Co-hydrolysis, waste repurposing methods for culture media and cultivation system

Abstract

The present invention relates to the production of culture medium comprising a co-hydrolysis of plant- based protein source by microorganisms. The invention further relates to the use of this culture medium for the cultivation of non-human metazoan cells used as a consumable product for human and / or animal consumption. Further, the invention provides novel methods for repurposing waste molecules and by- products associated with preparation of culture medium and cultivation of non-human metazoan cells.

Description

[0001] CO-HYDROLYSIS, WASTE REPURPOSING METHODS FOR CULTURE MEDIA AND CULTIVATION SYSTEM ABSTRACT

[0002] [1] The present invention relates to the production of culture medium comprising a co-hydrolysis of plantbased protein source by microorganisms. The invention further relates to the use of this culture medium for the cultivation of non-human metazoan cells used as a consumable product for human and / or animal consumption. Further, the invention provides novel methods for repurposing waste molecules and byproducts associated with preparation of culture medium and cultivation of non-human metazoan cells.

[0003] FIELD OF THE INVENTION

[0004] [2] The present invention relates to the field of cell cultivation and the innovative utilization of by-products generated during this process.

[0005] [3] The present invention relates to the field of cell cultures. In particular, it relates to systems and methods for production of culture medium for cell cultivation.

[0006] BACKGROUND OF THE INVENTION

[0007] [4] Cell cultures have been established and used to study animal cell behavior in vitro since the early 20th century. Over the past century, cell culturing techniques have advanced significantly, enabling the production and isolation of biological molecules derived from these cultures. Nowadays, these cell cultures have been used in various fields of research such as the biosimilar drug industry, regenerative medicine, cell-based therapies, vaccine and antibody production, and more recently in the production of cultured meat. These applications rely on highly precise and controlled cell culturing processes.

[0008] [5] A key challenge in this area is the formulation of culture media that is free of animal products (i.e., animal-free media) that is both nutritionally adequate and economically viable at an industrial scale. One promising approach is the use of plant-based protein hydrolysates, such as those derived from soy or pea protein, which can serve as a cost-effective and ethical source of amino acids and peptides.

[0009] [6] However, plant-derived protein sources often present significant drawbacks. These include the presence of anti-nutritional compounds, most notably inositol hexaphosphate (phytic acid) and its derivatives. Such compounds are known to chelate essential minerals, leading to the formation of precipitates in the culture medium. Furthermore, they can interfere with cell growth and metabolic activity, compromising the reproducibility and efficiency of cell culture processes. In addition, protein hydrolysates prepared from plant-based sources may exhibit batch-to-batch variability, incomplete hydrolysis, and other biochemical inconsistencies that undermine their utility in sensitive biotechnological applications.

[0010] [7] To address these challenges, the present invention introduces a novel and synergistic method for producing high-quality protein hydrolysates. This method involves the co-hydrolysis of two biologically distinct protein sources: (i) a plant -based protein isolate that is rich in protein but may contain undesirable compounds such as inositol hexaphosphate; and (ii) a microorganism, such as Saccharomyces cerevisiae, which not only contributes valuable proteins but also provides enzymes with specific activity during processing.

[0011] [8] Particular microorganisms are known to produce enzymes with specific activity (e.g. proteolytic activity and phytase activity), particularly during autolysis or metabolic activity. These enzymes can degrade complex protein structures and cleave phosphate groups from inositol hexaphosphate and / or its derivatives. When the particular microorganism is combined with the plant -based protein source under appropriate processing conditions, there are benefits from both exogenously added enzymes and endogenously produced enzymes. This combination results in a cooperative hydrolysis mechanism in which the total amount of exogenously supplied enzymes can be significantly reduced. Moreover,particular microorganisms are capable of undergoing autolysis through various endogenously produced enzymes, thereby providing an additional source of protein beyond the plant -based protein source.

[0012] [9] The synergistic action of endogenously produced enzymes by microorganisms and exogenously supplied enzymes results in more efficient breakdown of proteins into bioavailable peptides and amino acids while also removing inhibitory compounds such as inositol hexaphosphate. In parallel, the release of phosphate ions from the degradation of inositol hexaphosphate and its derivatives can help meet the phosphate requirements of cell culture media, potentially reducing or eliminating the need for the supplementation of additional inorganic phosphate.

[0013]

[0010] The combined action of endogenously produced enzymes by microorganisms and exogenously supplied enzymes leads to a more efficient breakdown of proteins into bioavailable peptides and amino acids. Each type of enzyme acts independently and contributes to the hydrolysis process in distinct ways:

[0014] (a) Endogenously produced enzymes (such as proteases, peptidases, and glycosidases) are naturally secreted by the microorganisms and catalyze the initial breakdown of the protein source, breaking down complex molecules into simpler peptides and amino acids.

[0015] (b) Exogenously supplied enzymes, which are added to the system, can complement or enhance the activity of the endogenous enzymes by targeting specific peptide bonds.

[0016] Additionally, the enzymatic breakdown of inositol hexaphosphate and its derivatives by both endogenous and exogenous enzymes facilitates the release of phosphate ions. These released phosphate ions can help meet the phosphate requirements of cell culture media, potentially reducing or eliminating the need for supplemental inorganic phosphate. This dual action also helps remove inhibitory compounds, such as inositol hexaphosphate, which could otherwise hinder cell growth and metabolism.

[0017]

[0011] Following enzymatic treatment, the resulting protein hydrolysate is typically subjected to separation and sterilization steps. These are necessary to remove insoluble residues and microbial contaminants, yielding a purified, non-toxic, and cell-compatible protein hydrolysate. The final product may then be combined with other defined components to formulate complete, animal-free media suitable for supporting the growth of various metazoan cells, including those used for cultured meat production, tissue engineering, and recombinant protein production.

[0018]

[0012] This co-hydrolysis approach enables a cost-effective, scalable, and nutritionally rich solution for nextgeneration culture media, overcoming limitations of single-source hydrolysates while reducing reliance on high-load enzyme supplementation and synthetic additives.

[0019]

[0013] The sources of protein, the foundation of the culture medium for the cultivation of non-human metazoan cells, may contain by-products which may not be further implemented for the use in the culture medium and are therefore removed from it.

[0020]

[0014] In addition, after cultivation of non-human metazoan cells, the waste medium comprises a significant concentration of waste molecules. These include both those generated by the cells during the cultivation and unused molecules from the culture medium that are not metabolized by the cells. These waste molecules are mixed with the culture media rendering it toxic, even though there are still essential nutrients in the waste culture medium. This results in a higher demand for culture medium and an excessive amount of culture medium needed for the proliferation of the cells, making the process highly inefficient.

[0021]

[0015] Additionally, these waste molecules are rich in nitrogen and phosphorus that pose a serious environmental risk if they are not properly reduced by wastewater treatment, as it can lead to eutrophication when discharged into natural water bodies, thereby damaging aquatic ecosystems.

[0022]

[0016] Additionally, the cultivation of non-human metazoan cells generates carbon dioxide, a greenhouse gas that may pose potential environmental risks and regulatory compliance.

[0023]

[0017] The present invention addresses these challenges by disclosing the methods for the repurposing of byproducts generated during the culture medium production, including waste molecules within the wastemedium comprising those generated by non-human metazoan cells and / or those from the components of culture medium not metabolized by the non-human metazoan cells.

[0024] BRIEF SUMMARY OF THE INVENTION

[0025]

[0018] The present intention relates to the field of culture medium for culturing non -human metazoan cells, comprising a plant-based protein material and microbial biomass. The natural production of phytases and proteases by microorganisms acts synergistically with exogenously added phytase and proteases to enhance the enzymatic hydrolysis of the plant -based protein source. This results in the generation of a high-quality protein hydrolysate, providing a highly functional culture medium suitable for supporting the growth and maintenance of non-human metazoan cells.

[0026]

[0019] In the present invention, the waste medium and / or concentrate of the waste medium from non-human metazoan cell cultivation are reused; waste medium and / or concentrate may be supplemented with nutritional additives or any other appropriate supplements to prepare culture medium for non-human metazoan cells (direct recycling), or by introducing converting organisms into the waste medium or waste medium concentrate (microorganism-assisted recycling / repurposing), or by any other suitable physico-chemical methods of conversion to culture medium for non-human metazoan cells or to other products (indirect recycling / repurposing).

[0027]

[0020] In cases where the reuse of the waste medium and / or concentrate is not feasible, safe, robust, and economical methods of waste medium disposal are required. The process of waste medium disposal is disclosed in the present invention.

[0028]

[0021] Culture medium for non-human metazoan cells, which is generated at least in part by direct recycling, microorganism-assisted recycling or indirect recycling of waste medium and / or concentrate of waste medium from non-human metazoan cell cultivation, is further referred to as rejuvenated culture medium. The process of generation and use of rejuvenated culture medium are disclosed in the present invention.

[0029]

[0022] Microorganisms may be cultivated in the waste medium and / or waste medium concentrate from non- human metazoan cell cultivation to produce microbial biomass, which may later be broken down into nutrients, such as amino acids, by enzymatic hydrolysis, autolysis, or other suitable processes; the process of breaking down microbial biomass into nutrients may happen directly in the waste medium and / or waste medium concentrate, or it may happen after separation of the microbial biomass from the waste medium and / or waste medium concentrate. The nutrients obtained therein, further referred to as microbial biomass lysate, and may be used to prepare culture medium for non-human metazoan cells. The processes of microbial cultivation to produce microbial biomass, the preparation of microbial biomass lysate, and the use of microbial biomass lysate for the preparation of culture media for and cultivation of non-human metazoan cells are disclosed in the present invention.

[0030]

[0023] In one aspect of the invention, the term lysate may refer to a hydrolysate obtained by the process of autolysis, enzymatic hydrolysis, thermal hydrolysis, acidic hydrolysis, or any other suitable method of hydrolysis.

[0031]

[0024] Microorganisms may be cultivated in the waste medium and / or waste medium concentrate, wherein the microorganisms may be used for the production of recombinant proteins, or to produce fungal mycelium further utilized with the non-human metazoan cells for food production.

[0032]

[0025] Microorganisms may produce biogas by utilizing waste molecules, wherein the biogas may be used as a renewable energy source.

[0033]

[0026] The use of waste medium concentrate as fertilizer in agriculture provides essential nutrients, such as nitrogen and phosphorus, to enhance soil fertility, offering a valuable alternative to synthetic fertilizers.

[0034]

[0027] All of the aforementioned aspects of the invention are described in the detailed description of the present invention.BRIEF DESCRIPTION OF THE DRAWINGS

[0035]

[0028] Fig. 1: Illustrates the scheme of the preparation of the culture medium with production of sediment and waste medium.

[0036]

[0029] Fig. 2: Illustrates the scheme of the reuse of the waste medium by the cultivation system.

[0037]

[0030] Fig. 3: Illustrates the scheme of an exemplary aspect of the invention illustrated in Fig. 2.

[0038]

[0031] Fig. 4: Illustrates the scheme of direct recycling.

[0039]

[0032] Fig. 5: Illustrates the scheme of an exemplary aspect of the invention illustrated in Fig. 4.

[0040]

[0033] Fig. 6: Illustrates the scheme of the reuse of the waste medium by the cultivation system selected for the cultivation of bacteria, microalgae, yeasts, plant cells and / or other converting organisms.

[0041]

[0034] Fig. 7: Illustrates the scheme of an exemplary aspect of the invention illustrated in Fig. 6.

[0042]

[0035] Fig. 8: Illustrates the scheme of the reuse of the waste medium by cultivation of plants, macroalgae, fungi or any other converting organism for the production of food / feed products.

[0043]

[0036] Fig. 9: Illustrates the scheme of an exemplary aspect of the invention illustrated in Fig. 8.

[0044]

[0037] Fig. 10: Illustrates the cultivation of non-human metazoan cells using three different variants of the culture medium prepared from soy or yeast hydrolysates.

[0045]

[0038] Fig. 11: Illustrates a comparison of cell densities of non-human metazoan cells cultured in the culture medium and rejuvenated culture medium.

[0046]

[0039] Fig. 12: Illustrates the composition of the waste medium.

[0047]

[0040] Fig. 13: Illustrates a schematic diagram of protein hydrolysate processing.

[0048]

[0041] Fig.14: Illustrates the scheme of a cultivation system used for cultivation of non-human metazoan cells.

[0049]

[0042] Fig. 15: Illustrates a schematic diagram of a cultivation system including waste medium recycling

[0043] Fig. 16: Illustrates an exemplary aspect of the invention according to the scheme in Fig. 15

[0050]

[0044] Fig. 17: Illustrates a schematic diagram of a cultivation system including waste medium recycling

[0045] Fig. 18: Illustrates an exemplary aspect of the invention according to the scheme in Fig. 17

[0051]

[0046] Fig. 19: Illustrates a schematic diagram of cultivation system including waste medium recycling

[0047] Fig. 20: Illustrates an exemplary aspect of the invention according to the scheme in Fig. 19

[0052]

[0048] Fig. 21: Illustrates an exemplary scheme of fermentation and co-hydrolysis processes.

[0053]

[0049] Fig. 22: Illustrates up-scaled cultivation system variant 1.

[0054]

[0050] Fig. 23: Illustrates a graphical aspect of the invention according to scheme in Fig. 22.

[0055]

[0051] Fig. 24: Illustrates up-scaled cultivation system variant 2.

[0056]

[0052] Fig. 25: Illustrates a graphical aspect of the invention according to scheme in Fig. 24.

[0057]

[0053] Fig. 26: Illustrates gas recycling system variant 1.

[0058]

[0054] Fig. 27: Illustrates a graphical aspect of the invention according to scheme in Fig. 26.

[0059]

[0055] Fig. 28: Illustrates gas recycling system variant 2.

[0060]

[0056] Fig. 29: Illustrates a graphical aspect of the invention according to scheme in Fig. 28.

[0061]

[0057] Fig. 30: Illustrates gas recycling system variant 3.

[0062]

[0058] Fig. 31: Illustrates a graphical aspect of the invention according to scheme in Fig. 30.

[0063]

[0059] Fig. 32: Illustrates gas recycling system variant 4.

[0064]

[0060] Fig. 33: Illustrates a graphical aspect of the invention according to scheme in Fig. 32.

[0065] CROSS-REFERENCE TO RELATED APPLICATIONS

[0066]

[0061] The PCT application PCT / IB2024 / 053805 filed 18 April 2024, and PCT application PCT / IB2024 / 059990 filed 11 October 2024, and U.S. Provisional Patent Application No. 63 / 589,661 filed October 12, 2023, and U.S. Provisional Patent Application No. 63 / 555,543 filed February 20, 2024, and U.S. Provisional Patent Application No. 63 / 570,973 filed March 28, 2024, and U.S. Provisional Patent Application No. 63 / 654,493 filed May 31, 2024, and U.S. Non -Provisional Patent Application No. 18 / 731,896 filed on June 3, 2024, and U.S. Non-Pro visional Patent Application No. 18 / 763,199 filed on July 3, 2024, and U.S. Provisional Patent Application No. 63 / 698,265 filed September 24, 2024, U.S.Provisional Patent Application No. 63 / 497,051 filed April 192023, U.S. Provisional Patent Application No. 63 / 737,932 filed December 23, 2024, U.S. Provisional Patent Application No. 63 / 760,147, U.S. Non-provisional Patent Application No. 18 / 927,171 filed October 25, 2024, U.S. Non-provisional Patent Application No. 19 / 028,830 filed January 17, 2025, U.S. Non-provisional Patent Application No.

[0067] 19 / 028,930 filed January 17, 2025, U.S. Non-provisional Patent Application No. 19 / 029,060 filed January 17, 2025, U.S. Provisional Patent Application No. 63 / 765,920, U.S. Provisional Application No.

[0068] 63 / 785,064 filed April 8, 2025, U.S. Provisional Application No. 63 / 785,075 filed April 8, 2025, U.S. Provisional Application No. 63 / 786,408 filed April 10, 2025, U.S. Non-Provisional Patent Application No. 19 / 175,419 filed April 10, 2025, U.S. Non-Provisional Patent Application No. 19 / 223,759 filed May 30, 2025, U.S. Provisional Patent Application No. 63 / 874,983 filed September 3, 2025, U.S. Provisional Patent Application No. 63 / 898,215 filed October 13, 2025, and the U.S: Provisional Application No.

[0069] 63 / 943,891 filed December 182025, are all incorporated herein by reference in their entirety.

[0070] DETAILED DESCRIPTION OF THE INVENTION

[0071]

[0062] In one aspect of the invention, the culture media for the cultivation of non -human metazoan cells may comprise protein hydrolysate generated by hydrolysis of a source of protein.

[0072]

[0063] As used herein, the term “source of protein” refers to at least one plant used for production of protein hydrolysate. The source of protein may comprise soy, pea, rice, wheat, wheat gluten, corn, fava beans, alfalfa, hemp, chickpea, potato, pumpkin, rapeseed, red lentil, rice, sunflower, water lentil, duckweed, mungbean, bean, and / or any other appropriate plants.

[0073]

[0064] In one aspect of the invention, the source of protein may comprise other substrates not originating from plants, such as brewer’s yeasts, Spirullina, Chlorella, microbial protein or any other appropriate source of protein derived from organisms other than plants.

[0074]

[0065] The source of protein, also referred to as substrate, source of protein, or source of amino acids, may comprise protein hydrolysate prepared from soy, pea, rice, wheat, wheat gluten, corn, faba beans, alfalfa, hemp, chickpea, potato, pumpkin, rapeseed, red lentil, Spirulina, Chlorella, sunflower, water lentil, mung beans, flax, baker's yeast, brewer spent grain, distillers spent grain (DDGS), or tomato pomace, in the form of a powder, lysate, concentrate, isolate, liquid, solid or any other appropriate form. The plant sources of protein disclosed herein often contain phytin, phytic acid, inositol hexaphosphate, or phytate and / or any related form of those compounds, which will be referred to as inositol hexaphosphate and its derivatives in this document.

[0075]

[0066] The protein hydrolysate processing depicted in Fig. 13 may comprise the following steps:

[0076] proteolysis of source of protein (401) by proteolytic enzymes (402) to result in formation of protein hydrolysate (403);

[0077] removal of inositol hexaphosphate and / or its derivatives from protein hydrolysate (403) by the addition of enzymes having phytase activity and / or a precipitating agent (404) resulting in the formation of modified protein hydrolysate (405); and

[0078] removal of solid residues (406) from the modified protein hydrolysate resulting in the formation of a purified protein hydrolysate (407).

[0079]

[0067] According to some aspects of the invention, the terms “source of amino acids”, “substrate”, “proteinous substrate”, “source of protein”, or “protein source” may be used interchangeably when referring to a component of the production of protein hydrolysate and / or the culture medium.

[0080]

[0068] The term “protein hydrolysate” as used herein refers to peptides and / or amino acids generated by hydrolysis of a source of protein.

[0081]

[0069] Hydrolysis is a chemical reaction in which an addition of water molecules breaks one or more chemical bonds, in this case peptide bonds.

[0070] Methods of hydrolysis of a source of protein may include acidic hydrolysis, basic hydrolysis, enzymatic hydrolysis, thermal hydrolysis, or autolysis.

[0082]

[0071] Before the hydrolysis of the source of protein, the source of protein may be pre-treated. This pretreatment may comprise heat treatment, mechanical treatment, enzymatic treatment, chemical treatment or any other suitable processing treatment technique.

[0083]

[0072] The hydrolysis may be performed on a protein isolate or concentrate, protein flour, protein meal, seed or grain from the source of protein, or on the whole biomass of the source of protein. The source of protein may be physically, mechanically, or chemically pretreated, steeped in water to induce germination, or subjected to methods such as soaking, blanching, removal of hull, husk or any other outer layer of the source of protein, milling, heat treatment or any other appropriate method to enhance the speed and efficiency of the hydrolysis process and to reduce the presence of antinutritional compounds. Saccharides, fats or other compounds may be removed from the source of protein to facilitate processing. Examples of suitable industrially scalable sources of amino acids may include soy, pea, rice, wheat, wheat gluten, corn, faba beans, alfalfa, hemp, chickpea, potato, pumpkin, rapeseed, red lentil, Spirulina, Chlorella, sunflower, water lentil, mung beans, flax, brewer spent grain, distillers spent grain (DDGS), tomato pomace, in form of powder, lysate, concentrate, isolate, liquid, solid or any other appropriate form. The present invention is not limited to the listed exemplary sources of amino acids.

[0084]

[0073] In one aspect of the invention, the incubation of the source of proteins in water to induce germination, or its pretreatment with soaking, blanching, milling, heat treatment or any other appropriate method may be performed for a specific incubation time, wherein the incubation time may differ according to used technique.

[0085]

[0074] In one aspect of the invention, the pretreatment of the source of protein may be performed to enhance the yield of cell biomass during the cultivation of non-human metazoan cells using the protein hydrolysate within the culture medium.

[0086]

[0075] The process of hydrolysis entails cleaving the original protein molecule into shorter peptide chains and / or single amino acids. As used herein, the term “protein hydrolysate” is understood to be a mix of amino acids, peptides and other molecules prepared from a suitable source of protein by any suitable method, including acidic, basic, or enzymatic hydrolysis, autolysis, thermal hydrolysis, or lysis by fermentation with a suitable microorganism able to break down the protein. The “protein hydrolysate” according to the present disclosure may be, for example, plant protein enzymatic hydrolysates, various types of yeast extracts or lysates (such as whole yeast autolysate), or algae acidic hydrolysate.

[0087]

[0076] In one aspect of the invention, the microorganism used for fermentation and co-hydrolysis may be selected from the group of Saccharomyces cerevisiae, Pichia pastoris, Schizosaccharomyces pombe, Candida utilis, Debaryomyces hansenii, Yarrowia lipolytica, Aspergillus oryzae, Aspergillus niger, Bacillus subtilis, Kluyveromyces lactis, Kluyveromyces marxianus, Streptococcus thermophilus, Lactobacillus helveticus, Bacillus licheniformis , or any other appropriate microorganism.

[0088]

[0077] Methods of protein hydrolysis may include acidic hydrolysis, basic hydrolysis, enzymatic hydrolysis, thermal hydrolysis or autolysis. Acidic hydrolysis subjects the source of protein to a very low pH, usually at an elevated temperature. The duration of the reaction may be in a range of 1 hour to 96 hours, in a range of 2 hours to 72 hours, in a range of 4 hours to 48 hours, in a range of 4 hours to 36 hours, in a range of 4 hours to 24 hours or in a range of 4 hours to 12 hours. Acidic hydrolysis unfortunately leads to significant degradation of several amino acids, most notably tryptophan, which would then have to be sourced separately at significant costs. Significant degradation of some amino acids also occurs during basic hydrolysis, which subjects the source of protein to a very high pH, usually at an elevated temperature. Additionally, the acid or base used for hydrolysis would have to be removed from the protein hydrolysate before it could be used to cultivate cells, presenting further complications. For example, when acidic hydrolysis is performed using hydrochloric acid, the acid may be removed by neutralization or evaporation. However, both processes are economically unfavorable because: i) theneutralization process results in an unfavorably high concentration of salts, which also need to be removed, and ii) evaporation is energy-intensive and the resulting HC1 vapors pose a health and environmental hazard that would need to be solved. Thermal hydrolysis subjects the source of protein to very high temperatures at which the peptide bonds in the protein will break. However, at these temperatures, undesirable chemical reactions may occur. For example, some amino acids may break down or react with other compounds in the hydrolysate, for example through Maillard reactions with saccharides. Additionally, thermal degradation of the reaction substrate may produce compounds harmful to the cultivated cells. The process of autolysis relies on the activity of the endogenous enzymes of the source organism to break down the source of protein, and this process is usually not very effective and does not generally result in sufficient hydrolysis of the source protein. Additionally, proteins can be broken down by fermentation with organisms such as Bacillus licheniformis, Saccharomyces cerevisiae or Aspergillus oryzae, which produce a large amount of proteolytic enzymes. However, with this approach, some of the amino acids from the source of protein may be consumed by the organism that was used to break down the protein during the process of fermentation. Also, metabolic waste products and other compounds from the fermenting organism may contaminate the resulting lysate and adversely affect its properties in respect to mammalian cell cultivation. Therefore, the fermentation approach may be more suitable to achieve a limited breakdown of the protein source and / or to accumulate enzymes which can function independently of the actual fermenting microorganism - as such, the possible negative effects of microbial presence are minimized, and other methods may subsequently be used to achieve a sufficient degree of protein hydrolysis of the substrate.

[0089]

[0078] The protein hydrolysate according to the invention may be obtained by enzymatic hydrolysis of a suitable source of protein. The use of an industrially scalable source of protein is advantageous. In one aspect of the invention, soy protein isolate may be used as the source of protein for enzymatic hydrolysis. Advantageously, soy protein isolate has a favorable ratio of most amino acids for the purpose of mammalian cell cultivation. However, to achieve optimal levels of certain amino acids that may be present in lower concentrations, these amino acids may need to be supplemented separately in the culture media.

[0090]

[0079] The source of protein for hydrolysis may be subjected to an initial thermal pretreatment in a solvent to improve solubility and susceptibility to hydrolysis. The temperature during the thermal pretreatment may be in the range of 75 °C to 95 °C, or in the range of 80 °C to 92.5 °C, or in the range of 85 °C to 90 °C for a time in the range of 5 minutes to 120 minutes, in the range of 15 minutes to 60 minutes, or in the range of 30 minutes to 45 minutes.

[0091]

[0080] In another aspect of the invention, the temperature during the thermal pretreatment may be in the range of 75 °C to 130 °C, or in the range of 80 °C to 120 °C, or in the range of 85 °C to 115 °C for a time in the range of 5 minutes to 120 minutes, in the range of 15 minutes to 60 minutes, or in the range of 30 minutes to 45 minutes.

[0092]

[0081] In another aspect of the invention, the temperature during the thermal pretreatment may be in the range of 70 °C to 99 °C, or in the range of 80 °C to 98 °C, or in the range of 85 °C to 97 °C for a time in the range of 5 minutes to 120 minutes, in the range of 15 minutes to 60 minutes, or in the range of 30 minutes to 45 minutes. In another aspect of the invention, the hydrolysis tank may be pressurized, therefore temperatures above 100 °C might be used as well.

[0093]

[0082] In some aspects, the hydrolysis reaction comprises an endoprotease. The concentration of the endoprotease, for example ALCALASE® serine endopeptidase enzyme, 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 of the concentration of enzyme to the concentration of protein in the reaction mixture. The concentration of the enzyme may be determined by the Bradford assay, BCA assay or other protein determination assays. The resulting solution has a basic to neutral pH, allowing for a high activity of ALCALASE® serine endopeptidase enzyme.

[0083] In some aspects of the invention, the endopeptidase enzyme may have endopeptidase activity in the range of 0.0001 to 300,000 TU, or in the range of 0.001 to 30,000 TU, or in the range of 0.01 to 3,000 TU per 1 g of enzyme preparation, wherein the one tyrosine unit (TU) is equal to amount of enzyme which liberates one micromole of tyrosine equivalents from casein at 40 °C and at pH 7. In other aspect of the invention, the endopeptidase enzyme may have endopeptidase activity in the range of 0.00001 to 4,000 TU, or in the range of 0.0001 to 40 TU, or in the range of 0.001 to 40 TU per 1 mg of protein in the enzyme preparation, wherein the one tyrosine unit (TU) is equal to amount of enzyme which liberates one micromole of tyrosine equivalents from casein at 40 °C and at pH 7.

[0094]

[0084] The amount of endoprotease, for example ALCALASE® serine endopeptidase enzyme, added to the reaction may be adjusted according to its activity so the resulting endoprotease activity in the reaction mixture may be in the range of 0.01 to 1,000, or 0.1 to 200, or 1 to 50 TU per 1 g of protein.

[0095]

[0085] The temperature for hydrolysis may be in the range of 40 °C to 75 °C, or in the range 45 °C to 70 °C, or in the range of 50 °C to 65 °C. Over a period of constant mixing, which may be in the range of 30 minutes to 24 hours, or in the range of 1 hour to 12 hours, or in the range of 2 hours to 8 hours, the pH of the solution decreases as a result of the hydrolysis of peptide bonds and increased number of carboxylic groups.

[0096]

[0086] This decreased pH allows for a high activity of an exoprotease (i.e., exopepsidase), or exoprotease and endoprotease mix, for example FLAVOURZYME® proteolytic enzyme blend. The concentration of the exoprotease (i.e., exopeptidase) or exoprotease and endoprotease mix 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 3 % expressed as a ratio of the concentration of enzyme to the concentration of protein in the reaction mixture. The concentration of the enzyme may be determined by the Bradford assay, BCA assay or other protein determination assays.

[0097]

[0087] In one aspect of the invention the exopeptidase enzyme may have activity in a range of 0.001 to 2,100,000 LAPU, or 0.01 to 210,000 LAPU, or 0.1 to 21,000 LAPU, wherein one LAPU unit is defined as amount of enzyme which liberates one micromole of p-nitroaniline from L-leucine-p-nitroanilide in one minute at 37 °C and pH 8, per 1 g of enzyme preparation of protein in enzyme solution.

[0098]

[0088] In another aspect of the invention the exopeptidase enzyme may have activity in a range of 0.000005 to 1,500 LAPU, or 0.00005 to 150 LAPU, or 0.0005 to 15 LAPU, wherein one LAPU unit is defined as amount of enzyme which liberates one micromole of p-nitroaniline from L-leucine-p-nitroanilide in one minute at 37 °C and pH 8, per 1 mg of protein in the enzyme preparation.

[0099]

[0089] The amount of exoprotease, for example FLAVOURZYME® proteolytic enzyme blend, added to the reaction may be adjusted according to its activity so the resulting exoprotease activity in the reaction mixture may be in the range of 0.1 to 1,500, or 1 to 500, or 5 to 100 LAPU per 1 g of protein.

[0100]

[0090] The resulting mixture may then be incubated for an additional time period in the range of 1 hour to 50 hours, or in the range of 5 hours to 40 hours, or in the range of 8 hours to 30 hours at temperature in the range of 30 °C to 80 °C, or in the range of 40 °C to 70 °C, or in the range of 50 °C to 60 °C, with constant mixing, after which the residual enzyme is thermally deactivated. With this procedure, 20 % to 100 %, or 25 % to 70 %, or 30 % to 60 % of the source protein may be converted into free amino acids.

[0101]

[0091] The ratio of enzyme to substrate may be optimized to decrease the amount of enzyme, which is the most expensive input of the culture medium production. For example, the total amount of proteolytic enzyme used may be in the range of 20 % to 0.0002 %, or in the range of 6 % to 0.05 %, or in the range of 3 % to 0.1 % of the total amount of source protein used.

[0102]

[0092] The hydrolysis by-products, including the sediment obtained from centrifugation and filtration, may be further utilized as components in subsequent applications. These by-products may be processed or refined to extract useful materials, which can serve as raw ingredients or additives in other industrial or biochemical processes, thereby enhancing the overall efficiency and sustainability of the production system. In one aspect of the invention, the sediment may be used for the production of edible products used for animal and / or human consumption.

[0093] The term “sediment” or “solid residues” refers to solid particles larger than 0.2 pm derived from the input of the hydrolysis tank, such as the source of protein or the enzymes entering the hydrolysis tank. In another aspect of the invention, the solid particles that may be subjected to filtration are not limited to the source of protein, enzymes or to other components entering the hydrolysis tank, but may also comprise solid particles formed in the process of preparing culture medium. It is also noted that, as an incidental effect of the solid residue removal process, particles smaller than 0.2 pm may be also removed.

[0103]

[0094] In one aspect of the invention, total amino acid composition of the sediment may comprise:

[0104] alanine in a range of 0.5 wt. % to 12 wt. %, in a range of 1 wt. % to 10 wt. %, or in a range of 2 wt. % to 8 wt. % of dry mass weight;

[0105] arginine in a range of 1 wt. % to 14 wt. %, in a range of 1.5 wt. % to 12 wt. %, or in a range of 2.5 wt. % to 10 wt. % of dry mass weight;

[0106] glycine in a range of 0.5 wt. % to 10 wt. %, in a range of 1 wt. % to 8 wt. %, or in a range of 1.5 wt. % to 6 wt. % of dry mass weight;

[0107] isoleucine in a range of 0.5 wt. % to 12 wt. %, in a range of 1 wt. % to 10 wt. %, or in a range of 2 wt. % to 8 wt. % of dry mass weight;

[0108] lysine in a range of 0.5 wt. % to 11 wt. %, in a range of 1 wt. % to 9 wt. %, or in a range of 1.5 wt. % to 7 wt. % of dry mass weight;

[0109] phenylalanine in a range of 1 wt. % to 13 wt. %, in a range of 1.5 wt. % to 11 wt. %, or in a range of 2.5 wt. % to 9 wt. % of dry mass weight;

[0110] proline in a range of 0.5 wt. % to 10 wt. %, in a range of 1 wt. % to 8 wt. %, or in a range of 1.5 wt. % to 6 wt. % of dry mass weight;

[0111] serine in a range of 0.5 wt. % to 12 wt. %, in a range of 1 wt. % to 10 wt. %, or in a range of 2 wt. % to 8 wt. % of dry mass weight;

[0112] threonine in a range of 0.5 wt. % to 10 wt. %, in a range of 1 wt. % to 8 wt. %, or in a range of 1.5 wt. % to 6 wt. % of dry mass weight;

[0113] tyrosine in a range of 0.5 wt. % to 10 wt. %, in a range of 1 wt. % to 8 wt. %, or in a range of 1.5 wt. % to 6 wt. % of dry mass weight;

[0114] valine in a range of 1.5 wt. % to 14 wt. %, in a range of 2 wt. % to 12 wt. %, or in a range of 2.5 wt. % to 10 wt. % of dry mass weight;

[0115] aspartic acid in a range of 1 wt. % to 16 wt. %, in a range of 2 wt. % to 14 wt. %, or in a range of 3 wt. % to 12 wt. % of dry mass weight;

[0116] glutamic acid in a range of 1 wt. % to 18 wt. %, in a range of 3 wt. % to 16 wt. %, or in a range of 3.5 wt. % to 14 wt. % of dry mass weight;

[0117] leucine in a range of 1 wt. % to 18 wt. %, in a range of 3 wt. % to 16 wt. %, or in a range of 3.5 wt. % to 14 wt. % of dry mass weight; andhistidine in a range of 0.15 wt. % to 8 wt. %, in a range of 0.5 wt. % to 6 wt. %, or in a range of 1 wt. % to 4 wt. % of dry mass weight.

[0118]

[0095] In one aspect of the invention, the concentration of saccharides in the sediment may be in a range of 15 wt. % to 80 wt. %, in a range of 18 wt. % to 75 wt. %, or in a range of 20 wt. % to 70 wt. % of dry mass weight.

[0119]

[0096] The method of enzymatic hydrolysis may use proteolytic enzymes in order to achieve protein hydrolysis at much milder conditions than acidic or basic hydrolysis, therefore preserving the amino acids of the original protein.

[0120]

[0097] The term “proteolytic enzymes” refers to enzymes from the group of proteases, peptidases, esterases and / or any other enzyme that is capable of cleaving of peptide bonds between amino acids and / or is capable of adding a water molecule to an ester to produce alcohol or an acid.

[0121]

[0098] Proteolytic enzymes may be derived from plants, animals, microorganisms, or any other appropriate source separately or in combination.

[0122]

[0099] Exemplary enzymes that may be used to catalyze the breakdown of peptide bonds are ALCALASE® serine endopeptidase enzyme (protease from Bacillus licheniformis), Subtilisin Carlsberg (protease from Bacillus licheniformis), FLAVOURZYME® proteolytic enzyme blend (protease from Aspergillus oryzae). PROTAMEX® enzyme preparation, NOVOPRO® D protease enzyme, PROTANA® Prime, THERMOASE® PC10FNA protease, Protease AN Amano 100SD, Protease A Amano 2SD, Protease M Amano SD, Protease P Amano 6SD, ProteAX, Peptidase R, Alkaline Protease, COROLASE®C 7089 enzyme preparation, COROLASE® 2TSN enzyme preparation, COROLASE® 8000 enzyme preparation, COROLASE® 7000 enzyme preparation, MAXIPRO® TNP protease, MAXIPRO® FPC protease, MAXIPRO® BAP protease, MAXIPRO® NPU protease, Papain, Bromelain, SUMIZYME® BNP-L enzyme, SUMIZYME® AP-L enzyme, SUMIZYME® LP-G enzyme, SUMIZYME® FP-G enzyme, SUMIZYME® FL-G enzyme, PROLYVE® Exo L enzyme, PROLYVE® ALK3000L enzyme (alkaline protease), PROLYVE® BS2 Liquid enzyme, PROLYVE® NP Liquid enzyme (neutral protease), PROLYVE® SMIX enzyme (protease blend), PROMOD® 24L enzyme (protease preparation), PROMOD® 295L enzyme (protease preparation), PROMOD® 327L enzyme (protease preparation), PROMOD® 295L enzyme, PROMOD® 327L enzyme, PROMOD® 987MDP enzyme (protease preparation), FLAVORPRO® 766MDP enzyme (protease preparation), FLAVORPRO® 766MDP enzyme, FLAVORPRO® 750MDP enzyme, FLAVORPRO® 091MDP enzyme, ENZECO® BROMELAIN 240 MCU enzyme (bromelain protease from Ananas comosus). ENZECO® FUNGAL ACID PROTEASE CONCENTRATE enzyme (acid protease from Aspergillus niger), ENZECO® PROTEASE 400 WF enzyme, ENZECO® FICIN 50K enzyme (protease from Ficus carica), and PANOL® 300 MCU enzyme (plant-derived protease preparation), or any other appropriate proteolytic enzyme, or any combination thereof.

[0123]

[0100] Proteolytic enzymes may be used either alone or in combination. The number of proteolytic enzymes or enzyme mixes used may be in a range of 1 to 6, or in a range of 2 to 5, or in a range of 2 to 4.

[0124]

[0101] In one aspect of the invention, at least one enzyme and / or enzyme mix may be used for the catalysis of the breakdown of peptide bonds in the source of protein.

[0125]

[0102] The duration of the treatment by proteolytic enzymes may differ according to the source of protein used. Proteolytic enzymes may be used for a portion of time in a range of 4 hours to 40 hours, in a range of 6 hours to 35 hours, in a range of 7 hours to 30 hours, or in a range of 10 hours to 25 hours. In one aspect of the invention, the proteolytic enzymes may be used for a portion of time of at least 2 hours, at least 2.5 hours, at least 3 hours, at least 3.5 hours, at least 4 hours, at least 4.5 hours, at least 5 hours, or at least 10 hours.

[0103] The temperature during the proteolysis treatment may depend on the temperature optimum of the selected proteolytic enzyme. This enzyme and / or enzymes may be used in a range of 25 °C to 80 °C, in a range of 30 °C to 70 °C, or in a range of 40 °C to 65 °C.

[0126]

[0104] Proteolytic enzymes may contain additives used to maintain pH and stability. Examples of additives include, but are not limited to water, citric acid, glycerol, potassium sorbate, sodium chloride, sorbitol, dextrin, sucrose or any other appropriate agent used for maintaining stability and pH.

[0127]

[0105] The enzymes may further comprise additives capable of maintaining stability and pH, wherein the total composition comprises additives in a range of 10 wt. % to 95 wt. % of total composition, 20 wt. % to 92 wt. % of total composition, 25 wt. % to 90 wt. % of total composition, or 30 wt. % to 88 wt. % of total composition.

[0128]

[0106] In one aspect of the invention, the concentration of the source of protein in the reaction mixture for hydrolysis may be in the range of 1 g / L to 150 g / L, or in the range of 30 g / L to 100 g / L, or in the range of 40 g / L to 80 g / L of the reaction mixture.

[0129]

[0107] In another aspect of the invention, the concentration of the source of protein in the reaction mixture for hydrolysis may be in the range of 0.5 g / L to 250 g / L, 1 g / L to 150 g / L, or in the range of 30 g / L to 100 g / L, or in the range of 40 g / L to 80 g / L of the reaction mixture.

[0130]

[0108] The concentration of the enzyme 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 of the concentration of enzyme to the concentration of protein in the reaction mixture. The concentration of the enzyme may be determined by the Bradford assay, BCA assay or other protein determination assays.

[0131]

[0109] One of the approaches to characterize protein hydrolysates may be size-exclusion chromatography (SEC) combined with mass spectrometry (MS) or photo diode array (PDA) which allows for the separation of the peptides from hydrolysate based on their molecular weights. The results may vary according to mobile phase composition, column type and its specification, flow rate, temperature, sample injection parameters, detector settings and / or any other parameter used.

[0132]

[0110] In one aspect of the invention, proteolytic enzymes endogenous to a source of protein may yield peptides in the purified protein hydrolysate with a molecular weight higher than 17 kDa, with a relative fraction size in a range of 0 % to 10 % of the purified protein hydrolysate, in a range of 0 % to 8 % of the purified protein hydrolysate, or in a range of 0 % to 6 % of the purified protein hydrolysate.

[0133]

[0111] In one aspect of the invention, proteolytic enzymes endogenous to a source of protein may yield peptides in the purified protein hydrolysate with a molecular weight ranging from 6.7 kDa to 17 kDa, with a relative fraction size in a range of 2 % to 35 % of the purified protein hydrolysate, in a range of 4 % to 30 % of the purified protein hydrolysate, or in a range of 5 % to 25 % of the purified protein hydrolysate.

[0134]

[0112] In another aspect of the invention, proteolytic enzymes endogenous to a source of protein may yield peptides in the purified protein hydrolysate with a molecular weight ranging from 6.7 kDa to 17 kDa, with a relative fraction size in a range of 1 % to 35 % of the purified protein hydrolysate, in a range of 4 % to 30 % of the purified protein hydrolysate, or in a range of 5 % to 25 % of the purified protein hydrolysate.

[0135]

[0113] In one aspect of the invention, proteolytic enzymes endogenous to a source of protein may yield peptides in the purified protein hydrolysate with a molecular weight ranging from 1.7 kDa to 6.7 kDa, with a relative fraction size in a range of 5 % to 40 % of the purified protein hydrolysate, in a range of 10 % to 35 % of the purified protein hydrolysate, or in a range of 15 % to 30 % of the purified protein hydrolysate.

[0136]

[0114] In another aspect of the invention, proteolytic enzymes endogenous to a source of protein may yield peptides in the purified protein hydrolysate with a molecular weight ranging from 1.7 kDa to 6.7 kDa, with a relative fraction size in a range of 1 % to 40 % of the purified protein hydrolysate, in a rangeof 10 % to 35 % of the purified protein hydrolysate, or in a range of 15 % to 30 % of the purified protein hydrolysate.

[0137]

[0115] In one aspect of the invention, proteolytic enzymes endogenous to a source of protein may yield peptides in the purified protein hydrolysate with a molecular weight ranging from 1 kDa to 1.7 kDa, with a relative fraction size in a range of 5 % to 40 % of the purified protein hydrolysate, in a range of 10 % to 35 % of the purified protein hydrolysate, or in a range of 20 % to 30 % of the purified protein hydrolysate.

[0138]

[0116] In another aspect of the invention, proteolytic enzymes endogenous to a source of protein may yield peptides in the purified protein hydrolysate with a molecular weight ranging from 1 kDa to 1.7 kDa, with a relative fraction size in a range of 1 % to 40 % of the purified protein hydrolysate, in a range of 10 % to 35 % of the purified protein hydrolysate, or in a range of 20 % to 30 % of the purified protein hydrolysate.

[0139]

[0117] In one aspect of the invention, proteolytic enzymes endogenous to a source of protein may yield free amino acids and / or peptides in the purified protein hydrolysate with a molecular weight less than 1 kDa, with a relative fraction size in a range of 20 % to 70 % of the purified protein hydrolysate, in a range of 22 % to 60 % of the purified protein hydrolysate, or in a range of 25 % to 55 % of the purified protein hydrolysate.

[0140]

[0118] In another aspect of the invention, proteolytic enzymes endogenous to a source of protein may yield free amino acids and / or peptides in the purified protein hydrolysate with a molecular weight less than 1 kDa, with a relative fraction size in a range of 20 % to 100 % of the purified protein hydrolysate, in a range of 22 % to 70 % of the purified protein hydrolysate, or in a range of 25 % to 60 % of the purified protein hydrolysate.

[0141]

[0119] In one aspect of the invention, proteolytic enzymes endogenous to a source of protein may yield free amino acids in the purified protein hydrolysate with a relative fraction size in a range of 30 % to 50 % of the purified protein hydrolysate, in a range of 35 % to 45 % of the purified protein hydrolysate, or in a range of 38 % to 42 % of the purified protein hydrolysate.

[0142]

[0120] In another aspect of the invention, proteolytic enzymes endogenous to a source of protein may yield free amino acids in the purified protein hydrolysate with a relative fraction size in a range of 20 % to 100 % of the purified protein hydrolysate, in a range of 25 % to 70 % of the purified protein hydrolysate, or in a range of 30 % to 60 % of the purified protein hydrolysate.

[0143]

[0121] In one aspect of the invention, an additional method may be used to further separate the small peptides with molecular weights ranging from 1,000 Da to 6,500 Da, or 600 Da to 1,000 Da, or 300 Da to 600 Da, or molecular weights lower than 300 Da.

[0144]

[0122] In one aspect of the invention, proteolytic enzymes endogenous to a source of protein may yield amino acids and / or peptides in the purified protein hydrolysate with a molecular weight ranging from 1,000 Da to 6,500 Da, with a relative fraction size in a range of 5 % to 50 %, in a range of 10 % to 40 %, or in a range of 15 % to 35 % of the measured fractions.

[0145]

[0123] In one aspect of the invention, proteolytic enzymes endogenous to a source of protein may yield amino acids and / or peptides in the purified protein hydrolysate with a molecular weight ranging from 1,000 Da to 6,500 Da, with a relative fraction size in a range of 1 % to 70 %, in a range of 5 % to 40 %, or in a range of 10 % to 35 % of the measured fractions.

[0146]

[0124] In one aspect of the invention, proteolytic enzymes endogenous to a source of protein may yield amino acids and / or peptides in the purified protein hydrolysate with a molecular weight ranging from 600 Da to 1,000 Da, with a relative fraction size in a range of 10 % to 70 %, in a range of 15 % to 60 %, or in a range of 20 % to 50 % of the measured fractions.

[0147]

[0125] In one aspect of the invention, proteolytic enzymes endogenous to a source of protein may yield amino acids and / or peptides in the purified protein hydrolysate with a molecular weight ranging from600 Da to 1,000 Da, with a relative fraction size in a range of 1 % to 60 %, in a range of 2 % to 40 %, or in a range of 5 % to 30 % of the measured fractions.

[0148]

[0126] In one aspect of the invention, proteolytic enzymes endogenous to a source of protein may yield amino acids and / or peptides in the purified protein hydrolysate with a molecular weight ranging from 300 Da to 600 Da, with a relative fraction size in a range of 10 % to 65 %, in a range of 15 % to 55 %, or in a range of 20 % to 45 % of the measured fractions.

[0149]

[0127] In one aspect of the invention, proteolytic enzymes endogenous to a source of protein may yield amino acids and / or peptides in the purified protein hydrolysate with a molecular weight ranging from 300 Da to 600 Da, with a relative fraction size in a range of 1 % to 60 %, in a range of 2 % to 40 %, or in a range of 5 % to 30 % of the measured fractions.

[0150]

[0128] In one aspect of the invention, proteolytic enzymes endogenous to a source of protein may yield amino acids and / or peptides in the purified protein hydrolysate with a molecular weight less than 300 Da, with a relative fraction size in a range of 10 % to 90 %, in a range of 15 % to 80 %, or in a range of 20 % to 70 % of the measured fractions.

[0151]

[0129] In one aspect of the invention, proteolytic enzymes endogenous to a source of protein may yield amino acids and / or peptides in the purified protein hydrolysate with a molecular weights less than 300 Da, with a relative fraction size in a range of 20 % to 100 %, in a range of 30 % to 80 %, or in a range of 40 % to 70 % of the measured fractions.

[0152]

[0130] In another aspect of the invention, a complementary method may be used to further separate the small peptides with molecular weights ranging from 1,500 Da to 7,000 Da, 1,000 Da to 1,500 Da, 300 Da to 1,000 Da, or molecular weights lower than 300 Da to provide a detailed evaluation of the peptide molecular weight distribution.

[0153]

[0131] In one aspect of the invention, proteolytic enzymes endogenous to a source of protein may yield peptides in the purified protein hydrolysate with a molecular weight ranging from 1,500 Da to 7,000 Da, with a relative fraction size in a range of 1 % to 50 %, in a range of 5 % to 40 %, or in a range of 10 % to 30 % of the measured fractions.

[0154]

[0132] In one aspect of the invention, proteolytic enzymes endogenous to a source of protein may yield peptides in the purified protein hydrolysate with a molecular weight ranging from 1 ,000 Da to 1 ,500 Da, with a relative fraction size in a range of 1 % to 60 %, in a range of 2 % to 50 %, or in a range of 5 % to 40 % of the measured fractions.

[0155]

[0133] In one aspect of the invention, proteolytic enzymes endogenous to a source of protein may yield peptides in the purified protein hydrolysate with a molecular weight ranging from 300 Da to 1 ,000 Da, with a relative fraction size in a range of 1 % to 60 %, in a range of 2 % to 40 %, or in a range of 5 % to 30 % of the measured fractions.

[0156]

[0134] In one aspect of the invention, proteolytic enzymes endogenous to a source of protein may yield amino acids and / or peptides in the purified protein hydrolysate with a molecular weight less than 300 Da, with a relative fraction size in a range of 20 % to 100 %, in a range of 30 % to 80 %, or in a range of 40 % to 70 % of the measured fractions.

[0157]

[0135] In one aspect of the invention, the water source may comprise distilled water, demineralized water, deionized water and / or tap water. In one aspect of the invention, the water source may be characterized by conductivity in a range of 1 pS-cm1to 600 pS-cm1, in a range of 5 pS-cm1to 100 pS-cm1or in a range of 10 pS-cm1to 50 pS-cm1.

[0158]

[0136] In one aspect of the invention, the proteolytic enzymes may be immobilized on a solid support.

[0159] This aspect sterically prevents the molecules of the enzyme from breaking each other down and allows the enzyme to be separated from the reaction mixture after the reaction and used again. The solid support may be present 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 fibrous structure, through which the reaction mixture is perfused. The enzyme may also be added in soluble (free) form. After hydrolysis is complete, the resulting proteinhydrolysate is separated from the solid support with immobilized enzyme by simply draining the reaction vessel (in the case of large solid structure) or removing the enzyme on solid support by filtration or sedimentation (in the case of suspended carriers). The reaction vessel may be formed, for example, by a hydrolysis tank. The filtration step may also remove any solid residues from the source protein, such as cell wall debris. Free enzymes may be removed from the protein hydrolysate by ultrafiltration or deactivated by elevated temperature when hydrolysis is complete. Ultrafiltration of the protein hydrolysate may additionally remove any larger peptide chains which were not digested by the enzyme; these relatively larger peptide chains may not be metabolized by the cells and cause harm to them and therefore their removal may be beneficial. The temperature elevation used to deactivate the enzyme may also sterilize the resulting protein hydrolysate.

[0160]

[0137] The protein hydrolysate may be thermally treated at the end of hydrolysis to deactivate enzymes and kill microorganisms. In one aspect of the invention, this treatment may take place at a “low temperature” in the range of 80 °C to 120 °C, in the range of 85 °C to 100 °C or in the range of 90 °C to 95 °C for time in the range of 15 minutes to 180 minutes or in the range of 20 minutes to 120 minutes or in the range of 25 minutes to 60 minutes. In another aspect of the invention, the thermal treatment may be performed at a “high temperature” in the range 100 °C to 180 °C, in the range of 120 °C to 170 °C, or in the range of 130 °C to 160 °C, or in the range of 135 °C to 150 °C for a time period in the range of 1 second to 60 seconds, in the range of 3 seconds to 30 seconds, or in the range of 5 seconds to 20 seconds, or in the range of 10 seconds to 15 seconds. Both the low temperature method and the high temperature method may be performed in a hydrolysis tank or in a hydrolysis tank configured as flash pasteurizer or another suitable continuous flow heating device.

[0161]

[0138] If the enzyme is removed by ultrafiltration, it may retain at least partial catalytic activity and thus may be recycled for another round of hydrolysis. Ultrafiltration or thermal deactivation may also be used to remove active enzyme molecules from hydrolysates prepared by immobilized enzymes, in the event that some of the enzyme detaches from the solid support and dissolves into the reaction mixture.

[0162]

[0139] The solid support may be formed by, for example, silica, epoxide resin, cellulose, chitosan, glass wool, alginate, or by other appropriate materials. The solid support may be in the form of porous or solid beads, sponge, fibers, or another suitable configuration. The solid support may have a large surface area to volume ratio to allow the binding of a large amount of enzyme. For example, beads of porous silica or any other suitable material with a diameter in the range of 1 pm to 10,000 pm, or in the range of 10 pm to 1,000 pm, or in the range of 20 pm to 500 pm, may be used as a solid support for enzyme immobilization. Immobilization may be achieved, for example, by functionalizing the silica bead surface with amino groups and using a crosslinking agent, such as glutaraldehyde, to bind the enzyme to the solid support. Other functional groups, like aldehyde or epoxy groups, may also be used for enzyme immobilization. The amino groups in this aspect of the invention are covalently bonded to glutaraldehyde, after which excess glutaraldehyde is removed and the enzyme is added. The amino groups on the surface of the enzyme then bind the remaining free aldehyde groups of the glutaraldehyde molecules on the silica bead surface. The immobilization may be performed in water or a suitable aqueous buffer. Due to the porous nature and large surface area of the silica beads, a relatively high amount of enzyme may be immobilized relative to the weight of the solid support.

[0163]

[0140] Water may be used to dissolve the source of protein for hydrolysis. Some proteins may require a buffer to adjust the pH to increase solubility. The pH may 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 at all, may be used so that the resulting protein hydrolysate may be added to the final culture media at high concentrations while minimizing its impact on media osmolality.

[0141] The buffer may include, for example, phosphate buffer, bicarbonate buffer, tris HC1 buffer, borate buffer, glycine -NaOH buffer, Good’s buffer or any other appropriate buffer, or a combination thereof.

[0164]

[0142] In one aspect of the invention, a concentration of potassium phosphate buffer in a range of 1 mM to 100 mM, in a range of 10 mM to 40 mM or in a range of 15 mM to 35 mM may be used for pH adjustment to dissolve soy protein to a concentration in a range of 1 g / L to 150 g / L, in a range of 20 g / L to 100 g / L, in a range of 30 g / L to 80 g / L, in a range of 40 g / L to 70 g / L, in a range of 50 g / L to 65 g / L or in a range of 55 g / L to 60 g / L. In another aspect of the invention, the soy protein is dissolved in distilled water to a concentration in a range of 1 g / L to 150 g / L, in a range of 20 g / L to 100 g / L, in a range of 30 g / L to 80 g / L, in a range of 30 g / L to 80 g / L, in a range of 40 g / L to 70 g / L, in a range of 50 g / L to 65 g / L or in a range of 55 g / L to 60 g / L, where the concentration is defined as the amount of source of protein per liter of the reaction multiplied by the percentage protein content in the source of protein.

[0165]

[0143] In another aspect of the invention, a concentration of potassium phosphate buffer in a range of 1 mM to 100 mM, in a range of 10 mM to 40 mM, or in a range of 15 mM to 35 mM may be used for pH adjustment to dissolve soy protein to a concentration in a range of 0.5 g / 1 to 250 g / L, or in a range of 1 g / L to 150 g / L, or in a range of 30 g / L to 100 g / L, or in a range of 40 g / L to 80 g / L. In another aspect of the invention, the soy protein is dissolved in distilled water to a concentration in a range of 0.5 g / L to 250 g / L, or in a range of 1 g / L to 150 g / L, or in a range of 30 g / L to 100 g / L, or in a range of 40 g / L to 80 g / L, where the concentration is defined as the amount of source of protein per liter of the reaction multiplied by the percentage protein content in the source of protein.

[0166]

[0144] Other concentrations of the source of protein may be used, however, very high concentrations of this source lead to incomplete dissolving of the protein and formation of a highly viscous colloidal solution, presenting problems for hydrolysis and further processing, while low concentrations of protein may limit the speed of the hydrolysis reaction. To ensure the best dissolution of the proteins in the reaction mixture a heat-treatment may be used. Temperatures below boiling may be used for extended periods of time in order to significantly increase the content of dissolved proteins and to deactivate potential inhibitors of proteases and other antinutritional compounds.

[0167]

[0145] In one aspect of the invention, the source of protein may be added at a higher concentration than the maximum soluble concentration. This additional protein may be dissolved after the protein concentration in the reaction mixture is decreased due to its hydrolysis by the enzyme. This results in high concentration of available substrate during the entire process, potentially improving hydrolysis efficiency. Multiple cycles of substrate addition into the same reaction mixture may be performed. In one aspect of the invention a base or a suitable buffer may be added to counteract changes in pH and keep the enzyme in its pH optimum or a pH stat may be used.

[0168]

[0146] A parameter by which the conversion of source of protein into bioa vailable products for animal cells may be evaluated, is the degree of hydrolysis (DH), defined as the percentage of peptide bonds in the source of protein that are hydrolyzed during the reaction. DH can be determined as the difference of amino nitrogen (AN) of hydrolysed substrate and amino nitrogen of substrate before hydrolysis (ANO) multiplied by factor (F) and divided by total nitrogen (TN). AN may be determined by formol titration of the hydrolysate sample, ANO may be determined by formol titration of the substrate solution before the process of hydrolysis, TN may be determined by Kjeldahl method. Factor F is a value calculated from empirical data based on amino acids composition of the particular source of protein and it represents the ratio of total nitrogen to alpha amino nitrogen in the sample. A higher degree of hydrolysis corresponds to a larger percentage of the source protein converted into free amino acids or short peptides, which are usable by mammalian cells as nutrition. Mammalian cells are generally incapable of absorbing and digesting proteins and longer peptides. Peptides longer than four amino acids, and / or heavier than approximately 500 Daltons, have poor absorption by mammalian cells. In various aspects of theinvention, the amount of the source of protein in the range of 20 % to 100 %, in the range of 30 % to 75 %, in the range of 35 % to 70 % or in the range of 40 % to 65 % may be converted into free amino acids, expressed as the percentage of mass concentration of amino acids to mass concentration of protein. The degree of hydrolysis, meaning the percentage of peptide bonds that undergo hydrolysis out of the total amount of peptide bonds present in the substrate at the start of the reaction, may be in the range of 10 % to 70 %, in the range 20 % to 60 % or in the range of 25 % to 50 %.

[0169]

[0147] Proteolytic enzymes are classified according to the basis of amino acid group present on the active site as serine proteases (subtilisin, trypsin), cysteine proteases (papain-like, trypsin-like), aspartic proteases (pepsin, cathepsin D), glutamic proteases (eqolisin), threonine proteases (ornithine, acetyltransferase) and metalloproteases (Myxobacter I and II). Another classification divides enzymes according to their enzymatic function into exoproteases and endoproteases. Exoproteases cleave the protein or peptide chains at the N or C terminal ends, whereas endoproteases can cleave peptide bonds in the middle of the protein or peptide chain. Exoproteases are classified according to the mechanism of action on aminopeptidases that act on the N terminal end and carboxypeptidases that act on the C terminal end. The present examples of proteases acting on the active side of related amino acids are not limited to the listed exemplary proteases.

[0170]

[0148] In one aspect of the invention, a combination of endoproteases and exoproteases may be used, since endoproteases may create more free ends of peptide chains, increasing the efficiency of exoproteases, and exoproteases are more efficient in hydrolyzing the protein to single amino acids.

[0171]

[0149] In one aspect of the invention, endoproteases and exoproteases may be used sequentially in this order to maximize hydrolysis efficiency.

[0172]

[0150] In one aspect of the invention, the terms "endoprotease" and "endopeptidase" are used interchangeably and may refer to enzymes that cleave peptide bonds within a polypeptide chain. Similarly, the terms "exoprotease" and "exopeptidase" are used interchangeably and may refer to enzymes that cleave peptide bonds from the terminal ends of a polypeptide chain.

[0173]

[0151] In one aspect of the invention, additional enzymes may be added to the reaction mixture after the beginning of hydrolysis. The additional enzymes may include the same enzyme that is used to begin hydrolysis or a different enzyme, and is used to counteract the gradual decrease in enzymatic activity due to degradation of the enzyme molecule. In one aspect of the invention, enzymes with a higher pH optimum may be added at the start of the hydrolysis, when pH is higher, and enzymes with a lower pH optimum may 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.

[0174]

[0152] In another aspect of the invention, additional sources of protein may be added to the reaction mixture after the beginning of hydrolysis. The advantages of this aspect may be, for example, easier dispersion and dissolution of additional sources of protein when the previous amount of source of protein is at least partially hydrolyzed.

[0175]

[0153] The addition of enzyme or substrate after the beginning of the hydrolysis process may be performed in a fed-batch (wherein additional reagents are added to the reaction mixture, and subsequently the whole reaction batch is harvested) or semi-continuous (wherein a portion of the reaction mixture, or certain additional reagents within the reaction mixture, is periodically removed and replaced with fresh components) or in continuous (wherein addition to and harvesting from the reaction mixture are both done continuously) reaction mode.

[0176]

[0154] 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 and protein hydrolysis steps. In one aspect of the invention, in the case of immobilized enzymes, mixing methods that minimize mechanical damage to the solid carriers should beused. These may include roller mixing, shaking, or low-shear impellers such as hydrofoil or elephant ear impellers. In the case of enzymes immobilized to a large solid support, sufficient perfusion of the support with the reaction mixture must be assured.

[0177]

[0155] The mixing of the source of protein, e.g. protein isolate, with water, dissolving the source of protein and the process of hydrolysis itself may be performed in the hydrolysis tank at a laboratory or industrial scale. In one aspect of the invention, an external -loop circulation pump may be used to improve protein powder dispersion and to draw protein powder closer to the agitator, wherein such external-loop circulation may be configured as externally driven submersible agitator. Such agitator may comprise a cylindrical housing with an internal set of blades. In another aspect of the invention, a hybrid powder mixer or any other suitable mixer might be used for the same effect accompanied with improved powder dissolution.

[0178]

[0156] In one aspect of the invention, the hydrolysis tank may be configured to provide an environment for the hydrolysis reaction. The hydrolysis tank may comprise a main body constructed from at least one material selected from stainless steel, glass-lined steel, titanium, polyethylene, polypropylene, polytetrafluoroethylene or any other suitable materials. The main body may comprise various shapes, such as cylindrical or rectangular or any other suitable geometries. The hydrolysis tank may comprise insulation configured as an outer jacket of the hydrolysis tank, wherein the space between the outer jacket and the wall of the hydrolysis tank may be filled with an appropriate insulation material or medium. The hydrolysis tank may further comprise at least one input and at least one output for loading and unloading the ingredients. The input of the hydrolysis tank may be configured as a shaft or funnel, wherein the shaft or funnel may be used for loading the ingredients. The hydrolysis tank may further comprise a heating system configured to heat the inner environment of the hydrolysis tank. The hydrolysis tank may comprise mixing mechanisms comprising at least one stirrer, paddle or any other instrument capable of mixing the protein hydrolysate. The sealing mechanisms of the hydrolysis tank may comprise materials such as silicone, ethylene propylene diene monomer, and polytetrafluoroethylene. The hydrolysis tank may be configured to withstand a maximum temperature of at least 80 °C, at least 90 °C, at least 100 °C, at least 105 °C, at least 110 °C, at least 120 °C, or at least 150 °C. The hydrolysis tank may further comprise auxiliary components selected from the group of pumps, pressure sensors, flow meters, valves and means for monitoring the hydrolysis reaction.

[0179]

[0157] The volume of the hydrolysis tank 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, or in the range of 0.3 L to 20,000 L, or in the range of 1 L to 10,000 L.

[0180]

[0158] In one aspect of the invention, the hydrolysis tank may be equipped with different types of sensors, for example, thermal sensor, pH probe, conductometer, or any other type of appropriate sensor according to the needs of the process of hydrolysis. The pH may be measured by various methods and devices including potentiometry, colorimetry, spectrophotometry, ion-selective electrodes, conductometry or any other measuring technique and / or device. The temperature in the reaction vessel may be measured by various devices including a resistance temperature detector, thermocouple, digital thermometer with insertion probe, infrared thermometer with fiber optic probe or any other appropriate device.

[0181]

[0159] A sampling system may be used for precise monitoring of the degree of hydrolysis. The degree of hydrolysis may be monitored by titration and / or by absorbance measurement, for example at a wavelength in a range of 190 nm to 350 nm, or in a range 190 nm to 230 nm.

[0182]

[0160] Water may enter the hydrolysis tank through a water purification unit. In one aspect of the invention, a water purification unit may provide at least one purification process selected from the group of reverse osmosis, deionization, electrodeionization, electrodialysis and distillation.

[0183]

[0161] The mixing may be provided by an appropriate stirring unit that may include, for example, a paddle impeller. In some aspects, an elephant-ear impeller may be used. The outer diameter of the stirreror impeller may be in the range of 1 / 10 to 9 / 10 of the inner reactor diameter, or in the range of 3 / 10 to 8 / 10 of the inner reactor diameter, or in the range of 4 / 10 to 7 / 10 of the inner reactor diameter. As an example, the outer diameter of stirrer / impeller may be 2 / 3 of the inner reactor diameter. The stirrer or impeller may be located in the center of the hydrolysis tank or outside of the center of the hydrolysis tank.

[0184]

[0162] In another aspect of the invention, the mixing in the hydrolysis tank may be provided by an appropriate stirring unit that may include, for example, a paddle impeller. In some aspects, an elephantear or rushton-like impeller may be used. The outer diameter of the stirrer or impeller may be in the range of 1 / 10 to 9 / 10 of the inner reactor diameter, or in the range of 3 / 10 to 8 / 10 of the inner reactor diameter, or in the range of 4 / 10 to 7 / 10 of the inner reactor diameter. As an example, the outer diameter of stirrer / impeller may be 2 / 3 of the inner reactor diameter. The stirrer or impeller may be located in the center of the hydrolysis tank or outside of the center of the hydrolysis tank. The hydrolysis tank may be equipped with baffles or other suitable features to prevent vortex formation and improve mixing efficiency.

[0185]

[0163] The reaction components may be added to the hydrolysis tank manually, or automatically by using a conveyor, loading tank or any other appropriate device used for transferring reaction components. The source of protein may be in a liquid solution or in the form of a powder.

[0186]

[0164] The automation system may use various types of pumps for feeding enzymes at precise temperatures during hydrolysis. Each enzyme may be pumped at a certain temperature. Both parameters may be set ahead of the operation in an automation profile setting for the hydrolysis process.

[0187]

[0165] The term “reaction components” may comprise a source of protein, proteolytic enzymes, or any other appropriate component necessary for effective hydrolysis by proteolytic enzymes.

[0188]

[0166] The hydrolysis tank may be equipped with a temperature control unit (TCU) that is configured to maintain the temperature of the culture medium at the desired temperature range. The desired temperature setpoint may be changed at least once during the process. The TCU may be equipped with heating and / or cooling exchangers and at least one pump.

[0189]

[0167] In one aspect of the invention, the temperature may be controlled automatically through a cascade control system, wherein the tank temperature acts as the primary control variable, and the temperature control unit (TCU) responds to maintain the hydrolysis tank at the desired setpoint. The cascade control system continuously adjusts the TCU heating and / or cooling output based on real-time feedback from the hydrolysis tank to ensure stable and precise temperature regulation during hydrolysis that takes place in the hydrolysis tank.

[0190]

[0168] In one aspect of the invention, the loading tank may be configured to provide proteolytic enzymes in liquid form into the hydrolysis tank. The loading tank may be configured to maintain the optimal conditions necessary for preserving the stability and activity of stored enzymes. The loading tank may comprise a main body constructed from at least one material selected from high-grade stainless steel, glass-lined steel, titanium, polyethylene, polypropylene, polytetrafluoroethylene or any other suitable materials. The main body may comprise various shapes, such as cylindrical or rectangular or any other suitable geometries. The interior surface of the loading tank may be polished to a mirror finish to minimize adhesion of enzyme residues and facilitate easy cleaning. The loading tank may be equipped with a cooling system to maintain a consistent temperature, essential for enzyme stability. The loading tank may comprise multiple temperature sensors to continuously monitor the internal temperature. The loading tank may be coupled with the hydrolysis tank, wherein the hydrolysis tank may further comprise at least one input and at least one output for loading and unloading the enzymes. The input of the loading tank may be configured as a shaft and / or funnel, wherein the shaft and / or funnel may be used for loading of the proteolytic enzymes. The loading tank may comprise at least one sealing mechanism, which may comprise materials such as silicone, ethylene propylene diene monomer, and polytetrafluoroethylene and / or any other appropriate material.

[0169] The loading tank may be made, for example, of stainless steel or glass. The volume of the loading tank may be in the range of 100 mL to 5 m3, or in the range of 2 L to 3 m3, or in the range of 500 L to 1 m3.

[0191]

[0170] In one aspect of the invention, another loading tank (126) may be configured to provide a source of protein into the hydrolysis tank with the same composition as the loading tank for the proteolytic enzymes.

[0192]

[0171] Many plant sources of protein comprise inositol hexaphosphate, which is an important compound found in plants and is often a crucial part of a plant metabolism. Its salt form, phytin, is the main storage compound of phosphate in plants. The inositol hexaphosphate or its derivatives and / or any related form of those compounds comprising phosphate from plant sources may significantly influence the downstream processes of the invention as well as metazoan cell proliferation and viability of the cell. Four aspects are disclosed herein in order to regulate the content and / or concentration of such compounds.

[0193]

[0172] The first aspect disclosed herein relates to the use of enzymes having phytase activity derived from an animal, plant, or microorganism source or any other appropriate source. Enzymes having phytase activity may be used separately or in combination.

[0194]

[0173] The term “enzyme having phytase activity” refers to enzymes from the group of phytases, phosphatases and / or any other enzymes capable of cleaving phosphate ester bonds.

[0195]

[0174] Activity of enzymes having phytase activity may differ according to the source of protein.

[0196] Enzymes having phytase activity may have different activity at different pH and / or temperature conditions, which may be measured by various methods and devices including potentiometry, colorimetry, spectrophotometry, ion-selective electrodes, conductometry and / or any other measuring technique and / or device. The temperature of the reaction mixture may be monitored in real time by various devices including a resistance temperature detector, thermocouple, digital thermometer with insertion probe, infrared thermometer with fiber optic probe or any other appropriate device.

[0197]

[0175] The enzyme kinetics of the reaction may be calculated based on the Michaelis -Menten model, which provides a foundational equation for describing the rate of enzymatic reaction to provide a product from a substrate. This model is integral for understanding the relationship between substrate concentration and reaction velocity, and its incorporation allows for the precise determination of key kinetic parameters, such as the maximum reaction velocity (Vmax) and the Michaelis constant (Km).

[0198]

[0176] Enzymes having phytase activity may contain additives used to maintain pH and stability.

[0199] Examples of additives include, but are not limited to water, citric acid, glycerol, potassium sorbate, sodium chloride, sorbitol, dextrin, sucrose or any other appropriate agent used for maintaining stability and pH.

[0200]

[0177] Enzymes having phytase activity may be used either individually or in combination, wherein the combination may comprise use of at least 2 or more enzymes having phytase activity.

[0201]

[0178] The duration of the treatment by enzymes having a phytase activity may differ according to the source of protein used. The duration of the treatment by enzymes having phytase activity may be in a range of 5 minutes to 320 minutes, in a range of 10 minutes to 180 minutes, or in a range of 30 minutes to 120 minutes.

[0202]

[0179] The temperature of the treatment by enzymes having a phytase activity may be in a range of 20 °C to 70 °C, in a range of 25 °C to 65 °C, or in a range of 30 °C to 60 °C.

[0203]

[0180] Enzymes having phytase activity may create complexes with a substrate. The substrate may comprise any inositol hexaphosphate or its derivatives that may undergo cleaving of phosphate ester bond connecting phosphate groups with inositol or its derivatives, in the presence of water. The function of enzymes having phytase activity according to this aspect of the invention is not limited to said substrates.

[0181] Enzymes having phytase activity may catalyze release of at least one organophosphate group from inositol hexaphosphate or its derivatives and may result in free inorganic phosphate and series of lower phosphoric esters as inositol pentaphosphate, inositol tetraphosphate, inositol triphosphate, inositol diphosphate, inositol monophosphate intermediates or inositol or a combination thereof.

[0204]

[0182] After each cleavage of a phosphate ester bond and release of inorganic phosphate and series of lower phosphoric esters as inositol pentaphosphate, inositol tetraphosphate, inositol triphosphate, inositol diphosphate, inositol monophosphate intermediates, inositol or a combination thereof, the enzyme having phytase activity may then bind to another inositol hexaphosphate or its derivatives or any related form of those compounds for further hydrolysis in the presence of water.

[0205]

[0183] In another aspect of the invention, additional sources of protein may be added to the reaction mixture after the beginning of hydrolysis. The advantages of this aspect may be, for example, easier dispersion and dissolution of additional sources of amino acids when the previous amount of source of protein is at least partially hydrolyzed.

[0206]

[0184] Addition of enzymes having phytase activity in a solid or liquid state or in a combination thereof, into a hydrolysis tank containing a source of protein and water source from loading tank, leads to cleavage of phosphate ester bonds and release of molecules at the specific pH and temperature.

[0207]

[0185] In one aspect of the invention, the hydrolysis tank may be composed of various materials, have a specific volume, and may be coupled with a water purification unit and / or loading tank and may comprise a stirring unit.

[0208]

[0186] In one aspect of the invention, the loading tank may be configured to provide a combination of enzymes having phytase activity in dry or liquid form or a combination thereof and proteolytic enzymes and / or source of protein into the hydrolysis tank.

[0209]

[0187] In another aspect of the invention, the cultivation system may comprise two or more hydrolysis tanks. When using two or more hydrolysis tanks, they may be referred to, for example, as first hydrolysis tank, second hydrolysis tank, third hydrolysis tank, and so on. Notations of hydrolysis tanks may be chosen according to the selected number of hydrolysis tanks. The first hydrolysis tank may be configured for hydrolysis of the source of protein by proteolytic enzymes, and the second hydrolysis tank may be configured for the hydrolysis of inositol hexaphosphate and / or its derivatives by enzymes having phytase activity. All of the hydrolysis tanks may be coupled with a mixing tank, water purification unit, at least one loading tank, at least one pump and / or at least one filtration unit.

[0210]

[0188] In one aspect of the invention, the hydrolysis tank may be configured to perform both hydrolysis of the source of protein by proteolytic enzymes and hydrolysis of inositol hexaphosphate and / or its derivatives.

[0211]

[0189] In one aspect of the invention, the concentration of enzymes having phytase activity may differ according to concentration of inositol hexaphosphate contained in the source of protein. The concentration of the enzymes having phytase activity may be in the range of 0.00001 % to 5 % or in the range of 0.0001 % to 2 %, or in the range of 0.0005 % to 0.5 % expressed as a ratio of the concentration of enzymes having phytase activity to the concentration of inositol hexaphosphate in the reaction mixture. The concentration of the enzyme may be determined by the Bradford assay, BCA assay or other protein determination assays. The concentration of inositol hexaphosphate or its derivatives may be determined by electrophoresis, liquid chromatography, enzymatic assay or any other suitable analytical method.

[0212]

[0190] The enzyme having phytase activity may have phytase activity in the range of 0.0001 to 3,000,000 FTU, or in the range of 0.001 to 300,000 FTU, or in the range of 0.01 to 30,000 FTU (one phytase unit is equal to amount of enzyme that liberates one micromole of phosphate in one minute from phytate at 37 °C and pH 5.5) per 1 gram of the enzyme preparation.

[0191] The amount of enzyme having phytase activity, for example Maxamyl, added to the reaction may be adjusted according to its activity so the resulting exoprotease activity in the reaction mixture may be equal to 0.001 to 1,000, or 0.01 to 500, or 1 to 100 FTU per 1 gram of protein source.

[0213]

[0192] The efficiency of cleavage by enzymes having phytase activity may be regulated by pH or temperature change. Efficiency of cleavage may be improved by addition of a pH regulating substance to obtain a pH in a range of 2 to 10, in a range of 3 to 9, in a range of 4 to 8, in a range of 5 to 9, or in a range of 6 to 7, or in a range of 5.6 to 8.5, or in a range of 5.2 to 7.5, or in a range of 5.1 to 6, wherein the substance can be an inorganic molecule comprising HC1, NaOH and / or NH40H or any other appropriate pH regulating compound. Efficiency of cleavage may be improved by changing the temperature. The temperature may be in a range of 20 °C to 70 °C, in a range of 25 °C to 65 °C, or in a range of 30 °C to 60 °C.

[0214]

[0193] In one aspect of the invention, the use of enzymes having phytase activity on protein hydrolysate may result in a modified protein hydrolysate. As used herein, the term “modified protein hydrolysate” refers to protein hydrolysate comprising cleaved or precipitated inositol hexaphosphate and its derivatives or a combination thereof.

[0215]

[0194] The modified protein hydrolysate may be subjected to thermal treatment at the end of hydrolysis to deactivate enzymes having phytase activity and / or kill microorganisms. In one aspect of the invention, this treatment may be at “low temperature” in the range of 80 °C to 120 °C, in the range of 85 °C to 100 °C, or in the range of 90 °C to 95 °C for a time in the range of 15 minutes to 180 minutes, or in the range of 20 minutes to 120 minutes, or in the range of 25 minutes to 60 minutes.

[0216]

[0195] In another aspect of the invention, thermal treatment may be performed at a “high temperature" in the range 80 °C to 160 °C, in the range of 100 °C to 155 °C, or in the range of 110 °C to 150 °C for a time is in the range of 1 second to 600 seconds, in the range of 3 seconds to 300 seconds, or in the range of 5 seconds to 60 seconds. The low temperature thermal treatment may be performed in a hydrolysis tank. Both the low temperature and high temperature thermal treatments may be performed in a hydrolysis tank configured as a flash pasteurizer or another suitable continuous flow heating device.

[0217]

[0196] In another aspect of the invention, thermal treatment may be performed at a “high temperature” in the range 100 °C to 180 °C, in the range of 120 °C to 170 °C, or in the range of 130 °C to 160 °C, or in the range of 135 °C to 150 °C for a time period in the range of 1 second to 60 seconds, in the range of 3 seconds to 30 seconds, or in the range of 5 seconds to 20 seconds, or in the range of 10 seconds to 15 seconds. The low temperature thermal treatment may be performed in a hydrolysis tank. Both the low temperature and high temperature thermal treatments may be performed in a hydrolysis tank configured as a flash pasteurizer or another suitable continuous flow heating device.

[0218]

[0197] If the enzyme is removed by ultrafiltration, it may retain at least partial catalytic activity and thus may be recycled for another round of hydrolysis. Ultrafiltration or thermal deactivation may also be used to remove active enzyme molecules from hydrolysates prepared by immobilized enzymes in the event that some of the enzyme detaches from the solid support and dissolves into the reaction mixture.

[0219]

[0198] In one aspect of the invention, the use of proteolytic enzymes and enzymes having phytase activity may be in one of two orders. In the first order, enzymes having phytase activity may be added to the hydrolyzed source of protein and water in the hydrolysis tank. The efficiency of cleavage of phosphate ester bonds may be regulated by pH and temperature changes. In the second order, proteolytic enzymes may be added to the source of protein, water and to previously cleaved phosphate and series of lower phosphoric esters as inositol pentaphosphate, inositol tetraphosphate, inositol triphosphate, inositol diphosphate, inositol monophosphate, inositol, intermediates of inositol derivatives or a combination thereof in the hydrolysis tank to generate protein hydrolysate free of inositol hexaphosphate and its derivatives. The second order is less preferable due to the disadvantageous step of pH change especially if the enzyme used has a pH optimum in the acidic region.

[0199] In one aspect of the invention, solid residues may be removed from modified protein hydrolysate by a filtration unit.

[0220]

[0200] In one aspect of the invention, the solid residues may be further used for the production of an edible product, wherein the edible product may be used for human or animal consumption.

[0221]

[0201] In one aspect of the invention, the hydrolysis tank may be connected to the filtration unit by a pump. This pump may be used for the transfer of protein hydrolysate to the filtration unit.

[0222]

[0202] In one aspect of the invention, the filtration unit may comprise at least one filter selected from the group of membrane filters, depth filters, mesh filters, activated carbon filters, ceramic filters, ultrafiltration filters, nanofiltration filters, ion exchange filters, crossflow (tangential flow) filters, adsorption filters or fiber filters. The filter of the filtration unit may comprise at least one material selected from the group of cellulose, glass fiber, polyethersulfone (PES), polyvinylidene fluoride (PVDF), nylon, polypropylene, polyethylene terephthalate (PET), polytetrafluoroethylene (PTFE), polyacrylonitrile (PAN), polyvinyl chloride (PVC), stainless steel, silica, alumina, silicon carbide, titanium dioxide, titanium carbide, silicon carbide, zeolites, or synthetic polymers. The filter may be housed in a housing configured to cover the whole filter, wherein the housing may comprise at least one material selected from the group of stainless steel, polycarbonate, polyethylene, or other suitable biocompatible and sterilizable materials. If the filtration unit is composed of multiple filters or includes filters with non-uniform pore size distribution across the depth of the filter, the filters may be arranged so that the filters with largest pores and / or, in the case of filters with non-uniform pore size distribution, the side with the largest pores, may be located upstream (in the direction of the hydrolysis tank) and the filters with the smallest pores and / or the side with the smallest pores may be located downstream (in the direction of the mixing tank within the filtration unit). In the case of using multiple filters or using filters with non-uniform pore size distribution across the depth of the filter, the maximal pore size of the last filter used of the filtration unit may be in a range of 0.1 pm to 1 pm, in a range of 0.2 pm to 1 pm, in a range of 0.3 pm to 1 pm, in a range of 0.4 pm to 1 pm, in a range of 0.5 pm to 1 pm, in a range of 0.6 pm to 1 pm, in a range of 0.7 pm to 1 pm, in a range of 0.8 pm to 1 pm or in a range of 0.9 pm to 1 pm. The size of the pore may vary according to the selected type of filter and the specific requirements of the filtration. The filtration unit may further include sealing mechanisms such as O-rings, gaskets, clamps or any other sealing mechanisms capable of preventing leakage and maintaining a sterile environment. The sealing mechanisms of the filtration unit may comprise materials such as silicone, ethylene propylene diene monomer, or polytetrafluoroethylene. The filtration unit may further comprise auxiliary components selected from the group of pumps, pressure sensors, flow meters, valves and means for monitoring the filtration process.

[0223]

[0203] In one aspect of the invention, the filtration unit may be configured to utilize centrifugal force to separate solid-phase particles from liquid phase. This separation process is facilitated by the implementation of centrifugal filters, which may be strategically designed and positioned within the filtration unit.

[0224]

[0204] In one aspect of the invention, the removal of solid residues from the modified protein hydrolysate by the filtration unit may result in a purified protein hydrolysate derived from modified protein hydrolysate. As used herein, the term “purified protein hydrolysate” refers to protein hydrolysate substantially free from solid residues.

[0225]

[0205] The second aspect disclosed herein relates to the use of precipitating agents to generate precipitates of inositol hexaphosphate and its derivatives. Precipitating agents comprise an organic or inorganic compound of 1) a suitable cation with high affinity to inositol hexaphosphate and its derivatives - e.g. bivalent cation of calcium, zinc, cobalt, manganese, trivalent cation of iron or any other suitable cation, and 2) an anion with a pKa that is higher than the pKa of inositol hexaphosphate and phosphoric acid - e.g. acetate, carbonate, or hydroxide or any other suitable anion, or any other combination of mentioned cations or anions.

[0206] Addition of salts or hydroxides into water results in dissolution of the mentioned salts or hydroxides and their dissociation to cations and anions. Cations, especially divalent or multivalent metal cations, interact with negative inositol hexaphosphate and its derivatives by binding to its phosphate groups through ionic interactions. This binding reduces the solubility of inositol hexaphosphate in water, leading to the formation of insoluble metal-phytate complexes resulting in the creation of precipitates.

[0226]

[0207] Addition of salts or hydroxides into hydrolysis tank containing source of protein and water from the loading tank may result in the generation of free metal ions from the before mentioned salts or hydroxides that may form metal-phytate complexes resulting in formation of precipitates in water containing the source of protein.

[0227]

[0208] In one aspect of the invention, the hydrolysis tank may be composed of various materials, may have a specific volume, and may be configured with a stirring unit, water purification unit, loading tank and / or storage tank.

[0228]

[0209] In another aspect of the invention, the precipitation process may take place in the second hydrolysis tank, wherein the second hydrolysis tank may be connected to the first hydrolysis tank for the source of protein and to the mixing tank.

[0229]

[0210] In one aspect of the invention, the concentration of precipitating agents may differ according to concentration of inositol hexaphosphate contained in the source of protein. The concentration of precipitating agents may be in the range of 150:1 to 1:1 or in the range of 90:1 to 3:1, or in the range of 60:1 to 10:1 expressed as a ratio of the molar concentration of precipitating agents to the molar concentration of inositol hexaphosphate and / or its derivatives in the reaction mixture.

[0230]

[0211] In one aspect of the invention, the use of precipitating agents on the protein hydrolysate may result in a modified protein hydrolysate.

[0231]

[0212] In one aspect of the invention, removal of solid residues from protein hydrolysate by a filtration unit may be used. In one aspect of the invention, the solid residues may be further used for the production of an edible product, wherein the edible product may be used for human or animal consumption.

[0232]

[0213] In one aspect of the invention, the hydrolysis tank may be connected to the filtration unit by a pump. This pump may be used for the transfer of protein hydrolysate to the filtration unit.

[0233]

[0214] In one aspect of the invention, the filtration of the solid residues formed by precipitation may be performed similarly to as described herein.

[0234]

[0215] In one aspect of the invention, the filtration of solid residues from the modified protein hydrolysate by the filtration unit may result in a purified protein hydrolysate.

[0235]

[0216] The third aspect disclosed herein relates to the combination of the previously mentioned two aspects: the use of enzymes having phytase activity and precipitating agents. Combining these aspects may significantly decrease the amount of inositol hexaphosphate and its derivatives.

[0236]

[0217] In one aspect of the invention, addition of enzymes having phytase activity in solid or liquid state or their combination thereof into hydrolysis tank containing a source of protein may decrease the amount of inositol hexaphosphate and its derivatives. The addition of precipitating agents in the hydrolysis tank after the addition of enzymes having phytase activity results in the precipitation of the remaining inositol hexaphosphate and its derivatives. In another aspect of the invention, the enzymes with phytase activity and precipitating agents may be used in one of two orders.

[0237]

[0218] In another aspect of the invention, the combination of treatment by enzymes having phytase activity and precipitation process may take place in a second hydrolysis tank, wherein the second hydrolysis tank may be connected to the hydrolysis tank for the source of protein and to the mixing tank.

[0238]

[0219] The addition of enzymes having phytase activity in a solid or liquid state or in a combination thereof into a hydrolysis tank containing the source of protein and water source from loading tank leads to cleavage of phosphate ester bonds at a specific pH and temperature.

[0220] The addition of precipitating agents into a hydrolysis tank containing the source of protein and a water source from the loading tank leads to binding of precipitating agents to inositol hexaphosphate and its derivatives causing the formation of precipitates.

[0239]

[0221] The first and second aspect may be combined, wherein inositol hexaphosphate and its derivatives may be removed by the first aspect at least 90 % and by the second aspect by up to 10 %, by the first aspect at least 80 % and by the second aspect by up to 20 %, by the first aspect at least 70 % and by the second aspect by up to 30 %, by the first aspect at least 60 % and by the second aspect by up to 40 %, by the first aspect at least 50 % and by the second aspect by up to 50 %, by the first aspect at least 40 % and by the second aspect by up to 60 %, by the first aspect at least 40 % and by the second aspect by up to 60 %, by the first aspect at least 30 % and by the second aspect by up to 70 %, by the first aspect at least 20 % and by the second aspect by up to 80 %, by the first aspect at least 10 % and by the second aspect by up to 90 %.

[0240]

[0222] In one aspect of the invention, the ratio of precipitating agent to enzymes having phytase activity may be selected according to the amount of inositol hexaphosphate and its derivatives.

[0241]

[0223] In one aspect of the invention, the use of enzymes having phytase activity and precipitating agents on protein hydrolysate may result in a modified protein hydrolysate. The term “modified protein hydrolysate” refers to protein hydrolysate comprising cleaved or precipitated inositol hexaphosphate and its derivatives or a combination thereof.

[0242]

[0224] In one aspect of the invention, solid residues may be removed from protein hydrolysate by a filtration unit. In one aspect of the invention, the solid residues may be further used for the production of an edible product, wherein the edible product may be used for human or animal consumption.

[0243]

[0225] In one aspect of the invention, the hydrolysis tank may be connected to the filtration unit by a pump. This pump may be used for the transfer of protein hydrolysate to the filtration unit.

[0244]

[0226] In one aspect of the invention, the filtration of the solid residues formed by using precipitation may be performed similarly to as described herein.

[0245]

[0227] In one aspect of the invention, the filtration of solid residues from the modified protein hydrolysate by the filtration unit may result in a purified protein hydrolysate.

[0246]

[0228] The purified protein hydrolysate is transferred to the mixing tank where the nutritional additives, shear protectants, anti-foaming agents and / or proliferation additives may be loaded from the loading tank for the preparation of culture medium.

[0247]

[0229] The term “purified protein hydrolysate” refers to a hydrolysate that is free from solid residues in a range of 90 % to 100 %, in a range of 95 % to 100 %, in a range of 98 % to 100 %. The term “culture medium” refers to a mix of at least one of purified protein hydrolysate, nutritional additive, shear protectant and / or anti-foaming agent.

[0248]

[0230] The nutritional additive may include at least one of saccharides, mineral compounds, vitamins, amino acids, peptides, organic amines, signaling compounds, oligonucleotides, fatty acids, phospholipids, organic micronutrients or any other appropriate nutritional additive according to the selected source of protein, wherein the selected source of protein may contain a low concentration of particular amino acids which are essential for the cell growth and metabolism. Those particular amino acids may be added subsequently as nutritional additives from a loading tank.

[0249]

[0231] In one aspect of the invention, the mixing tank may comprise a main body constructed from at least one material selected from stainless steel, glass-lined steel, titanium, polyethylene, polypropylene, polytetrafluoroethylene or any other suitable materials. The main body may comprise various shapes, such as cylindrical or rectangular or any other suitable geometries. The input of the mixing tank may be configured as a shaft or funnel, wherein the shaft or funnel may be used for loading the nutritional additives. The mixing tank may further comprise a heating system configured to heat the inner environment of the mixing tank. The mixing tank may comprise mixing mechanisms comprising at least one stirrer, paddle or any other instrument capable of mixing the nutritional additives with the purifiedprotein hydrolysate. The sealing mechanisms of the mixing tank may comprise materials such as silicone, ethylene propylene diene monomer, and polytetrafluoroethylene. The mixing tank may be configured to withstand a maximum temperature of at least 100 °C. The mixing tank may further comprise auxiliary components selected from the group of pumps, pressure sensors, flow meters, valves and means for monitoring the hydrolysis reaction.

[0250]

[0232] In one aspect of the invention, the loading tank may be configured to provide nutritional additives in dry or liquid form or a combination thereof, shear protectants, and anti-foaming agents into the mixing tank.

[0251]

[0233] In one aspect of the invention, the loading tank for the addition of nutritional additives may be composed of various materials and specific volumes described elsewhere herein.

[0252]

[0234] The first, the second and the third aspect may be applied to obtain a purified protein hydrolysate, wherein each aspect may prevent the formation of precipitates. This ensures that the filters are not blocked by precipitates and that the provided nutrients are more effectively processed by the cells, as opposed to the unusable precipitates.

[0253]

[0235] The first or the third aspect may be applied to obtain released free phosphates from the inositol hexaphosphate and its derivatives and may be used as nutrition for the cells and thus may not be added via loading tank into mixing tank with other nutritional additives.

[0254]

[0236] In one aspect of the invention, the amount of free phosphate provided from the cleaved inositol hexaphosphate and its derivatives is in a range of 50 % to 100 % of total phosphate ions in the culture medium, in a range of 60 % to 100 % of total phosphate ions in the culture medium, in a range of 70 % to 100 % of total phosphate ions in the culture medium, in a range of 80 % to 100 % of total phosphate ions in the culture medium, in a range of 90 % to 100 % of total phosphate ions in the culture medium, in a range of 60 % to 90 % of total phosphate ions in the culture medium, or in a range of 70 % to 80 % of total phosphate ions in the culture medium

[0255]

[0237] In one aspect of the invention, the amount of free phosphate provided from the cleaved inositol hexaphosphate and its derivatives is in a range of 50 % to 100 % of total phosphate ions in the protein hydrolysate, in a range of 60 % to 100 % of total phosphate ions in the purified protein hydrolysate, in a range of 70 % to 100 % of total phosphate ions in the purified protein hydrolysate, in a range of 80 % to 100 % of total phosphate ions in the purified protein hydrolysate, in a range of 90 % to 100 % of total phosphate ions in the purified protein hydrolysate, in a range of 60 % to 90 % of total phosphate ions in the purified protein hydrolysate, or in a range of 70 % to 80 % of total phosphate ions in the purified protein hydrolysate.

[0256]

[0238] In one aspect of the invention, solid residues may be removed from the culture medium by a filtration unit. In one aspect of the invention, the solid residues may be further used for the production of an edible or food product, wherein the edible or food product may be used for human or animal consumption. In another aspect of the invention, the solid residues may be removed by centrifugation. In another aspect of the invention, centrifugation and filtration may be used in sequence. Preferably, centrifugation comes before filtration to prolong the lifetime of the filter.

[0257]

[0239] In one aspect of the invention, a protein source used for hydrolysis may comprise enzymes from the group of endopeptidases, exopeptidases, phytases, beta-glucanases, lipases, nucleases, muramidase, or any other suitable enzyme. These endogenous enzymes may be released from the protein source contributing to the hydrolysis process. This process, further referred to as autolysis, may allow for hydrolysis with a reduced need for the addition of exogenous enzymes.

[0258]

[0240] In one aspect of the invention, the culture medium may be prepared by using two or more different types of protein sources. When utilizing these two or more different types of protein sources, they may be referred to as “first protein source”, “second protein source”, and so on. Enzymatic processing of these two or more protein sources is herein referred to as co-hydrolysis. In another aspect of the invention, the co-hydrolysis process may involve the combined action of endogenously producedenzymes, generated by microorganisms, and exogenously supplied enzymes added to the reaction mixture.

[0259]

[0241] In one aspect of the invention, co-hydrolysis may utilize at least one protein substrate comprising endogenously produced enzymes from the group of endopeptidases, exopeptidases, phytases, lipases, nucleases, beta-glucanases or any other suitable enzymatic activity. These enzymes may contribute to the hydrolysis of the protein source comprising the enzymes, as well as the hydrolysis of the other protein sources used. As in autolysis, this may allow for a reduced need of exogenously supplied enzymes. By combining a lower-cost protein source having little to no suitable enzymatic activity with another protein source having high suitable enzymatic activity, the nutrient yield relative to cost may be enhanced in comparison to the separate hydrolysis of either individual protein source.

[0260]

[0242] In one aspect of the invention, the exogenously supplied enzymes may comprise proteolytic enzymes, enzymes having phytase activity, or any other enzyme that may catalyze hydrolysis.

[0261]

[0243] The first protein source may comprise a plant-based protein source that is rich in protein and suitable for enzymatic hydrolysis. For example, the first protein source may comprise soy protein isolate, soy protein concentrate, soy flour, soybeans, pea protein, faba bean protein, rice protein, wheat, wheat gluten, and / or duckweed. The present list of examples is not limiting, and any plant-based protein source may be used. The first protein source may further comprise inositol hexaphosphate and / or its derivatives.

[0262]

[0244] The first protein source may comprise a plant -based protein source that is rich in protein and suitable for enzymatic hydrolysis. For example, the first protein source may comprise soy (soy protein concentrate and / or soy protein isolate), pea, rice, wheat, wheat gluten, corn, fava beans, alfalfa, hemp, chickpea, potato, pumpkin, rapeseed, red lentil, rice, sunflower, water lentil, duckweed, mungbean, bean, or any other appropriate protein source.

[0263]

[0245] The second protein source may comprise at least one protein-rich microbial biomass. The microorganisms may be capable of autolysis by production of different types of enzymes (e.g. endopeptidases, exopeptidases, beta-glucanases, nucleases, lipases) and / or capable of endogenous production of enzymes resulting in breaking-down of the peptide bonds (e.g. peptidases), phosphate ester bonds (e.g. phytases) and / or glycosidic bonds (e.g. beta-glucanase). The second protein source may contribute to the efficiency of the hydrolysis process. The microorganism may be from the group of yeasts, including but not limited to Saccharomyces cerevisiae, Pichia pastoris, Schizosaccharomyces pombe, Candida utilis, Debaryomyces hansenii, Kluyveromyces marxianus, Kluyveromyces lactis, Yarrowia lipolytica, or any other appropriate yeast.

[0264]

[0246] The second protein source may be undergoing autolysis, during which the endogenously produced enzymes degrade cellular components, including proteins. The autolysis process may be triggered by pH change, temperature change and / or addition of chemical agents. Endogenously produced enzymes may be released into the environment as a result of cell activity and / or autolysis and / or mechanical cell disruption, allowing the breakdown of plant-based proteins from the first protein source. Additionally, endogenously produced enzymes may catalyze the breakdown of phosphate ester bonds of the inositol hexaphosphate and / or its derivatives into phosphates, which may be utilized by non-human metazoan cells as nutrients. In another aspect of the invention, endogenously produced enzymes may breakdown nucleic acids into free nucleotides and / or nucleosides (including the deoxy variants) and / or free phosphates, which may be utilized by non-human metazoan cells as nutrients (notably as precursors for nucleic acid synthesis). In another aspect of the invention, endogenously produced enzymes may break down glycosidic bonds within carbohydrate molecules resulting in release of oligosaccharides, disaccharides, and / or monosaccharides, which may subsequently be utilized as energy sources or structural components by non-human metazoan cells.

[0265]

[0247] In one aspect of the invention, the cells forming the second protein source may be genetically modified and / or otherwise suitably engineered and / or grown under conditions optimized to facilitate an increased production of endogenous enzymes, preferably from the group of endopeptidases,exopeptidases, phytases and / or beta-glucanases. The genetic modification may comprise an insertion of at least one heterologous, or homologous gene encoding an enzyme for breaking down peptide bonds and / or at least one gene encoding an enzyme for breaking down phosphate ester bonds, or a combination thereof. Such modification may further increase the hydrolysis process and may reduce the need to add enzymes during the process.

[0266]

[0248] In one aspect of the invention, the terms “endogenously produced enzymes” and “production of endogenous enzymes” may be interchangeable.

[0267]

[0249] In another aspect of the invention, the expression of at least one inserted gene encoding an enzyme from the group of exopeptidases, endopeptidases, phytases, or beta-glucanases, or a combination thereof, in the genetically modified cells of the second protein source may be altered by a promoter.

[0268]

[0250] In one aspect of the invention, the endogenously produced enzymes may comprise one or more naturally occurring enzymes from the particular microorganism. These enzymes may comprise, for example, lipase, phosphoesterase, phytase, endopeptidase, exopeptidase, beta-glucanase, nuclease or any other naturally occurring enzyme within the particular microorganism. Additionally, the endogenously produced enzymes, whose genes were incorporated via genetic modification, may also comprise enzymes whose sequences were artificially designed and developed through computational methods, including machine learning techniques. These artificially designed enzymes are configured to perform substantially the same function as the naturally occurring enzyme described herein.

[0269]

[0251] The extent of enzyme production may vary depending on the strain of microorganism used as a second protein source, cultivation conditions, and nutrient availability. In some aspects of the invention, the second protein source may comprise enzymes having endopeptidase activity in the range of 0.0001 to 300,000 TU, or in the range of 0.001 to 30,000 TU, or in the range of 0.01 to 3000 TU, wherein one tyrosine unit (TU) is equal to amount of enzyme which liberates one micromole of tyrosine equivalents from casein at 40 °C and pH 7, enzymes having exopeptidase activity in the range of 0.0001 to 200,000 LAPU, or in the range of 0.001 to 20,000 LAPU, or in the range of 0.01 - 2,000 LAPU, wherein one LAPU unit is defined as amount of enzyme which liberates one micromole of p-nitroaniline from L-leucine-p-nitroanilide in one minute at 37 °C and pH 8, enzymes having phytase activity in the range of 0.0001 to 3,000,000 FTU, or in the range of 0.001 to 300,000 FTU or in the range of 0.01 to 30,000 FTU, wherein one phytase unit (FTU) is equal to amount of enzyme that liberates one micromole of phosphate in one minute from phytate under optimal conditions at 37 °C and pH 5.5, and enzymes having beta-glucanase activity in the range of 0.00001 to 10,000 FBG, or in the range of 0.0001 to 1,000 FBG, or in the range of 0.001 to 100 FBG, wherein one fungal beta-glucanase unit (FBG) is defined as amount of enzyme liberating reducing carbohydrates at a rate corresponding to one micromole of glucose per minute from beta-glucan at 37 °C and pH 5, per 1 g of the second protein source. These endogenously produced enzymes contribute to the partial hydrolysis of proteins derived from the first and / or the second protein source. Additionally, these endogenously produced enzymes may contribute to the degradation of inositol hexaphosphate and / or its derivatives, and beta-glucans and / or its derivatives. As a result, the endogenous enzyme production reduces the supplementation of any exogenously supplied enzymes, thereby decreasing the overall requirement for externally supplied enzymatic additives.

[0270]

[0252] In one aspect of the invention, the term “partial hydrolysis” refers to the breakdown of a protein or other macromolecule into smaller peptides, amino acids, or related fragments through the action of endogenously produced enzymes alone, such as those generated by the second protein source. Partial hydrolysis thus may reflect the level of protein degradation achieved prior to the introduction of any exogenously supplied enzymes, wherein the addition of exogenously supplied enzymes may further enhance, extend, or accelerate the hydrolytic process by providing additional enzymatic activities that complement or augment the endogenous enzymes.

[0271]

[0253] In another aspect of the invention, the preparation of the hydrolysis reaction mixture by the combination of the first and the second protein source may be carried out in water, optionallysupplemented with suitable additives such as acids, bases, salts or buffers. The first protein source and the second protein source may be present in the hydrolysis reaction mixture in the substrate ratios by pure protein content and / or by whole substrate weight. The ratio of the first protein source to the second protein source may be expressed as pure protein content and / or whole substrate content. The specific ratio may vary depending on the desired amino acid profile, enzymatic activity, the combination of protein sources used, and / or other parameters, such as temperature or pH.

[0272]

[0254] The first protein source and the second protein source may be in a ratio of substrates from 0.1:99.9 to 99.9:0.1 by pure protein content, or 0.5:99.5 to 99.5:0.5 by whole substrate weight, ratio of substrates from 1:99 to 99:1 by pure protein content or 2.5:97.5 to 97.5:2.5 by whole substrate weight, ratio of substrates from 2.5:97.5 to 97.5:2.5 by pure protein content or 5:95 to 95:5 by whole substrate weight. In one aspect of the invention, the plant -based protein source (first protein source) may provide the majority of amino acids to the final protein hydrolysate.

[0273]

[0255] In one aspect of the invention, to catalyze the breakdown of peptide and phosphate ester bonds from the first and second protein sources, the hydrolysis reaction mixture may be supplemented with exogenously supplied enzymes. The ratio of exogenously supplied enzyme to substrate may be expressed as a ratio of the concentration of enzyme to the concentration of protein in the reaction mixture. The concentration of the enzyme may be determined by the Bradford assay, BCA assay or other protein determination assays. This ratio may vary depending on the type of substrate, its amount, and the intended purpose of hydrolysis. The substrate may comprise, for example, the first and the second protein sources. The enzymes may comprise, for example, at least one endopeptidase, exopeptidase, beta- glucanase, phytase, any appropriate exogenous enzyme, or a combination thereof, with specific ratios optimized according to the particular application. The hydrolysis mixture may comprise, for example, soy protein as the first protein source and P. pastoris as the second protein source. The ratio of the exogenous enzyme or enzymes to the relevant substrate may be, for example, within the following ranges (the ratio of the peptidases is expressed to the final substrates mixture, the phytase is expressed to the first substrate and the beta-glucanase is expressed to the second substrate):

[0274] (a) the first set of ratios may be 0 % to 20 % of endopeptidases, 0 % to 20 % of exopeptidases, 0 % to 2 % of phytase and / or 0.01 % to 20 % of beta-glucanase;

[0275] (b) the second set of ratios may be 0.05 % to 10 % of endopeptidases, 0.05 % to 10 % of exopeptidases, 0.0005 % to 1 % of phytase and / or 0.05 % to 10 % of beta-glucanase; and

[0276] (c) the third set of ratios may be 0.1 % to 2 % of endopeptidases, 0.1 % to 2 % of exopeptidases, 0.001 % to 0.2 % of phytase and / or 0.01 % to 5 % of beta-glucanase.

[0277]

[0256] In one aspect of the invention, to catalyze the breakdown of peptide and phosphate ester bonds from the first and second protein sources, the hydrolysis reaction mixture may be supplemented with exogenously supplied enzymes. The ratio of the exogenously supplied enzyme to substrate may be expressed as a ratio of the concentration of enzyme to the concentration of protein in the reaction mixture. The concentration of the enzyme may be determined by the Bradford assay, BCA assay or other protein determination assays. This ratio may vary depending on the type of substrate, its amount, and the intended purpose of hydrolysis. The substrate may comprise, for example, the first and the second protein sources. The enzymes may comprise, for example, at least one endopeptidase, exopeptidase, beta- glucanase, phytase, any appropriate exogenous enzyme, or a combination thereof, with specific ratios optimized according to the particular application. The hydrolysis mixture may comprise, for example, soy protein as the first protein source and P. pastoris as the second protein source. The ratio of the exogenous enzyme or enzymes to the relevant substrate may be, for example, within the following ranges (the ratio of the peptidases is expressed to the final substrates mixture, the phytase is expressed to the first substrate and the beta-glucanase is expressed to the second substrate):

[0278] (a) the first set of ratios may be 0 % to 20 % (w / w) of endopeptidases, 0 % to 20 % (w / w) of exopeptidases, 0 % to 2 % (w / w) of phytase and / or 0.01 % to 20 % (w / w) of beta-glucanase;(b) the second set of ratios may be 0.05 % to 10 % (w / w) of endopeptidases, 0.05 % to 10 % (w / w) of exopeptidases, 0.0005 % to 1 % (w / w) of phytase and / or 0.05 % to 10 % (w / w) of beta- glucanase; and

[0279] (c) the third set of ratios may be 0.1 % to 2 % (w / w) of endopeptidases, 0.1 % to 2 % (w / w) of exopeptidases, 0.001 % to 0.2 % (w / w) of phytase and / or 0.01 % to 5 % (w / w) of beta-glucanase.

[0280]

[0257] In one aspect of the invention, the endogenously produced enzymes and / or exogenously supplied enzymes may catalyze the breakdown of the peptide bonds within the sources of protein resulting in the formation of the protein hydrolysate.

[0281]

[0258] Additionally, the endogenously produced enzymes and / or exogenously supplied enzymes may catalyze the breakdown of the phosphate ester bonds of the inositol hexaphosphate and / or its derivatives within the sources of protein.

[0282]

[0259] Additionally, the endogenously produced enzymes and / or exogenously supplied enzymes may catalyze the breakdown of the glycosidic bonds of the beta-glucan and / or its derivatives within the sources of protein.

[0283]

[0260] In one aspect of the invention, the breakdown of the peptide bonds within the sources of protein, the breakdown of the phosphate ester bonds of the inositol hexaphosphate and / or its derivatives and / or the breakdown of the glycosidic bonds of the beta-glucan or its derivatives may occur simultaneously and may result in formation of modified protein hydrolysate.

[0284]

[0261] The modified protein hydrolysate may be thermally treated at the end of hydrolysis to deactivate enzymes and / or microbial cells. In one aspect of the invention, this treatment may be at a “low temperature” in the range of 80 °C to 120 °C, in the range of 85 °C to 100 °C, or in the range of 90 °C to 95 °C.

[0285]

[0262] The modified protein hydrolysate may be thermally treated at the end of hydrolysis to deactivate enzymes and / or microbial cells. In one aspect of the invention, this treatment may be at “low temperature” for a duration in the range of 15 minutes to 180 minutes, or in the range of 20 minutes to 120 minutes, or in the range of 25 minutes to 60 minutes.

[0286]

[0263] In another aspect of the invention, the modified protein hydrolysate may be thermally treated at the end of hydrolysis to deactivate enzymes and / or microbial cells. In one aspect of the invention, this treatment may be at “high temperature” in the range 80 °C to 160 °C, or in the range of 100 °C to 155 °C, or in the range of 110 °C to 150 °C.

[0287]

[0264] In another aspect of the invention, the modified protein hydrolysate may be thermally treated at the end of hydrolysis to deactivate enzymes and / or microbial cells. In one aspect of the invention, this treatment may be at “high temperature” for a duration in the range of 3 seconds to 300 seconds, or in the range of 5 seconds to 60 seconds.

[0288]

[0265] The low temperature method may be performed in a hydrolysis tank. Both the low temperature and high temperature method may be performed in a hydrolysis tank configured as a flash pasteurizer or another suitable continuous flow heating device.

[0289]

[0266] Further, the solid residues from the modified protein hydrolysate may be removed to obtain a purified protein hydrolysate. The purified protein hydrolysate may subsequently be supplemented with nutritional additives to formulate a culture medium suitable for the cultivation of non -human metazoan cells.

[0290]

[0267] In one aspect of the invention, the terms “protein source contributing to the hydrolysis process”, “second protein source”, “source of protein comprising microbial biomass”, “protein source comprising microbial biomass”, “substrate”, “microorganism” or “microbial cells” may be interchangeable.

[0291]

[0268] In one aspect of the invention, the protein source comprising microbial biomass may be subjected to a cell-disruption process configured to release endogenously produced enzymes from within the microbial cells. The cell-disruption process may be performed by mechanical, chemical, or enzymatic means, or by any combination thereof, including but not limited to high-pressure homogenization (e.g.,French press or microfluidization), bead milling, ultrasonication, osmotic shock, enzymatic digestion, or detergent-assisted lysis, or any other appropriate method suitable for cell disruption. In some aspects of the invention, the extent of cell-disruption may be controlled to achieve partial or complete release of intracellular enzymes while maintaining enzyme activity for subsequent hydrolysis of the first protein source.

[0292]

[0269] In another aspect of the invention, the enzymes for the lysis and / or cell -disruption step may be from the group of muramidases, mutanolysin, lysostaphin, amylases, cellulases, hemicellulases, pectinases, pectin lyases, arabinanases, galactanases, [3-glucanases, [3-glycosidases, Viscozyme® enzyme, or other enzyme preparations capable of degrading cell wall components such as peptidoglycan, cellulose, hemicellulose, amylose, amylopectin, or [3-glucans, and / or any mixture thereof.

[0293]

[0270] In another aspect of the invention, the lysis and / or cell-disruption step applied to the protein source comprising microbial biomass may be configured to facilitate the release of intracellular and / or periplasmic enzymes naturally produced by the protein source comprising microbial biomass. These enzymes may comprise, but are not limited to, one or more proteolytic enzymes, peptidases, enzymes having phytase activity, carbohydrases, or other hydrolytic enzymes that contribute to the degradation of the protein source. The conditions of the lysis step may be optimized to promote enzyme release while preserving enzymatic activity for use in the subsequent hydrolysis or co-hydrolysis stage.

[0294]

[0271] In some aspects of the invention, the lysis and / or cell disruption step may be adjusted in intensity or duration to balance enzyme liberation and stability, thereby enhancing the overall efficiency of the hydrolytic conversion.

[0295]

[0272] In one aspect of the invention, the first protein source may be subjected to a fermentation process, wherein the first source of protein may be subjected to submerged fermentation or solid-state fermentation.

[0296]

[0273] In another aspect of the invention, the first protein source may be mixed with water to reach a concentration in a range from 0.1 to 250 g / L, in a range of 0.5 to 200 g / L or in a range of 1 to 150 g / L and then further subjected for the submerged fermentation. Other nutrients can be added to the submerged fermentation mixture to support growth of the microorganism and production of enzymes. In another aspect of the invention, the first protein source can be also left out from the submerged fermentation and replaced by other nutrients.

[0297]

[0274] In another aspect of the invention, the first protein source may be mixed with a limited amount of water, wherein the limited amount of water added to the source of protein may be in a range from 0.3 mL / g to 4 mL / g, or in a range from 0.4 mL / g to 3 mL / g, or in a range from 0.5 mL / g to 2 mL / g of source of protein and further subjected to solid-state fermentation.

[0298]

[0275] In another aspect of the invention, the first source of protein may be mixed with a limited amount of water in a ratio in a range of 0.25:1 to 0.33:1, in a range of 0.33:1 to 2.5:1, or in a range of 0.5:1 to 1:2 and then further subjected to solid-state fermentation.

[0299]

[0276] In one aspect of the invention, submerged and / or solid-state fermentation may be performed by mixing a first protein source with the protein source comprising microbial biomass, wherein the cells of microbial biomass may be viable.

[0300]

[0277] In another aspect of the invention, prior to submerged and / or solid-state fermentation and / or initiation of the hydrolysis reaction, a protein source comprising microbial biomass may be prepared as an inoculum and expanded in a culture medium to obtain a desired biomass level suitable for use as the second protein source in the fermentation and co-hydrolysis process.

[0301]

[0278] The inoculum of a protein source comprising microbial biomass may be provided in the form of a liquid suspension, a semi-solid form, a concentrated biomass slurry, and / or a dried or lyophilized powder that may be reconstituted in culture medium, water or buffer prior to use. In some aspects of the invention, the inoculum may comprise whole viable cells, resting cells, or lysed biomass containing active intracellular enzymes. The concentration, moisture content, and physiological state of theinoculum may be selected to ensure optimal conditions for the fermentation step and / or compatibility with the first protein source during the subsequent hydrolysis step.

[0302]

[0279] The term “optimal conditions for the fermentation step” refers to a set of physico-chemical and operational parameters adjusted to support the desired microbial growth, metabolic activity, and product formation in a manner that maximizes yield, productivity, and / or process efficiency. Such optimal conditions typically include, but are not limited to pH, temperature, nutrient composition in the fermentation mixture, osmotic and ionic conditions, and inoculum density.

[0303]

[0280] In another aspect of the invention, the expanded protein source comprising microbial biomass may be used for fermentation to achieve a desired enzymatic activity, usually a high enzymatic sactivity, before being combined with the first protein source. At least one of the physico-chemical conditions and / or nutrient composition in the fermentation mixture may be adjusted during fermentation to induce the production of at least one endogenously produced enzyme (e.g., proteases, peptidases, glycosidases, lipases, phytases). The adjusted physico-chemical conditions and / or nutrient composition in the fermentation mixture may include, but are not limited to, a specific temperature, pH, oxygen level, a nitrogen source, a carbon source, a phosphate source, mineral salts, trace elements, vitamins, growth factors, anti-foaming agents, acids and bases level and composition, the ratio of carbon to nitrogen source, the ratio of nitrogen to phosphate source, the ratio of carbon to phosphate source, and / or any combination thereof.

[0304]

[0281] In yet another aspect of the invention, the up-regulation of the production of at least one endogenously produced enzyme may be nutritionally triggered prior to mixing with the first protein source in one of the following ways:

[0305] (a) The amount of nitrogen source may be altered by introducing a signaling-level, low, medium or high dose of a protein material and / or additional nitrogen-containing nutrient and / or an equivalent inducer while not materially advancing the growth of the protein source comprising microbial biomass, thereby favoring expression of proteolytic enzymes. The proteinaceous nutrient, nitrogen-containing nutrient and equivalent inducer that increases protease production may be characterized by a final concentration in a range of 0.001 mg / L to 100,000 mg / L, a range of 0.005 mg / L to 60,000 mg / L, in a range of 0.01 mg / L to 30,000 mg / L in the culture media, wherein the proteinaceous nutrient, nitrogen-containing nutrient and equivalent inducer that elevates protease expression may be at least one of:

[0306] (i) protein materials and their derivatives, including, but not limited to proteins (such as casein, soy protein, collagen, albumins, plant, animal and / or microbial hydrolysates, tryptone, extract of microorganisms and / or plants) and / or peptides and / or amino acid mixture, wherein the final concentration may be in a range of 1,000 mg / L to 100,000 mg / L, in a range of 1 ,200 mg / L to 60,000 mg / L, or in a range of 1 ,500 mg / L to 30,000 mg / L in the culture media; and

[0307] (ii) nitrogen-containing nutrients, including but not limited to inorganic compounds (such as ammonium salts, like (NH^SCL, NH4Q, NH4NO3, (bTLjsPCL, (NH4)2HP04, (NH4)H2P04, nitrates, urea) and / or organic compounds (such as amines, amides, amino sugars, nucleotides, ureides), wherein the final concentration may be in a range of 0.001 mg / L to 10,000 mg / L, in a range of 0.005 mg / L to 9,000 mg / L, or in a range of 0.01 mg / L to 8,000 mg / L in the culture media.

[0308] (b) The amount of carbon source may be altered by introducing a signaling-level, low, medium, or high dose of a carbonaceous nutrient and / or an equivalent inducer while not materially advancing the growth of the protein source comprising microbial biomass, thereby favoring expression of hydrolytic, catabolic and proteolytic enzymes. The carbonaceous nutrient and the equivalent inducer that increases enzyme production may be characterized by a final concentration in a range of 0.001 mg / L to 60,000 mg / L, in a range of 0.005 mg / L to 30,000 mg / L, or in a range of 0.01 mg / L to 20,000mg / L in the culture media, wherein the carbonaceous nutrient and the equivalent inducer that may elevate enzyme production may be at least one of:

[0309] (i) Carbohydrates and / or their derivatives, including, but not limited to monosaccharides (e.g. glucose, fructose), disaccharides (e.g. sucrose, lactose), oligosaccharides, and polysaccharides (e.g., starches, dextrins, pectins, inulin, hemicelluloses, cellulose hydrolysates),

[0310] (ii) Complex carbohydrate sources (e.g. whey, rice bran, corn steep liquor, sugar syrups, molasses),

[0311] (iii) Organic acids and / or their salts (e.g., acetate, lactate, citrate, succinate), (iv) Alcohols (e.g., methanol, ethanol), and

[0312] (v) Lipids / fats, triglycerides, fatty acids, and plant or lignocellulosic extracts / hydrolysates (including food / feed side-streams containing assimilable carbon).

[0313] (c) The amount of phosphate source may be altered by a signaling-level, low, medium, or high dose of a phosphate-containing nutrient and / or an equivalent inducer while not materially advancing the growth of the protein source comprising microbial biomass, thereby favoring expression of enzymes having phytase activity. The phosphate-containing nutrient and the equivalent inducer that may elevate production of enzyme having phytase activity may be characterized by a final concentration in a range of 0.001 mg / L to 60,000 mg / L, in a range of 0.005 mg / L to 30,000 mg / L, or in a range of 0.01 mg / L to 20,000 mg / L in the culture media, wherein the phosphate-containing nutrient and the equivalent inducer that may increase production of at least one enzyme having phytase activity may be at least one of:

[0314] (i) Inorganic orthophosphates and / or their salts (including, without limitation, KH2PO4, K2HPO4, NaFLPCL, Na2HP04),

[0315] (ii) Polyphosphates (e.g., sodium or potassium polyphosphate),

[0316] (iii) Organophosphate compounds (including phosphorylated sugars such as glucose-6-phosphate, nucleotides / nucleosides and their salts, phospholipids, phosphoproteins and phosphopeptides, inositol phosphates), and

[0317] (iv) Food / feed side-streams containing assimilable phosphorus.

[0318] (d) The amount of sulfur source may be altered by introducing a signaling-level, low, medium, or high dose of a sulfur-containing nutrient and / or an equivalent inducer while not materially advancing the growth of the protein source comprising microbial biomass, thereby favoring expression of sulfatases, sulfonatases and / or hydrolytic / catabolic enzymes. The sulfur-containing nutrient and the equivalent inducer that increases enzyme production may be characterized by a final concentration in a range of 0.001 mg / L to 60,000 mg / L, in a range of 0.005 mg / L to 30,000 mg / L, or in a range of 0.01 mg / L to 20,000 mg / L in the culture media, wherein the sulfur-containing nutrient and the equivalent inducer may be at least one of:

[0319] (i) Inorganic sulfur sources including, but not limited to sulfate (e.g., Na2SC>4, BLSCh, (NH4)2S04), sulfite (e.g., Na2SCh), thiosulfate (e.g., Na2S2Ch),

[0320] (ii) Organosulfur compounds including sulfur-containing amino acids (e.g., cysteine, methionine), glutathione, taurine, sulfonates (e.g., p-toluene sulfonate, alkanesulfonates), sulfate esters (e.g., choline sulfate, aryl / alkyl sulfates), sulfoxides / sulfones (e.g., DMSO, DMSP, DSS), and

[0321] (iii) Food / feed side-streams containing assimilable sulfur.

[0322]

[0282] In one aspect of the invention, protein materials and their derivatives, including, but not limited to proteins (such as casein, soy protein, collagen, albumins, plant, animal and / or microbial hydrolysates, tryptone, extract of microorganisms and / or plants) and / or peptides and / or amino acid mixture, may have a final concentration may be in a range of 1,000 mg / L to 100,000 mg / L, a range of 1,200 mg / L to 60,000mg / L, or a range of 1 ,500 mg / L to 30,000 mg / L in the culture media, wherein the concentration may depend on the specific protein material used.

[0323]

[0283] In one aspect of the invention, nitrogen-containing nutrients, including but not limited to inorganic compounds (such as ammonium salts, like (NH^SC , NH4Q, NH4NO3, (NI ^PCh, (NH4)2HP04, (NH4)H2P04, nitrates, urea) and / or organic compounds (such as amines, amides, amino sugars, nucleotides, ureides), may have a the final concentration in a range of 0.001 mg / L to 10,000 mg / L, a range of 0.005 mg / L to 9,000 mg / L, or a range of 0.01 mg / L to 8,000 mg / L in the culture media, wherein the concentration may depend on the specific nitrogen-containing nutrient.

[0324]

[0284] In one aspect of the invention, the term “signaling-level dose” refers to a concentration of a nutrient, inducer, or substrate that is sufficient to activate or modulate one or more cellular regulatory pathways associated with nutrient metabolism, wherein the concentration may be in a range of 0.001 ng / pL to 100 ng / pL, or 0.005 ng / pL to 75 ng / pL, or 0.01 ng / pL to 50 ng / pL.

[0325]

[0285] In one aspect of the invention, the term “low dose” refers to a concentration of a nutrient, inducer, or substrate that is sufficient to activate or modulate one or more cellular regulatory pathways associated with nutrient metabolism, wherein the concentration may be in a range of 50 ng / pL to 1000 ng / pL, or 75 ng / pL to 750 ng / pL, or 100 ng / pL to 500 ng / pL.

[0326]

[0286] In one aspect of the invention, the term “medium dose” refers to a concentration of a nutrient, inducer, or substrate that elicits a measurable metabolic or physiological response greater than that induced by a low dose, but below the saturation or maximal response levels, wherein the concentration may be in a range of 500 ng / pL to 50,000 ng / pL, or 750 ng / pL to 20,000 ng / pL, or 1,000 ng / pL to 10,000 ng / pL.

[0327]

[0287] In one aspect of the invention, the term “high dose” refers to a concentration of a nutrient, inducer, or substrate that produces a near -maximal or maximal metabolic or physiological response, optionally approaching saturation of the relevant cellular pathways, wherein the concentration may be in a range of 10,000 ng / pL to 200,000 ng / pL, or 20,000 ng / pL to 150,000 ng / pL, or 50,000 ng / pL to 100,000 ng / pL.

[0328]

[0288] Submerged fermentation and / or solid-state fermentation may be performed under aerobic or anaerobic conditions. Fermentation may be performed in a fermentation vessel, such as but not limited to, a stirred-tank bioreactor, aerobic or anaerobic digester, or airlift bioreactor. Appropriate stirring, aeration, temperature, humidity and pH control may be provided in the vessel. Advantageously, anaerobic fermentation may not require aeration or significant stirring after the initial mixing of the inoculum and the first protein source, presenting simplified hardware requirements.

[0329]

[0289] In one aspect of the invention, the fermentation vessel used for cultivating the inoculum of the protein source comprising microbial biomass may comprise a container having an internal volume in a range from 0.01 L to 10,000 L, or in a range from 0.1 L to 5,000 L, or in a range from 0.5 L to 500 L. The vessel may be constructed from stainless steel, glass, polymeric materials, or any other material resistant to microbial, enzymatic, and chemical activity occurring during fermentation.

[0330]

[0290] In one aspect of the invention, the fermentation vessel may be equipped with suitable means of loading and mixing the substrate, inoculum, water and / or other ingredients. For example, a hybrid powder mixer may be used. In another aspect of the invention an external-loop circulation pump may be used to improve protein powder dispersion and to draw protein powder closer to the agitator.

[0331]

[0291] In one aspect of the invention, the fermentation vessel may be able to resist an internal overpressure of at least 1 barg.

[0332]

[0292] In one aspect of the invention, the fermentation vessel may be compatible with a clean-in-place (CIP) procedure using a solution of a base and / or solution of an acid.

[0333]

[0293] In one aspect of the invention, the fermentation vessel may be compatible with a sterilize-in- place (SIP) procedure using steam at >120 °C.

[0294] The fermentation vessel may be equipped with a temperature -control system configured to maintain the internal temperature in a range from 10 °C to 70 °C, or in a range from 20 °C to 60 °C, or in a range from 25 °C to 50 °C, depending on the microbial strain used in the inoculum.

[0334]

[0295] In one aspect of the invention, the fermentation vessel may comprise an agitation system including impellers, paddles, rotating shafts, or gas flow mechanisms, providing a mixing speed in a range from 10 rpm to 1000 rpm, or in a range from 20 rpm to 500 rpm. Anaerobic fermentation may be performed with minimal or no agitation after initial inoculum-substrate mixing. In another aspect of the invention, the fermentation vessel may be equipped with an impeller for mixing of very viscous fluids and / or semi-solids, such as a close-clearance helical ribbon impeller.

[0335]

[0296] The fermentation vessel may include sensors for monitoring pH, temperature, dissolved oxygen, pressure, and microbial density, wherein the pH may be controlled in a range from 3.0 to 9.0, or in a range from 4.0 to 8.0, depending on the microorganism used in the inoculum.

[0336]

[0297] In one aspect of the invention, the source of protein may be mixed with a limited amount of water to form a solid to semi-solid mixture. This mixture may optionally be inoculated with an inoculum of the protein source comprising microbial biomass, referred to as solid-state mixture. The mixture may be incubated at a temperature that allows the native microorganisms of the first protein source and / or the inoculated protein source comprising microbial biomass to ferment the first protein source and chemically alter it for further processing. This process will further be referred to as solid-state fermentation. A fermented first protein source may subsequently be used for hydrolysis.

[0337]

[0298] In one aspect of the invention, the ratio of the inoculum to the first protein source (wet microbial biomass to substrate dry matter) may be in the range 1:1,000,000 to 1:1, or in range 1:100,000 to 1:10, or in range 1:10,000 to 1:50. In another aspect of the invention, an existing batch of fermented first protein source comprising viable microorganisms may serve to inoculate the new batch. In another aspect of the invention, when inoculating a new batch of the first protein source with an existing batch of fermented first protein source, the ratio of the old to the new batch may be in the range of 1 : 1 ,000 to 1 : 1 , or in the range of 1:500 to 1:3, or in the range of 1:100 to 1:5.

[0338]

[0299] In one aspect of the invention, the mixture of the first protein source with water and an inoculum of the protein source comprising microbial biomass for the submerged fermentation, referred to as submerged fermentation mixture, may be used in a ratio, wherein the ratio of the volume of the first protein source with water to the volume of the inoculum of the protein source comprising microbial biomass may be in a range from 1:10,000 to 1:10, or in a range from 1:5,000 to 1:10, or in a range from 1:1,000 to 1:10, depending on the desired enzymatic activity and the properties of the inoculum.

[0339]

[0300] In one aspect of the invention, the solid-state fermentation mixture may comprise a moisture content in the range of 20 % to 80 % (w / w), or in a range of 25 % to 75 % (w / w), or in a range of 30 % to 70 % (w / w).

[0340]

[0301] In one aspect of the invention, solid-state fermentation may be performed at a water activity in the mixture in the range of 0.5 to 1, or in a range of 0.6 to 1, or in a range of 0.7 to 1.

[0341]

[0302] In another aspect of the invention, the solid-state fermentation mixture may be incubated at a temperature in a range of 15 °C to 60 °C, or in a range of 20 °C to 40 °C, or in a range of 22 °C to 35 °C for a period of 6 hours to 7 days, or in a range of 12 hours to 5 days, or in a range of 18 hours to 3 days under controlled humidity conditions suitable for the growth of the protein source comprising microbial biomass and / or enzyme secretion.

[0342]

[0303] In one aspect of the invention, the solid-state fermentation mixture may be supplemented with one or more of:

[0343] (a) A carbohydrate source, for example starch, wheat flour, or glucose (to promote microbial growth in substrates with low content of easily digestible carbon sources);

[0344] (b) A selective antimicrobial compound, for example s lysozyme (to selectively inhibit the growth of undesirable microorganisms, while leaving desirable microorganisms unaffected);(c) A pH-adjusting reagent, for example HCL, lactic acid, or NaOH; and

[0345] (d) A buffering agent, for example sodium citrate or sodium phosphate.

[0346]

[0304] In one aspect of the invention, during incubation, the protein source comprising microbial biomass may produce a complex of endogenously produced enzymes that may act in situ on the solid source of protein, resulting in partial hydrolysis and higher degree of solubility of the protein matrix.

[0347]

[0305] In one aspect of the invention, the endogenously produced enzymes produced by the protein source comprising microbial biomass during solid-state fermentation may remain active when the first protein source is introduced into a hydrolysis reaction, acting synergistically with exogenously supplied enzymes.

[0348]

[0306] In one aspect of the invention, the overall process may provide a synergistic effect, wherein the endogenous enzyme production, the first protein source softening, and partial hydrolysis during the solid- state fermentation may enhance the rate and degree of enzymatic hydrolysis during the following liquidphase stage, thereby increasing overall conversion efficiency.

[0349]

[0307] In another aspect of the invention, the synergistic effect of solid-state fermentation of the first protein source may result in an increase of the degree of hydrolysis in the step of enzymatic hydrolysis by 2 % to 60 %, or by 5 % to 50 %, or by 10 % to 40 %, or by 15 % to 35 % compared to unfermented substrate, and / or in an increase in the yield of free amino acids by 2 % to 60 %, or by 5 % to 50 %, or by 10 % to 40 %, or by 15 % to 35 % compared to an unfermented first protein source.

[0350]

[0308] In another aspect of the invention, the synergistic effect of solid-state fermentation and the protein source of microbial biomass may allow for a lower dose of at least one exogenously supplied enzyme during the hydrolysis process without reducing the degree of hydrolysis or amino acid yield in comparison to an unfermented first protein source.

[0351]

[0309] In one aspect of the invention, the solid-state fermentation may be performed in the same cultivation system as the hydrolysis or co-hydrolysis process. In another aspect of the invention, the solid-state fermentation process may be performed apart from the cultivation system.

[0352]

[0310] In one aspect of the invention, after completion of the solid-state fermentation, the solid-state fermentation mixture may be diluted with water, buffer, or any other suitable aqueous solution to obtain a protein hydrolysate. The volume of the water, buffer or any other suitable aqueous solution may be in a range of 1 L to 7,000 L, or in a range of 1 L to 5,000 L, or in a range of 1 L to 3,000 L, or in a range of 1 L to 1 ,500 L, depending on the capacity of the fermentation vessel used for cell cultivation.

[0353]

[0311] In another aspect of the invention, the volume of the water, buffer or any other suitable aqueous solution may be adjusted to achieve a hydrolysate concentration of approximately 90 g / L, corresponding, for example, to 450 kg of the solid-state fermentation mixture diluted in approximately 5,000 L of solvent, or an equivalent ratio in smaller or larger production scales.

[0354]

[0312] In another aspect of the invention, following the fermentation process, the mixture may be supplemented with exogenously supplied enzymes to initiate the co-hydrolysis process.

[0355]

[0313] In another aspect of the invention, a protein source comprising microbial biomass may be introduced into the hydrolysis reaction while the microbial cells are viable. Viable microbial cells may survive and / or grow in the hydrolysis mixture for some time, until the temperature and / or pH and / or any other physico-chemical factor reaches the deactivation limit of microbial cells and / or an enzyme enhancing lysis of microbial cells is introduced. This may allow for a higher amount of endogenous enzymes to be introduced into the hydrolysis mixture. The hydrolysis reaction mixture may be supplemented with nutritional additives to support the growth of the cells from the source of protein comprising microbial biomass.

[0356]

[0314] In one aspect of the invention, the overall process may be that depicted in the scheme of Fig.

[0357] 21. The present scheme flow comprises a fermentation process followed by a co-hydrolysis process. The process may begin with the preparation of an inoculum of the protein source comprising microbial biomass (1001) capable of production of endogenously produced enzymes suitable for fermentation andhydrolysis. The inoculum of protein source comprising microbial biomass may be mixed with a first protein source (1002) to form either (i) a submerged fermentation mixture, in which the inoculum and the protein source are combined with water (1003) in a predetermined volume resulting in submerged fermentation (1004), or (ii) a solid-state fermentation mixture, in which the inoculum and the protein source are combined with a limited amount of water resulting in solid-state fermentation (1005). In the solid-state fermentation mixture, optionally, an amount of exogenously supplied enzymes (1007) may be added to stimulate endogenously produced enzymes of the protein source comprising microbial biomass. The submerged fermentation mixture, the solid-state fermentation mixture, or both, may be subjected to fermentation under appropriate environmental conditions to enable microbial growth and endogenous enzyme secretion. Following solid-state fermentation, an additional volume of water may be added. Such additional volume of water may be in a range of 1 L to 7,000 L. After completion of fermentation in either mode, exogenously supplied enzymes may be introduced into the fermented first protein source. These exogenously supplied enzymes cooperate with the endogenously produced enzymes synthesized during fermentation to initiate and carry out a co-hydrolysis process, resulting in the breakdown of the protein source into peptides and / or amino acids.

[0358]

[0315] In one aspect, the invention provides a method comprising solid-state fermentation, wherein the method comprises:

[0359] (a) providing a first source of protein comprising a plant -based protein source; (b) mixing the first source of protein with limited amount of water and an inoculum comprising a protein source of microbial biomass;

[0360] (c) incubating the solid-state mixture for 12 hours to 7 days at temperatures ranging from 25 °C to 45 °C; and

[0361] (d) allowing endogenously produced enzymes from the protein source comprising a microbial biomass to act in situ on the solid protein source to achieve partial hydrolysis of the protein source.

[0362]

[0316] In one aspect, the invention provides a method comprising solid-state fermentation and hydrolysis, wherein the method comprises:

[0363] (a) providing a first source of protein comprising a plant -based protein source; (b) mixing the first source of protein with a limited amount of water and an inoculum comprising a protein source of microbial biomass;

[0364] (c) incubating the solid-state mixture for 12 hours to 7 days at temperatures ranging from 25 °C to 45 °C;

[0365] (d) allowing endogenously produced enzymes from the protein source comprising a microbial biomass to act in situ on the protein source to achieve partial hydrolysis of the protein source; and (e) adding exogenously supplied enzymes to the solid-state mixture to finish a hydrolysis process thereby generating a protein hydrolysate.

[0366]

[0317] In one aspect, the invention provides a culture medium obtained by a method of solid-state fermentation, wherein the method may comprise:

[0367] (a) providing a first source of protein comprising a plant -based protein source; (b) mixing the source of protein with limited amount of water and an inoculum comprising a protein source of microbial biomass;

[0368] (c) incubating the solid-state mixture for 12 hours to 7 days at temperatures ranging from 25 °C to 45 °C; and

[0369] (d) allowing endogenously produced enzymes from the protein source comprising a microbial biomass to act in situ on the solid protein source to achieve partial hydrolysis of the protein source.

[0370]

[0318] In one aspect, the invention provides a method comprising solid-state fermentation and hydrolysis, wherein the method comprises:

[0371] (a) providing a first source of protein comprising a plant -based protein source;(b) mixing the source of protein with limited amount of water and an inoculum comprising a protein source of microbial biomass;

[0372] (c) incubating the solid-state mixture for 12 hours to 7 days at temperatures ranging from 25 °C to 45 °C;

[0373] (d) allowing endogenously produced enzymes from the protein source comprising a microbial biomass to act in situ on the protein source to achieve partial hydrolysis of the protein source;

[0374] (e) optionally lysing the protein source comprising a microbial biomass to release endogenously produced enzymes;

[0375] (f) generating a protein hydrolysate.

[0376]

[0319] In one aspect, the invention provides a method comprising solid-state fermentation and hydrolysis, wherein the method comprises:

[0377] (a) providing a first source of protein comprising a plant -based protein source;

[0378] (b) mixing the source of protein with limited amount of water and an inoculum comprising a protein source of microbial biomass;

[0379] (c) incubating the solid-state mixture for 12 hours to 7 days at temperatures ranging from 25 °C to 45 °C;

[0380] (d) allowing endogenously produced enzymes from the protein source comprising a microbial biomass to act in situ on the solid protein source to achieve partial hydrolysis of the protein source;

[0381] (e) optionally lysing the protein source comprising a microbial biomass to release endogenously produced enzymes; and

[0382] (f) adding exogenously produced enzymes to the solid-state mixture to finish a hydrolysis process, thereby generating a protein hydrolysate.

[0383]

[0320] In one aspect, the invention provides a culture medium obtained by a method of solid-state fermentation, wherein the method may comprise:

[0384] (a) providing a first source of protein comprising a plant -based protein source;

[0385] (b) mixing the source of protein with limited amount of water and an inoculum comprising a protein source of microbial biomass;

[0386] (c) incubating the solid-state mixture for 12 hours to 7 days at temperatures ranging from 25 °C to 45 °C;

[0387] (d) allowing endogenously produced enzymes from the protein source comprising a microbial biomass to act in situ on the solid protein source to achieve partial hydrolysis of the protein source; and (e) adding at least one nutritional additive.

[0388]

[0321] In one aspect, the invention provides a culture medium obtained by a method of solid-state fermentation and hydrolysis, wherein the method may comprise:

[0389] (a) providing a first source of protein comprising a plant -based protein source; (b) mixing the source of protein with a limited amount of water and an inoculum comprising a protein source of microbial biomass;

[0390] (c) incubating the solid-state mixture for 12 hours to 7 days at temperatures ranging from 25 °C to 45 °C;

[0391] (d) allowing endogenously produced enzymes from the protein source comprising a microbial biomass to act in situ on the solid protein source to achieve partial hydrolysis of the protein source;

[0392] (e) adding exogenously supplied enzymes to the solid-state mixture to finish a hydrolysis process; and

[0393] (f) adding at least one nutritional additive.

[0394]

[0322] In one aspect, the invention provides a method of hydrolyzing a protein source comprising:

[0395] (a) preparing an inoculum comprising a protein source of microbial biomass;(b) optionally subjecting the inoculum comprising the protein source of microbial biomass to at least one physico-chemical condition and / or nutrient composition to enhance the production of endogenously produced enzymes;

[0396] (c) introducing the inoculum of the protein source comprising a microbial biomass into a hydrolysis reaction mixture or solid-state mixture to prepare a protein hydrolysate.

[0397]

[0323] In one aspect, the invention comprises a method of hydrolyzing a protein source comprising:

[0398] (a) preparing an inoculum comprising a protein source of microbial biomass;

[0399] (b) optionally subjecting the inoculum comprising the protein source of microbial biomass to at least one physico-chemical condition and / or nutrient composition to enhance the production of endogenously produced enzymes;

[0400] (c) introducing the inoculum comprising the protein source comprising a microbial biomass into a hydrolysis reaction mixture or solid-state mixture to prepare a protein hydrolysate; and

[0401] (d) adding exogenously supplied enzymes to the solid-state mixture to finish a hydrolysis process, thereby generating a protein hydrolysate.

[0402]

[0324] In one aspect of the invention, a method for co-hydrolyzing the protein source may comprise:

[0403] providing at least two protein sources to form hydrolysis reaction mixture, wherein

[0404] a first protein source comprises a plant-based protein source; and

[0405] a second protein source comprises at least one microorganism; and wherein

[0406] the second protein source comprises endogenously produced enzymes suitable for co-hydrolysis.

[0407]

[0325] In one aspect of the invention, a method for co-hydrolyzing the protein source may comprise:

[0408] (i) providing at least two protein sources to form hydrolysis reaction mixture, wherein:

[0409] (a) a first protein source comprises a plant-based protein source; and

[0410] (b) a second protein source comprises at least one microorganism;

[0411] wherein the second protein source comprises endogenously produced enzymes suitable for co- hydrolysis; and

[0412] (ii) combining the at least two protein sources to form a hydrolysis mixture to initiate the cohydrolysis with added exogenously supplied enzymes,

[0413] wherein the endogenously produced enzymes from the second protein source catalyze:

[0414] (a) the breakdown of peptide bonds to release peptides and amino acids; and

[0415] (b) the breakdown of phosphate ester bonds of inositol hexaphosphate and / or its derivatives to release phosphate ions.

[0416]

[0326] In one aspect of the invention, a method for co-hydrolyzing the protein source may comprise:

[0417] (i) providing at least two protein sources to form hydrolysis reaction mixture, wherein:

[0418] (a) a first protein source comprises a plant-based protein source; and

[0419] (b) a second protein source comprises at least one microorganism;

[0420] wherein the second protein source comprises endogenously produced enzymes suitable for cohydrolysis; and

[0421] (ii) combining the at least two protein sources to form a hydrolysis mixture to initiate the co- hydrolysis with added exogenously supplied enzymes,

[0422] wherein the endogenously produced enzymes from the second protein source catalyze:

[0423] (a) the breakdown of peptide bonds to release peptides and amino acids; and

[0424] (b) the breakdown of phosphate ester bonds of inositol hexaphosphate and / or its derivatives to release phosphate ions; and

[0425] (iii) providing exogenously supplied enzymes for the hydrolysis.

[0426]

[0327] In one aspect of the invention, a method for producing culture medium by co-hydrolysis of a protein source may comprise:

[0427] (i) providing at least two protein sources, wherein:

[0428] (a) a first protein source comprises plant-based protein source; and(b) a second protein source comprises at least one microorganism;

[0429] wherein the hydrolysis mixture is subjected to co-hydrolysis by endogenously produced enzymes from the second protein source; and

[0430] (ii) providing exogenously supplied enzymes for the hydrolysis;

[0431] wherein the exogenously supplied enzymes and endogenously produced enzymes exert a synergic effect in the generation of protein hydrolysate; and

[0432] (iii) purifying the resulting protein hydrolysate to prepare a culture medium for the cultivation of non-human metazoan cells.

[0433]

[0328] In one aspect of the invention, a culture medium may comprise:

[0434] (i) a purified protein hydrolysate, obtained by a method comprising at least two protein sources, wherein a first protein source comprises a plant-based protein source and a second protein source comprises microorganisms with endogenously produced enzymes; and

[0435] (ii) at least one nutritional additive.

[0436]

[0329] In one aspect of the invention, a culture medium may comprise:

[0437] (i) a purified protein hydrolysate, obtained by a method comprising at least two protein sources, wherein a first protein source comprises a plant-based protein source and a second protein source comprises microorganisms with endogenously produced enzymes; and

[0438] (ii) at least one nutritional additive,

[0439] wherein the culture medium comprises peptides, amino acids and phosphate ions, thereby promoting the proliferation of non-human metazoan cells.

[0440]

[0330] In one aspect of the invention, a cultivation system for the preparation of the culture medium by co-hydrolysis may comprise:

[0441] at least one culture medium tank, configured for the preparation of culture medium,

[0442] wherein the culture medium tank comprises at least one of: a mixing tank, a hydrolysis tank, a storage tank, a loading tank, and a waste medium tank,

[0443] wherein the enzymatic co-hydrolysis may be performed in the hydrolysis tank by a combination of at least two protein sources, wherein a second protein source comprises at least one microorganism with endogenously produced enzymes, which are combined with exogenously supplied enzymes, thereby resulting in the breakdown of peptide bonds of the protein source and the breakdown of phosphate ester bonds of inositol hexaphosphate and its derivatives.

[0444]

[0331] In one aspect of the invention, a cultivation system for the preparation of a culture medium by co-hydrolysis may comprise:

[0445] (i) at least one culture medium tank, configured for the preparation of the culture medium, wherein the culture medium tank comprises at least one of: a mixing tank, a hydrolysis tank, a storage tank, a loading tank, or a waste medium tank,

[0446] wherein the enzymatic co-hydrolysis may be performed in the hydrolysis tank by the combination of at least two protein sources, wherein a second protein source comprises at least one microorganism with endogenously produced enzymes, which are combined with exogenously supplied enzymes, thereby resulting in the breakdown of peptide bonds of the protein source and the breakdown of phosphate ester bonds of inositol hexaphosphate and its derivatives; and

[0447] (ii) a control unit

[0448]

[0332] In one aspect of the invention, a cultivation system for the preparation of the culture medium by co-hydrolysis may comprise:

[0449] at least one hydrolysis tank configured to perform enzymatic hydrolysis of two protein sources; a first protein source comprising at least one plant -based protein source;

[0450] a second protein source comprising biomass of at least one converting microorganism;wherein the second protein source endogenously produces enzymes which are combined with exogenously supplied enzymes, thereby resulting in the breakdown of peptide bonds of the protein source and the breakdown of phosphate ester bonds of inositol hexaphosphate and its derivatives; and wherein the at least one hydrolysis tank may be configured to thermally inactivate both protein sources (i.e. to stop autolysis of the first protein source and / or to stop proliferation of the second protein source) at the end of enzymatic hydrolysis to sterilize the protein hydrolysate and provide protein hydrolysate that is free of any active converting microorganism.

[0451]

[0333] In one aspect of the invention, the composition of the culture medium may be defined in terms of the total input of medium components into the cultivation process. In this aspect of the invention, summary amounts of components introduced into the cultivation process at any time point over its entire duration are provided. Furthermore, in this aspect of the invention, the provided concentration ranges for the individual medium components describe the total amount of the given component introduced into the cultivation process at any time point during the cultivation process in relation to the volume of spent culture medium which exits the process. The spent culture medium may exit the cultivation process together with the cultivated cells (harvesting), or separately from the cultivated cells (perfusion). The cultivation process may further have the characteristics of a batch process, where all of the components are introduced into the cultivation process at a single time point and the harvest is performed at a single time point, a fed-batch process, where some components may be introduced after the start of the process and the harvest is done at a single time point, a continuous process, where components may be introduced during the whole duration of cultivation and harvesting may be performed during the whole duration of cultivation, or a combination of the described characteristics. For brevity, this aspect of the invention will be referred to herein as “total input.

[0452]

[0334] In another aspect of the invention, the composition of the culture medium may be described in terms of the concentration of components which are present at a particular time point during the cell cultivation process in the culture medium. In this aspect of the invention, the provided concentration ranges for the individual medium components describe the concentrations present in the culture medium in the cultivation device at any time point during the cultivation process. For brevity, this aspect of the invention will be referred to herein as “momentary composition.”

[0453]

[0335] The total inputs into the culture medium according to the invention may comprise an optimized ratio of essential amino acids, which may be sourced from a protein hydrolysate, in combination with at least one type of compound selected from a group comprising: saccharides, vitamins and organic micronutrients, mineral compounds, iron supplementation compounds, organic amines, shear protectants, anti-foaming agents or a combination thereof. The media may also contain other compounds, like fatty acids, phospholipids, additional amino acids or oligonucleotides, for example. Media according to the invention with an optimized ratio of amino acids and other nutrients may facilitate efficient production of biomass and a low production of waste metabolites, such as ammonia or lactate, by the cells.

[0454]

[0336] An optimized ratio of essential amino acids is such that essential amino acids may be introduced into the cultivation process in any ratio where the percentage of essential amino acids that can be converted into cellular protein is in the range of 5 % to 100 %, or in the range of 20 % to 90 %, or in the range of 30 % to 80 %. The term “highest possible conversion efficiency” determines what percent of the essential amino acids provided to the 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.

[0455]

[0337] The highest possible conversion efficiency is determined by the essential amino acid that is the most limiting to the cells. It is calculated such as that for all individual essential amino acids added to the medium in any form at any time point during the cultivation process, the content of that particular essential amino acid in the culture media as a fraction of total essential amino acid content added in anyform at any time point to the culture media is divided by the content of that individual amino acid in cellular protein as a fraction of total content of essential amino acids in the lowest obtained ratio, in other words the ratio for the essential amino acid which forms the lowest percentage of the amino acids added to the medium in comparison to the percentage of that particular amino acid in cellular biomass, is then multiplied by 100 to obtain the highest possible conversion efficiency of the provided essential amino acids into cellular protein. All percentages in the calculation of highest possible conversion efficiency are percentages by weight.

[0456]

[0338] The amino acids in the culture media may be present in the form of free amino acids or peptides.

[0457] Non-essential amino acids are omitted in this calculation, as they can be synthesized by the cells and thus are not limiting in terms of the highest possible conversion efficiency. An example of possible essential amino acid content in cellular protein can be seen in Table 1 below.

[0458]

[0339] The above description may be summarized by the following equation:

[0459]

[0460] where

[0461] HEAA is the highest conversion efficiency for a particular amino acid,

[0462] AEAAM is the content of that particular essential amino acid in 100 g of protein in culture medium, EAEAAM is the total content of all essential amino acids in 100 g of protein in culture medium, AEAAC is the content of that particular essential amino acid in 100 g of cellular protein and EAEAAC is the total content of all essential amino acids in 100 g of cellular protein.

[0463]

[0340] An example calculation for the essential amino acid tryptophan would proceed as follows:

[0464] assuming that the total amount of tryptophan added to the culture media over the period of cultivation was 2 g, and the total amount essential amino acids added to the media over the same time period was 100 g. Table 1 shows that in 100 g of cellular protein, out of 44.7 g of total essential amino acids, 1.6 g are tryptophan.

[0465]

[0341] The calculation:

[0466]

[0467] shows that the highest conversion efficiency for tryptophan is 55.875 %. Now, this process is repeated for each of the nine individual essential amino acids. The lowest of nine numbers obtained is the final highest conversion efficiency.

[0468]

[0342] 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 cultivation process matches the amino acid composition of cellular protein. Because cells cannot synthesize essential amino acids, the essential amino acid with the lowest relative total input into the cultivation process in comparison to its content in cellular protein will limit maximal cell yield and therefore the maximal percentage of essential aminoacids converted to cellular protein (this can be understood as an application of Liebig's law of the minimum).

[0469]

[0343] The conversion efficiency for total essential amino acids may be in the range of 5 % to 100 %, 20 % to 100 %, 30 % to 100 %, or 50 % to 100 %, calculated by the above mentioned equation.

[0470]

[0344] If the essential amino acid composition of cellular protein according to the example mentioned in Table 1 is used, the resulting total inputs of each essential amino acid given as grams per 100 g of the total input of all essential amino acids may be in the ranges summarized in the Table 2.

[0471]

[0345] The ranges of concentrations of amino acids in grams per 100 g of total essential amino acids introduced into the cultivation process may be according to Table 2, regardless of whether the essential amino acid composition of cellular protein is according to Table 1 or not.

[0472]

[0346] It should be noted that for the purpose of this equation, it is necessary to consistently consider amino acid content either as free amino acids, or as amino acids that are part of a peptide chain (in which case the molecular weight of each amino acid must be considered lower by the weight of one water molecule, to account for the fact that water is a byproduct of a peptide bond formation). In the equation above and Tables 1-3, everything is counted as amino acids that form a peptide chain. Elsewhere in the present document, when amino acid input or concentration is discussed, these are calculated with the molecular weights of free amino acids, and when protein input or concentration is discussed, it is assumed that the amino acids are part of a peptide chain for any calculations.

[0473]

[0347] Table 1 - Example of possible essential amino acid content in the cellular protein.

[0474]

[0475]

[0476]

[0348] Table 2 - Ranges of concentrations of amino acids in grams per 100 grams of total essential amino acids introduced into the cultivation process.

[0477]

[0478]

[0349] However, the composition of cell biomass is somewhat variable, and therefore the values for each essential amino acid in terms of weight percentage of total essential amino acids used in the media may also be in the ranges summarized in the Table 3.

[0350] Table 3 - Ranges of weight percentage concentration of total essential amino acids introduced into the cultivation process.

[0479]

[0480]

[0351] Amino acids may be introduced into the cultivation process in the form of free amino acids, salts of amino acids, esters of amino acids, or any other suitable derivatives, as well as oligopeptides, for example dipeptides, tripeptides or tetrapeptides, or polypeptides.

[0481]

[0352] The total input of hydrolysate (expressed as protein dry weight) introduced into the culture medium in the cultivation process may be in the range of 1 g / L to 200 g / L, or 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.

[0482]

[0353] The total input of amino acids from hydrolysate, including amino acids in the form of short peptides or suitable bioavailable derivatives, for example phosphoesters, such phosphoserine, or other derivatives, such as methylglycine, is at least 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, or 95 % by weight of the total input of all amino acids into the culture medium.

[0354] 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 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.

[0483]

[0355] 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 with respect to the total amount of hydrolysate protein in the culture medium.

[0484]

[0356] 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 % with respect to the total amount of hydrolysate protein in the culture medium.

[0485]

[0357] 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 % with respect to the total amount of hydrolysate protein in the culture medium.

[0486]

[0358] 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 % with respect to the total amount of hydrolysate protein in the culture medium.

[0487]

[0359] 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 % with respect to the total amount of hydrolysate protein in the culture medium.

[0488]

[0360] 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 % with respect to the total amount of hydrolysate protein in the culture medium.

[0489]

[0361] 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 hydrolysate protein into the culture medium.

[0490]

[0362] 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 0 g / L to 30 g / L, or in the range 0.5 g / L to 20 g / L, 1 g / L to 10 g / L. The total input of ammonia sourced from hydrolysate may be in the range of 10 mg / L to 1,000 mg / L, or in the range of 20 mg / L to 500 mg / L, or in the range of 40 mg / L to 300 mg / L.

[0491]

[0363] At least one or any combination of saccharides selected from the group of glucose, fructose, galactose, sucrose, lactose, maltose, or a combination thereof, or any other appropriate saccharide may be introduced. The total input of saccharides may be in an amount 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.

[0492]

[0354] The media may contain at least one of 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, PO43, Zn2+, SeO32. The media may also contain trace amounts of other mineral compounds and elements, such as cobalt, iodine or manganese.

[0493]

[0364] As the media is prepared by dissolving different constituent compounds in water, any appropriate chemical compound may be used as long as it dissociates to the desired ions in aqueous solution. For example, NaCl and KC1 both produce a Cl ion when dissolved. As another example, CuSO4and MgCF or MgSO4and CuCF may be used to produce Cu2+, Mg2+, SO42and 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 may 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.

[0494]

[0365] The total input of Na+may be in the range of 20 mmol / L to 120 mmol / L, or in the range of 30 mmol / L to 100 mmol / L, or in the range of 40 mmol / L to 80 mmol / L.

[0366] The total input of Ca2+may be in the range of 0.01 mmol / L to 2 mmol / L, or in the range of 0.05 mmol / L to 1 mmol / L, or in the range of 0.1 mmol / L to 0.6 mmol / L.

[0495]

[0367] The total input of Cl may be in the range of 25 mmol / L to 130 mmol / L, or in the range of 35 mmol / L to 110 mmol / L, or in the range of 45 mmol / L to 90 mmol / L.

[0496]

[0368] The total input of Mg2+may be in the range of 0.3 mmol / L to 10 mmol / L, or in the range of 0.5 mmol / L to 8 mmol / L, or in the range of 1 mmol / L to 5 mmol / L.

[0497]

[0369] The total input of PO43may be in the range of 0.5 mmol / L to 12 mmol / L, or in the range of 0.7 mmol / L to 10 mmol / L, or in the range of 1 mmol / L to 6 mmol / L.

[0498]

[0370] The total input of SO42may be in the range of 0.1 mmol / L to 5 mmol / L, or in the range 0.3 mmol / L to 3 mmol / L, or in the range 0.6 mmol / L to 2 mmol / L.

[0499]

[0371] The total input of K+may be in the range of 2 mmol / L to 18 mmol / L, or in the range of 4 mmol / L to 15 mmol / L, or in the range of 6 mmol / L to 12 mmol / L.

[0500]

[0372] The culture media may contain at least one vitamin from the group of 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 appropriate 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 to bioactive vitamin B12 by the cells. As another example, thiamine hydrochloride (chloride salt form of thiamine) may be used instead of thiamine. The total input of vitamins introduced into the cultivation process, omitting the vitamins present in lysates or extracts, may be in the range of 0.1 mg / L to 1,000 mg / L, or in the range of 5 mg / L to 500 mg / L, or in the range of 20 mg / L to 300 mg / L.

[0501]

[0373] The total input of choline may be in the range of 10 mg / L to 1,000 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.

[0502]

[0374] The total input of niacinamide (or another vitamer of vitamin B3) may be in the range 3 mg / L to 150 mg / L, or in the range 6 mg / L to 100 mg / L, or in the range of 10 mg / L to 80 mg / L.

[0503]

[0375] At least one organic amine may be introduced to the cultivation process, and may be selected from: putrescine, ethanolamine, or a combination thereof, or any other appropriate amine. The total input of organic amines into the cultivation process may be in an amount in the range of 0.01 mg / L to 1,000 mg / L, or in the range of 0.1 mg / L to 100 mg / L, or in the range of 0.5 mg / L to 20 mg / L.

[0504]

[0376] 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 appropriate lysate or extract. Extract or lysate for supplementation of micronutrients may be added to the culture media 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.

[0505]

[0377] Iron may be supplemented to the culture medium in compounds with oxidation state iron (III) or iron (II). Iron may be present as free ions, or it may be chelated with a suitable chelating agent to improve its solubility and bioavailability. Chelating agents may include citrate, gluconate, ammonium citrate, EDTA, their combinations, or any other suitable chelating agent. Iron may be introduced into the culture medium bound to the chelating agent (for example, in the form ferric citrate), or iron and a chelating agent may be added separately (for example, in the form of ferric chloride and sodium citrate). The relative amount (w / w) of the total input of the chelating agent to the total input of iron may 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. The total input of iron may be in the amount 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.

[0506]

[0378] The culture medium may comprise a shear protectant to prevent cell damage from mechanical forces caused by mixing and / or sparging in the cultivation device. At least one shear protectant may be selected from: polyethylene glycol (PEG), methyl cellulose (MC), (hydroxypropyl)methyl cellulose (HPMC), hydroxypropyl cellulose (HPC), carboxymethyl cellulose (CMC), dextran sulfate, or any otherappropriate shear protectant, or their combination. Shear protectants may be present in the culture medium in 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.

[0507]

[0379] As described herein, the physico-chemical parameters and composition of the culture medium may be optimized to facilitate fast biomass production, efficient use of nutrients and low production of waste metabolites.

[0508]

[0380] The osmolality of the medium may be in the range of 200 mOsm / kg to 400 mOsm / kg, or range of 250 mOsm / kg to 350 mOsm / kg, or range of 280 mOsm / kg to 330 mOsm / kg. Osmolality may be adjusted before or after the culture medium is introduced into the cultivation device, or a combination of both, and it may be adjusted at a single time point or multiple timepoints. To increase osmolality, NaCl, KC1, glucose, any other appropriate osmolyte or their combination may be used. To decrease osmolality, water or any other appropriate dilute aqueous solution may be used.

[0509]

[0381] The pH of the culture medium in the cultivation 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. Adjustment of pH may be performed before or after the culture medium is introduced into the cultivation device, or a combination of both, and it may be adjusted at a single time point or multiple timepoints. NaOH, HC1, NaHCOs, or any other appropriate acid or base may be used to adjust the pH; alternatively, pH may be adjusted by changing the partial pressure of CO2 in the cultivation device (higher CO2 partial pressure will result in more CO2 being dissolved into the culture medium, leading to lower pH). The partial pressure of CO2 in the cultivation device may be adjusted by changing the percentage of CO2 in the sparging gas, changing the total pressure in the cultivation device, or changing the mixing and sparging rate in the cultivation device (reducing or increasing CO2 mass transfer coefficient), or any other appropriate method. The partial pressure of CO2 in the cultivation device may be in the range of 0.05 kPa to 100 kPa, or in the range 2 kPa to 60 kPa, or in the range 5 kPa to 30 kPa.

[0510]

[0382] The momentary concentration of saccharides in the medium may be in the range 0.005 g / L to 40 g / L, or in the range 0.1 g / L to 20 g / L, or in the range 0.5 g / L to 5 g / L.

[0511]

[0383] The momentary concentration of all amino acids (taking into account both amino acids sourced 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 0.1 g / L to 15 g / L, or in the range 0.5 g / L to 10 g / L.

[0512]

[0384] The composition of culture media as described herein may be suitable for cell lines that have been extensively adapted to conditions in vitro. However, some cell types may require additional components in the culture medium, for example protein growth factors, to survive and proliferate. In another aspect of the invention, the culture medium composition suitable for these growth factor dependent cell lines may be described as follows.

[0513]

[0385] The hydrolysates of protein isolates may be used as amino acid sources in culture media according to the invention. Recombinant protein production may be used in culture medium components preparation.

[0514]

[0386] The culture medium according to the invention may comprise macronutrients, micronutrients, signaling compounds and / or other components. The components may be dissolved, for example, in purified water, or in water with inorganic salts, for example phosphate buffer saline (PBS) or water or PBS with Bovine serum albumin (BSA), for example 1 % BSA in total.

[0515]

[0387] The signaling compounds may vary according to the specific cell type used in the cultivation in the cultivation device. Examples of those cells may be fibroblasts, myoblasts, adipocytes and their precursors or a combination thereof.

[0516]

[0388] The signaling compounds may or may not induce specific change in cell fate. Examples of these changes may be stimulation of proliferation and / or stimulation of differentiation. The signaling compounds may be used in a certain order during a certain time period. Examples of those may be theusage of a signaling compound for stimulation of proliferation which is then substituted in the media with a signaling compound for inducing differentiation. The precise order of dosing of signaling compounds may or may not be correlated or cross-linked with other tools which affect the cell fate during cultivation.

[0517]

[0389] Signaling compounds for various cell types aimed for stimulation of proliferation may comprise, for example, at least one of the following signaling proteins: FGF family ligands, insulin, insulin like growth factor (IGF) family ligands, TGF family ligands, or transferrin, or any other appropriate signaling compound.

[0518]

[0390] Signaling compounds for various cell types aimed for myogenic differentiation may comprise at least one of FGF, insulin, TGF, transferrin, IGF, epidermal growth factor (EGF), Bone morphogenic protein (BMP), interleukin 6 (IL-6), or IL-13, or any other appropriate signaling compound.

[0519]

[0391] The amino acids and their derivatives that may be supplied to the media are for example:

[0520] glycine, L-alanine, L-arginine, L-asparagine L-aspartic acid, L-cystine L-glutamic acid, L-glutamine, L- histidine, L-hydroxyproline, L-ornithine, L-citrulline, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-pyroglutamic acid, L-phosphoserine, L-tryptophan, L-tyrosine or L-valine. For the preparation of the culture medium, the given amino acid may be added in the pure form, or as part of a complex mixture of compounds (for example a hydrolysate), or the hydrates or salts (for example hydrochlorides or sodium salts) of amino acids may be used.

[0521]

[0392] In one aspect of the invention, the culture media may comprise protein hydrolysate as a 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 the purpose of cell cultivation, or some amino acids may be supplied to the media separately, for example L-methionine, which is found in very low concentrations in most scalable sources of protein. Other different individual amino acids may be supplied separately from a different source than a protein hydrolysate.

[0522]

[0393] The processes of cultivation of cells according to the invention may be performed in cultivation systems, equipment or culture vessels well-known and / or commonly used by any person skilled in the art.

[0523]

[0394] The processes of cultivation of cells according to the invention may be performed in a cultivation system. In one aspect of the invention, the cultivation system (1) is as depicted in Fig. 14.

[0524] The cultivation system (1) may comprise a seeding tank (2), a cultivation device (101), a harvesting device (102), a control unit (113), and sensors and analytical instruments (6) as depicted in Fig. 14.

[0525] Optionally, the cultivation system (1) may further comprise a device for preparing food products (not depicted in Fig. 14).

[0526]

[0395] The control unit may control and / or regulate every process taking place within the cultivation system. The control unit may be operated using at least one printed circuit board (PCB) and / or microprocessor with software capable of controlling the cultivation device, regardless of the extensions and scale of the system. The control unit may be connected to at least one central data storage. The cultivation system may comprise one or more subcontrol units.

[0527]

[0396] The culture medium may be prepared in a culture medium tank. The culture medium tank may comprise at least one of: mixing tank, hydrolysis tank, storage tank, loading tank or waste medium tank, or any other appropriate device. The media components may be mixed in a mixing tank, which may be made from stainless steel, glass, or any other suitable material. The mixing tank may be equipped with a stirring unit comprising, for example, a shaft with one or more impellers. The mixing tank may be equipped with a heating system. The temperature of the mixing tank may 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 may be connected to one or more storage tanks. The mixing tank may be connected to one or more cultivation devices, formed for example by a bioreactor. The culture medium components may be mixed directly in the cultivation device. The volume of the mixing tank may be in the range of 500 mL to 100 m3, or in therange 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. The storage tanks may be made from stainless steel, glass or any other suitable material. The volume of the storage tank may 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. The media components may be dosed into the mixing tank through a sterilization filter, or may be sterilized prior to the placement into the mixing tank or may be sterilized in the mixing tank. The mixing tank may be equipped with different types of sensors, such as, for example, thermal sensor, pH probe, conductometer, or any other type of appropriate sensor according to the needs of the process.

[0528]

[0397] In one aspect of the invention, the first protein source, the second protein source, or exogenously supplied enzymes, or a combination thereof, may be introduced into the hydrolysis tank by a loading tank. Alternatively, the input of the hydrolysis tank may be configured as a shaft or funnel.

[0529]

[0398] The hydrolysis of the first protein source using a second protein source and exogenously supplied enzymes may be performed within the hydrolysis tank.

[0530]

[0399] The culture media according to present invention may comprise protein hydrolysate as source of amino acids. The process of medium preparation may have the characteristics of a batch process, a continuous process, or a combination thereof.

[0531]

[0400] A more detailed description of the cultivation system, the culture medium, the protein hydrolysate, the cell cultivation methods, the products from the non-human metazoan cells, including specific methodologies, formulations, and system parameters, is available in International Patent Application No. PCT / IB2024 / 059990, which is hereby incorporated by reference in its entirety. The present disclosure provides a summary of key aspects relevant to the current invention, while additional details regarding the inventions can be found in the referenced document.

[0532]

[0401] During the production of the protein hydrolysate from sources of protein, various by-products may be generated. These by-products, referred to as solid residues, may include any portion of the source of protein not incorporated into the final culture medium formulation, such as leaves, peels, stems, roots or any other plant parts. Additionally, after the hydrolysis of source of protein, the protein hydrolysate may comprise sediments separated by filtration unit, wherein filtration unit may comprise suitable separation device, such as a centrifuge and / or a flotation device and / or at least one filter selected from the group of membrane filters, depth filters, mesh filters, activated carbon filters, ceramic filters, ultrafiltration filters, nanofiltration filters, ion exchange filters, crossflow (tangential flow) filters, adsorption filters and / or fiber filters.

[0533]

[0402] In one aspect of the invention, the filtration unit may be configured to utilize centrifugal force to separate solid-phase particles from liquid phase. This separation process is facilitated by the implementation of centrifugal filters, which may be strategically designed and positioned within the filtration unit.

[0534]

[0403] The filtration unit may further comprise auxiliary components selected from the group of pumps, pressure sensors, flow meters, valves and means for monitoring the filtration process.

[0535]

[0404] In one aspect of the invention, the protein hydrolysate may be subjected to enzymatic, chemical, or mechanical treatment to produce a modified protein hydrolysate.

[0536]

[0405] The protein hydrolysate may be modified by at least one enzyme, wherein the enzymes may comprise proteolytic enzymes and / or enzymes having phytase activity.

[0537]

[0406] In one aspect of the invention, the sequence of enzyme application may be interchangeable, wherein proteolytic enzymes may be applied prior to enzymes having phytase activity, or alternatively, the enzymes having phytase activity may be applied prior to the proteolytic enzymes.

[0538]

[0407] The term “proteolytic enzymes” refers to enzymes from the group of proteases, peptidases and / or any other enzyme that is capable of cleaving of peptide bonds between amino acids and / or is capable of addition of water molecule to an ester to produce alcohol or an acid.

[0408] The term “enzyme having phytase activity” refers to the enzymes from the group of phytases, phosphatases and / or any other enzymes capable of cleaving phosphate ester bonds.

[0539]

[0409] The sediment may comprise DNA, RNA, proteins, organic acids, phenolic compounds, carbohydrates, fatty acids, mineral compounds, and / or any other waste molecules depending on the used source of protein.

[0540]

[0410] The sediment generated during the processing of the protein hydrolysate, and the solid residues from the source of protein, may be separated from the produced modified protein hydrolysate using a filtration unit, thereby producing a purified protein hydrolysate.

[0541]

[0411] The purified protein hydrolysate may be combined with additional nutritional additives suitable for the cultivation of non-human metazoan cells.

[0542]

[0412] The nutritional additives may be selected from at least one of saccharides, mineral compounds, vitamins, amino acids, peptides, organic amines, signaling compounds, oligonucleotides, fatty acids, phospholipids, organic micronutrients, or any other appropriate nutritional additives.

[0543]

[0413] Amino acids may be supplied either by the hydrolysis of a source of protein and / or through direct addition to the protein hydrolysate. For example, some sources of protein may comprise lower content of particular amino acid in comparison to other sources of protein, therefore, there is a need to add such amino acid to provide sufficient nutrition to the non-human metazoan cells. For example, soy protein comprises a low content of the essential amino acid L-methionine, and therefore the supplementation of L-methionine may be necessary to maximize non-human metazoan cell culture growth.

[0544]

[0414] The nutritional additives may comprise at least one saccharide selected from the group of glucose, fructose, galactose, sucrose, lactose, maltose, and / or any other appropriate saccharides.

[0545]

[0415] The nutritional additives may comprise at least one mineral compound selected from the group of Ca2+, Cl , Cu2+, SO42, Fe3+, NO3, Fe2+, Mg2+, K+, Na+, CO32, HCO3, H2PO4, HPO42, PO43, Zn2+, SeO32, and / or any other appropriate mineral compound.

[0546]

[0416] The nutritional additives may comprise at least one vitamin selected from the group of alphatocopherol, ascorbic acid, cobalamin, biotin, choline, pantothenic acid, folic acid, niacinamide, pyridoxine, riboflavin, thiamine, i-inositol, and / or any other appropriate vitamin.

[0547]

[0417] The nutritional additives may comprise at least one amino acid and / or an amino acid derivative selected from the group consisting of glycine, L-alanine, L-arginine, L-asparagine L-aspartic acid, L- cystine, L-cysteine, 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, L-valine, and / or any other appropriate amino acid or their derivative.

[0548]

[0418] The nutritional additives may comprise at least one organic amine selected from the group of putrescine, ornithine, choline, ethanolamine and / or any other appropriate organic amine.

[0549]

[0419] The nutritional additives may comprise at least one signaling compound selected from the group of FGF family ligands, insulin and IGF family ligands, TGF family ligands, transferrin, and / or any other appropriate signaling compound.

[0550]

[0420] The nutritional additives may comprise at least one oligonucleotide selected from the group of single or double stranded chains of nucleic acids containing 10 nucleotides to 70 nucleotides, 10 nucleotides to 120 nucleotides, or 1 nucleotide to 1,000 nucleotides, and / or any other appropriate oligonucleotide.

[0551]

[0421] The nutritional additives may comprise at least one fatty acid selected from the group of linoleic acid, lipoic acid, and / or any other appropriate fatty acid.

[0552]

[0422] The prepared culture medium may be used for the cultivation of non-human metazoan cells.

[0553]

[0423] The non-human metazoan cells may have characteristics and / or properties of: hepatocytes, myocytes, myoblasts, osteoblasts, fibroblasts, lipoblasts, odontoblasts, keratinocytes, mesenchymal stemcells, multipotent progenitor cells, embryonic stem cells, myofibroblasts, myosatellite cells and / or any combinations thereof. The present invention is not limited by the present examples of cell types.

[0554]

[0424] The non-human metazoan cells may comprise bovine, avian, porcine, equine, piscine, cervine or cricetine cell lines. In another aspect of the invention, the non-human metazoan cells may comprise any other non-human metazoan cell line.

[0555]

[0425] The non-human metazoan cells may be genetically modified, may be subjected to non-genetic modification, and / or may be adapted to different conditions and environments.

[0556]

[0426] The non-human metazoan cells may be genetically modified. Genetic modifications may comprise permanent and / or transient genetic modifications, wherein such genetic modifications may be gain-of-function or loss-of-function modifications. The modifications may include point substitutions, point deletions, point insertions, larger deletions or larger insertions. The nucleic acid introduced into the cells may be naturally present within the species of the target cells, may be of the origin of another species, may be synthetic, or a combination thereof. Such genetic modifications may be performed using methods such as CRISPR / Cas9, ZFNs, TALENs, Cre-Lox recombination, RMCE and / or other tools. Other methods for genetic modification may comprise introduction by bacterial vectors, yeast vectors or viral vectors based on adenoviruses, adeno-associated viruses, retro / lentiviruses and / or vectors.

[0557]

[0427] The cultivation of the non-human metazoan cells may be performed using different work modes, such as batch cultivation, fed-batch cultivation, continuous cultivation, semicontinuous cultivation and / or perfusion cultivation, or any other appropriate cultivation mode, according to the selected non-human metazoan cells to be cultivated and / or preferred cell cultivation conditions.

[0558]

[0428] In the batch cultivation, all of the nutrients within the culture medium are provided at the beginning and there is no further nutrient addition or waste removal during the process. In the fed-batch cultivation, a portion of the nutrients within the culture medium and / or culture medium volume is provided at the beginning of the cultivation and then the other portion of the nutrients and / or culture medium volume is added during the cultivation in increments. In the semi -continuous cultivation, the whole culture medium and / or specific nutrients within the culture medium are periodically removed and replaced during the cultivation. In continuous cultivation, the whole culture medium and / or specific nutrients within the culture medium are continuously added and replaced. In addition to the continuous cultivation, a perfusion element may be implemented to retain at least some portion of the cultivated non-human metazoan cells that would otherwise be removed with waste medium.

[0559]

[0429] The non-human metazoan cells may be cultivated in the culture medium at a pH in the range of 3 to 12, or in the range of 5 to 10, or in the range of 6 to 9.

[0560]

[0430] The non-human metazoan cells may be cultivated in the culture medium at a temperature in the range of 20 °C to 45 °C, or in the range 25 °C to 40 °C, or in the range 35 °C to 39 °C.

[0561]

[0431] After cultivation, the cell biomass of non-human metazoan cells may be harvested using a non- human metazoan cells harvesting device.

[0562]

[0432] In one aspect of the invention, the cultivation system may comprise non-human metazoan cells harvesting device and / or converting organisms harvesting device, wherein these harvesting devices may be designed for the efficient collection and separation of cultivated biomass from the culture medium, rejuvenated culture medium or any appropriate liquid solution. In another aspect of the invention, the harvesting device may comprise at least one harvesting tank and / or at least one centrifugal separation system applying centrifugal force for the separation of cell biomass from the culture medium or any appropriate liquid solution.

[0563]

[0433] In one aspect of the invention, according to the scheme depicted in Fig. 1, the source of protein (001) is subjected to the hydrolysis process in the generation of purified protein hydrolysate (002) and solid residues and sediment (003), which are removed from the protein hydrolysate by a filtration unit. The purified protein hydrolysate (002) is mixed with additional compounds (004), such as nutritional additives, in generation of the culture medium (005), which is used for the cultivation of the non-humanmetazoan cells. Harvested cells (006) are separated from the waste medium (007) and the harvested cells (006) are used for the food or feed product production and / or the production of substances having therapeutic effect, such as pharmaceuticals, signaling compounds and / or nutritional additives.

[0564]

[0434] Furthermore, the culture medium derived from sources of protein and / or waste medium generated after the cultivation with non-human metazoan cells within the culture medium may comprise a high concentration of molecules either produced by or not utilized by the non-human metazoan cells, collectively referred to as waste molecules.

[0565]

[0435] The waste medium may contain waste molecules selected from bioactive compounds, such as glucosinolates, saponins, terpenes, phenolic compounds, phytosterols, salts (for example NaCl, KC1 and other salts), organic acids (for example lactic acid, acetic acid, citric acid and other acids), trace metals (Zn, Fe, Co, Cu, Mg, Se and other metals) and other unused nutritional additives added to the culture media (vitamins, signaling compounds, growth factors, mineral compounds, amino acids, oligonucleotides). Additionally, the waste medium may comprise other bioactive compounds such as monosaccharides (for example glucose, glucosamine and / or any other monosaccharides), polysaccharides (fibers and / or any other polysaccharides), organic acids (lactic acid, citric acid, succinic acid and / or other organic acids), nitrogen-rich molecules (ammonia, nitrates, nucleic acids, amino acids, peptides, proteins and / or any other nitrogen-rich molecules) and phosphorus-rich molecules (phosphates, phospholipids, phytates, nucleic acids and / or any other phosphorus -rich molecules) and other waste molecules depending on the source of protein. In one aspect of the invention, the waste medium may comprise cell debris derived from non-human metazoan cells, wherein the cell debris may include cellular fragments, such as lipid membrane segments and membrane-associated proteins, and / or organelles, fragments and / or remnants of organelles. The cell debris may further comprise cytoplasmic components, including cytoskeletal fragments, cytoplasmic proteins, lipids, and / or carbohydrates.

[0566]

[0436] Additionally, during the cultivation process, non-human metazoan cells produce carbon dioxide as a metabolic by-product, also referred to as a waste molecule.

[0567]

[0437] These waste molecules, solid residues or sediments hold potential as nutrient sources for the cultivation of other organisms, including specific microorganisms, algae or plants, which thrive on nitrogen and phosphorus compounds, organic acids, phenolic compounds, mineral compounds and / or any other waste molecules. By repurposing these waste molecules, solid residues, or sediments as substrates, specific microorganisms, such as bacteria, archaea or fungi, algae, microalgae and / or plants may be cultivated to produce valuable biomaterials, bioactive compounds, or serve as nutrient sources for the cultivation of non-human metazoan cells.

[0568]

[0438] In one aspect of the invention, the waste medium may have an osmolality in the range of 200 to 400 mOsm / kg, or in the range of 250 to 350 mOsm / kg, or in the range of 270 to 320 mOsm / kg. The waste medium may have a pH in the range of 5 to 10, or in the range of 6 to 9, or in the range of 6.5 to 8.5 at room temperature and atmospheric CO2 concentration. The waste medium may contain chloride ions in the range of 1,000 to 5,000 mg / L, or in the range of 1,500 mg / L to 4,000 mg / L, or in the range of 2,000 mg / L to 3,500 mg / L. The waste medium may contain total nitrogen in the range of 100 to 2,000 mg / L, or in the range of 200 to 1 ,500 mg / L, or in the range of 300 to 1 ,200 mg / L.

[0569]

[0439] The waste medium may comprise viable non-human metazoan cells, microbial contaminants and / or viral contaminants. Those viable cells and / or contaminants may be subjected to a treatment capable of killing and / or otherwise rendering incapable of further growth such cells and / or contaminants. This process may be referred to as "inactivation" of the waste medium. The inactivation of the waste medium may result in the generation of inactivated waste medium.

[0570]

[0440] In one aspect of the invention, waste medium may be processed in the same way as regular wastewater.

[0441] The waste medium may be subjected to a treatment to reduce the content of organic compounds; the technology to remove organic compounds may be selected from microbial treatment, solid phase adsorption, or any other suitable method.

[0571]

[0442] The waste medium may be subjected to a treatment to remove solid residues; the technology to remove solid residues may be selected from filtration, flotation, centrifugation or any other suitable method. If a treatment to reduce the content of organic compounds is employed, the treatment to remove solid residues should preferably come after the treatment to reduce the content of organic compounds.

[0572]

[0443] The waste medium may be subjected to a treatment to reduce water content, thereby concentrating waste molecules and producing waste medium concentrate. The treatment may comprise evaporation, such as vacuum distillation, membrane processes, such as reverse osmosis, and / or any other treatment for the reduction of water content. For example, during the evaporation process, water is vaporized and subsequently condensed back into liquid form, resulting in recovered water.

[0573]

[0444] The waste medium concentrate may have characteristics of a liquid, liquid with suspended solids, or a solid depending on the degree of water removal.

[0574]

[0445] In one aspect of the invention, the waste medium concentrate may be disposed of as dangerous waste. The methods of disposal may include incineration at a dedicated facility, or any other appropriate method for disposing of dangerous waste.

[0575]

[0446] In one aspect of the invention, water recovered in the production of waste medium concentrate may be repurposed for various applications comprising preparation of new culture media, equipment washing, heating or cooling systems, steam generation, and / or any other appropriate uses.

[0576]

[0447] The waste medium may be also subjected to crystallization, precipitation, ultrafiltration, solid phase extraction, liquid phase extraction, electrochemical treatment or any other suitable process to get rid of waste molecules from the waste medium and obtain recovered water and / or recovered waste molecules.

[0577]

[0448] In one aspect of the invention, the recovered water may be further purified by using reverse osmosis, precipitation, coagulation, filtration, ultrafiltration, distillation and / or any other appropriate purification technique to clean the recovered water.

[0578]

[0449] In one aspect of the invention, the waste medium may comprise 95 % to 99.9 % of water, or 97 % to 99.8 % of water, or 98 % to 99.5 % of water prior to being subjected to the concentration device.

[0579]

[0450] In one aspect of the invention, the waste medium may comprise 5 % to 0.1 % of waste molecules, or 3 % to 0.2 % of waste molecules, or 2 % to 0.5 % of waste molecules prior to being subjected to the concentration device.

[0580]

[0451] In one aspect of the invention, the waste medium concentrate may be characterized by the concentration factor. The concentration factor is defined as the mass of the waste medium before concentration, divided by the mass of the resulting waste medium concentrate. The concentration factor may be in the range of 2 to 100, or in the range of 5 to 50, or in the range of 8 to 20.

[0581]

[0452] In one aspect of the invention, the pH of the waste medium and / or waste medium concentrate may be changed by addition of pH regulating agents comprising HC1, NaOH, KOH or any other pH regulating agent. Advantageously, the pH may be adjusted to >8 to mitigate the corrosion of hardware that comes into contact with the waste medium and / or waste medium concentrate.

[0582]

[0453] In one aspect of the invention, the pH of the waste medium and / or waste medium concentrate may be regulated during the cultivation of converting organisms. The pH of the waste medium or waste medium concentrate during the cultivation of converting organisms may be in the range of 3 to 12, or in the range of 5 to 10, or in the range of 6 to 8. For example, the optimal pH for growth of Lactobacillus species may be in the range from 5.5 to 6.5.

[0583]

[0454] In one aspect of the invention, waste medium and / or waste medium concentrate may be supplemented with hydrolysate and / or nutritional additives and / or food industry by-products, in a process of direct recycling, to make a rejuvenated culture medium.

[0455] In one aspect of the invention, hydrolysate and / or nutritional additives may be supplemented into waste medium in the form of complete fresh culture medium to make a rejuvenated culture medium.

[0584]

[0456] The rejuvenated culture medium prepared by direct recycling may comprise 10 to 90 %, or 20 % to 80 %, or 30 % to 70 % waste medium by volume.

[0585]

[0457] In one aspect of the invention, the rejuvenated culture medium may comprise waste medium concentrate, wherein the volume of the waste medium concentrate used multiplied by the concentration factor of the waste medium concentrate may be in the range of 10 % to 90 %, or 20 % to 80 %, or 30 to 70 %.

[0586]

[0458] In one aspect of the invention, the rejuvenated culture medium may be supplemented with hydrolysate in the range of 0.1 g / Lto 100 g / L, or 0.5 g / L to 50 g / L, or 1 g / L to 10 g / L (hydrolysate concentrations given in grams of protein in source of protein per liter of culture medium), and / or choline in the range of 0.1 mg / L to 500 mg / L, or 0.5 mg / L to 250 mg / L, or 1 mg / L to 125 mg / L, and / or cysteine in the range of 5 mg / L to 1 ,000 mg / L, or in the range of 10 mg / L to 500 mg / L, or in the range of 20 mg / L to 250 mg / L, and / or methionine in the range of 1 mg / L to 500 mg / L, or 2 mg / L to 250 mg / L, or 5 mg / L to 125 mg / L, and / or glucose in the range of 0.5 g / L to 100 g / L, or 1 g / L to 50 g / L, or, 2 g / L to 20 g / L, and / or phosphate in the range of 5 mg / L to 1,500 mg / L, or 10 mg / L to 1,000 mg / L, or 20 mg / L to 500 mg / L, and / or any other appropriate additives. All additive amounts are listed as the total input, where the total input is defined as the amount added to the medium before or during cell cultivation divided by the volume of waste medium at the end of the process.

[0587]

[0459] In another aspect of the invention, the waste medium resulting from cultivated non-human metazoan cells in rejuvenated culture medium may be processed and used in all the ways of processing and using waste medium described elsewhere in the present invention, including repeated recycling.

[0588]

[0460] In another aspect of the invention, the waste medium and / or waste medium concentrate may be utilized for the cultivation of converting organisms in a process of microorganism-assisted reuse / recycling, wherein a portion of the waste medium and / or waste medium concentrate may be used for the cultivation of specific converting organisms, while another portion may be used for the cultivation of different converting organisms. For example, one portion of the waste medium and / or concentrate may be used for cultivating microbial organisms, while another portion may be utilized for the cultivation of plant organisms.

[0589]

[0461] The waste medium and / or concentrate may be supplemented with additional compounds to facilitate converting organism cultivation. The waste medium and / or waste medium concentrate may be supplemented with nutritional additives, technological and / or processing additives, solid residues, sediments, and / or food industry by-products. The technological and / or processing additives may comprise anti-foaming agents, shear protectants, anti-oxidants, or any other appropriate compounds.

[0590]

[0462] In one aspect of the invention, the anti-foaming agents may comprise silicone -based antifoaming agents, polyethylene glycol (PEG), poly vinyl alcohol (PVA), polydimethylsiloxane, polysorbate 80, vegetable oils, or any other appropriate anti-foaming agent, or a combination thereof. The concentration of the anti-foaming agent in the culture medium may be in the range of 0.001 % to 5 %, or in the range of 0.01 % to 1 %, or in the range of 0.1 % to 0.5 % by weight.

[0591]

[0463] In another aspect of the invention, the shear protectants may comprise polyethylene glycol (PEG), methyl cellulose (MC), hydroxypropyl cellulose (HPC), carboxymethyl cellulose (CMC), dextran sulfate, or any other appropriate shear protectant or a combination thereof. The shear protectant may be added to the culture media in an amount in the range of 0 % to 5 %, 0.01 % to 2 %, or 0.02 % to 1 % by weight.

[0592]

[0464] In one aspect of the invention, the nutritional additives supplemented in the waste medium and / or waste medium concentrate may be identical to those used in the production of the culture medium. The food industry by-products may comprise corn steep liquor, molasses, spent grain, bagasse, sedimentobtained from protein hydrolysis or any other appropriate by-product from food and / or agricultural industry.

[0593]

[0465] In another aspect of the invention, the food industry by-products may comprise agricultural byproducts.

[0594]

[0466] The converting organisms may comprise bacteria, archaea or fungi, algae (microalgae, macroalgae) and / or plants and / or their cells or any other organism suitable for the recycling of the waste molecules.

[0595]

[0467] The biomass of the converting organisms may be synonymous to microbial biomass.

[0596]

[0468] The microbial biomass may be used as a source of protein in the process of preparing a hydrolysate as described previously. A hydrolysate using microbial biomass as a source of protein may be synonymous to microbial biomass lysate. Microbial biomass lysate may be used for the preparation of culture media for non-human cells as described previously, or for any other appropriate purpose.

[0597]

[0469] The microbial biomass may be separated from the waste medium and / or waste medium concentrate before hydrolysis, or hydrolysis may take place directly in the waste medium and / or concentrate. To separate the microbial biomass from the waste medium and / or concentrate, centrifugation, filtration, flotation, or any other appropriate method or combination may be used. In the case that hydrolysis takes place directly in the waste medium and / or waste medium concentrate, the final product is still considered as a microbial biomass lysate.

[0598]

[0470] The waste medium and / or waste medium concentrate after the separation of microbial biomass may be referred to as microorganism-digested medium and / or microorganism-digested concentrate.

[0599]

[0471] Microorganism-digested medium and / or microorganism-digested concentrate may be supplemented with nutritional additives, technological and / or processing additives, solid residues, sediments, and / or food industry by-products to create a rejuvenated culture medium.

[0600]

[0472] In another aspect of the invention, the waste medium and / or waste medium concentrate may be used for extraction of specific waste molecules contained therein by implementing at least one precipitation, ultrafiltration, electrical field separation, and / or any other separation technique and / or their combination.

[0601]

[0473] In one aspect of the invention, selected converting organisms may be used for utilization of waste molecules within the waste medium and / or in waste medium concentrate. Alternatively, they may utilize carbon dioxide that was produced by non-human metazoan cells during cultivation in the culture medium.

[0602]

[0474] The converting organisms may proliferate by utilizing waste molecules and may provide alternative ways for their use during the cultivation process or after their harvesting by converting organisms harvesting devices.

[0603]

[0475] The converting organisms may proliferate by utilizing waste molecules and may provide alternative pathways for their utilization during the cultivation process or after harvesting by devices designed for harvesting converting organisms. By "alternative pathways," it is meant that the harvested converting organisms can be used for the production of a food product and / ;or may be utilized in the production of a protein hydrolysate.

[0604]

[0476] In another aspect of the invention, bacteria, archaea, fungi and algae may be referred to as microorganisms .

[0605]

[0477] In another aspect of the invention, bacteria, archaea, fungi and algae may be referred to as converting organisms, wherein terms “microorganisms”, “converting organisms" and “converting microorganisms” may be interchangeable.

[0606]

[0478] Archaea utilized for the cultivation in the waste medium and / or waste medium concentrate may be selected from the archaeal families including, but not limited to Methanobacteriaceae, Methanococcaceae, Methanomicrobiaceae, Methanosarcinaceae, and Methanospirillaceae.

[0479] Bacterial species utilized for cultivation in the waste medium and / or waste medium concentrate may be selected from bacterial families including, but not limited to Methylomonadaceae, Methylococcaceae, Methylothermacease, Crenotrichaceae, Beijerinckiaceae, Methylocystaceae, Methylacidiphilaceae, Enterobacteriaceae, Bacillaceae, Pseudomonadaceae, Burkholderiaceae, Desulfobacteraceae, Geobacteraceae, Alcaligenaceae, Corynebacteriaceae, , Bacteroidaceae, Bifidobacteriaceae, Lactobacillaceae, Streptococcaceae, Lactobacillaceae, Rhodobacteraceae, Lactobacillaceae, Propionibacteriaceae, Nocardiaceae, Piscirickettsiaceae, B . radyrhizobiaceae, Aquificaceae, Helicobacteraceae, Piscirickettsiaceae, Gordoniaceae, Micrococcaceae, Streptomycetaceae, Xanthobacteraceae, Nitrosomonadaceae, Nitrosococcaceae, Bradyrhizobiaceae, Nitrospinaceae, Nitrospiraceae, Enterococcaceae , and Rhizobiaceae.

[0607]

[0480] In one aspect of the invention, the bacterial species cultured in the waste medium and / or waste medium concentrate may be utilized as the protein source for hydrolysis, wherein the protein hydrolysate within the culture medium may be used for the cultivation of non-human metazoan cells. In another aspect of the invention, the bacterial species may be used for synthesis of amino acids, vitamins or any other suitable nutritional additives. Bacterial species may be also used for synthesis of biogas and / or as fertilizer components.

[0608]

[0481] The biogas may comprise methane, carbon dioxide, hydrogen sulfide, nitrogen, oxygen, ammonia, hydrogen, water vapor, trace amounts of other simple organic compounds and / or simple inorganic compounds. The biogas may be further used, for example, as a fuel, for heating, for electricity generation and / or may be used for any other appropriate application.

[0609]

[0482] Species of fungi utilized for cultivation in the waste medium and / or waste medium concentrate may be selected from fungal families including, but not limited to Phaffomycetaceae, Saccharomycetaceae, Debaryomycetaceae, Dipodascaceae, Metschnikowiaceae, Trichomonascaceae, Sporidiobolaceae, Candidaceae, Cryptococcaceae, Filobasidiaceae, Lipomycetaceae, Dipodascaceae, Pichiaceae, Hericiaceae, Tricholomataceae, and Pleurotaceae.

[0610]

[0483] In one aspect of the invention, the species of fungi cultured in the waste medium and / or waste medium concentrate may be utilized as the substrate for the cultivation of non-human metazoan cells, fertilizers and / or as components for the production of food products and / or for the production of food products in the combination with non-human metazoan cells.

[0611]

[0484] Macroalgae and / or microalgae utilized for cultivation in the waste medium and / or waste medium concentrate may be selected from algae families including, but not limited to Ulvaceae, Laminariaceae, Fucaceae, Gracilariaceae, Gigartinaceae, Sargassaceae, Caulerpaceae, Chlorellaceae, Diatomaceae, Scenedesmaceae, Nannochloropsaceae, Haematococcaceae, Eustigmataceae, Dunaliellaceae, Botryococcaceae, Chlamydomonadaceae , and Volvocaceae.

[0612]

[0485] Plants utilized for cultivation in the waste medium and / or waste medium concentrate may be selected from plant families including, but not limited to Acanthaceae, Amaranthaceae, Poaceae, Tamaricaceae, and Plumbaginaceae.

[0613]

[0486] In one aspect of the invention, the macroalgae, microalgae and / or plants may be utilized as the substrate for the cultivation of non-human metazoan cells, fertilizers and / or as components for the production of food products and / or for the production of food products in the combination with non- human metazoan cells.

[0614]

[0487] Cultivation conditions of converting organisms to utilize waste molecules may vary according to each selected species.

[0615]

[0488] Microorganisms, specifically bacteria, archaea and / or fungi, may be cultivated in aerobic or anaerobic conditions depending on the selected species.

[0616]

[0489] Microorganisms, specifically bacteria, archaea and / or fungi, may be cultivated at a temperature in the range of 20 °C to 60 °C, or in the range of 25 °C to 55 °C, or in the range of 30 °C to 45 °C, or in the range of 35 °C to 40 °C.

[0490] Microorganisms, specifically bacteria, archaea or fungi, may be cultivated for a time in the range of 1 to 20 days, or in the range of 2 to 18 days, or in the range of 5 to 15 days, or in the range of 9 to 12 days.

[0617]

[0491] Microorganisms, specifically bacteria, archaea or fungi, may be cultivated at a pH in the range of 3 to 11 , or in the range of 4 to 9, or in the range of 6 to 7.

[0618]

[0492] Algae, including microalgae and / or macroalgae, may be cultivated at a temperature in the range of 10 °C to 35 °C, or in the range of 15 °C to 30 °C, or in the range of 20 °C to 25 °C.

[0619]

[0493] Algae may be cultivated for a time in the range of 1 to 70 days, or in the range of 5 to 60 days, or in the range of 10 to 50 days, or in the range of 15 to 40 days, or in the range of 20 to 30 days.

[0620]

[0494] Algae may be cultivated at a pH in the range of 3 to 11 , or in the range of 4 to 9, or in the range of 6 to 7.

[0621]

[0495] Plants, their seeds and / or plant cells may be cultivated at temperature in the range of 10 °C to 45 °C, or in the range of 15 °C to 40 °C, or in the range of 20 °C to 35 °C, or in the range of 25 °C to 30°C.

[0622]

[0496] Seed germination of plants used for utilization of waste or residual molecules may take time in the range of 5 to 25 days, or in the range of 10 to 20 days, or in the range of 13 to 15 days.

[0623]

[0497] The time required to obtain a fully grown plant may vary depending on the plant species, but may be in the range of 1 to 700 days, or in the range of 50 to 600 days, or in the range of 150 to 450 days, or in the range of 250 to 350 days.

[0624]

[0498] Plants, their seeds and / or plant cells may be cultivated at a pH in the range of 3 to 11, or in the range of 4 to 9, or in the range of 6 to 7.

[0625]

[0499] Plants may be cultivated with the use of illumination by implementing grow lights radiating ultraviolet, blue, red or far-red light and by implementing a photoperiod duration in the range of 0 to 18 hours, or in the range of 2 to 16 hours, or in the range of 6 to 10 hours.

[0626]

[0500] Converting organisms may be cultivated together, create consortiums involving symbiotic or mutualistic relationships among these species and may be able to utilize the same or different waste molecules.

[0627]

[0501] At least two converting organisms may be cultivated together, creating a consortium involving symbiotic or mutualistic relationships among these species, and may be able to utilize the same or different waste molecules.

[0628]

[0502] In one aspect of the invention, the converting organisms may be cultivated together to form consortiums involving symbiotic or mutualistic relationships among these species.

[0629] (a) A symbiotic relationship is defined as a close and long-term interaction between two different biological organisms, where at least one of the organisms benefits from the association, and the other organism may or may not benefit, or may even be harmed. In the context of the present invention, symbiotic relationships between converting organisms may involve the exchange of nutrients, enzymes, or other substances that facilitate mutual survival and growth.

[0630] (b) A mutualistic relationship is a type of symbiotic relationship where both organisms involved benefit from the association. For example, in a mutualistic relationship, one organism may provide nutrients or a suitable environment for the other, while the other organism may provide essential metabolic by-products or protection from environmental stresses. In the context of this invention, mutualistic relationships may involve converting organisms that utilize similar or complementary waste molecules, enhancing the efficiency of the hydrolysis process or nutrient cycling.

[0631]

[0503] The converting organisms, whether in a symbiotic or mutualistic relationship, may utilize the same or different waste molecules during their cultivation, depending on the specific organisms and the metabolic pathways involved.

[0632]

[0504] The cultivation conditions for the consortium may be selected according to the species selected for the consortium.

[0505] In one aspect of the invention, the consortium may be formed from the converting organisms within the same domain, including, but not limited to the following examples:

[0633] a consortium formed from species within the domain archaea , for example combined cultivation of Methanoculleus marisnigri and Methano spirillum hungatei or any other combination of species from the families mentioned in previous paragraphs;

[0634] a consortium formed from species within the domain bacteria, for example combined cultivation of Pseudomonas stutzeri and Cupriavidus Metallidurans or Pseudomonas aeruginosa and E. coli or any other species from the families mentioned in previous paragraphs;

[0635] a consortium formed from species within the domain eukaryote, for example combined cultivation of Saccharomyces cerevisiae and Candida albicans or Sargassum muticum and Saccharina latissima or any other species from the families mentioned in previous paragraphs;

[0636] a consortium formed by combination across mentioned domains, for example combined cultivation of Methanosarcina barkeri and Geobacter sulfurreducens or Saccharomyces cerevisiae and Lactobacillus plantarum or Chlorella vulgaris and Nitrosomonas europaea or Zea mays and Rhizobium species or any combination of other species from the families mentioned in previous paragraphs.

[0637]

[0506] In another aspect of the invention, the consortium may comprise at least 2 converting organisms.

[0638]

[0507] In one aspect of the invention, the microbial consortium may comprise naturally occurring consortia commonly found in environments like activated sludge systems or biogas production facilities. For example, these consortia may include bacterial species of Methanobacterium formicicum, Methano spirillum hungatei, or Geobacter sulfurreducens, as well as other microorganisms commonly associated with anaerobic digestion or organic matter degradation.

[0639]

[0508] Microorganisms and / or plant cells may be genetically modified to produce at least one recombinant protein that may be utilized as a culture medium component.

[0640]

[0509] The incorporated genes used for production of recombinant proteins by the cells may encode at least one signaling compound, wherein the signaling compound may comprise FGF family ligands, insulin, IGF family ligands, TGF family ligands, EGF family ligands, transferrin or any other appropriate signaling compound.

[0641]

[0510] The genetic modifications may comprise permanent and / or transient genetic modifications, wherein such genetic modifications may be gain-of-function or loss-of-function modifications. The modifications may include point substitutions, point deletions, point insertions, larger deletions or larger insertions. The nucleic acid introduced into the cells may be naturally present within the species of the target cells, may originate from another species, may be synthetic, or a combination thereof. Such genetic modifications may be performed using methods such as CRISPR / Cas9, ZFNs, TALENs, Cre-Lox recombination, RMCE, genomic integration by homologous recombination or non -homologous end joining, and / or other tools. Other methods for genetic modification may comprise introduction by bacterial vectors, yeast vectors or viral vectors based on adenoviruses, adeno-associated viruses, re tro / lenti viruses and / or vectors.

[0642]

[0511] After the cultivation process, the cultivated microorganisms and / or plant cells may be harvested by a converting organisms harvesting device to separate the cell biomass from microorganism-digested medium, whose waste molecules may be concentrated in the concentration device generating microorganism-digested concentrate .

[0643]

[0512] The harvested cell biomass of microorganisms and / or plant cells may be inactivated by a sterilization unit and further lysed by applying thermal, mechanical, chemical, or enzymatic treatment, or a combination thereof, and used as a nutrient source for the cultivation of non-human metazoan cells. Alternatively, the harvested cell biomass of microorganisms and / or plant cells may be utilized for food production.

[0513] In another aspect of the invention, the lysis of microorganisms and / or plant cells may be performed before the inactivation of the cultivated microorganisms by downstream sterilization unit.

[0644]

[0514] The microorganism-digested medium may be processed in the same way as the waste medium derived from non-human metazoan cell cultivation. For example, the species of fungi may be cultivated in the waste medium and / or waste medium concentrate and after the cultivation, the species of fungi may be harvested by a converting organisms harvesting device and the microorganism-digested medium generated by the species of fungi may be processed the same way as the waste medium derived from non-human metazoan cells cultivation. This includes storing microorganism-digested medium generated by the species of fungi in the waste medium tank, followed by its transfer into the concentration device to remove the water and produce microorganism-digested concentrate, which may be supplemented with nutritional additives, solid residues, sediments and / or food industry by-products, and subsequently used for the cultivation of converting organisms. In another example, the bacterial species may be cultivated in the waste medium and / or waste medium concentrate and after the cultivation, the bacterial species may be harvested by a converting organisms harvesting device and microorganism-digested medium generated by bacterial species may be processed the same way as the waste medium derived from non- human metazoan cells cultivation. This includes storing the microorganism-digested medium generated by bacterial species in the waste medium tank, followed by its transfer into the concentration device to remove the water and produce microorganism-digested concentrate, which may be supplemented with nutritional additives, solid residues, sediments and / or food industry by-products, and subsequently used for the cultivation of converting organisms.

[0645]

[0515] In another aspect of the invention, the grown microorganisms and / or plant cells may be lysed directly within the waste medium and / or waste medium concentrate by applying thermal, mechanical, chemical, or enzymatic treatment, or a combination thereof. This hydrolysate from lysed grown microorganisms may be utilized for the cultivation of non-human metazoan cells.

[0646]

[0516] In another aspect of the invention, converting organisms may produce nutrients during their cultivation that can be utilized by non-human metazoan cells. For example, Saccharomyces cerevisiae may produce minerals, trace metals, and fat-soluble vitamins, which may serve as valuable components for supporting the growth and proliferation of non-human metazoan cells.

[0647]

[0517] In another aspect of the invention, nutrients produced by converting organisms may accumulate in the microorganism-digested medium or microorganism-digested concentrate, or may remain within the cells themselves. These cells, containing the nutrients, may subsequently be utilized for the production of protein hydrolysate.

[0648]

[0518] In one aspect of the invention, all converting organisms may be harvested after cultivation in the waste medium and / or waste medium concentrate, and may be processed by applying thermal, mechanical, chemical, or enzymatic treatment or a combination thereof to produce a protein hydrolysate. This protein hydrolysate may be mixed with nutritional additives, technological and / or processing additives and / or food industry by-products and / or other hydrolysates to produce a culture medium for the cultivation of non-human metazoan cells.

[0649]

[0519] The non-human metazoan cells may be cultivated in the rejuvenated culture medium under the same conditions as those specified for the cultivation of these cells in the culture medium mentioned in previous paragraphs.

[0650]

[0520] Cultivated cell biomass of non-human metazoan cells may be harvested from the rejuvenated culture medium by non-human metazoan cells harvesting device or any other appropriate device for harvesting of cells.

[0651]

[0521] Harvested cell biomass of non-human metazoan cells may be combined with harvested cell biomass of converting organisms to make food products.

[0652]

[0522] As used herein, the terms 'couple,' 'coupled,' 'connect,' and 'connected' are intended to broadly encompass any direct or indirect physical, fluidic, pneumatic, electrical, mechanical, communicative, oroperational relationship between components. This includes but is not limited to rigid connections, flexible connections, permanent connections, detachable connections, wireless or wired communicative links, and / or any combination thereof. Unless explicitly stated otherwise, these terms do not imply a particular type of connection or coupling method.

[0653]

[0523] For example, a control unit may be communicatively and operatively coupled with any component within the cultivation system to enable monitoring, control, and / or operation of the cultivation system as a whole and / or individual components thereof.

[0654]

[0524] For yet another example, the loading tank may be coupled to a cultivation device through various means, including but not limited to, by a tube for automated transfer of mass, by a dispenser and / or by a manual hand-dosing using an operator.

[0655]

[0525] Cultivation of cell cultures may be performed in the cultivation system, wherein the cultivation system comprises at least one upstream process device, at least one cultivation device, and at least one downstream process device. Additionally, the cultivation system may comprise support equipment for cleaning in place (CIP) and sterilization in place (SIP) processes.

[0656]

[0526] The cultivation system is controlled by a control unit, preferably a PC unit with a specifically designed software, which can be operated by a skilled operator to ensure total control of all processes.

[0657]

[0527] The upstream process device may comprise at least one of: a hydrolysis tank, a mixing tank, an upstream sterilization unit, and a culture medium storage tank.

[0658]

[0528] The upstream process device may further comprise: a plurality of loading tanks for the addition of nutritional additives, food industry by-products, solid residues, sediments, technological and / or processing additives or any other appropriate supplements, a plurality of valves, a plurality of sensors, a plurality of tubes, or any other appropriate components.

[0659]

[0529] In another aspect of the invention, the harvested converting organisms may be subjected to hydrolysis in the hydrolysis tank to generate the protein hydrolysate.

[0660]

[0530] In one aspect of the invention, the hydrolysis tank may be configured to provide an environment for the hydrolysis reaction. The hydrolysis tank may comprise a main body constructed from at least one material selected from stainless steel, glass-lined steel, titanium, polyethylene, polypropylene, polytetrafluoroethylene or any other suitable materials. The main body may comprise various shapes, such as cylindrical or rectangular or any other suitable geometries. The hydrolysis tank may comprise insulation configured as an outer jacket of the hydrolysis tank, wherein the space between the outer jacket and the wall of the hydrolysis tank may be filled with an appropriate insulation material or medium. The hydrolysis tank may further comprise at least one input and at least one output for loading and unloading the ingredients. The input of the hydrolysis tank may be configured as a shaft or funnel, wherein the shaft or funnel may be used for loading the ingredients. The hydrolysis tank may further comprise a heating and a cooling system configured to regulate the temperature of the inner environment of the hydrolysis tank. The hydrolysis tank may comprise mixing mechanisms comprising at least one stirrer, paddle or any other instrument capable of mixing the protein hydrolysate. The sealing mechanisms of the hydrolysis tank may comprise materials such as silicone, ethylene propylene diene monomer, and polytetrafluoroethylene. The hydrolysis tank may be configured to withstand a maximum temperature of at least 80 °C, at least 90 °C, at least 100 °C, at least 105 °C, at least 110 °C, at least 120 °C, or at least 150 °C. The hydrolysis tank may further comprise auxiliary components selected from the group of pumps, pressure sensors, flow meters, valves and means for monitoring the hydrolysis reaction.

[0661]

[0531] In one aspect of the invention, the heating of the hydrolysis tank may be achieved by hot water and / or steam circulation in the jacket, and / or by passing the content of the hydrolysis tank through an external heat exchanger, and / or by direct steam injection, and / or by any other suitable methods or their combinations.

[0532] In one aspect of the invention, the cooling of the hydrolysis tank may be achieved by cold water circulation in the jacket, and / or by passing the content of the hydrolysis tank through an external heat exchanger, and / or by any other suitable methods or their combinations.

[0662]

[0533] The working volume of the hydrolysis tank 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.

[0663]

[0534] The reaction components may be added to the hydrolysis tank manually, or automatically using a conveyor, loading tank or any other appropriate device used for transfer of reaction components. The source of protein may be in a liquid solution or in the form of a powder.

[0664]

[0535] The term “reaction components” may comprise a source of protein, proteolytic enzymes, or any other appropriate component necessary for effective hydrolysis by proteolytic enzymes.

[0665]

[0536] In one aspect of the invention, the hydrolysis tank may be equipped with different types of sensors, for example, a thermal sensor, pH probe, conductometer, or any other type of appropriate sensor according to the needs of the process of hydrolysis. The pH may be measured by various methods and devices comprising potentiometry, colorimetry, spectrophotometry, ion-selective electrodes, conductometry or any other measuring technique and / or device. The temperature in the reaction vessel may be measured by various devices comprising resistance temperature detector, thermocouple, digital thermometer with insertion probe, infrared thermometer with fiber optic probe or any other appropriate device.

[0666]

[0537] In one aspect of the invention, a filtration unit may comprise a suitable separation device, such as a centrifuge and / or a flotation device and / or at least one filter selected from the group of membrane filters, depth filters, mesh filters, activated carbon filters, ceramic filters, ultrafiltration filters, nanofiltration filters, ion exchange filters, crossflow (tangential flow) filters, adsorption filters and / or fiber filters.

[0667]

[0538] In one aspect of the invention, the filtration unit may be configured to utilize centrifugal force to separate solid-phase particles from liquid phase. This separation process is facilitated by the implementation of centrifugal filters, which may be strategically designed and positioned within the filtration unit.

[0668]

[0539] The filtration unit may further comprise auxiliary components selected from the group of pumps, pressure sensors, flow meters, valves and means for monitoring the filtration process.

[0669]

[0540] In one aspect of the invention, the filtration unit may be installed downstream of the hydrolysis tank to make purified protein hydrolysate.

[0670]

[0541] In one aspect of the invention, the cultivation system may comprise at least one mixing tank, wherein the mixing tank may comprise a main body constructed from at least one material selected from stainless steel, glass-lined steel, titanium, polyethylene, polypropylene, polytetrafluoroethylene or any other suitable materials. The main body may comprise various shapes, such as cylindrical or rectangular or any other suitable geometries. The input of the mixing tank may be configured as a shaft or funnel, wherein the shaft or funnel may be used for loading the nutritional additives. The mixing tank may be coupled with at least one loading tank for the addition of nutritional additives. The mixing tank may further comprise a temperature control system. The mixing tank may comprise mixing mechanisms comprising at least one stirrer, paddle or any other instrument capable of mixing the nutritional additives with the purified protein hydrolysate. The sealing mechanisms of the mixing tank may comprise materials such as silicone, ethylene propylene diene monomer, and polytetrafluoroethylene. The mixing tank may be configured to withstand a maximum temperature of at least 100 °C. The mixing tank may further comprise auxiliary components selected from the group of pumps, pressure sensors, flow meters, valves and means for monitoring the hydrolysis reaction.

[0671]

[0542] In another aspect of the invention, the mixing tank may comprise a thermometer and / or conductometer and / or a pH meter, the mixing tank may further comprise at least one shaft for loading the dry ingredients and at least one stirring unit.

[0543] The culture medium may be subjected to sterilization using a sterilization unit, further referred to as the upstream sterilization unit.

[0672]

[0544] The upstream sterilization unit may employ methods of sterilization selected from: thermal sterilization, filter sterilization, electron beam sterilization, any other appropriate sterilization method or their combination.

[0673]

[0545] In one aspect of the invention, the upstream sterilization unit may comprise a UHT pasteurization unit; the UHT pasteurization by the upstream sterilization unit may be performed at target temperatures in the range of 120 °C to 180 °C, in the range of 130 °C to 170 °C, or in the range of 135 °C to 160 °C.

[0674]

[0546] During the process of UHT pasteurization performed by the upstream sterilization unit, the culture medium may be kept at the target temperature for a portion of time in the range of 0.5 seconds to 60 seconds, in the range of 1 second to 30 seconds and / or in the range of 2 seconds to 20 seconds.

[0675]

[0547] In one aspect of the invention, the UHT treatment may reduce microbial contamination by a factor of at least 103, or at least 108, or at least 1013.

[0676]

[0548] In one aspect of the invention, one or multiple components of the culture medium may be sterilized by a different process than UHT. For example, vitamin B12 solution may be sterilized using a 0.1 pm rated filter and later added to the rest of the culture medium, which was sterilized by UHT.

[0677]

[0549] In one aspect of the invention, the upstream sterilization unit may comprise a filtration unit, preferably comprising a 0.2 pm rated filter and / or a 0.1 pm rated filter.

[0678]

[0550] After sterilization performed by upstream sterilization unit, the culture medium may be allowed to rest for a time, which may be in the range of 16 hours to 48 hours, in the range of 24 hours to 44 hours, or in the range of 32 hours to 40 hours within a culture medium storage tank. During this period of time the waste medium may be subjected to the measuring of the contamination by optical density at 600 nm, flow cytometry, sampling and detection of colony forming units, pH measurement, oxygen concentration measurement, or any other appropriate methods for detection of bacterial contamination.

[0679]

[0551] The culture medium for the cultivation of non -human metazoan cells may be stored in the culture medium storage tank, wherein the culture medium storage tank may comprise a main body constructed from at least one material selected from stainless steel, glass-lined steel, titanium, polyethylene, polypropylene, polytetrafluoroethylene or any other suitable materials. The culture medium storage tank may further comprise auxiliary components selected from the group of pumps, pressure sensors, flow meters, valves and means for monitoring the state of the culture medium. The culture medium storage tank may further comprise at least one input and at least one output for loading and unloading the ingredients. The culture medium storage tank may further comprise mixing mechanisms comprising at least one stirrer, paddle or any other instrument capable of mixing the culture medium. The sealing mechanisms of the storage tank may comprise materials such as silicone, ethylene propylene diene monomer, or polytetrafluoroethylene. The culture medium storage tank may comprise a heating system configured to heat the inner environment of the storage tank or may comprise a cooling system configured to cool the inner environment.

[0680]

[0552] In one aspect of the invention, the working volume of the culture medium storage tank may be in the range of 100 L to 100 m3, or in the range of 200 L to 50 m3, or in the range of 300 L to 30 m3.

[0681]

[0553] The cultivation device may comprise at least one of: a culture vessel, a gas sparging and recycling system, and a stirring unit.

[0682]

[0554] The cultivation device may comprise at least one culture vessel made from food -grade stainless steel, stainless steel, glass, or any other suitable material that is not toxic to said metazoan cells and at the same time is inert to the culture medium, cell metabolites and other substances considered. The culture vessel may be cylindrical, cubic, rounded cubic, round-bottom cylindrical, or another suitableshape, and may comprise a stirred tank, bubble column tank, airlift tank, packed bed tank, rotating -wall tank, wheel-tank, fixed-bed tank, perfusion tank or hollow fiber tank.

[0683]

[0555] The mixing may be provided by the appropriate stirring unit that may comprise, for example, one or more impellers, baffles, couplings, motors, shafts. In some aspects, an axial flow impeller or mixed flow impeller, for example a wide -blade hydrofoil impeller or an elephant ear impeller, may be used. The outer diameter of the impeller or impellers may be in the range of 1 / 10 to 9 / 10 of the inner reactor diameter, or in the range of 3 / 10 to 8 / 10 of the inner reactor diameter, or in the range of 4 / 10 to 7 / 10 of the inner reactor diameter. As an example, the outer diameter of the impeller may be 2 / 3 of the inner reactor diameter. The stirring shaft may be located in the center of the cultivation device or outside of the center of the cultivation device.

[0684]

[0556] In one aspect of the invention, the gas sparging and recycling system may be configured to deliver gaseous nutrients to the cultured cells while simultaneously recycling spent gases. The system may provide essential gases, such as oxygen and / or carbon dioxide, to maintain optimal growth conditions, and it may remove or recycle exhaust gases to enhance efficiency and minimize waste during the cultivation process.

[0685]

[0557] The gas sparging and recycling system may comprise one or more spargers, for example a ring sparger and / or a sintered sparger. The gas sparging and recycling system may further comprise one or more mass flow controllers or other suitable devices to control the flow of gasses into the sparger. The gasses delivered to the sparger may comprise air, oxygen, CO2, N2, recycled exhaust gas from the bioreactor, or any other appropriate gasses. The gas sparging and recycling system may further comprise one or more filters, compressors, dehumidifiers, and / or any other appropriate components.

[0686]

[0558] The cultivation device may further comprise a plurality of sensors and analytical instruments located inside or outside the culture vessel to provide real-time data about the metazoan cell processes and the parameters, such as pH, total pressure in the culture vessel, concentrations, or partial pressures of important gasses such as O2 and CO2, temperature, nutrient concentration, and cell density.

[0687]

[0559] In one aspect of the invention, the cultivation device and / or a collateral cultivation device may comprise at least one outlet and / or at least one inlet for transferring of mass in solid or liquid form.

[0688]

[0560] In another aspect of the invention, the cultivation device and / or a collateral cultivation device may comprise an inoculation port configured to inoculate at least one cell line.

[0689]

[0561] The cultivation of non-human metazoan cells within the culture medium and / or rejuvenated culture medium may take place in a cultivation device.

[0690]

[0562] The downstream process device may comprise at least one of: a non-human metazoan cells harvesting device (comprising harvesting tank and centrifuge), non-human metazoan cells biomass storage tank, waste medium tank, concentration device, downstream sterilization unit, collateral cultivation device, converting organisms harvesting device, converting organisms biomass storage tank, and digested medium storage tank.

[0691]

[0563] In one aspect of the invention, the downstream process device may comprise a non-human metazoan cells harvesting device, wherein this harvesting device may be designed for the efficient collection and separation of cultivated cell biomass of non-human metazoan cells from the culture medium. In another aspect of the invention, the non-human metazoan cells harvesting device may comprise at least one harvesting tank and / or at least one centrifugal separation system applying centrifugal force for the separation of non-human metazoan cell biomass from the culture medium or any appropriate liquid solution. The harvesting tank may comprise a vessel of a cylindrical, cubical, or any other appropriate shape. The working volume of the harvesting tank may be in the range of 100 L to 200 m3, or in the range of 200 L to 100 m3, or in the range of 500 L to 20 m3. The surfaces of the harvesting tank that come into contact with the liquid may be made of stainless steel, for example 316L stainless steel, or another appropriate material. The internal surface of the tank may have a smooth surface finish, for example 0.8 Ra, to facilitate cleaning. The harvesting tank may be constructed in sucha way as to withstand steam sterilization, at, for example, a temperature of at least 120 °C and total internal pressure of at least 2 atm. The harvesting tank may further comprise one or more impellers, and / or one or more valves, and / or one or more cleaning spray balls, and / or any other appropriate components. In another aspect of the invention, the harvested cell biomass may be used for the production of any substance having therapeutic effect, such as pharmaceuticals, signaling compounds and / or nutritional additives.

[0692]

[0564] In one aspect of the invention, the centrifugal separation system may comprise at least one of:

[0693] a disc stack centrifuge or a decanter centrifuge. Preferably, the centrifugal separation system may allow for continuous and / or intermittent discharge of both the liquid and solid fraction without needing to stop the centrifugal separation system.

[0694]

[0565] In another aspect of the invention, the centrifugal force of the centrifugal separation system for the harvesting of the cell biomass of non-human metazoan cells may be in the range of 100 G to 200,000 G, or in a range of 200 G to 100,000 G, or in a range of 500 G to 20,000 G, depending on the sensitivity and type of non-human metazoan cells being harvested.

[0695]

[0566] In one aspect of the invention, the waste medium with cell biomass of non-human metazoan cells may be stored at a temperature in the range of 1 °C to 35 °C, or in a range 5 °C to 25 °C, or in a range of 12 °C to 20 °C in the harvesting tank.

[0696]

[0567] After the cultivation of non-human metazoan cells, the cell biomass may be harvested using a non-human metazoan cells harvesting device. The separated cell biomass may be stored in a non-human metazoan cells biomass storage tank, wherein the non-human metazoan cells biomass storage tank may comprise a main body constructed from at least one material selected from stainless steel, glass -lined steel, titanium, polyethylene, polypropylene, polytetrafluoroethylene or any other suitable materials. The main body may comprise various shapes, such as cylindrical or rectangular or any other suitable geometries. The sealing mechanisms of the non-human metazoan cells biomass storage tank may comprise materials such as silicone, ethylene propylene diene monomer, and polytetrafluoroethylene. The biomass storage tank may be configured to withstand a maximum temperature of at least 100 °C. The biomass storage tank may further comprise auxiliary components selected from the group of pumps, pressure sensors, flow meters, valves and means for monitoring the hydrolysis reaction.

[0697]

[0568] The working volume of the non-human metazoan cells biomass storage tank may be in the range of 10 L to 10 m3, or in the range of 20 L to 5 m3, or in the range of 30 L to 3 m3.

[0698]

[0569] The harvested cell biomass of non-human metazoan cells may be stored in at least one biomass storage tank.

[0699]

[0570] In one aspect of the invention, the harvested cell biomass of non-human metazoan cells may be stored at a temperature in the range of 1 °C to 30 °C, or in the range 5 °C to 25 °C, or in the range of 12 °C to 19 °C.

[0700]

[0571] The waste medium and / or waste medium concentrate may be subjected to sterilization using a sterilization unit, further referred to as the downstream sterilization unit.

[0701]

[0572] The downstream sterilization unit may employ methods of sterilization selected from: thermal sterilization, filter sterilization, electron beam sterilization, any other appropriate sterilization method or their combination.

[0702]

[0573] The waste medium from the cultivation of non-human metazoan cells may be subjected to a heat sterilization treatment to inactivate viruses, bacteria, or any other appropriate microorganisms in the waste medium, while maintaining the activity of waste molecules.

[0703]

[0574] The UHT pasteurization of waste medium may be performed by a sterilization device capable of providing inactivated waste medium.

[0704]

[0575] In one aspect of the invention, the UHT pasteurization performed by the downstream sterilization unit may be performed at target temperatures in the range of 40 °C to 160 °C, in a range of 50 °C to 150 °C, or in a range of 60 °C to 145 °C.

[0576] In another aspect of the invention, the waste medium tank may be configured for the storage of the waste medium and may have the same material composition as the non-human metazoan cells biomass storage tank or the converting organisms storage tank. Additionally, the waste medium tank may comprise mixing mechanisms comprising at least one stirrer, paddle or any other instrument capable of mixing the waste medium.

[0705]

[0577] The waste medium may be subjected to a treatment to reduce volume of water content thereby concentrating materials in the waste medium and generating waste medium concentrate. In one aspect of the invention, the water content may be reduced by implementation of concentration device, wherein the concentration device may comprise a main body constructed from at least one material selected from stainless steel, glass-lined steel, titanium, polyethylene, polypropylene, polytetrafluoroethylene or any other suitable materials. The main body may comprise various shapes, such as cylindrical or rectangular or any other suitable geometries. The concentration device may comprise insulation configured as an outer jacket of the concentration device, wherein the space between the outer jacket and the wall of the concentration device may be filled with an appropriate insulation material or medium. The sealing mechanisms of the concentration device may comprise materials such as silicone, ethylene propylene diene monomer, and polytetrafluoroethylene. The concentration device may be configured to withstand a maximum temperature of at least 80 °C, at least 90 °C, at least 100 °C, at least 105 °C, at least 110 °C, at least 120 °C, or at least 150 °C.

[0706]

[0578] In one aspect of the invention, at least one antifoaming agent may be used to reduce foaming during the hydrolysis process. Such anti-foaming agents must not be cytotoxic and must exhibit antifoaming activity under process conditions. Such anti-foaming agents may comprise polyalkylene glycol, polyglycol ethers of aliphatic alcohols, a combination of vegetable esters with mono / diglycerides of edible fatty acids and vegetable oils, hydrophobic silica or alkoxylated fatty acid ester of aliphatic alcohols or any other appropriate anti-foaming agents. Such anti-foaming agents may be added in a concentration in a range of 250 ppm to 8,000 ppm, in a range of 350 ppm to 5,000 ppm, in a range of 500 ppm to 3,500 ppm, in a range of 700 ppm to 2,000 ppm, in a range of 800 ppm to 1,300 ppm, depending on selected protein substrate and anti-foaming agent to be used. Exemplary anti- foaming agents may be Struktol SB 2121, Struktol J647 or SB 420.

[0707]

[0579] In some aspects of the invention, the concentration device may be coupled with at least one loading tank, wherein the loading tank may supply a solution of anti-foaming agent in case the waste medium produces too much foam during the concentration process. The at least one concentration device may be further coupled with at least one antifoaming agent tank, at least one acidic agent tank and at least one alkaline agent tank, each configured to supply an antifoaming agent, an acidic agent or an alkaline agent to the concentration device and / or to a waste medium and / or to a waste medium concentrate processed therein. The antifoaming agent tank may be configured to provide an antifoaming agent to reduce or prevent foam formation in the concentration device during evaporation of water from the waste medium and / or from the waste medium concentrate. The acidic agent tank and the alkaline agent tank may be configured to provide the acidic agent and the alkaline agent, respectively, to adjust physicochemical properties of the waste medium and / or of the waste medium concentrate, in particular pH, osmolality, and / or conductivity, for example when the waste medium concentrate cannot be further used and must be discarded, in order to meet external regulations for the discharge of such waste medium concentrate. The use of the acidic agent tank and the alkaline agent tank for the adjustment of the pH, the osmolality and / or the conductivity of the waste medium and / or of the waste medium concentrate may be independent of cleaning-in-place and / or sterilization-in-place procedures in the cultivation system, which may be carried out separately.

[0708]

[0580] The waste medium recycling system may be employed within the cultivation system. The cultivation system may comprise a water source. The water source may be coupled with at least one water storage tank. The water storage tank may comprise a cylindrical housing. The housing may havean inner volume of at least 1 m3, at least 2 m3, at least 5 m3, at least 10 m3, at least 20 m3, or at least 50 m3. In other aspects of the invention, the volume of the housing of the water storage tank may be in the range of 1 m3to 50 m3, in a range of 5 m3to 45 m3, in a range of 10 m3to 30 m3, in a range of 15 m3to 25 m3. The water storage tank may further include at least one input and at least one output, wherein such input may be used for a coupling with the water source, or for a coupling with a concentration device, and wherein such output may be used for a coupling with a hydrolysis tank, a mixing tank, and / or a cooling system of the cultivation system. In some aspects of the invention, at least one water purification unit may be employed to demineralize and / or deionize the water for subsequent use in culture medium production that takes place upstream, in a hydrolysis tank, a mixing tank, or any other culture medium tank. The water storage tank may be coupled with at least one pump to transfer water within the waste medium recycling system.

[0709]

[0581] The waste medium recycling system may comprise at least one waste medium tank. The waste medium tank may comprise a cylindrical housing. The housing may have an inner volume of at least 1 m3, at least 2 m3, at least 5 m3, at least 10 m3, at least 20 m3, or at least 50 m3. In other aspects of the invention, the volume of the housing of the waste medium tank may be in the range of 1 m3to 50 m3, in a range of 5 m3to 45 m3, in a range of 10 m3to 30 m3, in a range of 15 m3to 25 m3. The waste medium tank may be coupled with at least one pump to transfer waste medium.

[0710]

[0582] In some aspects, the water storage tank may be made of stainless steel, such as AISI 304 or AISI 316, or of a food-grade polymeric material, optionally comprising an inner coating or liner suitable for contact with water intended for culture medium preparation. The water storage tank may comprise one or more level sensors, such as float switches or pressure-based level transmitters, configured to enable automatic control of filling and emptying operations. The water storage tank may further comprise at least one temperature sensor and, optionally, a heating and / or cooling jacket or an external heat exchanger, configured to maintain the water within a predetermined temperature range suitable for culture medium preparation. In some aspects of the invention, the water storage tank may be equipped with at least one manhole or inspection port, and one or more cleaning-in-place (CIP) and / or sterilization- in-place (SIP) nozzles to facilitate sanitation of the inner surfaces. The at least one pump coupled with the water storage tank may be at least one of centrifugal pumps, positive displacement pumps, and / or diaphragm pumps, and may be controlled by a process control system, such as a programmable logic controller (PLC), based on signals from one or more sensors.

[0711]

[0583] The waste medium recycling system may comprise at least one waste medium concentrate storage tank. The waste medium concentrate storage tank may be constructed as a cylindrical housing. The housing may have an inner volume of at least 1 m3, at least 2 m3, at least 5 m3, at least 10 m3, at least 20 m3, or at least 50 m3. In other aspects of the invention, the volume of the housing of the waste medium concentrate storage tank may be in the range of 1 m3to 50 m3, in a range of 5 m3to 45 m3, in a range of 10 m3to 30 m3, in a range of 15 m3to 25 m3. The waste medium concentrate storage tank may comprise at least one output for exhaust air to relieve pressure or any undesired gases in the housing. The housing of the waste medium concentrate storage tank may be made of material that is non-corrosive when in contact with the waste medium concentrate, which may be harsh to most materials. The waste medium concentrate storage tank may comprise at least one output for coupling with at least one collateral cultivation device, or for discarding if the waste medium concentrate cannot be further used. The waste medium concentrate storage tank may further comprise at least one input for receiving the waste medium concentrate from the concentration device.

[0712]

[0584] In some aspects of the invention, the waste medium concentrate storage tank may be configured to handle fluids with high viscosity, and may therefore comprise a reinforced agitation system and / or a conical or sloped bottom to facilitate complete drainage. The exhaust air output may be equipped with a gas treatment unit, such as an activated carbon filter, a scrubber, or a condenser, configured to remove volatile compounds or odors prior to releasing the exhaust air to the environment. The waste mediumconcentrate storage tank may comprise one or more CIP and / or SIP ports to enable automated cleaning and sterilization between processing cycles. In some aspects of the invention, the waste medium concentrate storage tank may be operatively connected to a control system configured to determine, based on one or more measured parameters (e.g., concentration of organic matter, conductivity, pH, or volume), whether the waste medium concentrate may be directed to the collateral cultivation device or to a disposal line. The waste medium concentrate storage tank may additionally comprise one or more sampling ports allowing sterile or controlled sampling of the concentrate for analytical purposes, such as monitoring nutrient content, contaminants, or microorganism load. In further aspects of the invention, any of the water storage tank, the waste medium tank, and the waste medium concentrate storage tank may be arranged in parallel or in series to increase total capacity, allow redundancy, or enable operation in batch, fed-batch, or continuous modes, thereby improving flexibility and scalability of the waste medium recycling system within the cultivation system.

[0713]

[0585] In one exemplary aspect of the invention, the cultivation system may comprise at least one cultivation device configured to cultivate at least one non-human metazoan cell line in a culture medium based on protein hydrolysate, at least one pre -harvest vessel coupled with the at least one cultivation device, at least one first harvesting device configured to separate a waste medium from a cell biomass, at least one waste medium tank configured to store the waste medium, at least one concentration device configured to evaporate water from the waste medium to provide a waste medium concentrate, wherein the evaporated water may be returned to at least one water storage tank, at least one waste medium concentrate storage tank configured to store the waste medium concentrate, at least one collateral cultivation device configured to use the waste medium concentrate as substrate for cultivation of at least one converting microorganism providing a proteinaceous biomass, and at least one second hydrolysis tank configured to hydrolyse the proteinaceous biomass to provide a hydrolysed proteinaceous biomass for production of the culture medium based on protein hydrolysate.

[0714]

[0586] In one exemplary aspect of the invention, the cultivation system may comprise at least one water storage tank coupled with a water source and with at least one concentration device, wherein the at least one concentration device may be coupled with at least one waste medium tank and may be configured to evaporate water from a waste medium to provide a waste medium concentrate and to return evaporated water to the at least one water storage tank, at least one waste medium concentrate storage tank coupled with the at least one concentration device, at least one collateral cultivation device coupled with the at least one waste medium concentrate storage tank and configured to use the waste medium concentrate as substrate for cultivation of at least one converting microorganism providing a proteinaceous biomass, and at least one second hydrolysis tank configured to hydrolyse the proteinaceous biomass to provide a hydrolysed proteinaceous biomass usable for production of a culture medium based on protein hydrolysate.

[0715]

[0587] In one exemplary aspect of the invention, the cultivation system may comprise at least one cultivation device configured to cultivate at least one non-human metazoan cell line in a culture medium based on protein hydrolysate, at least one first harvesting device configured to separate a waste medium from a cell biomass and to direct the waste medium to at least one waste medium tank, at least one concentration device configured to process the waste medium from the at least one waste medium tank to obtain a waste medium concentrate and evaporated water, wherein the evaporated water may be returned to at least one water storage tank, and at least one collateral cultivation device and at least one second hydrolysis tank configured to convert the waste medium concentrate via at least one converting microorganism into a hydrolysed proteinaceous biomass for re-use in the culture medium based on protein hydrolysate.

[0716]

[0588] In one exemplary aspect of the invention, the cultivation system may comprise a waste medium recycling system including at least one waste medium tank configured to receive a waste medium from at least one first harvesting device, at least one concentration device configured to evaporate water fromthe waste medium and to provide a waste medium concentrate, at least one waste medium concentrate storage tank configured to store the waste medium concentrate, at least one collateral cultivation device configured to use the waste medium concentrate as substrate for cultivation of at least one converting microorganism providing a proteinaceous biomass, and at least one second hydrolysis tank configured to hydrolyse the proteinaceous biomass to provide a hydrolysed proteinaceous biomass for preparation of a culture medium based on protein hydrolysate.

[0717]

[0589] In one exemplary aspect of the invention, the cultivation system may be configured such that a waste medium originating from at least one cultivation device may be collected in at least one waste medium tank, concentrated in at least one concentration device to obtain a waste medium concentrate and evaporated water, wherein the evaporated water is returned to at least one water storage tank, and wherein the waste medium concentrate is stored in at least one waste medium concentrate storage tank and used in at least one collateral cultivation device and at least one second hydrolysis tank to provide a hydrolysed proteinaceous biomass for use in production of a culture medium based on protein hydrolysate.

[0718]

[0590] In one aspect of the invention, the collateral cultivation device may be configured to facilitate the co-hydrolysis process.

[0719]

[0591] In one aspect of the invention, the cultivation system may be as depicted in the scheme of Fig.

[0720] 15. An exemplary aspect of the invention according to the scheme of Fig. 15 may be as depicted in Fig.

[0721] 16. The cultivation system may comprise:

[0722] a water source coupled with a water storage tank (728);

[0723] the water storage tank (728) coupled with the water purification unit (710);

[0724] the water purification unit (710) coupled with at least one culture medium tank (714);

[0725] wherein the at least one culture medium tank (714) is coupled to at least one loading tank (107) and is configured to produce culture medium for the cultivation of non -human metazoan cells; wherein the at least one loading tank (107) may be configured to load a protein substrate, a proteolytic enzyme, an enzyme having phytase activity, and / or at least one nutritional additive;

[0726] a cultivation device (101) coupled with the at least one culture medium tank (714);

[0727] a pre -harvest vessel (702) coupled with the cultivation device (701);

[0728] a harvesting device (102) coupled with the pre -harvest vessel (702);

[0729] the harvesting device (102) coupled with a waste medium tank (103);

[0730] the waste medium tank (103) coupled with a concentration device (106);

[0731] the concentration device (106) coupled with a waste medium concentrate storage tank (730) and also coupled with the water storage tank (728); and

[0732] the waste medium concentrate storage tank (730) coupled with a collateral cultivation device (731).

[0733]

[0592] In one aspect of the invention, the cultivation system may be as depicted in the scheme of Fig.

[0734] 17. An exemplary aspect of the invention may be as depicted in Fig. 18. The cultivation system may comprise:

[0735] a water source coupled with a water storage tank (728);

[0736] the water storage tank (728) coupled with the water purification unit (710);

[0737] the water purification unit (710) coupled with at least one hydrolysis tank (110);

[0738] the at least one hydrolysis tank (110) coupled with at least one mixing tank (110b);

[0739] wherein the hydrolysis tank (110) may be configured to perform hydrolysis of a protein source; a sterilization unit (105) coupled with the at least one mixing tank (110b);

[0740] the sterilization unit (105) coupled with the cultivation device (101);

[0741] the cultivation device (101) coupled with the at least one culture medium tank (714);

[0742] a pre -harvest vessel (702) coupled with the cultivation device (101);

[0743] a harvesting device (102) coupled with the pre -harvest vessel (702);

[0744] the harvesting device (102) coupled with a waste medium tank (103);the waste medium tank (103) coupled with a concentration device (106);

[0745] the concentration device (106) coupled with a waste medium concentrate storage tank (730) and also coupled with the water storage tank (728); and

[0746] the waste medium concentrate storage tank (730) coupled with a collateral cultivation device (104).

[0747]

[0593] In one aspect of the invention, the cultivation system may be as depicted in the scheme of Fig.

[0748] 19. An exemplary aspect of the invention may be as depicted in Fig. 20. The cultivation system may comprise:

[0749] a water source coupled with at least one water storage tank;

[0750] wherein the at least one water storage tank may be coupled with at least one water purification unit; wherein the at least one water storage tank may be coupled with at least one first hydrolysis tank and / or at least one mixing tank;

[0751] wherein the at least one first hydrolysis tank may be coupled with the at least one mixing tank; wherein the at least one first hydrolysis tank may be configured to provide a protein hydrolysate, and wherein the at least one mixing tank may be configured to receive the protein hydrolysate from the at least one hydrolysis tank and to mix with at least one other nutritional additive to provide a culture medium based on protein hydrolysate;

[0752] at least one sterilization unit coupled with the at least one mixing tank;

[0753] wherein the at least one sterilization unit may be configured to sterilize the culture medium;

[0754] at least one culture medium storage tank coupled with at least one cultivation device;

[0755] wherein the at least one culture medium storage tank may be configured to store the culture medium based on protein hydrolysate;

[0756] wherein the at least one cultivation device may be coupled with at least one pre -harvest vessel; wherein the at least one cultivation device may be configured to cultivate at least one non -human metazoan cell line in the culture medium based on protein hydrolysate, and

[0757] wherein the at least one pre -harvest vessel may be configured to receive a cell biomass of the at least one non-human metazoan cell line;

[0758] at least one first harvesting device coupled with the at least one pre -harvest vessel, configured to separate a waste medium from the cell biomass of at least one non-human metazoan cell line;

[0759] at least one waste medium tank coupled with the at least one first harvesting device,

[0760] wherein the at least one waste medium tank may be configured to store the waste medium;

[0761] at least one concentration device coupled with the at least one waste medium tank,

[0762] wherein the at least one concentration device may be configured to evaporate water from the waste medium and to provide a waste medium concentrate;

[0763] wherein the evaporated water may be returned to the at least one water storage tank;

[0764] at least one waste medium concentrate storage tank coupled with the at least one concentration device, wherein the at least one waste medium concentrate storage tank may be configured to store the concentrate;

[0765] at least one collateral cultivation device coupled with the at least one concentrate storage tank, wherein the at least one collateral cultivation device may be configured to use the concentrate as substrate for cultivation of at least one converting microorganism;

[0766] wherein the at least one converting organism may provide a proteinaceous biomass;

[0767] at least one second hydrolysis tank coupled with the at least one collateral cultivation device, wherein the at least one second hydrolysis tank may be configured to hydrolyse the proteinaceous biomass to provide a hydrolysed proteinaceous biomass; and

[0768] wherein the hydrolysed proteinaceous biomass may be used for production of a culture medium based on protein hydrolysate, which may be subsequently used for cultivation of the at least one non-human metazoan cell line in the at least one cultivation device.

[0594] The cultivation system (100) may comprise at least one CIP unit (116) which may be configured to clean-in-place; and at least one SIP unit (117) which may be configured to sterilize -in-place.

[0769]

[0595] In one aspect of the invention, the centrifuge unit and harvesting device (102) may serve similar functions and / or may substitute each other in some applications.

[0770]

[0596] In one aspect of the invention, the cultivation system may be expanded to increase its capacity by incorporating additional components, devices or units, such as more hydrolysis tanks or cultivation devices, effectively multiplying the output while maintaining continuous operation. This modular configuration may include a specific arrangement for coupling or connecting particular components in an efficient and integrated manner to optimize performance. Alternatively, the capacity of the cultivation system may increase by increasing the volumes or size of such components, such as larger hydrolysis tanks, larger mixing tanks, or larger cultivation devices. These scalable design options enable flexibility in adapting the cultivation system to different production demands while maintaining operational efficiency.

[0771]

[0597] In one aspect of the invention, the cultivation system may comprise two or more hydrolysis tanks. When employing at least two hydrolysis tanks, the capacity for protein hydrolysate production is increased, allowing for continuous operation. This configuration also provides practical advantages, such as the ability to sanitize one hydrolysis tank while the other remains in use, ensuring uninterrupted production. Additionally, the increased hydrolysate production capacity makes it beneficial to employ multiple bioreactors, thereby further enhancing the overall throughput of the cultivation system. When employing two or more hydrolysis tanks, they may be referred to as “first hydrolysis tanks”, “second hydrolysis tank” and / or “third hydrolysis tank”.

[0772]

[0598] In one aspect of the invention, the cultivation system may comprise two or more cultivation devices. The cultivation devices may be arranged in series, where a smaller volume cultivation device is utilized initially, followed by one or more cultivation devices of progressively larger volumes, enabling a stepwise increase in cultivation scale. In another aspect of the invention, the cultivation devices may be arranged in parallel, where the culture medium from at least one mixing tank is distributed into multiple separate lines, each equipped with one or more cultivation devices operating simultaneously. In some aspects of the invention, a combination of both series and parallel arrangements may be employed, such as two or more parallel cultivation lines, each comprising a series of cultivation devices with increasing volumes. This flexible and modular setup allows for optimized scalability and efficient utilization of available resources.

[0773]

[0599] In one aspect of the invention, the cultivation system as depicted in the Fig. 22 and graphical illustration from Fig. 23 may comprise:

[0774] a water purification unit (710) configured to purify water;

[0775] wherein the purified water may be stored in a water storage tank (1101);

[0776] wherein the water storage tank (1101) may be coupled with a hydrolysis tank (110);

[0777] wherein the hydrolysis tank (110) may be coupled with a centrifuge (1103);

[0778] a non-sterile protein hydrolysate storage tank (1105) coupled with the centrifuge (1103);

[0779] wherein the non-sterile hydrolysate storage tank (1105) may be coupled with a sterilization unit (105a); a sterile protein hydrolysate storage tank (1106) coupled with a mixing tank (110b);

[0780] wherein the mixing tank (110b) and the hydrolysis tank (110) may be coupled with a loading tank (107); wherein the mixing tank (110b) may be coupled with a sterilization unit (105b);

[0781] a fresh culture medium storage tank (1107) coupled with the sterilization unit (105b);

[0782] a cultivation device (101) coupled with the fresh culture medium storage tank (1107);

[0783] a pre -harvest vessel (1108) coupled with the cultivation device (101);

[0784] a harvesting device (102) coupled with the pre -harvest vessel (1108);

[0785] wherein the harvesting device (102) may be coupled with a waste medium tank (103) and / or a postprocessing (1109).

[0600] The cultivation system according to the scheme depicted in Fig. 24 is an exemplary variant of the up-scaled cultivation system from the scheme depicted in Fig.22 and graphical illustration from Fig.

[0786] 23. The cultivation system according to the scheme in Fig. 24 and graphical illustration from Fig. 25 may comprise:

[0787] a water purification unit (710) configured to purify water;

[0788] wherein the purified water may be stored in a water storage tank (1101);

[0789] wherein the water storage tank (1101) may be coupled with a first mixing tank (1104a) and a second mixing tank (1104b);

[0790] a first hydrolysis tank (1102a) and a second hydrolysis tank (1102b) coupled with the water storage tank (110a);

[0791] wherein the first hydrolysis tank (104a) and the second hydrolysis tank (104b) are coupled with the centrifuge (1103);

[0792] the centrifuge (1103) coupled with a non-sterile hydrolysate storage tank (1105);

[0793] wherein the non-sterile hydrolysate storage tank (1105) is coupled with a sterilization unit (105); wherein the sterilization unit (105) may be configured to perform UHT thermal treatment of the protein hydrolysate;

[0794] a sterile protein hydrolysate storage tank (1106) coupled with the sterilization unit (105);

[0795] the first mixing tank (1104a) and the second mixing tank (1104b) both coupled with a sterilization unit (105a) and sterilization unit (105b);

[0796] wherein the first mixing tank (1104a) and the second mixing tank (1104b) are configured to mix the sterile protein hydrolysate with at least one additional compound;

[0797] wherein the at least one additional compound may be loaded from at least one loading tank (107); a first fresh culture medium storage tank (1107a) and a second fresh culture medium storage tank (1107b) coupled with the sterilization unit (105a) and sterilization unit (105b);

[0798] wherein the sterilization unit (105a) and sterilization unit (105b) may be configured to perform UHT thermal treatment of the culture medium;

[0799] a first cultivation device (101a) and a second cultivation device (101b);

[0800] wherein the first cultivation device (101a) and the second cultivation device (101b) may be configured to cultivate at least one non-human metazoan cell line;

[0801] a first pre-harvest vessel (1108a) coupled with the first cultivation device (101a) and a second preharvest vessel (102b) coupled with the second cultivation device (101b);

[0802] wherein the first pre -harvest vessel (1108a) and a second pre -harvest vessel (1108b) may be configured to continuously harvest a portion of cell biomass from the at least one cultivation device; wherein the first pre -harvest vessel (1108a) and a second pre -harvest vessel (1108b) may be coupled with a harvesting device (102);

[0803] wherein the harvesting device (102) may be coupled with a waste medium tank (103) and with a postprocessing device (1109).

[0804] at least one CIP unit (116) and / or at least one SIP unit (117);

[0805] wherein the at least one CIP unit (116) may be coupled to at least one component of the cultivation system (100) and may be configured to clean-in-place; and

[0806] wherein the at least one SIP unit (117) may be coupled to at least one component of the cultivation system (100) and may be configured to sterilize-in-place.

[0807]

[0601] In one aspect of the invention, the cultivation system may be designed to be scalable in both capacity and operational complexity. The cultivation system, as depicted in Fig. 22, may serve as a foundational model, which may be adapted or expanded depending on specific production requirements. For example, up-scale implementation may comprise increasing working volumes of components, such as hydrolysis tanks, mixing tanks, or culture vessels of a cultivation device.

[0602] In one aspect of the invention, the cultivation system may further comprise one or more parallel process lines, wherein the upstream section, or at least part of the upstream section where sterile protein hydrolysate is prepared, may be coupled to multiple core cultivation sections and their respective downstream sections.

[0808]

[0603] Unless stated otherwise, terms used herein are intended to be interpreted broadly to encompass various configurations, connections, and associations. For example, the term "coupled" may refer to any operative connection, including but not limited to fluidly coupled, mechanically connected, physically linked via tubing, piping, or other conduits, and / or electronically or functionally integrated to facilitate the intended operation of the cultivation system. In the absence of specific limitations, the disclosed components, methods, and features should not be construed as being restricted to a singular implementation but rather as encompassing various possible alternatives, modifications, and equivalents.

[0809]

[0604] In one aspect of the invention, the cultivation system may comprise an external physical stimulation mechanism, which is capable of influencing the biological, biochemical and chemical reactions inside the cultivation device. The exposure of cultivated non-human metazoan cells to an external physical stimulation may influence cell proliferation, differentiation, cell cycle progression, growth rate, enzyme activities, membrane structure and cellular transformation. The external physical influence is capable of permeating through cells and changing the electric field of the cell membrane, which can cause biological changes, especially changes in the ion efflux between the inner and outer space of the cells. The external physical stimulation mechanisms are based on exposure to at least one source of energy selected from the group of acoustic waves, electromagnetic waves, electric current, magnetic fields and / or any other energy source. The external physical stimulation mechanisms may be positioned inside and / or outside the cultivation device and may be applied globally or locally to a cultivated non-human metazoan cell population, wherein local application refers to application to a volume of the cultivation device that is smaller than the volume of the whole cultivation device. In addition, ultrasound may be used to externally stimulate the cultivated non-human metazoan cell population and may also mitigate the formation of foam above the liquid phase in the cultivation device, i.e. in the non-working volume of the cultivation device.

[0810]

[0605] The cells may be stimulated using magnetic fields comprising impulses, wherein the impulse may be a magnetic stimulus in the form of monophasic, biphasic or polyphasic shape. The impulse duration may be in a range of 1 microsecond to 1,000 microseconds, 10 microseconds to 1,000 microseconds, 15 microseconds to 950 microseconds, 100 microseconds to 900 microseconds, 100 microseconds to 700 microseconds, or 150 microseconds to 500 microseconds.

[0811]

[0606] The impulses may be applied in a continual or pulsed form. The pulse may be defined as an impulse followed by no magnetic stimulus. The pulse may be applied with a frequency of 0.1 Hz to 500 Hz, 1 Hz to 300 Hz, 1 Hz to 150 Hz, 1 Hz to 100 Hz, 2 Hz to 80 Hz, 5 Hz to 80 Hz, or 5 Hz to 50 Hz. The impulses and / or pulses may be assembled in a train. The impulses within the train may be modulated in amplitude or frequency to create various envelopes e.g. rectangular, triangle, trapeze and / or staircase.

[0812]

[0607] The train duration may be in the range of 0.1 seconds to 120 seconds, or in a range of 0.5 seconds to 50 seconds, or in a range of 1 second to 20 seconds.

[0813]

[0608] The repetition rate of impulses may be in a range of 1 Hz to 300 Hz and the intensity of the field may be in the range of 0.01 mT to 7 T, or in the range of 0.1 mT to 6 T, or in the range of 0.5 mT to 5 T, or in the range of 0.8 mT to 4 T, wherein the intensity is measured on the coil surface.

[0814]

[0609] The magnetic field may be applied by at least one magnetic field generating coil, which is coupled to a switch (e.g. such as a diode, pin diode, MOSFET, JFET, IGBT, BJT, thyristor and / or a combination thereof) and an energy storage device (e.g. a capacitor) wherein the energy storage device is coupled to a power source. The switch and the energy storage device may be coupled to a control unit (e.g. PCB, PC) which controls and / or regulates the operation and application of a magnetic field.

[0610] The control unit may command to repeatedly switch on / off the switch and discharge the energy storage device to the magnetic field generating coil in order to generate the magnetic field.

[0815]

[0611] The capacitance of the energy storage device may be in the range of 5 nF to 100 mF, or in the range of 25 nF to 50 mF, or in the range of 100 nF to 10 mF, or in the range of 1 pF to 1 mF, or in the range of 5 pF to 500 pF or in the range of 10 pF to 180 pF, or in the range of 20 pF to 80 pF.

[0816]

[0612] The energy storage device may be charged on a voltage in a range from 250 V to 50 kV, 700 V to 5 kV, 700 V to 3 kV, or 1 kV to 1.8 kV.

[0817]

[0613] The energy storage device may provide a current pulse discharge in a range from 100 A to 5 kA, 200 A to 3 kA, 400 A to 3 kA, or 700 A to 2.5 kA. The current may correspond with a value of the peak magnetic flux density generated by the magnetic field generating device.

[0818]

[0614] Further, the energy storage device may provide a current pulse discharge in a range from 1,000 A to 10,000 A, 2,000 A to 8,000 A or 2,500 to 7,500 A.

[0819]

[0615] The inductance of the magnetic field generating coil may be up to 1 H, or in the range of 1 nH to 500 mH, 1 nH to 50 mH, 50 nH to 10 mH, 500 nH to 1 mH, or in the range of 1 pH to 500 pH or in the range of 10 pH to 60 pH.

[0820]

[0616] The maximal value of the magnetic flux density derivative may be in the range of 0.3 kT / s to 900 kT / s, 0.5 kT / s to 400 kT / s, 1 kT / s to 300 kT / s, 1.5 kT / s to 250 kT / s, 2 kT / s to 200 kT / s, or 2.5 kT / s to 150 kT / s.

[0821]

[0617] The cells may also be stimulated using mechanical stimulation e.g. acoustic waves having characteristics of ultrasound, infrasound and / or audible sound. The acoustic waves, infrasound and / or ultrasound may be characterized by a frequency in the range of 0.01 Hz to 2000 MHz, 20 kHZ to 100 MHz, 20 kHZ to 20 MHz, or 20 kHZ to 1 MHz.

[0822]

[0618] The acoustic waves, infrasound and / or ultrasound may be characterized by a power density in the range of 1 mW-cm2to 10 W-cm2, 0.001 W-cm2to 500 W-cm2, or 0.005 W-cm2to 350 W-cm2, or 0.05 W-cm2to 250 W-cm2.

[0823]

[0619] The mechanical stimulation may be applied by ultrasound transducer (e.g. a piezoelectric transducer or capacitive transducer), or a piezoelectric, electrohydraulic or electromagnetic wave generator coupled to the control unit, which controls and / or regulates the operation and application of mechanical stimulation.

[0824] [0620...

Claims

CLAIMS:

1. A rejuvenated culture medium for cultivating non -human metazoan cells, the rejuvenated culture medium comprising:(a) waste molecules derived from a waste medium, a waste medium concentrate, or a combination thereof, generated during cultivation of non-human metazoan cells; and(b) at least one nutritional additive,wherein the rejuvenated culture medium is configured to support cultivation of non-human metazoan cells.

2. The rejuvenated culture medium according to claim 1 , wherein the waste medium, waste medium concentrate or a combination thereof are supplemented with hydrolysate, nutritional additives, food industry by-products or a combination thereof, in a process of direct recycling, to make a rejuvenated culture medium.

3. The rejuvenated culture medium according to claim 2, wherein the rejuvenated culture medium may be supplemented with a protein hydrolysate in the range of 0.1 g / L to 100 g / L.

4. The rejuvenated culture medium according to claim 2, wherein the nutritional additives include at least one of saccharides, mineral compounds, vitamins, amino acids, peptides, organic amines, signaling compounds, oligonucleotides, fatty acids, phospholipids or organic micronutrients, wherein the nutritional additives are added into the waste medium and / or waste medium concentrate from a loading tank.

5. The rejuvenated culture medium according to claim 1 , wherein the waste medium has an osmolality in the range of 200 to 400 mOsm / kg, a pH in the range of 5 to 10 at room temperature and atmospheric CO2 concentration, and contains chloride ions in the range of 1,000 to 5,000 mg / L.

6. The rejuvenated culture medium according to claim 1, wherein the waste medium is subjected to a treatment to remove solid residues, wherein the treatment comprises at least one of filtration, flotation, or centrifugation.

7. The rejuvenated culture medium according to claim 1, wherein the waste medium comprises 95 % to 99.9 % of water prior to being subjected to the concentration device to generate the waste medium concentrate.

8. The rejuvenated culture medium according to claim 1, wherein the waste medium is subjected to a treatment to reduce water content, thereby concentrating waste molecules and producing waste medium concentrate, the treatment comprising evaporation, vacuum distillation, reverse osmosis, or a combination thereof; and wherein recovered water is obtained during the treatment.

9. The rejuvenated culture medium according to claim 8, wherein the waste medium concentrate has a volume, and wherein the volume of the waste medium concentrate multiplied by the concentration factor of the waste medium concentrate is in the range of 10 % to 90 %, and wherein the waste medium concentrate is characterized by a concentration factor, defined as the mass of the waste medium before concentration, divided by the mass of the resulting waste medium concentrate, the concentration factor being in the range of 2 to 100.

10. The rejuvenated culture medium according to claim 1, wherein the waste medium, waste medium concentrate, or a combination thereof, is configured for the cultivation of converting organisms.

11. The rejuvenated culture medium according to claim 1, wherein the non-human metazoan cells comprise at least one of hepatocytes, myocytes, myoblasts, osteoblasts, fibroblasts, lipoblasts, odontoblasts, keratinocytes, mesenchymal stem cells, multipotent progenitor cells, embryonic stem cells, myofibroblasts, or myosatellite cells, and have characteristics of bovine, avian, porcine, equine, piscine, cervine or cricetine cell lines.

12. A method for cultivating converting organisms, the method comprising:(a) providing a waste medium generated from cultivation of non-human metazoan cells;(b) concentrating waste molecules present in the waste medium to produce a waste medium concentrate; and(c) cultivating converting organisms in the waste medium concentrate.

13. The method according to claim 1, wherein the converting organisms comprise bacteria, archaea, fungi, algae (microalgae, macroalgae), plants, and / or their cells.

14. The method according to claim 13, wherein the converting organisms proliferate by utilizing waste molecules.

15. The method according to claim 14, wherein the cell biomass of converting organisms is used as a source of protein in the process of preparing the protein hydrolysate, and wherein the protein hydrolysate is used for the preparation of culture medium for non-human metazoan cells.

16. The method according to claim 13, wherein the at least two converting organisms are cultivated together to form consortiums, and wherein the converting organisms utilize the same or different waste molecules.

17. A method for co-hydrolyzing protein sources, the method comprising:(a) providing at least two protein sources, comprising:(i) a first protein source comprising a plant-based protein source ; and(ii) a second protein source comprising at least one microorganism,wherein the second protein source comprises endogenously produced enzymes for co-hydrolysis; (b) combining the at least two protein sources to form a hydrolysis mixture and thereby initiating cohydrolysis with added exogenously supplied enzymes; and(c) generating a protein hydrolysate.

18. The method according to claim 1, wherein the first protein source comprises a plant -based protein source that is rich in protein and suitable for enzymatic hydrolysis, selected from soy (soy protein concentrate and / or soy protein isolate), pea, rice, wheat, wheat gluten, corn, fava beans, alfalfa, hemp, chickpea, potato, pumpkin, rapeseed, red lentil, rice, sunflower, water lentil, duckweed, mungbean, or bean.

19. The method according to claim 1, wherein the second protein source comprises at least one microorganism, and wherein the microorganism is at least one of Saccharomyces cerevisiae, Pichia pastoris, Schizosaccharomyces pombe, Candida utilis, Debaryomyces hansenii, Yarrowia lipolytica, Aspergillus oryzae, Aspergillus niger, Bacillus subtilis, Kluyveromyces lactis, Kluyveromyces marxianus, Streptococcus thermophilus, Lactobacillus helveticus, or Bacillus licheniformis, wherein the microorganism is capable of autolysis20. The method according to claim 19, wherein the at least one microorganism is capable of producing different types of enzymes and / or capable of endogenous production of enzymes resulting in breaking-down of peptide bonds, phosphate ester bonds and / or glycosidic bonds.

21. The method according to claim 1, wherein the endogenously produced enzymes comprise one or more naturally occurring enzymes selected from lipase, phosphoesterase, an enzyme having phytase activity, endopeptidase, exopeptidase, beta-glucanase, or nuclease.

22. The method according to claim 1 , wherein the second protein source comprises enzymes having endopeptidase activity in the range of 0.0001 to 300,000 TU, exopeptidase activity in the range of 0.0001 to 200,000 LAPU, phytase activity in the range of 0.0001 to 3,000,000 FTU, and beta- glucanase activity in the range of 0.00001 to 10,000 FBG per 1 g of the second protein source.

23. A cultivation system for preparing a culture medium by co-hydrolysis, the system comprising: (a) at least one culture medium tank configured for preparation of the culture medium, wherein the culture medium tank comprises at least one of a mixing tank, a hydrolysis tank, a storage tank, at least one loading tank, and a waste medium tank;(b) a hydrolysis tank configured to perform enzymatic co-hydrolysis by receiving and combining at least two protein sources and exogenously supplied enzymes,wherein the first protein source comprises a plant-based protein source, and the second protein source comprises at least one endogenously produced enzyme;c) a cultivation device for cultivating non-human metazoan cells in the culture medium prepared from a protein hydrolysate produced by enzymatic co-hydrolysis.

24. A cultivation system of claim 23, wherein the at least one loading tank is configured to provide the first protein source, the second protein source, exogenously supplied enzymes, or a combination thereof.

25. A cultivation system of claim 23, wherein the protein hydrolysate prepared by enzymatic cohydrolysis is sterilized by at least one sterilization unit.

26. A method of a solid-state fermentation and hydrolysis, comprising:(a) providing a first protein source;(b) mixing the first protein source with water in amount of 0.3 mL / g to 4 mL / g and an inoculum comprising a second protein source to form a solid-state mixture;(c) incubating the solid-state mixture;(d) allowing endogenously produced enzymes from the second protein source present in the inoculum to act in situ on the solid protein source to achieve partial hydrolysis of the first protein source; and (e) adding exogenously supplied enzymes to the solid-state mixture to finish a hydrolysis process, thereby generating a protein hydrolysate.

27. The method according to claim 24, wherein the inoculum of the second protein source is provided in the form of a liquid suspension, a semi-solid form, a concentrated biomass slurry, and / or a dried or lyophilized powder.

28. The method according to claim 24, wherein the second protein source is subjected to at least one physico-chemical condition and / or nutrient composition during fermentation to induce the production of at least one endogenously produced enzyme, wherein the physico-chemical condition and / or nutrient composition include a specific temperature, pH, oxygen level, nitrogen source, a carbon source, a phosphate source composition, a ratio of carbon to nitrogen source, a ratio of nitrogen to phosphate source, or a ratio of carbon to phosphate source.

29. The method according to claim 24, wherein the production of endogenously produced enzymes by the second protein source is nutritionally triggered prior to mixing with the first protein source.

30. The method according to claim 24, wherein the at least one exogenously supplied enzyme is selected from endopeptidase, exopeptidase, beta-glucanase or an enzyme having phytase activity.

31. The method according to claim 1, wherein the solid-state mixture has a moisture content in the range of 10 % to 40 % (w / w).

32. The method according to claim 1, wherein the solid-state mixture is incubated in a temperature range of 15 °C to 60 °C for a period of 6 hours to 7 days under controlled humidity and aeration conditions suitable for the growth of the second protein source and enzyme secretion.

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