WO2023212122A1 - Processes and compositions for preparation of whole-cut meat analogues - Google Patents

Abstract

The present disclosure provides production materials, methods, techniques, cell lines, and strategies to simulate the structure of whole cuts of meat through a meat weaving technique which immerses and binds aligned protein or starch fibers within a polymerized solution. The aligned protein fibers simulate skeletal muscle cells and the immersion-binding solution simulates extracellular matrix (ECM).

Description

PROCESSES AND COMPOSITIONS FOR PREPARATION OF WHOLE-CUT MEAT

ANALOGUES

CROSS REFERENCE

[00001] This application claims priority to U.S. Provisional Application No. 63/335,346 filed April 27, 2022 and U.S. Provisional Application No. 63/460,569 filed April 19, 2023, which applications are herein incorporated by reference in their entireties for all purposes.

COLOR DRAWINGS

[00002] The patent or application file contains at least one drawing executed in color.

Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.

BACKGROUND

[00003] In the 1940’s and 50’s, the high cost of animal meat made it inaccessible to many populations of lower socioeconomic status throughout the world. Thus synthetic meat analogues were thought to be a way to make meat products cheaper, especially in periods of war and economic instability. Moreover, intensive animal agriculture was becoming increasingly industrialized, making animal meat production cheaper and more efficient.

SUMMARY

[00004] Recognized herein is a need to diversify protein production and lessen the demand for animal meat. The present disclosure provides materials, methods, techniques, cell lines, and strategies to produce to simulate the structure of whole cuts of meat through via a meat weaving technique which immerses and binds aligned protein fibers within a polymerized solution. Further, the aligned protein fibers simulate skeletal muscle cells and the immersion-binding solution simulates extracellular matrix (ECM).

[00005] An aspect of the disclosure provides a method for meat production, comprising: (a) providing a meat phase; (b) fabricating meat fibers from the meat phase; (c) aligning the meat fibers to obtain aligned meat fibers; (d) providing a matrix phase; (e) immersing the aligned meat fibers in the matrix phase; and (f) using one or more gelling or cross-linking agents to promote the matrix phase to gel or polymerize to form a solid matrix comprising the meat libers, wherein: (i) in (a), the meat phase comprises cultured animal cells; or (ii) in (b), the matrix phase comprises cultured animal cells. In some embodiments, the method for meat production further comprises processing the solid matrix into a final meat product. In some embodiments, the method for meat production further comprises sectioning the final meat product into individual whole meat cuts. [00006] In some embodiments, in (a) the meat phase comprises cultured animal cells. In some embodiments, in (a) the matrix phase comprises cultured animal cells. In some embodiments, (i) in (a), the meat phase comprises cultured animal cells; and (ii) in (b), the matrix phase comprises cultured animal cells. In some embodiments, the meat phase comprises recombinant animal protein or animal- derived ingredients. In some embodiments, the meat phase comprises products of fermentation. In some embodiments, the meat phase comprises myoglobin. In some embodiments, the matrix phase comprises recombinant animal protein or animal-derived ingredients. In some embodiments, the matrix phase comprises products of fermentation. In some embodiments, the matrix phase comprises myoglobin. In some embodiments, the meat phase and the matrix phase are separately formulated. In some embodiments, the cultured animal cells are skeletal muscle cells or fat cells. In some embodiments, the method for meat production comprises functionalizing the cultured animal cells or processing the cultured animal cells to uptake biological materials. In some embodiments, the method for meat production comprises functionalizing the cultured animal cells or processing the cultured animal cells to uptake red pigments. In some embodiments, the method for meat production comprises rinsing the cultured animal cells with water or a salt solution to wash away cell culture media or functionalization components. In some embodiments, the water is removed from the cultured animal cells using filters, centrifugation, or gravity separation. In some embodiments, the method for meat production further comprises culturing animal cells to obtain the cultured animal cells. In some embodiments, the method for meat production further comprises storing the cultured animal cells after culturing in cold storage and adding the cultured animal cells to the meat phase prior to or during (a) or to the matrix phase prior to (d).

[00007] In some embodiments, in (a), the meat phase comprises a fiber solution comprising one or more of proteins, hydrocolloids, or other ingredients. In some embodiments, the meat phase comprises one or more recombinant proteins produced from plants, fungi, insects; or a protein produced from fermentation. In some embodiments, the method for meat production further comprises treating the meat fibers or aligned meat fibers with chemical, biochemical, mechanical, or temperature treatments, hi some embodiments, (b) comprises using wet spinning to fabricate meat fibers. In some embodiments, (b) comprises using melt spinning to fabricate meat fibers, hi some embodiments, in (a), the meat phase comprises a soy protein isolate dissolved in an alkaline solution.

[00008] In some embodiments, the method for meat production further comprises extruding the soy protein isolate through a spinneret and into an acid bath. In some embodiments, (b) further comprises mixing plant proteins or starches in powdered material form with alkaline water and plasticizing at high temperatures to obtain plasticized mixture, and extruding plasticized mixture through dies to generate meat fibers. In some embodiments, (b) further comprises dry spinning, rotary spinning, gel spinning, dry jet wet spinning, or electrospinning to generate meat fibers. In some embodiments, (b) comprises melt blowing, interfacial polyelectrolyte complexation, extrusion or stereolithography-based three-dimensional (3D)-printing, high moisture extrusion (HME), shear cell technology, or fermentation. In some embodiments, (b) comprises 3D-printing or adding edible photoinitiators to the meat phase to initiate photopolymerization of reactive monomers to generate the meat fibers. In some embodiments, (b) comprises using 3D-printing to produce meat fibers and using hydrocolloids or enzymes mixed with the meat phase to cross-link or polymerize the meat fibers. In some embodiments, (b) comprises using 3D-printing utilizing stereolithography and in (d) the matrix phase comprises an edible photo-initiator. In some embodiments, (b) comprises using HME or shear cell technology and the method further comprises separating the meat fibers using a mechanical, thermal, chemical, or biochemical process that results in shredding or peeling of the meat fibers. In some embodiments, the aligned meat fibers have micron, submicron or nanometer dimensions.

[00009] In some embodiments, (b) further comprises processing the meat fibers to modulate one or more of texture, elastic modulus, tensile strength, elasticity, flexibility, brittleness, porosity, and mechanical stability of meat fibers. In some embodiments, the method for meat production further comprises treating the meat fibers or aligned meat fibers with enzymes to break covalent bonds or to catalyze covalent cross-linking of the meat fibers or aligned meat fibers. In some embodiments, the method for meat production further comprises treating the meat fibers or aligned meat fibers with enzymes to break covalent bonds and wherein the enzymes comprise trypsin, papain proteases, or amylases. In some embodiments, the method for meat production further comprising treating the meat fibers or aligned meat fibers with enzymes to catalyze covalent cross-linking of the meat fibers or aligned meat fibers wherein the enzymes comprise transglutaminase, other cross-linking enzymes, or derivatives thereof.

[00010] In some embodiments, the method for meat production further comprises chemical treatments to stabilize or destabilize cross-linking, strength, or elasticity of the meat fibers or aligned meat fibers. In some embodiments, the chemical treatments comprise treatment with acetic anhydride, acetaldehyde, glyoxal, glutaraldehyde, or a combination of glutaraldehyde and acetic anhydride.

[00011] In some embodiments, the method for meat production further comprises freezing, fractional freezing, or drying the meat fibers or aligned meat fibers. In some embodiments, drying comprises hot air drying (AD), freeze drying (FD), infrared drying (IR), microwave drying (MV), or vacuum drying (VD).

[00012] In some embodiments, the method for meat production further comprises stretching or twisting the meat fibers or aligned meat fibers. In some embodiments, the method for meat production further comprises marinating the meat fibers or aligned meat fibers. In some embodiments, the method for meat production further comprises treating the meat fibers or aligned meat fibers with a solution comprising one or more of a coloring agent, a flavorant; cultured animal cells; a recombinant animal protein, and a fat.

[00013] In some embodiments, (c) comprises using an automated mechanical process, wherein the automated mechanical process comprises: (i) weaving protein fibers through a hollow cylindrical apparatus having a first end and a second end; and (ii) threading the protein fibers through a first mechanical grid or membrane fixed at the first end of the cylindrical apparatus and a second mechanical grid or membrane fixed at the opposing second end of the cylindrical apparatus. In some embodiments, the matrix phase comprises one or more of salts, acids, or bases. In some embodiments, (e) comprises adding the matrix phase into an apparatus comprising the aligned meat fibers and applying a vacuum or compression.

[00014] An aspect of the disclosure provides a composition comprising: (i) cultured animal cells; (ii) a polymerizing, crosslinking, binding, gelling, or emulsifying agent; and (iii) a recombinant animal protein or an animal-derived protein. In some embodiments, the composition further comprises plant, animal, fungi, insect, algae, or microbial derived ingredients.

[00015] In some embodiments, the cultured animal cells comprise skeletal muscle cells, fat cells, or a combination thereof. In some embodiments, the composition further comprises one or more ingredients derived from soy, pea, fava bean, potato, carrot, chickpea, lentils, lupin, mung bean, wheat gluten, sunflower, rapeseed, mycelium, common mushrooms, yeast, bacteria, or red algae. In some embodiments, the composition further comprises one or both of a stabilizer or a preservative.

[00016] In some embodiments, the composition further comprises one or more of a vitamin, a nutrient, antioxidants, an ion, an amino acid, a peptide, a salt, a phosphate salt, an acid, a base, a polyvinyl alcohol, a polyacrylamide, a polyethylene oxide, a glycerol, or a solvent. In some embodiments, the composition further comprises one or more of myoglobin, gelatin, collagen, caseinate, albumin, or egg white proteins. In some embodiments, the composition further comprises a pigment. In some embodiments, the pigment comprises a betalain, an anthocyanin, a phycoerythrin, or a carotenoid, hi some embodiments, the composition further comprises a starch. In some embodiments, the starch comprises a modified starch or maltodextrin. In some embodiments, the composition further comprises one or more of a hydrocolloid, a monosaccharide, a disaccharide, or a polysaccharide. In some embodiments, the composition further comprises a gum. In some embodiments, the gum comprises Arabic gum, xanthan gum, locust bean gun, gellan gum, or cellulose gum. In some embodiments, the composition further comprises one or more of an alginate, an agar, a carrageenans, a micro-fiber or a nanofiber. In some embodiments, the composition further comprises one or more protein flours, isolates, concentrates, or extracts. In some embodiments, the composition further comprises oils or fats. In some embodiments, the oils or fats comprise triglycerides, free fatty acids, or omega 3-6-9 fatty acids.

[00017] In some embodiments, the polymerizing, crosslinking, binding, gelling, or emulsifying agent is selected from the group consisting of a saponin, a protein, a phospholipid, and a polysaccharide. In some embodiments, the polymerizing, crosslinking, binding, gelling, or emulsifying agent comprises a photo-initiator, In some embodiments, the polymerizing, crosslinking, binding, gelling, or emulsifying agent comprises a cross-linking enzyme. In some embodiments, the cross-linking enzyme is a transglutaminase or a derivative thereof.

[00018] An aspect of the disclosure provides a meat phase for fabricating meat fibers comprising cultured animal cells, proteins, and hydrocolloids. In some embodiments, the meat phase further comprises an emulsifier, a gelator, a stabilizer, a preservative, or a combination thereof. In some embodiments, the proteins are recombinant proteins produced from plants, fungi, insects; produced from fermentation; or produced from animal-derived proteins. In some embodiments, the meat phase further comprises soy protein isolate or plant protein dissolved in an alkaline aqueous solution, the alkaline aqueous solution being at least 25% alkaline water. In some embodiments, the meat phase further comprises edible photoinitators. In some embodiments, the meat phase further comprises a fiber solution.

[00019] An aspect of the disclosure provides a matrix phase for binding meat fibers comprising:

(i) cultured animal skeletal muscle or fat cells; (ii) recombinant animal proteins or animal-derived proteins; and (iii) plant, fermentation, or fungi-based ingredients. In some embodiments, the matrix phase further comprises one or more of an emulsifier, a gelling or crosslinking agent, a stabilizer, a preservative, a salt, an acid, or a base. In some embodiments, the gelling or crosslinking agent comprises a hydrocolloid-based gelling agent. In some embodiments, the matrix phase further comprises a gelling agent selected from the group consisting of agar, pectin, and alginate. In some embodiments, the matrix phase further comprises an edible photo-initiator. In some embodiments, the edible photo-initiator comprises riboflavin or a polymer substrate. In some embodiments, the plant or fungi ingredients are in the form of a powder, a liquid, or a solid. In some embodiments, the plant or fungi ingredients are in the form of a solid, and wherein the solid is a pulverized and a shredded protein.

[00020] In some embodiments, the matrix phase further comprises transglutaminase or derivatives thereof. In some embodiments, the matrix phase further comprises cultured chicken skeletal muscle cells and cultured fat cells, wherein the matrix phase comprises a higher amount of cultured chicken skeletal muscle cells to cultured fat cells, and wherein the matrix phase has a higher amount of protein than it does fat. [00021] In some embodiments, the matrix phase further comprises cultured bovine skeletal muscle cells and cultured fat cells, wherein the matrix phase comprises a higher amount of cultured fat cells to cultured bovine skeletal muscle cells and a higher amount of cultured cells to other ingredients. In some embodiments, the matrix phase further comprises skeletal muscle cells lacking red pigments. In some embodiments, the matrix phase further comprises skeletal muscle cells comprising red pigments.

BRIEF DESCRIPTION OF THE DRAWINGS

[00022] FIG. 1 is an example schematic of meat weaving operations;

[00023] FIG. 2 is an example diagram of meat weaving technique;

[00024] FIG. 3 is a diagram of fiber spinning techniques;

[00025] FIG. 4 is a comparison of different fiber spinning techniques;

[00026] FIG. 5 is an example of composition of muscle and matrix liquid solutions;

[00027] FIG. 6 is an example of techniques for meat fiber processing; and

[00028] FIG. 7 is an example of meat fiber immersion and binding principles.

DETAILED DESCRIPTION

[00029] The present disclosure provides methods of simulating the structure of whole cuts of meat. The present disclosure provides methods of immersing aligned protein fibers into a polymerized solution to facilitate binding and resulting in a meat analogue that is comparable to animal derived meat products. For example, the aligned protein fibers simulate skeletal muscle cells and the immersion-binding solution simulates extracellular matrix (ECM).

[00030] Further, the present disclosure provides meat analogue compositions as well as meat phase and matrix phase (e.g., liquid matrix solution).

[00031] Definitions

[00032] By “3D printing” is meant a technique to fabricate structures through periodic arrangement of various materials in a precisely controlled manner. The structures are printed from an extruder head as a liquid or gel and solidify or polymerize as a solid.

[00033] By “stereolithography” is meant a type of 3D printing that uses a laser to activate a molecule that reacts to photons to initiate a reaction that polymerizes a network from monomers in a sample.

[00034] By “cross-linking” is meant any reaction that involves the covalent binding between macromolecules of a polymer, while coupling is generated in blends of two or more polymers. [00035] By enhanced sensory properties is meant the taste and look of cell derived meat is improved over cell derived meat made by alternate methods or that the taste and look of the cell derived meat is close or identical to natural meat.

[00036] By “meat” is meant food products composed of animal flesh or non-animal ingredients that simulate the experience of animal flesh, that comprises water, protein, and fat.

[00037] By “nutritional profiles” is meant the unique composition of nutrients, including protein, fat, carbohydrates, vitamins, and electrolytes.

[00038] By “network” or polymer network is meant a network of macromolecules or an assembly of interacting macromolecules. A network may be formed by the reaction of monomer or prepolymer molecules to give a covalent network or by the intermolecular association of linear or branched polymers, commonly through hydrogen bonding, to give a physical network.

[00039] By “organoleptic” is meant any aspects of food, water or other substances that an individual experiences via the senses.

[00040] By “oxidizer” is meant any substance that oxidizes another substance by accepting electrons and the fuel supplied.

[00041] By “photocatalyst” or “photo-initiator” is meant any substance that absorbs light in the ultraviolet-visible spectral range, generally about 250-450 nm, and convert this light energy into chemical energy in the form of reactive intermediates, such as free radicals and reactive cations, which subsequently initiate polymerization.

[00042] By “physiochemical properties” is meant physical properties (such as, but not limited to, texture, elastic modulus, tensile strength, elasticity, flexibility, brittleness, or porosity of fibers), solvation properties related to interactions with different media, and properties or molecular attributes that define intrinsic chemical reactivity.

[00043] By a “bioprocess” is meant a process that uses complete living cells or their components (e.g., bacteria, enzymes, chloroplasts, etc.) to obtain desired products.

[00044] By “upstream bioprocess” is meant a process that can include early cell isolation and cultivation, cell banking and culture expansion of the cells until final harvest, the termination of the culture and collection of the live cell batch.

[00045] Animal meat is a staple of Western diets. Beef and pork are popular types of meat in western diets, and other animals such as goat, deer, rabbit, and sheep are consumed as well. Animal meat has specific sensory properties associated with its appearance and taste. The sensory properties of the meat arise from the muscle and fat cells of the meat. Analogue meat product meant to replace or compete with animal meat products can possess similar sensory properties. Consequently, materials and methods described herein can recreate the experience of eating animal meat. [00046] Meat Weaving Process

[00047] In one aspect, the disclosure provides a method for producing meat analogues by meat weaving. The method may comprise (a) providing a meat phase, (b) fabricating meat fibers from the meat phase, (c) aligning the meat fibers to obtain aligned meat fibers; (d) providing a matrix phase; (e) immersing the aligned meat fibers in the matrix phase; and (f) using one or more gelling or crosslinking agents to promote the matrix phase to gel and polymerize to form a solid matrix comprising the meat fibers, wherein: (i) in (a), the meat phase comprises cultured animal cells; or (ii) in (b), the matrix phase comprises cultured animal cells. In some embodiments, the meat phase or the matrix phase comprises recombinant proteins or products of cellular agriculture. In some embodiments, the meat phase or the matrix phase comprises myoglobin or gelatin protein.

[00048] Cultured animal cells may be collected from an upstream bioprocess and prepared for meat weaving. The cultured animal cells may be skeletal muscle cells or fat cells. The cultured animal cells may be processed prior to addition to the meat phase or the matrix phase or to a combination of both. Cell processing may comprise cell functionalization, promoting the cultured animal cells to uptake red pigments, rinsing the cells to remove cell culture media or functionalization components, removing water from the cells, or storing the cells in cold storage.

[00049] The meat phase (e.g., liquid meat solution) and matrix phase (e.g., liquid matrix solution) may be separately formulated. Both the meat phase (e.g., liquid meat solution) and the matrix phase (e.g., liquid matrix solution) may have plant and/or fungi ingredient base formulas that may be further modified by the addition of cultured animal cells, recombinant animal proteins, animal derived ingredients, or combinations thereof. The recombinant proteins may be produced from plants, fungi, insects, or fermentation.

[00050] The meat phase (e.g., liquid meat solution) may comprise one or more of cultured animal cells, proteins, starches, hydrocolloids, emulsifiers, gelators, stabilizers and preservatives. Meat fibers may be fabricated from the meat phase. The meat phase (e.g., liquid meat solution) can be processed in any manner that produces separated, elongated protein fibers (e.g., meat fibers). For example, the meat phase (e.g., liquid meat solution) may be processed via wet or melt spinning or 3D printing. A subsequent meat fiber processing operation can further modify meat fibers to augment their physiochemical, material, sensory, and nutritional profiles. The meat fibers may be aligned (e.g., collected and organized in parallel). The meat fibers may simulate cellular meat fibers or extracellular matrix fibers.

[00051] The matrix phase may function as both a binding system for the meat fibers and a way to deliver cultured cells to the product, which may improve sensory and nutritional properties. The matrix phase may comprise cultured cells (e.g., skeletal muscle, fat cells, or a ratio of both), gelling agents, and emulsifiers. The matrix phase (e.g., liquid matrix solution) may be further modified by the addition of free meat fibers. The meat fibers may simulate cellular meat fibers or extracellular matrix fibers. The matrix phase (e.g., liquid matrix solution) may be added to (e.g., poured between) aligned meat fibers (referred to as meat fiber immersion). After meat fiber immersion, the matrix phase may gel or polymerize to form a solid matrix, After meat fiber immersion, the matrix phase may bind the meat fibers together. Gelling or cross-linking agents may be used to promote the matrix phase to gel and polymerize to form a solid matrix. A final processing operation may prepare the product for packaging, storage, and distribution.

[00052] In meat weaving, species-specific properties, meat cut-specific properties, and nutritional properties of cultivated meat products may be customized by changing ingredient compositions of the meat phase and matrix phase, meat fiber fabrication and processing methods, and/or the polymerization and emulsification mechanisms of the matrix. For example, in lean cuts of poultry, the density of meat fibers may be increased, decreasing the volume available for matrix phase (e.g., liquid matrix solution). The matrix phase (e.g., liquid matrix solution) may contain a higher ratio of cultured chicken skeletal muscle cells to fat cells. The initial meat phase may include skeletal muscle cells (e.g., chicken skeletal muscle cells) or may be modified during the fiber processing operation where muscle cells may be deposited into the meat fibers, enriching the fibers. The resulting product composition may have more protein and less fat.

[00053] In a wagyu beef example, the matrix phase may contain a high ratio of bovine fat cells to skeletal muscle cells and a high ratio of cultured cells to other ingredients combined with a lower density of aligned meat fibers. This strategy provides a mechanism to generate visible intramuscular fat, or marbling. A lower meat fiber density and a higher volume ratio of bovine fat cells increases in the matrix increases the percentage of marbling and overall fat content of the product.

[00054] Meat fibers may be produced from a diverse range of processes. These processes include fiber spinning such as melt, dry, wet, rotary, gel, dry jet wet, or electrospinning; melt blowing; interfacial polyelectrolyte complexation; extrusion or stereolithography-based 3D printing; high moisture extrusion (HME); shear cell technology, or fermentation-based systems that produce protein from carbon metabolism of microbial species. In techniques that produce whole cut meat analogues, such as HME and shear cell technology, the meat fibers are separated in a mechanical, thermal, chemical, or biochemical process that results in shredding of the fibers. Fiber spinning technologies have high potential for meat weaving since separate fibers are produced and collected in a fashion similar to yam in the textile industry. [00055] The meat weaving method may be used to produce meat analogues that are ready to consume. Cells harvested from an upstream production run may be mixed with other ingredients without subsequent culturing operations aimed at optimizing the activity or properties of living cell populations, making the meat weaving process less complex and more efficient than tissue engineering cultivated meat products. Meat weaving may be used to produce meat analogues for meat products from any species, including cattle, sheep, swine, goats, fish, chicken, turkeys, ducks, geese, ratites, mammoth, and squab. Meat weaving may be used to produce meat analogues for Wagyu beef. May weaving may be used to produce meat analogues for mammoth meat. Meat weaving methods described herein may be adapted for bioengineered tissue and organ applications.

[00056] Emulsifiers

[00057] The meat phase or the matrix phase may comprise emulsifiers. An emulsion is a stable mixture of immiscible fluids, such as oil and water, achieved by finely dividing one phase into very small droplets. Emulsifiers may dissolve in both oil and water and produce an emulsion by mediating the stable segregation of immiscible phases. The emulsifiers form an interfacial layer between oil and water phases, preventing droplets of the discontinuous phase from merging. Generally, emulsifiers act in the interfacial region by direct absorption in the interface or integration in the hydrophobic/hydrophilic region, which increases water viscosity and thus, emulsion stability.

[00058] The lipid droplets in oil-in-water emulsions may affect food’s desirable physicochemical and sensory attributes, such as appearance, texture, stability, aroma, taste, and mouthfeel. For example, turbidity and viscosity of an aqueous solution may be increased by the addition of lipid droplets. Making a successfill emulsion can result in a homogenous eating product that has a long shelf life and does not have separation of the components over time. Lipid droplets in emulsions may also serve as a delivery system to encapsulate non-polar hydrophobic ingredients, such as colors, flavors, vitamins, nutrients, and antioxidants.

[00059] Non-limiting examples of food-grade emulsifiers include proteins, polysaccharides, phospholipids, and small molecule surfactants. Proteins are naturally amphiphilic, with hydrophobic and hydrophilic domains with high emulsifying, water/oil holding capacity and their gelling properties. Proteins have surface active molecules that may adsorb to oil-water interfaces during homogenization and form a protective coating around droplets that provide stability during storage. The use of phosphate salts may increase the solubility of proteins, which improve the emulsion stability.

[00060] Phospholipids are amphiphilic, polar lipid molecules composed of two fatty acid chains bound to glycerol and a phosphoric acid group. Phospholipids can form monolayers around oil droplets, and in other circumstances they may form multiple bilayers. Phospholipid-based emulsifiers used in commercial food products include lecithins, which may be isolated from soybeans, eggs, milk, rapeseed, canola seed, cottonseed, and sunflower. Lecithins can contain a combination of various phospholipids and other lipophilic materials like triglycerides, glycolipids, and sterols, can also be fractionated to create more refined ingredients.

[00061] Saponins are small molecule surfactants that can be derived from tree bark which include a mixture of different amphiphilic components that form micelles in water. The saponins are amphiphilic because they have hydrophilic regions containing sugar groups and hydrophobic regions containing phenolic groups. An example of a saponin is Q-Naturale. Saponins are particularly efficacious emulsifiers due to their physio chemical properties: they may produce nano-emulsions and are stable over a wide range of pH, ionic strength, and temperature.

[00062] Polysaccharides are natural biopolymers that include one or more types of monosaccharide linked together by glycosidic bonds. Polysaccharides vary considerably in molar mass, degree of branching, electrical charge, hydrophobicity, and polarity, which alter their physicochemical attributes and functional performance. Good polysaccharide emulsifiers include those that covalently bound to non-polar, hydrophilic groups, such as methylated groups, polypeptides (glycoproteins), or lipids (glycolipids). An example of a polysaccharide emulsifier is Gum Arabic, which contains a non-polar polypeptide backbone and polar polysaccharide side chains. Modified starch and modified cellulose are also good emulsifiers, which have non-polar hydrocarbon chains covalently attached to polysaccharide chains.

[00063] Emulsifiers may serve a number of functions, including: they can facilitate the initial formation of fine lipid droplets during homogenization and they can enhance the stability of the lipid droplets once they have been formed. Homogenization can be an initial operation in producing a stable emulsion. Homogenization disrupts and mixes the oil and water phases leading to the production of fine lipid droplets. High-energy methods of emulsion use mechanical systems like high shear mixers, colloid mills, high-pressure valve homogenizers, micro fluidizers, and sonicators. Polysaccharides or proteins may be depolymerized or denatured within sonicators due to the high local temperature and pressure gradients generated. Globular proteins may also be denatured and aggregate within high- pressure homogenizers or microfluidizers. Low- energy homogenization relies on the spontaneous formation of emulsions when the reaction environment or composition changes. These approaches include phase inversion temperature (PIT), spontaneous emulsification (SE), and emulsion inversion point (EIP) methods. [00064] An emulsifier may be dissolved in the aqueous phase, and the aqueous and oil phases may be mixed using a high shear mixer to produce an emulsion with droplets (e.g., droplets greater than 1 μm in diameter) coated in emulsifier. Remaining emulsifier may be mixed into the aqueous phase. The emulsion may be pumped through a high-pressure valve which further breaks up the droplets into smaller diameters.

[00065] Homogenization can increase surface area for emulsifiers to cover. If the surface of the lipid droplet may be completely covered in short time by the emulsifier, then newly formed lipid droplets may not have the opportunity to coalesce into larger droplets. Lipid droplet surfaces may be saturated with emulsifier before droplet coalescence, which can be modulated by controlling emulsifier adsorption rate. If the emulsifier adsorption rate is higher than the lipid droplet collision rate, then a stable emulsion may form.

[00066] Additionally, the emulsifier concentration and surface load impact droplet size. Droplet diameters can decrease with increasing emulsifier concentration, and the minimum droplet size that may be produced at a given emulsifier concentration can increase with increasing adsorption rate. In some cases, the adsorption rate of natural emulsifiers follows the order: small molecule surfactants (e.g., saponins) < globular proteins (e.g., whey protein) < flexible proteins (e.g., caseinate) < polysaccharides (e.g., gum Arabic). Many amphiphilic polysaccharides have relatively large molecular weights and dimensions and may have high adsorption rates. High concentrations may be used to produce small droplets during homogenization. For example, a 1:1 mass ratio of emulsifier- to-oil may be used form small droplets using gum Arabic compared to less than 1:10 for whey proteins.

[00067] In some cases, a natural emulsifier may rapidly adsorb to the oil droplet surfaces generated during homogenization, appreciably decrease the oil-water interfacial tension to facilitate droplet fragmentation, and generate a protective coating to inhibit droplet coalescence within the homogenizer. An emulsifier may keep lipid droplets stable during different operations in production, transport, storage, and cooking, Additionally, the formation and stabilization of emulsions may be improved by combining emulsifying systems, like protein-polysaccharide, surfactant-protein, or surfactant-polysaccharide combinations. Emulsifiers may not necessarily polymerize three- dimensional cross-linked networks.

[00068] Gelling / crosslinking agents

[00069] The meat phase or matrix phase may comprise a gelling agent, a crosslinking agent or a combination thereof. Gelation may comprise association of polymer chains. The association may be mediated by ionic bonding. The association may be mediated by covalent bonding. In some embodiments, the polymer chains are crosslinked. Gelation may form a three-dimensional network of polymer chains. In some embodiments, the network of polymer chains is continuous. This network of polymer chains may trap and immobilize liquid within it to form a rigid structure that is resistant to flow. Gelation may comprise a phase transition process. Gelation may comprise the process of forming a gel. A gel may be mostly liquid by mass and volume but behave like solids because of a three-dimensional cross-linked network within the liquid. The crosslinking within the fluid may give a gel its structure and biomechanical characteristics, such as tensile and elastic strength. A gel may be a dispersion of molecules of a liquid within a solid medium.

[00070] A gelling agent may be used for gelation. A gelling agent may share properties with thickening agents, emulsifiers, or stabilizers. A gelling agent may be or comprise an emulsifier, a stabilizer, a thickening agent, a cross-linking agent, or any combination thereof.

[00071] In some embodiments, a gelling agent comprises an emulsifier. Some compounds may function as both an emulsifier and a gelator. In some embodiments, a gelling agent comprises a stabilizer. In some embodiments, a gelling agent comprises a thickening agent. The stabilizer or thickening agent may be a hydrocolloid. The hydrocolloid may be a hydrophilic polysaccharide with excellent ability to hold water in the gel network. The hydrocolloid may contribute to increasing gel consistency.

[00072] Gelling agents may be or comprise crosslinking agents. The crosslinking agents may cause crosslinking of polymer chains. Crosslinking occurs through various electron sharing mechanisms between molecular species. Crosslinking reactions may be initiated by heat, pressure, change in pH, light irradiation of photoreactive compounds, or enzymes. Chemical bonds and chemical crosslinking may occur via covalent bonding, where electron pairs are equally shared between atoms of similar electronegativity. A solid formed from a network of covalently bonded atoms may be regarded as a single, large molecule. Covalent cross-links can be mechanically and thermally stable, so once formed are difficult to break. Such networks can be strong, stiff, and brittle with a high melting point. Covalent crosslinking may be initiated by chemical crosslinking agents, mediated by free radicals in oxidative crosslinking and photo-crosslinking, or catalyzed by enzymes. Examples of covalent bonding includes redox reactions, disulfide bond formation, and isopeptide bond formation.

[00073] A free radical is an atom, molecule, or negatively charged ion that has at least one unpaired valence electron, and these unpaired electrons make radicals highly chemically reactive. Radicals may be generated in a number of ways, including in redox reactions, ionizing radiation, heat, electrical discharges, and electrolysis. Polymerization can occur when an initial radical reduces a non-radical, which can then produce new radicals in a radical chain reaction. [00074] Oxidative cross-linking can occur during redox reactions, when reducing agents are exposed to oxidizers, such as atmospheric oxygen or free radicals, and lose electrons. Undesirable polymerization reactions may be slowed or stopped with antioxidants that inhibit the formation of oxidative cross-links. When formation of oxidative cross-links by oxidation is desirable, an oxidizer may be used.

[00075] Photo-oxidization involves the formation of oxygen radicals upon exposure to visible or ultraviolet light, followed by a reduction-oxidation reaction. Light irradiation of a photo catalyst, or photo-initiator, can result in a highly reactive radical. Monomers with functional groups that accept electrons, such as vinyl bonds, can be polymerized into a hydrogel in a chain growth mechanism when reduced by radicals. Some amino acids demonstrate considerable intrinsic redox properties, such as tyrosine and tryptophan, with the oxidative coupling of two adjacent residues. Oxidation of tyrosine residues catalyzes the formation of di-tyrosine bonds. Photo-oxidization efficiency can also be associated with the overall content and special distribution of the redox amino acids. Other materials that lack intrinsic redox potential may be chemically functionalized for photo-crosslinking, such as, for example, hydrocolloids.

[00076] Photopolymerizable hydrogels made of edible polymers, such as alginate, chitosan, and gelatin, have wide industrial and clinical applications. They provide certain advantages compared with hydrogels that are cross-linked by other methods (temperature, chemically, or others). The ability to spatially localize the light path and alter the photo-flux provides both physical and chemical approaches to control hydrogel polymer crosslinking efficiency, which impacts tensile and elastic strength. Photopolymerization by laser direct writing can be used to develop complex 2D and 3D structures, such as with tens of nanometers of resolution. An example of an edible photo-initiators is the nutrient riboflavin or vitamin B2.

[00077] The oxidation of sulfhydryl (— SH) groups mediates disulfide bonding. An organic disulfide bond refers to a functional group with the structure R—S-S—R'. The linkage is also called an SS-bond or a disulfide bridge. Disulfide bonds may be formed under oxidizing conditions and play an important role in the folding and stability of some proteins, including proteins secreted to the extracellular medium. In protein biology, disulfide bonds are formed between thiol groups in two sulfur-containing cysteine residues and can be e an important component of the secondary and tertiary structure of proteins. A variety of oxidants can participate in this reaction, including oxygen and hydrogen peroxide, which can be under equilibrium control, especially in the presence of a catalytic amount of base. Alternatively, enzymes may catalyze disulfide bond formation, such as protein disulfide isomerase (PDI) in eukaryotes. Gluten changes structure of foods by undergoing oxidative crosslinking by changing disulfide bonding. Disulfide bonding is disrupted in high temperature protein extrusion, where proteins are denatured when plasticized and then cooled, allowing new disulfide bond formation between proteins.

[00078] An isopeptide bond is an amide bond that may form for example between a carboxyl group of one amino acid and an amino group of another. At least one of these joining groups can be part of the side chain of one of these amino acids. Bond formation may be either enzyme catalyzed, as can be the case for the isopeptide bond formed between lysine and glutamine catalyzed by transglutaminases, other enzymes, or derivatives thereof, or it may form spontaneously. Transglutaminase catalyzes the formation of an isopeptide bond between γ-carboxamide groups of glutamine residue side chains and the ε-amino groups of lysine residue side chains or other species with primary amine group with subsequent release of ammonia. Transglutaminase or derivatives thereof can be used to bond proteins together. Spontaneous isopeptide bond formation may involve the presence of another residue, glutamic acid, which catalyzes bond formation in a proximity induced manner.

[00079] In ionic bonding, when oppositely electrostatically charged ion species interact, electrons are unequally shared between atoms. An ionic bond results from the transfer of electrons from a metal to a non-metal in order to obtain a full valence shell for both atoms. This electron transfer forms positively and negatively charged ions, which then are subject to electrostatic attraction to form a solid. Ionic compounds can have a high melting point, depending on the charge of the ions they consist of. The larger the electronegativity difference, the stronger the cohesive forces and the higher the melting point. Materials which form ionic cross-linked gels include gelatin and many hydrocolloids. In some embodiments, hydrocolloid gelators involve heat for gelation; which provides energy to alter the structure of the molecule, making it more amenable to cross-linking. Some gels are thermally reversible, like gelatin, and gels vary in their optical characteristics. Specific ions may be used to complete the gelling process.

[00080] Gelling agents may be or comprise proteins or starches. Gelation may be an important functional property for various food applications used to make products such as cheese or tofu. There are different mechanisms to trigger protein gelation, including adding salt or other types of adjuvants, acidification, enzymatic reactions, or applying high pressure, but the most common technique is heating or cooling. Multiple protein species may individually form discrete networks simultaneously or complex together to form a heterogenous network.

[00081] Aggregation and gelation of globular proteins may be initiated by heating that renders the peptide chain more mobile allowing amino acids to interact with and bind to other proteins. Heat- induced gelation may involve denaturation, aggregation, and an above a critical concentration, formation of a continuous protein network. Non-covalent interactions (electrostatic, hydrophobic interactions, and hydrogen bonds) and covalent interactions (disulfide bonds) can both be involved in gelation.

[00082] Plant-based meat fabrication techniques can involve purified protein ingredients stripped of carbohydrates and phenols, such as, for example, with high moisture extrusion or fiber spinning. Some protein flours can have good emulsification and gelling properties. For example, in the case of fava bean and pea protein gels, the level of starch can affect gel firmness. Flours may produce strong, elastic gels that have high fat binding and water holding capacity.

[00083] A gelling agent may be thermostable above the cooking temperature of meat, between about 63-74°C (145° - 165° F), inclusively, where “about” is +/- 5° C. A gelling agent may be thermostable above room temperature. A gelling agent may be thermostable at a temperature of at least -25°C, at least -15°C, at least -5°C, at least 0°C, at least 5°C, at least 15°C, at least 25°C, at least 35°C, at least 45°C, at least 55°C, at least 45°C, at least 55°C, at least 65°C, at least 75°C, at least 85°C, at least 95°C, at least 105°C, at least 115°C, at least 125°C, at least 135°C, or at least 145°C.

[00084] Gel Emulsification

[00085] In some embodiments, the meat phase or matrix phase may comprise a gel. In some embodiments, the meat phase or matrix phase may comprise ingredients that promote the formation of a gel. A gel may be an emulsion gel or an emulsion with a gel-like lattice and solid mechanical properties. The formation of the emulsion gel may involve using emulsifying agents and gelling agents in two operations: 1) the production of a stabilized emulsion, and 2) gelation of the continuous phase. Several gelling agents maybe used in the development of emulsion hydrogels. The gelling agents may be proteins (such as protein isolates or gelatin), polysaccharides (such as alginates, agar, carrageenan, or inulin), or combinations thereof

[00086] Emulsion gels can be used in reformulated meat products where animal fat is replaced by fats derived from plants to make a healthier product and in plant-based meats where addition of fat may improve sensory and nutritional profiles. The emulsion gels in reformulated ground burger products can be formed with polysaccharide-stabilized gels, such as those which use alginate, carrageenan, and gellan gum.

[00087] Sausages may involve different gel compositions. Salt and water allow solubilization of meat proteins, which may link insoluble, crushed meat fibers and homogenized fat particles in a continuous protein-gel network after heating. [00088] Emulsion hydrogels used in sausage reformulation may be based on protein- or polysaccharide/protein-stabilized emulsions that include soy protein, gelatin, and caseinate. Additionally, other compounds like phosphate salts, inulin, carrageenan or bamboo fiber may be used to stabilize the emulsion hydrogel.

[00089] Meat fiber Fabrication Methods

[00090] Fiber Spinning Methods

[00091] Edible protein fibers may be formed by forming a protein solution that is spun into a polymer using a spinning method. Spinning methods may comprise one or more of wet spinning, dry spinning, melt spinning, and centrifugal or rotary spinning. These fibers may be collected in like threads wrapped around spools. The edible protein fibers may be derived from animal, plant, fermentation, or fungal sources. These fibers may have micron, submicron and/or nanometer dimensions. A general process diagram for melt spinning, dry spinning, and wet spinning to produce spun fiber is shown in FIG. 3. Example characteristics of each method of spinning are summarized in FIG. 4.

[00092] Melt spinning may be used for polymers that may be melted easily. Melt spinning uses heat to melt the polymer to a viscosity suitable for extrusion. This type of spinning may be used for polymers that are not decomposed or degraded by the temperatures necessary for extrusion. In melt spinning, polymer may be heated and the polymer may melt to form a liquid spinning solution or dope. Melt spinning may involve air and steam to produce spun fiber from heated polymer chips (FIG. 3). Chips of polymers may be fed to a hopper which is heated. There may be a grid (e.g., sieve) at the base which permits only molten liquid to pass through. The solution may be purified by filter, The molten polymer may be extruded at high pressure and constant rate through a spinneret into a relatively cooler air stream that solidifies the fibers. The fiber may be immediately wound onto bobbins or further treated for certain desired characteristics or end use. An advantage of melt spinning is the high spinning speed (e.g., 2500-3000 ft/min). Melting spinning also does not require solvents, is non-toxic, and may not involve purification problems. Melt spinning may be used for fiber and staple production and involve high productivity and low investments costs (FIG. 4).

[00093] Both dry and wet spinning involve solvent spinning, where polymers are dissolved in solvents and undergo precipitation and/or aggregation (e.g., coagulation and flocculation) upon removal of the solvent (FIG. 3). Dry spinning involves dissolving the polymer into a solvent solution that may be easily evaporated. A volatile solvent may be used to dissolve the raw materials (e.g., polymer chips) and form a salutation. The solution may be purified by filter. The solution may be extruded through a spinneret into a warm air chamber where the solvent evaporates, solidifying the fine fibers. The fiber may be immediately wound onto bobbins or further treated for certain desired characteristics or end use. The solvent may be recovered. Dry spinning may be used for fiber production, do not require purification, and involve high productivity (FIG. 4).

[00094] Wet Spinning may be used when the polymer does not melt and dissolves in non-volatile or thermal unstable solvents, where the solvent may not be evaporated and is removed by chemical processing (e.g., using a coagulant and separating the solvent and coagulant from a solvent coagulant mixture) (FIG. 3). In wet spinning, a non-volatile solvent may be used to convert the raw material (e.g., polymer chips) into a solution. The polymer solution may be extruded through the spinneret into a spinning bath, After extrusion, the solvent may be removed in a liquid coagulation medium (FIG. 3). Finally, the fiber either may be immediately wound onto bobbins or may be further treated for certain desired characteristics or end use. In wet spinning, large tows may be handled. Low temperatures may be used during the spinning process. Wet pinning may be used for fiber and staple production and generally involve low investment costs (FIG. 4).

[00095] Centrifugal forces may also be applied to fiber spinning methods. Rotary Jet-Spinning (RJS) may modify dry spinning methods. In RJS, a liquid polymer solution may be loaded into a reservoir and pushed out through a tiny opening by centrifugal force as the device spins. As the solution leaves the reservoir, the solvent evaporates and the polymers may solidify and elongate into small, thin fibers. For polymers requiring solvents that don’t evaporate easily, centrifugal forces may be applied to wet spinning techniques. Such systems may use precipitation rather than evaporation to separate the solvent from the polymer. In this system, called immersion Rotary Jet-Spinning (iRJS), when the polymer solution shoots out of the reservoir, it passes through an area of open air, where the polymers elongate and the chains align. The solution hits a liquid bath that removes the solvent and precipitates the polymers to form solid fibers. Since the bath is also spinning — like water in a salad spinner — the nanofibers follow the stream of the vortex and wrap around a rotating collector at the base of the device.

[00096] In gel spinning, also known as dry-wet or semi-melt spinning, a polymer solution or plasticised gel may be extruded from the spinnerets and pass through air and cooled further in a liquid bath composed of solvent or water before being stretched into gel fiber by ultra-high extension. Gel spinning can be used for producing high-strength and high-elastic modulus fibers in the gel state. The polymer may not be in a true liquid state during extrusion. The polymer chains may not be completely separated, as in true solution. The polymer chains may be bound together at various points in liquid crystal form. This produces strong inter-chain forces in the resulting fibers that can significantly increase the tensile strength of the fibers. In addition, the liquid crystals are aligned along the fiber axis by the shear forces during extrusion. The fibers emerge with an unusually high degree of orientation relative to each other, further enhancing strength.

[00097] In dry jet wet spinning, the polymer may be dissolved in an appropriate solvent to make the fiber solution. This solution may be extruded under heat and pressure into an air gap before it enters a coagulation bath. The produced fiber may be washed and dried before it is heat treated and drawn. This may be an alternative method to wet spinning. An inert atmosphere may prevent oxidation in some polymers. Fibers may be extruded into a nitrogen atmosphere. This method may be used for high performance fibers with a liquid crystal structure. These fibers may be dissolved in an appropriate solvent.

[00098] Spun fibers may be processed by lubricants (FIG. 3). Spun fibers may be processed by drawing, crimping, cutting, or a combination of the above to produce a tow or staple. Spun fibers may be processed by draw texturizing or draw twisting. Spun fibers may be processed by twisting and coning to produce a continuous fiber yam (FIG. 3).

[00099] Meat Weaving Technique

[00100] The meat weaving technique may comprise one or more operations of the following: cell processing, meat phase preparation, meat fiber fabrication, meat fiber processing, matrix phase (e.g., liquid matrix) preparation, meat fiber immersion, matrix binding, and product processing (FIG. 1 ). Cultured animal cells and/or recombinant animal proteins and/or animal derived ingredients can be added to enrich the product during one or more of the operations. For example, cultured animal cells may be processed and added to the meat phase and/or the matrix phase during meat phase preparation and/or matrix phase preparation (FIG. 2). The meat phase may be used for meat fiber fabrication, and fabricated meat fibers may be further processed during meat fiber processing. Meat fibers may be aligned during meat fiber processing (FIG. 2). Meat fiber fabrication and processing may occur before or after matrix phase preparation. Free meat fibers may be added to the matrix during matrix phase preparation. The matrix phase may be added to the aligned meat fibers during meat fiber immersion. The matrix phase may gel or polymerize to form a solid matrix binding the meat fibers during matrix binding. The solid matrix may be processed (e.g., sectioned into individual whole meat cuts) to yield a final product during product processing (FIG. 2).

[00101] Cell Processing

[00102] Cells may be isolated from any animal including any one or more of cattle, sheep, swine, goats, fish, chicken, turkeys, ducks, geese, ratites, and squab. In some embodiments, the cells are isolated from the longissimus thoracis muscle of a Wagyu calf. In some embodiments, the cells are isolated from the longissimus thoracis muscle of a chick. In some embodiments, the cells may be skeletal muscle cells. In some embodiments, the cells may be preadipocytes or fat cells. The cells may be myoblast stem cells. The cells may be engineered cells. The cells may be genetically modified. The cells may undergo development to increase mitotic capacity for scaleup. The cells may be grown in single cell suspension for scaleup. The cells may undergo a differentiation operation. Differentiation may increase myoglobin, myosin heavy chain, and other myogenic protein expression by meat fibers. A cell processing operation may comprise customized methods of cell functionalization, cell harvest, and preparation of cellular material as a food ingredient. Cell functionalization may occur in an in situ bioreactor or as part of a downstream processing operation. Cell processing may comprise processing cells to uptake pigments (e.g., red pigments).

[00103] Cells may be harvested after cell functionalization and further processed, which can occur in in situ bioreactors or as part of downstream processing. The cells can be rinsed with water or salt solution to wash away cell culture media or functionalization components. Water may be removed from the cell solution using filters, centrifugation, or gravity separation. This may produce a viscous, syrup like material. The cells may be stored in cold storage or other conditions that make them suitable for mixing with liquid muscle or matrix phases.

[00104] Meat phase preparation

[00105] A meat phase (e.g., liquid meat solution) may be prepared to fabricate meat fibers. The composition of the meat phase (e.g., liquid meat solution) may vary depending on the fiber fabrication method. The meat phase may be a homogenous or heterogenous mixture. The meat phase may be a solution or a suspension. In some embodiments, the meat phase (e.g., liquid muscle solution) may predominantly contain protein. In other embodiments, the meat phase may contain a more heterogenous mixture of proteins, starches, hydrocolloids, or other ingredients. In some embodiments, the meat phase comprises cells. In other embodiments, the meat phase does not comprise cells (FIG. 5). In other embodiments, the meat phase contains products of fermentation. The meat phase (e.g., liquid meat solution) may comprise ingredients that improve the physiochemical properties of the protein fibers, such as emulsifiers, gelators, stabilizers, and preservatives. The meat phase (e.g., liquid meat solution) may also comprise ingredients that enhance the nutritional and sensory properties of the meat, such as cultured animal cells; recombinant proteins produced from plants, fungi, insects, or fermentation; or animal derived ingredients. In some embodiments, the meat phase (e.g., liquid meat solution) comprises soy protein. In some embodiments, the meat phase (e.g., liquid meat solution) comprises a soy protein percentage of between 5% and 35%, 10 and 30%, or 15% and 25%. In some embodiments, the meat phase (e.g., liquid meat solution) comprises a soy protein percentage of at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, or at least 60%. The meat phase may be mixed with an alkaline aqueous solution. In some embodiments, the alkaline aqueous solution is at least 0.25%, at least 0.5%, at least 1.5%, at least 5%, at least 10%, at least 15%, at least 20%, or at least 25% alkaline water.

[00106] For example, when using wet spinning, the meat solution may be composed of soy protein isolate dissolved in an alkaline aqueous solution, where the soy protein will polymerize and precipitate once extruded through a spinneret into an acid bath. In a melt spinning example, plant proteins in powdered material form can be mixed with -25% alkaline water, plasticizing at high temperatures, and then extruding through dies that create the fiber form, In a 3D printing application, edible photoinitators can be added to the liquid protein solution to initiate photopolymerization of reactive monomers to fabricate or contribute to the fabrication of protein fibers

[00107] Meat fiber fabrication

[00108] Meat fibers can be produced from fiber spinning methods, such as one or more of melt, dry, wet, rotary, gel, dry jet wet, or electrospinning; melt blowing; interfacial polyelectrolyte complexation; extrusion or stereolithography-based 3D printing; high moisture extrusion (HME); shear cell technology; or fermentation-based systems that produce protein from carbon metabolism of microbial species.

[00109] 3D printing techniques that produce individual protein fibers may involve crosslinking agents to polymerize the fiber. In extrusion 3D printing, hydrocolloids or enzymes like transglutaminase can be mixed with the liquid meat solution. In 3D printing utilizing stereolithography, the meat solution can contain an edible photo initiator, such as riboflavin or B2 vitamin, with polymer substrates that react with free radicals produced during photo-oxidation.

[00110] In techniques that produce whole cut meat analogues without separated fibers, like HME and shear cell technology, the meat fibers can be separated in a mechanical, thermal, chemical, or biochemical process that results in shredding or peeling of the fibers.

[00111] Separate protein fibers are collected by winding around spools or similar collection instruments. The mechanical collection process produces aligned fibers like a spool of thread. These fibers can have micron, submicron and nanometer dimensions, and their diameter is determined by the fiber production method. Protein fibers may or may not contain cells at this stage.

[00112] Meat fiber processing and alignment

[00113] Meat fiber processing operations can further modify meat fibers to augment their physiochemical, material, sensory, and nutritional profiles, and collect and organize fibers in parallel. Physio chemical processing changes the material properties of the protein fibers in preparation of downstream manufacturing operations. These treatments may be used to augment and improve material properties such as texture, elastic modulus, tensile strength, elasticity, flexibility, brittleness, or porosity of fibers. Enhancing the mechanical stability of meat fibers may improve their handling throughout the manufacturing process, as well as bite and mouthfeel of the final product. Increasing porosity of the fibers may allow sub-surface penetration of compounds during organoleptic processing and may increase surface area for matrix phase (e.g., liquid matrix) immersion and binding, which may improve sensory qualities.

[00114] Physiochemical processing can include chemical, biochemical, mechanical, or temperature treatments (FIG. 6). Chemical treatments can stabilize or destabilize polymer crosslinking, strength, or elasticity via the addition of inorganic (such as iron or aluminum salts) or organic (such as cationic, anionic, and non-ionic polymers) chemicals, such as treatment with acetic anhydride, acetaldehyde, glyoxal, glutaraldehyde, a combination of glutaraldehyde and acetic anhydride. Biochemical treatments use enzymes that break covalent bonds (such as trypsin and papain proteases, or amylases) to increase fiber porosity or catalyze covalent crosslinking (such as transglutaminase). Temperature treatments can disrupt covalent bonds in fibers via freezing, fractional freezing, or drying: hot air drying (AD), freeze drying (FD), infrared drying (IR), microwave drying (MV), vacuum drying (VD). Mechanical treatments can influence the anisotropic molecular orientation of molecules in meat fibers with stretching or twisting. Increasing anisotropic molecular orientation can improve fiber strength, elasticity, and texture.

[00115] Organoleptic processing follows a meat marinating principle, where meat fibers are marinated with compounds that improve sensory qualities. Meat fibers can be treated with a solution containing any combination of the following: coloring agents, such as heme-containing proteins or red food dyes, or pigments (e.g., betanin); synthetic or natural flavorants; ions or salts (e.g., Ca²⁺ ions); vitamins (e.g., vitamin B12); cultured animal cells; recombinant animal proteins or animal-derived proteins or fats produced via molecular farming or fermentation; animal-derived ingredients; and any other plant or fungi-based ingredients that in some way improve sensory properties (FIG. 6). In some embodiments, the meat fibers are marinated with solution comprising myoglobin. The percentage myoglobin of the solution may be between 2% to 20%, 5% to 15%, or 7.5% to 12.5%. The percentage myoglobin of the solution may be at least 2%, at least 5%, at least 7.5%, at least 10%, or at least 12.5%.

[00116] Meat fiber alignment can be achieved using a mechanical process (FIG. 6). In some embodiments, the mechanical process is automated. The protein fibers can be woven or looped through a vessel (e.g., a hollow cylindrical apparatus) (FIG. 6). The ends of the fibers may be threaded through two mechanical grids or membranes, similar to a thread through canvas, which are fixed at both ends of the vessel (e.g., hollow cylindrical apparatus). This controls spatial distribution between aligned fibers where fiber density can be increased or decreased. The meat fiber density in the vessel may be between 10% to 90%, 20% to 80%, 30% to 70%, 40% to 60%, or 45% to 55%. The meat fiber density in the vessel may be at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%.

[00117] Moreover, meat fiber alignment can be performed at any time during the fiber processing operation either before or after any combination of physiochemical and organoleptic processing treatments. The timing of the fiber alignment operation can be customized for variations in process design.

[00118] Matrix phase preparation

[00119] A matrix phase (e.g., liquid matrix solution) can be prepared that can immerse and bind aligned meat fibers. Binding occurs when one or more gelling and/or crosslinking agents within the matrix phase (e.g., liquid matrix solution) catalyze covalent and/or ionic crosslinking that polymerizes a network of food polymer ingredients. The polymerized emulsion matrix can simulate extracellular matrix in muscle tissue and binds aligned protein fibers together. The preparation of the matrix phase may comprise more than one binding or processing operation.

[00120] The matrix phase can include ingredients that enhance the nutritional and sensory properties of the meat, such as cultured animal skeletal muscle and/or fat cells, recombinant animal proteins, animal-derived ingredients, and plant- or fungi-based ingredients. In some embodiments, the matrix phase comprises muscle cells. In some embodiments, the matrix phase comprises fat cells. In some embodiments, the matrix phase comprises fibroblast cells. In some embodiments, the matrix phase comprises muscle cells and fat cells and fibroblast cells (FIG. 5). The matrix phase may comprise recombinant protein, animal-derived ingredients, plant- or fungi-based ingredients, or combinations thereof. In some embodiments, the matrix phase comprises fava bean protein-rich flour (FPR). An FPR solution can be made from dry fractionated FPR produced by milling/air classification. In some embodiments, the matrix phase (e.g., liquid matrix solution) comprises an FPR percentage of between 5% and 50%, between 10% and 40%, or between 15% and 35%. In some embodiments, the matrix phase (e.g., liquid matrix solution) comprises an FPR percentage of at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%. In some embodiments, the matrix phase comprises fat cells, skeletal muscle cells, or a combination thereof. In some embodiments, the matrix phase comprises a ratio of fat cells to skeletal muscle cells of about 5:1, about 4:1, about 3:1, about 2:1, about 3:2, about 4:3, about 1 :1, about 3:4, about 2:3, about 1:2, about 1 :3, about 1:4, or about 1:5. In some embodiments, percentage of fat cells in the total cells is at least 5%, at least 10%, at least at least 20%, at least 30%, at least 40%, at least, at least 50%, or at least 60%. In some embodiments, skeletal muscle cells that are processed to increase red pigmentation are not added to matrix phase formulations that simulate marbling. The whiteish-yellow color of intramuscular fat is an important visual characteristic beef, and skeletal muscle cells containing red pigments may change the color of the matrix to pink or red. Skeletal muscle cells that are not processed to uptake red pigments can be added to the matrix phase (e.g., liquid matrix solution). In some embodiments, the matrix phase is a suspension of cells, starch, and protein solution. In some embodiments, the ratio of cells to protein solution is about 5:l, about 4:1, about 3:1, about 2:1, about 3:2, about 4:3, about 1:1, about 3:4, about 2:3, about 1:2, about 1:3, about 1:4, or about 1:5. In some embodiments, the percentage of cells in the suspension by volume is at least 5%, at least 10%, at least at least 20%, at least 30%, at least 40%, at least, at least 50%, or at least 60%. Animal, plant, or fungi ingredients can be added to the matrix phase in the form of powders, liquids, or solids, where the solids can be pulverized and shredded protein that was processed from any of the previously discussed fiber fabrication techniques, in addition to tomographic volumetric 3D printing (TVP).

[00121] The matrix phase (e.g., liquid matrix solution) may comprise colloidal particles, hydrocolloid mixtures, emulsion gels, water in oil emulsions, oil in water emulsions, or combinations thereof. The matrix phase may comprise emulsifiers, phospholipids, biosurfactants, gelling or crosslinking agents, stabilizers, preservatives, various salts acids, and bases, or combinations thereof. The matrix phase may comprise random coil biopolymers, compact biopolymers, nanofiber matrices, microfiber matrices, millifiber matrices, or combinations thereof. Hydrocolloid-based gelling agents that form a thermo-reversible polymer network showing temperature hysteresis above the normal cooking temperature range of animal meat, about 145-165°F (63-74°C) can be used. Agar is an example, which sets around about 95°F (35°C) but melts at around about 194°F (90°C). Thermo- irreversible gels, such as pectin or alginate, can also be used to form a gel thermostable above cooking temperature.

[00122] In some embodiments, high animal-, plant-, fermentation-, or fungi-based protein concentrations provide a basis for network polymerization using crosslinking and transglutaminase (TG) and/or other cross-linking enzymes and/or derivatives thereof. In some embodiments, the crosslinking is ionic. In some embodiments, the crosslinking is covalent. The enzymes may bind amine to amine groups, amine groups to starch groups, or starch groups to starch groups. Crosslinking can be enhanced with addition of plant-based albumin. In matrix phases (e.g., liquid matrix solutions) containing a high % of cultured fat cells to simulate marbling, emulsifiers may be included to form a stable emulsion within the polymerized matrixed. [00123] Salts, acids, or bases may also be added to the solution to adjust pH, provide ions for gelation, and stabilize emulsions, color, flavor, or crosslinking efficacy of the gelling agent.

[00124] Free matrix fibers can be added to the matrix phase for various applications. Fibers of different width and length can be used, such as millifibers, microfibers, and/or nanofibers can be mixed with matrix phase to provide additional structural scaffolding for binding events. This can improve cell encapsulation during enzyme binding through increasing the number of active sites for polymerization within the network. In some embodiments, these free matrix fibers are not woven or arranged in the meat phase. Free matrix fibers can be produced from a melt-blown process, shredded or mechanically dissociated, and mixed with cells and binder. This provides additional binding sites for the enzyme and improves polymerization through the matrix phase and integration of cells and fibers into the product. This can modify the mechanical and material properties of the cultivated meat product. Free matrix fibers may be randomly oriented or undergo an alignment process. For example, a flow process for the matrix may create shear forces that align fibers in parallel with fibers in the meat phase. This may yield superior texture and product quality compared to nanofibers of random alignment.

[00125] In some embodiments, the matrix phase is liquid. The liquid phase matrix phase may exhibit flow characteristics. In other embodiments, the matrix phase is a gel. A gel may be or comprise a polymerized material comprising high water content. The gel matrix phase may be or comprise a hydrated gel that does not exhibit flow characteristics.

[00126] In some embodiments, the matrix phase is semi-liquid or semi-gel. The matrix phase may comprise a heterogeneous mixture of polymerized and unpolymerized matrix components. In some embodiments, the matrix components may be polymerized before matrix binding. A semi-liquid or semi-gel phase may be used to optimize flow while creating optimal binding and product qualities. For example, nanofibers may be partially polymerized in aggregates upon addition to the meat phase but can flow with the rest of the unpolymerized fraction and undergo a final binding operation upon immersion.

[00127] In some embodiments, the matrix phase is solid. The solid matrix phase may comprise polymerized materials. In some embodiments, the polymerized materials do not associate with water molecules or water content. In some embodiments, the matrix phase is semi-solid. The semi-solid matrix phase may comprise liquid components and solid components. The solid components may associate through covalent bonding. The semi-solid matrix phase may have a lower water content than a gel matrix phase. In some embodiments, the matrix phase is a hydrated solid matrix. The hydrated solid matrix may comprise liquid components and solid components. The solid components may associate through covalent bonding. The hydrated solid matrix may comprise water. In some embodiments, the hydrated solid matrix does not form a gel from water content. In some embodiments, the matrix phase is a dehydrated solid matrix. The dehydrated solid matrix may be or comprise a polymerized network or powder. The dehydrated solid matrix may be generated by freeze drying or other methods of dehydration that can be useful for shipping or storage purposes. The dehydrated matrix may be hydrated post-assembly to create the final meat product. This hydration may improve the sensory and nutritional properties. In some embodiments, the dehydrated matrix may be hydrated through the addition of cells to the dehydrated matrix. The addition of cells to the dehydrated matrix may comprise delivery of cells in water to acellular or cellular meat fibers in the meat phase or the matrix phase. The acellular or cellular meat fibers can serve as a scaffold for encapsulating cells and water without the process of tissue engineering. The preparation of the matrix phase may include more than one binding operation where the muscle and matrix phases may be assembled and polymerized prior to a second polymerization operation once water and/or cells are added.

[00128] In some embodiments, the matrix phase comprises mixed phases. The matrix phase may comprise one or more of a liquid component, a semi-liquid component, a semi-gel component, a gel component, a semi-solid component, a hydrated solid component, and/or a dehydrated solid component.

[00129] Meat fiber immersion

[00130] In some embodiments, the matrix phase (e.g., liquid matrix solution) is added to the meat fibers during meat fiber immersion (FIG. 7). In some embodiments, a liquid matrix phase (e.g., liquid matrix solution) is poured into the apparatus containing aligned fibers. A vacuum may be applied to remove air bubbles and gas from the product (FIG. 7). In some embodiments, a gel matrix phase is added to the meat fibers and the fibers may be mechanically pressed or folded into the gel matrix phase to encapsulate the fibers. In some embodiments, the mechanical pressing or folding breaks the gel components of the gel matrix phase. In other embodiments, the mechanical pressing or folding does not break the gel components of the gel matrix phase. The gel matrix phase may be repolymerized through mechanical, chemical, or biochemical processing such as with enzymes. For example, a gel matrix phase comprising cells, nanofibers, and enzymes may be pre-polymerized and meat fibers may be mechanically lowered into the gel. Then active enzyme within the gel may repolymerize broken gel polymers and initiate a final polymerization operation between matrix and meat phases during matrix binding. In some embodiments, the meat fiber immersion occurs at a temperature of between 30°C and 70°C, between 45°C and 60°C, or between 50°C and 55°C. In some embodiments, the meat fiber immersion occurs at a temperature of at least 20°C, at least 30°C, at least 40°C, at least 50°C, at least 60°C, or at least 70°C.

[00131] Matrix binding

[00132] The gelling / crosslinking agent or agents that can be included in the matrix can react to promote matrix binding. In some embodiments, the gelling / crossing linking agent or agents promote the matrix to form a 3D network (FIG. 7). In some embodiments, the gelling / crosslinking agent or agents convert the liquid matrix phase (e.g., liquid matrix solution) to a solid gel over a defined reaction period. In some embodiments, the gelling / crosslinking agent or agents bind partially polymerized aggregates of nanofibers upon immersion. In some embodiments, the gelling / crosslinking agent or agents can repolymerize broken gel polymers and initiate a final polymerization operation between matrix and meat phases.

[00133] Multiple enzymes may be used together for binding, which includes transglutaminase, other enzymes, or derivatives thereof that bind amine groups to starch groups, or starch groups to starch groups. Additionally, improvements to natural enzyme sequences can be used to create more highly active enzymes that need lower concentrations than the wild type enzyme, therefore saving manufacturing costs while improving quality. In some embodiments, a solution of transglutaminase, other enzymes, or derivatives thereof is added to the meat fibers before meat fiber immersion, The percentage enzyme in the solution may be between 10% to 40%, 15% to 35%, or 20% to 30%. The percentage enzyme in the solution may be at least 5%, least 10%, at least 15%, at least 20%, at least 25%, at least 30, or at least 35%.

[00134] In some embodiments, a vacuum or compression pressure is applied to remove air bubbles and promote network polymerization or binding between the meat fibers and matrix phase (FIG. 7). In some embodiments, the matrix binding occurs at a temperature of between 0°C and 15°C, between 2°C and 10°C, or between 3°C and 7°C. In some embodiments, the matrix binding occurs at a temperature of at most 2°C, at most 4°C, at most 7°C, at most 10°C, at most 15°C, or at most 25°C.

[00135] Product processing

[00136] Meat weaving products can be sectioned into individual whole meat cuts, packaged, and placed in cold storage. Products can be packaged in air- and leak-proof packages under vacuum. In some cases, a specific gas will be pumped into the package to preserve flavor and quality. Packages that are airtight and leak proof can enable juices accumulate in the package without evaporating, in contrast with plastic wrapped packaging typically used by most supermarkets. [00137] Meat Phase and Matrix Phase Compositions

[00138] The composition of the meat phase and matrix phase and processes involved in cell functionalization and fiber processing can be fine-tuned to impart specific sensory and nutritional properties in alignment with species-specific and product-specific characteristics.

[00139] The meat phase or matrix phase may be composed of, but is not limited to, any of the following ingredients in any combination or concentration: Cultured animal cells including skeletal muscle cells, fat cells, fibroblast cells; Synthetic or chemical sources of, for example, any synthetic compound that affects flavor, color, gelation or crosslinking efficacy, or impart other useful functional qualities, polymerizing, crosslinking, binding, gelling, or emulsifying agents, vitamins, nutrients, antioxidants, or ions (such as calcium or zinc), nucleotides, amino acids or peptides, salts, phosphate salts, acids, bases, polyvinyl alcohols, polyacrylamides, polyethylene oxides, glycerols, or other chemicals, solvents, such as water; Plant, animal, algae, fungi, insect, or microbe derived ingredients, for example, recombinant or animal-derived proteins that impart functional, organoleptic, or nutritional value, such as myoglobin, gelatin, collagen, caseinate, albumin, keratin, or egg white proteins, biological pigments, such as betalains, anthocyanins, phycoerythrin, or carotenoids, starches, modified starches, maltodextrins, hydrocolloids, monosaccharides, disaccharides, polysaccharides, gums (such as Arabic gum, xanthan gum, locust bean gun, gellan gum, cellulose gum), alginates or agars, carrageenans, such as kappa, lambda, or iota carrageenan, pectins, micro- or nanofibers, such as cellulose, methylcellulose, carboxymethylcellulose, or zein, protein flours, isolates, concentrates, or extracts, oils or fats, such as triglycerides, free fatty acids, or omega 3-6-9 fatty acids, biosurfactants, emulsifying agents, such as saponins, proteins, phospholipids (like lechithins), or polysaccharides, polymerizing or binding agents such as enzymes, gelling agents, or other crosslinking agents such as photo-initiators (e.g., riboflavin), transglutaminase, other cross-linking enzymes, or derivatives thereof, , vitamins, nutrients, antioxidants, ions (such as calcium or zinc), nucleases and/or nucleotides (e.g., Flippase / FRT system), amino acids or peptides any other natural compound that affects flavor, color, gelation or crosslinking efficacy, or impart other useful functional qualities. Plant, animal, fungi, insect, algae, or microbial derived ingredients may be from any species, for example, plant species including soy, pea, fava bean, potato, carrot, chickpea, lentils, lupin, mung bean, wheat gluten, sunflower, rapeseed, fungal species including mycelium, common mushrooms, microbial species including yeast, bacteria, and algae including unicellular or multicellular algae, red algae; and cultivated cell line ingredients from any species, for example, any animal species living or extinct, such as cow, pig, lamb, goat, bison, deer, turkey, chicken, duck, fish, salmon, tuna, tilapia, shark, crab, shrimp, lobster, marine mammal, dolphin, whale, dog, cat, lion, or mammoth. [00140] The meat phase or matrix phase may comprise colloidal particles, hydrocolloid mixtures, emulsion gels, water in oil emulsions, oil in water emulsions, or combinations thereof.

[00141] These methods canbe used to produce any meat product from any breed of any species living or extinct: such as beef, pork, mutton, poultry, chicken (e.g., chicken breast), turkey (e.g., turkey breast), sushi, filet, wagyu, angus, kobe, or mammoth.

[00142] The meat phase or matrix phase may comprise polymers or fibers or combinations thereof. The polymers may be random coil biopolymers or compact biopolymers. The fibers may be nanofibers, microfibers, millifibers, or combinations thereof. The meat phase or matrix phase may comprise nanofiber matrices, microfiber matrices, or millifiber matrices providing structure throughout.

[00143] Meat fibers and matrix formulations can be prepared from any, but are not limited to, of the following techniques: Fiber spinning methods, such as melt, dry, wet, rotary, gel, dry jet wet, or electrospinning; melt blowing; interfacial polyelectrolyte complexation; extrusion or stereolithography-based 3D printing; high moisture extrusion (HME); shear cell technology; fermentation-based systems that produce protein from carbon metabolism of microbial species; gelation; polymerization; or emulsification.

[00144] Fibers produced from wet and melt spinning can simulate different aspects of meat products, such as cells or extracellular matrix.

[00145] Spun wet fibers can compose or simulate the “cellular” material of meat. The spun wet fibers may utilize different protein & starch profiles than melt spun fibers because high temperatures are not involved as in a plastification process in melt spinning. The spun wet fibers can yield higher protein content than melt spun fibers. Furthermore, wet fiber spinning can simulate the protein content of meat fibers. Natural or synthetically derived ingredients can be used to color the final wet fibers, such as myoglobin. Myoglobin protein can be sourced from non-genetically engineered sources or synthesized using chemical or biological engineering. Myoglobin can be produced recombinantly from an expression system and can be mixed with the wet fiber “dope” and spun into various wet fibers. The myoglobin may be polymerized within the dope and serve as a flavoring, nutritional, and coloring agent for meat fibers. Myoglobin can also be used in a melt spinning process to color fibers.

[00146] Melt spun fibers can compose or simulate extracellular matrix. Melt spun fibers may simulate extracellular matrix proteins, such as collagens or elastin that control the texture and mouthfeel of meat products. Furthermore, melt spun fibers can be woven into various 2D and 3D shapes to yield customizable material properties that simulate macroscopic layers and structures of extracellular matrix. An example is taking a starch-based melt spun fiber and weaving it into a 2D mesh that can be used to assemble and layer meat fibers together. Melt blown fibers can be used similarly, where a fibrous blown layer and be used as a film or mesh to assemble meat components. Additionally, melt spun fibers may comprise monocomponent, bicomponent, tricomponent, or multicomponent ingredients. Melt spun fibers may comprise proteins, starches, or other ingredients. Using more than one component may change the material properties of the fiber and may improve tensile strength, elasticity, and/or sensory properties. Mono component, bicomponent, tricomponent, or multicomponent ingredients can also be used to simulate different types of extracellular matrix.

[00147] Free matrix fibers (e.g., meat fibers) can be added to the matrix phase for various applications. Fibers of different width and length can be used, such as millifibers, micro fibers, and/or nanofibers can be mixed with matrix phase to provide additional structural scaffolding for binding events. This can improve cell encapsulation during enzyme binding through increasing the number of active sites for polymerization within the network. Free matrix fibers (e.g., meat fibers) can be wet spun fibers or melt spun fibers or a combination thereof. In some embodiments, these free matrix fibers are not woven or arranged in the meat phase.

[00148] Nanofibers are fibers that can be produced from a spinning process that are of nanoscale width and between 1 nm and 1,000 nm in length. Microfibers are fibers that can be produced from a spinning process that are either nanoscale or microscale in width and between 1 ,000 nm and 1 ,000,000 nm in length. Millifibers are fibers that can be produced from a spinning process that are either nanoscale, microscale, or milliscale in width and over 1,000,000 nm in length.

[00149] For example, nanofibers can be produced from a melt-blown process, shredded or mechanically dissociated, and mixed with cells and binder. This can provide additional binding sites for the enzyme and improve polymerization through the matrix phase and integration of cells and fibers into the product. This can also modify the mechanical and material properties of the cultivated meat product.

[00150] Free matrix fibers may be randomly oriented or undergo an alignment process. For example, a flow process for the matrix may create shear forces that align fibers in parallel with fibers in the meat phase. This may yield superior texture and product quality compared to nanofibers of random alignment.

EXAMPLES

[00151] Wagyu Ribeye steak

[00152] Determine target product characteristics: In this example, designing a cell-based Wagyu rib eye steak first involves assessing meat characteristics that influence sensory properties, such as meat fiber type composition, fatty acid composition, IMF%, and chemical composition of the longissimus thoracis. The average IMF of the thoracic muscle is 31.5%. The chemical composition of high quality wagyu is about 22% crude protein, 31% crude fat, and 47% water. Adipocytes and skeletal muscle cells is grown to volumes that yield a 2:3 ratio of skeletal muscles to fat cells. Cultured cells may not need to compose the entire 22% of crude protein and 31% of crude fat, since plant-based ingredients can add bulk and texture. However, since IMF is a hallmark of Wagyu beef, cell-based products may be held up to the JMGA standard of BMS, and require higher % of crude fat.

[00153] Design bioprocess to meet product characteristics: The cell isolation, cell line development strategy, culture process, and product formulation is determined by the previous characteristics. First, skeletal muscle and preadipocytes are isolated from the longissimus thoracis muscle of a Wagyu calf. These two cell lines undergo development to increase mitotic capacity for scale up. The skeletal muscle cells are grown in single cell suspension for scale up, then undergo a differentiation operation. The cells first undergo cell aggregation into micro tissues with low agitation rates inside the vessel. Then after sufficient tissue synthesis (e.g., below ~ 300um in thickness), differentiation media are supplemented to the culture. Differentiation may increase myoglobin, myosin heavy chain, and other myogenic protein expression by meat fibers.

[00154] Cell processing — skeletal muscle and fat cells are harvested from a bioprocess and stored in conditions making them suitable for mixing with liquid muscle or matrix fractions.

[00155] Liquid meat preparation - a solution of soy protein is dissolved in an alkaline aqueous solvent containing NaOH, such as a composition of 15-25% soy protein isolate, 0.5-1.5% NaOH, 0.05-0.3% xanthate or xanthate reaction product, and recombinant myoglobin.

[00156] Matrix phase (e.g., liquid matrix) preparation - Dry fractionated fava bean protein-rich flour (FPR) is produced by milling/air classification, A 30% solution of FPR is dissolved into 10 mM phosphate buffer (pH 7) under agitation and heated at about 90°C for about 30 min and cooled to about 53°C, which is below the lower end of the cooking temperature of meat (for example, about 145°F or 63°C) and fat rendering (for example, about 130- 140°F or 54-60°C) but above gelation onset temperature for FPR (for example, about 52.5-59.5°C). A slurry of cultivated fat and skeletal muscle cells at a 3:1 ratio is slowly dispersed into the protein solution while maintaining a temperature of about 53°C under low agitation speed until there is a 50-50 ratio of cells to protein solution volume, giving a 15% final FPR concentration. Importantly, the matrix phase (e.g., liquid matrix solution) does not undergo a high energy homogenization operation, which may rupture the cells.

[00157] Meat fiber fabrication - a wet spinning method is used to develop meat fibers from the liquid meat solution. First, the liquid meat solution is extruded through a spinneret in an acid coagulation bath between about pH 3-4 composed of about 2% H₂SO₄, 18%, Al₂(SO₄)₃, 12% NaCl and 68% H₂O. This produces fibers that polymerize once the alkaline solvent reacts with the acid bath and leeches out from the fiber. The soy fibers are then stretched to about 400% their original length along spools in the coagulation bath, and the fiber is run through a washing bath that increases the pH to 5.6-6.4 (the typical pH range for meat), composed of 12% NaCl, 1% Al₂(SO₄)₃, and 87% H₂O. Finally, the fibers are then run through a bath of acetic anhydride/acetic acid (9:1 ratio) to increase tenacity, elongation, and flexibility of fibers and improve handling qualities.

[00158] Meat fiber processing - Meat fiber alignment is achieved using an automated mechanical process where the protein fibers are woven or looped through a hollow cylindrical apparatus called the meat weaving vessel. In the case of wagyu steak, the fiber density is approximately 50% of the volume of the cylinder is occupied by meat fibers, leaving the other 50% of volume for the matrix phase (e.g., liquid matrix).

[00159] The meat fibers are processed with papain enzyme solution to increase porosity of fibers. The papain solution is drained and the fibers are rinsed with water. A marination solution containing 10% myoglobin in water is pumped into the chamber and the fibers are soaked for 1 -5 hours, then the marinade solution is drained and fibers are rinsed once with water. The temperature of the meat weaving vessel is cooled to 4°C and 25% solution of transglutaminase in water is then pumped into the chamber to soak into the fibers for 5 minutes, and then the TG solution is drained. The low temperature of the vessel lowers the kinetic activity of the TG to prevent heat setting.

[00160] Meat fiber immersion - the matrix phase (e.g., liquid matrix solution), held at about 53°C, is pumped into the cooled meat weaving vessel.

[00161] Matrix binding - A vacuum is applied to remove air bubbles and promote binding between fibers and matrix and the vessel is held at 4°C overnight. The temperature of the vessel cools the matrix, promoting gelling within the matrix and enzymatic TG activity that binds the fibers and the protein in the matrix.

[00162] Chicken Breast

[00163] Determine target product characteristics: In this example, designing a cell-based chicken breast first involves assessing meat characteristics that influence sensory properties, such as meat fiber type composition, fatty acid composition, IMF%, and chemical composition of the longissimus thoracis. Chicken is a lean meat that doesn’t contain IMF, unlike the marbling found in beef, and is 70% water, 20% of crude protein, and 5% of crude fat. Therefore, chicken cell lines can be primarily composed of skeletal muscle cells. Cultured cells may not need to compose the entire 20% of crude protein and 5% of crude fat, since plant-based ingredients can add bulk and texture. Chicken contains low levels of myoglobin compared to beef.

[00164] Design bioprocess to meet product characteristics: The cell isolation, cell line development strategy, culture process, and product formulation is determined by the previous characteristics. First, skeletal muscle cells is isolated from the longissimus thoracis muscle of a chick. These cell lines undergo development to increase mitotic capacity for scale up. The skeletal muscle cells is grown in single cell suspension for scale up, then undergo a differentiation operation. The cells first undergo cell aggregation into micro tissues with low agitation rates inside the vessel. Then after sufficient tissue synthesis (e.g., below ~300um in thickness), differentiation media is supplemented to the culture. Differentiation may increase myoglobin, myosin heavy chain, and other myogenic protein expression by meat fibers.

[00165] Cell processing - chicken skeletal muscle cells is harvested from a bioprocess and stored in conditions making them suitable for mixing with liquid muscle or matrix solutions.

[00166] Liquid meat preparation - a solution of soy protein is dissolved in an alkaline aqueous solvent containing NaOH, such as a composition of 15-25% soy protein isolate, 0.5-1.5% NaOH, and 0.05-0.3% xanthate or xanthate reaction product.

[00167] Matrix preparation — Dry fractionated fava bean protein-rich flour (FPR) is produced by milling/air classification. A 30% solution of FPR is dissolved into 10 mM phosphate buffer (pH 7) under agitation and heated at about 90°C for about 30 min and cooled to about 53°C, which is below the lower end of the cooking temperature of meat (for example, about 145°F or 63°C) and fat rendering (for example, about 130-140°F or 54-60°C) but above gelation onset temperature for FPR (for example, about 52.5-59.5°C). A slurry of cultivated chicken skeletal muscle cells is slowly dispersed into the protein solution while maintaining a temperature of about 53°C under low agitation speed until there is a 50-50 ratio of cells to protein solution volume, giving a 15% final FPR concentration. Importantly, the matrix phase (e.g., liquid matrix solution) does not undergo a high energy homogenization operation, which may rupture the cells.

[00168] Meat fiber fabrication - a wet spinning method is used to develop meat fibers from the liquid meat solution. First, the solution is extruded through a spinneret in an acid coagulation bath between pH 3-4 composed of 2% H₂SO₄, 18%, Al₂(SO₄)₃, 12% NaCl and 68% H₂O. This produces fibers that polymerize once the alkaline solvent reacts with the acid bath and leeches out from the fiber. The soy fibers are then stretched to 400% their original length along spools in the coagulation bath, and the fiber is run through a washing bath that increases the pH to 5.6-6.4 (the typical pH range for meat), composed of 12% NaCl, 1% Al₂(SO₄)₃, and 87% H₂O. Finally, the fibers are then run through a bath of acetic anhydride/acetic acid (9:1 ratio) to increase tenacity, elongation, and flexibility of fibers and improve handling qualities.

[00169] Meat fiber processing - Meat fiber alignment is achieved using an automated mechanical process where the protein fibers are woven or looped through a hollow cylindrical apparatus called the meat weaving vessel. In the case of chicken breast, the fiber density is approximately 70% of the volume of the cylinder is occupied by meat fibers, leaving the other 30% of volume for the matrix phase.

[00170] The meat fibers are processed with papain enzyme solution to increase porosity of fibers. The papain solution is drained and the fibers are rinsed with water. A marination solution containing 1 % myoglobin and 5% chicken skeletal muscle cells boiled in salt water, to form a type of chicken broth, is pumped into the chamber and the fibers are soaked for about 1-5 hours, then the marinade solution is drained and fibers are rinsed once with water. The temperature of the meat weaving vessel is cooled to about 4°C and 25% solution of transglutaminase in water is then pumped into the chamber to soak into the fibers for 5 minutes, and then the TG solution is drained. The low temperature of the vessel lowers the kinetic activity of the TG to prevent heat setting.

[00171] Meat fiber immersion - the matrix phase (e.g., liquid matrix solution), held at 53°C, is pumped into the cooled meat weaving vessel.

[00172] Matrix (e.g., liquid matrix) binding - A vacuum is applied to remove air bubbles and promote binding between fibers and matrix and the vessel is held at about 4°C overnight. The temperature of the vessel cools the matrix, promoting gelling within the matrix and enzymatic TG activity that binds the fibers and the protein in the matrix.

[00173] To clarify the use of and to hereby provide notice to the public, the phrases "at least one of <A>, <B>, ... and <N>" or "at least one of <A>, <B>, ... or <N>" or "at least one of <A>, <B>, ... <N>, or combinations thereof or "<A>, <B>, ... and/or <N>” are defined by the Applicant in the broadest sense, superseding any other implied definitions hereinbefore or hereinafter unless expressly asserted by the Applicant to the contrary, to mean one or more elements selected from the group comprising A, B, ... and N. In other words, the phrases mean any combination of one or more of the elements A, B, ... or N including any one element alone or the one element in combination with one or more of the other elements which may also include, in combination, additional elements not listed. Unless otherwise indicated or the context suggests otherwise, as used herein, "a" or "an" means "at least one" or "one or more."

[00174] The disclosure is not restricted by the examples given in the specification. The disclosure comprises any feature or combination of features described in the specification and claims, even if not explicitly depicted in this specific combination.

[00175] The foregoing disclosure is not intended to be limiting.

[00176] Other objects, advantages and novel features of the present invention are apparent from the foregoing detailed description of the one or more preferred embodiments, examples and aspects. It should be recognized that the one or more examples in the disclosure are non-limiting examples and that the present disclosure is intended to encompass variations and equivalents of these examples.

Claims

CLAIMS:

1. A method for meat production, comprising:

(a) providing a meat phase;

(b) fabricating meat fibers from the meat phase;

(c) aligning the meat fibers to obtain aligned meat fibers;

(d) providing a matrix phase;

(e) immersing the aligned meat fibers in the matrix phase; and

(f) using one or more gelling or cross-linking agents to promote the matrix phase to gel or polymerize to form a solid matrix comprising the meat fibers, wherein: (i) in (a), the meat phase comprises cultured animal cells; or (ii) in (b), the matrix phase comprises cultured animal cells.

2. The method of claim 1, further comprising processing the solid matrix into a final meat product.

3. The method of claim 2, further comprising sectioning the final meat product into individual whole meat cuts.

4. The method of claim 1 , wherein, in (a) the meat phase comprises cultured animal cells.

5. The method of claim 1, wherein in (a) the matrix phase comprises cultured animal cells.

6. The method of claim 1, wherein: (i) in (a), the meat phase comprises cultured animal cells; and (ii) in (b), the matrix phase comprises cultured animal cells.

7. The method of claim 1, wherein the meat phase comprises recombinant animal protein or animal-derived ingredients.

8. The method of claim 1 , wherein the meat phase comprises products of fermentation.

9. The method of claim 8, wherein the meat phase comprises myoglobin.

10. The method of claim 1, wherein the matrix phase comprises recombinant animal protein or animal-derived ingredients.

11. The method of claim 1 , wherein the matrix phase comprises products of fermentation.

12. The method claim 11 , wherein the matrix phase comprises myoglobin.

13. The method of claim 1 , wherein the meat phase and the matrix phase are separately formulated.

14. The method of claim 1, wherein the cultured animal cells are skeletal muscle cells or fat cells.

15. The method of claim 1, comprising functionalizing the cultured animal cells or processing the cultured animal cells to uptake biological materials.

16. The method of claim 1, comprising functionalizing the cultured animal cells or processing the cultured animal cells to uptake red pigments.

17. The method of claim 1, further comprising rinsing the cultured animal cells with water or a salt solution to wash away cell culture media or functionalization components.

18. The method of claim 17, wherein the water is removed from the cultured animal cells using filters, centrifugation, or gravity separation.

19. The method of claim 1 , further comprising culturing animal cells to obtain the cultured animal cells.

20. The method of claim 19, further comprising storing the cultured animal cells after culturing in cold storage and adding the cultured animal cells to the meat phase prior to or during (a) or to the matrix phase prior to (d).

21. The method of claim 1, wherein, in (a), the meat phase comprises a fiber solution comprising one or more of proteins, hydrocolloids, or other ingredients.

22. The method of claim 1, wherein the meat phase comprises one or more recombinant proteins produced from plants, fungi, insects; or a protein produced from fermentation.

23. The method of claim 1 , further comprising treating the meat fibers or aligned meat fibers with chemical, biochemical, mechanical, or temperature treatments.

24. The method of claim 1, wherein (b) comprises using wet spinning to fabricate meat fibers.

25. The method of claim 1 , wherein (b) comprises using melt spinning to fabricate meat fibers.

26. The method of claim 1, wherein, in (a), the meat phase comprises a soy protein isolate dissolved in an alkaline solution.

27. The method of claim 26, further comprising extruding the soy protein isolate through a spinneret and into an acid bath.

28. The method of claim 1, wherein (b) further comprises mixing plant proteins or starches in powdered material form with alkaline water and plasticizing at high temperatures to obtain plasticized mixture, and extruding plasticized mixture through dies to generate meat fibers.

29. The method of claim 1, wherein (b) further comprises dry spinning, rotary spinning, gel spinning, dry jet wet spinning, or electrospinning to generate meat fibers.

30. The method of claim 1, wherein (b) comprises melt blowing, interfacial polyelectrolyte complexation, extrusion or stereolithography-based three-dimensional (3D)-printing, high moisture extrusion (HME), shear cell technology, or fermentation.

31. The method of claim 1, wherein (b) comprises 3D-printing or adding edible photoinitators to the meat phase to initiate photopolymerization of reactive monomers to generate the meat fibers.

32. The method of claim 1, wherein (b) comprises using 3D-printing to produce meat fibers and using hydrocolloids or enzymes mixed with the meat phase to cross-link or polymerize the meat fibers.

33. The method of claim 1, wherein (b) comprises using 3D-printing utilizing stereolithography and in (d) the matrix phase comprises an edible photo-initiator.

34. The method of claim 1, wherein (b) comprises using HME or shear cell technology and the method further comprises separating the meat fibers using a mechanical, thermal, chemical, or biochemical process that results in shredding or peeling of the meat fibers.

35. The method of claim 1 , wherein the aligned meat fibers have micron, submicron or nanometer dimensions.

36. The method of claim 1, wherein (b) further comprises processing the meat fibers to modulate one or more of texture, elastic modulus, tensile strength, elasticity, flexibility, brittleness, porosity, and mechanical stability of meat fibers.

37. The method of claim 1 , further comprising treating the meat fibers or aligned meat fibers with enzymes to break covalent bonds or to catalyze covalent cross-linking of the meat fibers or aligned meat fibers.

38. The method of claim 37, further comprising treating the meat fibers or aligned meat fibers with enzymes to break covalent bonds and wherein the enzymes comprise trypsin, papain proteases, or amylases.

39. The method of claim 37, further comprising treating the meat fibers or aligned meat fibers with enzymes to catalyze covalent cross-linking of the meat fibers or aligned meat fibers wherein the enzymes comprise transglutaminase, other cross-linking enzymes, or derivatives thereof.

40. The method of claim 1, further comprising chemical treatments to stabilize or destabilize cross -linking, strength, or elasticity of the meat fibers or aligned meat fibers.

41. The method of claim 40, wherein the chemical treatments comprise treatment with acetic anhydride, acetaldehyde, glyoxal, glutaraldehyde, or a combination of glutaraldehyde and acetic anhydride.

42. The method of claim 1, further comprising freezing, fractional freezing, or drying the meat fibers or aligned meat fibers.

43. The method of claim 42, wherein drying comprises hot air drying (AD), freeze drying (FD), infrared drying (IR), microwave drying (MV), or vacuum drying (VD).

44. The method of claim 1, further comprising stretching or twisting the meat fibers or aligned meat fibers.

45. The method of claim 1, further comprising marinating the meat fibers or aligned meat fibers.

46. The method of claim 1 , further comprising treating the meat fibers or aligned meat fibers with a solution comprising one or more of a coloring agent, a flavorant; cultured animal cells; a recombinant animal protein, and a fat.

47. The method of claim 1, wherein (c) comprises using an automated mechanical process, wherein the automated mechanical process comprises: (i) weaving protein fibers through a hollow cylindrical apparatus having a first end and a second end; and (ii) threading the protein fibers through a first mechanical grid or membrane fixed at the first end of the cylindrical apparatus and a second mechanical grid or membrane fixed at the opposing second end of the cylindrical apparatus.

48. The method of claim 1, wherein the matrix phase comprises one or more of salts, acids, or bases.

49. The method of claim 1, wherein (e) comprises adding the matrix phase into an apparatus comprising the aligned meat fibers and applying a vacuum or compression.

50. A composition comprising:

(i) cultured animal cells;

(ii) a polymerizing, crosslinking, binding, gelling, or emulsifying agent; and

(iii) a recombinant animal protein or an animal-derived protein.

51. The composition of claim 50, further comprising plant, animal, fungi, insect, algae, or microbial derived ingredients.

52. The composition of claim 50, wherein the cultured animal cells comprise skeletal muscle cells, fat cells, or a combination thereof.

53. The composition of claim 50, further comprising one or more ingredients derived &om soy, pea, fava bean, potato, carrot, chickpea, lentils, lupin, mung bean, wheat gluten, sunflower, rapeseed, mycelium, common mushrooms, yeast, bacteria, or red algae.

54. The composition of claim 50, further comprising one or both of a stabilizer or a preservative.

55. The composition of claim 50, further comprising one or more of a vitamin, a nutrient, antioxidants, an ion, an amino acid, a peptide, a salt, a phosphate salt, an acid, a base, a polyvinyl alcohol, a polyacrylamide, a polyethylene oxide, a glycerol, or a solvent.

56. The composition of claim 50, further comprising one or more of myoglobin, gelatin, collagen, caseinate, albumin, or egg white proteins.

57. The composition of claim 50, further comprising a pigment.

58. The composition of claim 57, wherein the pigment comprises a betalain, an anthocyanin, a phycoerythrin, or a carotenoid.

59. The composition of claim 50, further comprising a starch.

60. The composition of claim 59, wherein the starch comprises a modified starch or maltodextrin.

61. The composition of claim 50, further comprising one or more of a hydrocolloid, a monosaccharide, a disaccharide, or a polysaccharide.

62. The composition of claim 61, further comprising a gum.

63. The composition of claim 62, wherein the gum comprises Arabic gum, xanthan gum, locust bean gun, gellan gum, or cellulose gum.

64. The composition of claim 50, further comprising one or more of an alginate, an agar, a carrageenans, a micro-fiber or a nano fiber.

65. The composition of claim 50, further comprising one or more protein flours, isolates, concentrates, or extracts.

66. The composition of claim 50, further comprising oils or fats.

67. The composition of claim 66, wherein the oils or fats comprise triglycerides, free fatty acids, or omega 3-6-9 fatty acids.

68. The composition of claim 50, wherein the polymerizing, crosslinking, binding, gelling, or emulsifying agent is selected from the group consisting of a saponin, a protein, a phospholipid, and a polysaccharide.

69. The composition of claim 50, wherein the polymerizing, crosslinking, binding, gelling, or emulsifying agent comprises a photo-initiator.

70. The composition of claim 50, wherein the polymerizing, crosslinking, binding, gelling, or emulsifying agent comprises a cross-linking enzyme.

71. The composition of claim 70, wherein the cross-linking enzyme is a transglutaminase or a derivative thereof.

72. A meat phase for fabricating meat fibers comprising cultured animal cells, proteins, and hydrocolloids.

73. The meat phase of claim 72, further comprising an emulsifier, a gelator, a stabilizer, a preservative, or a combination thereof.

74. The meat phase of claim 72, wherein the proteins are recombinant proteins produced from plants, fungi, insects; produced from fermentation; or produced from animal-derived proteins.

75. The meat phase of claim 72, further comprising soy protein isolate or plant protein dissolved in an alkaline aqueous solution, the alkaline aqueous solution being at least 25% alkaline water.

76. The meat phase of claim 72, further comprising edible photoinitators.

77. The meat phase of claim 72, further comprising a fiber solution.

78. A matrix phase for binding meat fibers comprising:

(i) cultured animal skeletal muscle or fat cells;

(ii) recombinant animal proteins or animal-derived proteins; and

(iii) plant, fermentation, or fimgi-based ingredients.

79. The matrix phase of claim 78, further comprising one or more of an emulsifier, a gelling or crosslinking agent, a stabilizer, a preservative, a salt, an acid, or a base.

80. The matrix phase of claim 78, wherein the gelling or crosslinking agent comprises a hydrocolloid-based gelling agent.

81. The matrix phase of claim 78, further comprising a gelling agent selected from the group consisting of agar, pectin, and alginate.

82. The matrix phase of claim 78, further comprising an edible photo-initiator.

83. The method of claim 82, wherein the edible photo-initiator comprises riboflavin or a polymer substrate.

84. The matrix phase of claim 78, wherein the plant or fungi ingredients are in the form of a powder, a liquid, or a solid.

85. The matrix phase of claim 84, wherein the plant or fungi ingredients are in the form of a solid, and wherein the solid is a pulverized and a shredded protein.

86. The matrix phase of claim 78, further comprising transglutaminase or derivatives thereof.

87. The matrix phase of claim 78, further comprising cultured chicken skeletal muscle cells and cultured fat cells, wherein the matrix phase comprises a higher amount of cultured chicken skeletal muscle cells to cultured fat cells, and wherein the matrix phase has a higher amount of protein than it does fat.

88. The matrix phase of claim 78, further comprising cultured bovine skeletal muscle cells and cultured fat cells, wherein the matrix phase comprises a higher amount of cultured fat cells to cultured bovine skeletal muscle cells and a higher amount of cultured cells to other ingredients.

89. The matrix phase of claim 78, further comprising skeletal muscle cells lacking red pigments.

90. The matrix phase of claim 78, further comprising skeletal muscle cells comprising red pigments.