Heat-induced meat flavors

Abstract

This disclosure describes methods for improving umami and meaty flavors of cell-based food products. In particular, the disclosed method includes an amino acid integration method where cultured cells and exogenous amino acids (e.g., glutamic acid and aspartic acid) are combined and heated, resulting in a synergistic improvement of umami flavors. The disclosed method further includes a browning method by which a cell suspension is dehydrated and heated at high temperatures (e.g., greater than 140C) to induce non-enzymatic browning and caramelization reactions in the cell suspension. The cell-based flavoring product can be combined with a food product as a flavor, aroma, and color enhancer. The disclosed methods may use one or both of the amino acid integration method and the browning method to improve organoleptic properties of cell-based food products.

Description

HEAT-INDUCED MEAT FLAVORSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 659,658, filed on June 13, 2024, which is incorporated herein by reference in its entirety.BACKGROUND

[0002] As the world’s population continues to grow, cell-based or cultured food products (e.g., cell-based meat products) for consumption have emerged as an attractive alternative (or supplement) to conventional meat from animals. For instance, cell-based, cultivated, or cultured meat represents a technology that could address the specific dietary needs of humans. Cell-based food products can be prepared from a combination of cultivated adherent and suspension cells derived from a non-human animal. Because the cells for cell-based meat are made in a food cultivation facility, cell masses are often formed and shaped to mimic familiar forms of conventional meat.

[0003] In addition to addressing dietary needs, cell-based food products help alleviate several drawbacks linked to conventional food products for humans, livestock, and the environment. For instance, conventional meat production involves controversial practices associated with animal husbandry, slaughter, and harvesting. Other drawbacks associated with harvested or slaughtered meat production include low conversion of caloric input to edible nutrients, microbial contamination of the product, emergence and propagation of veterinary and zoonotic diseases, relative natural resource requirements, and resultant industrial pollutants, such as greenhouse gas emissions and nitrogen waste streams.

[0004] Despite advances in creating cell-based food products, existing methods or systems for cultivating and processing cell-based food products face several shortcomings, such as challenges or failures to mimic the organoleptic properties (e.g., taste, aroma, appearance, etc.) of slaughtered or harvested meat. Some existing methods or systems often produce cell-based food products with undesirable taste or that taste different than slaughtered meats. For instance, existing systems often face significant challenges in achieving the complex flavor profile of slaughtered meat. In particular, existing systems often grow cells lacking naturally occurring flavor compounds found in slaughtered meat. Existing systems may produce cells that lack the distinct umami flavors found in slaughtered meats. In another example, some cells grown using existing methods often lack the same composition of compounds that contribute significantly to the flavor of slaughtered meat during cooking.

[0005] In addition to taste challenges, existing methods or systems of forming cell-based or cultured meat often form cell-based meats that appear less appealing than slaughtered meat. Due, in part, to the deficiency of naturally occurring compounds found in slaughtered meats, cells grown using existing methods may fail to brown or exhibit reduced browning during cooking. For example, slaughtered meat contains a well-developed structure of muscle fibers and proteins, which contribute to the Maillard reaction that contributes to browning and additional flavor. In contrast, many existing cell-based meats lack sufficient protein and muscle fiber density, leading to a reduced or uneven Maillard reaction. The difference in composition between existing cell-based meat and slaughtered meats often results in cell-based meat appearing paler and less appetizing when cooked.

[0006] Moreover, several existing methods or systems of forming cell-based or cultured meat often produce cells that smell less appealing or less familiar than slaughtered meat. More specifically, due to differences in composition and the absence or presence of certain volatile compounds, existing cell-based meats often lack the same levels of specific compounds required for aromatic reactions during cooking. Thus, many existing cell-based meats can be less intense and complex and lacking savory notes that make cooked slaughtered meat appealing.

[0007] These, along with additional problems and issues exist in existing methods for cultivating cell-based food products.BRIEF SUMMARY

[0008] This disclosure generally describes methods for improving the umami and meaty flavors in cell-based-meat food products. This disclosure describes two methods that, individually or combined, enhance the flavor, aroma, and color of cell-based-meat food products. In particular, this disclosure describes an amino acid integration method by which amino acids (e.g., aspartic acid and glutamic acid) are introduced to cultivated cells. The amino acid integration method includes heating the mixture to create an umami taste through heat-induced cleavage, degradation, and regeneration of peptide bonds between exogenous amino acids and endogenous cell components. This disclosure further describes a browning method by which cultivated cells are heated to induce non-enzymatic Maillard and caramelization reactions. More specifically, cells grown in suspension can be moisture adjusted to a target concentration then heated at target times and temperatures to produce a meat-flavored cell-based flavoring product. The cell-based flavoring product can be incorporated as a flavor and aroma enhancer to food products.

[0009] Additional features and advantages of one or more embodiments of the present disclosure will be set forth in the description that follows.BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Various embodiments will be described and explained with additional specificity and detail through the use of the accompanying drawings, which are summarized below.

[0011] FIG. 1 illustrates an overview of combining non-human cultivated animal cells and exogenous amino acids resulting in a synergistic improvement of umami flavor in accordance with one or more embodiments of the present disclosure.

[0012] FIG. 2 illustrates the effects of varying concentrations of glutamic acid and aspartic acid in amino acid blends in accordance with one or more embodiments of the present disclosure.

[0013] FIG. 3 illustrates an overview of forming a cell-based flavoring product comprising heating a cell suspension using a non-enzymatic browning reaction in accordance with one or more embodiments of the present disclosure.

[0014] FIG. 4 illustrates results of a technical tasting reflecting improved meaty flavors when the cell-based flavoring product was mixed with water in accordance with one or more embodiments of the present disclosure.

[0015] FIG. 5 illustrates results of a technical tasting reflecting improved meaty flavors when the cell-based flavoring product was mixed with baked cell suspension in accordance with one or more embodiments of the present disclosure.

[0016] FIG. 6 illustrates results of a technical tasting reflecting improved meaty flavors when the cell-based flavoring product was mixed into a chopped and formed patty in accordance with one or more embodiments of the present disclosure.

[0017] FIG. 7 illustrates an overview of a combined method that incorporates the amino acid integration method and browning method in accordance with one or more implementations of the present disclosure.

[0018] FIG. 8 illustrates a series of acts for combining an amino acid blend with non-human cultivated animal cells to synergistically improve umami flavor in accordance with one or more embodiments of the present disclosure.

[0019] FIG. 9 illustrates a series of acts for creating a cell-based flavoring product to improve meaty flavors in accordance with one or more embodiments of the present disclosure.

[0020] FIG. 10 illustrates a series of acts for combining an amino acid blend with non-human cultivated animal cells and subsequently heating the mixture to a browning temperature to create a cell-based flavoring product in accordance with one or more embodiments of the present disclosure.

[0021] FIGS. 11A-11D illustrate an overview diagram of growing and processing different types of cells in accordance with one or more embodiments of the present disclosure.DETAILED DESCRIPTION

[0022] This disclosure describes one or more implementations of a method for imparting stronger umami and savory flavors to cell-based food products. In one or more embodiments, the disclosed method comprises adding exogenous compounds, such as aspartic acid and glutamic acid, to non-human cultivated animal cells. The disclosed method further comprises heating the nonhuman cultivated animal cells and the exogenous compounds to stimulate various endogenous cell components interacting with the exogenous compounds to create an umami taste. The disclosed method further includes a browning method to create a meaty flavor compound by dehydrating and heating non-human cultivated animal cells at high temperatures to induce non-enzymatic Maillard and caramelization reactions via inherent reactants in cultivated meat suspensions. The meaty flavor compound can be reintroduced to cultivated meat food products to enhance the flavor, aroma, and color of the cultivated meat food products.

[0023] As indicated above, the disclosed method may comprise an amino acid integration method. The amino acid integration method may comprise formulating a cell mixture by combining an amino acid blend with non-human cultivated animal cells and heating the cell mixture to a peptide-forming temperature for a peptide-forming time. Additionally, the disclosed method may comprise a browning method. In one or more embodiments, the browning method comprises moisture adjusting non-human cultivated animal cells to a target cell concentration and heating the non-human cultivated animal cells to a browning reaction temperature for a browning reaction time, wherein the non-human cultivated animal cells are heated without fat. The browning method can further comprise incorporating the cell-based flavoring product into a cell mass. The disclosed method may use one or both of the amino acid integration method and the browning method to enhance the meat flavor, aroma, and appearance of cell-based food products.

[0024] As mentioned, the disclosed method can use an amino acid integration method to improve the umami flavor of cultivated cells. In particular, the amino acid integration method introduces free amino acids in combinations at different percentages to non-human cultivated animal cells. For instance, the free amino acids can be introduced to non-human cultivated animal cells in suspension. The amino acid integration method can further include subjecting the non- human cultivated animal cells and the amino acids to a heating treatment to induce the umami peptide formation by heat-induced cleavage, degradation, and regeneration.

[0025] Additionally, and as mentioned, the disclosed methods can include a browning method. In particular, the browning method can include moisture adjusting a cell suspension to a target cell concentration. For example, the browning method can include using various techniques to increase the percentage of total solids of non-human cultivated animal cells relative to a cell suspension. Additionally, the browning method can include heating the cell suspension to abrowning reaction temperature to form a heated cell suspension that can be used as a flavoring compound. In some examples, the browning reaction temperature is high (e.g., equal to or greater than 140C) to induce non-enzymatic browning. The heated cell suspension can be milled or powdered to generate a cell-based flavoring product.

[0026] Furthermore, the disclosed method can use a combination of the amino acid integration method and the browning method to intensify desirable flavors in a cell-based flavoring product. In particular, the disclosed method can comprise formulating a cell mixture by combining an amino acid blend with non-human cultivated animal cells and heating the cell mixture to a peptide-forming temperature. The disclosed method further comprises moisture adjusting the cell mixture to a target cell concentration and heating the cell mixture to a browning reaction temperature to form a cell-based flavoring product.

[0027] The disclosed method provides several benefits relative to existing methods for growing cell-based meats. In particular, the disclosed method improves the flavor profile of cultivated meat cells. More specifically, the amino acid integration method introduces amino acids that, together with endogenous cell components, synergistically improve the umami flavor of non- human cultivated animal cells. Additionally, the disclosed methods can introduce enhanced meat flavors by creating a flavor compound comprising heated cell suspension that has undergone non- enzymatic Maillard and caramelization reactions. The flavor compound can be incorporated into a cell mass as an additive to enhance the flavor of the cell-based food product.

[0028] Additionally, the disclosed method can improve the appearance of cell-based food products. In contrast to existing systems that produce cultivated meats that appear pale when cooked, the disclosed method can introduce the cell-based flavoring product to a cell-based food product. More specifically, the cell-based flavoring product produced by the browning method can be substantially evenly incorporated throughout a cell-based food product to improve the color of the cell-based food product. Furthermore, when cooked, the cell-based food product can exhibit more even browning with the incorporation of the cell-based flavoring product.

[0029] The disclosed method can further improve the aroma of cell-based food products. In particular, by introducing amino acids and other compounds to the non-human cultivated animal cells and heating the mixture, the disclosed method can improve the composition of cultured cells. Furthermore, the disclosed methods can create a flavoring compound comprising a heated cell suspension that has undergone a browning process that intensifies the meaty aromas of the heated cell suspension. The improved composition of the non-human cultivated animal cells together with aromatic heated cell suspension can impart a more intensely meaty and complex aroma to the cellbased food product.

[0030] As illustrated by the foregoing discussion, the present disclosure utilizes a variety of terms to describe features and advantages of the disclosed method. Additional detail is now provided regarding the meaning of such terms. As used herein, the term “cells” (or “non-human cultivated animal cells”) refers to cells that form food products (e.g., meat products). Generally, non-human cultivated animal cells may comprise at least one of muscle cells, muscle progenitor cells, or muscle support cells. In particular, non-human cultivated animal cells may comprise different cell types, such as one or more of myoblasts, mesoangioblasts, myofibroblasts, mesenchymal stem cells, hepatocytes, fibroblasts, pericytes, adipocytes, epithelial cells, chondrocytes, osteoblasts, osteoclasts, pluripotent cells, somatic stem cells, endothelial cells, embryonic stem cells, induced pluripotent stem cells, or other similar cell types. Furthermore, cells may comprise different types of progenitor cells, including myogenic progeny and progenitors, adipogenic progeny or progenitors, mesenchymal progeny or progenitors, or other types of progenitor cells. In some instances, the cells may comprise cells from distinct lineages, such as ectoderm or endoderm lineages, that have been transdifferentiated into cells useful for forming a cell-based food product for consumption, such as those cell types described above. In some embodiments, the disclosed method includes seeding telomerase reverse transcriptase (TERT) immortalized chicken fibroblasts or other immortalized cells, spontaneously immortalized or otherwise.

[0031] As used herein, the term “suspension culture” (or “cell suspension”) refers to cells growing in an at least partially liquid growth medium in which cells grow, multiply, and / or maintain nourishment. In particular, a suspension includes an agitated growth medium that is housed in a container in which single cells or small aggregates of cells grow, multiply, and / or maintain nourishment from the nutrients of the agitated growth medium. Cells grown in suspension are not attached to a substrate and therefore differ from an adherent culture.

[0032] Also, as used herein, the terms “cell culture media” or “culture media” refer to a liquid or gel comprising compounds that support the growth of cells. In particular, cell culture media comprises sources of energy and compounds to regulate the cell cycle. For example, a cell culture media can contain amino acids, vitamins, inorganic salts, glucose, dissolved gases, serum, growth factors, hormones, and attachment factors. The cell media may also help maintain pH and osmolarity during cell growth and proliferation.

[0033] As used herein, the term “cell mass” refers to a mass comprising cells of meat. In particular, a cell mass refers to cells of cultured meat gathered into a collective mass. As discussed below, a cell mass may comprise different cell types, such as one or more of myoblasts, mesangioblasts, myofibroblasts, mesenchymal stem cells, hepatocytes, fibroblasts, pericytes, adipocytes, epithelial, chondrocytes, osteoblasts, osteoclasts, pluripotent cells, somatic stem cells,endothelial cells, or other similar cell types. For example, a cell mass can include a cell sheet of cultured meat growing within an enclosure, such as a chamber, housing, container, etc.

[0034] As used herein, the term “acid” refers to a chemical substance that can donate a proton (H+ion) or form a covalent bond with an electron pair in a reaction. Acids are characterized by their ability to increase the concentration of hydrogen ions (H+) in an aqueous solution, enabling pH values less than 7. Common properties of acids include a sour taste, the ability to turn blue litmus paper red, and reactivity with bases to form salts and water. Acids play a critical role in various chemical reactions and industrial processes.

[0035] As used herein, the term “food acid” refers to acids that occur naturally in foods or are added to them to impart a sour or tart taste, act as preservatives, or maintain / modify the pH balance. Food acids are generally safe for consumption and play important roles in food processing and flavoring. Example food acids include citric acid, acetic acid, lactic acid, tartaric acid, malic acid, phosphoric acid, ascorbic acid, fumaric acid, sorbic acid, and benzoic acid.

[0036] As used herein, the term “amino acid” refers to organic compounds that constitute the fundamental building blocks of proteins. An amino acid molecule comprises an amino group (-NH2) and a carboxyl group (-COOH), along with a distinct side chain (R group) that imparts unique properties and functions to the amino acid. Amino acids, commonly incorporated into proteins, plays a crucial role in various biological processes, including protein synthesis, enzymatic activity, and metabolic pathways. Amino acids are categorized into essential amino acids, which must be acquired through dietary intake, and non-essential amino acids, which can be synthesized endogenously by the organism. Example amino acids include alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys), glutamic acid (Glu), glutamine (Gin), glycine (Gly), histidine (His), isoleucine (He), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), and tryptophan (Trp).

[0037] As further used herein, the term “glutamic acid” refers to an amino acid. For example, glutamic acid can be the hydrochloride salt of glutamic acid (C5H9NO4 HCL). In some cases, glutamic acid improves the umami flavor.

[0038] As used herein, the term “peptide-forming temperature” refers to a temperature or range of temperatures at which amino acids effectively bond to form peptides. Peptides comprise chains of amino acids linked by peptide bonds. In particular, the term peptide-forming temperature refers to a temperature or range of temperatures that facilitates the coupling reactions between amino acids while preserving the integrity of the reactants. In some examples, the peptide-forming temperature comprises a temperature between 20C and 100C. Relatedly, as used herein, the term “peptide-forming time” refers to a duration required for amino acids to bond and form peptides ata peptide-forming temperature. For example, a peptide-forming time may comprise 10, 15, 30, or more minutes.

[0039] As used herein, the term “browning reaction temperature” refers to a temperature or range of temperatures at which browning occurs. In particular, browning reaction temperature can comprise a range of temperatures at which the Maillard reaction occurs. The Maillard reaction comprises a chemical reaction between amino acids and reducing sugars to produce a variety of flavor compounds, aromas, and brown pigments. In some examples, the browning reaction temperature is a temperature that results in the formation of melanoidins, which are responsible for brown coloring. For example, a browning reaction temperature typically begins around 140C to 165C. Relatedly, as used herein, the term “browning reaction time” refers to the duration required for a browning reaction to occur. More particularly, browning reaction time refers to a time required for the browning reaction for non-human cultivated animal cells to reach a desired level of browning, flavor, and aroma.

[0040] As used herein, the term “heated cell suspension” refers to a browned cell suspension used to impart or enhance organoleptic properties of a cell-based food product. In particular, heated cell suspension comprises non-human cultivated animal cells that enhance the taste, appearance, smell, or other properties of a cell-based food product. For example, a heated cell suspension may comprise non-human cultivated animal cells grown in suspension or on a substrate that have undergone a heating process to have more intense meat-like characteristics. For example, a heated cell suspension can be formed by applying a Maillard reaction to a cell suspension. In some examples, the heated cell suspension has previously been moisture adjusted such that the heated cell suspension has a dry texture. A heated cell suspension can be further milled into a powdered heated cell suspension and incorporated within a cell mass.

[0041] As used herein the term “fat” refers to any ester of fatty acids. In particular, fats can refer to oils or fatty tissues that can be used in cooking. For example, fat can refer to vegetable oils used for cooking (e.g., canola oil, olive oil, avocado oil, etc.) and animal fats used for cooking (e.g., butter, lard, tallow, ghee, etc.).

[0042] As used herein, the term “cell-based food product” refers to a food product comprising non-human animal cells grown in vitro. For instance, the cell-based food product can include isolated cells from animals combined with other ingredients or additives such as, but not limited to, plant proteins, salts, flavorings, acids. Such products are interchangeably referred to as in vitro meat product, in vitro food product, lab grown meat, cultured meat, cultured food, or slaughter free meat, depending on context.

[0043] Additional detail will now be provided regarding disclosed methods in relation to illustrative figures portraying example embodiments and implementations of the disclosed methodsand apparatuses. As mentioned previously, the disclosed method can include an amino acid integration method by which peptide bonds are formed between exogenous amino acids and endogenous cell compounds to enhance the umami flavor of non-human cultivated animal cells. FIG. 1 illustrates an overview of combining cultured cells and exogenous amino acids resulting in a synergistic improvement of umami flavor in accordance with one or more embodiments of the present disclosure. As shown in FIG. 1, the disclosed method performs a series of acts 100 to enhance umami flavor in a cell-based food product. By way of overview, the series of acts 100 includes an act 102 of combining an amino acid blend with non-human cultivated animal cells, an act 104 of heating a cell mixture to a peptide-forming temperature for a peptide-forming time, an act 106 of combining the flavor-enhanced cell mixture with a cell mass, and an act 108 of moisture adjusting the flavor-enhanced cell mixture to form the cell-based food product.

[0044] Umami is one of the five basic tastes, alongside sweet, sour, bitter, and salty. Umami is often described as a savory or meaty flavor and is primarily attributed to the presence of the amino acid glutamate — or glutamic acid — as well as nucleotides inosinate and guanylate. The disclosed methods can include an amino acid integration method by which amino acids are integrated and / or added to cultivated cells to impart an umami flavor of the cultivated cells. Examples of high umami food products include mushrooms, chicken broth, and soy sauce.

[0045] As shown in FIG. 1, the disclosed method comprises the act 102 of combining an amino acid blend with non-human cultivated animal cells. In some implementations, the act 102 comprises preparing an amino acid blend. The amino acid blend can comprise a solvent, one or more amino acids, and additional compounds. In particular, the solvent comprises a substance that dissolves one or more solutes to form a solution. In some implementations, the solvent comprises water, cell culture media, broth, or another fluid. Furthermore, in one or more implementations, the amino acid blend does not include a solvent. For example, the amino acid blend can comprise a solid or powdered blend that is added and mixed into non-human cultivated animal cells to form a cell mixture.

[0046] The amino acid blend further comprises one or more amino acids. For example, amino acids may comprise different concentrations of aspartic acid, glutamic acid, or sulfur- containing amino acids (e.g., methionine, cysteine, homocysteine, and taurine). Additionally, and as mentioned, the amino acid blend can comprise additional compounds. For instance, in some implementations, the amino acid blend further includes ketoses, which are monosaccharides that contain ketones within their molecular structures. In still further implementations, the amino acid blend includes one or more additional non-amino acids. For example, in one or more implementations, the amino acid blend includes one or more food acids, such as citric acid or lactic acid.

[0047] In some implementations, the disclosed method comprises preparing the amino acid blend prior to combining the amino acid blend with the non-human cultivated animal cells. For example, in some implementations, the amino acid blend is prepared by mixing components of the amino acid blend in predetermined percentages. The percentages provided when describing the amino acid blend are percentage by weight of the cell mixture. In one or more implementations, the amino acid blend comprises between 0.1 percent by weight and 5.0 percent by weight of the cell mixture. In alternative implementations, the amino acid blend comprises between 0.3 percent by weight and 4.0 percent by weight of the cell mixture. In still further implementations, the amino acid blend comprises between 0.5 percent by weight and 3.0 percent by weight of the cell mixture.

[0048] The amino acid blend can comprise different compounds. As illustrated in FIG. 1, an amino acid blend can comprise a combination of aspartic acid (Asp) and glutamic acid (Glu). In some implementations, the amino acid blend contains only one of aspartic acid or glutamic acid. The disclosed methods may comprise forming an amino acid blend containing different concentrations of glutamic acid and aspartic acid. For instance, the amino acid blend can comprise a first concentration by weight of aspartic acid and a second concentration by weight of glutamic acid. In some embodiments, the concentrations of aspartic acid and glutamic acid are equal within the amino acid blend. For example, an amino acid blend may comprise 0.5% aspartic acid and 0.5% glutamic acid. In other embodiments, the amino acid blend contains concentrations of aspartic acid and glutamic acid that are different from each other. For instance, an amino acid blend may contain a higher concentration of aspartic acid and a lower concentration of glutamic acid, or vice versa. For instance, an amino acid blend may contain 0.25% aspartic acid and 1.5% glutamic acid. In one or more embodiments, the amino acid blend comprises between 0.1% to 2.5% aspartic acid and between 0.1% to 2.5% glutamic acid. FIG. 2 illustrates the effects of varying concentrations of glutamic acid and aspartic acid in amino acid blends in accordance with one or more embodiments of the present disclosure.

[0049] As further shown in FIG. 1 , in some implementations, the amino acid blend comprises a combination of ketoses and sulfur-containing amino acids. Ketoses comprise monosaccharaides with a ketone group. Fructose is an example of a ketose. Ketoses, when combined with sulfur- containing amino acids — such as methionine, cysteine, homocysteine, and taurine — can significantly enhance the umami flavor in cultivated cells. The combination of ketoses and sulfur- containing amino acids can impart an umami flavor to cultivated cells. In some examples, the combinations of ketoses and sulfur-containing amino acids enhances umami flavors through the Maillard reaction during heating. The amino acid blend can comprise different concentrations of ketoses and sulfur-containing amino acids. For instance, the amino acid blend can compriseconcentrations of ketoses between 0.25% to 1.5% ketoses and 0.25% to 1.5% sulfur-containing amino acids.

[0050] As shown in FIG. 1, the disclosed method comprises combining cells 116 with an amino acid blend. The cells 116 comprise non -human cultivated animal cells in suspension or as adherent cells. In one or more embodiments, the cells 116 are grown in a culture medium. In some examples, the cells 116 comprise cells that have been grown and harvested. For instance, the cells 116 may comprise cells that have been removed from cell culture media and washed. In some examples, the cells 116 are resuspended in a buffer or media meant for human consumption.

[0051] The cells 116 contain endogenous cell components that form peptide bonds with amino acids in the amino acid blend. For example, in some implementations, the cells 116 comprise purine-5’ -nucleotides. Purine-5’ -nucleotides comprise organic molecules that consist of a purine base attached to a ribose sugar, which in turn is linked to a phosphate group at the 5’ position of the sugar. Examples of purine-5 ’-nucleotides include adenosine-5’-monophosophate (5 ’-AMP), inosine-5’ -monophosphate (5 ’-IMP), and guanosine-5 ’-monophosphate (5’-GMP).

[0052] As illustrated in FIG. 1, the disclosed method includes the act 104 of heating a cell mixture to a peptide-forming temperature for a peptide-forming time. In particular, the disclosed method comprises heating the cell mixture to induce the umami peptide formation through heat- induced cleavage, degradation, and regeneration. The peptide-forming temperature comprises a range of temperatures at which exogenous amino acids in the amino acid blend bond with endogenous cell components (e.g., purine-5 ’-nucleotides). For example, the peptide-forming temperature can comprise a temperature or range of temperatures between 20C and 100C. More specifically, in one or more embodiments, the peptide-forming temperature is between 85C and 95C. For instance, the peptide-forming temperature can equal 90C. In some examples, exogenous amino acids included in an amino acid blend may also be present within the cells.

[0053] Additionally, the cell mixture can be heated for a peptide-forming time. The peptide- forming time refers to the duration at which the cell mixture must be held at the peptide-forming temperature for exogenous amino acids to form bonds with endogenous cell components. For example, the peptide-forming time can comprise 10, 15, 30, 90, 120, or more minutes. In some implementations, and as shown in FIG. 1, the cell mixture can be stirred during the peptide-forming time to facilitate peptide formation.

[0054] As explained above, heating of the cell mixture (the combination of the amino acid blend and the non-human cultivated animal cells) at the peptide-forming temperature for the peptide forming time, synergistically generates an umami flavor in the cell mixture. Furthermore, synergistically generating the umami flavor in the cell mixture also reduces the metallic / livery taste common to cultivated meat cells. In other words, heating of the cell mixture at the peptide-formingtemperature for the peptide forming time produces a flavor-enhanced cell mixture. The flavor- enhanced cell mixture can be used to create a cell-based food product as explained below.

[0055] In addition to the synergically created flavor (umami plus less metallic / livery taste), heating of the cell mixture at the peptide-forming temperature for the peptide forming time produces a color more consistent with a meat-based broth. For example, in connection with nonhuman cultivated chicken cells, the flavor-enhanced cell mixture had a whiter color consistent with chicken broth compared to the cells prior adding of the amino acid blend and heating.

[0056] As mentioned above, in some embodiments, the disclosed methods include utilizing the flavor-enhanced cell mixture to improve the flavor of a cell-based food product. For example, FIG 1 illustrates the optional act 106 of combining the flavor-enhanced cell mixture with a cell mass. In particular, the flavor-enhanced cell mixture can be used as a flavoring compound to flavor a cell mass. For example, a flavor-enhanced cell mixture 110 is added to a cell mass 112 to boost the umami flavor of the cell mass 112. The flavor-enhanced cell mixture 110 comprises the cells as well as all components of the amino acid blend. The disclosed method uses the combined flavor- enhanced cell mixture 110 and the cell mass 112 as the cell-based food product.

[0057] In some implementations, instead of using the flavor-enhanced cell mixture as a flavoring compound, the act 106 uses cells in the flavor-enhanced cell mixture as a base for creating a cell-based food product. In particular, the disclosed method comprises the act 108 of moisture adjusting the flavor-enhanced cell mixture to form the cell-based food product. In embodiments where the cells 116 comprise cells in suspension, the act 106 removes the media or other suspension fluid to obtain a cell mass 114. In some embodiments, the disclosed method comprises washing the flavor-enhanced cell mixture 110 to remove compounds, such as excess amino acid blend compounds, from the cells.

[0058] As mentioned, the disclosed method may comprise combining different combinations of aspartic acid and glutamic acid to achieve different sensory goals. FIG. 2 illustrates different concentrations of glutamic acid and aspartic acid influencing the flavor of cell-based food products in accordance with one or more embodiments of the present disclosure. FIG. 2 illustrates a table 200 showing the correlation between sensory notes and varying concentrations of glutamic acid and aspartic acid when heated with cells grown in suspension. The combination of cultured cells and exogenous amino acids result in a synergistic improvement of meaty flavor.

[0059] FIG. 2 shows flavor notes for combinations of suspension cells, glutamic acid, and aspartic acid. The concentrations of glutamic acid and aspartic acid are shown relative to the total volume of the cell mixture (i.e., the amino acid blend combined with the cells). The concentrations of glutamic acid and aspartic acid can indicate the percentage of the cell mixture by weight or volume. In samples treated using the amino acid integration method, higher concentrations ofglutamic and aspartic concentrations were associated with decreased culture cell off flavors. Specifically, cells grown in suspension often have metallic or liver-like flavors. As shown, a sample containing suspension cells, 0.25% glutamic acid, and 0.25% aspartic acid results in a synergistic savory flavor. However, lower concentrations of glutamic and aspartic acids also correspond with a stronger metallic aftertaste. A sample containing suspension cells, 0.5% glutamic acid, and 0.5% aspartic acid results in the cells having a savory flavor with some sourness with reduced metallic taste. A sample containing suspension cells, 1.5% glutamic acid, and 1.5% aspartic acid results in the cells having a savory flavor with some sourness and reduced cultured cell off flavors.

[0060] As mentioned, the disclosed method can include a browning method to impart stronger meat aroma and flavors to non-human cultivated animal cells. FIG. 3 illustrates an overview of forming a flavoring compound comprising a heated cell suspension for incorporation into a cell mass by using a non-enzymatic browning reaction in accordance with one or more embodiments of the present disclosure. By way of overview, FIG. 3 illustrates a series of acts 300 comprising an act 302 of moisture adjusting a cell suspension, an act 304 of heating the cell suspension to a browning reaction temperature for a browning reaction time, and an act 306 of comminuting the browned cell suspension to generate a cell-based flavoring product.

[0061] As shown in FIG. 3, the disclosed method comprises the act 302 of moisture adjusting a cell suspension. The disclosed method comprises moisture adjusting a cell suspension comprising cells to a target cell concentration. For example, cells within the cell suspension comprise cells grown in suspension. The cells have been separated from growth media, and in some embodiments, washed. The disclosed method comprises an optional conditioning step of moisture adjusting the cell suspension to remove excess moisture from growth media, washing media, nutrient media, or other types of media. For example, the cell suspension can be moisture adjusted using vacuum concentration, rotary evaporator (rotovap), belt-spray drying, air drying, spray drying, centrifugal evaporation, or other techniques. In another embodiment, the act 302 of moisture adjusting cell suspension comprises drying the cell suspension to a powder and then reconstituting the powdered cell suspension to an appropriate moisture content for heating and further processing.

[0062] As just mentioned, the disclosed method comprises moisture adjusting the cell suspension to a target cell concentration. More specifically, the disclosed method increases the concentration of cells relative to a total volume or weight of the cell suspension. For example, in one or more implementations, the initial cell suspension comprises between 1% to 20% total solids (e.g., cells) by weight. In one or more implementations, the initial cell suspension comprises between 4% to 8% total solids by weight. In some examples, the target concentration comprises a concentration of 10%-50% total solids by weight or volume. Thus, in one or more implementations,the act 302 of moisture adjusting a cell suspension comprises at least doubling a concentration of cells in the cell suspension by removing moisture (e.g., water) from the cell suspension.

[0063] Additionally, in some examples, the target concentration comprises a concentration of 15% to 40% total solids by weight relative to the cell suspension. In experiments, cell suspensions that have been adjusted to 10%-l 5% total solids by weight scored high in pleasant browning smells. In some embodiments, the disclosed method moisture adjusts the cells at low temperatures, for example, below 65C to avoid denaturing the cells. In yet other examples, the target concentration comprises a concentration of 20% to 40% total solids by weight relative to the cell suspension. Higher percentage total solids by weight (e.g., greater than 30%) begin exhibiting browning characteristics and may still have pleasant browning smells. However, higher than 40% solids by weight began to test as having metallic or burnt smells.

[0064] As shown in FIG. 3, the disclosed method comprises the act 304 of heating the cell suspension to a browning reaction temperature for a browning reaction time. In particular, the disclosed method comprises heating the cell suspension to induce the Maillard reaction and develop flavor in the cells. In some examples, the browning reaction temperature comprises a temperature between 150C and 190C. More specifically, in some implementations the browning reaction temperature comprises a temperature between 160C and 180C. In yet another implementation, the browning reaction temperature comprises a temperature between 165C and 177C.

[0065] The cell suspension is kept at the browning reaction temperature for a browning reaction time. The browning reaction time comprises a duration required for the cell suspension to reach a target level of browning. For example, the browning reaction time can comprise a duration of between 5 to 120 minutes. More specifically, in some implementations the browning reaction time can comprise a duration of between 10 to 60 minutes. In yet another implementation, browning reaction time can comprise a duration of between 10 to 40 minutes. In a still further implementation, browning reaction time can comprise a duration of between 30 to 40 minutes.

[0066] In some implementations, different browning reaction temperatures and browning reaction times can be used to achieve different flavor profiles. For example, the disclosed method can develop a roast chicken flavor in the cell-based flavoring product by heating the cell suspension to a browning reaction temperature between 140C to 173C for a browning reaction time between 20 to 30 minutes. Additionally, or alternatively, the disclosed method imparts a tangy, cheesy, or parmesan flavor to the cell-based flavoring product by heating the cell suspension to a browning reaction temperature between 145C to 165C for 12 to 18 minutes. As a particular example, the browning reaction temperature lower than 163C may lead to increased parmesan flavor.

[0067] The temperature and duration of heating may be adjusted and / or optimized to control the degree of Maillard reaction, thus controlling the intensity and type of flavor of the cell-basedflavoring product. For example, the heating temperature and duration may be optimized for each combination of animal cell type to maintain maximum aroma and umami in the cell-based flavoring product or provide the desired intensity and type of flavor.

[0068] The disclosed method may use several techniques to heat the cell suspension to the browning reaction temperature for the browning reaction time. For instance, and as illustrated in FIG. 3, the disclosed method may heat the cell suspension using an oven. More specifically, in some embodiments, the disclosed method includes using a convection oven with a fan to heat or cook the cell suspension. Additionally, in some implementations, the disclosed method comprises heating the cell suspension by conduction heating. For instance, the cell suspension may be heated to the browning reaction temperature by pan frying the cell suspension. The disclosed methods can include other techniques for heating the cell suspension to the browning reaction temperature. For instance, the disclosed method may include heating the cell suspension by microwaving, grilling, or other heating techniques.

[0069] In addition to adjusting the browning reaction temperatures and browning reactions times, the disclosed method can modify pH, oven fan speed, ionic strength, and additives to the cell suspension to increase or modify flavor intensity. To illustrate, the following table includes various browning reaction heating parameters that yield heated cell suspensions with a desirable chicken taste.

[0070] The disclosed method can include heating the cell suspension to the browning reaction temperature with or without fats. Heating the cell suspension to the browning reaction temperature with fats often results in undesirable organoleptic properties. For instance, heating the cell suspension by pan frying the cell suspension in canola oil for 1 to 2 minutes resulted in stronger cultured cell off flavors and undesirable textures when compared with pan frying the cell suspension without oil. Furthermore, pan frying the cell suspension in canola oil for a longer duration (e.g., 2 to 3 minutes) resulted in more desirable texture, however the cultured cell offflavors persisted. Additionally, when the cell suspension was pan fried with more oil (e.g., 4 tablespoons versus 1 tablespoon), the heated cell suspension had a burnt appearance and taste.

[0071] Heating the cell suspension by baking the cell suspension in the presence or absence of fats yielded similar results to those described above. More specifically, the heated cell suspension, when baked with 10% oil, was less crispy and had a less intense meaty flavor when compared to cell suspension baked without oil.

[0072] In summary, heating the cell suspension without fats, results in a fuller, meaty flavor. Indeed, in some embodiments, heating the cell suspension without fats, i.e. exogenous fats, synergistically improved the flavor by providing clearer and more distinct meaty flavors. In other words, heating the cell suspension without fats synergistically results in a more intense meaty and savory flavor. This is an unexpected result as typically fats improve the flavor of meat-based food products. The synergistic effects of heating the cell suspension without fats is likely due to the unique properties of suspension-based non-human cultivated animal cells. Additionally, heating the cell suspension without fats synergistically improved the texture by resulting in a crispier product. Thus, in a preferred embodiment, the cell suspension is heated to browning reaction temperatures without the addition of fats. In other implementations, the cell suspension is heated to browning reaction temperatures with the addition of fats, at a cost to the intensity and quality of the flavor, in order to increase the caloric content of the cell-based flavoring product.

[0073] In addition to suspension cells, one or more additional ingredients may be added to alter the flavor or aroma of the cell-based flavoring product. For example, the flavor and aroma of the cell-based flavoring product may be controlled by the addition of food-grade ingredients, thereby altering flavor notes in the cell-based flavoring product. The additional food ingredients may modulate the intensity of meat-specific flavors in the cell-based flavoring product. Suitable food ingredients may include protein ingredients (such as soy protein powder and whey powder), sugars, nucleotides, amino acids, food acids, proteins, fatty acids, salts, synthetic flavors, plantbased proteins, etc.

[0074] As shown in FIG. 3, the disclosed method comprises the act 306 of comminuting the browned cell suspension to generate a cell-based flavoring product. Specifically, as shown in FIG. 3, the browned cell suspension 310 is further moisture reduced due to heating and comprises a film or sheet of browned cell suspension. To produce the cell-based flavoring product 312, act 306 comprises comminuting the browned cell suspension (e.g., reducing the browned cell suspension to particles). In one or more implementations, act 306 involves milling, powdering, pulverizing, or grinding the browned cell suspension. Thus, the cell-based flavoring product 312 can comprise a powder in one or more implementations. In another implementation, the cell-based flavoring product 312 comprises particles with irregular and fragmented geometries as shown in FIG. 3. Theparticles of the cell-based flavoring product 312 can comprise tiny cubes, thin shreds, or small, jagged chunks. Additionally, the particles of the cell-based flavoring product 312 can comprise non-uniform color. In other words, the color of the particles of the cell-based flavoring product 312 can range from light brown, to golden brown, to dark brown, depending upon how long and how thick the cell suspension is when brought to the browning reaction temperature.

[0075] In some embodiments, the cell-based flavoring product 312 is used to enhance the flavor of a cell-based food product. For example, one or more implementations involve mixing the cell-based flavoring product 312 throughout a cell mass of non-human cultivated animal cells to form a final cell-based food product. In some embodiments, the cell-based flavoring product 312 is added as a coating to a cell mass of non-human cultivated animal cells. In some implementations, a combination of methods is performed whereby the cell-based flavoring product 312 is incorporated throughout a cell mass of non-human cultivated animal cells and is also added as a coating to the cell mass.

[0076] The disclosed methods can comprise incorporating different concentrations of the cell-based flavoring product 312 into a cell mass of non-human cultivated animal cells. Generally, greater concentrations of the cell-based flavoring product 312 within the cell mass leads to stronger browned and meat flavoring. In some examples, the cell-based flavoring product 312 comprises between 0.2% to 10% of the cell-based food product by weight.

[0077] As mentioned, in some embodiments, the cell mass comprises non-human cultivated animal cells. The cell mass comprises cells grown in suspension, adherent cells, or a combination. In some examples, the disclosed method comprises adding additional compounds to the cell-based flavoring product 312 and the cell mass to form the cell-based food product. For example, the disclosed method may comprise adding binding agents such as methylcellulose or soy protein isolate. Flavor enhancers like yeast extract, hydrolyzed vegetable protein, or umami-rich compounds such as monosodium glutamate (MSG) can further be added to the cell mass. In some implementations, nutritional additives like vitamins and minerals are added to the cell mass. The disclosed methods can further comprise adding preservatives and colorants to the cell mass.

[0078] In some implementations, the disclosed method further comprises adding a second cell-based flavoring product to a cell mass. As mentioned, cell-based flavoring products can have different flavor profiles (e.g., chicken or cheesy) based on different heating parameters. The disclosed method can include incorporating two or more cell-based flavoring products to the same cell mass to introduce different combinations of flavors to the cell mass. The disclosed methods can further include additional acts of moisture adjusting a second cell suspension to a second target cell concentration, heating the second cell suspension to a second browning reaction temperature for a second browning reaction time, comminuting the browned second cell suspension to form asecond cell-based flavoring product, and incorporating the second cell-based flavoring product into the cell mass.

[0079] In addition to the foregoing, one or more implementations involve using the cellbased flavoring product 312 in a variety of culinary and food production applications to enhance or replicate the taste of meat without using slaughtered meat. For example, one or more implementations involve adding the cell-based flavoring product 312 to plant-based foods to provide a meaty flavor, adding the cell-based flavoring product 312 to broth, soups, or stews to enrich their savory profiles, adding the cell-based flavoring product 312 to sauces or gravies to provide a robust and savory taste, adding a powdered version the cell-based flavoring product 312, to chips, crackers, instant noodles, ready-to-eat meals, canned items etc. to enhance taste and replicate the flavor of meat. Still further implementations involve using the cell-based flavoring product 312 in seasonings, rubs, marinades, or brines.

[0080] Technical tasting tests show improved flavors from the cell-based flavoring product 312 heated to a browning reaction temperature. FIGS. 4-6 illustrate improved flavor profiles resulting from incorporation of the cell-based flavoring product 312 into different mediums in accordance with one or more embodiments of the present disclosure. FIG. 4 illustrates results of a technical tasting when a powdered cell-based flavoring product was mixed with water in accordance with one or more embodiments of the present disclosure. FIG. 5 illustrates results of a technical tasting when powdered cell-based flavoring product was mixed with baked cell suspension in accordance with one or more embodiments of the present disclosure. FIG. 6 illustrates results of a technical tasting when powdered cell-based flavoring product was mixed into a chopped and formed patty in accordance with one or more embodiments of the present disclosure.

[0081] As mentioned, FIG. 4 illustrates results of a technical tasting when powdered cellbased flavoring product was mixed with water. FIG. 4 illustrates a powdered cell-based flavoring product 402 comprising milled cell-based flavoring product. FIG. 4 further includes a bar graph 404 displaying responses to various sensory attributes of 6% powdered cell-based flavoring product in water. As shown in FIG. 4, 100% of tasters noted a browning and chicken skin flavors. 80% of tasters noted roasted chicken and umami flavors. 60% of tasters brothy and chicken fat flavors. As shown, the powdered cell-based flavoring product, even when mixed with water, demonstrated desirable flavor profiles.

[0082] As mentioned, FIG. 5 illustrates results of a technical tasting when powdered cellbased flavoring product was mixed with baked cell suspension. The baked cell suspension had not been moisture adjusted and was simply a liquid cell suspension that was baked. Powdered cellbased flavoring product was added to the baked cell suspension and homogenized at a 6% concentration to form a spiked heated cell substrate solution 502. The control of the technicaltasting comprised a baked cell suspension without the powdered cell-based flavoring product. As demonstrated by horizontal bar graph 504, 80% of tasters preferred the spiked heated cell substrate solution 502 to the control. 60% of tasters noted more browning in the spiked heated cell substrate solution, and 40% of tasters noted that the spiked heated cell substrate solution 502 had less halide and animalic flavors, e.g. barnyard, pet cage, chicken coop, etc. In summary, the spiked heated cell substrate solution 502 intensified certain desirable sensory attributes like browning and roasted chicken while diminishing less desirable attributes such as halide, animalic flavors, and plant-based notes.

[0083] FIG. 6 illustrates results of a technical tasting when powdered cell-based flavoring product was mixed into a chopped and formed patty. FIG. 6 illustrates a spiked patty 602 containing 10% powdered cell-based flavoring product and control patty 604 containing no powdered cellbased flavoring product. The spiked patty 602 was formed by adding the powdered cell-based flavoring product to the dry patty blend prior to chopping and forming. As shown by FIG. 6, the spiked patty 602 demonstrates significantly better browning than the control patty 604. Furthermore, and as shown by horizontal bar graph 606, 100% of tasters preferred the spiked patty 602 to the control patty 604. 100% of tasters noted more browning, 80% of tasters noted more roasted chicken and chicken skin flavors, 60% of tasters noted more umami and dry flavors in the spiked patty 602 when compared to the control patty 604. Additionally, 80% of tasters noted less vegetable oil and cardboard flavors and 60% of tasters noted less soy flavors in the spiked patty 602 when compared with the control patty 604. In sum, the powdered cell-based flavoring product, when added to a chopped and formed patty, intensified certain desirable sensory attributes like roasted chicken, chicken skin, and umami flavors while diminishing less-desirable flavors including waxy, moist, beany, and cardboard flavors.

[0084] In some implementations, the disclosed method uses a combination of the amino acid integration method illustrated in FIG. 1 and the browning method illustrated in FIG. 3. FIG. 7 illustrates an overview of a combined method that incorporates the amino acid integration method and browning method in accordance with one or more implementations of the present disclosure.

[0085] As shown in FIG. 7, the disclosed method comprises an act 702 of formulating a cell mixture by combining an amino acid blend with cells. As described previously with respect to FIG. 1, the amino acid blend can include aspartic acid and glutamic acid or ketoses and sulfur-containing amino acids. The amino acid blend can be combined with suspension cells or adherent cells. Furthermore, the cells may comprise cells that ultimately become the cell mass to which a flavor compound is added.

[0086] The disclosed method further includes an act 704 of heating a cell mixture to a peptide-forming temperature for a peptide-forming time. As described previously, the amino acidblend comprises amino acids that, when heated in combination with cells, interact with endogenous cell components in a heat induced synergistic way to create an umami taste.

[0087] As illustrated in FIG. 7, the disclosed method includes the act 706 of moisture adjusting the cell mixture. In some embodiments, the disclosed method comprises moisture adjusting the cell mixture to a target cell concentration. For example, the cell mixture can be moisture adjusted to a target cell concentration of 10%- 15% solids relative to the cell mixture.

[0088] The disclosed method further includes an act 708 of heating the cell mixture to a browning reaction temperature. In particular, the act 708 comprises heating the cell mixture to a browning reaction temperature for a browning reaction time to form a browned cell mixture. The disclosed method heats the cell mixture at high temperatures (e.g., greater than 140C) to induce non-enzymatic Maillard reactions to form a browned cell mixture. The browning reaction temperature is greater than the peptide-forming temperature.

[0089] As shown in FIG. 7, the disclosed method comprises the act 710 of comminuting the browned cell mixture to generate a cell-based flavoring product 714. Specifically, the browned cell mixture is further moisture reduced due to heating and comprises a film or sheet of browned cell mixture. To produce the cell-based flavoring product 714, act 710 comprises comminuting the browned cell mixture (e.g., reducing the browned cell mixture to particles). In one or more implementations, act 710 involves milling, powdering, pulverizing, or grinding the browned cell mixture. Thus, the cell-based flavoring product 714 can comprise a powder in one or more implementations. In another implementation, the cell-based flavoring product 714 comprises particles with irregular and fragmented geometries as shown in FIG. 7. The particles of the cellbased flavoring product 714 can comprise tiny cubes, thin shreds, or small, jagged chunks. Additionally, the particles of the cell-based flavoring product 714 can comprise non-uniform color. In other words, the color of the particles of the cell-based flavoring product 714 can range from light brown, to golden brown, to dark brown, depending upon how long and how thick the cell suspension is when brought to the browning reaction temperature. The disclosed method further includes an act of incorporating the cell-based flavoring product 714 into a food product (e.g., a cell mass comprising non-human cultivated animal cells) as described above.

[0090] FIGS. 1-7, the corresponding text, and the examples provide several different systems, methods, techniques, components, and / or devices relating to improving organoleptic properties of cultured cells in accordance with one or more implementations. In addition to the above description, one or more implementations can also be described in terms of flowcharts including acts for accomplishing a particular result. FIGS. 8-10 illustrate such flowcharts of acts. The acts described herein may be repeated or performed in parallel with one another or in parallel with different instances of the same or similar acts.

[0091] FIG. 8 illustrates a series of acts 800 comprising an act 802 of formulating a cell mixture and an act 804 of heating the cell mixture to a peptide-forming temperature. As illustrated in FIG. 8, the series of acts 800 includes the act 802 of formulating a cell mixture. In particular, the act 802 comprises formulating a cell mixture by combining an amino acid blend with cells.

[0092] The series of acts 800 illustrated in FIG. 8 also includes the act 804 of heating the cell mixture to a peptide-forming temperature. In particular, the act 804 comprises heating the cell mixture to a peptide-forming temperature for a peptide-forming time. In certain embodiments, the amino acid blend comprises at least one of glutamic acid or aspartic acid. Additionally, in certain embodiments, the cells contain endogenous purine-5’ -nucleotides comprising at least one of adenosine-5 ’-monophosphate, inosine-5’ -monophosphate, or guanosine-5 ’-monophosphate. In particular embodiments, the peptide-forming temperature is between 20C and 100C. Additionally, in some embodiments, the peptide-forming temperature is between 85-95C. In some implementations, the amino acid blend comprises a concentration of 0.25% to 1.5% glutamic acid. Furthermore, the amino acid blend can comprise a concentration of 0.25% to 1.5% aspartic acid. In one or more embodiments, the amino acid blend comprises at least one of ketoses or sulfur- containing amino acids.

[0093] FIG. 9 illustrates a series of acts 900 comprising an act 902 of moisture adjusting a cell suspension, an act 904 of heating the cell suspension to a browning reaction temperature, and an act 906 of incorporating the cell-based flavoring product into a cell mass. As illustrated in FIG. 9, the series of acts 900 includes the act 902 of moisture adjusting a cell suspension. In particular, the act 902 comprises moisture adjusting a cell suspension to a target cell concentration.

[0094] The series of acts 900 includes the act 904 of heating the cell suspension to a browning reaction temperature. In particular, the act 904 comprises heating the cell suspension to a browning reaction temperature for a browning reaction time, wherein the cells are heated without fat. FIG. 9 illustrates the act 906 of comminuting the browned cell suspension to generate a cellbased flavoring product. In particular, the act 906 comprises milling the browned cell suspension into particles.

[0095] In certain embodiments, the browning reaction temperature comprises a temperature between 165C and 177C. Additionally, in some implementations, the target cell concentration comprises a concentration of 10%-15% total solids relative to the cell suspension. In some embodiments, the browning reaction time comprises a time between 30 to 40 minutes. In some embodiments, the cell suspension comprises cells grown in a bioreactor.

[0096] In some embodiments, the series of acts 900 further comprises incorporating the cellbased flavoring product into a food product (e.g., a cell mass comprising non-human cultivated animal cells) such that the heated suspension comprises between 0.5% to 10% of the cell-basedfood product. In some embodiments, the series of acts 900 further comprises moisture adjusting a second cell suspension to a second target cell concentration, heating the second cell suspension to a second browning reaction temperature for a second browning reaction time, comminuting the second browned cell suspension to generate a second cell-based flavoring product, and incorporating the second cell-based flavoring product into the cell-based food product.

[0097] In some embodiments, the series of acts 900 further comprises imparting a roast chicken flavor to the cell-based flavoring product by heating the cell suspension to a browning reaction temperature between 168C to 173 C for a browning reaction time between 20 to 30 minutes.

[0098] Additionally, in some embodiments, the series of acts 900 comprises imparting a cheesy flavor to the cell-based flavoring product by heating the cell suspension to a browning reaction temperature between 163C to 165C for abrowning reaction time between 12 to 30 minutes.

[0099] FIG. 10 illustrates a series of acts 1000 comprising an act 1010 of formulating a cell mixture, an act 1020 of heating the cell mixture to a peptide-forming temperature, an act 1030 of moisture adjusting the cell mixture, an act 1040 of heating the cell mixture to a browning reaction temperature, and an act 1060 of incorporating the browned cell mixture into a cell mass.

[0100] The act 1010 illustrated in FIG. 10 comprises formulating a cell mixture. In particular, the act 1010 comprises formulating a cell mixture by combining an amino acid blend with cells.

[0101] The series of acts 1000 comprises the act 1020 of heating the cell mixture to a peptide- forming temperature. In particular, the act 1020 comprises heating the cell mixture to a peptide- forming temperature for a peptide-forming time.

[0102] The series of acts 1000 comprises the act 1030 of moisture adjusting the cell mixture. In particular, the act 1030 comprises moisture adjusting the cell mixture to a target cell concentration.

[0103] The series of acts 1000 comprises the act 1040 of heating the cell mixture to a browning reaction temperature. In particular, the act 1040 comprises heating the cell mixture to a browning reaction temperature for a browning reaction time to form a browned cell mixture.

[0104] As illustrated in FIG. 10, the series of acts 1000 comprises the act 1060 comminuting the browned cell mixture to generate a cell-based flavoring product. To produce the cell-based flavoring product, act 1060 comprises comminuting the browned cell mixture by reducing the browned cell mixture to particles. In one or more implementations, act 1060 involves milling, powdering, pulverizing, or grinding the browned cell mixture.

[0105] In some embodiments, the peptide-forming temperature comprises a temperature between 20C and 100C, and the browning reaction temperature comprises a temperature between 163C and 173C. Additionally, in some implementations, the cell mixture and the cell mass comprise cells grown in a bioreactor.

[0106] In some embodiments, the above-described methods for flavoring a cell-based food product can be expressed in terms of a cell-based food product. For example, a cell-based food product can comprise a cell mass and a flavor compound comprising cells heated to a browning reaction temperature for a browning reaction time, wherein the cells are heated without fat. In one or more embodiments, the cells comprise suspension cells grown in a bioreactor. Furthermore, in particular embodiments, the browning reaction temperature comprises a temperature between 168C and 173C.

[0107] The paragraphs above-describe methods for flavoring a cell-based food product. FIGS. 11A-11D and the following accompanying paragraphs describe procurement of cells and growth of cells into a cell tissue mass in accordance with one or more embodiments. Generally, FIGS. 11 A-l ID illustrate a process of collecting cells from an animal, growing cells in a favorable environment, banking successful cells, and collecting cells into a cell tissue mass followed by dewetting and / or other treatments.

[0108] As illustrated by step 1102 in FIG. 11 A, tissue is collected from a living animal via biopsy. In particular, stem cells, mesenchymal progeny, ectoderm lineage, and / or endoderm lineages can be isolated from the removed tissue. In some implementations of the present disclosure, tissue, such as fat and others, are processed to isolate stem cells, mesenchymal, ectoderm, and / or endoderm progeny or lineage cells. As illustrated, tissue 1104 is removed from an animal. In some examples, the tissue 1104 is removed from a living animal by taking a skin sample from the living animal. For instance, skin or muscle samples may be taken from a chicken, cow, fish, shellfish, or another animal.

[0109] Cells may be extracted from the tissue 1104 that was removed from the animal. More specifically, the tissue 1104 is broken down by enzymatic and / or mechanical means. To illustrate, FIG. 11 A includes digested tissue 1106 that comprises the cells to be grown in cultivation.

[0110] Cells in the digested tissue 1106 may be proliferated under appropriate conditions to begin a primary culture. As illustrated in FIG. 11 A, cells 1108 from the digested tissue 1106 are spread on a surface or substrate and proliferated until they reach confluence. As shown in FIG. 11 A, in some cases, cells 1112 have reached confluence when they start contacting other cells in the vessel, and / or have occupied all the available surface or substrate.[OHl] In some examples, cells are stored and frozen (i.e., banked) at different steps along the cell culture process. Cryopreservation generally comprises freezing cells for preservation and long-term storage. In some implementations, tissue and / or cells are removed from a surface or substrate, centrifuged to remove moisture content, and treated with a protective agent for cryopreservation. For example, as part of cryopreservation, tissues and cells are stored attemperatures at or below -80C. The protective agent may comprise dimethyl sulfoxide (DMSO) or glycerol.

[0112] Cells stored through cryopreservation may be used to replenish working cell stock. For instance, while a portion of the digested tissue 1106 is used as the cells 1108 spread on a surface or substrate, the remaining or excess digested tissue 1106 is transferred to cryovials 1110 for storage. Furthermore, the cells 1112 may be banked once reaching confluence and stored in cryovials 1114.

[0113] Once the cells 1112 have reached confluence, or just before the cells 1112 have reached confluence (e.g., occupation of about 80% of the substrate), the disclosed process comprises a series of cell passage steps. During cell passage, the cells 1112 are divided into one or more new culture vessels for continued proliferation. To illustrate, the cells 1112 may be diluted or spread on one or more surfaces or substrates to form the cells 1118. The cells 1118 are then grown 1116 to confluence, or just before confluence.

[0114] The cycle of dividing the cells 1112 into the cells 1118 for continued proliferation in new culture vessels may be repeated for a determined number of cycles. Typically, cell lines derived from primary cultures have a finite life span. Passaging the cells allows cells with the highest growth capacity to predominate. In one example, cells are passaged for five cycles to meet a desired genotypic and phenotypic uniformity in the cell population.

[0115] In some implementations, the disclosed method comprises immortalizing cells that have been grown and passaged for the determined number of cycles. For instance, the cells 1118 may be immortalized. As shown in FIG. 11B, cells 1120 have demonstrated a preferred growth capacity to proceed to immortalization. To achieve immortalization, the disclosed process transfects the cells 1120 with genes of interest. In one example telomerase reverse transcriptase (TERT) is introduced to the cells 1120. In some embodiments, the cells may be subjected to a selection process as known by those skilled in the art. The cells 1120 may then be passaged for a predetermined set of passaging cycles. In one example passaging cycle, the cells 1120 are grown to (or near) confluence 1124, then they are reseeded in new growth vessels, preserved in vials 1122, or some combination of both. The disclosed process may include any number of passaging cycles to ensure that the cells have reached immortality (e.g., can passage 60+ times without senescing), a target growth capacity, and / or a target quantity for banking. For example, cells may be passaged until they have reached a passage level of 100 (e.g., have been passaged for 100 passaging cycles). In another example, cells are passaged until they reach a population doubling level of 100.

[0116] Cells that have reached immortality or a target growth capacity by living through a target passage level may be adapted to suspension culture. In one example, a suspension culture media and agitation of cells in this suspension environment help cells to adapt and start proliferatingin the new growth environment. The cells adapted to suspension 1126 may be stored in cryovials 1128 for cryopreservation and banking. Cells in suspension 1126 will begin to proliferate and the process begins a series of dilute and expand steps.

[0117] During dilution and expansion, cells are moved from growth vessels into newer, and progressively larger, growth vessels. For example, cells in suspension 1126 may begin in a single tube. The cells will proliferate and increase in cellular density. Once the cells have reached a target cell number (i.e., viable cell density (VCD) at desired volume), they are diluted and moved to a larger growth vessel. Optionally, the cells are banked in cryovials throughout expansion. For example, once cells in suspension reach a maximum VCD, the cells may begin to leave exponential growth due to overcrowding. After reaching a target density, the suspension cells may be transferred to a larger vessel 1130 and diluted with additional media. The dilute-and-expand steps are repeated using progressively larger vessels (e.g., the vessel 1131 and the vessel 1132) and / or progressive dilution until the cells reach a production-ready volume. For example, cells may be production ready at about a 1,000 - 100,000-liter scale at 5 million cells per mL. The cells may be banked in cryovials at any of the dilution and expansion cycles.

[0118] As part of preparing cells to form a comestible food product, the disclosed process comprises growing the cells on microcarriers in a suspension. The cells grown in suspension may remain in the vessel 1132 or may be transferred to a different bioreactor.

[0119] FIG. 11C illustrates a bioreactor system comprising a plurality of adherent bioreactors 1148 connecting in parallel to a media vessel 1140. Adherent bioreactors provide an optional finishing step for cells grown in suspension conditions, whereby free-floating cells adhere to substrates and form tissue. The media vessel 1140 holds the cells grown in suspension media. In some implementations, cells from the vessel 1132 are transferred directly to a cell culture media (or just “media”) vessel 1140. In one example, the media vessel 1140 comprises the vessel 1132. As shown, a plurality of valves 1144 is secured to the plurality of adherent bioreactors 1148 to enable individual use and access of each of the adherent bioreactors 1148. For instance, to limit flow to only a first bioreactor of the plurality of adherent bioreactors 1148, the valve 1144 of the first bioreactor is opened while the remaining valves 1144 are closed. Furthermore, the bioreactor system can include a directional valve 1142 for changing between flow directions.

[0120] In some implementations, and as illustrated in FIG. 11C, cells (e.g., suspension cells) are prepared by flowing cells suspended in media (e.g., cell culture media) into the plurality of adherent bioreactors 1148. Cells and media that flowed through the adherent bioreactors 1148 are cycled back to the media vessel 1140. The media and cells can be cycled through the adherent bioreactors 1148 until a target adhered cell volume is reached. For instance, in someimplementations, the disclosed method comprises measuring a cell density of outflow from the adherent bioreactors 1148 to infer a seeded cell volume.

[0121] Prior to optionally finishing the cells of the present disclosure in the adherent bioreactors 1148, the cells are grown in suspension conditions to grow into cell tissue adhering to the microcarriers. Once they have grown to a target density, either according to a learned timing or according to a measured fluctuation in cell metabolism of components such as glucose and oxygen, then the cell tissue is ready for removal or further processing. The removal process of the disclosed method uses filters to separate the cell tissue from the media. The wash buffer 1156 and cell tissue are flowed through a filter 1152 where the cell tissue is collected into one or more cell tissue masses 1154.

[0122] As described above and depicted in FIGS. 1 and 3A-3B, for example, this disclosure describes a method of growing non-human cultivated animal cells on chickpea microcarriers suspended in cell culture media as part of forming cell-based food products. In one or more embodiments, however, the disclosed process comprises growing the non-human cultivated animal cells on the chickpea microcarriers in an adherent culture. For example, the non-human cultivated animal cells, chickpea microcarriers, and cell culture media may be transferred for growth on a substrate. For instance, the non-human cultivated animal cells can be transferred from a suspension bioreactor to a plurality of adherent bioreactors. In one or more cases, the chickpea microcarriers are added to the adherent bioreactors. In some embodiments, the adherent bioreactors comprise pipe-based bioreactors attached to a plurality of valves that enable individual use and access of each of the adherent bioreactors. For instance, to limit flow to only a first bioreactor of the plurality of adherent bioreactors, the valve of the first bioreactor is opened while the remaining valves are closed. Furthermore, the bioreactor system can include a directional valve for changing between flow directions.

[0123] In some implementations, the non-human cultivated animal cells are prepared by flowing the non-human cultivated animal cells and chickpea microcarriers suspended in cell culture media across substrates in the plurality of adherent bioreactors. More particularly, the non-human cultivated animal cells and chickpea microcarriers from the suspension bioreactor vessel may contact or land on the substrates in the plurality of adherent bioreactors. The non-human cultivated animal cells, chickpea microcarriers, and cell culture media that flowed through the adherent bioreactors are cycled back to the suspension bioreactor vessel. The cell culture media, non-human cultivated animal cells and chickpea microcarriers can be cycled through the adherent bioreactors until a target adherent cell volume is reached. For instance, in some implementations, the disclosed method comprises measuring a cell density of outflow from the adherent bioreactors to infer an adherent cell volume.

[0124] The non-human cultivated animal cells grow into adherent textured cell tissue within the adherent bioreactors. Once they have grown to a target volume or quality, either according to a learned timing or according to a measured fluctuation in cell metabolism of components such as glucose and oxygen, then the adherent textured cell tissue is ready for removal. The removal process of the disclosed method uses a high-pressure flow to shear the adherent textured cell tissue comprising the non-human cultivated animal cells and chickpea microcarriers off the substrate surfaces. In one example, wash buffer from a wash tank is flowed across the substrates in the adherent bioreactors. The wash buffer and textured cell tissue mixture are flowed through a filter where the textured cell tissue is collected into one or more cell tissue masses.

[0125] The cell tissue masses 1154 may be further processed to adjust moisture content. FIG. 1 ID illustrates an example apparatus for reducing moisture content in the cells. In particular, FIG. 1 ID illustrates a pressure apparatus 1160 that compresses the cell tissue masses 1158a and 1158b. While FIG. 11D illustrates a mechanical method for adjusting moisture content of the cell tissue masses 1158a and 1158b, other methods may be used to adjust moisture content. For example, the cell tissue masses 1158a and 1158b may be mixed with a drying agent, vacuum dried, centrifuged, or otherwise dried. A moisture-adjusted-cell tissue mass may be transferred to a container 1162 for additional processing. For example, the cell tissue masses 1158a or 1158b may be removed from the container 1162 to be formed into a cell-based food product.

[0126] In accordance with common practice, the various features illustrated in the drawings may not be drawn to scale. The illustrations presented in the present disclosure are not meant to be actual views of any particular apparatus (e.g., device, system, etc.) or method, but are merely idealized representations that are employed to describe various embodiments of the disclosure. Accordingly, the dimensions of the various features may be arbitrarily expanded or reduced for clarity. In addition, some of the drawings may be simplified for clarity. Thus, the drawings may not depict all of the components of a given apparatus (e.g., device) or all operations of a particular method.

[0127] Terms used herein and especially in the appended claims (e.g., bodies of the appended claims) are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including, but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes, but is not limited to,” etc.).

[0128] Additionally, if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases “at least one” and “one or more” to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claimrecitation by the indefinite articles “a” or “an” limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases “one or more” or “at least one” and indefinite articles such as “a” or “an” (e.g., “a” and / or “an” should be interpreted to mean “at least one” or “one or more”); the same holds true for the use of definite articles used to introduce claim recitations.

[0129] In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the bare recitation of “two recitations,” without other modifiers, means at least two recitations, or two or more recitations). Furthermore, in those instances where a convention analogous to “at least one of A, B, and C, etc.” or “one or more of A, B, and C, etc.” is used, in general such a construction is intended to include A alone, B alone, C alone, A and B together, A and C together, B and C together, or A, B, and C together, etc. For example, the use of the term “and / or” is intended to be construed in this manner.

[0130] Further, any disjunctive word or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” should be understood to include the possibilities of “A” or “B” or “A and B.”

[0131] However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles “a” or “an” limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases “one or more” or “at least one” and indefinite articles such as “a” or “an” (e.g., “a” and / or “an” should be interpreted to mean “at least one” or “one or more”); the same holds true for the use of definite articles used to introduce claim recitations.

[0132] Additionally, the use of the terms “first,” “second,” “third,” etc., are not necessarily used herein to connote a specific order or number of elements. Generally, the terms “first,” “second,” “third,” etc., are used to distinguish between different elements as generic identifiers. Absent a showing that the terms “first,” “second,” “third,” etc., connote a specific order, these terms should not be understood to connote a specific order. Furthermore, absent a showing that the terms “first,” “second,” “third,” etc., connote a specific number of elements, these terms should not be understood to connote a specific number of elements. For example, a first widget may be described as having a first side and a second widget may be described as having a second side. The use of the term “second side” with respect to the second widget may be to distinguish such side of the second widget from the “first side” of the first widget and not to connote that the second widget has two sides.

[0133] All examples and conditional language recited herein are intended for pedagogical objects to aid the reader in understanding the invention and the concepts contributed by the inventor to furthering the art and are to be construed as being without limitation to such specifically recited examples and conditions. Although embodiments of the present disclosure have been described in detail, it should be understood that the various changes, substitutions, and alterations could be made hereto without departing from the spirit and scope of the present disclosure.

[0134] The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. Indeed, the described embodiments are to be considered in all respects only as illustrative and not restrictive. For example, the methods described herein may be performed with less or more steps / acts or the steps / acts may be performed in differing orders. Additionally, the steps / acts described herein may be repeated or performed in parallel to one another or in parallel to different instances of the same or similar steps / acts. The scope of the invention is, therefore, indicated by the appended claims rather than by the foregoing description. All changes that come within the meaning and range of equivalency of the claims are to be embraced within their scope.

Claims

CLAIMSWhat is claimed is:

1. A method for flavoring a cell-based food product comprising: formulating a cell mixture by combining an amino acid blend with non-human cultivated animal cells; and heating the cell mixture to a peptide-forming temperature for a peptide-forming time to synergistically generate an umami flavor in the cell mixture.

2. The method of claim 1, wherein the amino acid blend comprises at least one of glutamic acid or aspartic acid.

3. The method of claim 1, wherein the non-human cultivated animal cells contain endogenous purine-5’ -nucleotides comprising at least one of adenosine-5’ -monophosphate, inosine-5’ -monophosphate, or guanosine-5 ’-monophosphate.

4. The method of claim 1, wherein the peptide-forming temperature is between 20C and 100C.

5. The method of claim 1, wherein the peptide-forming temperature is between 85- 95C.

6. The method of claim 1, wherein cell mixture comprises a concentration by weight of 0.25% to 1.5% glutamic acid from the amino acid blend.

7. The method of claim 1, wherein the cell mixture comprises a concentration by weight of 0.25% to 1.5% aspartic acid from the amino acid blend.

8. The method of claim 1, wherein the amino acid blend comprises at least one of ketoses or sulfur-containing amino acids.

9. A method of forming a food product comprising: moisture adjusting a cell suspension comprising non-human cultivated animal cells to a target cell concentration; heating the moisture-adjusted cell suspension to a browning reaction temperature for a browning reaction time to generate a browned cell suspension, wherein the moisture-adjusted cell suspension is heated without fat; and comminuting the browned cell suspension to generate a cell-based flavoring product.

10. The method of claim 9, wherein the browning reaction temperature comprises a temperature between 165C and 177C.

11. The method of claim 9, wherein the target cell concentration comprises a concentration of 10%-l 5% total solids relative to the cell suspension.

12. The method of claim 9, wherein the browning reaction time comprises a time between 30 and 40 minutes.

13. The method of claim 9, further comprising incorporating the cell-based flavoring product into a cell-based food product.

14. The method of claim 13, wherein the cell-based flavoring product comprises between 0.5% and 10% by weight of the cell-based food product.

15. The method of claim 13, further comprising: moisture adjusting a second cell suspension to a second target cell concentration; heating the second cell suspension to a second browning reaction temperature for a second browning reaction time; and incorporating the heated second cell suspension into the cell-based food product.

16. The method of claim 9, further comprising imparting a roast chicken flavor to the cell-based flavoring product by heating the moisture-adjusted cell suspension to a browning reaction temperature between 168C and 173C for a browning reaction time between 20 and 30 minutes.

17. The method of claim 9, further comprising imparting a cheesy flavor to the cellbased flavoring product by heating the moisture-adjusted cell suspension to a browning reaction temperature between 163C and 165C for a browning reaction time between 12 to 30 minutes.

18. A method of forming a food product comprising: formulating a cell mixture by combining an amino acid blend with non-human cultivated animal cells; heating the cell mixture to a peptide-forming temperature for a peptide-forming time; moisture adjusting the cell mixture to a target cell concentration; heating the moisture-adjusted cell mixture to a browning reaction temperature for a browning reaction time to form a browned cell mixture; and comminuting the browned cell mixture to generate a cell-based flavoring product.

19. The method of claim 18, wherein: the peptide-forming temperature comprises a temperature between 20C and 100C; and the browning reaction temperature comprises a temperature between 163C and 173C.

20. The method of claim 18, wherein the amino acid blend comprises at least one of glutamic acid or aspartic acid.

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