterilize the resulting hydrolysates.

[0049] By heat-treating the hydrolysates at the end of hydrolysis, the enzymes can be inactivated and the microorganisms killed. In one aspect of the present invention, this treatment may be carried out at a lower temperature setting in the range of 80-120°C, or 85-100°C, or 90-95°C, for a longer duration in the range of 15-180 minutes, or 20-120 minutes, or 25-60 minutes. In another aspect of the present invention, this treatment may be carried out at a higher temperature in the range of 80-160°C, or 100-155°C, or 110-150°C, for a shorter duration in the range of 1-600 seconds, or 3-300 seconds, or 5-60 seconds. Methods with lower temperature settings may be carried out in a reactor setting, and either method may be carried out in a flash pasteurizer or another suitable continuous flow heating device.

[0050] If the enzyme is removed by ultrafiltration, it may retain at least partially its catalytic activity and therefore can be reused for another hydrolysis. Alternatively, if some of the enzyme separates from the solid support and dissolves in the reaction mixture, the active enzyme molecule can be removed from the hydrolysate prepared by the immobilized enzyme using ultrafiltration or thermal deactivation.

[0051] The solid support may be formed from, for example, silica, epoxide, cellulose, chitosan, glass wool, alginate, or other suitable material. The solid support may be in the form of porous or solid beads, sponge, fibrous, or another suitable configuration. The solid support may have a large surface area-to-volume ratio to allow for the binding of large amounts of enzyme. For example, porous silica beads, or any other suitable material having a diameter in the range of 1 to 10,000 micrometers, or 10 to 1,000 micrometers, or 20 to 500 micrometers, can be used as a solid support for enzyme immobilization. Immobilization may be achieved, for example, by functionalizing the surface of the silica beads with amino groups and using a crosslinking agent such as glutaraldehyde to bind the enzyme to the solid support. Other functional groups such as aldehyde groups or epoxy groups may also be used for enzyme immobilization. In this embodiment of the present invention, the amino group is covalently bonded with glutaraldehyde, and then the excess glutaraldehyde is removed and the enzyme is added. Next, the amino groups on the surface of the enzyme bind to the remaining free aldehyde groups of the glutaraldehyde molecule on the surface of the silica beads. Immobilization can be carried out in water or a suitable aqueous buffer. Thanks to the porosity and large surface area of ​​the silica beads, a relatively large amount of enzyme can be immobilized relative to the weight of the solid support.

[0052] The enzymes according to the present invention may be, for example, Alcalase (a protease derived from Bacillus licheniformis), Flavorzyme (a protease derived from Aspergillus oryzae), Protamex, Novo-Pro D, Thermoase PC10FNA, Protease AN Amano 100SD, Protease A Amano 2SD, Protease M Amano SD, Protease P Amano 6SD, ProteAX, Peptidase R, Alkali Protease, Colorase 7089, Colorase 2TSN, Colorase 8000, MaxiproTNP, MaxiproFPC, Papain, Bromelain, or any other suitable proteolytic enzyme, or a combination thereof.

[0053] The protein source for hydrolysis can be dissolved using water or a suitable aqueous buffer. Some proteins may require buffer to adjust the pH to a level with better solubility. The pH may be in the range of 2–12, 5–10, or 6–8.5. A highly diluted buffer may be used, or no buffer may be used at all, and the resulting hydrolysate can be added to the final culture medium at high concentrations while minimizing its effect on the osmotic pressure of the medium.

[0054] Examples of buffer solutions include phosphate buffer, bicarbonate buffer, Tris-HCl buffer, borate buffer, glycine-NaOH buffer, Good's buffer, or any other suitable buffer, or a combination thereof.

[0055] The concentration of protein in the reaction mixture for hydrolysis may range from 1 to 150 grams / liter, or from 20 to 100 grams / liter, or from 30 to 80 grams / liter per liter of the reaction mixture.

[0056] The enzyme concentration, expressed as the ratio of the enzyme concentration to the protein concentration in the reaction mixture (also known as the enzyme / protein ratio), may be in the range of 0.01–10%, 0.05–5%, or 0.1–1%. The enzyme concentration can be determined by the Bradford assay, BCA assay, or other protein determination assay.

[0057] In one aspect of the present invention, soy protein can be dissolved to concentrations ranging from 1 to 150 grams / liter, 20 to 100 grams / liter, or 30 to 80 grams / liter by adjusting the pH of potassium phosphate buffer in the range of 1 to 100 mM, 10 to 40 mM, or 15 to 35 mM. In another aspect of the present invention, soy protein is dissolved in distilled water to concentrations ranging from 1 to 150 grams / liter, 20 to 100 grams / liter, or 30 to 80 grams / liter.

[0058] While other concentrations of source protein may be used, very high concentrations of source protein lead to incomplete dissolution of the protein and the formation of a highly viscous colloidal solution, presenting problems with hydrolysis and further processing, while low concentrations of protein may limit the rate of the hydrolysis reaction. Heat treatment may be used to ensure optimal dissolution of the protein in the reaction mixture. Temperatures below boiling point may be used for extended periods to significantly increase the content of dissolved protein and inactivate potential inhibitors of proteases and other antitrophic compounds.

[0059] In one aspect of the present invention, the source protein may be added at a concentration higher than the maximum soluble concentration. This additional protein may dissolve after the protein concentration in the reaction mixture has decreased due to its hydrolysis by the enzyme. This results in a high concentration of available substrate throughout the process, potentially improving hydrolysis efficiency. Multiple cycles of substrate addition to the same reaction mixture may be performed. In one aspect of the present invention, the changes may be counteracted by adding a base or a suitable buffer to maintain the enzyme at its optimal pH, or a pH stat may be used.

[0060] A key parameter for evaluating the conversion of substrate proteins into bioavailable products for animal cells is the degree of hydrolysis, defined as the percentage of peptide bonds in the source protein that are hydrolyzed during the reaction. A higher degree of hydrolysis corresponds to a greater percentage of the source protein converted into free amino acids or shorter peptides that can be used as nutrition by mammalian cells. Mammalian cells generally cannot absorb and digest proteins and longer peptides. Peptides longer than 4 amino acids, in other words, heavier than about 500 daltons, are very poorly absorbed by mammalian cells. In various embodiments of the present invention, when expressed as the mass concentration of amino acids relative to the mass concentration of protein, amounts ranging from 20% to 100%, 30% to 70%, 35% to 65%, or 40% to 60% of the source protein can be converted into free amino acids. The degree of hydrolysis (DH) refers to the percentage of peptide bonds that undergo hydrolysis out of the total amount of peptide bonds present in the substrate at the start of the reaction, and can range from 10% to 60%, 20% to 50%, or 25% to 40%.

[0061] Enzymes used for hydrolysis can be classified into two general categories: exoproteases and endoproteases. Exoproteases can cleave protein or peptide chains at their ends, while endoproteases can cleave peptide bonds in the middle of the chain. In one aspect of the present invention, a combination of endoproteases and exoproteases may be used because the endoprotease generates more free ends in the peptide chain, increasing the efficiency of the exoprotease, and the exoprotease is more efficient at hydrolyzing proteins to single amino acids. In one aspect of the present invention, endoproteases and exoproteases may be used sequentially in this order to maximize hydrolysis efficiency.

[0062] In one aspect of the present invention, additional enzymes may be added to the reaction mixture after the start of hydrolysis. This may be done using the same enzyme, mainly to counteract the gradual decrease in its enzymatic activity due to the decomposition of the enzyme molecule. In one aspect of the present invention, when the pH is higher, an enzyme with an optimal value at a higher pH may be added at the start of hydrolysis, and when the pH is lower, an enzyme with an optimal value at a lower pH may be added later, thus maximizing the efficiency of each enzyme. pH tends to decrease naturally during hydrolysis due to the increasing number of carboxylic acid groups.

[0063] In another aspect of the present invention, additional substrates may be added to the reaction mixture after the start of hydrolysis. An advantage of this approach may be that the additional substrates are more easily dispersed and dissolved when the previous amount of substrate is at least partially hydrolyzed.

[0064] The addition of enzymes or substrates after the start of the hydrolysis process can be carried out in fed-batch mode (additional reagents are added to the reaction mixture, and then the entire reaction batch is collected) or in continuous mode (both addition to the reaction mixture and collection from the reaction mixture are carried out continuously).

[0065] Whether immobilized or free enzymes are used, thorough mixing of the reaction mixture is crucial for achieving high efficiency. In the case of immobilized enzymes, this applies to both the enzyme immobilization step and the protein hydrolysis step. In one aspect of the present invention, in the case of immobilized enzymes, a mixing method that minimizes mechanical damage to the solid support should be used. These may include roller mixing, shaking, or low-shear impellers such as hydrofoils or elephant ear impellers. In the case of enzymes immobilized on large solid supports, sufficient perfusion between the support and the reaction mixture must be ensured.

[0066] The processes of mixing a protein source, such as a protein isolate, with water or a suitable aqueous buffer, dissolving the protein source, and hydrolysis itself can be carried out in a suitable reaction vessel on a laboratory or industrial scale. The reaction vessel may be formed, for example, by a hydrolysis tank.

[0067] Examples of the reaction vessel for hydrolysis may include a batch reactor, a continuous stirred tank reactor, or a plug flow reactor. The volume of the reaction vessel may be in the range of 0.1 L to 100,000 L, or in the range of 0.3 L to 15,000 L, or in the range of 1 L to 5,000 L.

[0068] Mixing can be provided by a suitable stirring unit that may include, for example, a paddle impeller. An elephant ear impeller can be used. The outer diameter of the stirrer or impeller may be in the range of 1 / 10 to 9 / 10 of the inner diameter of the reactor, or in the range of 3 / 10 to 8 / 10 of the inner diameter of the reactor, or in the range of 4 / 10 to 7 / 10 of the inner diameter of the reactor, for example 2 / 3 of the inner diameter of the reactor. The stirrer or impeller can be arranged at the center of the reaction vessel or outside the center of the reaction vessel.

[0069] The reaction components can be added to the reaction vessel manually, or based on gravity from a storage tank connected to the reaction vessel, or using a pump system. The source protein can be in the form of a liquid solution or powder and can be added to the reaction vessel manually or automatically.

[0070] The storage tank can be made of, for example, stainless steel or glass. The volume of the storage tank can be within the range of 100 ml to 5 m 3 within the range of 2 L to 3 m 3 within the range of 500 L to 1 m 3 and can be in the range of.

[0071] The reaction vessel, such as a hydrolysis tank, can be equipped with different types of sensors, such as a thermal sensor, a pH probe, a conductivity meter, or any other suitable type of sensor according to the requirements of the hydrolysis process. The pH can be monitored throughout the procedure by a pH electrode. The temperature inside the reaction vessel can be adjusted using, for example, a reactor thermal jacket that may include a heating coil and / or a heating / cooling medium.

[0072] A sampling system may be used to accurately monitor the degree of hydrolysis. The degree of hydrolysis can be monitored by titration and / or absorbance measurement at wavelengths in the range of 190–350 nm or 190–230 nm.

[0073] After the hydrolysis process, the phytic acid content can be reduced using another treatment. Phytic acid is an important compound in plant metabolism, and its salt form, phytic acid, is the main phosphorus storage compound in plants. On the other hand, phytic acid is one of the antinutrient compounds in leguminous plants and can significantly affect downstream processes as well as cell proliferation and viability. Enzymatic treatment or any other method can be used to reduce the phytic acid content. When enzymatic treatment is used, the selection of an appropriate phytase enzyme is as important as appropriate conditions with respect to pH and temperature. This process may involve the addition of a phytase enzyme in an enzyme / substrate ratio and concentration correlated, where the substrate means the source of the protein used. The enzyme / substrate ratio is 1.1·10 -11 A range of % to 1%, or 1.1·10 -10 A range of % to 0.001%, or 1.1·10 -9 The concentration may be in the range of % to 0.0001%. The temperature may be in the range of 20°C to 80°C, 30°C to 70°C, or 40°C to 60°C. After enzyme treatment for a time ranging from 20 minutes to 4 hours, 30 minutes to 3 hours, or 1 to 2 hours, heat treatment for enzyme deactivation may be performed. Deactivation may be carried out, for example, at 90°C for 30 minutes or at 95°C for 15 minutes.

[0074] For filtration purposes, such as removing impurities, separating enzymes immobilized on a carrier from reaction mixtures, or separating larger peptide hydrolysates, a suitable filtration unit equipped with a filtration material may be used. The filtration material may be, for example, filter cloth, ceramic, glass, membrane, or other suitable material. The pore size in the filtration material may be, but is not limited to, 500 μm to 10 μm for filtration, 10 μm to 0.1 μm for microfiltration, 0.1 μm to 1 nm for ultrafiltration, or 1 nm to 0.1 nm for nanofiltration. Membranes characterized in the range of 60 kDa to 500 Daa may be used. As a step prior to filtration, centrifugation may be used to facilitate the filtration process. Hydrolysis by free enzymes

[0075] In one aspect of the present invention, hydrolysis by free enzymes may be carried out by dissolving a protein substrate in a reaction vessel formed by a hydrolysis tank, for example. This protein substrate may be, for example, whole biomass, protein concentrate, protein powder, raw protein powder, soybeans, peas, rice, wheat, corn, broad beans, alfalfa, hemp, chickpeas, potatoes, pumpkins, rapeseed, red lentils, spirulina, chlorella, sunflowers, duckweed, mango beans, flax, or yeast, or protein extracts isolated from another suitable protein source.

[0076] The protein concentration in the reaction mixture may range from 1 g / L to 150 g / L, or from 20 g / L to 100 g / L, or from 30 to 80 g / L. For a given volume of the reaction mixture, alcalase may be added in an amount calculated according to the enzyme / protein ratio, in the range of 0.01 to 10%, 0.05 to 3%, or 0.1 to 0.8%, in terms of enzyme concentration / protein concentration. The resulting mixture has a neutral to basic pH, allowing for high activity of alcalase. The temperature may range from 50°C to 70°C, or from 55°C to 65°C, or from 58°C to 63°C. Over a certain mixing period, which may range from 30 minutes to 24 hours, or from 1 to 12 hours, or from 2 to 8 hours, the pH of the mixture decreases as a result of hydrolysis of peptide bonds and an increase in the number of carboxyl groups.

[0077] This enables high activity of the flavorzyme, which can be added in calculated amounts depending on the enzyme / protein ratio, ranging from 0.01 to 10%, 0.1 to 2%, or 0.2 to 1% relative to the reaction mixture. The resulting mixture can then be incubated at temperatures ranging from 30 to 80°C, 40 to 70°C, or 45 to 60°C for a further time ranging from 1 to 48 hours, 5 to 24 hours, or 8 to 20 hours, after which the residual enzyme may be thermally deactivated. This procedure can convert 20% to 100%, 30% to 70%, or 40% to 60% of the source protein into free amino acids. Hydrolysis by immobilized enzymes

[0078] The protein hydrolysis process may be carried out in a reaction vessel formed, for example, by a hydrolysis tank. Protein hydrolysis may be carried out using immobilized enzyme in amounts ranging from 0.01 g to 10 g, or from 0.25 g to 1.8 g, or from 0.5 g to 1.5 g on 10 g of enzyme carrier. The enzyme carrier may be prepared from glass, porous silica, alginate, epoxy methacrylate, chitosan, or any other suitable material, in the form of beads, wool, sponge, fibrous material, or any other suitable form. The enzyme carrier may be formed, for example, by glass beads, porous silica beads, alginate beads, epoxy methacrylate beads, glass wool, chitosan, or any other suitable enzyme carrier. Suitable enzyme carriers are described in more detail in the section entitled “Preparation of Hydrolyzates - General Description” herein. For example, 1 g of immobilized enzyme may be used on 10 g of porous silica beads.

[0079] The immobilized enzyme can be prepared by suspending a set weight of NH2-functionalized porous silica microbeads in a set weight of distilled water. The set weight ratio of NH2-functionalized porous silica microbeads to distilled water may be in the range of 1:1 to 1:10000, or 1:10 to 1:1000, or 1:20 to 1:100. The silica beads are further activated by the addition of glutaraldehyde. The amount of glutaraldehyde added to the reaction mixture may be in the range of 0.01 to 70 mmol, or 0.05 to 40 mmol, or 0.1 to 10 mmol per gram of silica beads. Excess glutaraldehyde is washed away, and the silica beads are resuspended, for example, to half of their original volume. Then, the alcalase is added to the final concentration while stirring constantly. This allows 10 to 100%, 60 to 90%, or 70 to 80% of the enzyme used to be immobilized on the silica beads. This can correspond to 10-100 grams, 30-60 grams, or 40-50 grams of immobilized enzyme per kilogram of silica beads.

[0080] In one aspect of the present invention, silica beads having immobilized alcalase can be added to a mixture of soy protein and distilled water. The amount of silica beads having immobilized alcalase may be, for example, in the range of 10-20 g / L, or 12-18 g / L, or 14-16 g / L, or any other suitable amount. After hydrolysis by alcalase, the beads bound to the alcalase can be removed by centrifugation. Silica beads immobilized with flavorzyme are added in amounts, for example, in the range of 4-40 g / L, or 5-30 g / L, or 10-20 g / L. The appropriate time for hydrolysis may be, for example, in the range of 10 minutes to 24 hours, or 30 minutes to 12 hours, or 1 to 6 hours.

[0081] The hydrolysis temperature may be in the range of 10–90°C, 25–80°C, or 50–70°C. In another aspect of the present invention, the alcalase beads may not be removed in this step, but may instead be removed at the end of the process. In yet another aspect of the present invention, the alcalase and flavorzyme beads may be of various sizes to facilitate their separation after removal from the solution. In another aspect of the present invention, the flavorzyme beads may be added at the start of hydrolysis or at any other point during hydrolysis. After further hydrolysis, the flavorzyme beads are removed by centrifugation while constantly mixing at a temperature that may be in the range of 20–90°C, 30–80°C, or 40–60°C, for a time that may be in the range of 1–24 hours, 6–20 hours, or 10–18 hours, and the resulting hydrolysate is heat-sterilized, which also inactivates any enzymes that may be detached from the solid support. After filtering to remove solid residue, the hydrolysate may be used to prepare a culture medium. This method allows for the conversion of source protein amounts ranging from 20-100%, 30-95%, or 40-90% into cell-usable products, i.e., free amino acids or peptides with a density of 500 Da or less.

[0082] Alcalases and flavorzymes are highly stable in their immobilized forms and can therefore be recycled in the hydrolysate production process according to the present invention. In one aspect of the present invention, silica beads having immobilized alcalases can be used for hydrolysis cycles of 2-50, 5-40, or 10-30 while maintaining about half of their original catalytic activity. In another aspect of the present invention, silica beads having immobilized flavorzymes can be used for hydrolysis cycles of 2-50, 5-40, or 10-30 while maintaining sufficient catalytic activity. Generally, immobilized enzymes tend to be more stable than free enzymes, but their enzymatic activity decreases with use. Therefore, in later cycles, the reaction duration or enzyme-to-protein ratio may be altered to maintain consistent quality of the resulting hydrolysates. Culture medium composition

[0083] In one aspect of the present invention, the composition of a culture medium can be defined with respect to the total input of medium components to the culture process. In this aspect of the present invention, the total content of components introduced into the culture process at any point in time throughout its entire duration is provided. Furthermore, in this aspect of the present invention, the concentration range provided for individual medium components is expressed as the total content of a given component introduced into the culture process at any point in the culture process relative to the amount of used culture medium leaving the process. The used culture medium may be removed from the culture process together with the cultured cells (recovered) or separately from the cultured cells (perfused). The culture process may further have the characteristics of a batch process in which all components are introduced into the culture process at a single time point and recovered at a single time point, a fed-batch process in which some components may be introduced after the start of the process and recovered at a single time point, a continuous process in which components may be introduced throughout the entire duration of the culture and recovered throughout the entire duration of the culture, or a combination of the characteristics described. For brevity, this aspect of the present invention is referred to herein as “total input”.

[0084] In another aspect of the present invention, the composition of a culture medium may be expressed in terms of the concentration of components present in the culture medium at a specific point in time during the cell culture process. In this aspect of the present invention, the concentration range provided for individual medium components represents the concentration present in the culture medium in the culture apparatus at any point in time during the culture process. For brevity, this aspect of the present invention is referred to herein as “instantaneous composition”. Total input

[0085] The total input to the culture medium according to the present invention may include an optimized essential ratio of amino acids, which may be derived from protein hydrolysates, in combination with at least one compound selected from the group including sugars, vitamins and organic micronutrients, mineral compounds, iron supplementation compounds, organic amines and shear protectants, or combinations thereof. The culture medium may also contain other compounds, such as fatty acids, phospholipids, or nucleic acids. The culture medium according to the present invention, having an optimized ratio of amino acids and other nutrients, can promote efficient biomass production by cells and low production of waste metabolites such as ammonia or lactate.

[0086] An optimized ratio of essential amino acids is one in which essential amino acids can be introduced into the culture process in any ratio in which the proportion of essential amino acids that can be converted into cellular proteins ranges from 5% to 100%, 20% to 90%, or 30% to 80%. The term "highest possible conversion efficiency" determines what percentage of the essential amino acids supplied to the cell can be converted into cellular proteins, assuming no loss of amino acids due to catabolism, conversion to other compounds (such as nucleic acids), or spontaneous degradation.

[0087] The highest possible conversion efficiency is determined by the most limited essential amino acids for the cell. For every individual essential amino acid added to the culture medium in any form at any point in the culture process, the content of that particular essential amino acid in the culture medium as part of the total essential amino acid content added to the culture medium in any form at any point in time is divided by the content of that individual amino acid in the cellular protein as part of the total essential amino acid content obtained at the lowest ratio. In other words, the ratio for the essential amino acid that forms the lowest proportion of amino acids added to the culture medium is multiplied by 100 compared to the proportion of that particular amino acid in the cellular biomass to obtain the highest possible conversion efficiency of the provided essential amino acid to the cellular protein. All percentages in the calculation of the highest possible conversion efficiency are weight percentages.

[0088] Amino acids in the culture medium can exist in the form of free amino acids or peptides. Non-essential amino acids can be synthesized by cells and are therefore not limiting in terms of the highest possible conversion efficiency, and are therefore omitted from this calculation. Examples of possible essential amino acid content in cellular proteins can be seen in Table 1 below.

[0089] The above explanation can be summarized by the following formula:

number

[0090] An example of calculating the essential amino acid tryptophan is as follows: Assume that the total amount of tryptophan added to the culture medium over the culture period is 2 grams, and the total amount of essential amino acids added to the culture medium over the same period is 100 grams. Table 1 shows that in 100 grams of cell protein, 1.6 grams of tryptophan is present out of 44.7 grams of total essential amino acids.

[0091] Calculation:

number

[0092] The amount of essential amino acids that can be converted into cellular proteins is determined by how closely the total input of essential amino acids to the culture process matches the amino acid composition of the cellular proteins. Since cells cannot synthesize essential amino acids, the essential amino acid whose total input to the culture process is lowest compared to its content in the cellular proteins limits the maximum cellular yield, i.e., the maximum percentage of essential amino acids that can be converted into cellular proteins (this can be understood as the application of Liebig's law of minimums).

[0093] The total conversion efficiency of essential amino acids can range from 5% to 100%, 20% to 100%, 30% to 100%, or 50% to 100%, as calculated by the above formula.

[0094] When the essential amino acid compositions for cellular proteins shown in Table 1 are used, the resulting total input for each essential amino acid, given as grams per 100 grams of the total input for all essential amino acids, may be within the range summarized in Table 2.

[0095] The range of amino acid concentrations per gram per 100 grams of total essential amino acids introduced into the culture process may follow Table 2, regardless of whether the essential amino acid composition of the cellular protein follows Table 1 or not.

[0096] It should be noted that for the purpose of this formula, amino acid content must be consistently considered either as free amino acids or as amino acids that are part of a peptide chain (in this case, the molecular weight of each amino acid must be considered lower by the weight of one water molecule to account for the fact that water is a byproduct of peptide bond formation). In the formula above and Tables 1-3, all are counted as amino acids that form a peptide chain. Elsewhere in this specification, when amino acid inputs or concentrations are discussed, these are calculated using the molecular weight of free amino acids, and when protein inputs or concentrations are discussed, amino acids are assumed to be part of a peptide chain for any calculation. [Table 1] [Table 2]

[0097] However, the composition of cellular biomass is somewhat variable, and therefore, the values ​​of each essential amino acid in relation to the total weight percentage of essential amino acids used in the culture medium may also be within the ranges summarized in Table 3. [Table 3]

[0098] Amino acids can be introduced into the culture process in the form of free amino acids, amino acid salts, amino acid esters, or any other suitable derivatives, as well as oligopeptides, such as dipeptides, tripeptides, or tetrapeptides, or polypeptides. The culture medium according to the present invention may include, as described above, soy protein enzyme hydrolysate, or any other suitable scalable hydrolysate as described in the description of the hydrolysate and its preparation. For example, suitable industrially scalable protein sources for hydrolysate preparation may include soybeans, peas, rice, wheat, corn, broad beans, alfalfa, hemp, chickpeas, potatoes, pumpkins, rapeseed, red lentils, spirulina, chlorella, sunflowers, duckweed, mango beans, or yeast. The present invention is not limited to the exemplary protein sources listed.

[0099] The total input of hydrolysates (expressed as dry weight of protein) introduced into the culture medium during the culture process may range from 1 g / L to 200 g / L, or from 3 g / L to 100 g / L, or from 10 g / L to 60 g / L, or from 8 g / L to 50 g / L.

[0100] The total amino acid input from hydrolysates of short peptides or suitable bioavailable derivatives, such as phosphoesters (e.g., phosphoserine), or other derivatives, such as amino acids in the form of methylglycine, is at least 75%, 80%, 85%, 90%, or 95% by weight of the total amino acid input to the culture medium.

[0101] The culture medium according to the present invention may contain amino acids added separately from the hydrolysate, such as L-methionine, L-cysteine, or L-ornithine. The total input of amino acids added separately from the hydrolysate may be in the range of 0.02 g / L to 30 g / L, or in the range of 0.05 g / L to 15 g / L, or in the range of 0.1 g / L to 10 g / L.

[0102] The total L-cysteine ​​content in the culture medium may be in the range of 0.1 to 10% by weight, 0.5 to 7% by weight, or 1 to 5% by weight, relative to the total hydrolyzate protein content in the culture medium.

[0103] The total L-ornithine content in the culture medium is in the range of 0-5%, 0.0001-3%, or 0.001-0.5% relative to the total hydrolyzate protein content in the culture medium.

[0104] The total L-methionine content in the culture medium may be in the range of 0.05–6%, 0.1–3%, or 0.2–2% relative to the total hydrolyzate protein content in the culture medium.

[0105] The total L-tryptophan content in the culture medium may be in the range of 0.05-6%, 0.1-3%, or 0.2-2% relative to the total hydrolyzate protein content in the culture medium.

[0106] The total L-histidine content in the culture medium may be in the range of 0.03-4%, 0.07-2%, or 0.15-1.5% relative to the total hydrolyzate protein content in the culture medium.

[0107] The total L-threonine content in the culture medium may be in the range of 0.1–7%, 0.2–5%, or 0.3–3% of the total hydrolyzed protein content in the culture medium.

[0108] The total amino acid input added to the culture medium separately from the hydrolysate may be in the range of 0.2% to 25%, 0.5% to 15%, or 1% to 10%, expressed as a percentage of the total hydrolysate protein input to the culture medium.

[0109] The culture medium according to the present invention may contain a biologically available inorganic source of nitrogen, such as ammonia. The total input of the inorganic nitrogen source may be in the range of 0 g / L to 30 g / L, or in the range of 0.5 g / L to 20 g / L, or 1 g / L to 10 g / L.

[0110] As the sugar, glucose, fructose, galactose, sucrose, lactose, maltose, or a combination thereof, or at least one compound selected from any other suitable group of sugars may be used. The total sugar input may be in the range of 1 g / L to 350 g / L, or in the range of 2 g / L to 100 g / L, or in the range of 3 g / L to 20 g / L.

[0111] The culture medium contains the following mineral compounds: Ca 2+ Cl - Cu 2+ SO4 2- Fe 3+ NO3 - Fe 2+ Mg 2+ , K + kaNa + CO3 2- , HCO3 - H2PO4 - HPO4 2- , PO4 3- Zn 2+ SeO3 2- The culture medium may contain at least one or any combination of the following. The culture medium may also contain trace amounts of other mineral compounds and elements such as cobalt, iodine, or manganese.

[0112] Since culture media are prepared by dissolving different constituent compounds in water, any suitable compound can be used as long as it dissociates into the desired ion in aqueous solution. For example, both NaCl and KCl dissociate into Cl when dissolved. - It generates ions. Another example is using CuSO4 and MgCl2 or MgSO4 and CuCl2 to produce Cu 2+ Mg 2+ SO4 2- and Cl - Ions may be generated. Assuming equimolar amounts, the resulting aqueous solution will have the same composition for both combinations of compounds used. The total input of mineral compounds introduced into the culture process may range from 0.1 g / L to 50 g / L, or from 1 g / L to 20 g / L, or from 3 g / L to 10 g / L.

[0113] Na + The total input may be in the range of 20-120 mmol / L, 30-100 mmol / L, or 40-80 mmol / L.

[0114] Cl - The total input may be in the range of 25–130 mmol / L, 35–110 mmol / L, or 45–90 mmol / L.

[0115] Mg 2+ The total input may be in the range of 0.3 to 10 mmol / L, or 0.5 to 8 mmol / L, or 1 to 5 mmol / L.

[0116] PO4 3- The total input may be in the range of 0.5 to 12 mmol / L, or 0.7 to 10 mmol / L, or 1 to 6 mmol / L.

[0117] SO4 2- The total input may be in the range of 0.1 to 5 mmol / L, or 0.3 to 3 mmol / L, or 0.6 to 2 mmol / L.

[0118] K + The total input may be in the range of 2–18 mmol / L, 4–15 mmol / L, or 6–12 mmol / L.

[0119] The culture medium may contain at least one of the following vitamins: alpha-tocopherol (vitamin E), ascorbic acid (vitamin C), vitamin B12, biotin, choline, pantothenic acid, folic acid, niacinamide, pyridoxine, riboflavin, thiamine, i-inositol, or a combination thereof. Any suitable bioactive derivative or precursor of these compounds may be used. For example, cyanocobalamin can be used as a substitute for vitamin B12 because it is readily converted to bioactive vitamin B12 by cells. As another example, thiamine hydrochloride (the chloride salt form of thiamine) may be used as a substitute for thiamine. The total vitamin input introduced into the culture process, excluding vitamins present in the lysates or extracts, may range from 0.1 mg / L to 1,000 mg / L, or from 5 mg / L to 500 mg / L, or from 20 mg / L to 300 mg / L.

[0120] The total choline input may range from 10 mg / L to 1,000 mg / L, or from 20 mg / L to 500 mg / L, or from 30 mg / L to 200 mg / L.

[0121] The total input of niacinamide (or another vitamin B3) may range from 3 mg / L to 150 mg / L, or from 6 mg / L to 100 mg / L, or from 10 mg / L to 80 mg / L.

[0122] As the organic amine, at least one compound selected from putrescine, ethanolamine, or a combination thereof, or any other suitable amine may be used. The total input of the organic amine to the culture process may be in the range of 0.01 mg / L to 1,000 mg / L, or in the range of 0.1 mg / L to 100 mg / L, or in the range of 0.5 mg / L to 20 mg / L.

[0123] Vitamins and organic amines, or their respective precursors or derivatives, may be supplied in the form of lysates or extracts, such as autolyzed yeast extracts or any other suitable lysates or extracts. Extracts or lysates for micronutrient supplementation may be added to the culture medium in amounts ranging from 0.01 g / L to 20 g / L, or from 0.1 g / L to 10 g / L, or from 0.5 g / L to 5 g / L.

[0124] Iron can be supplemented in the culture medium with compounds containing iron(III) or iron(II) in an oxidized state. Iron may be present as a free ion or chelated with a suitable chelating agent to improve its solubility and bioavailability. Examples of chelating agents include citrates, glucons, ammonium citrate, EDTA, combinations thereof, or any other suitable chelating agent. Iron may be introduced into the culture medium conjugated with the chelating agent (e.g., in the form of iron(III) citrate), or iron and the chelating agent may be added separately (e.g., in the form of ferric trichloride and sodium citrate). The relative amount (w / w) of the total input of chelating agent to the total input of iron may be in the range of 10,000:1 to 1:100, or 1,000:1 to 1:10, or 10:1 to 1:1. The total iron input may be in the range of 0.00001 g / L to 0.5 g / L, or in the range of 0.0001 g / L to 0.1 g / L, or in the range of 0.001 g / L to 0.05 g / L.

[0125] The culture medium may contain at least one shear protectant, such as polyethylene glycol (PEG), Pluronic F68, Pluronic F127, methylcellulose (MC), (hydroxypropyl)methylcellulose (HPMC), hydroxypropylcellulose (HPC), carboxymethylcellulose (CMC), dextran sulfate, or any other suitable shear protectant or combination thereof. The total input of shear protectants may range from 0 g / L to 50 g / L, or from 0.02 g / L to 10 g / L, or from 0.1 g / L to 5 g / L.

[0126] In one aspect of the present invention, the culture medium may include the total input composition described in Table 6. instantaneous composition

[0127] According to the present invention, the physicochemical parameters and composition of the culture medium can be optimized to promote rapid biomass production, efficient use of nutrients, and low production of waste metabolites.

[0128] The osmotic pressure of the culture medium may be in the range of 200 mOsm / kg to 400 mOsm / kg, or 250 mOsm / kg to 350 mOsm / kg, or 280 mOsm / kg to 330 mOsm / kg. The osmotic pressure may be adjusted at a single time point or at multiple time points, either before or after the culture medium is introduced into the culture apparatus, or a combination of both. To increase the osmotic pressure, NaCl, KCl, glucose, any other suitable osmoregulator, or a combination thereof may be used. To decrease the osmotic pressure, water or any other suitable diluted aqueous solution may be used.

[0129] The pH of the culture medium in the culture apparatus may be in the range of 6–8, 6.5–7.5, or 6.8–7.3. pH adjustment may be performed before introducing the culture medium into the culture apparatus, after introducing it, or a combination of both, at a single time point or at multiple time points. pH may be adjusted using NaOH, HCl, NaHCO3, or any other suitable acid or base, or by changing the partial pressure of CO2 in the culture apparatus (higher CO2 partial pressure results in greater CO2 dissolution into the culture medium and a lower pH). The partial pressure of CO2 in the culture apparatus may be adjusted by changing the proportion of CO2 in the sparging gas, changing the total pressure in the culture apparatus, changing the mixing and sparging rates in the culture apparatus (decreasing or increasing the CO2 mass transfer coefficient), or by any other suitable method. The partial pressure of CO2 in the culture apparatus may be in the range of 0.05 kPa–100 kPa, 2 kPa–60 kPa, or 5 kPa–30 kPa.

[0130] The culture medium may contain a shear protectant to prevent cell damage caused by mechanical forces resulting from mixing and / or sparging within the culture apparatus. The shear protectant may be at least one of polyethylene glycol (PEG), methylcellulose (MC), (hydroxypropyl)methylcellulose (HPMC), hydroxypropylcellulose (HPC), carboxymethylcellulose (CMC), dextran sulfate, or any other suitable shear protectant, or a combination thereof. The shear protectant may be present in the culture medium at concentrations ranging from 0 g / L to 50 g / L, or from 0.02 g / L to 10 g / L, or from 0.1 g / L to 5 g / L.

[0131] The transient concentration of sugar in the culture medium may be in the range of 0.005 g / L to 40 g / L, or in the range of 0.1 g / L to 20 g / L, or in the range of 0.5 g / L to 5 g / L.

[0132] The transient concentrations of all amino acids (including both amino acids derived from hydrolysates and separately added amino acids and bioavailable derivatives, such as esters) and peptides in the culture medium may range from 0.005 g / L to 30 g / L, or from 0.1 g / L to 15 g / L, or from 0.5 g / L to 10 g / L.

[0133] The above culture medium composition may be suitable for cell lines that are well adapted to in vitro conditions. However, some cell types may require additional components in the medium, such as protein growth factors, for survival and proliferation. In another aspect of the present invention, a culture medium composition suitable for these growth factor-dependent cell lines may be described as follows. Hydrolyzed plant protein isolates can be used as an amino acid source in the culture medium according to the present invention. Recombinant protein production can be used in the preparation of culture medium components.

[0134] The culture medium according to the present invention may contain macronutrients and micronutrients, other components that modulate the properties of the basic culture medium (osmotic pressure and micronutrient availability), and signaling components. The components can be dissolved, for example, in purified water, or in water containing inorganic salts, such as phosphate-buffered saline (PBS), or in water or PBS containing a total of 1% bovine serum albumin (BSA), such as 1% BSA.

[0135] The signaling compounds may vary depending on the specific cell type used for culture in the bioreactor. Examples of these cells may include fibroblasts, myoblasts, adipocytes and their precursors, or combinations thereof.

[0136] Signaling compounds may or may not induce specific changes in cell fate. Examples of these changes may include stimulation of proliferation and / or differentiation. Signaling compounds may be used in a specific order during a specific period. Examples of these may include the use of a signaling compound for growth stimulation followed by its use in a culture medium replaced with a signaling compound for differentiation induction. The precise order of administration of signaling compounds may or may not be correlated or cross-linked with other tools that affect cell fate during culture.

[0137] Examples of signaling compounds for various cell types intended to stimulate proliferation include, for example, the following signaling proteins: FGF family ligands, insulin, insulin-like growth factor 1 (IGF-1), TGF family ligands, or transferrin, or at least one of any other suitable signaling compounds.

[0138] Examples of signaling compounds for various cell types intended for myogenic differentiation include FGF, insulin, TGF, transferrin, IGF, epidermal growth factor (EGF), bone morphogenetic protein (BMP), interleukin-6 (IL-6) or IL-13, or at least one of any other suitable signaling compounds.

[0139] The culture medium according to the present invention may contain amino acids (AA) or a source thereof in combination with at least one compound selected from the group including sugars, fatty acids, vitamins and organic micronutrients, mineral compounds such as inorganic salts, supplements such as iron supplements, organic amines, signaling compounds such as growth factors or signaling proteins or oligonucleotides, shear protectants, additional compounds or compounds for manipulation, or any other suitable compounds, or combinations thereof. The medium may also contain other compounds such as phospholipids or nucleic acids. The amino acids may be supplied, for example, from protein hydrolysates.

[0140] Amino acids and their derivatives that can be supplied to the culture medium include, for example, glycine, L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cystine, L-glutamic acid, L-glutamine, L-histidine, L-hydroxyproline, L-ornithine, L-citrulline, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-pyroglutamic acid, L-phosphoserine, L-tryptophan, L-tyrosine, or L-valine. For the preparation of the culture medium, a given amino acid may be added in its pure form, as part of a complex mixture of compounds (e.g., hydrolysate), or as an amino acid hydrate or salt (e.g., hydrochloride or sodium salt).

[0141] The culture medium according to the present invention may contain protein hydrolysates as the main source of amino acids. Protein hydrolysates may function as a source of all important amino acids in the culture medium according to the present invention for the purpose of cell culture, or some amino acids, such as L-methionine, which is found in very low concentrations in the most scalable protein source, may be supplied separately to the medium. Other different individual amino acids may be supplied separately from sources other than protein hydrolysates.

[0142] In one aspect of the present invention, the culture medium may contain at least one of the amino acids listed in Table 4. Table 4 also discloses, but is not limited to, possible exemplary concentrations of at least one amino acid that may be used in the culture medium according to the present invention. [Table 4]

[0143] The culture medium may contain at least one of the following organic micronutrient compounds: spermine, spermidine, putrescine, thymidine, L-ornithine, ethanolamine, myo-inositol, choline, and / or any other suitable organic micronutrient compounds.

[0144] Signaling compounds, such as growth factors, may be used in the culture medium according to the present invention. For example, at least one of transferrin, insulin, FGF (e.g., FGF-1, FGF-2), TGF (e.g., TGFβ1), IGF, or any other suitable compound may be used as a signaling compound.

[0145] In one aspect of the present invention, the content of signaling compounds, such as growth factors, such as FGF, TGFβ1, insulin, or transferrin, or other signaling compounds, may be reduced. The concentration of TGFβ1 may be in the range of 0 to 0.002 mg / L. The concentration of transferrin in the culture medium of the present invention may be in the range of 0 to 10 mg / L, or in the range of 0.1 to 8 mg / L, or in the range of 0.5 to 5 mg / L. In one aspect of the present invention, the amount of reduced transferrin may be in the range of 0 to 0.01 mg / L.

[0146] The insulin concentration in the culture medium may be in the range of 0 to 2 g / L, or in the range of 0.1 mg / L to 1 g / L, or in the range of 0.5 mg to 500 mg / L. In one embodiment of the present invention, the amount of insulin reduced may be in the range of 0 to 0.1 mg / L.

[0147] The concentration of FGF-2 in the culture medium may be in the range of 0 to 1 mg / L, or in the range of 0.1 to 0.8 mg / L, or in the range of 0.2 to 0.5 mg / L. In one embodiment of the present invention, the amount of FGF-2 reduced may be in the range of 0 to 0.01 mg / L.

[0148] The concentration of TGFβ1 in the culture medium may be in the range of 0 to 0.2 mg / L, or in the range of 0.01 to 0.15 mg / L, or in the range of 0.05 to 0.1 mg / L. In one embodiment of the present invention, the reduced amount of TGFβ1 may be in the range of 0 to 0.001 mg / L.

[0149] In one aspect of the present invention, the culture medium may not contain any signaling compounds, such as growth factors. The culture medium according to the present invention may be serum-free and / or protein-free.

[0150] The culture medium may contain an antifoaming agent, such as a silicone-based antifoaming agent, polyethylene glycol (PEG), polyvinyl alcohol (PVA), polydimethylsiloxane, polysorbate 80, or vegetable oil, or any other suitable antifoaming agent, or a combination thereof. The concentration of the antifoaming agent in the culture medium may be in the range of 0.001% to 5% by weight, or in the range of 0.01% to 1%, or in the range of 0.1% to 0.5%.

[0151] In one aspect of the present invention, the content of the culture medium components may be within the range shown in Table 5. [Table 5] TIFF2026516676000008.tif165170

[0152] In another aspect of the present invention, the culture medium may contain signal transduction molecules or nucleic acids. Nucleic acid-nucleotide

[0153] In one aspect of the present invention, oligonucleotides can be used as components of a culture medium for cell culture. Oligonucleotides may have single or double strands of nucleic acid containing 10 to 70 nucleotides, 10 to 120, or 1 to 1,000 nucleotides.

[0154] In one aspect of the present invention, oligonucleotides may be added to the culture medium at molar concentrations in the range of 5 nM / L to 100 nM / L, 5 nM / L to 500 nM / L, or 50 nM / L to 50 mM / L, or the concentration may fluctuate during culture such that a peak of lower concentration follows a peak of higher concentration. The peak of higher concentration may occur at 1 to 10 hours or 10 to 72 hours of culture.

[0155] In one aspect of the present invention, the oligonucleotide may be one of the components of a cell type-specific signaling compound, or it may be added to the culture medium independently of other components.

[0156] Examples of oligonucleotides that function as AONs include oligonucleotides that target the mRNA of target genes. Examples of these target genes may include ferroportin, myostatin, p53, and miRNA140.

[0157] Examples of oligonucleotides (aptamers) that act as ligands for appropriate proteins include oligonucleotides that can bind to target proteins such as FGF-2 receptors, TGF-β receptors, TrF receptors, and insulin receptors.

[0158] As additional compounds, at least one of hypoxanthine, putrescine, pyruvate, thymidine, ethanolamine, salts or derivatives thereof, such as sodium hypoxanthine or putrescine dihydrochloride, or any other suitable additional compound may be used.

[0159] Hypoxanthine, such as hypoxanthine sodium, can be used in the culture medium according to the present invention at concentrations ranging from 0 to 239 mg / L, 10 to 200 mg / L, or 50 to 100 mg / L.

[0160] Putrescine, for example, putrescine dihydrochloride, can be used in the culture medium according to the present invention at concentrations ranging from 0 to 8.1 mg / L, or from 1 to 6 mg / L, or from 2 to 5 mg / L.

[0161] Pyruvate, such as sodium pyruvate, can be used in the culture medium according to the present invention at concentrations ranging from 0 mg / L to 5.5 g / L, from 100 mg / L to 3 g / L, or from 500 mg / L to 1 g / L.

[0162] Thymidine can be used in the culture medium according to the present invention at concentrations ranging from 0 to 36.5 mg / L, 5 to 25 mg / L, or 10 to 20 mg / L.

[0163] Recombinantly prepared signaling compounds can be used in the culture medium according to the present invention. The signaling compounds can be stabilized to prevent degradation, such as thermal degradation or proteolysis. They can be secreted into the culture medium or accumulated in cells or intracellular compartments. Then, in the recovery process, they may or may not be recovered, may be purified and separated, or the entire culture may be recovered. Various fractions (parts) can be separated from the entire cultured culture and recovered in the form of easily handleable pellets. These pellets may be further processed and may function as direct compounds added to the culture medium. The pellets may be dissolved, lysed, or reconstituted in a suitable solvent before being applied to the culture medium.

[0164] In one aspect of the present invention, the production of recombinant signaling compounds can be used as a culture medium component. Examples of recombinant protein production include the following expression systems: bacteria (e.g., Escherichia coli, Bacillus subtilis), brewer's yeast (budding yeast), non-conventional yeasts (e.g., Pichia pastoris, Hansenula polymorpha, Yarrowia lipolytica), filamentous fungi (e.g., Aspergillus species, Trichoderma reesei), plants (e.g., Nicotiana tabacum, Hordeum vulgare, Zea may), insect cells or mammalian cell lines (e.g., HEK293, CHO-K1), or any other suitable expression system. The production of recombinant proteins, and the subsequent cell lysis and induction of pellets or other recombinant protein-rich derivatives, can be used in various strains of Lactobacillus species, such as Streptococcus thermophilus, S. cerevisiae, P. pastoris, and Lactobacillus acidophilus, Lactobacillus plantarum, and Lactobacillus casei.

[0165] In one aspect of the present invention, a culture medium for cell culture for food preparation may have a total input of hydrolysates ranging from 8 g / L to 50 g / L, expressed as the dry protein weight introduced into the culture medium during the culture process.

[0166] The total amino acid input from hydrolysates, including short peptides or suitable bioavailable derivative forms of amino acids, may be at least 75% by weight of the total amino acid input to the culture medium.

[0167] The substrate source for hydrolysis may be selected from at least one of the following: phototrophic organisms such as land plants, green algae, red algae, brown algae, or other phototrophic eukaryotes; phototrophic prokaryotes such as cyanobacteria; or cultured heterotrophic prokaryotes or eukaryotes such as bacteria or yeast.

[0168] The substrate source for hydrolysis may be selected from at least one of the following: soybeans, peas, rice, wheat, corn, broad beans, alfalfa, hemp, chickpeas, potatoes, pumpkins, rapeseed, red lentils, spirulina, chlorella, sunflowers, duckweed, mango beans, or baker's yeast.

[0169] The substrate source for hydrolysis may be at least one of soybeans, broad beans, or peas.

[0170] In one aspect of the present invention, the culture medium may further include supplemental L-methionine, or at least one of L-cysteine, L-cystine, L-ornithine, L-tryptophan, L-histidine, and L-threonine, which are added separately from the hydrolysate.

[0171] The total amino acid input added to the culture medium separately from the hydrolysate may be in the range of 0.1 g / L to 10 g / L.

[0172] The total amino acid input added to the culture medium separately from the hydrolysates may range from 0.5% to 15% of the total input of hydrolyzed proteins to the culture medium, expressed as a percentage.

[0173] The total L-cysteine ​​content in the culture medium may be in the range of 1 to 5% by weight relative to the total hydrolyzate protein content in the culture medium.

[0174] The total content of L-ornithine in the culture medium may be in the range of 0.001 to 0.5% relative to the total content of hydrolyzed proteins in the culture medium.

[0175] The total L-methionine content in the culture medium may be in the range of 0.2 to 2% relative to the total hydrolyzate protein content in the culture medium.

[0176] The total L-tryptophan content in the culture medium may be in the range of 0.2 to 2% relative to the total hydrolyzate protein content in the culture medium.

[0177] The total L-histidine content in the culture medium may be in the range of 0.15 to 1.5% relative to the total hydrolyzate protein content in the culture medium.

[0178] The total L-threonine content in the culture medium may be in the range of 0.3 to 3% relative to the total hydrolyzed protein content in the culture medium.

[0179] In one aspect of the present invention, the culture medium may further contain at least one compound from the group consisting of vitamins, sugars, minerals, organic amines, micronutrients, iron supplementation compounds, shear protectants, and low-level organic compounds, or combinations thereof.

[0180] The vitamins may include at least one of alpha-tocopherol (vitamin E), ascorbic acid (vitamin C), vitamin B12, biotin, choline, pantothenic acid, folic acid, niacinamide, pyridoxine, riboflavin, thiamine, i-inositol, or derivatives thereof, or combinations thereof.

[0181] The sugar may include at least one of glucose, fructose, galactose, sucrose, lactose, maltose, or a combination thereof.

[0182] The organic amine may contain at least one of putrescine or ethanolamine.

[0183] Micronutrients may include at least one of spermine, spermidine, putrescine, thymidine, L-ornithine, ethanolamine, myo-inositol, or choline.

[0184] The iron supplementation compound may contain at least one compound of iron(III) or iron(II) in an oxidized state.

[0185] The shear protectant may include at least one of polyethylene glycol (PEG), Pluronic F68, Pluronic F127, methylcellulose (MC), (hydroxypropyl)methylcellulose (HPMC), hydroxypropylcellulose (HPC), carboxymethylcellulose (CMC), dextran sulfate, or a combination thereof.

[0186] In one aspect of the present invention, the total vitamin input per liter of culture medium may be in the range of 20 mg / L to 300 mg / L, excluding vitamins present in the lysate or extract. The total sugar input per liter of culture medium can range from 3 g / L to 20 g / L. The total iron input per liter of culture medium may range from 0.001 g / L to 0.05 g / L.

[0187] The total choline input may range from 20 mg / L to 500 mg / L.

[0188] The total input of niacinamide or another vitamin B3 may range from 6 mg / L to 100 mg / L.

[0189] The total input of mineral compounds introduced into the culture process may range from 1 g / L to 20 g / L.

[0190] Na + The total input may be in the range of 30 to 100 mmol / L.

[0191] Cl - The total input may be in the range of 35-110 mmol / L.

[0192] Mg 2+The total input ranges from 0.5 to 8 mmol / L.

[0193] PO4 3- The total input ranges from 0.7 to 10 mmol / L.

[0194] SO4 2- The total input is in the range of 0.3 to 3 mmol / L.

[0195] K + The total input ranges from 4 to 15 mmol / L.

[0196] The total input of shear protection agent may range from 0.1 g / L to 5 g / L.

[0197] In one aspect of the present invention, the culture medium may contain an exogenous signaling protein at a low concentration. The concentration of the exogenous signaling protein may be 0, or it may be in the range of 0 to 50 mg / L.

[0198] The cells used for culture may be non-human metazoan cells.

[0199] In one aspect of the present invention, the cells for culture may be non-human vertebrate cells.

[0200] In one aspect of the present invention, cells for culture may be selected from the following: Embryonic stem cells (ESCs) and stem cells including other cell types derived from blastocysts or other early embryos; Muscle stem cells, e.g., muscle satellite cells, mesenchymal stem cells; or cells whose stem characteristics have been established in vitro, e.g., induced pluripotent stem cells (iPSCs); Or cells having the following characteristics: myoblasts, myocytes, fibroblasts, myofibroblasts, fibroadipogenic progenitor cells, preadipocytes, adipocytes, epithelial cells, chondrocytes, chondrocytes, macrophages, keratinocytes, hepatocytes, or Sertoli cells.

[0201] In one aspect of the present invention, cells for culture may be scaffold-independent, meaning that cells may survive and proliferate in a suspension state without adhering to a surface, and / or cells for culture may survive and proliferate as a suspension of cell aggregates, cell aggregates, spheroids or organoids, or a combination thereof.

[0202] The culture medium according to the present invention can be used for suspension culture, which means that in the culture process, cells are cultured in a stirred tank or other suitable type of bioreactor in which the majority of cells exist as a suspension of single cells or cell aggregates.

[0203] In one aspect of the present invention, cells for culture can be immortalized.

[0204] In one aspect of the present invention, the pH of the culture medium may be in the range of 6.8 to 7.3.

[0205] In one aspect of the present invention, the culture medium may contain a biologically available inorganic nitrogen source, and the total input of the inorganic nitrogen source may be in the range of 1 g / L to 10 g / L.

[0206] In one aspect of the present invention, the culture medium preparation process may have the characteristics of continuous culture medium preparation, the culture medium components may be introduced separately into the culture method, and the method may include the following steps: a) A step of introducing concentrated basic medium into the culture apparatus at a specified flow rate, where the basic medium is concentrated to X times the desired concentration in the final medium, and the basic medium is introduced at a flow rate of 1 / X times the total discharge flow rate from the culture apparatus. b) A step of introducing a concentrated sugar solution into the culture apparatus, where the flow rate is controlled so that the sugar concentration in the culture medium is maintained at a desired set point. c) A step of introducing the hydrolysate and amino acid solution into the culture apparatus, where the flow rate is controlled so that the concentration of amino acids in the culture medium is maintained at a desired set point. d) Step of introducing a concentrated NaCl solution into the culture apparatus, where the flow rate is controlled so that the total osmotic pressure of the culture medium is maintained at a desired set point, for example, 310 mOsm / kg. e) A step of introducing desalinated water into the culture device, in which the flow rate is adjusted to be equal to the difference between the total output flow rate from the culture device and the sum of all other input flow rates, thereby maintaining a constant volume of liquid in the bioreactor.

[0207] In one aspect of the present invention, the culture medium preparation process may include pH adjustment by changing the partial pressure of CO2.

[0208] In one aspect of the present invention, the process of preparing a culture medium containing a protein hydrolysate may include a hydrolysis step of a protein substrate source selected from at least one of phototrophic organisms such as land plants, green algae, red algae, brown algae or other phototrophic eukaryotes, phototrophic prokaryotes such as cyanobacteria, or cultured heterotrophic prokaryotes or eukaryotes such as bacteria or yeast, or the protein substrate source for hydrolysis may be selected from at least one of soybeans, peas, rice, wheat, corn, broad beans, alfalfa, hemp, chickpeas, potatoes, pumpkins, rapeseed, red lentils, spirulina, chlorella, sunflowers, water lentils, mango beans or yeast.

[0209] The protein concentration in the reaction mixture can range from 30 to 130 g / L.

[0210] In one aspect of the present invention, the protein substrate for hydrolysis may be subjected to an initial heat pretreatment to improve its solubility and sensitivity to hydrolysis, the temperature of which may be in the range of 80 to 95°C for 15 to 60 minutes.

[0211] In one aspect of the present invention, the culture medium preparation process may include the preparation of a protein hydrolysate by enzymatic hydrolysis, wherein the enzymes used for hydrolysis may include at least one endoprotease and at least one exoprotease.

[0212] In one aspect of the present invention, the process of preparing the culture medium may include hydrolysis using at least one of the following types of enzymes: serine proteases (e.g., subtilisin-like proteases), cysteine ​​proteases, metalloproteases, glutamate proteases, or aspartate proteases.

[0213] The culture medium preparation process may involve hydrolysis using at least one enzyme from among alcalase, flavorzyme, protamex, Novo-Pro D, Thermoase PC10FNA, protease AN Amano 100SD, protease A Amano 2SD, protease M Amano SD, protease P Amano 6SD, ProteAX, peptidase R, alkaline protease, colorase 7089, colorase 2TSN, colorase 8000, maxipro TNP, maxipro FPC, papain, or bromelain.

[0214] The enzyme concentration, expressed as the ratio of the enzyme concentration to the substrate protein concentration in the reaction mixture, can range from 0.05 to 5%.

[0215] The enzymatic hydrolysis process can take place at temperatures ranging from 30 to 65°C. The pH can be in the range of 5 to 9, and the duration can be in the range of 10 to 26 hours.

[0216] Enzymes can be immobilized on microcarriers.

[0217] In one aspect of the present invention, in the culture medium preparation process, the amount of source protein converted to free amino acids may be in the range of 35 to 65% when expressed as the mass concentration of amino acids relative to the mass concentration of protein.

[0218] The degree of hydrolysis, defined as the percentage of the total peptide bonds present in the substrate at the start of the hydrolysis reaction that have been hydrolyzed, can range from 20% to 50%.

[0219] In one aspect of the present invention, the culture medium preparation process may include monitoring hydrolysis by measuring conductivity.

[0220] In one aspect of the present invention, in the culture medium preparation process, the hydrolysate may be further treated with phytase to remove phytic acid.

[0221] In one aspect of the present invention, the hydrolysate may be heat-treated at the end of the hydrolysis process to inactivate the enzymes and kill the microorganisms.

[0222] The heat treatment temperature for the hydrolysate may be in the range of 85 to 100°C for a time range of 20 to 120 minutes, or in the range of 100 to 155°C for a time range of 3 to 300 seconds.

[0223] The cell culture process of the present invention may use the culture medium described above.

[0224] In one aspect of the present invention, the cells used in the cell culture process may be non-human metazoan cells.

[0225] In one aspect of the present invention, cells used in the cell culture process may exhibit at least one of the following characteristics: a) Immortality (no Hayflick limit), b) Ability to survive and reproduce in the absence of attachment to a solid surface (foothold-independent), c) Glutamine synthesis ability, d) Asparagine synthesis ability, e) Proline synthesis ability, f) Survival and proliferation ability in the absence of growth factors, or g) Iron uptake ability in the absence of transferrin.

[0226] The culture medium described above can be used for cell culture for the purpose of preparing food. [Examples]

[0227] Example 1 - Enzymatic hydrolysis by free enzymes Hydrolysis with free enzymes was performed in a hydrolysis tank by dissolving the soy protein isolate in distilled water at a concentration of 10 g / L and adding alcalase at a concentration of 0.05 g / L. The alcalase used was supplied by Novozymes. The resulting solution had a basic pH, and high activity of alcalase was achieved at 62°C. Over 2 hours with constant mixing, the pH of the solution decreased as a result of hydrolysis of peptide bonds and an increase in the number of carboxyl groups. These conditions allowed for high activity of flavorzyme added to a concentration of 0.15 g / L. The resulting mixture was then incubated at 62°C for a further 20 hours with constant mixing, after which the residual enzymes were thermally inactivated. In this procedure, 43% of the source protein was converted to free amino acids.

[0228] Table 6 summarizes the results of HPLC analysis of amino acid content using UV detection (cysteine ​​was not measured in this analysis). [Table 6] Example 2 - Enzymatic hydrolysis by immobilized enzymes

[0229] The immobilized enzyme was prepared by suspending 600 mg of NH2-functionalized porous silica microbeads in 50 ml of distilled water. 0.003% by volume of glutaraldehyde was added to further activate the silica beads. After 30 minutes, the excess glutaraldehyde was washed away with distilled water, and the silica beads were suspended to half their original volume. Alcalase supplied by Novozymes was then added with constant stirring until a final concentration of 0.1% was reached. This procedure resulted in the immobilization of 80% of the enzyme used onto the silica beads, equivalent to 4 grams of enzyme per kilogram of silica beads.

[0230] Silica beads containing immobilized alcalase were added to a mixture of 13 g / L soy protein in distilled water at a bead density of 10 g / L. Hydrolysis was carried out in a hydrolysis tank at 62°C for 2 hours with constant mixing. After removing the alcalase-containing beads by centrifugation, 40 g of silica beads immobilized with flavorzyme were added. Further hydrolysis was carried out at 62°C for 20 hours with constant mixing. After removing the flavorzyme beads by centrifugation, the resulting hydrolysate was heat-sterilized at 130°C and 2.5 atm for 20 minutes to inactivate any enzymes that may have detached from the solid support. After filtering to remove solid residue, the hydrolysate was used to prepare a culture medium. In this method, 5% of the source protein was converted to free amino acids.

[0231] Table 7 summarizes the results of HPLC (UV detection) analysis of amino acid content (cysteine ​​was not measured in this analysis). [Table 7] Example 3 - Culture medium composition

[0232] The culture medium was prepared with the compositions shown in Tables 8 and 9. [Table 8] [Table 9] Example 4 - Mixing of culture medium batches:

[0233] The culture medium components for batch mixing were prepared. Solution 1, containing vitamins and micronutrients, was prepared by dissolving the compounds shown in Table 10 in distilled water so that the final volume of the solution was 10 L. [Table 10]

[0234] Solution 2 (basic solution) was prepared by dissolving the compounds listed in Table 11 in distilled water until the final volume of the solution was 80 L.

Table 11

[0235] Hydrolysate - For example, as described in Example 1, 10 liters of soy protein hydrolysate having a protein concentration of 10 g / L as described in the chapter on hydrolysate preparation was prepared according to an appropriate hydrolysis procedure.

[0236] The medium components were mixed: 1 liter of Solution 1, 80 liters of Solution 2 and 10 liters of the hydrolysate were mixed in a 120 L mixing tank.

[0237] The pH of the solution was adjusted to 7.2 using 1 M NaOH or 1 M HCl.

[0238] The total volume of the solution was adjusted to 100 L using distilled water.

[0239] The final medium solution was filtered through a 0.1 μm candle filter. The sterilized medium was stored in a sterilized storage tank directly connected to the culture apparatus. Example 5 - Continuous mixing of the culture medium

[0240] Five types of medium component solutions were prepared (the hydrolysate was prepared according to the procedure for preparing the hydrolysate, and the other solutions were prepared by dissolving the components in distilled water at the required concentrations).

[0241] The basic medium solution was prepared with the composition shown in Table 12.

[0242]

Table 12

[0243]

Table 13

[0244] Furthermore, a soy protein hydrolysate solution was prepared with a protein concentration of 10 g / L.

[0245] A sugar solution was prepared with the composition shown in Table 14. [Table 14]

[0246] Salt solutions were prepared with the compositions shown in Table 15. [Table 15]

[0247] All component solutions were prepared in separate mixing tanks and equilibrated to pH 7.2 using a 1M solution of NaOH or a 1M solution of HCl.

[0248] The mixture was filtered using a 0.1 μm candle filter, and all components were placed into individual sterile storage tanks.

[0249] Sterile culture medium components were sequentially introduced into the culture device in volumes of 0.33, 0.1, 0.1, 0.05, and 0.05 times the daily working volume (vvd) of the culture device. Furthermore, sterile distilled water was introduced into the culture device at 0.37 (vvd). Example 6: Composition of culture medium

[0250] A culture medium for cell culture was prepared containing the following types of culture medium components: a) Signaling compounds b) Basic culture medium compounds c) Nutritional compounds.

[0251] Concentrated stock solutions of these three culture medium components were prepared and stored separately. The final culture medium was prepared by mixing them together at the final concentration per liter according to the required concentration, before culturing the cells.

[0252] An example of the culture medium composition is shown in Table 16. This culture medium composition contains a nutritional mixture of soy protein hydrolysate, fatty acids and sugars, vitamins, inorganic salts, additional compounds and growth factors. [Table 16] TIFF2026516676000020.tif54170 [Table 17] TIFF2026516676000022.tif187170

[0253] Another example of a culture medium composition is shown in Table 17. This culture medium composition contains a nutritional mixture of raw food-grade amino acids, fatty acids and sugars, D-glucose, vitamins, inorganic salts, additional compounds and growth factors. [Industrial applicability]

[0254] The culture medium according to the present invention may be suitable, for example, for cell culture in the production of cultured meat or pet food. The present invention also provides advantageous processes for preparing culture media, as well as processes for protein hydrolysis to shorter peptide chains and / or single amino acids.

Claims

1. A culture medium for cell culture for food preparation, comprising a protein hydrolysate as an amino acid source.

2. A culture medium for cell culture for the preparation of food according to claim 1, wherein the total input of hydrolysates, expressed as dry protein weight, introduced into the culture medium for cell culture for the preparation of food during the culture process is in the range of 8 g / L to 50 g / L.

3. The culture medium according to claim 1, wherein the total input of amino acids from the hydrolysate, which includes amino acids in the form of short peptides or suitable bioavailable derivatives, is at least 75% by weight of the total input of all amino acids to the culture medium.

4. The culture medium according to claim 1, wherein the substrate source for hydrolysis is selected from at least one of phototrophic organisms such as land plants, green algae, red algae, brown algae or other phototrophic eukaryotes, phototrophic prokaryotes such as cyanobacteria, or cultured heterotrophic prokaryotes or eukaryotes such as bacteria or yeast.

5. The culture medium according to claim 4, wherein the substrate source for the hydrolysis is at least one of soybeans, peas, rice, wheat, corn, broad beans, alfalfa, hemp, chickpeas, potatoes, pumpkins, rapeseed, red lentils, spirulina, chlorella, sunflowers, duckweed, mango beans, or baker's yeast.

6. The culture medium according to claim 4, wherein the substrate source for the hydrolysis is at least one of soybeans, broad beans, or peas.

7. The culture medium according to claim 1, wherein the total essential amino acid conversion efficiency is in the range of 30% to 100% when calculated using formula 1 with respect to Table 1: [Math 1] During the ceremony, H ＥＡＡ This is the highest conversion efficiency for a specific amino acid, A ＥＡＡＭ This refers to the content of a specific essential amino acid in 100g of protein in the culture medium. ΣA ＥＡＡＭ This is the total content of total essential amino acids in 100g of protein in the culture medium. A ＥＡＡＣ This refers to the content of a specific essential amino acid in 100g of cellular protein. ΣA ＥＡＡＣ This represents the total content of total essential amino acids in 100g of cellular protein. 【number】

8. The culture medium according to any one of claims 1 to 7, further comprising supplemental L-methionine, or at least one of L-cysteine, L-cystine, L-ornithine, L-tryptophan, L-histidine, and L-threonine, added separately from the hydrolysate.

9. The culture medium according to claim 8, wherein the total input of amino acids added to the culture medium separately from the hydrolysate is in the range of 0.1 g / L to 10 g / L.

10. The culture medium according to claim 8, wherein the total input of amino acids added to the culture medium separately from the hydrolysate is in the range of 0.5 to 15% when expressed as a percentage of the total input of hydrolysate proteins to the culture medium.

11. The culture medium according to claim 8, wherein the total content of L-cysteine ​​in the culture medium is in the range of 1 to 5% by weight relative to the total content of hydrolyzed proteins in the culture medium.

12. The culture medium according to claim 8, wherein the total content of L-ornithine in the culture medium is in the range of 0.001 to 0.5% relative to the total content of hydrolyzed proteins in the culture medium.

13. The culture medium according to claim 8, wherein the total content of L-methionine in the culture medium is in the range of 0.2 to 2% relative to the total content of hydrolyzed proteins in the culture medium.

14. The culture medium according to claim 8, wherein the total content of L-tryptophan in the culture medium is in the range of 0.2 to 2% relative to the total content of hydrolyzed proteins in the culture medium.

15. The culture medium according to claim 8, wherein the total content of L-histidine in the culture medium is in the range of 0.15 to 1.5% relative to the total content of hydrolyzed proteins in the culture medium.

16. The culture medium according to claim 8, wherein the total content of L-threonine in the culture medium is in the range of 0.3 to 3% relative to the total content of hydrolyzed proteins in the culture medium.

17. A culture medium according to any one of claims 1 to 16, further comprising at least one compound from the group consisting of vitamins, sugars, minerals, organic amines, micronutrients, iron supplementation compounds, shear protectants, and low-level organic compounds, or combinations thereof.

18. The culture medium according to claim 17, wherein the vitamin comprises at least one of alpha-tocopherol (vitamin E), ascorbic acid (vitamin C), vitamin B12, biotin, choline, pantothenic acid, folic acid, niacinamide, pyridoxine, riboflavin, thiamine, i-inositol, or derivatives thereof, or combinations thereof.

19. The culture medium according to claim 17, wherein the sugar comprises at least one of glucose, fructose, galactose, sucrose, lactose, maltose, or a combination thereof.

20. The culture medium according to claim 17, wherein the organic amine comprises at least one of putrescine or ethanolamine.

21. The culture medium according to claim 17, wherein the micronutrient comprises at least one of spermine, spermidine, putrescine, thymidine, L-ornithine, ethanolamine, myo-inositol, or choline.

22. The culture medium according to claim 17, wherein the iron supplementation compound comprises at least one compound of oxidized iron(III) or iron(II).

23. The culture medium according to claim 17, wherein the shear protective agent comprises at least one of polyethylene glycol (PEG), Pluronic F68, Pluronic F127, methylcellulose (MC), (hydroxypropyl)methylcellulose (HPMC), hydroxypropylcellulose (HPC), carboxymethylcellulose (CMC), dextran sulfate, or a combination thereof.

24. Excluding vitamins present in the lysate or extract, the total vitamin input per liter of culture medium is in the range of 20 mg / L to 300 mg / L. The total sugar input per liter of culture medium is in the range of 3 g / L to 20 g / L. The culture medium according to claim 17, wherein the total iron input per liter of culture medium is in the range of 0.001 g / L to 0.05 g / L.

25. The culture medium according to claim 17, wherein the total choline input is in the range of 20 mg / L to 500 mg / L.

26. The culture medium according to claim 17, wherein the total input of niacinamide or another vitamin B3 is in the range of 6 mg / L to 100 mg / L.

27. The culture medium according to claim 17, wherein the total input of mineral compounds introduced into the culture process is in the range of 1 g / L to 20 g / L.

28. Na ＋ The culture medium according to claim 17, wherein the total input is in the range of 30 mmol / L to 100 mmol / L.

29. Cl － The culture medium according to claim 17, wherein the total input is in the range of 35 mmol / L to 110 mmol / L.

30. The Mg ２＋ The culture medium according to claim 17, wherein the total input is in the range of 0.5 mmol / L to 8 mmol / L.

31. PO ４ ３－ The culture medium according to claim 17, wherein the total input of PO is in the range of 0.7 mmol / L to 10 mmol / L.

32. SO ４ ２－ The culture medium according to claim 17, wherein the total input is in the range of 0.3 mmol / L to 3 mmol / L.

33. K ＋ The culture medium according to claim 17, wherein the total input is in the range of 4 mmol / L to 15 mmol / L.

34. The culture medium according to claim 17, wherein the total input of the shear protective agent is in the range of 0.1 g / L to 5 g / L.

35. It contains a low concentration of exogenous signaling proteins, The concentration of the exogenous signaling protein is 0, Alternatively, the culture medium according to any one of claims 1 to 34, wherein the concentration of the exogenous signaling protein is 0 to 50 mg / L.

36. The culture medium according to any one of claims 1 to 35, wherein the cells to be cultured are non-human metazoan cells.

37. The culture medium according to claim 36, wherein the cells to be cultured are non-human vertebrate cells.

38. The cells that are cultured are Embryonic stem cells (ESCs) and other cell types derived from blastocysts or other early embryos; Muscle stem cells, e.g., muscle satellite cells, mesenchymal stem cells; or cells whose stem characteristics have been established in vitro, e.g., induced pluripotent stem cells (iPSCs); A culture medium according to any one of claims 1 to 37, selected from cells having the following characteristics: myoblasts, myocytes, fibroblasts, myofibroblasts, fibroadipogenic progenitor cells, preadipocytes, adipocytes, epithelial cells, chondrocytes, chondrocytes, macrophages, keratinocytes, hepatocytes, or Sertoli cells.

39. The cultured cells are anchorage-independent, meaning they can survive and proliferate in a suspension state without attaching to any surface. and / or The culture medium according to any one of claims 1 to 38, wherein the cultured cells can survive and proliferate as a suspension of cell aggregates, cell aggregates, spheroids or organoids, or a combination thereof.

40. The culture medium according to any one of claims 1 to 39, wherein the culture medium is used for suspension culture, that is, the cells are cultured in a stirred tank or other suitable type of bioreactor in which the majority of the cells exist as a suspension of single cells or cell aggregates during the culture process.

41. A culture medium according to any one of claims 1 to 40, wherein the cells to be cultured are immortalized.

42. The culture medium according to any one of claims 1 to 41, wherein the pH of the culture medium is in the range of 6.8 to 7.

3.

43. A culture medium containing a biologically available inorganic nitrogen source, wherein the total input of the inorganic nitrogen source is in the range of 1 g / L to 10 g / L.

44. A culture medium preparation process having the characteristics of continuous culture medium preparation, wherein the culture medium components are introduced separately into the culture process, and the process is, a) A step of introducing a concentrated basic medium into the culture apparatus at a specified flow rate, wherein the basic medium is concentrated by X times the desired concentration in the final medium, and the basic culture medium is introduced at a flow rate of 1 / X times the total discharge flow rate from the culture apparatus; b) A step of introducing a concentrated sugar solution into the culture apparatus, wherein the flow rate is controlled so that the concentration of the sugar in the culture medium is maintained at a desired set point; c) A step of introducing a hydrolyzate and amino acid solution into a culture apparatus, wherein the flow rate is controlled so that the concentration of amino acids in the culture medium is maintained at a desired set point. d) A step of introducing a concentrated NaCl solution into the culture apparatus, wherein the flow rate is controlled so that the total osmotic pressure of the culture medium is maintained at a desired set point, for example, 310 mOsm / kg. e) A preparation process comprising the step of introducing desalinated water into the culture apparatus, wherein the flow rate is adjusted to be equal to the difference between the total output flow rate from the culture apparatus and the sum of all other input flow rates, thereby keeping the volume of liquid in the bioreacoo constant.

45. CO ２ A process for preparing a culture medium according to claim 44, wherein the pH is adjusted by changing the partial pressure.

46. A process for preparing a culture medium containing a protein hydrolysate, wherein the process includes a hydrolysis step of a protein substrate source selected from at least one of phototrophic organisms such as land plants, green algae, red algae, brown algae or other phototrophic eukaryotes, phototrophic prokaryotes such as cyanobacteria, or cultured heterotrophic prokaryotes or eukaryotes such as bacteria or yeast. Alternatively, a process in which the source of protein substrate for hydrolysis is selected from at least one of soybeans, peas, rice, wheat, corn, broad beans, alfalfa, hemp, chickpeas, potatoes, pumpkins, rapeseed, red lentils, spirulina, chlorella, sunflowers, duckweed, mango beans, or yeast.

47. The culture medium preparation process according to claim 46, wherein the protein concentration in the reaction mixture is 30 to 130 g / L.

48. The culture medium preparation process according to claim 46, wherein the protein substrate for hydrolysis is subjected to an initial heat pretreatment at a temperature in the range of 80 to 95°C for 15 to 60 minutes in order to improve its solubility and sensitivity to hydrolysis.

49. The process for preparing a culture medium according to claim 46, wherein the protein hydrolysate is prepared by enzymatic hydrolysis, and the enzyme used in the hydrolysis is at least one endoprotease and at least one exoprotease.

50. The culture medium preparation process according to claim 46, wherein the enzyme used in the hydrolysis is at least one of serine protease, cysteine ​​protease, metalloprotease, glutamate protease, and aspartate protease.

51. A process for preparing a culture medium according to claim 46, wherein at least one of Alcalase, Flavorzyme, Protamex, NovoPro D, Thermoase PC10FNA, Protease AN Amano 100SD, Protease A Amano 2SD, Protease M Amano SD, Protease P Amano 6SD, ProteAX, Peptidase R, Alkaliprotease, Colorase 7089, Colorase 2TSN, Colorase 8000, MaxiPro TNP, MaxiPro FPC, Papain, or Bromelain is used for hydrolysis.

52. The culture medium preparation process according to claim 46, wherein the concentration of the enzyme is in the range of 0.05 to 5% when expressed as the ratio of the concentration of the enzyme to the concentration of the substrate protein in the reaction mixture.

53. The process for preparing a culture medium according to claim 46, wherein the enzymatic hydrolysis process is carried out at a temperature in the range of 30 to 65°C, a pH in the range of 5 to 9, and a time in the range of 10 to 26 hours.

54. The culture medium preparation process according to claim 46, wherein the enzyme is immobilized on a microcarrier.

55. The amount of source protein converted to free amino acids is in the range of 35-65% when expressed as the mass concentration of amino acids relative to the mass concentration of protein. The culture medium preparation process according to claim 45, wherein the degree of hydrolysis, defined as the proportion of hydrolyzed peptide bonds out of the total amount of peptide bonds present in the substrate at the start of the hydrolysis reaction, is in the range of 20% to 50%.

56. A process for preparing a culture medium according to claim 46, comprising monitoring hydrolysis by measuring conductivity.

57. The process for preparing a culture medium according to claim 46, further comprising treating the hydrolyzed product with phytase to remove phytic acid.

58. The process for preparing a culture medium according to claim 46, wherein the hydrolyzed product is heat-treated at the end to inactivate the enzyme and kill the microorganisms.

59. The process for preparing a culture medium according to claim 58, wherein the heat treatment temperature of the hydrolyzed product is in the range of 85 to 100°C for a time in the range of 20 to 120 minutes, or in the range of 100 to 155°C for a time in the range of 3 to 300 seconds.

60. A cell culture process using the culture medium described in any one of claims 1 to 59.

61. The cell culture process according to claim 60, wherein the cells are non-human metazoan cells.

62. The cell culture process according to claim 60, wherein the cells exhibit at least one of the following characteristics: a) Immortality (no Hayflick limit), b) Ability to survive and reproduce in the absence of attachment to a solid surface (foothold-independent), c) Glutamine synthesis ability, d) Asparagine synthesis ability, e) Proline synthesis ability, f) Survival and proliferation ability in the absence of growth factors, g) Iron uptake ability in the absence of transferrin.

63. Use of the culture medium according to any one of claims 1 to 62 in food production using the cell culture.

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