Continuous drawing and collection of plant protein isolate hollow fibers

Abstract

The present invention is directed toward methods of manufacture of edible plant protein isolate-based membranes useful for the production of cultured meat products.

Description

Attorney Docket No: P25-032-SEC-W001CONTINUOUS DRAWING AND COLLECTION OF PLANT PROTEIN ISOLATE HOLLOW FIBERSRelated Applications

[0001] The present application claims the benefit of priority of U.S.Provisional Patent Application No.: 63 / 754,862, filed February 6, 2025, the entire content of which is incorporated herein by reference.Background

[0002] Membrane dimensions, integrity, and pore properties are paramount for effective use in membrane-based bioreactors.Membranes need to be self-supporting to allow for the transfer of media and nutrients through the membrane without interfering support structures and to allow for greater surface area for the culturing of adherent cells. Further, for the production of edible food stuffs, membranes need to be made of materials generally recognized as safe (GRAS). Still further, making membranes which are edible, both from a technical aspect ( / .e., non-toxic and digestible) and from a practicable, consumer acceptable aspect ( / .e., having texture and mouth feel acceptable to consumers) has not been achieved in the art.

[0003] The production of such membranes, whether flat sheet (for example, nano porous membranes) or fibers (for example, hollow fibers or solid fibers) has been elusive. This is because the generation of the desired mechanical integrity along with the desired characteristics necessary for cell growth and tissue formation and for the generation of a cultured meat product acceptable to the consumer must often times align competing requirements with physical production and material constraints. To the best of our knowledge no one has successfully navigated this complex and intertwined journey to produce a membrane having the desired and needed characteristics for the production of a cultured meat product.

[0004] What is needed are membranes and methods of production thereof, of edible membranes useful in membrane-based bioreactors that are suitable for production of a structured cultured meat product.Attorney Docket No: P25-032-SEC-W001Summary of the Invention

[0005] The present invention is directed toward methods of production of membranes, particularly hollow fiber membranes, that have the physical characteristics necessary for use in the production of cultured meat, particularly, structured cultured meat, and methods of use therewith.

[0006] The membranes of the present invention are plant protein isolate-based or predominately plant protein isolate-based. The formation of membranes, especially hollow fiber membranes, having the desired characteristics necessary for use in the production of consumable cultured meat (e.g., structured cultured meat) has been elusive in the art. A structured cultured meat product needs to have a certain and desired texture, mouth feel, firmness, density, etc., to evoke the pleasures and gratifications of eating natural meat. Part of this equation is the framework on which the meat cells (and other cells) are grown. It must give the cells the proper foundation in which to grow in a manner that mirrors natural meat while at the same time give the mouth feel a consumer would expect when eating a natural meat product. In other words, the “scaffolding” or “framework” on which the cultured meat is grown must be able to satisfy many competing requirements for commercial production cell culture, both structurally and physiologically, while also being edible and being acceptable to the general public.

[0007] The plant protein-based hollow fibers of the present invention we believe give the best opportunity to meet the desired requirements of commercial production growth of cells, digestibility and mouth feel in a cultured meat product. However, the production of these fibers has not been without significant developmental problems. Traditional hollow fiber production methods, when used for the production of plant protein-based hollow fibers, have resulted in inconsistent results at best, and disastrous results at worst. Traditional production methods have often resulted in collapsed hollow fibers and / or hollow fibers of poor strength ( / .e., walls to thin or weak to support cell growth orAttorney Docket No: P25-032-SEC-W001support a dense cell mass) or restrictive wall porosity ( / .e., pores being too small to allow for the efficient and necessary transfer of media for the required cell growth and density). Collapsed hollow fibers are not capable of providing the required nutrients to the cell mass to generate a dense meat product because culture media cannot flow through them.

[0008] The invention disclosed herein provides methods useful and suitable to produce a plant protein-based hollow fiber of suitable dimensions, structure and porosity for the production of a structured cultured meat product. The present invention also provides hollow fibers ( / .e., hollow fiber membranes) made with the processes and methods of the present invention. Furthermore, the processes of the present invention can also be used to solid fiber and produce sheet membranes with similar characteristics for strength, porosity and texture.

[0009] We teach herein that, surprisingly, the rate of coagulation of membranes as well as their mechanical stability throughout formation, specifically plant protein-based hollow fiber membranes, is a predictor of the scalability of the process. For plant-based protein isolate membranes, the rate of coagulation is typically low due to the nature of the materials used and that the mechanical integrity of the partially formed membranes is too weak to sustain the production process {i.e., the drawing of the membrane) without the fiber collapsing or breaking. We have developed in this invention a new and non-obvious method of processing that allows for a faster rate of dehydration of the protein membrane which enables and facilitates membrane production that results in suitable production speeds and the desired mechanical integrity of the membrane. In this nascent field, we have developed this crucial and previously unknown process for manufacturing edible plant protein-based membranes.

[0010] In this regard, this invention relates to a process for the manufacture of such a product. In prior art scalable production processes, membranes are typically made out of synthetic organic polymers. Whether in flat sheet or fiber format, the formation of theAttorney Docket No: P25-032-SEC-W001membranes occurs through a liquid-to-solid transition of the polymer solution in a formulation bath. In the case of inorganic polymers, the time-scale of such physical transition can be highly tuned to regulate both pore size and also the stability of the membrane during its formation steps. For such reasons, inorganic polymers dominate the filtration and the size exclusion membrane industry.

[0011] No edible plant-based protein isolate membranes of the production characteristics desired in this application exist today to the best of our knowledge. W02024 / 209200 to Kalvotech teaches a process for making edible hollow fibers. However, their process is complicated and costly involving multiple post processing steps including inducing required. Further, their process is performed without the processing components utilized in the present invention.

[0012] WO2023 / 021213 to Merck Patent GmbH teaches methods of making edible hollow fibers that utilize one or more energy sources to enable crosslinking of the fibers.

[0013] We started our work with the idea that in the food and cultured meat industry plant-based proteins are better materials of choice for membranes. Protein structures can have hydrophilic and hydrophobic regions, making it more stable in application. However, their processing into membranes is extremely challenging to those in the art, as the limited choice of solvents available does not allow for the liquid- to-solid transition at the same fast rate of the synthetic analogues. As a result, prior art protein-based membranes low mechanical reliability is a limiting factor for their scalable production. This is even more crucial in the case of hollow fiber membranes, which, in contrast to flat-sheet membranes, cannot benefit from a camer / support web during the coagulation step. Due to these problems, generation of protein-based hollow fiber membranes at scalable production speeds suitable for food production has been elusive. This invention provides for improved production methods for the generation of fibers (both hollow and solid) and sheet membranes with the characteristics desired for structured cultured meat production.Attorney Docket No: P25-032-SEC-W001

[0014] In certain scenarios of the present invention, the dope solution is alkaline and the formation bath used for coagulation is acidic. One could also envision a system in the reverse where the dope is in an acidic environment and the formation bath is alkaline. (Also referred to as pH Induced Phase Separation (pH IPS); See, e.g., WO2023 / 021213 to Merck Patent GmbH). However, in either of these scenarios, there’s insufficient protein solubility and inadequate molecular chain entanglement for physical integrity sufficient for processing or postprocessing. Therefore, a more advanced and innovative coagulation system is required to generate useful hollow fibers especially at production scale.

[0015] In one aspect, the process of the present invention utilizes multiple mechanisms to solve these prior art problems. As the membranes are formed, they undergo a dehydration step. As the water is replaced or partially replaced with ethanol and glycerin, the molecular mobility of the proteins is lowered, resulting in a more ridged final structure. While the present invention is not limited to theory, it is believed that glycerin stabilizes the protein's tertiary and quaternary structures by forming hydrogen bonds with polar groups on the protein molecules. This stabilization can enhance the firmness of the protein matrix. Glycerol is also known to shift the native protein ensemble to more compact states. (Vagenende V, Yap MG, Trout BL. Mechanisms of protein stabilization and prevention of protein aggregation by glycerol. Biochemistry. 2009 Nov 24;48(46):11084-96). This stabilization is essential for the fiber to, for example, move over rollers during production without collapsing.

[0016] The second is the use of a non-solvent induced phase separation (NIPS) process. By lowering the water content and increasing the alcohol content within coagulating protein dope as the membranes are being formed, the protein in the membrane will denature and become more solid. While not limited to theory, we believe that for the proteins we are working with this mechanism is driven by the protein denaturing. The denaturing can be an effect of either the disruption of the hydrogen bonds and hydrophobicAttorney Docket No: P25-032-SEC-W001interactions that maintain a protein's native structure and / or the effect of ethanol acting as a dehydrating agent, which can remove water molecules from the protein matrix. This dehydration can further denature the protein structure, leading to conformational changes. (See, Yangyang, et al., Food Hydrocolloids, Ethanol induced changes in structural, morphological, and functional properties of whey proteins isolates: Influence of ethanol concentration, Vol. 111, article 106379).

[0017] Within the system described for the present invention, a polysaccharide such as alginate can be also used, if desired. Most of the polysaccharides used in membrane formation will not be soluble in ethanol. So, an additional benefit in structure forming is seen as the polysaccharide goes through a NIPS process aiding in maintaining structural integrity. In a preferred embodiment, alginate is optionally proved in the formation dope at about 1.0% or less, preferably at about 0.1%.

[0018] We found that if the formation bath was an aqueous buffer, the coagulation would be too slow, and the scalability of the process would be significantly limited with the afore mentioned problems with the fibers. If only ethanol is used, the cost associated with the production area needed would increase 10X due to safety precautions necessary due to the low flash point of ethanol. If only glycerol is used the viscosity of the bath is too high, lowering the rate of diffusion and electron transfer. Additionally, this high viscosity would put a considerable amount of drag on the fiber being pulled through the formation bath, leading to breakage. We found, surprisingly and unexpectedly, the blending of buffer, glycerin and ethanol in the formation bath allows for targeted fluid densities and viscosities which aids in the sustained membrane and fiber formation that is necessary for the manufacture of a membrane suitable for the production of a cultured meat product. This formation bath formulation also provides for the production of membranes with the required rigidity and stability for production of a cultured meat product. In one embodiment, the concentration of glycerol is about 30%, the concentration of ethanol is about 20% (or less) in an aqueous solution such as a buffer (v:v).Attorney Docket No: P25-032-SEC-W001However, one of skill in the art, armed with the teachings of this specification, will be able to adapt the formulation for specific uses. In this regard, the concentration of glycerol may range from about 20% - 40% or about 25% - 35%, ethanol may range from about 10% - 20% or from about 15% - 20%, with the remainder of the formulation being buffer. In a preferred embodiment, the final pH of the formulation bath is below 6 and comprises sodium acetate, acid (e.g., hydrochloric acid) and calcium chloride. In a more preferred embodiment, the final pH of the formulation bath is about 4 to about 5 and comprises 0.16-0.2M sodium acetate, 0.088-0.11M acid (e.g., hydrochloric acid) and 0.36- 0.45M calcium chloride.

[0019] The present invention contemplates a process for the manufacture of edible membranes, the process comprising: providing: one or more plant protein isolates; admixing the plant protein isolate with NaOH and urea to make an alkaline plant protein isolate dope; forming the plant protein isolate dope into a fiber or flat sheet membrane into a formation bath, wherein the formation bath comprises about 20% to about 40% glycerol, about 10% to about 20% ethanol in an aqueous buffer at a pH below 6 to make a plant protein isolate membrane; processing the membrane through one or more glycerol baths wherein the final bath is over 100 °C and said membrane is in the glycerol bath for a sufficient length of time to cause any water to evaporate; resuspending the membrane in a glycerol and water bath, and; drying the membrane in an oven.

[0020] Further, the present invention contemplates a process for the manufacture of edible hollow fibers, comprising: providing: one or more plant protein isolates; admixing the plant protein isolate with NaOH and urea to make an alkaline plant protein isolate dope; extruding the plant protein isolate dope from a hollow fiber nozzle into a formation bath, wherein the formation bath comprises about 20% to about 40% glycerol, about 10% to about 20% ethanol in an aqueous buffer at a pH below 6 to make a plant protein isolate hollow fiber; processing the hollow fiber through one or more glycerol baths wherein the final bath is over 100 °C and said hollow fiber is in the glycerol bath for aAttorney Docket No: P25-032-SEC-W001sufficient length of time to cause any water to evaporate; resuspending the hollow fiber in a glycerol and water bath, and; drying the hollow fiber in an oven.

[0021] Further still, the present invention contemplates that the pH of the formation bath is about 3.0 - about 6.0, about 3.0 - about 5.0 or about 4.5.

[0022] Further still, the present invention contemplates that the dope further comprises a polysaccharide.

[0023] Further still, the present invention contemplates that the polysaccharide is alginate or pectin.

[0024] Further still, the present invention contemplates that the concentration of the polysaccharide in the plant protein isolate dope is less than 5%, less than 2%, less than 1% or less than 0.2%.

[0025] Further still, the present invention contemplates that the plant protein isolate is admixed for over 1 hour, for 1 to 48 hours, for 1 to 24 hours, for 1 to 12 hours, for 1 to 6 hours for 1 to 4 hours or from 1 to 2 hours to make the plant protein isolate dope.

[0026] Further still, the present invention contemplates that the plant protein isolate dope has a pH of about 9 or higher.

[0027] Further still, the present invention contemplates that the plant protein isolate dope has a pH of about 11 - 12.5.

[0028] Further still, the present invention contemplates that the aqueous buffer comprises 0.16 M - 0.2 M sodium acetate, 0.088 M - 0.11 M hydrochloric acid, and 0.36 M - 0.45 M calcium chloride and has a pH of about 4.0 - 5.0.

[0029] Further still, the present invention contemplates that the plant protein isolate is selected from one or more of mung bean, soybean, lentil, red lentil, pea, rice, faba and chickpea.

[0030] Further still, the present invention contemplates that when forming hollow fibers the extruded plant protein isolate is formed into a hollow fiber having an outer diameter of about 50 pm to about 1000 pm, about 400 pm to about 600 pm and a wall thickness of about 75 pm to about 300 pm, about 100 pm to about 200 pm, or is formed intoAttorney Docket No: P25-032-SEC-W001a hollow fiber having an outer diameter of about 450 pm to about 550 pm and a wall thickness of about 125 pm to about 175 pm.

[0031] Further still, the present invention contemplates that the plant protein isolate dope is formed into membranes with micro and or nano pores, i.e., micro or nano porous membranes. Micro and nano pore sizes are known to one of skill in the art. See, for example, T.J. Mays, A new classification of pore sizes, Studies in Surface Science and Catalysis, Elsevier, Volume 160, 2007, Pages 57-62 where nanopore is defined as pore size between 0.1 and 100 nm, micropore is defined as pore size between 0.1 and 100 pm and Millipore size is defined as between 0.1 and 100 mm.

[0032] Further still, the present invention contemplates that the formed membrane is a hollow tube-like (i.e., a hollow fiber) or a solid cylindrical-like fiber or a flat sheet.

[0033] Further still, the present invention contemplates that the aqueous buffer comprises one or more salts generally recognized as safe (GRAS) by the Food and Drug Administration (FDA).

[0034] Further still, the present invention contemplates that the aqueous buffer comprises one or more salts selected from the group consisting of calcium chloride, potassium chloride, sodium chloride and calcium lactate.

[0035] Further still, the present invention contemplates that the formulation bath has a density lower than the density of the plant protein isolate membrane.

[0036] Further still, the present invention contemplates that the aqueous buffer comprises one or more acids generally recognized as safe (GRAS) by the FDA.

[0037] Further still, the present invention contemplates that the aqueous buffer comprises one or more GRAS acids selected from the group consisting of sodium acetate, citric acid, hydrochloric acid, acetic acid and sodium citrate.

[0038] Further still, the present invention contemplates that the glycerol content of the glycerol and water bath is about 40% - about 50% glycerol.Attorney Docket No: P25-032-SEC-W001

[0039] Further still, the present invention contemplates that the oven is at a temperature of 40 °C or higher and the membrane is in the oven for a period of time sufficient to make the membrane dry to the touch.

[0040] Further still, the present invention contemplates a process for the manufacture of edible membranes, comprising: providing: one or more plant protein isolates and, optionally, a polysaccharide; admixing the plant protein isolate with acid and urea to make an acidic plant protein isolate dope with a pH of about 2 -5; forming the plant protein isolate dope into a fiber or flat sheet membrane into a formation bath, wherein the formation bath comprises about 20% to about 40% glycerol, about 10% to about 20% ethanol in an aqueous buffer as a pH of about 8 - 11 to make a plant protein isolate membrane; processing the membrane through one or more glycerol baths wherein the final bath is over 100 °C and said membrane is in the glycerol bath for a sufficient length of time to cause any water to evaporate; resuspending the membrane in a glycerol and water bath, and; drying the membrane in an oven.

[0041] Further still, the present invention contemplates that the formation bath comprises about 30% glycerol and about 20% ethanol in an aqueous buffer.Description of the Figures

[0042] Fig. 1 shows three images of the hollow fibers made with the process of the present invention.

[0043] Fig. 2 shows hollow fibers made with differing concentrations of ethanol. Hollow fibers made in 20% ethanol are translucent. Hollow fibers made in 80% ethanol are opaque.

[0044] Fig. 3 shows a contour plot of strain at max stress of hollow fibers made with varying percentages of ethanol and glycerol in the formulation bath.

[0045] Fig. 4 shows strain at ultimate strength of the hollow fibers made with varying percentages of glycerol and ethanol in a second bath.Attorney Docket No: P25-032-SEC-W001

[0046] Fig. 5 shows the elastic modulus of hollow fibers made by the process of the present invention with increasing concentrations of glycerol in the second dehydration bath.

[0047] Fig. 6 shows the breaking stress of hollow fibers made by the process of the present invention with increasing concentrations of glycerol in the second dehydration bath.

[0048] Fig. 7 shows changes in hollow fiber lumen diameter in relation to differing percentages of ethanol in the formation bath.

[0049] Fig. 8 shows the flash point of the formation bath with varying concentrations of ethanol.Detailed Description of the Invention

[0050] Definitions

[0051] The term “plant protein isolate” shall refer to protein isolated from plant sources by methods known to those of skill in the art.

[0052] The terms “dehydration” and “dehydration steps” shall refer to the removal of water from a reaction mixture. In the context of the present invention, the source material ( / .e., the reaction mixture; here the plant protein isolate dope and / or the membranes made from said dope) is dehydrated by exposing the reaction mixture to increasing concentrations of non-aqueous fluids, for example ethanol and / or glycerol. Dehydration can be partial or complete. Partial dehydration is from about 25% to 95%. In preferred embodiment of the present invention partial dehydration is about 50%. Complete dehydration shall refer to the removal of over 98% of the water from the reaction mixture. “Dehydration steps” shall refer to the process of exposing the reaction mixture ( / .e., protein isolate dope) or formed membranes to successive increasing concentrations of non-aqueous solutions, for example and without limitation, ethanol, glycerin and or mixtures of the ethanol and glycerin. “Reverse dehydration steps” shall refer to the process of exposing the reaction mixture or formed membranes to successive decreasing concentrations of non-aqueous solutions to increase the water content of the reaction mixture or formed membranes. Although ethanol and / or glycerin are preferred in the present invention asAttorney Docket No: P25-032-SEC-W001dehydration agents, other dehydrating agents are known to one of skill in the art and may be substituted in the procedures of the present invention.

[0053] The term “elastic modulus” (also known as modulus of elasticity) is the unit of measurement of an object's or substance's resistance to being deformed elastically (i.e., non-permanently) when a stress is applied to it.

[0054] The term "tensile strength” (also called TS, ultimate tensile strength, UTS) is the maximum stress that a material can withstand while being stretched or pulled before breaking.Structured Meat Products

[0055] The present invention contemplates edible membranes including, but not limited to, fibers (e.g., hollow fibers or solid fibers) of suitable integrity for use in bioreactors for the production, for example, of structured clean meat, and methods of production of structured clean meat therewith and the structured clean meat produced with the membranes of the present invention. Clean meat (also known in the art as “cultured meat” or “lab grown meat”) is defined in the art as meat or a meat-like product (referred to collectively herein as “clean meat,” “clean meat product,” “cultured meat,” or “cultured meat product”) grown from cells in a laboratory, factory or other production facility suitable for the large-scale culture of cells.

[0056] A “structured meat product,” “structured clean meat product,” “structured cultured meat” or “structured cultured meat product” is a meat product or clean meat product having a texture and structure like, similar to or suggestive of natural meat from animals. The structured meat product of the present invention has a texture and structure that resembles natural meat 1) in texture and appearance, 2) in handleability when being prepared for cooking and consumption (e.g., when being sliced, ground, cooked, etc.) and 3) in mouth feel when consumed by a person. The materials and methods of the present invention, when used in the production of structured clean meat, achieve at least one of these criteria, two of these criteria or all three ofAttorney Docket No: P25-032-SEC-W001these criteria. The prior art technology is unable to produce a structured meat product sufficiently meeting any of these criteria.

[0057] The structured meat product of the present invention meets these criteria by culturing suitable cells (discussed, infra) in a bioreactor (also, discussed, infra) comprising the hollow fibers of the present invention. The membranes (e.g. hollow fibers) of the present invention, at least in part or in substantial part, provide the structure and texture to the final structured clean meat product that provides the desired appearance, handleability and mouth feel of the product.Further, the membranes (e.g., hollow fibers) of the present invention aid in providing a suitable environment for the growth of the cells into a structured clean meat product. In this context, the membranes of the present invention provide at least a surface suitable for the attachment of the cultured cells, elongation of the cells into morphologies resembling myocytes or myocyte-like cells ( / .e., substantially resembling myocytes in structure and appearance), and formation of the myocytes into myotubule or myotubule-like structures ( / .e., substantially resembling myotubules in structure and appearance).Production of Membranes of the Present Invention

[0058] It is understood that in the present invention the term “membrane” or “membranes” refers to any porous membrane structure produced by the methods of the present invention including, but not limited to, hollow fiber membranes, solid fiber membranes and sheet ( / .e., flat) membranes. Unless specifically indicated otherwise, reference to “membranes,” “hollow fibers,” “hollow fiber membranes” and “sheet membranes” will be understood to inclusive of any membrane structure produced by the methods of the present invention regardless of shape, form or appearance, unless the language makes it clear that a specific membrane type is being discussed.

[0059] It is contemplated that the edible and / or dissolvable hollow fibers, solid fibers and sheet membranes of the present invention may be made from one or more of plant isolates (e.g., soy / zein / wheat / mung / soy / lentil), hydrocolloids ( / .e., polysaccharidesAttorney Docket No: P25-032-SEC-W001such as xanthan, methyl cellulose(s), alginate, agar, pectin, gelatin, carrageenan, cellulose / gellan / guar / tara / bean / other gums), proteins (e.g., polypeptides, peptides, glycoprotein and amino acids; for example, various starches (corn / potato / rice / wheat / sorghum), lipids, (e.g., free fatty acids, triglycerides, natural waxes, and phospholipids), alcohols (e.g., polyalcohol), carbohydrates and other natural substances such as alginate. In a preferred embodiment, the membranes of the present invention are made from one or more protein isolates and one or more polysaccharides.

[0060] In a preferred embodiment, it is contemplated that the plant protein isolates are sourced from the legume family (i.e. Fabaceae), especially the Viceae & Phaseoleae tribes. The polysaccharide can be included at less than 20%, less than 5%, less than 1% or less than 0.5% by weight.

[0061] In one preferred formulation, the protein-based dope of the present invention comprises, consists essentially of or consists of a mixture of about 14 wt% protein isolate, about 1% alginate wt% and about 2 wt% urea adjusted to a pH of about 11 with sodium hydroxide or other base. However, the protein isolate concentration may range from about 10% to about 20%, the polysaccharide concentration may range from about 0.01% to about 5% and the urea concentration may range from about 0.5% to about 5%. The pH may be alkaline and be from about 9 to about 13. One of skill in the art, armed with the teachings of this specification, will be able to adjust the formulation as necessary for achieving desired membrane characteristics without undue experimentation.

[0062] Further, it is contemplated that other materials may be added to the membranes (e.g., hollow fibers) or coated on to the membranes that aid in cell attachment and cell growth. For example, it is contemplated that the hollow fiber additive or coating is one or more of proteins, hydrogels, or other coatings known by one of skill in the art including extra cellular matrix (ECM) components and extracts, poly-D- lysine, laminin, collagen (e.g., collagen I and collagen IV), gelatin, fibronectin, plant-based ECM materials, collagen-like, fibronectin-likeAttorney Docket No: P25-032-SEC-W001and laminin-like materials known to one of ordinary skill in the art that are isolated from a plant or synthesized from more simple substances. The overall result is that the fibers of the present invention impart the texture and structure of meat and meat products giving the structured clean meat product produced by the present invention a texture, appearance, handleability and mouth feel similar to real meat.

[0063] It is noted by the Inventors of the present invention that soy, lentil, and mung bean protein isolates confer several of the desired characteristics to the membranes produced by the methods of the present invention. It is also noted by the Inventors that both soybean (Glycine max) and mung bean (Vigna radiata) are from the same classification family related to legumes (i.e., peas or beans), Fabaceae. Doyle, J. J., Leguminosae, Encyclopedia of Genetics, 2001, 1081 - 1085. Although the present invention is not limited by theory, it is believed that other members of this family, especially the Millettioids and Phaseoloids including the geneses Glycine and Vigna, will work substantially similar to soy and mung bean protein isolates

[0064] It is further contemplated that the hollow fibers of the present invention may comprise one or more of cellulose, chitosan, collagen, zein, alginate, agar, inulin, gluten, pectin, legume protein, methyl cellulose(s), gelatin, tapioca, xanthan / guar / tara / bean / other gums, proteins (e.g., polypeptides, peptides, glycoprotein and amino acids including, but not limited to, various forms of corn / potato / rice / wheat / sorghum starches, plant isolates and soy / zein / casein / wheat protein, all of which are known to one of skill in the art), lipids, (for example, free fatty acids, triglycerides, natural waxes, and phospholipids). Cellulosic polymers may include cellulose acetate-butyrate, cellulose propionate, ethyl cellulose, methyl cellulose, nitrocellulose, etc. More specifically, the hollow fibers of the present invention may comprise a mixture of one or more legume proteins and hydrocolloids (e.g., polysaccharides).

[0065] In an embodiment, it is contemplated that the hollow fibers of the present invention are edible, dissolvable, or edible and dissolvable. In other words, the fibers may be either edible or dissolvable or both.Attorney Docket No: P25-032-SEC-W001Further still, for fibers that are dissolvable, there may be differing degrees of dissolvability. For example, some fibers may be readily dissolvable upon exposure to a suitable solvent (e.g., a non-toxic solvent that is generally recognized as safe by the Food and Drug Administration (FDA) or other organization recognized as being qualified to assess the safety of consumable substances). Other fibers may be less readily dissolvable. In this regard, the less readily dissolvable fibers may be partly dissolved after the cells being cultured have reached the requisite level of confluency thereby leaving enough of the fiber to provide for a desired mouth feel and texture to the structured clean meat of the present invention but not an excess of fiber that may make the structured clean meat product of the present invention seem tough or chewy. Dissolvable hollow fiber constituents are known to those of skill in the art. For example, alginate is dissolvable upon exposure to a Ca2+ chelator. In an embodiment of the present invention, it is contemplated that the hollow fibers of the present invention comprise an amount of alginate to render the fibers partially dissolvable and / or a percentage of fibers in a device comprising the hollow fibers of the present invention comprise alginate.

[0066] While the preferred embodiments of the membranes of the present invention do not use crosslinkers, for some uses of the membranes of the present invention they may be desired. Thus, in an embodiment of the present invention, it is contemplated that one or more crosslinkers are used in the hollow fibers of the present invention. Crosslinkers, as the name implies, bind one or more of the other constituents of the hollow fiber to strengthen the fiber. In an embodiment of the present invention, the crosslinker may be the dissolvable component or one of the dissolvable components of the hollow fibers of the present invention. Exemplary crosslinkers and crosslinking mechanisms as contemplated by the present invention, include but are not limited to, covalently bonded ester crosslinks (U.S. Patent No. 7,247,191) and UV-crosslinking (U.S. Patent No.8,337,598), both of which are incorporated herein by reference in their entirety. Further, use of crosslinkers in the production of hollow fibersAttorney Docket No: P25-032-SEC-W001is known to one of skill in the art. See, for example, US Patent Nos.: 9,718,031; 8,337,598; 7,247,191 ; 6,932,859 and 6,755,900, all of which are incorporated herein in their entirety.

[0067] The membranes and fibers of the present invention are produced from a blend of protein(s) and polysaccharide(s). The ratio of protein to polysaccharide is contemplated to be from approximately 1 :99 to approximately 99: 1 , approximately 1 : 10 to 10: 1 , approximately 2:5 to 5:2, approximately 3:7 to 7:3, approximately 4:6 to 6:4 or approximately 1 :1 , or any ratio within the stated rations. In a preferred embodiment, the protein content of the mixture is higher than the polysaccharide content. In a preferred embodiment, the protein isolate content is about 90%, 95%, 98%, 99%, 99.9% or100%. Protein isolate is approximately 80 - 90% protein.

[0068] Hollow fiber manufacturing techniques, in particular, and membrane manufacturing techniques, in general, are known to one of skill in the art. (See, for example, Vandekar, V.D., Manufacturing of Hollow Fiber Membrane, Int’l J Sci & Res, 2015, 4:9, pp. 1990 - 1994, and references cited therein). Like flat sheet membranes, known methods of fiber manufacturing typically include some technique of phase separation. Common methods of nonsolvent induced phase separation include thermally induced phase separation, vapor induced phase separation, heat induced phase separation (see, for example U.S. Patent No. 5,444,097 to MilliporeSigma, which is incorporated herein by reference), or a combination thereof. However, other techniques like thermal extrusion and stretching can be used for hollow fiber and membrane formation. Typically, one would destabilize the polymer in solution by means of nonsolvent, thermal destabilization, or removal of the solvent. As described in here, dissolutions of the polymer (polysaccharides and proteins in this case) will be followed by the gelation or solidification via multiple crosslinking processes. The fibers may be further stretched to produce fibers with diameters less than 100 pm and a wall thickness as thin as 10 pm.

[0069] In some aspects of the present invention, pH induced phase separation (“pH Induced Phase Separation (pHIPS)” or “ProtonAttorney Docket No: P25-032-SEC-W001Induced Phase Separation” (Satoru Tokutomi, Kazuo Ohki, Shun-ichi, Ohnishi, Proton-induced phase separation in phosphatidylserine / phosphatidylcholine membranes, Biochimica et Biophysica Acta (BBA), Biomembranes, Volume 596, Issue 2, 28 February 1980, Pages 192-200) is used in the manufacture of the membranes (i.e., hollow fibers and sheet membranes) of the present invention. pH induced phase separation is exemplified in the Examples section, infra, and in WO 2022 / 038240A2 to MilliporeSigma, which is incorporated herein by reference and is representative of what one of skill in the art understood at the time of its filing. Liquid phase separation of macromolecules controlled by pH is studied in cellular physiology (Adame-Arana, 0., et al., Liquid Phase Separation Controlled by pH, 2020 Oct 20; 119(8): 1590-1605; Epub 2020 Sep 16). The use of pH induced phase separation confers unexpected and surprising benefits on the membranes of the present invention; namely mechanical integrity, pore size, and porosity are enhanced over conventional processes.

[0070] Dry spinning involves dissolving the polymer in a very volatile solvent. The solvent / polymer mixture is heated after extrusion and evaporation of the solvent the polymer solidifies. In contrast, wet spinning is more versatile since the process involves a larger number of parameters that can be varied. The polymer and solvent mixture is extruded into a nonsolvent bath where demixing and / or phase separation occurs because of the exchange of solvent and nonsolvent. Between the extrusion and the nonsolvent bath there is an air gap where the hollow fiber membrane formation begins.

[0071] A technique that can eliminate or minimize the use of solvents is melt spinning with cold stretching (MSCS). This approach leads to cost effective production, but may sacrifice structure control and potential degradation of the food materials. In this technique the materials are heated for extrusion and then pulled as they are cooled as to mechanically form pores in the hollow fiber wall. All of three of these techniques have been widely studied and are known in the art they wellAttorney Docket No: P25-032-SEC-W001summarized (see, Tan, XM. and Rodrigue, D., Polymers (Basel), 2019, Aug 5:11(8)).

[0072] Modifications of these techniques are also known to one of skill in the art. See, for example, WO2011 / 108929 (incorporated herein by reference in its entirety) where a modified wet spinning extrusion process for the production of hollow fibers comprised of multiple polymers and polymer layers is disclosed. Manufacture of hollow fibers from non-synthetic materials is also known to one of skill in the art. See, for example, US Patent No. 4,824,569 to Suzuki, which is incorporated herein in its entirety. ]Hollow Fiber Membranes of the Present Invention for the Production of a Structured Meat Product

[0073] The macroscopic structure of the hollow fibers of the present invention, in an embodiment, is contemplated to promote the orientation of the cells along the fibers. In this regard, it is desired by the present invention that the orientation of the component molecules from which the hollow fiber is constructed be oriented parallel, essentially parallel or predominately parallel to the length of the hollow fibers. In is further contemplated that the component molecules create a surface texture at least on the outer surface of the hollow fiber that aids in cell attachment and aids in cell orientation. Thus, in an embodiment, it is contemplated that the surface texture of the hollow fibers of the present invention create attachment points for cell attachment. In another embodiment, it is further contemplated that the cells grown on the hollow fibers of the present invention (in particular, the myocytes, myocyte-like cells or cells having characteristics of myocytes) orient and extend along the length of the hollow fiber similar to and resembling myocytes in vivo.

[0074] Thus, the orientation of the surface structure of the scaffold directly correlates to the alignment of the myotubes during formation. It can be thought of as if skeletal muscle wants to form along a preexisting structure. It can be envisioned that a bundle of fibers closely mimics skeletal muscle structure for the formation of alignedAttorney Docket No: P25-032-SEC-W001myotubes. Therefore, a hollow fiber bioreactor doesn’t only achieve the tissue-like cell densities, but it also achieves the myotube alignment that other technologies do not, resulting in the most realistic mouth feel of all discussed technologies. The alignment phenomena can be better understood by reviewing: My mistake: Decellularized Apium graveolens Scaffold for Cell Culture and Guided Alignment of C2C12 Murine Myoblast - Santiago Campuzano, 2020, Ph.D. thesis, University or Ottawa, pp 58-59.

[0075] With regard to producing a structured clean meat product, it is contemplated that the hollow fibers of the present invention have a range of sizes over which they will be suitable for the present invention. It is also contemplated that the hollow fibers of the present invention are spaced such that the cells grown on the hollow fibers achieve a density similar to that of real meat and with a minimum of void space between the cells. In one embodiment, it is contemplated that the hollow fibers of the present invention have an outer diameter of about 0.1 mm to about 3.0 mm, a porosity of about 0% porosity (making it diffusion based) to about 75%, and a wall thickness of about .008 to about 0.5 mm or about 0.01 mm to about 0.2 mm or any thickness between .008 mm to 0.5 mm not specifically iterated above. It was found by the present inventors that this size is suitable for the transport of media through the lumen of the fiber and permit the adequate flow of media through the wall of the hollow fiber while at the same time being rigid enough to support cell growth and, further, provide for the desired final product structure, texture, handleability and mouth feel. However, depending on the desired structured clean meat product (e.g., beef, poultry, fish, pork, etc.) other embodiments with regard to variations of the diameter, wall thickness and porosity of the fibers are contemplated; discussed infra.

[0076] Fiber porosity. The hollow fibers of the present invention need to have a porosity that allows for adequate flow of media though the wall of the fiber while at the same time ensuring a suitable surface for cell growth and cell support. The porosity of the hollow fibers is related, in part, to the thickness of the wall of the hollow fiber and to theAttorney Docket No: P25-032-SEC-W001composition of the hollow fiber. If the wall is thin enough, then about 0% porosity may suffice allowing the media diffusing through the hollow fiber wall. The porosity of the hollow fibers of the present invention may be as high as 75% or 90%. Thus, the range of porosity of the hollow fibers of the present invention is from 0% to about 90%, from about 10% to about 75%, from about 30% to about 60%, or any percentage value between 0% and 75% not specifically iterated above.

[0077] The hollow fibers of the present invention may also be subject to a pore forming step. The pore forming mechanism will be one of the following techniques, well known in the art of membrane formation: TIPS = thermally induced phase separation, NIPS = non-solvent induced phase separation, VIPS = vapor-induced phase separation, pH induced phase separation, MSCS = melt-spinning combined with stretching, (see, Review on Porous Polymeric Membrane Preparation. Part II: Production Techniques with Polyethylene, Polydimethylsiloxane, Polypropylene, Polyimide, and Polytetrafluoroethylene, Xue Mei Tan, 1, 2, 2019). In all scenarios the polymer will be in a liquid phase either by thermally melting it or chemical dissolution. From there, the polymer is extruded into a cylindrical shape, and drawn onto a spindle. During the extrusion step, a bore fluid can be used to prevent the hollow fiber from collapsing on itself. Between the extruding nozzle and the rewind spindle, there may also be a pore forming chamber, such as a water bath or an atmospheric environmental chamber.

[0078] The present invention also contemplates the configuration of the hollow fibers of the present invention in a bioreactor. Fiber configuration may include one or both of fiber positioning and spacing. Fibers may be configured in any configuration that permits growth of the cell population with a minimum of void space between cells at confluency. For example, the fibers can be oriented in square / rectangle (rows and columns) or triangle / hexagonal (honeycomb) packing modes. Thus, in one embodiment it is contemplated that the fibers are arranged such that the fibers, when viewed on end, form an ordered pattern of rows and columns. InAttorney Docket No: P25-032-SEC-W001another embodiment, it is contemplated that the fibers, when viewed on end, form a honeycomb pattern. In another embodiment, it is contemplated that the fibers of the present invention are arranged randomly or semi-randomly. In another embodiment, it is contemplated that the hollow fibers are arranged in an ordered or semi-ordered pattern of varying densities.

[0079] The hollow fibers can range from about 0.1 mm to about 3.0 mm, about 0.5 mm to about 2.0 mm and about 0.8 mm to about 1.3 mm in outer diameter, and any value in between the cited values. A 1.0 mm hollow fiber assumes about 0.3 mm to about 0.5 mm of meat growth around the outer diameter. An end diameter of approximately 1.1 mm can result in meat with about 85 hollow fibers / cm2

[0080] In another embodiment, it is contemplated that the fibers have varying degrees or amounts of space between fibers. For example, having rows of fibers at a higher density interspersed between fibers at a lower density may be used to produce changes in the texture of the final structured clean meat product, such as is common in natural fish meat. Further still, it is contemplated that fibers of varying diameters, porosities and wall thicknesses may be used in the same hollow fiber cartridge, again, to simulate the appearance, texture, handleability and mouth feel of natural meat.

[0081] In any configuration, the fibers are spaced such that the spacing between the fibers is of a distance that permits an adequate flow of media (and the nutrients, growth factors, etc., contained therein) to reach all of the cell mass. This, of course, will be related at least in part on flow rate of the media and porosity of the hollow fiber walls but is related in greater part on physical distance from the surface of the outer wall of the hollow fiber to the cells. In other words, media and nutrients will only travel or defuse a limited distance through a cell mass. It is currently thought that the maximum for diffusion of oxygen and nutrients is 200 pm. Rouwkema, J. , etal., (2009) Supply of Nutrients to Cells in Engineered Tissues, Biotechnology and Genetic Engineering Reviews, 26:1, 163-178. Thus, spacing between fibers should be about, should be no more than, 400 pm from the outer wallAttorney Docket No: P25-032-SEC-W001of one fiber to the outer wall of a neighboring fiber. In culture conditions where media flows both through the hollow fibers and through the spacing between the hollow fibers the spacing can be greater. For example, spacing could be 800 pm from the outer wall of one fiber to the outer wall of a neighboring fiber. These figures are if the culture process relies on diffusion alone. However, use of a pump (for example) will create a flow of media from the hollow fibers, through the cell culture space between the hollow fibers and to the housing exits (rather than relying on diffusion alone) allowing the fibers to be spaced further apart. For example, in some embodiments it is contemplated the maximum distance between fibers is from about 0.05 mm (50 pm) to about 5.0 mm; about 0.1 mm to about 3.0 mm; about 0.1 mm to about 2.0 mm; about 0.1 mm to about 1.0 mm or about 0.2 mm to about 0.5 mm or any distance between the stated values. While it is a preferred embodiment that media flows from the center of the hollow fibers through the culture to the housing exits, it is also contemplated that the media flow can be in the reverse direction or can be alternated from one direction to the other, as desired. Alternating the direction of the media flow is believed to assist in ensuring all cells have an adequate media supply.

[0082] It is an embodiment of the present invention that a degree of randomness will be inherent in the distancing of the hollow fibers of the present invention. The figures given in the previous paragraph are average fiber-to-f iber distances for a given assembly. In an embodiment of the present invention, spacers and / or assembly techniques may be used to ensure, normalize or control the distances between the fibers. See, for example, Han G, Wang P, Chung TS., Highly robust thin-film composite pressure retarded osmosis (PRO) hollow fiber membranes with high power densities for renewable salinity-gradient energy generation, Environ Sci Technol. 2013 Jul 16;47(14):8070-7. Epub 2013 Jun 28 or Chun Feng Wana, Bofan Li a, Tianshi Yang a, Tai-Shung Chung, Design and fabrication of inner- selective thin-film composite (TFC) hollow fiber modules for pressureAttorney Docket No: P25-032-SEC-W001retarded osmosis (PRO), Separation and Purification Technology, 172:32 -42, 2017.

[0083] Once the cell density becomes too dense or the thickness of the cell mass becomes too thick, the ability of the media to reach the cells furthest away from the hollow fiber becomes difficult. A lack of media to these cells may result in dead cells in the reactor and / or dead spaces where cells cannot grow. The corollary is that the media needs to flow through the hollow fiber cartridge to the housing exits. That is, a flow of media needs to be maintained at least until confluency is reached and the structured clean meat product is harvested. One of skill in the art, armed with the teachings of this specification, will be able to calculate the correct spacing of and porosity of the fibers of the present invention for a given desired structured clean meat product.

[0084] The hollow fibers of the present invention can be arranged and secured in what is referred to herein as a “hollow fiber cartridge.” In one embodiment, it is contemplated that the hollow fiber cartridge is made by having the ends of the hollow fibers are secured in an end piece in the desired arrangement. For example, each fiber has a first end and a second end. Each end is secured in an end piece, that is, a first and a second end piece. An end piece can be, for example, a resin or plastic that is known in the art to be inert and non-toxic to cells. At least one of the first or second ends of the hollow fibers is positioned in the end piece such that the interior lumen of the hollow fiber is in fluid communication with the exterior environment. Thus, with this positioning of the hollow fibers in the end piece, media can be caused to flow from the exterior environment of the hollow fiber ( / .e., outside of the hollow fiber but inside of, for example, a sterile bioreactor) into the inner lumen of the hollow fiber.

[0085] One of skill in the art understands how to assemble hollow fibers into a module or cartridge. These techniques are applicable to the hollow fibers of the present invention. In brief, after spinning, the hollow fibers are cut to length and the ends of the fibers encased ( / .e., potted) in a resin that will flow around the fiber ends and solidify.Sometimes, the section of the fibers may be encased in a substanceAttorney Docket No: P25-032-SEC-W001(e.g., Plaster of Paris or other easily removable material known to one of skill in the art) to close the pores of the fibers so that the “potting solution,” i.e., the liquid resin, does not enter or plug the pores in the fibers. See, for example, Vandekar, V.D., Manufacturing of Hollow Fiber Membrane, Int’l J Sci & Res, 2015, 4:9, pp. 1990 - 1994, and references cited therein. In the present invention, one or both of the ends of the “potted” bundle are trimmed or cut to expose the open ends of the fibers to permit the flow of media once the bundle is inserted into a housing for use in the production of the structured clean meat of the present invention.

[0086] Further still, it is contemplated in some embodiments that the hollow fiber cartridge of the present invention has securing devices to maintain a desired distance between the first and second end piece. This may be necessary or preferred, for example, for easier insertion of the hollow fiber cartridge of the present invention into, e.g., a bioreactor housing.

[0087] Thus, it is contemplated that in one embodiment the hollow fiber cartridge of the present invention contains a plethora of hollow fibers arranged in a desired arrangement. The hollow fibers of the present invention have a first end and a second end. The arrangement is maintained by securing the first end and the second end of the hollow fibers in a first and a second end piece. The hollow fibers, once secured as describe, are then positioned parallel, substantially parallel or essentially parallel to each other. Further, the first and second end pieces are positioned parallel, substantially parallel or essentially parallel to each other. Further still, the hollow fibers of the hollow fiber cartridge of the present invention are positioned perpendicular, substantially perpendicular or essentially perpendicular to the end pieces of the hollow fiber cartridge of the present invention. The diameter and length of the hollow fiber cartridge will depend on the desired structured clean meat product being produced and bioreactor configurations.

[0088] In an embodiment of the present invention, it is contemplated that the hollow fibers of the hollow fiber cartridge of the presentAttorney Docket No: P25-032-SEC-W001invention are at an average density of about 40 - about 120 per cm2, at an average density of about 60 - about 100 per cm2, at an average density of about 70 - about 90 per cm2or any value between the values given above but not specifically iterated.

[0089] In an embodiment of the present invention, it is contemplated that the hollow fibers in the hollow fiber cartridge of the present invention have a void space between the hollow fibers prior to the addition of cells and, the void space between the hollow fibers is about 25% - about 75% of the total area of the hollow fiber cartridge or about 40% - about 60% of the total area of the hollow fiber cartridge or any value between the values given above but not specifically iterated.

[0090] In an embodiment of the present invention, it is contemplated that the hollow fiber cartridge of the present invention is designed to be removably inserted into a housing. That is, the cartridge can be inserted into the housing at the beginning of a production run and removed, i.e. , harvested, at the end of the production run for any further desired processing of the structured clean meat product of the present invention. After harvesting of the structured clean meat product, a new hollow fiber cartridge of the present invention may be inserted into the housing and the process repeated. In this regard, the housing for the hollow fiber cartridge of the present invention is part of a bioreactor or bioreactor system.

[0091] Reactor configuration. The present invention is not limited to any particular reactor configuration or reactor system configuration so long as adequate media flow can be maintained through the culture and waste products removed. Hollow fiber reactors are typically tubular in shape although they can be oval, flat (sheet-like), rectangular or any other shape. In a preferred embodiment, the reactor comprises an insertable / removable insert that comprises the hollow fibers of the present invention. After confluent cell growth (as defined herein) is reached the insert can be removed and product finalized by removal of the insert ends and any further desired processing. Further processing may take the form of, for example, slicing, surface texturing, adding flavors, etc. Alternatively, further meat enhancement can take placeAttorney Docket No: P25-032-SEC-W001before the harvest and disassembly of the device. For example, the media can be flushed out of the hollow fiber device and then the additives would be pumped directly into or around the fibers.

[0092] Non-limiting examples of suitable reactor systems. The most suitable type of reactor system is the feed batch system although it is contemplated that any available reactor will be suitable for use with the hollow fibers and hollow fiber cartridge of the present invention. For example, the MOBIUS® system (MilliporeSigma, Bedford, MA) is an example of a commercial system that can easily be converted to use with the present invention. The bioreactor in which the structured clean meat product is produced ( / .e., the reactor comprising the hollow fibers of the present invention) may be seeded with cells grown in another bioreactor. The bioreactor that is seeding the hollow fiber device (a reactor suitable for cell growth (proliferation) and cell expansion) can be an existing commercial reactor, for example, a stirred tank or wavetype reactor. The proliferation / expansion bioreactor is contemplated to be, for example, a stirred tank or wave-type reactor (as are known to one of ordinary skill in the art) and to be a suspension, agglomerated biomass, microcamer culture, or other suitable reactor known to one of ordinary skill in the art. It is contemplated that the production bioreactor ( / .e., the reactor comprising the hollow fibers of the present invention) may be, for example, single use, multi-use, semi-continuous or continuous. The present invention further contemplates a manifold of multiple reactors comprising the hollow fiber of the present invention.

[0093] Thus, it is contemplated that an exemplary reactor system of the present invention comprises one of more hollow fiber cartridges of the present invention, a housing sized to hold said hollow fiber cartridge; a medium source fluidly connected to one or more inlets in said housing; one or more medium outlets in said housing; and one or more pumps to supply the medium to and / or remove waste medium from said hollow fiber cartridge through said medium inlet(s) and / or outlet(s). Further still, the inlets are fluidly connected to the interior of the hollow fibers. Yet further still, the hollow fiber bioreactor may comprise an automated controller or automatically controlled system.Attorney Docket No: P25-032-SEC-W001

[0094] The present invention also contemplates a process for producing a meat product, comprising; seeding a void space between the hollow fibers in a hollow fiber reactor of the present invention with one or more of myocytes, myocyte-like cells or engineered cells expressing one or more myocyte-like characteristics at a density of, for example, 100,000 cells to 100,000,000 (105- 108) (Radisic, et al., Biotechnol Bioeng, 2003 May 20:82(4):403-414.) and culturing the cells until achieving about 80% - about 99% confluency, 85% - about 99% confluency, about 90% - about 99% confluency, about 95% - about 99% confluency, about 98% - about 99% confluency or about 100% confluency (or any value in between the recited percent values), removing said first holding device and said second holding device from the first ends and second ends, respectively, of said hollow fibers.

[0095] After seeding, the hollow fiber cartridge has media supplied to the cells through one or both of the first end and second end of the hollow fibers into the interior of the hollow fibers, through the wall of the hollow fibers into the void space between the hollow fibers where said cells are seeded and through one or more of said outlets in said housing. In another embodiment, it is contemplated that media can also flow between fibers from both the inlet(s) and outlet(s) of device. For example, one fluid path is through fiber wall and the second fluid path is around the fibers. It is contemplated that the device may have multiple inlets and outlets. After the cells achieve confluency, flushing out any residual media and waste products and infusing the interior of the hollow fibers and / or any remaining void space between the cells with one or more of fats, flavors, colors, salts and preservatives.

[0096] Fats suitable for addition to the structured clean meat product of the present invention include, but are not limited to: saturated, monounsaturated, polyunsaturated fats such as com oil, canola oil, sunflower oil, and safflower oil, olive oil, peanut oil, soy bean, flax seed oil, sesame oil, canola oil, avocado oil, seed oils, nut oil, safflower and sunflower oils, palm oil, coconut oil, Omega-3, fish oil(s), lard, butter, processed animal fat, adipose tissue, or cellular agriculture derived fat, or combinations thereof. Synthetic fats such as oleoresin may also beAttorney Docket No: P25-032-SEC-W001used. In fact, any fat recognized by the Food and Drug Administration (FDA) is suitable for use in the present invention and contemplated for use in the structured clean meat product of the present invention. On the FDAs food additive list, natural substances and extractives (NAT), Nutrient (NLITR), Essential oil and / or oleoresin (solvent free) (ESO).

[0097] Flavors suitable for use in the structured clean meat product of the present invention include, but are not limited to, any flavor documented on the FDA’s food additive list. These may be documented as natural flavoring agents (FLAV), essential oils and / or oleoresin (solvent fee) (ESO), enzymes (ENZ), natural substances and extractives (NAT), non-nutritive sweetener (NNS), nutritive sweetener (NUTRS), spices, other natural seasonings & flavorings (SP), synthetic flavor (SY / FL), fumigant (FUM), artificial sweeteners including aspartame, sucralose, saccharin and acesulfame potassium and yeast extract, or combinations thereof, are contemplated for use in the structured clean meat product of the present invention.

[0098] Texture Enhancers suitable for use in the structured clean meat product of the present invention include, but are not limited to, pureed plant material, guar gum, cellulose, hemicellulose, lignin, beta glucans, soy, wheat, maize or rice isolates and beet fiber, pea fiber, bamboo fiber, plant derived fiber, plant derived gluten, carrageenan, xanthan gum, lecithin, pectin, agar, alginate, and other natural polysaccharides, grain husk, calcium citrate, calcium phosphates, calcium sulfate, magnesium sulfate and salts, or any combination thereof, are contemplated for use in the structured clean meat product of the present invention. These may be documented on the FDA’s food additive list as solubilizing and dispersing agents (SDA), and natural substances and extractives (NAT).

[0099] Nutritional Additives suitable for use in the structured clean meat product of the present invention include, but are not limited to, vitamins, trace elements, bioactive compounds, endogenous antioxidants such as A, B-complex, C, D, E vitamins, zinc, thiamin, riboflavin, selenium, iron, niacin, potassium, phosphorus, omega-3, omega-6, fatty acids, magnesium, protein and protein extracts, amino acids salt, creatine,Attorney Docket No: P25-032-SEC-W001taurine, carnitine, carnosine, ubiquinone, glutathione, choline, glutathione, lipoicacid, spermine, anserine, linoleic acid, pantothenic acid, cholesterol, Retinol, folic acid, dietary fiber, amino acids, and combinations thereof, are contemplated for use in the structured clean meat product of the present invention. Any food additive or additives that are generally recognized as safe (GRAS) or approved by the FDA are contemplated for use in the structured clean meat product of the present invention and incorporated herein. See, for example: www.fda.gov / food / food-additives-petitions / food-additive-status-list.

[0100] Any food coloring or colorings, natural or artificial, that are Generally Recognized As Safe (GRAS) or approved by the FDA are contemplated for use in the structured clean meat product of the present invention. See, for example: www.fda.gov / industry / color- additive-inventories / color-additive-status-list.

[0101] Prophetic cell types. The hollow fibers of the present invention are designed to be used to grow specific cell types suitable for the production of in vitro or lab grown meat and meat products, i.e. , the structured clean meat of the present invention. Therefore, while many different types of cells can grow on the hollow fibers (and in the hollow fiber cartridges of the present invention, if desired), the fibers were developed to be used to grow muscle cells (i.e., myocytes), or cells with the characteristics of muscle cells or engineered to have the characteristics of muscle cells (collectively referred to herein as muscle cells or myocytes), to confluency and to mimic the natural structure of muscle (i.e., meat). Preferably, the muscle is skeletal muscle. That is, the hollow fibers of the present invention are designed by the inventors to be suitable to grow myocytes to obtain muscle fibers or myofibrils. Further, other types of cells may be grown on the hollow fibers of the present invention and in reactors comprising the hollow fibers of the present invention. These cells may be grown independently or in combination with muscle cells. For example, adipocytes or cells having the characteristics of adipocytes or engineered to have the characteristics of adipocytes (collectively referred to herein as adipocytes) may be cultured with the muscle cells to achieve an endAttorney Docket No: P25-032-SEC-W001product resembling natural muscle or meat. The hollow fibers of the present invention are also suitable for including other cells to be cocultured with the muscle cells of the present invention, for example, fibroblasts, cells having the characteristics of fibroblasts or cells engineered to have the characteristics of fibroblasts.

[0102] With specific regard to a co-culture of muscle cells and adipocytes, the ratio of muscle cells to adipocytes may be 99:1, 95:5, 92:8, 90:10, 88:12, 85:1582:18, 80:20, 75:25 or any ratio from 100:0 to 75:25, inclusive.

[0103] The cells that are suitable for use with the present invention may be obtained from or derived from any animal from which food is now obtained. Prominent examples are bovine, porcine, ovine, piscine (e.g., fish such as tuna, salmon, cod, haddock, shark, etc.), shellfish, avian (e.g., chicken, turkey, duck, etc.). More exotic sources of cells may also be used, such as from animals that are traditionally hunted rather than farmed (e.g., deer, elk, moose, bear, rabbit, quail, wild turkey, etc.) or, for unique tastes, combinations thereof.

[0104] Cells used in the present invention may be derived by any manner suitable for the generation of differentiated cells having the characteristics desired. For example, any procedure suitable for deriving cells with differentiated myocyte-like characteristics, adipocytelike characteristics, etc. Such characteristics for myocytes include, for example, but not necessarily limited to, having an appearance of a long, tubular cell and with large complements of myosin and actin. Myocytes also have the ability to fuse with other myocytes to form myofibrils, the unit of muscle that helps to give muscle, i.e., meat, its distinctive texture. Such characteristics for adipocytes (also referred to in the art as lipocytes and fat cells) include, for example, but not necessarily limited to, having large lipid vacuoles that may take up as much as 90 % or more of the volume of the cell. The hollow fibers of the present invention provide, at least in part, a replacement of the connective tissue (referred to as “fascia” in the art) typically found in skeletal muscle.Attorney Docket No: P25-032-SEC-W001

[0105] Cells useful in the present invention include, but are not limited to, cells that are derived from mesenchymal stem cells (e.g., adult stem cells) or induced pluripotent stem cells (iPSC). iPSCs are cells engineered to revert to their pluripotent state from which numerous cell types can be derived. In other words, iPSCs are pluripotent stem cells that can be generated directly from a somatic cell. The technology was first reported in 2006 (Takahashi K, Yamanaka S, 25 August 2006, "Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors" Cell, 126 (4): 663-76), has advanced from that point on (see, for example: Li, et al., 30 April 2014, “Generation of pluripotent stem cells via protein transduction” Int. J. Dev. Biol., 58: 21 - 27), includes the generation of muscle cells (see, for example: Rao, etal., 9 January 2018, “Engineering human pluripotent stem cells into a functional skeletal muscle tissue” Nat Commun., 9 (1): 1 - 12) and is well known to one of ordinary skill in the art.

[0106] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0107] When introducing elements of the present disclosure or the preferred embodiments(s) thereof, the articles "a," "an," "the" and "said" are intended to mean that there are one or more of the elements. The terms "comprising," "including" and "having" are intended to be inclusive and mean that there may be additional elements other than the listed elements.

[0108] Furthermore, the transitional phrases “comprising,” “consisting essentially of” and “consisting of” have the meanings as given in MPEP 2111.03 (Manual of Patent Examining Procedure; United States Patent and Trademark Office, 9thEd., Revision Feb 2023 [R-07.2022]). Any claims using the transitional phrase “consisting essentially of” will be understood as reciting only essential elements ( / .e., the basic and novel characteristics) of the invention and any other elements recited in dependent claims are understood to be non-essential to the inventionAttorney Docket No: P25-032-SEC-W001recited in the claim from which they depend. Likewise, any additional elements over those claimed that are described in a prior art reference(s) are excluded from the claims by use of the transitional phrase “consisting essentially of” as being non-essential to the claimed invention.

[0109] All ranges recited herein include all values within the cited range including all whole, fractional and decimal numbers, inclusive.Exemplification

[0110] Example 1 - Exemplary Methods of Production of Membranes of the Present Invention

[0111] Legume bean protein isolate (e.g., mung bean protein isolate;Green Boy Group, Loa Angeles, CA) was mechanically mixed with 4- 10mL of 10N NaOH per 500mL dope and 0.5-5% urea for 3-28 hours, to form the dope with a pH of 10-12. The dope is then extruded from a hollow fiber nozzle with a wall thickness of 150um, and OD of 500 pm. The nozzle was submerged in the formation bath in some cases. The formation bath was adjusted to pH 4-5 with an acetate buffer and contained 50% buffer (0.16 - 0.20 M sodium acetate, 0.088 - 0.11 M acid (e.g., hydrochloric acid) and 0.36 - 0.45 calcium chloride), 30% glycerol, and 20% ethanol prior to use. Other acids may be used in the formation bath. For example, citric acid or otehr acid generally recognized as safe (GRAS) by the FDA may be used. The fiber was drawn continuously on to the collection spool in the formation bath.

[0112] With the batch process, after the fiber collection (approximately 1 hour), the spool was removed from the formation bath and submerged in one or more heated baths, the first of glycerol at 60-90 °C for about 10 minutes and then a glycerol bath at 110-130 °C for 5- 60 minutes. The first bath removes the formatin bath solution. The second bath allows water to evaporate. It is envisioned that is certain scenarios more baths with differing temperatures may be used so long as the last bath removes water from the fibers. (When the process is run continuously, the fiber is not spooled but directly transferred viaAttorney Docket No: P25-032-SEC-W001rollers into multiple baths. Each bath has an increasing glycerol content ending at 100% increasing and temperature.) From there the spool was placed in one or more baths that contain above 20%, preferably 40% to 50% (v:v) glycerol in water (e.g., Milli-Q® water or equivalent; MilliporeSigma, Burlington, MA). Finally, the resulting mung bean hollow fibers were dried in an oven at 50 °C for 2 or more hours.Examples of hollow fibers made with this method are shown in Fig. 1.

[0113] Example 2 - Experiments Demonstrating the Plasticizing of Plant-Protein Membranes

[0114] The strain value at ultimate tensile strength (UTS) point indicates how much the material can elongate or compress under maximum load before it starts to fail. This is an important parameter for understanding the ductility and toughness of a material.

[0115] Independently increasing the content of glycerol in the formation bath resulted in membranes that elongated more to reach its maximum tensile strength, indicating a more flexible, pliable material. Neither buffer nor ethanol alone was unable to achieve this. See, Figs. 3 and 4. The dots on Fig. 3 are data points. The contour graph was generated based on these data points. Darker regions indicate a higher strain was tolerated. Lighter regions indicate a lower strain was tolerated. The zeros on the sides of the triangle indicate the concentration of the component located across the triangle is zero. That is, the zero located between Ethanol and Glycerol indicates a buffer concentration of “0” at that point.

[0116] The strain value at ultimate tensile strength (UTS) point indicates how much the material can elongate or compress under maximum load before it starts to fail. This is an important parameter for understanding the ductility and toughness of a material.

[0117] Further, the mixture contour plot of Fig. 3 is of modeled and predicted strain at ultimate tensile strength for membranes in a formulation bath comprised of various proportions of ethanol, glycerol, and / or buffer. The proportion of each component is represented by the distance from the point that is labeled with the respective component (1), and the opposite edge (0). Black dots represent tested data points.Attorney Docket No: P25-032-SEC-W001The rest of the region within gray parallelogram represents interpolated strain values of our process window. The area outside the parallelogram includes extrapolated data points. A trend exists between an increasing strain and an increasing concentration of glycerol, as well as a decreasing concentration of ethanol.

[0118] Fig. 4 shows a response trace plot of the modeled strain at ultimate tensile strength for variations from centroid in tested formulations (.2 Ethanol, .3 Glycerol, .5 Buffer). The slope of each components curve indicates the modeled response in strain as the proportion of the component is changed from the reference blend. The positive slope of glycerol indicates a positive correlation between glycerol concentration and strain at ultimate tensile strength. The negative slope of the ethanol curve indicates a negative correlation between the strain and ethanol concentration. The buffer has a lesser negative slope, indicating a small negative correlation.

[0119] Example 3 - Un-Annealed Membrane Glycerol Content, Dehydration is the Driver of Mechanical Handleability

[0120] In an experiment to demonstrate the effectiveness of glycerol content in the formation bath. Flat sheet membrane was hand cast via doctoral blade onto the surface of PTFE (Teflon) film. The flat sheet membrane on the film was submerged into two different formation bath conditions. Both had 20% ethanol, but the remaining 80% of formation bath was 40% to 60% and 20% to 80% glycerol to buffer (v:v), respectively. We used 0.16-0.2 M sodium acetate, 0.088-0.11 M hydrochloric acid, and 0.36-0.45 M calcium chloride as the aqueous portion of the glycerol baths. We believe other salts and / or acids would also be effective in the present invention.

[0121] After 3 minutes of submersion, the flat sheet membranes were removed. As in this state membranes are unhandleable and impossible to remove from the PTFE without damaging the membranes. The partially formed membrane samples were submerged into a 60% ethanol and 40% glycerol bath and placed into an oven at 50 °C for 2 hours. The 40% glycerol prevented the membrane from collapsing.Attorney Docket No: P25-032-SEC-W001

[0122] In the fully dehydrated state, the dehydrated membrane was integral enough to cut into multiple sheets. The sheets were sorted into different saturation baths with various amounts of water and glycerol. Water was used instead of buffer to prevent further coagulation. The samples that were placed into 100% water were rehydrated and returned to an unhandleable state; in this state, they broke into pieces upon retrieval. The other blends of buffer and glycerol increased glycerol content to 20%, 40%, and 60%. From there, the 1x3 inch samples we tensile tested (ZwickRoell LP Kennesaw, GA 30144).

[0123] It is seen in Figs. 6 & 7 that both the elastic modulus and breaking stress (respectively) increased with increasing glycerol content. As expected from Example 2, it is seen that the absolute breaking stress increased with increased buffer concentration. This example demonstrated that 0% glycerol resulted in materials that were not able to be handled without breaking (data not shown). Furthermore, this example demonstrated that increased glycerol content in not yet annealed and partially coagulated plant protein-based membrane directly resulted in improved mechanical properties.

[0124] Example 4 - Ethanol Content Increases Coagulation to a degree; beyond that, unfavorable properties are seen.

[0125] Ethanol acts as a coagulant and nonsolvent for proteins due to its unique chemical properties and interactions with protein molecules. These reasons are mainly due to hydrophobic interactions, reduced solubility, denaturation induction and dehydration-based precipitation. In this example, plant-based protein isolate membrane dope was extruded through a hollow fiber nozzle into 100% ethanol. The fiber extruded turned white / off white and was no longer opaque / transparent upon entering the ethanol. Those skilled in the art of membrane formation would conclude that this was due to the change in light refraction of the forming membrane, which was indicative of material solidification. See, Fig. 2.

[0126] The images were taken with a Keyance (500 Park Boulevard, Suite 200, Itasca, IL 60143) VHX digital microscope. These images demonstrate ethanol’s ability to solidify the protein matrix. ImagesAttorney Docket No: P25-032-SEC-W001depict the change in light refraction of the structure. During the hollow fiber phase inversion or coagulation step, the initial clearer solution may undergo phase separation. As the polymer precipitates and forms a porous structure, the increased scattering of light by the newly formed protein-rich regions can make the membrane appear white or off-white.

[0127] Further analysis of the samples showed the effect that ethanol had on the dimensional stability of the protein matrix. As the ethanol content is increase, as defined as percent concentration in acetate buffer, the overall geometry of the fiber decreases. More specifically, a total of 30 percent change is observed from an ethanol-free bath to the ethanol-exclusive bath; which is undesirable. This is quantified in Fig.7.

[0128] For a manufacturing facility that needs to be built explosion proof (XP) the cost of the facility can increase by more than a multiple of 10. The standard can be found in 29 CFR 1910.106, which outlines the requirements for flammable and combustible liquids; NFPA (National Fire Protection Association): NFPA 30, the "Flammable and Combustible Liquids Code."

[0129] It could also be theorized that by increasing the concentration of glycerol, the boiling point and flash point of the described formation bath would increase. However, independent of the glycerol content and boiling point of the solution, the flash point does not change. One skilled in the art may assume that by adding glycerol to the formulation bath would lower its flashpoint and increase the safety in handling. Surprisingly, glycerol-buffer-ethanol flash point curve is very similar to that of the water-Ethanol flashpoint curve. This is quantified in Fig. 8.

[0130] In scaling such a process that maximizes the rate of coagulation, more ethanol is beneficial. However, in a balanced process that considers safety, efficiency, final product structure, and membrane structure, along with the maximum coagulation, it’s realized to have around or less than 20% ethanol at room temperature and when paired with buffer and glycerol.Attorney Docket No: P25-032-SEC-W001

[0131] Example 5 - Density of Formation Bath and Buoyancy of Fiber

[0132] If the hollow fiber floats while in the formation bath, the surface porosity and pore structure will vary around the circumference of the fiber. The air exposed side will see different diffusion of the formation bath resulting in a changing the rate of electron exchange for the PhlPS process; which would lead to varying pore properties around the circumference.

[0133] As seen in Example 3, increased glycerol aids in the integrity and mechanical properties of the plant-based protein isolate fiber. In a scenario of 40% Glycerol and 60% buffer, the solution density would be 1 ,155g / cm3, which is denser than the dope and therefore would result in floating fiber. In another scenario of 30% Glycerol, 20% ethanol, and 50% buffer, the solution density would be 1.102 g / cm3, which is lower than the fiber making it sink in the formation bath; resulting in a more homogenous fiber.

Claims

Attorney Docket No: P25-032-SEC-W001ClaimsWhat is claimed is:

1. A process for the manufacture of edible membranes, comprising: a. providing: one or more plant protein isolates;b. admixing the plant protein isolate with NaOH and urea to make an alkaline plant protein isolate dope;c. forming the plant protein isolate dope into a fiber or flat sheet membrane into a formation bath, wherein the formation bath comprises about 20% to about 40% glycerol, about 10% to about 20% ethanol in an aqueous buffer at a pH below 6 to make a plant protein isolate membrane;d. processing the membrane through one or more glycerol baths wherein the final bath is over 100 °C and said membrane is in the glycerol bath for a sufficient length of time to cause any water to evaporate;e. resuspending the membrane in a glycerol and water bath, and; f. drying the membrane in an oven.

2. A process for the manufacture of edible hollow fibers, comprising: a. providing: one or more plant protein isolates;b. admixing the plant protein isolate with NaOH and urea to make an alkaline plant protein isolate dope;c. extruding the plant protein isolate dope from a hollow fiber nozzle into a formation bath, wherein the formation bath comprises about 20% to about 40% glycerol, about 10% to about 20% ethanol in an aqueous buffer at a pH below 6 to make a plant protein isolate hollow fiber;d. processing the hollow fiber through one or more glycerol baths wherein the final bath is over 100 °C and said hollow fiber is in the glycerol bath for a sufficient length of time to cause any water to evaporate;e. resuspending the hollow fiber in a glycerol and water bath, and; f. drying the hollow fiber in an oven.Attorney Docket No: P25-032-SEC-W0013. The process of Claim 1 or 2, wherein said dope further comprises a polysaccharide.

4. The process of Claim 3, wherein the polysaccharide is alginate or pectin.

5. The process of Claim 3 or 4, wherein the concentration of the polysaccharide in the plant protein isolate dope is less than 5%, less than 2%, less than 1% or less than 0.2%.

6. The process of any one of the preceding claims, wherein the plant protein isolate is admixed for 1 to 48 hours.

7. The process of any one of the preceding claims, wherein the plant protein isolate dope has a pH of 9 or higher.

8. The process of any one of the preceding claims, wherein the plant protein isolate dope has a pH of about 11 - 12.5.

9. The process of any one of the preceding claims, wherein said aqueous buffer comprises 0.16 M - 0.2 M sodium acetate, 0.088 M - 0.11 M hydrochloric acid, and 0.36 M - 0.45 M calcium chloride and has a pH of about 4.0 - 5.0.

10. The process of any one of the preceding claims, wherein the plant protein isolate is selected from one or more of mung bean, soybean, lentil, red lentil, pea, rice, faba and chickpea.11.The process of any of claims 2-10, wherein said extruded plant protein isolate is formed into a hollow fiber having an outer diameter of about 400 pm to about 600 pm and a wall thickness of about 100 pm to about 200 pm.

12. The process of any of claims 2-10, wherein said extruded plant protein isolate is formed into a hollow fiber having an outer diameter of about 450 pm to about 550 pm and a wall thickness of about 125 pm to about 175 pm.40Attorney Docket No: P25-032-SEC-W00113. The process of any one of the preceding claims, wherein said plant protein isolate dope forms micro and or nano porous membranes.

14. The process of any one of the preceding claims, wherein said formed membrane is a hollow tube-like or a solid cylindrical-like fiber or a flat sheet.

15. The process of any one of the preceding claims, wherein said aqueous buffer comprises one or more salts generally recognized as safe (GRAS) by the FDA.

16. The process of any one of the preceding claims, wherein said aqueous buffer comprises one or more salts selected from the group consisting of calcium chloride, potassium chloride, sodium chloride and calcium lactate.

17. The process of any one of the preceding claims, wherein the formation bath has a density lower than the density of the plant protein isolate membrane.

18. The process of any one of the preceding claims, wherein said aqueous buffer comprises one or more acids generally recognized as safe (GRAS) by the FDA.

19. The process of any one of the preceding claims, wherein the aqueous buffer comprises one or more GRAS acids selected from the group consisting of sodium acetate, citric acid, hydrochloric acid, acetic acid and sodium citrate.

20. The process of any one of the preceding claims, wherein the glycerol content of the glycerol and water bath is about 40 - 50% glycerol.

21. The process of any one of the preceding claims, wherein said oven is at a temperature of 40 °C or higher and the membrane is in the oven for a period of time sufficient to make the membrane dry to the touch.

22. A process for the manufacture of edible membranes, comprising:Attorney Docket No: P25-032-SEC-W001a. providing: one or more plant protein isolates and, optionally, a polysaccharide;b. admixing the plant protein isolate with acid and urea to make an acidic plant protein isolate dope with a pH of about 2 -5; c. forming the plant protein isolate dope into a fiber or flat sheet membrane into a formation bath, wherein the formation bath comprises about 20% to about 40% glycerol, about 10% to about 20% ethanol in an aqueous buffer as a pH of about 8 - 11 to make a plant protein isolate membrane;d. processing the membrane through one or more glycerol baths wherein the final bath is over 100 °C and said membrane is in the glycerol bath for a sufficient length of time to cause any water to evaporate;e. resuspending the membrane in a glycerol and water bath, and; f. drying the membrane in an oven.

23. The process of any one of the preceding claims, wherein said formation bath comprises about 30% glycerol and about 20% ethanol in an aqueous buffer.

24. The process of any one of the preceding claims, wherein the pH of the formation bath about 3.0 - about 6.0, about 3.0 - about 5.0 or about

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