Culture media based on protein hydrolyzation and their preparation process

Description

1 / 67 Culture media based on protein hydrolysates and their preparation process. FIELD OF THE INVENTION

[0001] The invention relates to a culture medium based on a protein hydrolysate suitable for cell culture and a process for preparing the same. The culture media according to the invention can be used, for example, for cell culture to prepare a food product composed of cellular biomass for animal or human nutrition. BACKGROUND OF THE INVENTION

[0002] Mammalian cells are composed of a variety of chemical compounds. An important component of cellular biomass is protein, which generally represents 60% to 80% of the dry mass of cells. Proteins are long polymers of amino acids. There are 20 proteinogenic amino acids, 9 of which are dietary essential in mammals, meaning they cannot be synthesized by the organism and must be obtained from food or, in the case of cultured cells, from culture media. Additionally, some amino acids may be considered semi-essential or conditionally essential, because they can only be synthesized from a specific essential amino acid, or their synthesis tends to be very slow under certain conditions.

[0003] Spontaneous mutations specific to the cell line or deliberate modifications can cause cells to lack the ability to synthesize one or more amino acids, and therefore these amino acids need to be supplied in the culture medium (auxotrophy). Cells can, through spontaneous mutations or deliberate modification, gain or regain the ability to synthesize a particular amino acid. Even non-essential amino acids, which cells have the ability to synthesize, can be important for the culture process, since cells can also exhibit better growth or metabolic characteristics when supplied with some non-essential amino acids. Cells can also Petition 870250094777, dated 10 / 16 / 2025, page 19 / 103 2 / 67 have the ability to use other sources of nitrogen, mainly ammonia, to synthesize non-essential amino acids.

[0004] In general, amino acids primarily serve as building blocks for protein synthesis and are therefore consumed in proportions to the amino acid composition of the cellular protein. However, some amino acids may be consumed by cells at a higher rate than would correspond to their abundance in cellular proteins, as they can be used in energy metabolism, nucleic acid synthesis, and other similar processes. To provide cultured cells with adequate amino acids for protein synthesis, commonly used culture media formulations contain individual amino acids in different concentration ratios. These amino acids are generally produced by fermentation processes with microorganisms engineered to produce a specific amino acid. 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 applications and the production of therapeutic proteins, it is generally too expensive for the production of cultured meat.

[0005] Therefore, there is a need for culture media with an alternative and more economically advantageous source of amino acids. This culture medium must be suitable for cell cultivation and economically viable. BRIEF SUMMARY OF THE INVENTION

[0006] The disadvantages of solutions according to the state of the art are resolved by the present invention which provides suitable culture media for cell cultivation and processes for preparing them.

[0007] Culture media can be prepared by dissolving the medium components in water or in a suitable aqueous buffer. The complete medium can be prepared outside the cell culture device, formed, for example, by a bioreactor, and subsequently introduced into the device. Petition 870250094777, dated 10 / 16 / 2025, page 20 / 103 3 / 67 of cultivation; alternatively, constituent solutions of one or more components may be prepared outside the cultivation device and introduced separately into the cultivation device; alternatively, the individual components or mixtures of components may be introduced and dissolved directly in the cultivation device; alternatively, a combination of the above-mentioned methods may be used.

[0008] Sterilization of culture media is crucial to avoid contamination of the culture device, such as a bioreactor, by undesirable microorganisms. The culture medium can be sterilized after being introduced into the culture device; alternatively, the complete culture medium, the constituent solutions of one or more components, individual components or their mixtures can be sterilized before being introduced into the culture device; alternatively, a combination of the above-mentioned methods can be used.

[0009] The culture media according to the present invention may comprise protein hydrolysate as a source of amino acids. The protein hydrolysate may serve as a source of all amino acids in culture media according to the invention for cell culture purposes, or some amino acids may be supplied to the media separately. The advantageous process of hydrolyzing proteins into shorter peptide chains and / or single amino acids is also provided by the present invention. BRIEF DESCRIPTION OF THE DRAWING

[0010] Figure 1 describes the cultivation system according to the invention. DETAILED DESCRIPTION OF THE INVENTION

[0011] The disadvantages of solutions according to the state of the art are resolved by the present invention which provides suitable culture media for cell cultivation and processes for preparing them.

[0012] The culture media according to the invention can be used, for example, for cell culture for the purpose of using the cell biomass for animal or human nutrition. The culture media according to the invention Petition 870250094777, dated 10 / 16 / 2025, page 21 / 103 4 / 67 can be used for the production of cultivated meat.

[0013] The cell types used for culture processes in culture media according to the present invention may comprise many types of non-human metazoan cells, for example, stem cells comprising embryonic stem cells (ESCs) and other cell types derived from blastocysts or other early-stage embryos, muscle stem cells such as myosatelite cells, mesenchymal stem cells or cells derived from bone marrow, adipose tissue, subcutaneous tissue or other tissues, or cells where stem character is induced or established later, such as induced pluripotent stem cells (iPSCs).Other types of cells used may include, for example, myoblasts, myocytes, fibroblasts, fibroadipogenic progenitors, pre-adipocytes, adipocytes, epithelial cells, cartilage cells and tendon-derived cells such as chondroblasts and chondrocytes, macrophages, keratinocytes, hepatocytes, testicular cells, Sertoli cells or any other suitable cells.

[0014] The cell lines used for cultivation processes in the culture media according to the invention may include, for example, Chinese hamster ovary (CHO) cells, for example, CHO-K1 or CHO-DG44, C2C12, Madin-Darby bovine kidney cells (MDBKs), Madin-Darby canine kidney cells (MDC), UMNSAH / DF-1 or any other suitable cell lines.

[0015] The cells used for cultivation processes in the culture media according to the invention may be any suitable non-human metazoan cells. The cells for cultivation may be non-human vertebrate cells. The cells may be, for example, bovine, swine, fish (piscians), game (cervids), avian, rodent (cricetids, murines), equine or any other suitable cells. The cells for cultivation may be selected, without limitation, from at least one of the following animals: cattle (Bos taurus), chicken (Gallus domesticus), domestic pig (Sus domesticus), house cricket (Acheta domesticus), common snail (Helix Petition 870250094777, dated 10 / 16 / 2025, page 22 / 103 5 / 67 pomatia), common carp (Cyprinus carpio), horse mackerel (Equus ferus), spider crab (Cancer pagurus), Iberian green frog (Pelophylax ridibundus), common octopus (Octopus vulgaris), gilthead bream (Sparus aurata), roe deer (Capreolus capreolus), common sea urchin (Echinus esculentus), common seal (Phoca vitulina), African stag (Lucanus cervus), African elephant (Loxodonta africana), house mouse (Mus musculus), green turtle (Chelonia mydas), or any other suitable animals.

[0016] In one aspect of the invention, the cultured cells can be bovine cells. Bovine cells can be selected from the stem cell group, which includes embryonic stem cells and other cell types derived from blastocysts or other early-stage embryos, muscle stem cells such as myosatelite cells, mesenchymal stem cells or stem cells derived from bone marrow, adipose tissue, subcutaneous tissue or other tissues, or cells whose stem character is induced or established subsequently, such as induced pluripotent stem cells.Other types of bovine cells used may include bovine myoblasts, myocytes, fibroblasts, fibroadipogenic progenitors, preadipocytes, adipocytes, epithelial cells, cartilage and tendon-derived cells such as chondroblasts and chondrocytes, macrophages, keratinocytes, hepatocytes, testicular cells, Sertoli cells, mesenchymal stem cells, myosatelite cells, or a combination thereof.

[0017] In one aspect of the invention, the cells used for cultivation processes in the culture media according to the invention may be in at least one form of: a single cell; cell aggregates, which may take the form of cell clusters (weakly bound aggregates), spheroids (compact homogeneous aggregates) and / or organoids (compact heterogeneous aggregates); cells connected to carriers such as microcarriers, macrocarriers or microfragments; or in any other suitable cell form. The cells used for cultivation processes in the culture media according to the invention may be immortalized. Preparation of culture media - general description Petition 870250094777, dated 10 / 16 / 2025, page 23 / 103 6 / 67

[0018] Culture media can be prepared by dissolving the individual components of the medium in water or in a suitable aqueous buffer. The components can be sterilized by a suitable sterilization method to remove fungi, bacteria, viruses, and other possible contaminants. Sterilization can occur before or after dissolving the medium component. Sterilization can be performed 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 influence. Additionally, for aqueous solutions of media components that are solid, aqueous solutions of media components that are liquid, or separate media components that are liquid, filtration can be used as a sterilization method.Advantageously, physical sterilization methods (including filtration) can be used, as they minimize the risk of contamination of the final product with residues of chemical disinfectants. Advantageously, some medium components or their solutions can be mixed before sterilization, thus reducing the number of materials that need to be sterilized separately.

[0019] For filter sterilization of a culture medium or its components, the filter material may be polyethersulfone, cellulose acetate, ceramic, or any other suitable filter material. The size of the larger pores 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 performed as outletless filtration, tangential flow filtration, alternating flow filtration, or any other suitable filtration configuration.

[0020] The medium may be stored as a complete final solution of culture medium, or as a set of at least one solid component or mixtures of solid components, or as a set of at least one concentrated solution of one or more components, or as a set of a Petition 870250094777, dated 10 / 16 / 2025, page 24 / 103 7 / 67 or more liquids, or a combination of the above.

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

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

[0023] The volume of the mixing tank may be in the range of 500 mL to 100 m3, or in the range of 1 L to 10 m3, or in the range of 2 L to 5 m3, or in the range of 500 L to 3 m3.

[0024] Storage tanks can be made of stainless steel, glass, or any other suitable material. The tank volume can be in the range of 500 mL to 100 m3, or in the range of 1 L to 5 m3, or in the range of 2 L to 3 m3, or in the range of 500 L to 1 m3.

[0025] The media components can be dosed into the mixing tank through the sterilization filter, or they can be sterilized before being placed in the mixing tank, or they can be sterilized in the mixing tank.

[0026] The mixing tank can be equipped with different types of sensors, such as a thermal sensor, pH probe, conductivity meter, or any other type of sensor suitable according to the needs of Petition 870250094777, dated 10 / 16 / 2025, p. 25 / 103 8 / 67 process.

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

[0028] The control unit can control and / or regulate all processes that occur within the cultivation system. The control unit can be operated using at least one printed circuit board (PCB) and / or microprocessor with software that has the ability to control the cultivation device, regardless of the system's extensions and scale. The control unit can be connected to at least one central data storage. The cultivation system may comprise one or more sub-control units.

[0029] The culture media according to the present invention may comprise protein hydrolysate as a source of amino acids.

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

[0031] In the batch process of medium preparation, the complete culture medium can be prepared, introduced into the culture device and subsequently inoculated with cells.

[0032] The complete culture medium can be mixed in a mixing tank, or the culture medium components can be mixed directly in the culture device, as described above.

[0033] The complete medium can be sterilized in the mixing tank, in Petition 870250094777, dated 10 / 16 / 2025, page 26 / 103 9 / 67 culture device or during transfer from the mixing tank to the culture device, if they are separate. Alternatively, the individual culture medium components or the mixture of these components can be sterilized before being added to the mixing tank or during transfer to the mixing tank, producing a sterile complete culture medium. Preparation of media - continuous process

[0034] In the continuous process of preparing the culture medium, the preparation of the medium can occur simultaneously with the cell culture.

[0035] Individual components of the culture medium or mixture of components can be introduced into the culture device during cell culture. The components can be sterilized before or during transfer to the culture device. The components can be introduced into the culture device in any form, for example, they can be introduced as aqueous solutions, allowing convenient sterilization of the filter and rapid mixing of all culture medium components in the culture device.

[0036] The rate of addition of culture medium components may be fixed or the rate of addition may be adjusted to ensure ideal conditions for cell culture in the culture device. These ideal conditions may include, but are not limited to, the pH, osmolality, shear protector concentration, sugar concentration, and amino acid concentration ranges, as specified in this document in the section entitled Culture Medium Composition.

[0037] The conditions of the culture device can be determined by: a pH probe, a conductor, an osmometer, a glucose probe, a refractometer, a UV-Vis spectrometer, a Raman spectrometer, or any other suitable measurement method. In appropriate cases, the concentration of specific compounds in the culture device can also be approximated by a mass equilibrium equation. Petition 870250094777, dated 10 / 16 / 2025, page 27 / 103 10 / 67

[0038] To achieve the desired conditions in the culture device, the flow rates of the medium components can be adjusted using a suitable control mechanism, for example, a PID control circuit.

[0039] In one aspect of the invention, the continuous mixing process of the medium may comprise the following steps: - Introduction of a concentrated basal medium into the culture device at a defined flow rate; for example, if the basal medium is X times more concentrated than the desired concentration in the final medium, the basal medium is introduced at a flow rate 1 / X times the total outflow rate of the culture device. - Introduction of a concentrated sugar solution into the culture device where the flow rate is controlled so that the sugar concentration in the culture medium is maintained at a desirable set point, for example, 1 g / L. - Introduction of a hydrolysate and amino acid solution into the culture device, where the flow rate is controlled so that the amino acid concentration in the medium is maintained at a desirable set point. - Introduction of a concentrated NaCl solution into the culture device where the flow rate is controlled so that the total osmolality of the medium is maintained at a desirable set point, for example, 310 mOsm / kg; - Introduction of demineralized water into the culture device where the flow rate is adjusted to be equal to the difference between the total outflow rate of the culture device and the sum of all other inflow rates, thus maintaining a constant volume of liquid in the bioreactor. Hydrolysate preparation - general description

[0040] The protein source for hydrolysis can be selected from an industrially scalable protein source. Industrially scalable protein sources include phototrophic organisms such as land plants, green algae, red algae, brown algae or other phototrophic eukaryotes, phototrophic prokaryotes such as cyanobacteria, or prokaryotes Petition 870250094777, dated 10 / 16 / 2025, page 28 / 103 11 / 67 Cultured heterotrophs or eukaryotes, such as bacteria or yeast. The organism used as a protein source may have the ability to synthesize all amino acids from inorganic nitrogen sources, such as ammonia ions, nitrate ions, or molecular nitrogen. Hydrolysis can be carried out, for example, in a hydrolysis tank or any other suitable device.

[0041] Hydrolysis can be performed on an isolate or concentrate of protein from the source organism, or on the entire biomass of the source organism. The source organism can be pre-treated mechanically or chemically to enhance the speed and efficiency of the hydrolysis process. Saccharides, fats, or other compounds can be removed from the biomass of the source organism to facilitate processing. Examples of suitable industrially scalable protein sources may include soybeans, peas, rice, wheat, corn, broad beans, alfalfa, hemp, chickpeas, potatoes, pumpkin, rapeseed, red lentils, Spirulina, Chlorella, sunflower, duckweed, mung beans, flax, or baker's yeast. The present invention is not limited to the exemplary protein sources listed.

[0042] Protein hydrolysates or multiple hydrolysates from the same source organism or from different source organisms can serve as a source of all important amino acids in culture media for cell culture purposes, or some amino acids can be supplied separately, for example, methionine, which is found in very low concentrations in most scalable protein sources. Other different individual amino acids can be supplied separately from a source other than a protein hydrolysate. Typically, methionine and some non-essential amino acids, such as asparagine or glutamic acid, are commercially available wholesale (food grade) at prices compatible with use in industrial-scale cell culture. However, most of the essential amino acid content of the medium, according to the invention, can be derived from hydrolysates.This approach may be more economically viable on a large scale than using individual free amino acids, as is currently the case. Petition 870250094777, dated 10 / 16 / 2025, page 29 / 103 12 / 67 is commonly done in the biopharmaceutical industry or in 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, which includes acid, basic, or enzymatic hydrolysis, autolysis, or lysis by fermentation with a suitable microorganism that has the ability to break down protein. Protein hydrolysate according to the present disclosure may be, for example, enzymatic hydrolysates of plant proteins, various types of yeast extracts or lysates (such as whole yeast autolysate), or acid hydrolysate of algae.

[0044] Protein hydrolysis methods can include acid hydrolysis, base hydrolysis, enzymatic hydrolysis, or autolysis. Acid hydrolysis subjects the protein source to a very low pH, usually at a high temperature. The reaction duration can be hours or days. Unfortunately, acid hydrolysis results in significant degradation of several amino acids, primarily tryptophan, which would have to be obtained separately at significant cost. Significant degradation of some amino acids also occurs during base hydrolysis, which subjects the protein source to a very high pH, ​​usually at a high temperature. Additionally, the acid or base used for hydrolysis would have to be removed from the hydrolysate before it could be used to culture cells, presenting further complications. For example, when acid hydrolysis is performed using hydrochloric acid, the acid can be removed by neutralization or evaporation.However, both processes are economically unfavorable because: i) the neutralization process results in unfavorably high concentrations of salts, which must also be removed, and ii) evaporation consumes a lot of energy and the resulting HCl vapors pose a health and environmental risk that would otherwise be necessary. Petition 870250094777, dated 10 / 16 / 2025, page 30 / 103 13 / 67 to be solved. The autolysis process depends on the activity of the source organism's endogenous enzymes to break down the protein source, and this process is generally not very efficient and usually does not result in sufficient hydrolysis of the source protein. Additionally, proteins can be broken down by fermentation with organisms such as Bacillus licheniformis or Aspergillus oryzae, which produce a large amount of proteolytic enzymes. However, with this approach, some of the amino acids from the source protein may be consumed by the organism that was used to break down the protein during the fermentation process. Additionally, metabolic waste and other compounds from the fermenting organism can contaminate the resulting lysate and adversely affect its properties in relation to mammalian cell culture.

[0045] The hydrolysate according to the invention can be obtained by enzymatic hydrolysis of a suitable protein source. An industrially scalable protein source is advantageous. In one aspect of the 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 mammalian cell culture purposes, with the exception of methionine, which is present in a relatively low concentration. However, methionine can be added to the media separately, as mentioned above.

[0046] The protein substrate for solvent hydrolysis may be subjected to an initial heat pretreatment to enhance solubility and susceptibility to hydrolysis. The temperature during the heat pretreatment may be in the range of 75 to 95 °C, or in the range of 80 to 92.5 °C, or in the range of 85 to 90 °C for a time interval of 5 to 120 minutes, or 15 to 60 minutes, or for a time interval of 30 to 45 minutes.

[0047] The enzymatic hydrolysis method can use a so-called protease, an enzyme that catalyzes the breaking of peptide bonds to achieve protein hydrolysis under much milder conditions than acid or base hydrolysis. Petition 870250094777, dated 10 / 16 / 2025, p. 31 / 103 14 / 67 therefore, preserving all the amino acids of the original protein.

[0048] In one aspect of the invention, the enzyme used for hydrolysis can be immobilized on a solid support. This approach sterically prevents the enzyme molecules from breaking down and allows the enzyme to be separated from the reaction mixture after the reaction and reused. The solid support can be in the form of solid carriers suspended in the reaction mixture, or a solid structure with a large surface area, such as a sponge or a fibrous structure, through which the reaction mixture is perfused. The enzyme can also be added in soluble (free) form. After the hydrolysis is complete, the resulting hydrolysate is separated from the solid support with the immobilized enzyme 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 suspended carriers). The reaction vessel can be formed, for example, by a hydrolysis tank.The filtration step can also remove any solid residues from the source protein, such as cell wall debris. Free enzymes can be removed from the hydrolysate by ultrafiltration or deactivated with elevated temperature once hydrolysis is complete. Ultrafiltration of the hydrolysate can 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 elevated temperature used to deactivate the enzyme can also sterilize the resulting hydrolysate.

[0049] The hydrolysate can be heat-treated at the end of hydrolysis to deactivate enzymes and kill microorganisms. In one aspect of the invention, this treatment can be carried out with lower temperature settings in the range of 80 to 120 °C, or 85 to 100 °C, or in the range of 90 to 95 °C with longer times in the range of 15 to 180 minutes, or 20 to 120 minutes, or in the range of 25 to 60 minutes. In another aspect of the invention, this treatment can be carried out with higher temperatures in the range of 80 to 160 °C, or 100 to 155 °C, or in the range of 110 to 150 °C with shorter times in the range of 1 to 600 seconds, or 3 to 300 seconds. Petition 870250094777, dated 10 / 16 / 2025, page 32 / 103 15 / 67 seconds, or in the range of 5 to 60 seconds. The method with the lower temperature setting can be performed in the reactor configuration, and both methods can be performed in a flash pasteurizer or other suitable continuous flow heating device.

[0050] If the enzyme is removed by ultrafiltration, it may retain at least partial catalytic activity and therefore can be recycled to another hydrolysis cycle. Ultrafiltration or thermal deactivation can also be used to remove active enzyme molecules from hydrolysates prepared by immobilized enzymes, in the event that part of the enzyme detaches from the solid support and dissolves in the reaction mixture.

[0051] The solid support may be formed, for example, from silica, epoxy resin, cellulose, chitosan, glass wool, alginate, or other suitable materials. The solid support may be in the form of porous or solid microspheres, sponge, fibers, or other suitable configuration. The solid support may have a large surface area relative to its volume to allow the binding of a large number of enzymes. For example, porous silica microspheres or any other suitable material with a diameter in the range of 1 to 10000 micrometers, or in the range of 10 to 1000 micrometers, or in the range of 20 to 500 micrometers, may be used as a solid support for enzyme immobilization. Immobilization may be achieved, for example, by functionalizing the surface of the silica microsphere with amino groups and by using a crosslinking agent, such as glutaraldehyde, to bind the enzyme to the solid support.Other functional groups, such as aldehyde or epoxy groups, can also be used for enzyme immobilization. The amino groups in this aspect of the invention are covalently linked to glutaraldehyde, after which the excess glutaraldehyde is removed and the enzyme is added. The amino groups on the surface of the enzyme then bind to the remaining free aldehyde groups of the glutaraldehyde molecules on the surface of the silica microsphere. Immobilization can be carried out in water or in a suitable aqueous buffer. Due to the porous nature and large surface area of ​​silica microspheres, a quantity... Petition 870250094777, dated 10 / 16 / 2025, p. 33 / 103 16 / 67 a relatively high amount of enzymes can be immobilized relative to the weight of the solid support.

[0052] The enzymes according to the invention may be, for example, Alcalase (Bacillus licheniformis protease), Flavourzyme (Aspergillus oryzae protease), 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, Corolase 7089, Corolase 2TSN, Corolase 8000, Maxipro TNP, Maxipro FPC, Papain, Bromelain or any other suitable proteolytic enzyme, or a combination thereof.

[0053] Water or a suitable aqueous buffer can be used to dissolve the protein source for hydrolysis. Some proteins may require a buffer to adjust the pH to a level where they have better solubility. The pH can be in the range of 2 to 12, or in the range of 5 to 10, or in the range of 6 to 8.5. A very dilute buffer, or no buffer, can be used so that the resulting hydrolysate can be added to the final culture medium at high concentrations while minimizing its impact on the osmolarity of the medium.

[0054] The buffer may comprise, for example, 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 protein concentration in the reaction mixture for hydrolysis can be in the range of 1 to 150 grams per liter, or in the range of 20 to 100 grams per liter, or in the range of 30 to 80 grams per liter of the reaction mixture.

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

[0057] In one aspect of the invention, the phosphate buffer concentration of Petition 870250094777, dated 10 / 16 / 2025, page 34 / 103 17 / 67 potassium in the range of 1 to 100 mM, or in the range of 10 to 40 mM, or in the range of 15 to 35 mM can be used for pH adjustment to dissolve soy protein at a concentration in the range of 1 to 150 grams per liter, or in the range of 20 to 100 grams per liter, or in the range of 30 to 80 grams per liter. In another aspect of the invention, soy protein is dissolved in distilled water at a concentration in the range of 1 to 150 grams per liter, or in the range of 20 to 100 grams per liter, or in the range of 30 to 80 grams per liter.

[0058] Other concentrations of the source protein can be used, however very high concentrations of source protein lead to incomplete dissolution of the protein and the formation of a highly viscous colloidal solution, presenting problems for hydrolysis and subsequent processing, while low protein concentrations can limit the rate of the hydrolysis reaction. To ensure the best dissolution of the proteins in the reaction mixture, heat treatment can be used. Temperatures below boiling can be used for long periods of time to significantly increase the content of dissolved proteins and deactivate potential protease inhibitors and other antinutritional compounds.

[0059] In one aspect of the invention, the source protein can be added at a concentration higher than the maximum soluble concentration. This additional protein can be dissolved after the decrease in protein concentration in the reaction mixture due to its hydrolysis by the enzyme. This results in a high concentration of available substrate throughout the process, potentially enhancing the efficiency of the hydrolysis. Multiple cycles of substrate addition can be performed in the same reaction mixture. In one aspect of the invention, a suitable base or buffer can be added to neutralize the change and maintain the enzyme at its ideal pH, or a pH stat can be used.

[0060] The key parameter by which the conversion of substrate protein into bioavailable products for animal cells can be evaluated is the degree of hydrolysis, defined as the percentage of peptide bonds in the protein of Petition 870250094777, dated 10 / 16 / 2025, page 35 / 103 18 / 67 origin that are hydrolyzed during the reaction. A higher degree of hydrolysis corresponds to a greater percentage of the origin protein converted into amino acids or short free peptides, which are usable by mammalian cells as nutrition. Mammalian cells generally do not have the capacity to absorb and digest longer proteins and peptides. Peptides with more than four amino acids, that is, heavier than approximately 500 Daltons, have very low absorption by mammalian cells. In various aspects of the invention, the amount of origin protein in the range of 20% to 100%, in the range of 30% to 70%, or in the range of 35% to 65%, or in the range of 40% to 60%, can be converted into free amino acids, expressed as a mass concentration of amino acids in a mass concentration of protein.The degree of hydrolysis (DH), that is, the percentage of peptide bonds that undergo hydrolysis out of the total number of peptide bonds present in the substrate at the beginning of the reaction, can be in the range of 10% to 60%, in the range of 20% to 50%, or in the range of 25% to 40%.

[0061] The enzymes used for hydrolysis can fall into two general categories: exoproteases and endoproteases. Exoproteases cleave protein or peptide chains at the ends, while endoproteases can cleave peptide bonds in the middle of the chain. In one aspect of the invention, a combination of endoproteases and exoproteases can be used, since endoproteases can create more free ends of peptide chains, increasing the efficiency of exoproteases, and exoproteases are more efficient at hydrolyzing the protein into single amino acids. In one aspect of the invention, endoproteases and exoproteases can be used sequentially in this order to maximize hydrolysis efficiency.

[0062] In one aspect of the invention, additional enzymes can be added to the reaction mixture after the start of hydrolysis. This can be done with the same enzyme, mainly to counteract the gradual decrease in its enzymatic activity due to the degradation of the enzyme molecule. In another aspect of the invention, enzymes with a higher ideal pH can be added to the Petition 870250094777, dated 10 / 16 / 2025, page 36 / 103 19 / 67 The start of hydrolysis, when the pH is higher, and enzymes with a lower optimum pH can be added later, when the pH is lower, thus maximizing the efficiency of the respective enzymes. The pH tends to decrease naturally during hydrolysis due to the increase in the number of carboxylic groups.

[0063] In another aspect of the invention, additional substrate can be added to the reaction mixture after the start of hydrolysis. The advantages of this approach may be, for example, easier dispersion and dissolution of the additional substrate when the previous amount of substrate is at least partially hydrolyzed.

[0064] The addition of enzyme or substrate after the start of the hydrolysis process can be carried out in a fed-batch mode (additional reagents are added to the reaction mixture and subsequently the entire reaction batch is harvested) or in continuous reaction mode (the addition and harvesting of the reaction mixture are done continuously).

[0065] Regardless of whether immobilized or free enzyme is used, sufficient mixing of the reaction mixture is important to achieve high efficiency. In the case of immobilized enzymes, this applies to both the enzyme immobilization steps and protein hydrolysis. In one aspect of the invention, in the case of immobilized enzymes, mixing methods should be used that minimize mechanical damage to the solid carriers. These may include roller, agitation, or low-shear mixing impellers, such as hydrofoil or elephant ear type impellers. In the case of enzymes immobilized on a large solid support, sufficient perfusion of the support with the reaction mixture must be ensured.

[0066] The mixing of the protein source, for example, protein isolate, with water or a suitable aqueous buffer, dissolving the protein source and the hydrolysis process itself can be carried out in a reaction vessel suitable for laboratory or industrial scale. The reaction vessel can be formed, for example, by a hydrolysis tank. Petition 870250094777, dated 10 / 16 / 2025, page 37 / 103 20 / 67

[0067] The reaction vessel for hydrolysis may comprise, for example, 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] The mixture may be supplied by the appropriate agitation unit which may comprise, for example, a paddle impeller. An elephant ear type impeller may be used. The outer diameter of the agitator or impeller may be in the range of 1 / 10 to 9 / 10 of the reactor's inner diameter, or in the range of 3 / 10 to 8 / 10 of the reactor's inner diameter, or in the range of 4 / 10 to 7 / 10 of the reactor's inner diameter, for example, 2 / 3 of the reactor's inner diameter. The agitator or impeller may be located in the center of the reaction vessel or off-center in the reaction vessel.

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

[0070] The storage tank can be made, for example, of stainless steel or glass. The volume of the storage tank can be in the range of 100 mL to 5 m3, in the range of 2 L to 3 m3 or in the range of 500 L to 1 m3

[0071] The reaction vessel, for example, a hydrolysis tank, can be equipped with different types of sensors, such as a thermal sensor, pH probe, conductor, or any other type of sensor appropriate according to the needs of the hydrolysis process. The pH can be monitored throughout the procedure by a pH electrode. The temperature in the reaction vessel can be regulated, for example, with a reactor thermal jacket, which can be equipped with a heating coil and / or heating / cooling medium.

[0072] For precise monitoring of the degree of hydrolysis, it may be Petition 870250094777, dated 10 / 16 / 2025, page 38 / 103 21 / 67 a sampling system was used. The degree of hydrolysis can be monitored by titration and / or by absorbance measurement, for example, at a wavelength in the range of 190 to 350 nm, or 190 to 230 nm.

[0073] After the hydrolysis process, another treatment can be used to reduce the phytic acid content. Phytic acid is an important compound in plant metabolism; its salt form – phytin – is the main phosphorus storage compound in plants. On the other hand, phytic acid represents one of the antinutrient compounds of legumes, which can significantly influence downstream processes, as well as cell proliferation and viability. An enzymatic treatment or any other method can be used to reduce the phytic acid content. If enzymatic treatment is used, the selection of the appropriate phytase enzyme is crucial, as well as the appropriate conditions regarding pH and temperature. The process may involve adding the phytase enzyme in correlation with the enzyme / substrate ratio, where substrate means the protein source used.The enzyme / substrate ratio can be in the range of 1.1 · 10⁻¹¹% to 1%, or in the range of 1.1 · 10⁻¹⁰% to 0.001%, or in the range of 1.1 · 10⁻⁹% to 0.0001%. The temperature can be in the range of 20 °C to 80 °C, in the range of 30 °C to 70 °C, or in the range of 40 °C to 60 °C. After enzymatic treatment for a period of time in the range of 20 minutes to 4 hours, or in the range of 30 minutes to 3 hours, or in the range of 1 to 2 hours, heat treatment for enzymatic deactivation can be applied. Deactivation can be carried out, for example, for 30 minutes at 90 °C or 15 minutes at 95 °C.

[0074] For filtration purposes, for example, to remove impurities, to separate enzyme immobilized in a carrier from the reaction mixture, or to separate larger peptides from the hydrolysate, an appropriate filtration unit equipped with filtration materials may be used. The filtration material may be, for example, fabrics, ceramics, glass, filtration membranes, or other suitable materials. The pore size in the filtration material may be, for example, but not limited to, 500 μm - 10 μm for filtration, 10 μm to Petition 870250094777, dated 10 / 16 / 2025, page 39 / 103 22 / 67 0.1 μm for microfiltration, 0.1 μm to 1 nm for ultrafiltration, and 1 nm to 0.1 nm for nanofiltration. Membranes characterized with a range of 60 kDa to 500 Da can be used. As a step prior to filtration, centrifugation can be used to facilitate the filtration process. Free enzyme hydrolysis

[0075] In one aspect of the invention, hydrolysis by free enzymes can be carried out by dissolving the protein substrate in the reaction vessel formed, for example, by a hydrolysis tank. This protein substrate can be, for example, whole biomass, protein concentrate, protein flour, raw protein flour, protein isolate, protein extractor of soy, pea, rice, wheat, corn, broad beans, alfalfa, hemp, chickpea, potato, pumpkin, rapeseed, red lentil, Spirulina, Chlorella, sunflower, duckweed, mung bean, flax or yeast or other suitable protein source.

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

[0077] This allows for high Flavourzyme activity, which can be added in amounts calculated according to the enzyme / protein ratio in the range of 0.01 to 10%, or in the range of 0.1 to 2%, or in the range of 0.2 to 1% enzyme / protein ratio for the reaction mixture. The resulting mixture can then be incubated for an additional period of time. Petition 870250094777, dated 10 / 16 / 2025, page 40 / 103 23 / 67 in the range of 1 hour to 48 hours, or in the range of 5 to 24 hours, or in the range of 8 to 20 hours at temperatures in the range of 30 to 80 °C, or in the range of 40 °C to 70 °C, or in the range of 45 to 60 °C, with constant mixing, after which the residual enzyme is thermally deactivated. With this procedure, 20% to 100%, 30% to 70%, or 40% to 60% of the source protein can be converted into free amino acids. Hydrolysis by immobilized enzyme

[0078] The protein hydrolysis process can be carried out in a reaction vessel formed, for example, by a hydrolysis tank. Protein hydrolysis can be carried out with immobilized enzyme in an amount in the range of 0.01 g in 10 g, or in the range of 0.25 to 1.8 g, or in the range of 0.5 to 1.5 g in 10 grams of enzyme carrier. The enzyme carrier can be made of glass, porous silica, alginate, epoxy methacrylate, chitosan, or any other suitable material, in the form of microspheres, wool, sponge, fibers, or any other suitable form. The enzyme carrier can be, for example, formed by glass microspheres, porous silica microspheres, alginate microspheres, epoxy methacrylate microspheres, glass wool, chitosan, or any other suitable enzyme carrier. Suitable enzyme carriers are described in more detail in the section entitled Hydrolysate Preparation - General Description.For example, 1 gram of enzyme immobilized in 10 grams of porous silica microspheres can be used.

[0079] Immobilized enzymes can be prepared by suspending a fixed weight of NH2-functionalized porous silica microspheres in a defined weight of distilled water. The ratio of the defined weight of NH2-functionalized porous silica microspheres to distilled water can be in the range of 1:1 to 1:10000, or in the range of 1:10 to 1:1000, or in the range of 1:20 to 1:100. The silica microspheres are activated by the addition of glutaraldehyde. The amount of glutaraldehyde added to the reaction mixture can be in the range of 0.01 to 70 mmol, or in the range of 0.05 to 40 mmol, or in the range of 0.1 to 10 mmol of glutaraldehyde per 1 g of silica microspheres. The excess glutaraldehyde is washed away and the silica microspheres are resuspended, for example, in half. Petition 870250094777, dated 10 / 16 / 2025, page 41 / 103 24 / 67 of the original volume. Alcalase is then added to a final concentration with constant stirring. This procedure can immobilize 10 to 100%, 60 to 90%, or 70 to 80% of the enzyme used in the silica microspheres. This can correspond to 10 to 100 grams, 30 to 60 grams, or 40 to 50 grams of enzyme immobilized per 1 kilogram of silica microspheres.

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

[0081] The hydrolysis temperature may be in the range of 10 to 90 °C, in the range of 25 to 80 °C, or in the range of 50 to 70 °C. In another aspect of the invention, the Alcalase microspheres may not be removed at this stage and instead may be removed at the end of the process. In another aspect of the invention, the Alcalase and Flavourzyme microspheres may have different sizes, facilitating their separation after removal from the solution. In another aspect of the invention, the Flavourzyme microspheres may be added at the beginning of the hydrolysis or at any other point during the hydrolysis.After further hydrolysis, during a period of time that may range from 1 to 24 hours, or from 6 to 20 hours, or from 10 to 18 hours, at a temperature that may range from 20 to 90 °C, or from 30 to 80 °C, or from 40 to 60 °C, with constant mixing, the Flavourzyme microspheres are removed by centrifugation and the resulting hydrolysate is thermally sterilized, which also deactivates any enzyme that may have detached from the solid support. After filtration. Petition 870250094777, dated 10 / 16 / 2025, page 42 / 103 25 / 67 to remove solid debris, the hydrolysate can be used to prepare culture media. With this method, the amount of protein from the source in the range of 20% to 100%, or in the range of 30% to 95%, or in the range of 40% to 90%, can be converted into products usable in cells, i.e., free amino acids or peptides of 500 Da or less.

[0082] Since Alcalase and Flavourzyme are quite stable in their immobilized form, they can be recycled in the hydrolysate production process according to the invention. In one aspect of the invention, silica microspheres with immobilized Alcalase can be used for 2 to 50, 5 to 40, or 10 to 30 hydrolysis cycles, maintaining about half of their original catalytic activity. In another aspect of the invention, silica microspheres with immobilized Flavourzyme can be used for 2 to 50, 5 to 40, or 10 to 30 hydrolysis cycles, maintaining sufficient catalytic activity. In general, although immobilized enzymes tend to be more stable than free enzymes, their enzymatic activity decreases with use. Therefore, in subsequent cycles, the reaction duration or the enzyme / protein ratio can be altered to maintain a consistent quality of the resulting hydrolysate. Composition of culture media

[0083] In one aspect of the invention, the culture medium composition can be defined in terms of the total input of medium components into the cultivation process. In this aspect of the invention, summary quantities of components introduced into the cultivation process at any time during its duration are provided. Additionally, in this aspect of the invention, the concentration ranges provided for the individual medium components describe the total amount of the given component introduced into the cultivation process at any time during the cultivation process relative to the volume of culture medium expended exiting the process. The expended culture medium may exit the cultivation process together with the cultured cells (harvesting) or separately from the cultured cells (perfusion). The cultivation process may additionally have the characteristics of a process Petition 870250094777, dated 10 / 16 / 2025, page 43 / 103 26 / 67 discontinuous, in which all components are introduced into the cultivation process at a single time and harvesting is carried out at a single time; a fed-batch process, in which some components may be introduced after the start of the process and harvesting is done at a single time; a continuous process, in which components may be introduced throughout the duration of cultivation and harvesting may be carried out throughout the duration of cultivation; or a combination of the described characteristics. To summarize, this aspect of the invention will be referred to herein as total input.

[0084] In another aspect of the invention, the composition of the culture medium can be described in terms of the concentration of components that are present at a given time during the cell culture process in the culture medium. In this aspect of the invention, the concentration ranges provided for the individual components of the medium describe the concentrations present in the culture medium in the culture device at any time during the culture process. For summary, this aspect of the invention will be referred to herein as the momentary composition. Full entry

[0085] The total inputs in the culture medium according to the invention may comprise an optimized essential ratio of amino acids, which may be derived from a protein hydrolysate, in combination with at least one type of compound selected from a group comprising: sugars, vitamins and organic micronutrients, mineral compounds, iron supplementation compounds, organic amines and shear protectors or a combination thereof. The medium may also contain other compounds, such as fatty acids, phospholipids or nucleic acids, for example. Media according to the invention with an optimized ratio of amino acids and other nutrients may facilitate efficient biomass production and low production of residual metabolites, such as ammonia or lactate, by the cells.

[0086] An optimized ratio of essential amino acids is such that essential amino acids can be introduced into the cultivation process in Petition 870250094777, dated 10 / 16 / 2025, page 44 / 103 27 / 67 any ratio in which the percentage of essential amino acids that can be converted into cellular proteins is between 5% and 100%, between 20% and 90%, or between 30% and 80%. The term highest possible conversion efficiency determines what percentage of the essential amino acids supplied to cells can be converted into cellular protein, assuming no loss of amino acids to catabolism, conversion to other compounds (nucleic acids, for example), or spontaneous degradation.

[0087] The highest possible conversion efficiency is determined by the essential amino acid that is most limiting for the cells. It is calculated that, for all individual essential amino acids added to the medium, in any form, at any time during the culture process, the content of that specific essential amino acid in the culture media, as a fraction of the total content of essential amino acids added in any form and at any time to the culture medium, is divided by the content of that individual amino acid in the cell protein as a fraction of the total content of essential amino acids at the lowest ratio obtained, that is, the ratio of the essential amino acid that forms the smallest percentage of the amino acids added to the medium compared to the percentage of that specific amino acid in the cell biomass, is then multiplied by 100 to obtain the highest possible conversion efficiency of the supplied essential amino acids into cell protein.All percentages in the calculation of the highest possible conversion efficiency are percentages by weight.

[0088] Amino acids in culture media may be present in the form of free amino acids or peptides. Non-essential amino acids are omitted from this calculation because they can be synthesized by cells and are therefore not limiting in terms of the highest possible conversion efficiency. An example of a possible essential amino acid content in cellular protein can be seen in Table 1 below.

[0089] The description above can be summarized by the following equation: Petition 870250094777, dated 10 / 16 / 2025, page 45 / 103 28 / 67 EAAM ^EAAM EAA EAAC ya EAAC * 100, where Heaa is the highest conversion efficiency for a given amino acid. Aeaam is the content of this specific essential amino acid in 100 g of protein in culture medium. ΣΑεααμ is the total content of all essential amino acids in 100 g of protein in culture medium. Aeaac is the content of this specific essential amino acid in 100 g of cellular protein and ZAeaac is the total content of all essential amino acids in 100 g of cellular protein.

[0090] An example calculation for the essential amino acid tryptophan would proceed as follows: assuming that the total amount of tryptophan added to the culture medium during the cultivation period was 2 grams, and the total amount of essential amino acids added to the medium during the same time period was 100 grams. Table 1 shows that in 100 grams of cell protein, out of 44.7 grams of total essential amino acids, 1.6 grams are tryptophan.

[0091] The calculation: Heaa = 4^- * 100 = 55.875% 1.6 447 shows that the highest conversion efficiency for tryptophan is 55.875%. Petition 870250094777, dated 10 / 16 / 2025, page 46 / 103 29 / 67 Now, this process is repeated for each of the nine individual essential amino acids. The lowest of the nine numbers obtained is the highest final conversion efficiency.

[0092] The amount of essential amino acids that can be converted into cellular protein is determined by how closely the total input of essential amino acids into the culture process corresponds to the amino acid composition of the cellular protein. Because cells cannot synthesize essential amino acids, the essential amino acid with the lowest relative total input into the culture process compared to its content in cellular protein will limit the maximum cell yield and therefore the maximum percentage of essential amino acids converted into cellular protein (this can be understood as an application of Liebig's law of the minimum).

[0093] The conversion efficiency for the total essential amino acids can be in the range of 5% to 100%, 20% to 100%, 30% to 100% or 50% to 100%, calculated by the equation mentioned above.

[0094] If the essential amino acid composition of the cellular protein is used according to the example mentioned in Table 1, the resulting total inputs of each essential amino acid, expressed in grams per 100 grams of the total input of all essential amino acids, may be in the ranges summarized in Table 2.

[0095] The ranges of amino acid concentrations in grams per 100 grams of total essential amino acids introduced into the cultivation process may conform to Table 2, regardless of whether the essential amino acid composition of the cell protein conforms to Table 1 or not.

[0096] It should be noted that, for the purpose of this equation, it is necessary to consistently consider the amino acid content as either free amino acids or amino acids that are part of a peptide chain (in which case the molecular weight of each amino acid must be considered less than the weight of a water molecule, to account for the fact that Petition 870250094777, dated 10 / 16 / 2025, page 47 / 103 30 / 67 that water is a byproduct of the formation of a peptide bond). In the equation above and in Tables 1-3, everything is counted as amino acids that form a peptide chain. Elsewhere in this document, when amino acid input or concentration is discussed, these are calculated using the molecular weights of free amino acids, and when protein input or concentration is discussed, it is assumed that amino acids are part of a peptide chain for any calculations. TABLE 1 - Example of possible essential amino acid content in cellular protein. Amino acid content [g / 100 g of cellular protein] His 2.7 Ly 5.1 Leu 8.9 Lys 8.2 Met 2.9 Phe 4.7 Thr 4.8 Trp 1.6 Vai 5.8 Soma 44.7 TABLE 2 - Concentration ranges of amino acids in grams per 100 grams of total essential amino acids introduced in the cultivation process. Amino acid range 1 range 2 range 3 His 0.30 to 6.04 1.21 to 5.44 1.81 to 4.83 He 0.57 to 11.41 2.28 to 10.27 3.42 to 9.13 Leu 1.00 to 19.91 3.98 to 17.92 5.97 to 15.93 Lys 0.92 to 18.34 3.67 to 16.51 5.50 to 14.68 Met 0.32 to 6.49 1.30 to 5.84 1.95 to 5.19 Phe 0.53 to 10.51 2.10 to 9.46 3.15 to 8.41 Thr 0.54 to 10.74 2.15 to 9.66 3.22 to 8.59 Trp 0.18 to 3.58 0.72 to 3.22 1.07 to 2.86 Go 0.65 to 12.98 2.60 to 11.68 3.89 to 10.38 Petition 870250094777, dated 10 / 16 / 2025, p. 48 / 103 31 / 67

[0097] However, the composition of cell biomass is somewhat variable and therefore the values ​​of each essential amino acid in terms of percentage by weight of the total essential amino acids used in the medium may also be within the ranges summarized in Table 3. TABLE 3 - Concentration ranges of total essential amino acids introduced by weight in the cultivation process. Amino acid band 4 band 5 band 6 His 0.2 to 7.9 0.8 to 7.1 1.2 to 6.3 Ile 0.3 to 14.9 1.5a 13.4 2.3a 11.9 Leu 0.7 to 25.9 2.7 to 23.3 4.1 to 20.8 Lys 0.6 to 23.9 2.5a21.5 3.8 to 19.1 Met 0.2 to 8.5 0.9 to 7.6 1.3a6.8 Phe 0.3 to 13.7 1.4a 12.3 2.2 to 11.0 Thr 0.3 to 14.0 1.5a 12.6 2.2 to 11.2 Trp 0.1 to 4.7 0.5 to 4.2 0.7 to 3.8 Go 0.4 to 16.9 1.8 to 15.2 2.7 to 13.5

[0098] Amino acids can be introduced into the cultivation process in the form of free amino acids, amino acid salts, amino acid esters, or any other suitable derivatives, as well as oligopeptides, for example, dipeptides, tripeptides, or tetrapeptides or polypeptides. The culture medium according to the invention may comprise enzymatic hydrolysate of soy protein, or any other suitable scalable hydrolysate according to the description of the hydrolysates and their preparation, as mentioned above. For example, suitable industrially scalable protein sources for hydrolysate preparation may include soy, pea, rice, wheat, corn, broad beans, alfalfa, hemp, chickpea, potato, pumpkin, rapeseed, red lentil, Spirulina, Chlorella, sunflower, duckweed, mung bean, or yeast. The present invention is not limited to the exemplary protein sources listed.

[0099] The total amount of hydrolysate (expressed as dry weight of protein) introduced into the culture medium during the culture process may be in the range Petition 870250094777, dated 10 / 16 / 2025, page 49 / 103 32 / 67 of 1 g / L and 200 g / L, in the range of 3 g / L to 100 g / L or in the range of 10 g / L to 60 g / L or in the range of 8 g / L to 50 g / L.

[0100] The total amino acid input of the hydrolysate, including amino acids in the form of short peptides or suitable bioavailable derivatives, for example, phosphosterols such as phosphoserine, or other derivatives such as methylglycine, is at least 75%, 80%, 85%, 90% or 95%, by weight, of the total input of all amino acids in the culture medium.

[0101] The culture medium according to the invention may comprise amino acids added separately from the hydrolysate, for example, 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 amount of L-cysteine ​​in the culture medium may be in the range of 0.1 to 10, or 0.5 to 7, or 1 to 5% by weight relative to the total amount of hydrolyzed protein in the culture medium.

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

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

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

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

[0107] The total amount of L-threonine in the culture medium may be in the range of 0.1 to 7, or 0.2 to 5, or 0.3 to 3% in relation to the total amount of hydrolyzed protein in the culture medium. Petition 870250094777, dated 10 / 16 / 2025, pp. 50 / 103 33 / 67

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

[0109] The culture medium according to the invention may comprise an inorganic source of bioavailable nitrogen, for example, ammonia. The total input of 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, 1 g / L to 10 g / L.

[0110] As sugar, at least one compound selected from the following group may be used: glucose, fructose, galactose, sucrose, lactose, maltose, or a combination thereof or any other suitable saccharide. The total sugar intake 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 media may contain at least one or any combination of the following ions as a mineral compound: Ca2+, Cl-, Cu2+, SO42-, Fe3+, NO3-, Fe2+, Mg2+, K+, Na+, CO32-, HCO3-, H2PO4-, HPO42-, PO4 3-, Zn2+, SeO32-. The medium may also contain traces of other mineral compounds and elements, such as cobalt, iodine, or manganese.

[0112] Since the medium is prepared by dissolving different constituent compounds in water, any suitable chemical compound can be used, provided it dissociates into the desired ions in aqueous solution. For example, NaCl and KCl produce a Cl- ion when dissolved. As another example, CuSO4 and MgCl2 or MgSO4 and CuCl2 can be used to produce Cu2+, Mg2+, SO42- and Cl- ions. 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 cultivation process can be in the range of 0.1 g / L to 50 g / L, or in the range of 1 g / L to 20 g / L, or in the range of 3 g / L to 10 g / L.

[0113] The total Na+ intake may be in the range of 20 to 120 mmol / L, or in the range of 30 to 100 mmol / L, or in the range of 40 to 80 mmol / L. Petition 870250094777, dated 10 / 16 / 2025, pp. 51 / 103 34 / 67

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

[0115] The total Mg2+ intake may be in the range of 0.3 to 10 mmol / L, or in the range of 0.5 to 8 mmol / L or in the range of 1 to 5 mmol / L.

[0116] The total PO43- intake may be in the range of 0.5 to 12 mmol / L, or in the range of 0.7 to 10 mmol / L or in the range of 1 to 6 mmol / L.

[0117] The total SO42- intake may be in the range of 0.1 to 5 mmol / L, or in the range of 0.3 to 3 mmol / L or in the range of 0.6 to 2 mmol / L.

[0118] Total K+ intake may be in the range of 2 to 18 mmol / L, or in the range of 4 to 15 mmol / L or in the range of 6 to 12 mmol / L.

[0119] The medium may contain at least one vitamin from: 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 derivatives or precursors of these compounds may be used. For example, cyanocobalamin may be used instead of vitamin B12, as it can be readily converted into bioactive vitamin B12 by cells. As another example, thiamine hydrochloride (thiamine chloride salt form) may be used instead of thiamine. The total intake of vitamins introduced in the cultivation process, omitting vitamins present in lysates or extracts, can range from 0.1 mg / L to 1000 mg / L, or from 5 mg / L to 500 mg / L, or from 20 mg / L to 300 mg / L.

[0120] Total choline intake may be in the range of 10 mg / L to 1000 mg / L, or in the range of 20 mg / L to 500 mg / L, or in the range of 30 mg / L to 200 mg / L.

[0121] The total intake of niacinamide (or other vitamin B3 vitamer) may be in the range of 3 mg / L to 150 mg / L, or in the range of 6 mg / L to 100 mg / L, or in the range of 10 mg / L to 80 mg / L.

[0122] Since organic amines can be used, at least one compound selected from: putrescine, ethanolamine or a combination thereof, or any other suitable amine. The total input of organic amines in Petition 870250094777, dated 10 / 16 / 2025, pp. 52 / 103 35 / 67 cultivation process can be in an amount in the range of 0.01 mg / L to 1000 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 a lysate or extract, for example, autolysed yeast extract or any other suitable lysate or extract. The extract or lysate for micronutrient supplementation may be added to the culture medium in an amount in the range of 0.01 g / L to 20 g / L, or in the range of 0.1 g / L to 10 g / L, or in the range of 0.5 g / L to 5 g / L.

[0124] Iron can be supplemented to the culture medium in compounds with the oxidation state of iron(III) or iron(II). Iron can be present as free ions or can be chelated with a suitable chelating agent to enhance its solubility and bioavailability. Chelating agents may comprise citrate, gluconate, ammonium citrate, EDTA, combinations thereof, or any other suitable chelating agent. Iron can be introduced into the culture medium bound to the chelating agent (e.g., in the form of ferric citrate) or iron and the chelating agent can be added separately (e.g., in the form of ferric chloride and sodium citrate). The relative amount (w / w) of the total chelating agent input to the total iron input can be in the range of 10000:1 to 1:100, or in the range of 1000:1 to 1:10, or in the range of 10:1 to 1:1. Total iron intake 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 medium may contain at least one shear guard of: polyethylene glycol (PEG), Pluronic F68, Pluronic F127, methylcellulose (MC), (hydroxypropyl)methylcellulose (HPMC), hydroxypropylcellulose (HPC), carboxymethylcellulose (CMC), dextran sulfate or any other suitable shear guard or combination thereof. The total shear guard input may be in the range of 0 g / L to 50 g / L, or in the range of 0.02 g / L to 10 g / L, or in the range of 0.1 g / L to 5 g / L.

[0126] In one aspect of the invention, the culture medium may comprise a composition of the total inputs, as described in Table 6. Petition 870250094777, dated 10 / 16 / 2025, pp. 53 / 103 36 / 67 momentary composition

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

[0128] The osmolality of the medium may be in the range of 200 mOsm / kg to 400 mOsm / kg, or in the range of 250 mOsm / kg to 350 mOsm / kg, or in the range of 280 mOsm / kg to 330 mOsm / kg. Osmolality may be adjusted before or after the culture medium is introduced into the culture device, or a combination of both, and may be adjusted at a single time or at multiple times. To increase osmolality, NaCl, KCl, glucose, any other suitable osmolyte, or a combination thereof may be used. To decrease osmolality, water or any other suitable dilute aqueous solution may be used.

[0129] The pH of the culture medium in the culture device may be in the range of 6 to 8, or in the range of 6.5 to 7.5, or in the range of 6.8 to 7.3. pH adjustment may be performed before or after the introduction of the culture medium into the culture device, or a combination of both, and may be adjusted at a single point in time or at multiple points in time. NaOH, HCl, NaHCO3, or any other appropriate acid or base may be used to adjust the pH; alternatively, the pH may be adjusted by altering the partial pressure of CO2 in the culture device (a higher partial pressure of CO2 will result in more CO2 dissolving in the culture medium, leading to a lower pH).The partial pressure of CO2 in the cultivation device can be adjusted by altering the percentage of CO2 in the spray gas, altering the total pressure in the cultivation device, or altering the mixing and spraying rate in the cultivation device (reducing or increasing the CO2 mass transfer coefficient), or any other suitable method. The partial pressure of CO2 in the cultivation device can be in the range of 0.05 kPa to 100 kPa, or in the range of 2 kPa to 60 kPa, or in the range of 5 kPa to 30 kPa.

[0130] The culture medium may comprise a shear guard Petition 870250094777, dated 10 / 16 / 2025, pp. 54 / 103 37 / 67 to prevent cell damage caused by mechanical forces resulting from mixing and / or spraying in the culture device. As a shear guard, at least one of the following may be used: polyethylene glycol (PEG), methylcellulose (MC), (hydroxypropyl)methylcellulose (HPMC), hydroxypropylcellulose (HPC), carboxymethylcellulose (CMC), dextran sulfate, or any other suitable shear guard, or a combination thereof. Shear guards may be present in the culture medium at a concentration in the range of 0 g / L to 50 g / L, or in the range of 0.02 g / L to 10 g / L, or in the range of 0.1 g / L to 5 g / L.

[0131] The instantaneous concentration of sugars in the 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 instantaneous concentration of all amino acids (considering both amino acids from the hydrolysate and amino acids added separately and biologically available derivatives, such as esters) and peptides in the medium may be in the range of 0.005 g / L to 30 g / L, or in the range of 0.1 g / L to 15 g / L or in the range of 0.5 g / L to 10 g / L.

[0133] The culture media composition described above may be suitable for cell lines that have been extensively adapted to in vitro conditions. However, some cell types may require additional components in the medium, for example, protein growth factors, to survive and proliferate. In another aspect of the invention, a medium composition suitable for these growth factor-dependent cell lines can be described as follows. Hydrolysates of plant protein isolates can be used as amino acid sources in culture media according to the invention. Recombinant protein production can be used in the preparation of culture medium components.

[0134] The culture medium according to the invention may comprise macronutrients and micronutrients, other components that adjust the properties of the basal medium (osmolality and availability of micronutrients) Petition 870250094777, dated 10 / 16 / 2025, pp. 55 / 103 38 / 67 and signaling components. The components can be dissolved, for example, in purified water or in water with inorganic salts, for example, phosphate-buffered saline (PBS) or water or PBS with bovine serum albumin (BSA), for example, BSA at 1% total.

[0135] Signaling compounds can vary depending on the specific cell type used in the bioreactor culture. Examples of these cells may be fibroblasts, myoblasts, adipocytes and their precursors, or a combination thereof.

[0136] Signaling compounds may or may not induce specific changes in cell fate. Examples of such changes may be stimulation of proliferation and / or stimulation of differentiation. Signaling compounds may be used in a specific order over a specific period of time. Examples of this may be the use of a signaling compound for proliferation stimulation which is then replaced in the medium by the signaling compound for differentiation induction. The precise order of dosing of signaling compounds may or may not be correlated or cross-linked with other tools that affect cell fate during cultivation.

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

[0138] Signaling compounds for various cell types destined for myogenic differentiation may comprise at least one FGF, insulin, TGF, transferrin, IGF, epidermal growth factor (EGF), bone morphogenetic protein (BMP), interleukin 6 (IL-6) or IL-13, or any other appropriate signaling compound.

[0139] The culture medium according to the invention may comprise amino acids (AA) or their sources, in combination with at least one type of compound that may be selected from a group comprising: Petition 870250094777, dated 10 / 16 / 2025, pp. 56 / 103 39 / 67 Saccharides, fatty acids, vitamins and organic micronutrients, mineral compounds, for example, inorganic salts, supplements, for example, iron supplementation compounds, organic amines, signaling compounds, for example, growth factors or signaling proteins or oligonucleotides, shear guards, additional compounds or compounds for manipulation, or any other suitable compounds or a combination thereof. The medium may also contain other compounds, such as phospholipids or nucleic acids, for example. Amino acids may be obtained, for example, from a protein hydrolysate.

[0140] The amino acids and their derivatives that can be supplied to the medium are, for example: glycine, L-alanine, L-arginine, L-asparagine, L-laspartic 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, the specific amino acid can be added in pure form or as part of a complex mixture of compounds (e.g., a hydrolysate), or hydrates or salts (e.g., hydrochloride or sodium salts) of amino acids can be used.

[0141] Culture media according to the present invention may comprise protein hydrolysate as the main source of amino acids. The protein hydrolysate may serve as a source of all important amino acids in culture media according to the invention for cell culture purposes, or some amino acids may be supplied to the medium separately, for example, L-methionine, which is found in very low concentrations in most scalable protein sources. Other different individual amino acids may be supplied separately from a source other than a protein hydrolysate.

[0142] In one aspect of the invention, the culture medium may comprise at least one of the amino acids listed in Table 4. Table 4 also discloses a possible illustrative, but not limited, concentration of at least Petition 870250094777, dated 10 / 16 / 2025, pp. 57 / 103 40 / 67 minus one amino acid that can be used in the culture medium according to the invention. TABLE 4 Amino Acids Concentration mg / L Glycine 0-1875 L-Alanine 0-445 L-Arginine Hydrochloride 0-14750 L-Asparagine-H2O 0-750 L-Aspartic Acid 0-665 L-Cysteine ​​Hydrochloride-H2O 0-1756 L-Cystine-2HCl 0-3129 L-Glutamic Acid 0-735 L-Glutamine 0-36500 L-Histidine Hydrochloride 0-3148 L-Isoleucine 0-5447 L-Leucine 0-5905 L-Lysine Hydrochloride 0-9125 L-Methionine 0-1724 L-Phenylalanine 0-3548 L-Proline 0-1725 L-Serine 0-2625 L-threonine 0-5345 L-tryptophan 0-902 L-tyrosine disodium salt dihydrate 0-5579 L-Valine 0-5285

[0143] The culture medium may comprise 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, for example, growth factors, can be used in the culture medium according to the invention. For example, at least one of: transferrin, insulin, FGF (e.g., FGF-1, FGF-2), TGF (e.g., TGF beta 1), IGF or any other suitable compounds. Petition 870250094777, dated 10 / 16 / 2025, pp. 58 / 103 41 / 67 can be used as a signaling compound.

[0145] In one aspect of the invention, the content of signaling compounds, for example, the content of growth factors such as FGF, TGF beta 1, insulin or transferrin or other signaling compounds, can be reduced. The concentration of TGF beta 1 can be in the range of 0 to 0.002 mg / L. The concentration of transferrin in the culture medium according to the invention can 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 invention, the reduced amount of transferrin can 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 0.5 mg to 500 mg / L. In one aspect of the invention, the reduced amount of insulin 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 0.2 to 0.5 mg / L. In one aspect of the invention, the reduced amount of FGF-2 may be in the range of 0 to 0.01 mg / L.

[0148] The concentration of TGF beta 1 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 0.05 to 0.1 mg / L. In one aspect of the invention, the reduced amount of TGF beta 1 may be in the range of 0 to 0.001 mg / L.

[0149] In one aspect of the invention, the culture medium may be free of any signaling compounds, for example, growth factors. The culture medium according to the invention may be serum-free and / or protein-free.

[0150] The culture medium may comprise antifoaming agents, for example, silicone-based antifoaming agents, polyethylene glycol (PEG), polyvinyl alcohol (PVA), polydimethylsiloxane, polysorbate 80 or vegetable oils, or any other suitable antifoaming agent or combination thereof. The concentration of the antifoaming agent in the culture medium may be in the range of Petition 870250094777, dated 10 / 16 / 2025, pp. 59 / 103 42 / 67 0.001% and 5%, or in the range of 0.01% to 1% or in the range of 0.1% to 0.5% by weight.

[0151] In one aspect of the invention, the content of the culture medium components may be within the ranges according to Table 5. TABLE 5 - Concentration ranges of culture media components Component of media Concentration (mg / L) Supplement Transferrin 0-10 Insulin 0-2000 FGF2 0-1 TGF beta 1 0-0.2 Selenium sodium 0-1.4 Ascorbate 0-6400 Sugars D-Glucose (dextrose) 0-315100 Fatty acids Linoleic acid 0-4.2 Lipoic acid 0-10.5 Amino acids Glycine 0-1875 L-Alanine 0-445 L-Arginine hydrochloride 0-14750 L-Asparagine-H2O 0-750 L-Aspartic acid 0-665 L-Cysteine ​​hydrochloride-H2O 0-1756 L-Cystine-2HCl 0-3129 L-Glutamic acid 0-735 L-Glutamine 0-36500 L-Histidine Hydrochloride 0-3148 L-Isoleucine 0-5447 L-Leucine 0-5905 L-Lysine Hydrochloride 0-9125 L-Methionine 0-1724 L-Phenylalanine 0-3548 L-Proline 0-1725 L-Serine 0-2625 L-threonine 0-5345 Petition 870250094777, dated 10 / 16 / 2025, pp. 60 / 103 43 / 67 L-tryptophan 0-902 L-tyrosine disodium salt dihydrate 0-5579 L-Valine 0-5285 Vitamins Biotin 0-0.35 Choline chloride 0-898 ​​Calcium D-pantothenate 0-224 Folic acid 0-265 I-Inositol 0-1260 Niacinamide 0-202 Pyridoxine hydrochloride 0-201.3 Riboflavin 0-21.9 Thiamine hydrochloride 0-217 Vitamin B12 0-68 Inorganic Salts Calcium chloride (CaCl2) (anhydrous) 0-11660 Cupric sulfate (CuSO4-5H2O) 0-0.13 Ferric nitrate (Fe(NO3)3-9H2O) 0-5 Ferric sulfate (FeSO4-7H2O) 0-41.7 Magnesium chloride (anhydrous) 0-2864 Magnesium sulfate (MgSO4) (anhydrous) 0-4884 Potassium chloride (KCl) 0-31180 Sodium bicarbonate (NaHCO3) 0-243800 Sodium chloride (NaCl) 0-699550 Dibasic sodium phosphate (Na2HPO4) (anhydrous) 0-7102 Monobasic sodium phosphate (NaH2PO4H2O) 0-6250 Zinc sulfate (ZnSO4-7H2O) 0-43.2 Additional compounds Hypoxanthine 0-239 Putrescine 2HCl 0-8.1 Sodium pyruvate 0-5500 Thymidine 0-36.5

[0152] In other aspects of the invention, the culture medium may comprise signaling molecules or nucleic acids. Petition 870250094777, dated 10 / 16 / 2025, pp. 61 / 103 44 / 67 Nucleic acids - oligonucleotides

[0153] In one aspect of the invention, oligonucleotides can be used as constituent components of a culture medium for cell cultivation. The oligonucleotides can be single- or double-stranded nucleic acid chains containing 10 to 70 nucleotides, or 10 to 120, or 1 to 1000 nucleotides.

[0154] In one aspect of the invention, oligonucleotides can be added to the culture medium in molar concentrations in the range of 5 to 100 nM / L, or in the range of 5 to 500 nM / L, or in the range of 50 nM / L to 50 nM / L, or the concentration can vary during cultivation, where a peak of higher concentration can be followed by a lower concentration. The peak of high concentration can be from 1 to 10 hours or 10 to 72 hours of cultivation.

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

[0156] Examples of oligonucleotides serving as AONs can be oligonucleotides whose target is the mRNA of target genes. Examples of such target genes can be ferroportin, myostatin, p53, miRNA140, or others.

[0157] Examples of oligonucleotides that serve as a ligand for the appropriate protein (aptamers) may be oligonucleotides that have the ability to bind to target proteins, such as FGF-2 receptor, TGFbeta receptor, TrF receptor, insulin receptor, or others.

[0158] As an additional compound, at least one of the following compounds may be used: hypoxanthine, putrescine, pyruvate, thymidine, ethanolamine, the salts or their derivatives, for example, sodium hypoxanthine or putrescine dihydrochloride, or any other suitable additional compound.

[0159] Hypoxanthine, for example, sodium hypoxanthine, can be used in the culture medium according to the invention at concentrations in the range of 0 to 239 mg / L, or in the range of 10 to 200 mg / L or in the range of 50 to 100 mg / L. Petition 870250094777, dated 10 / 16 / 2025, pp. 62 / 103 45 / 67

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

[0161] Pyruvate, for example, sodium pyruvate, can be used in the culture medium according to the invention at concentrations in the range of 0 mg / L to 5.5 g / L, or in the range of 100 mg / L to 3 g / L or in the range of 500 mg / L to 1 g / L.

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

[0163] Recombinantly prepared signaling compounds can be used in the culture medium according to the invention. The signaling compounds can be stabilized to prevent degradation, for example, thermal degradation or proteolytic degradation. They can be secreted into the culture medium or accumulated in the cellular or subcellular compartment. Then, in the harvesting process, they may or may not be collected, purified and separated, or the whole culture can be collected. From the entire culture, various fractions (parts) can be divided and collected in the form of easily handled pellets. These pellets can be further processed and can serve as a direct compound to be added to the culture medium. The pellets can be dissolved, lysed or reconstituted before application to the culture medium in a suitable solvent.

[0164] In one aspect of the invention, a production of recombinant signaling compounds can be used as components of culture media. The production of recombinant proteins can comprise the following expression systems: bacteria (e.g., Escherichia coli, Bacillus subtilis), brewer's yeast (Saccharomyces cerevisiae), unconventional yeasts (e.g., Pichia pastoris, Hansenula polymorpha, Yarrowia lipolytica), filamentous fungi (e.g., Aspergillus spp., Trichoderma reesei), plants (e.g., Nicotiana tabacum, Hordeum vulgare, Zea may), insect cells or mammalian cell lines (e.g., HEK293, Petition 870250094777, dated 10 / 16 / 2025, pp. 63 / 103 46 / 67 CHO-K1) or any other appropriate expression systems. Recombinant protein production followed by cell lysis and derivatization of pellets or other derivatives rich in recombinant proteins can be used, for example, in Streptococcus thermophilus, S. cerevisiae, P. pastoris and various strains of Lactobacillus spp., such as Lactobacillus acidophilus, Lactobacillus plantarum and Lactobacillus casei.

[0165] In one aspect of the invention, the culture medium for cell cultivation for the preparation of food products may have the total hydrolysate input expressed as weight of dry protein introduced into the culture medium within the cultivation process in the range of 8 g / L to 50 g / L.

[0166] The total amino acid input of the hydrolysate, which includes amino acids in the form of short peptides or suitable bioavailable derivatives, may be at least 75%, by weight, of the total input of all amino acids in 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 yeasts.

[0168] The substrate source for hydrolysis may be selected from at least one of the following: soybean, pea, rice, wheat, corn, broad beans, alfalfa, hemp, chickpea, potato, pumpkin, rapeseed, red lentil, Spirulina, Chlorella, sunflower, duckweed, mung bean or baker's yeast.

[0169] The substrate source for hydrolysis can be at least one of the following: soybean, broad bean or pea.

[0170] In one aspect of the invention, the culture medium may further comprise at least one of: L-methionine or L-cysteine, L-cystine, Lornithine, L-tryptophan, L-histidine, L-threonine, added separately from the hydrolysate.

[0171] The total input of amino acids added to the culture medium Petition 870250094777, dated 10 / 16 / 2025, pp. 64 / 103 47 / 67 separately from the hydrolysate may be in the range of 0.1 g / L to 10 g / L.

[0172] The total input of amino acids added to the culture medium separately from the hydrolysate can be in the range of 0.5% to 15%, expressed as a percentage of the total input of hydrolyzed proteins in the culture medium.

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

[0174] The total amount of L-ornithine in the culture medium may be in the range of 0.001% to 0.5% in relation to the total amount of hydrolyzed protein in the culture medium.

[0175] The total amount of L-methionine in the culture medium can be in the range of 0.2% to 2% in relation to the total amount of hydrolyzed protein in the culture medium.

[0176] The total amount of L-tryptophan in the culture medium may be in the range of 0.2% to 2% in relation to the total amount of hydrolyzed protein in the culture medium.

[0177] The total amount of L-histidine in the culture medium may be in the range of 0.15% to 1.5% in relation to the total amount of hydrolyzed protein in the culture medium.

[0178] The total amount of L-threonine in the culture medium can be in the range of 0.3% to 3% in relation to the total amount of hydrolyzed protein in the culture medium.

[0179] In one aspect of the invention, the culture medium may comprise at least one compound from a group consisting of: vitamins, sugars, minerals, organic amines, micronutrients, iron supplementation compounds, shear protectors and low-abundance organic compounds, or a combination thereof.

[0180] The vitamin may comprise 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 Petition 870250094777, dated 10 / 16 / 2025, pp. 65 / 103 48 / 67 inositol or its derivatives, or a combination thereof.

[0181] Sugar may comprise at least one of: glucose, fructose, galactose, sucrose, lactose, maltose, or a combination thereof.

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

[0183] The micronutrient may comprise at least one of: spermine, spermidine, putrescine, thymidine, L-ornithine, ethanolamine, myo-inositol, or choline.

[0184] The iron supplementation compound may comprise at least one compound in the oxidation state of iron(III) or iron(II).

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

[0186] In one aspect of the invention, the total vitamin input per liter of culture medium, except for vitamins present in lysates or extracts, may be in the range of 20 mg / L to 300 mg / L, the total sugar input per liter of culture medium may be in the range of 3 g / L to 20 g / L, the total iron input per liter of culture medium may be in the range of 0.001 g / L to 0.05 g / L.

[0187] Total choline intake may be in the range of 20 mg / L to 500 mg / L.

[0188] The total intake of niacinamide, or other vitamin B3 vitamer, may be in the range of 6 mg / L to 100 mg / L.

[0189] The total input of mineral compounds introduced in the cultivation process can be in the range of 1 g / L to 20 g / L.

[0190] Total Na+ intake may be in the range of 30 to 100 mmol / L.

[0191] Total Cl- intake may be in the range of 35 to 110 mmol / L.

[0192] The total Mg2+ intake is in the range of 0.5 to 8 mmol / L.

[0193] The total PO4 3- intake is in the range of 0.7 to 10 mmol / L.

[0194] The total SO4 2- intake is in the range of 0.3 to 3 mmol / L. Petition 870250094777, dated 10 / 16 / 2025, pp. 66 / 103 49 / 67

[0195] Total K+ intake is in the range of 4 to 15 mmol / L.

[0196] The total shear protection input can be in the range of 0.1 g / L to 5 g / L.

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

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

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

[0200] In one aspect of the invention, the cells for cultivation can be selected from: Stem cells comprising embryonic stem cells (ESCs) and other cell types derived from blastocysts or other early-stage embryos; Muscle stem cells, such as myosatelite cells, mesenchymal stem cells; or cells in which stem characteristics are established in vitro, such as induced pluripotent stem cells (iPSCs); or cells with the characteristics of: myoblasts, myocytes, fibroblasts, myofibroblasts, fibroadipogenic progenitors, pre-adipocytes, adipocytes, epithelial cells, chondroblasts, chondrocytes, macrophages, keratinocytes, hepatocytes, or Sertoli cells.

[0201] In one aspect of the invention, the culture cells can be anchor-independent, meaning that the cells have the ability to survive and grow in suspension conditions without attachment to any surface, and / or the culture cells can survive and grow as a suspension of cell clusters, cell aggregates, spheroids or organoids or a combination thereof.

[0202] The culture medium according to the invention can be used for Petition 870250094777, dated 10 / 16 / 2025, pp. 67 / 103 50 / 67 suspension culture, which means that the cultivation process involves growing the cells in a stirred tank or other suitable type of bioreactor, where most of the cells are present as a suspension of single cells or cell aggregates.

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

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

[0205] In one aspect of the invention, the culture medium may comprise a source of bioavailable inorganic nitrogen, wherein the total input of inorganic nitrogen source may be in the range of 1 g / L to 10 g / L.

[0206] In one aspect of the invention, the culture medium preparation process may have the characteristics of continuous medium preparation, wherein the medium components may be introduced into the cultivation process separately, the process may comprise the following steps: a) introduction of a concentrated basal medium into the culture device at a defined flow rate, wherein the basal medium is X times more concentrated than the desired concentration in the final medium and the basal medium is introduced at a flow rate 1 / X times the total outflow rate of the culture device, b) introduction of a concentrated sugar solution into the culture device, where the flow rate is controlled so that the sugar concentration in the culture medium is maintained at a desirable set point, c) introduction of a hydrolysate and amino acid solution into the culture device, where the flow rate is controlled so that the amino acid concentration in the medium is maintained at a desirable set point, d) introduction of a concentrated NaCl solution into the culture device, where the flow rate is controlled so that the total osmolality of the medium is maintained at a desirable setpoint, for example, 310 mOsm / kg, (e) introduction of demineralized water into the culture device, in Petition 870250094777, dated 10 / 16 / 2025, pp. 68 / 103 51 / 67 that the flow rate is adjusted to be equal to the difference between the total outflow rate of the culture device and the sum of all other inflow rates, thus maintaining a constant volume of liquid in the bioreactor.

[0207] In one aspect of the invention, the process for preparing the culture medium may comprise adjusting the pH by altering the partial pressure of CO2.

[0208] In one aspect of the invention, the process for preparing the culture medium comprising the protein hydrolysate may comprise the step of hydrolyzing the 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 yeasts, or the protein substrate source for hydrolysis may be selected from at least one of: soybean, pea, rice, wheat, corn, broad beans, alfalfa, hemp, chickpea, potato, pumpkin, rapeseed, red lentil, Spirulina, Chlorella, sunflower, duckweed, mung bean or yeast.

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

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

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

[0212] In one aspect of the invention, the process for preparing the culture medium may comprise hydrolysis using at least one of the following types of enzymes: serine protease (for example, a protease similar to Petition 870250094777, dated 10 / 16 / 2025, pp. 69 / 103 52 / 67 subtilisin), cysteine ​​protease, metalloprotease, glutamic protease or aspartic protease.

[0213] The culture medium preparation process may involve hydrolysis using at least one enzyme from among: Alcalase, Flavourzyme, 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, Corolase 7089, Corolase 2TSN, Corolase 8000, Maxipro TNP, Maxipro FPC, Papain or Bromelain.

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

[0215] The enzymatic hydrolysis process can occur at a temperature in the range of 30 to 65 °C. The pH can be in the range of 5 to 9 and the time interval can be from 10 to 26 hours.

[0216] The enzyme can be immobilized on microcarriers.

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

[0218] The degree of hydrolysis, defined as the percentage of peptide bonds hydrolyzed in relation to the total amount of peptide bonds present in the substrate at the beginning of the hydrolysis reaction, can be in the range of 20% to 50%.

[0219] In one aspect of the invention, the process for preparing the culture medium may comprise monitoring the hydrolysis by conductometry.

[0220] In one aspect of the invention in the process of preparing the culture medium, the hydrolysate can be further processed with phytase to remove phytic acid.

[0221] In one aspect of the invention, the hydrolysate can be heat-treated at the end of the hydrolysis to deactivate enzymes and kill micro-Petition 870250094777, dated 10 / 16 / 2025, page 70 / 103 53 / 67 organisms.

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

[0223] In the cell culture process according to the invention, the culture medium described above can be used.

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

[0225] In one aspect of the invention, the cells used in the cell culture process may exhibit at least one of: a) immortalization (absence of Hayflick limit), b) ability to survive and multiply in the absence of attachment to a solid surface (anchorage independence), c) ability to synthesize glutamine, d) ability to synthesize asparagine, e) ability to synthesize proline, f) ability to survive and proliferate in the absence of growth factors, or g) capacity for iron absorption in the absence of transferrin.

[0226] The culture medium described above can be used for growing cells for the purpose of preparing the food product. Examples Example 1 - Enzymatic hydrolysis by free enzyme

[0227] Free enzyme hydrolysis was performed in the hydrolysis tank by dissolving 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, allowing for high Alcalase activity at 62 °C. Over 2 hours with constant mixing, the pH of the solution decreased as a result of the hydrolysis of the Petition 870250094777, dated 10 / 16 / 2025, pp. 71 / 103 54 / 67 peptide bonds and an increased number of carboxylic groups. These conditions allowed for high Flavourzyme activity, which was added at a concentration of 0.15 g / L. The resulting mixture was then incubated for a further 20 hours at 62 °C with constant mixing, after which the residual enzyme was thermally deactivated. With this procedure, 43% of the original protein was converted into free amino acids.

[0228] The results of the HPLC analysis of amino acid content using UV detection (cysteine ​​was not measured in this analysis) are summarized in Table 6. TABLE 6 - HPLC analysis of amino acid content in the hydrolysate Amino acid mg / L Asp 136.45 Glu 253.40 Asn 388.74 Ser 248.59 Gin 170.42 His 147.57 Gly 80.27 Thr 226.47 Arg 426.90 Ala 138.12 Tyr 210.82 Met 56.30 Vai 286.39 Cystine 8.46 Trp 60.30 Phe 296.79 He 260.56 Leu 469.37 Lys 381.21 Pro 31.25 Soma 4288.91 Example 2 - Enzymatic hydrolysis by immobilized enzyme Petition 870250094777, dated 10 / 16 / 2025, pp. 72 / 103 55 / 67

[0229] The immobilized enzymes were prepared by suspending 600 mg of NH2-functionalized porous silica microspheres in 50 mL of distilled water. The silica microspheres were activated by adding 0.003% by volume of glutaraldehyde. After 30 minutes, the excess glutaraldehyde was washed off with distilled water, and the silica microspheres were suspended at half their original volume. Alcalase, supplied by Novozymes, was then added at a final concentration of 0.1% with constant stirring. This procedure immobilized 80% of the enzyme used in the silica microspheres, corresponding to 4 grams of immobilized enzyme per 1 kilogram of silica microspheres.

[0230] Silica microspheres with immobilized Alcalase were added to a mixture of 13 g / L of soy protein in distilled water at a density of 10 grams of microspheres per liter. After hydrolysis in the hydrolysis tank for 2 hours at 62 °C with constant mixing, the microspheres with Alcalase were removed by centrifugation and 40 grams of silica microspheres with immobilized Flavourzyme were added. After further hydrolysis for 20 hours at 62 °C with constant mixing, the Flavourzyme microspheres were removed by centrifugation and the resulting hydrolysate was thermally sterilized for 20 minutes at 130 °C and a pressure of 2.5 atmospheres, which also deactivated any enzyme that may have detached from the solid support. After filtration to remove solid debris, the hydrolysate was used to prepare culture media. With this method, 5% of the original protein was converted into free amino acids.

[0231] The results of the HPLC (UV detection) analysis of amino acid content (cysteine ​​was not measured in this analysis) are summarized in Table 7. TABLE 7 - HPLC analysis of amino acid content in hydrolysate Soybean amino acid, water (mg / L) Asp 15.33 Glu 0.00 Petition 870250094777, dated 10 / 16 / 2025, pp. 73 / 103 56 / 67 Asn 1.51 Ser 5.86 Gin 10.57 His 14.04 Gly 68.39 Thr 0.00 Arg 21.31 Ala 32.71 Tyr 36.01 Met 11.58 Vai 36.01 Cystine 4.33 Trp 8.08 Phe 123.73 He 33.87 Leu 204.85 Lys 51.05 Pro 1.35 Sum 680.59 Example 3 - composition of culture media

[0232] The culture media were prepared with the compositions according to Table 8 and Table 9. TABLE 8: Example of total input of relevant compounds into the culture medium according to the invention. Compound Category Concentration in Medium [mg / L] Biotin Vitamins and organic compounds of low abundance 0.0035 Choline chloride Vitamins and organic compounds of low abundance 8.9800 Calcium D-pantothenate Vitamins and organic compounds of low abundance 2.2400 Petition 870250094777, dated 10 / 16 / 2025, pp. 74 / 103 57 / 67 Folic acid vitamins and low-abundance organic compounds 2.6500 Niacinamide vitamins and low-abundance organic compounds 2.0200 Pyridoxine hydrochloride vitamins and low-abundance organic compounds 2.0130 Riboflavin vitamins and low-abundance organic compounds 0.2190 Thiamine hydrochloride vitamins and low-abundance organic compounds 2.1700 Vitamin B12 vitamins and low-abundance organic compounds 0.6800 I-Inositol vitamins and low-abundance organic compounds 12.6000 Calcium chloride (CaCl2) (anhydrous) mineral compounds 116.6000 Cupric sulfate (CuSO4.5H2O) mineral compounds 0.0800 Ferric nitrate (Fe(NO3)3.9H2O) mineral compounds 0.0500 Ferrous sulfate (FeSCUJhW) mineral compounds 0.4170 Magnesium chloride (anhydrous) Mineral compounds 28.6400 Magnesium sulfate (MgSCM) (anhydrous) Mineral compounds 48.8400 Potassium chloride (KCl) Mineral compounds 311.8000 Sodium bicarbonate (NaHCO3) Mineral compounds 2438.0000 Sodium chloride (NaCl) mineral compounds 6995.5000 Dibasic sodium phosphate (Na2HPO4) (anhydrous) mineral compounds 71.0200 Monobasic sodium phosphate (NaH2PO4.H2O) mineral compounds 62.5000 Zinc sulfate (ZnSCMJhW) mineral compounds 0.4320 Sodium selenite mineral compounds 0.0300 Enzymatic hydrolysate of soy protein (expressed as dry mass of protein) Amino acids 1000.0000. Petition 870250094777, dated 10 / 16 / 2025, pp. 75 / 103 58 / 67 Ferric citrate iron supplementation 120.0000 Glucose sugars 3150.0000 Putrescine organic amines 1.0000 Ethanolamine organic amines 3.0000 TABLE 9: Example of the momentary composition of relevant compounds in the culture medium according to the invention. Compound Category Concentration in Medium [mg / L] Biotin Vitamins and organic compounds of low abundance 0.0035 Choline chloride Vitamins and organic compounds of low abundance 8.9800 Calcium D-pantothenate Vitamins and organic compounds of low abundance 2.2400 Folic acid Vitamins and organic compounds of low abundance 2.6500 Niacinamide Vitamins and organic compounds of low abundance 2.0200 Pyridoxine hydrochloride Vitamins and organic compounds of low abundance 2.0130 Riboflavin Vitamins and organic compounds of low abundance 0.2190 Thiamine hydrochloride Vitamins and organic compounds of low abundance 2.1700 Vitamin B12 Vitamins and organic compounds of low abundance 0.6800 I-Inositol Vitamins and organic compounds of low abundance 12.6000 Petition 870250094777, dated 10 / 16 / 2025, pp. 76 / 103 59 / 67 Calcium chloride (CaCl2) (anhydrous) mineral compounds 116.6000 Cupric sulfate (CuSCM.ShhO) mineral compounds 0.0800 Ferric nitrate (Fe(NO3)3.9H2O) mineral compounds 0.0500 Ferrous sulfate (FeSCUThW) mineral compounds 0.4170 Magnesium chloride (anhydrous) mineral compounds 28.6400 Magnesium sulfate (MgSCM) (anhydrous) mineral compounds 48.8400 Potassium chloride (KCl) mineral compounds 311.8000 Sodium bicarbonate (NaHCO3) mineral compounds 2438.0000 Sodium chloride (NaCl) mineral compounds 6995.5000 Dibasic sodium phosphate (Na2HPO4) (anhydrous) mineral compounds 71.0200 Monobasic sodium phosphate (NaH2PO4.H2O) mineral compounds 62.5000 Zinc sulfate (ZnSCMThEO) mineral compounds 0.4320 Sodium selenite mineral compounds 0.0300 Enzymatic hydrolysate of soy protein (expressed as dry mass of protein) Amino acids 1000.0000 Ferric citrate iron supplementation 120.0000 Glucose sugars 3150.0000 Putrescine organic amines 1.0000 Ethanolamine organic amines 3.0000. Example 4 - Batch mixing of culture medium:

[0233] Batch mixing medium components were prepared. Solution 1, comprising vitamins and micronutrient components, was prepared by dissolving the compounds according to Table 10 in distilled water, so that the final volume of the solution was 10 L. TABLE 10 Compound mg Biotin 3.5 Choline chloride 8980 Calcium D-pantothenate 2240 Petition 870250094777, dated 10 / 16 / 2025, pp. 77 / 103 60 / 67 Folic acid 2650 Niacinamide 2020 Pyridoxine hydrochloride 2013 Riboflavin 219 Thiamine hydrochloride 2170 Vitamin B12 680 I-Inositol 12600 Cupric sulfate (CuSO4.5H2O) 80 Ferric nitrate (Fe(NO3)3.9H2O) 50 Sodium selenite 30 Ferric citrate 120000 Putrescine 1200 Ethanolamine 3200

[0234] Solution 2 (basic solution) was prepared by dissolving compounds according to Table 11 in distilled water, so that the final volume of the solution was 80 L. TABLE 11 Compound g Calcium chloride (CaCl2) (anhydrous) 116.6 Ferrous sulfate (FeSO4.7H2O) 0.417 Magnesium chloride (anhydrous) 28.64 Magnesium sulfate (MgSO4) (anhydrous) 48.84 Potassium chloride (KCl) 311.8 Sodium bicarbonate (NaHCO3) 2438 Sodium chloride (NaCl) 6995 Dibasic sodium phosphate (Na2HPO4) (anhydrous) 71.02 Monobasic sodium phosphate (NaH2PO4.H2O) 62.5 Zinc sulfate (ZnSO4.7H2O) 0.432 Glucose 3150

[0235] Hydrolysate - 10 liters of soy protein hydrolysate with a protein concentration of 10 g / L were prepared according to a suitable hydrolysis procedure, as described in the chapter on Petition 870250094777, dated 10 / 16 / 2025, pp. 78 / 103 61 / 67 preparation of hydrolysate, for example, as described in Example 1.

[0236] The media components were mixed: 1 liter of solution 1, 80 liters of solution 2 and 10 liters of 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 solution of the medium was filtered through 0.1 µm candle filters. The sterile medium was stored in a sterile storage tank, which was connected directly to a culture device. Example 5 - continuous mixing of the culture medium

[0240] Five solutions of medium components were prepared (the hydrolysate was prepared according to the hydrolysate preparation procedure, other solutions were prepared by dissolving the components in distilled water at the required concentration).

[0241] The basal medium solution was prepared with the composition according to table 12. TABLE 12 Compound mg / L Biotin 0.0105 Choline chloride 26.94 Calcium D-pantothenate 6.72 Folic acid 7.95 Niacinamide 6.06 Pyridoxine hydrochloride 6.039 Riboflavin 0.657 Thiamine hydrochloride 6.51 Vitamin B12 2.04 I-Inositol 37.8 Calcium chloride (CaCl2) (anhydrous) 349.8 Petition 870250094777, dated 10 / 16 / 2025, pp. 79 / 103 62 / 67 Cupric sulfate (CuSO4.5H2O) 0.24 Ferric nitrate (Fe(NO3)3.9H2O) 0.15 Ferrous sulfate (FeSO4.7H2O) 1.251 Magnesium chloride (anhydrous) 85.92 Magnesium sulfate (MgSO4) (anhydrous) 146.52 Potassium chloride (KCl) 935.4 Zinc sulfate (ZnSO4.7H2O) 1.296 Sodium selenite 0.09 Ferric citrate 360 ​​Putrescine 3.6 Ethanolamine 9.6

[0242] The buffer solution was prepared with the composition according to table 13. TABLE 13 Compound mg / L Sodium bicarbonate (NaHCO3) 24380 Dibasic sodium phosphate (Na2HPO4) (anhydrous) 710.2 Monobasic sodium phosphate (NaH2PO4,H2O) 625

[0243] Additionally, the soy protein hydrolysate solution was prepared with a protein concentration of 10 g / L.

[0244] The sugar solutions were prepared with the composition according to table 14. TABLE 14 Compound mg / L Glucose 63000

[0245] The saline solutions were prepared with the composition according to table 15. TABLE 15 Compound mg / L Sodium chloride (NaCl) 139900 Petition 870250094777, dated 10 / 16 / 2025, pp. 80 / 103 63 / 67

[0246] All component solutions were prepared in individual mixing tanks and balanced to a pH of 7.2 using a 1 M NaOH solution or a 1 M HCl solution.

[0247] All components were filtered in individual sterile storage tanks using 0.1 µm candle filters.

[0248] The sterile medium components were introduced into the culture device in volumes of, in order, 0.33, 0.1, 0.1, 0.05 and 0.05 times the working volume of the culture device per day (vvd). Additionally, sterile distilled water is introduced into the culture device at 0.37 (vvd). Example 6: Culture medium composition

[0249] The culture medium for cell cultivation was prepared comprising the following types of medium components: a) signaling compounds b) basal medium compounds c) nutritional compounds.

[0250] Concentrated stock solutions of these three types of media components were prepared and stored individually. The final culture medium was prepared by mixing them before culturing the cells at the final concentration per liter according to the requested concentration.

[0251] An example of the composition of culture media is in accordance with Table 16. This culture medium composition comprises a nutritional mixture of soy protein hydrolysate, fatty acids and saccharides combined with vitamins, inorganic salts, additional compounds and growth factors. TABLE 16 - composition of the medium Component of media Concentration (mg / L) Growth factors Transferrin 0.100 Insulin 20,000 FGF2 0.100 Petition 870250094777, dated 10 / 16 / 2025, pp. 81 / 103 64 / 67 TGF beta 1 0.002 Saccharides D-Glucose (dextrose) 3 151.000 Fatty acids Linoleic acid 0.042 Lipoic acid 0.105 Nutritional blend hydrolyzed soy 10.000 Vitamins Biotin 0.004 Choline chloride 8.980 Calcium D-pantothenate 2.240 Folic acid 2.650 I-Inositol 12.600 Niacinamide 2.020 Pyridoxine hydrochloride 2.013 Riboflavin 0.219 Thiamine hydrochloride 2.170 Vitamin B12 0.680 Ascorbate 64.000 Inorganic Salts Sodium selenium 0.014 Calcium chloride (CaCl2) (anhydrous) 116.600 Sulfate Cuphoc (CuSO4-5H2O) 0.001 Ferric nitrate (Fe(NO3)3-9H2O) 0.050 Ferric sulfate (FeSO4-7H2O) 0.417 Magnesium chloride (anhydrous) 28.640 Magnesium sulfate (MgSO4) (anhydrous) 48.840 Potassium chloride (KCl) 311.800 Sodium bicarbonate (NaHCO3) 2,438.000 Sodium chloride (NaCl) 6,995.500 Dibasic sodium phosphate (Na2HPO4) (anhydrous) 71.020 Monobasic sodium phosphate (NaH2PO4H2O) 62.500 Petition 870250094777, dated 10 / 16 / 2025, pp. 82 / 103 65 / 67 Zinc sulfate (ZnSO4-7H2O) 0.432 Additional compounds Hypoxanthine 2.390 Putrescine 2HCl 0.081 Sodium pyruvate 55.000 Thymidine 0.365 TABLE 17 - Culture medium composition Component of media Concentration (mg / L) Growth factors Transferrin 0.100 Insulin 20.000 FGF2 0.100 TGF beta 1 0.002 LIF 0.050 Saccharides D-Glucose (dextrose) 1,000.000 Amino acids Glycine 18.750 L-Alanine 4.450 L-Arginine hydrochloride 147.500 L-Asparagine-H2O 7.500 L-Aspartic acid 6.650 L-Cysteine ​​hydrochloride-H2O 17.560 L-Cystine-2HCl 31.290 L-Glutamic acid 7.350 L-Glutamine 365.000 L-Histidine hydrochloride 31.480 L-Isoleucine 54,470 L-Leucine 59,050 L-Lysine Hydrochloride 91,250 L-Methionine 17,240 L-Phenylalanine 35,480 L-Proline 17,250 Petition 870250094777, dated 10 / 16 / 2025, pp. 83 / 103 66 / 67 L-Serine 26,250 L-Threonine 53,450 L-Tryptophan 9,020 L-Tyrosine Dihydrate Disodium Salt 55,790 L-Valine 52,850 Fatty Acids Linoleic Acid 0.042 Lipoic Acid 0.105 Vitamins Biotin 0.004 Choline Chloride 8,980 Calcium D-Pantothenate 2,240 Folic Acid 2,650 I-Inositol 12,600 Niacinamide 2,020 Pyridoxine Hydrochloride 2,013 Riboflavin 0.219 Thiamine Hydrochloride 2,170 Vitamin B12 0.680 Ascorbate 64,000 Inorganic Salts Sodium Selenium 0.014 Calcium Chloride (CaCl2) (anhydrous) 116,600 Cupric sulfate (CuSO4-5H2O) 0.001 Ferric nitrate (Fe(NO3)3-9H2O) 0.050 Ferric sulfate (FeSO4-7H2O) 0.417 Magnesium chloride (anhydrous) 28,640 Magnesium sulfate (MgSO4) (anhydrous) 48,840 Potassium chloride (KCl) 311,800 Sodium bicarbonate (NaHCO3) 2,438,000 Sodium chloride (NaCl) 6,995,500 Dibasic sodium phosphate (Na2HPO4) (anhydrous) 71,020 Monobasic sodium phosphate (NaH2PO4-H2O) 62,500 Zinc sulfate (ZnSO4-7H2O) 0.432 Petition 870250094777, dated 10 / 16 / 2025, pp. 84 / 103 67 / 67 Additional compounds: Hypoxanthine 2.390 mg, Putrescine 2HCl 0.081 mg, Sodium pyruvate 55.000 mg, Thymidine 0.365 mg

[0252] Another example of the composition of culture media is in accordance with Table 17. This culture medium composition comprises a nutritional mixture of raw food-grade amino acids, fatty acids and D-glucose saccharide combined with vitamins, inorganic salts, additional compounds and growth factors. INDUSTRIAL APPLICABILITY

[0253] The culture medium according to the invention may be suitable for cell cultivation, for example, in the production of cultured meat or in the production of pet food. Advantageous processes for preparing culture media and the process of hydrolyzing proteins into shorter peptide chains and / or single amino acids are also provided by the present invention. Petition 870250094777, dated 10 / 16 / 2025, pages 85 / 103

Claims

1 / 5 CLAIMS 1. Culture medium for cell cultivation, characterized in that it comprises: a protein hydrolysate as a source of amino acids, wherein the protein hydrolysate is prepared by enzymatic hydrolysis of a protein substrate, wherein a total amino acid input from the protein hydrolysate, including amino acids in the form of suitable bioavailable peptides or derivatives, is at least 75% by weight of the total input of all amino acids in the culture medium; and wherein the cells are non-human metazoan cells.

2. Culture medium, according to claim 1, characterized in that at least two types of enzymes are used for enzymatic hydrolysis, wherein the at least two types of enzymes comprise: i) at least one endoprotease and at least one exoprotease, or ii) at least two of the following types of enzymes: serine protease, cysteine ​​protease, metalloprotease, glutamic protease or aspartic protease.

3. Culture medium, according to any one of claims 1 to 2, characterized in that the protein substrate for the protein hydrolysate comprises at least one of soybean, pea, rice, wheat, corn, broad beans, alfalfa, hemp, chickpea, potato, pumpkin, rapeseed, red lentil, Spirulina, Chlorella, sunflower, duckweed, mung bean or baker's yeast.

4. Culture medium, according to any one of claims 1 to 3, characterized in that it additionally comprises one or more of: vitamins, sugars, minerals, organic amines, micronutrients, iron supplementation compounds, shear protectors or low-abundance organic compounds.

5. Culture medium, according to any one of claims 1 to 4, characterized in that it further comprises at least one exogenous signaling protein of a ligand from the FGF family, insulin, insulin-like growth factor 1, TGF family ligand or transfemna; wherein the concentration of the exogenous signaling protein is up to 50 mg / L.

6. Culture medium for cell cultivation, characterized in that it comprises: a protein hydrolysate as a source of amino acids, wherein the protein hydrolysate is prepared by enzymatic hydrolysis of a protein substrate, wherein the total input of supplemented amino acids added to the culture medium separately from the protein hydrolysate is in a range of 0.1 g / L to 10 g / L; and wherein the cells are non-human metazoan cells.

7. Culture medium according to claim 6, characterized in that the culture medium comprises at least one essential amino acid, wherein the highest possible conversion efficiency of the total essential amino acids is in the range of 30% to 100%, as calculated by equation (1): EAA A EA AM ΣA[·ααμ EAAC T4 EAAC * 100, where: Heaa is the highest possible conversion efficiency for a given amino acid, Aeaam is the content of that specific essential amino acid in 100 g of protein in the culture medium, ΣAεααμ is the total content of all essential amino acids in 100 g of protein in the culture medium, Aeaac is the content of that specific essential amino acid in 100 g of Petition 870250094777, of 10 / 16 / 2025, p.100 / 103 3 / 5 cellular protein, and wherein the amino acid concentrations in grams per 100 grams of total essential amino acids introduced into the cultivation process are 0.3 g 6.04 g for His, 0.57 g 11.41 g for Ile, 1.00 g 19.91 g for Leu, 0.92 g 18.34 g for Lys, 0.32 g 6.49 g for Met, 0.53 g 10.51 g for Phe, 0.54 g 10.74 g for Thr, 0.18 g 3.58 g for Trp, 0.65 g 12.98 g for Val; and wherein the highest possible conversion efficiency is determined by the most limiting essential amino acid.

8. Culture medium, according to any one of claims 5 to 6, characterized in that it additionally comprises a supplemented amino acid comprising at least one of: L-methionine, L-cysteine, L-cystine, L-ornithine, L-tryptophan, L-histidine or L-threonine, added separately from the protein hydrolysate.

9. Culture medium, according to any one of claims 5 to 7, characterized in that the culture medium comprises an organic amine, wherein the organic amine is at least one of putrescine or ethanolamine.

10. Culture medium, according to any one of claims 5 to 8, characterized in that the culture medium comprises a micronutrient, wherein the micronutrient comprises at least one of: spermine, spermidine, putrescine, thymidine, L-ornithine, ethanolamine, myo-inositol or choline.

11. Culture medium, according to any one of claims 5 to 9, characterized in that the culture medium comprises a shear guard, wherein the shear guard comprises at least one of: polyethylene glycol (PEG), methylcellulose (MC), (hydroxypropyl)methylcellulose (HPMC), hydroxypropylcellulose (HPC), carboxymethylcellulose (CMC) or dextran sulfate.

12. Culture medium for cell cultivation, characterized in that it comprises: Petition 870250094777, dated 10 / 16 / 2025, page 101 / 103 4 / 5 a protein hydrolysate as a source of amino acids, wherein the protein hydrolysate is prepared by enzymatic hydrolysis of a protein substrate, wherein a total input of protein hydrolysate, expressed as weight of dry protein introduced into the culture medium, is in the range of 8 g / L to 50 g / L; and wherein the cells are non-human metazoan cells.

13. Culture medium, according to claim 12, characterized in that enzymatic hydrolysis occurs in a hydrolysis reaction mixture comprising enzymes and protein substrate, wherein the total enzyme concentration in the hydrolysis reaction mixture is in the range of 0.05 to 5%, expressed as a ratio between the enzyme concentration and the protein substrate concentration in the reaction mixture.

14. Culture medium, according to any one of claims 12 to 13, characterized in that the degree of enzymatic hydrolysis of the protein substrate, defined as the percentage of peptide bonds hydrolyzed in relation to the total amount of peptide bonds present in the substrate at the beginning of the hydrolysis reaction, is in the range of 10% to 60%.

15. Culture medium, according to any one of claims 12 to 14, characterized in that an amount of the amino acid source in the range of 20% to 100% is converted during hydrolysis into free amino acids and short peptides smaller than 500 Da.

16. Culture medium, according to any one of claims 12 to 15, characterized in that enzymatic hydrolysis occurs in a hydrolysis reaction mixture comprising the protein substrate and the enzyme, wherein the protein substrate is at a concentration in the range of 30 g / L to 130 g / L.

17. Culture medium, according to any one of claims 12 to 16, characterized in that the protein substrate is additionally enzymatically treated by a phytase enzyme. Petition 870250094777, dated 10 / 16 / 2025, pp. 102 / 103 5 / 5 18. Culture medium, according to any one of claims 12 to 17, characterized in that the protein hydrolysate is heat-treated at the end of hydrolysis to deactivate enzymes and kill microorganisms.

19. Culture medium, according to any one of claims 12 to 18, characterized in that the culture medium is additionally filtered using a filter with a pore size in the range of 0.001 µm to 10 µm.

20. Culture medium, according to any one of claims 12 to 19, characterized in that the protein substrate is subjected to an initial heat pretreatment to improve solubility and susceptibility to hydrolysis. Petition 870250094777, dated 10 / 16 / 2025, p. 103 / 103

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