Edible protein thread, edible food and its manufacturing method

Abstract

The present invention relates to a method for producing an edible protein thread, the method comprising the steps of: - preparing a first composition (130), wherein said first composition comprises: animal protein, wherein the animal protein is derived from cultured non-human animal cells; and · either polyvalent ions or polyelectrolytes; - preparing a second composition (140), wherein said second composition comprises: the multivalent ions, if the step of preparing the first composition (130) includes the addition of the polyelectrolyte, or the polyelectrolyte, if the step of preparing the first composition (130) includes the addition of the multivalent ion; and - contacting (160) said first composition with said second composition to form edible protein threads, wherein said first composition has a temperature below 65°C.

Description

[Technical Field]

[0001] The present invention relates to the food field. In particular, the present invention relates to the meat substitute field. In particular, the present invention relates to cultured cell-based meat. In particular, the present invention can provide new edible protein threads that enable the production of edible products with a meat-like texture. This edible product with a meat-like texture can be considered as a substitute for traditional meat. [Background technology]

[0002] It is estimated that the world's population will increase by 2 billion people between 2020 and 2050 (United Nations, 2015). Humanity will face major challenges, including food production, including meat production. Traditional meat production is a resource-intensive process that places a heavy burden on the environment. Livestock are raised in agricultural environments, requiring large amounts of freshwater, feed, land, and other resources (Mark J Post: "Cultured meat from stem cells: Challenges and prospects," Meat science, Elsevier Science, GB, vol. 92, no. 3, 3 April 2012, pages 297-301). As a result, food production is already thought to account for approximately 26% of global greenhouse gas (GHG) emissions, of which livestock and fisheries account for 31%.Reducing traditional meat consumption globally could lead to significant reductions in greenhouse gas emissions related to climate change (Martin & Brandao, 2017. Evaluating the environmental consequences of Swedish food consumption and dietary choices. Sustainability, 9(12), 2227), especially if reductions are made in countries where meat consumption is high or increasing (Stoll-Kleemann & Schmidt, 2017. Reducing meat consumption in developed and transition countries to counter climate change and biodiversity loss: A review of influence factors. Regional Environmental Change, 17(5), 1261-1277.; Collier et al. 2021. Identifying barriers to decreasing meat consumption and increasing acceptance of meat substitutes among Swedish consumers; Appetite 167 (2021) 105643.). Furthermore, animal welfare is also a growing concern. For example, the European Union has enacted various animal welfare laws since 1986. Various directives have laid down rules for the protection of laying hens (1986 and 1988), calves and pigs (1991), and in 1998 Council Directive 98 / 58 / EC on the protection of animals kept for agricultural purposes established general rules for the protection of animals regardless of species.

[0003] Several meat substitutes have been developed from insects, plant components, and / or cultured animal, fungal, or plant cells (i.e., cell technology). Cell technology, in particular, is rapidly developing to meet emerging consumer demands.

[0004] To increase people's willingness to consume meat substitutes, it is important that the products mimic the aesthetic and sensory qualities of meat, including size, appearance, flavor, texture, and texture (Macdiarmid et al., 2016, "Eating like there's no tomorrow: Public awareness of the environmental impact of food and reluctance to eat less meat as part of a sustainable diet." Appetite. 2016 Jan 1;96:487-493). Although texturing techniques to improve the texture and taste of these products are continually improving, meat analogues still differ from traditional meat in terms of mouthfeel and flavor (Samard & Ryu, 2019, "A comparison of physicochemical characteristics, texture, and structure of meat analogues and meats." Journal of the Science of Food and Agriculture, 99(6), 2708-2715). Furthermore, consumers are unfamiliar with how to prepare meals using meat substitutes and report that preparing meals using meat substitutes is more difficult and time-consuming than preparing meals using meat (Elzerman et al., 2013; Exploring meat substitutes: Consumer experiences and contextual factors. British Food Journal, 115(5), 700-710).

[0005] Texture is considered one of the most important qualities of meat analogues (Sha & Xiong, 2020. Plant protein-based alternatives of reconstructed meat: Science, technology, and challenges. Trends in Food Science & Technology. Volume 102, August 2020, Pages 51-61). Many methods have been proposed to improve the texture of meat analogues. For example, combining oat and pea proteins has been proposed as a viable alternative to soy and gluten proteins (Kaleda et al. 2021. Physicochemical, textural, and sensorial properties of fibrous meat analogues from oat-pea protein blends extruded at different moistures, temperatures, and screw speeds. Future Foods 4 (2021) 100092). Biosurfactant-based emulsions have also been proposed for the production of 3D-printed foods (Shahbazi et al. 2021. Construction of 3D printed reduced-fat meat analogues by emulsion gels. Part II: Printing performance, thermal, tribological, and dynamic sensory characterization of printed objects. Food Hydrocolloids 121 (2021) 107054). According to this study, replacing oil with a biopolymer surfactant is recommended for producing fibrous 3D-printed reduced-fat meat analogues, and the printed reduced-fat structures provide a desirable sensory profile. However, many meat analogues still lack satisfying, flavorful fats.The combination of biopolymer surfactants and hydrocolloids cannot mimic all of the sensory properties typical of animal-derived meat, i.e., texture, taste, and aroma. It has also been proposed to produce extruded foods containing cultivated animal cells (WO2022047263). However, the resulting extruded foods bear little resemblance to what consumers expect from meat products, especially when the appearance of a raw product and the flavor trapped within the meat pieces are desired.

[0006] The use of scaffolds for culturing myoblasts has also been proposed, and it has been demonstrated that scaffolds prepared with non-mammalian biomaterials, such as salmon gelatin, alginate, agarose, and glycerol, can be used for in vitro meat formulation. However, regulatory issues and obvious technical obstacles exist before such technologies can be translated into large-scale production. Summary of the Invention [Problem to be solved by the invention]

[0007] Therefore, it is important to find satisfactory alternatives to animal slaughter and intensive meat production methods to produce edible foods with the pleasant flavor, texture, and cooking methods expected of meat. In particular, there is a need for edible protein threads that can be used to manufacture meat analogs to mimic the texture of conventional meat. The texture expected of animal meat before and after rendering cooking is also desired so that consumers can prepare the product in the same way as conventional meat and experience the same taste qualities. [Means for solving the problem]

[0008] The following presents a simplified summary of selected aspects, embodiments, and examples of the invention in order to provide a basic understanding of the invention. However, this summary is not intended to constitute an extensive overview of all aspects, embodiments, and examples of the invention. Its sole purpose is to present selected aspects, embodiments, and examples of the invention in a concise form as a prelude to the more detailed description of the aspects, embodiments, and examples of the invention that follows the summary.

[0009] The present invention aims to overcome the drawbacks of the prior art. In particular, the present invention proposes a method for producing edible protein threads, comprising the following steps: - preparing a first composition, wherein said first composition comprises: animal protein, wherein the animal protein is derived from cultured non-human animal cells; and · either polyvalent ions or polyelectrolytes; - preparing a second composition, wherein said second composition comprises: the multivalent ions, if the step of preparing the first composition includes the addition of the polyelectrolyte, or the polyelectrolyte, if the step of preparing the first composition includes the addition of the multivalent ion; and - contacting said first composition with said second composition to form edible protein threads, wherein said first composition has a temperature of less than 65°C.

[0010] Such edible protein yarns have a texture similar to that of meat fibers (eg, when incorporated into an edible product, can have a texture similar to that of a rump steak fillet).

[0011] Furthermore, protein yarns according to the invention can exhibit a variety of shapes and sizes, unlike high moisture extrusion typically used to structure plant-based products, and the method according to the invention can produce 3D products and larger yarns containing the flavors consumers expect.

[0012] In particular, the method of the present invention allows for the production of edible protein threads and, therefore, raw edible foods. These advantages allow consumers to cook the product in the same way as conventional meat and experience the same sensory qualities. The method is scalable and can produce aligned threads with a wider range of fats and proteins (compared to extrusion methods) without oxidation during processing.

[0013] Thus, the edible protein yarns of the present invention can be considered both a component and a step in the production of edible products that are alternatives to traditional meat products. Such edible protein yarns can be engineered to mimic a wide variety of meat fibers, including, but not limited to, beef, scallop, crab, chicken breast, duck breast, tuna, and salmon, and have a matching meaty or fishy flavor. Indeed, the presence of cultured cells or extracts thereof introduces complex and desirable flavors into the protein yarns, which can be tailored depending on the type of cultured cells used.

[0014] Advantageously, the first composition comprises at least 10% total protein weight compared to the total wet weight of the first composition. As shown in the examples, such a concentration of protein in the first composition can improve the mechanical properties of the yarn, such as strain at break, load at break, or tensile stress.

[0015] Furthermore, the first composition advantageously comprises at least 5% non-human animal protein by weight of total protein, said animal protein being derived from cultured non-human animal cells. In particular, the first composition comprises at least 0.5% animal protein derived from cultured non-human animal cells, relative to the total wet weight of the first composition. Indeed, as shown in the examples, such enrichment can improve strain at break, load at break, tensile stress, Young's modulus, or meat appearance, texture, or flavor.

[0016] According to other optional features of the edible food product according to the invention, it may optionally comprise one or more of the following features, alone or in combination: The first composition is not heated to a temperature of 60°C or above for more than 10 minutes. Indeed, as illustrated in the examples, heating the first composition at high temperatures for at least 10 minutes can degrade the properties of the edible protein thread. Preferably, the first composition is not heated to a temperature of 50°C or above for more than 10 minutes. - the animal cell is selected from a cell of the kingdom Animalia, preferably the animal cell is selected from a mammalian cell, an avian cell, an Actinopterygian cell, a Malacostraca cell, and combinations thereof; for example, the mammalian cell can be a Bovidae cell, a Cervidae cell, a Leporidae cell, or a Sardinidae cell; the avian cell can be an Anatidae cell or a Phasianidae cell; the Actinopterygian cell can be a Gadidae cell, a Hake cell, a Pleuronectinidae cell, a Salmonidae cell, or a Scombridae cell; the Malacostraca cell can be a Palaemonidae cell; the molluscan cell can be a cephalopod cell or a bivalve cell. The above-mentioned cells are particularly suitable for producing edible protein threads according to the method of the present invention and thus for preparing an edible food product. The non-human cultured animal cells include cells selected from the following: stem cells such as embryonic stem cells, satellite cells, induced pluripotent stem cells, germ cell, fibroadipogenic progenitor cells, muscle cells such as skeletal muscle cells, cardiac cells, and smooth muscle cells; myoblasts, muscle cells, hepatocytes, fibrocytes, fibroblasts, adipocytes, chondrocytes, chondroblasts, keratinocytes, melanocytes, bone cells, osteoblasts, Merkel cells, Langerhans cells, glial cells, Schwann cells, red blood cells, and white blood cells, and combinations thereof. The above cells are particularly suitable for producing edible protein threads according to the method of the present invention, and are therefore suitable for preparing edible foods. Preferably, the non-human animal cells include cells selected from stem cells, muscle cells, fibroblasts, adipocytes, red blood cells, and combinations thereof. The animal protein preferably comprises at least 0.25% myofibrillar protein by weight compared to the total protein weight. Indeed, as shown in the examples, if an edible protein yarn is made exclusively with plant proteins that contain no or only small amounts of myofibrillar protein, the properties of the edible protein yarn may be reduced. The first composition comprises extracellular matrix non-human animal molecules, preferably at least 0.1% by weight of the non-human animal molecules compared to the total weight of proteins. Preferably, the non-human animal molecules are selected from collagen, elastin, fibronectin, laminin, heparan sulfate, chondroitin sulfate, keratan sulfate, hyaluronic acid, or a combination thereof. Indeed, as shown in the examples, when the first composition comprises elastin and / or collagen, the properties of the edible protein thread are improved. In particular, the elastin and / or collagen must be added in a soluble form. - the first composition has a dry content of at least 12% by weight, preferably at least 15% by weight, more preferably at least 20% by weight. The polyelectrolyte is a thermostable gel-forming polyelectrolyte. Indeed, as shown in the examples, the use of a thermostable gel-forming polyelectrolyte improves the properties of the edible protein thread. In fact, the polyelectrolyte is advantageously selected from among polyelectrolytes that are capable of complexing with multivalent ions to form a thermostable gel. The polyelectrolyte is a polysaccharide. Polysaccharides as polyelectrolytes according to the invention are particularly suitable for producing edible protein threads according to the method of the invention and are therefore also suitable for preparing edible food products. The polyelectrolyte is selected from low methoxyl pectin, low methoxyl pectin derivatives, high methoxyl pectin, high methoxyl pectin derivatives, alginate, alginate derivatives, xanthan, xanthan derivatives, chitosan, chitosan derivatives, ionic carboxymethylcellulose derivatives, ionic pullulan derivatives, ionic dextran derivatives, ionic starch derivatives, or combinations thereof. The above-mentioned polysaccharides are particularly suitable for producing edible protein threads according to the method of the present invention, and therefore suitable for the production of edible food products. The first or second composition, preferably the first composition, further comprises an oxidoreductase such as peptidase, α-galactosidase, alcalase, thermolysin, pepsin, trypsin, chymotrypsin, asparaginase, elastase, subtilisin, glucose oxidase, laccase, transglutaminase, pectinesterase, sortase, tyrosinase, lysyl oxidase and peroxidase, genipin, riboflavin, monoamine oxidase, or a combination thereof, preferably laccase, transglutaminase, or a combination thereof. As shown in the examples, the method of the present invention allows the use of enzymes to improve the mechanical properties of edible protein yarns. The first composition further comprises a plant protein, an algae protein and / or a fungal protein, preferably the plant protein is selected from sunflower protein, soy protein, pea protein, canola protein, mung bean protein, chickpea protein, fava bean protein, lentil protein, seaweed protein, potato protein, quinoa protein, nut protein, wheat protein, chickpea protein, bambara bean protein, dulse protein, mesquite bean protein, duckweed protein, chickpea protein, dried fava bean protein, dried cowpea protein, lupin protein, jackfruit protein, amaranth protein, millet protein, oat protein, chia protein, hemp seed protein, rice protein, or a combination thereof. The first composition comprises at least 10% by weight of total protein relative to the total wet weight of the first composition. As shown in the examples, when the first composition comprises plant protein, the presence of non-human animal protein derived from non-human cultured animal cells is necessary to obtain the desired properties of the edible protein yarn. Thus, preferably, the first composition comprises at least 5% by weight of non-human animal protein derived from non-human cultured animal cells relative to the total weight of protein in the first composition. The preparation of the first composition further comprises the addition of a fat, preferably a vegetable fat or a fat obtained by fermentation, in particular at least 1% fat compared to the total wet weight of the first composition. As shown in the examples, the addition of fat to the first composition improves the properties of the edible protein thread. The preparation of the first composition includes homogenization, preferably at least 100 rpm for at least 30 seconds. Homogenization improves the properties of the edible protein fiber and, when combined with the addition of fat, can produce an emulsion, which can improve the properties of the edible protein fiber produced. - the preparation of the first composition comprises the addition of disrupted and / or intact cultured non-human animal cells. Thus, the preparation of the first composition can comprise the addition of disrupted and / or intact cultured non-human animal cells. Preferably, the preparation of the first composition comprises the addition of intact cultured non-human animal cells. -the first composition comprises: at least 10% by weight total protein based on total wet weight of the first composition, wherein the first composition comprises at least 5% by weight non-human animal protein and at least 0.25% by weight myofibrillar protein; and at least 0.1% by weight of a polyelectrolyte, based on the total wet weight of the first composition; -the first composition comprises: at least 10% by weight total protein based on the total wet weight of the first composition, wherein the first composition comprises at least 5% by weight non-human animal protein based on the total protein weight, and the animal protein is derived from non-human cultured animal cells; and at least 0.1% by weight of a polyelectrolyte, based on the total wet weight of the first composition; -the first composition comprises: at least 10% by weight total protein based on the total wet weight of the first composition, wherein the first composition comprises at least 5% by weight non-human animal protein based on the total protein weight, and the animal protein is derived from non-human cultured animal cells; at least 0.1 wt. % of a polyelectrolyte, based on the total wet weight of the first composition; and At least one cross-linking molecule.

[0017] The present invention also relates to methods of producing edible food products. In particular, the methods of producing edible food products include producing edible protein threads according to the methods of the present invention. The methods of producing edible food products can also include assembling one or more of the produced edible protein threads into an edible food product.

[0018] The present invention also relates to edible protein yarns obtainable from the methods of the present invention. In particular, the present invention relates to edible protein yarns obtainable from the methods of the present invention. Preferably, the edible protein yarns according to the present invention comprise an animal protein and a polyelectrolyte that forms a thermostable gel, wherein the animal protein is a cultured non-human animal cell protein.

[0019] The present invention also relates to edible food products comprising the edible protein thread according to the present invention.

[0020] The above and other objects, features and advantages of the present invention will become more apparent from the following detailed description taken in conjunction with the accompanying drawings. [Brief explanation of the drawings]

[0021] [Figure 1] 1 is a schematic diagram of a method for producing an edible food product having a meat-like texture according to one embodiment of the present invention. [Figure 2] 1 is a photograph of edible protein thread. [Figure 3] This is a photo of edible food. DETAILED DESCRIPTION OF THE INVENTION

[0022] Some aspects of the present invention are disclosed with reference to flow diagrams and / or block diagrams of methods and devices.

[0023] When present in the figures, flow diagrams and / or block diagrams illustrate the architecture, functionality, and possible implementations of devices, systems, or methods according to some embodiments of the present invention.

[0024] In some embodiments, the functions associated with the boxes may appear out of the order shown in the figures. For example, two boxes shown in succession may execute substantially simultaneously or may execute in reverse order, depending on the functions involved.

[0025] Exemplary embodiments of the present invention are described below.

[0026] As used herein, the expression "protein yarn" can refer to a long, thin thread of material that contains at least 5% by weight, preferably at least 7.5%, and more preferably at least 10% by weight of protein relative to the wet weight of the thread. Preferably, the protein yarn has a diameter of at least 0.01 mm, preferably at least 0.05 mm, and an aspect ratio (length / diameter) of at least 100, preferably at least 200. Preferably, the protein yarn has a diameter of at most 2 mm, more preferably at most 1 mm, and even more preferably at most 0.5 mm.

[0027] The expression "edible protein yarn" as used herein relates to a protein yarn suitable for animal consumption, preferably a protein yarn suitable for human consumption.

[0028] The expressions "edible product" or "edible food" as used herein relate to a product suitable for animal consumption, preferably a product suitable for human consumption. An edible product according to the invention may be a ready-to-eat (i.e. finished) food product or an intermediate product in the production chain of a finished food product. As described below, edible products according to the invention may be produced in the form of snacks that can be pressed, fried and / or toasted; sauces; spreads; pasta; pastes; processed meat analogues or specialities such as sausages and cured sausages, pâtés, foie gras; meat dough; soups; smoothies; seafood; and unprocessed meat analogues such as "meat-like" products.

[0029] In the following description, the term "meat" may refer to edible parts of an animal, such as animal tissues harvested from a slaughtered animal. Thus, meat may refer to the liver and other visceral tissues, fat tissue, and muscle tissues typically found in animals. Dead animals refer to all species in the animal kingdom, excluding humans, and preferably to all edible species, such as non-human vertebrates (e.g., livestock, fish, and birds), insects, crustaceans (e.g., shrimp, prawns, crabs, crayfish, and / or lobsters), and mollusks (e.g., octopus, squid, cuttlefish, scallops, and snails). Thus, for example, the present invention enables the production of edible products with a meat-like texture, such as products that mimic foie gras, marbled beef, or salmon meat.

[0030] As used herein, the expression "by weight" generally refers to the weight of one component compared to the weight of another component or to the weight of the composition as a whole, and can consider either wet weight or dry weight. Preferably, percentages are disclosed with reference to wet weight.

[0031] As used herein, the terms "cultured cells" or "farmed cells" are used interchangeably. They may refer to cells grown and / or propagated using a culture medium, preferably in a controlled environment. This particularly refers to cells whose growth has been controlled by humans, such as in an industrial process, as opposed to traditional meat cells grown in vivo or cells grown in a natural environment (such as mushrooms grown in a forest). Cultured cells may refer to cells belonging to the Animalia kingdom with respect to proteins, but may also refer to cells belonging to the Bacteria, Green Plants, or Fungi kingdoms to provide additional proteins and fats. Cultured cells may be produced from cells of any origin, such as biopsy cells, stem cells, or stem cells themselves. More specifically, a protein yarn derived from cultured cells may refer to a protein yarn composed primarily of proteins derived from cultured cells. For example, a cultured cell-based protein yarn comprises at least 5 wt. % of protein derived from cultured cells, preferably at least 10 wt. % of protein derived from cultured cells, more preferably 20 wt. % of protein derived from cultured cells, and even more preferably 30 wt. % of protein derived from cultured cells, relative to the total weight of protein.

[0032] As used herein, the term "cultured cell extract" can refer to any part of disrupted cells, such as a protein extract of cultured cells, or any purified or partially purified biological material recovered from disrupted cells. Disrupted cells can be cells whose cell walls are partially or completely disrupted. In the protein context, a cultured cell extract can include both disrupted cells and biological materials recovered from the disrupted cells. A cultured cell extract can be obtained, for example, by separation and purification of biological materials recovered from the disrupted cells. Thus, the extract can be obtained, for example, after at least a drying step, a precipitation step, or a solvent extraction step. An intact cultured animal cell can refer to a cultured animal cell whose membrane is intact as can be evaluated microscopically.

[0033] As used herein, the term "polyelectrolyte" may refer to a polymer that, when dissolved in a polar solvent such as water, has covalently attached charged groups. Generally, polyelectrolytes have a variety of such groups. Homogeneous polyelectrolytes have only one type of charged group, such as only carboxylate groups.

[0034] As used herein, the expression "myofibrillar protein" can refer to actin, myosin, tropomyosin, troponin, α-actinin, connectin, titin, nebulin, C protein, M protein, desmin, or a combination thereof.

[0035] The term "thermostable gel" as used herein can refer to, for example, a gel formed from a polyelectrolyte and a polyvalent ion that is not liquid at temperatures below 80°C. Preferably, it refers to a gel that is not liquid at temperatures below 100°C. The term "thermostable gel-forming polyelectrolyte" as used herein can refer to a polyelectrolyte that forms a thermostable gel when combined with an appropriate polyvalent ion. In particular, the thermostable gel-forming polyelectrolyte is combined with a polyvalent ion former that can complex with the polyelectrolyte to form a thermostable complex. For example, when the second composition contains a polyelectrolyte, the first composition contains a polyvalent ion, or when the second composition contains a polyvalent ion, the first composition contains a polyelectrolyte.

[0036] As used herein, the expression "biodegradable polymer" may refer to a polymer that can be broken down or decomposed into harmless products by the biological activity of living organisms (such as microorganisms).

[0037] As used herein, the phrase "fermentation-derived fat" or "fermented fat" may refer to lipid molecules produced, for example, by microbial fermentation in a growth reactor. The fermentation-derived lipid molecules may be chemically identical to fats produced by plants or animals.

[0038] As used herein, the term "flavor" generally refers to the quality of a product that affects taste and / or aroma. Thus, "meaty flavor" can refer to a flavor that is close to or approximates the flavor of a related conventional meat product.

[0039] The term "texture" as used herein may be considered as "the combination of rheological and structural (geometric and surface) properties of a food perceived by the mechanical, tactile, and, where appropriate, visual and auditory receptors" as defined in 2008 by the International Organization for Standardization (ISO, 2008, Terminology of Sensory Analysis, Vol. 1-107, p. 5492). Accordingly, "meat-like texture" may refer to the texture and structural (geometric and surface) properties of a food that are close to or approximate the texture of related conventional meat products (i.e., meat products obtained from the slaughter of animals). Edible foods having a meat-like texture and meat-like flavor according to the present invention may be considered as substitutes for meat products.

[0040] As used herein, the term "about" may allow for a degree of variation in a value or range, for example, within 10%, within 5%, or within 1% of the stated limit of the stated value or range.

[0041] As used herein, the term "substantially" refers to a majority or majority, such as at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or at least about 99.999% or more. Thus, a composition in which cells are preserved substantially intact refers to a composition that contains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 99.99% intact cells.

[0042] As mentioned above, producing tasty meat-like products without slaughtering animals presents challenges for both animal welfare and the environment. In addition to animal welfare and environmental protection, it seems necessary to produce foods that exhibit quality close to that of conventional products and meet consumer expectations, especially by producing protein fibers found in meat.

[0043] A new method has been developed to produce edible protein threads with a meat-like texture without containing slaughtered animal tissue.

[0044] Thus, according to a first aspect, the present invention relates to a method 100 for producing an edible protein thread.

[0045] The manufacturing method 100 of the present invention preferably allows for the production of edible protein threads that have a meat-like texture.

[0046] As shown in FIG. 1, the method includes the steps of preparing a first composition 130, preparing a second composition 140, and contacting the first composition with the second composition 160 to form an edible protein thread.

[0047] The method for producing edible protein yarns or edible foods can include several other steps, such as: blanching, heat sterilization, evaporation and distillation, dehydration, smoking, baking and roasting, frying, high pressure processing, pulsed electric field (PEF) processing, ultrasound, cavitation, shock wave processing, pasteurization, application of cold plasma, dielectric, ohmic and infrared processing, microwave heating, assisted extraction, food irradiation, UV microbial inactivation, pulsed light technology, supercritical extraction, extrusion, freezing, cooling, modified atmosphere, drying techniques (lyophilization, membrane), fermentation, homogenization, mincing, grinding, chopping, salting, tumbling, brine injection. Notably, the method of the present invention can also include additional steps, such as culturing non-human animal cells 110, processing non-human animal proteins 120, adding food additives 150, converting the edible protein yarn 170, and / or conditioning the edible food 180.

[0048] As shown in FIG. 1, the method of the present invention can include a step 110 of culturing non-human animal cells.

[0049] This step is specifically designed to produce an animal protein source without slaughtering animals.

[0050] The animal cells can be selected from, for example, any non-human cell found in animals that are commonly farmed, hunted, or fished. Thus, the cultured non-human animal cells can be selected from avian cells, bovine cells, seafood cells, porcine cells, and ovine cells. In the text, when this document refers to animal cells, it clearly refers to non-human animal cells.

[0051] Preferably, the animal cell is selected from a cell from the kingdom Animalia, in particular the animal cell is selected from a mammalian cell, an avian cell, an actinopterygian cell, a malacostraca cell, a molluscan cell, and combinations thereof.

[0052] For example, but not limited to, a mammalian cell can be a bovine cell, a cervid cell, a leporidae cell, or a sardine cell; an avian cell can be an anseriform cell or a pheasant cell; an actinopterygian cell can be a gadidae cell, a hake cell, a pleuronectin cell, a salmonid cell, or a mackerel cell; a malacostraca cell can be a palaemonid cell, and a mollusk cell can be a cephalopod or a bivalve cell.

[0053] Preferably, the non-human animal cells include bovine cells, cervidae cells, leporidae cells, sardine cells, anseriidae cells, pheasantidae cells, gadidae cells, hake cells, pleuronectid cells, salmonidae cells, salmonid cells and / or pelagic cells.

[0054] More preferably, the non-human animal cell comprises a bovine cell, a sardine cell, an anseriform cell, a pheasant cell, a gadidae cell, a hake cell, a salmonid cell, or a salmonid cell.

[0055] More preferably, the non-human animal cells include bovine cells, sardine cells, anseriform cells, pheasant cells, and mackerel cells.

[0056] Any number of cell types can be used in this step 110 of culturing, regardless of origin.

[0057] For example, non-human animal cells include cells selected from among embryonic stem cells, satellite cells, induced pluripotent stem cells, germ layer cells, stem cells such as fibroadipogenic progenitor cells, muscle cells such as skeletal muscle cells, cardiac cells, smooth muscle cells, myoblasts, muscle cells, hepatocytes, fibrocytes, fibroblasts, adipocytes, chondrocytes, chondroblasts, keratinocytes, melanocytes, osteocytes, osteoblasts, Merkel cells, Langerhans cells, glial cells, Schwann cells, erythrocytes (red blood cells) and leukocytes, and combinations thereof.

[0058] Preferably, the non-human animal cells include cells selected from stem cells, muscle cells, fibroblasts, adipocytes, red blood cells, and combinations thereof.

[0059] There are many methods for culturing cells. While these methods are often performed in the laboratory, many methods are suitable for large-scale production and human consumption. Below, we describe some of the methods adapted for culturing non-human cells in the context of the present invention.

[0060] During the step 110 of culturing the non-human animal cells, the cells can be cultured in suspension or in an adherent state.

[0061] Advantageously, cultured non-human animal cells can be considered as cells grown in a culture medium, regardless of their origin. Advantageously, the culture medium does not contain fetal bovine serum or growth factors. The culture medium can be supplemented, preferably gradually, with hydrolysates such as vegetable hydrolysates or yeast hydrolysates. Such serum-free media can reduce or eliminate the need for animal-derived components.

[0062] Various media formulations are optionally used to maintain self-renewal capacity, such as during expansion of the cell population. As discussed, media formulations can be modified from traditional media to eliminate the need for fetal bovine serum, animal substitutes for bovine serum, or growth factors. Rather, media may include plant or yeast hydrolysates. Examples of plant-based formulations include soy-based and plant hydrolysate-based media formulations. Some media formulations may further include at least one component to enhance the nutritional content of the cultured cells.

[0063] Furthermore, the medium contains all the components and nutrients necessary for cell growth, such as salt, glucose, water, salt minerals, and amino acids.

[0064] In one embodiment, the culture medium can contain a scaffold. Cells can be cultured in an incubator at, for example, a temperature of 37°C, 5% CO2, pH 7, and at least 95% moisture.

[0065] Additionally, the step 110 of culturing non-human animal cells can include differentiating the cultured cells. There are many methods for cell differentiation. While these methods are often performed in a laboratory, many methods are suitable for mass production and human consumption. Below, we describe some adapted methods for differentiating non-human cells in the context of the present invention or using already differentiated cells. Cell differentiation may include the production of specific proteins that contribute to the texture in a specific way, particularly relevant to a particular edible product.

[0066] Differentiation of non-human embryonic stem cells The cells in the first composition may be obtained from the differentiation of non-human embryonic stem cells. Differentiation is the total process by which undifferentiated or pre-differentiated cells achieve their function. The stem cells are isolated from the embryo and cultured using a specific culture medium to allow for cell proliferation and maintenance of an undifferentiated state. This is particularly advantageous for achieving sufficient cell density or number. In one embodiment, the medium formulation uses a defined serum-free medium.

[0067] Embryonic stem cells can then be induced to differentiate into, for example, hepatocytes, fibroblasts, keratinocytes, myocytes, or adipocytes. Differentiation can be triggered by exposure to specific factors (e.g., growth factors or proteins) or culture conditions (e.g., shear stress). For example, to obtain hepatocytes, non-human embryonic stem cells are induced into definitive endoderm cells, preferably using specific growth factors such as activin A, WNT, FGF, or BMP, or other components that affect differentiation, such as insulin, transferrin, selenium, rapamycin, KOSR, or sodium butyrate. Definitive endoderm cells are then specified into hepatic endoderm cells and then hepatoblasts, preferably using specific factors such as HGF, FGF, FGF, and BMP. Hepatoblasts are differentiated into hepatocytes by differentiation induced by a combination of factors, preferably HGF, oncostatin M, dexamethasone, and TGF-β. The differentiated hepatocytes are then cultured and expanded to the required cell mass.

[0068] Differentiation of non-human induced pluripotent stem cells. The cells in the first composition may be obtained from the differentiation of non-human induced pluripotent stem cells.

[0069] By employing an episomal reprogramming strategy, for example, avian skin fibroblasts isolated from geese, ducks, or chickens, induced pluripotent stem cells can be generated from fibroblasts without using traditional viral reprogramming techniques.

[0070] Induced pluripotent stem cells can be cultured using optimized media substrates and formulations to achieve sustained cell proliferation and maintenance of a dedifferentiated state. The media formulation preferably uses a defined serum-free medium. Cells are preferably cultured in a pathogen-free cell culture system. The pluripotent stem cells can then be stimulated to differentiate into specific lineages as hepatocytes and expanded to the desired cell mass.

[0071] Transdifferentiated non-human isolated cells. The cells in the first composition may be derived from transdifferentiated, non-human isolated cells. Transdifferentiation refers to the differentiation of one differentiated cell type into another, preferably in a single step. Transdifferentiation can refer to a method of changing the differentiated phenotype or developmental potential of a cell without forming a pluripotent intermediate cell; i.e., the cell does not first need to be dedifferentiated (or reprogrammed) and then differentiated into another cell type. Instead, the cell type is simply "switched" from one cell type to another without undergoing a less differentiated phenotype. Transdifferentiation can include a first step of exposing a first cell with a first cell fate to conditions that generate a second cell (i.e., a less differentiated cell) that can differentiate into a second cell fate, and a second step of exposing the less differentiated cell to conditions that induce differentiation into a cell with the second cell fate (e.g., a hepatocyte).

[0072] For example, non-human cells such as embryonic fibroblasts, embryonic stem cells, satellite cells, or muscle cells are isolated using techniques known in the art of cell biology as set forth above and cultured in a medium containing a basal medium, antibiotics, non-essential amino acids, as well as reducing agents, serum, minerals, and growth factors.

[0073] Immortalized mature non-human cardiomyocytes or hepatocytes. The cells in the first composition can be selected from immortalized mature non-human differentiated cells. Cells can be immortalized using classical techniques such as transformation or spontaneous immortalization by successively subculture the cells until spontaneous mutations that result in immortalization occur. For example, in the case of hepatocytes, the uniqueness of immortalized mature non-human hepatocytes lies in the fact that the cells can divide indefinitely. Mature avian hepatocytes can be isolated from the livers of ducks, geese, or chickens. Immortalized myocytes or hepatocytes can be grown to the required cell mass and grown in a culture medium.

[0074] Differentiated cells obtained from the differentiation of non-human progenitor cells. The cells in the first composition can be selected from differentiated cells derived from non-human progenitor cells. The progenitor cells can be grown using an optimized medium substrate and medium formulation to achieve sustained cell proliferation and maintenance of a pluripotent state. The medium formulation can include a synthetic serum-free medium. In the case of satellite cells, the satellite cells are induced to differentiate into mature skeletal muscle cells and expanded to the required cell number thanks to specific well-known differentiation factors.

[0075] As shown in FIG. 1, the method of the present invention can include processing 120 different animal proteins.

[0076] This step is specifically designed to prepare the animal protein produced during the cell culture step for subsequent steps.

[0077] For example, the animal protein used in the present invention can be incorporated into the composition in intact cultured cells, or the animal protein can be introduced into the composition together with disrupted cultured cells, or the animal protein can be extracted from cultured cells, e.g., disrupted cultured cells.

[0078] Thus, after the culturing step, the cultured cells may be preserved substantially intact or may be substantially disrupted, for example, by homogenization, extrusion, mixing, blending, or melt-blowing, electrospinning, centrifugal spinning, or blow-spinning.

[0079] If the cultured cells are disrupted, the method of the present invention can include a step of extracting specific compounds after disruption. For example, the method of the present invention can include a step of extracting proteins from the cultured cells. Proteins can be purified or separated into protein fractions according to specific physicochemical properties.

[0080] As shown in FIG. 1, a method 100 for making edible protein yarns according to the present invention includes preparing a first composition 130.

[0081] This step is specifically designed to define the main components of the protein yarn and its mechanical and organoleptic properties, and therefore such a step contributes to solving the problem solved by the present invention.

[0082] The dry content of the first composition can have a significant effect on the mechanical properties of the protein yarn obtained using said first composition.

[0083] Thus, the dry content of the first composition is preferably at least 12% by weight. The dry content of the first composition is preferably at least 15% by weight, more preferably at least 20% by weight. For example, the first composition has a dry content of at most 90% by weight. Preferably, the first composition has a dry content of at most 85% by weight, more preferably at most 80% by weight.

[0084] Furthermore, since this composition is the basis for the construction of protein yarns, it is preferable that the composition contain a substantial amount of protein. Thus, the first composition may contain at least 10% by weight of protein relative to the total wet weight of the first composition. Preferably, the first composition may contain at least 15% by weight of protein relative to the total wet weight of the first composition, more preferably at least 20% by weight, and even more preferably at least 25% by weight. The first composition may contain up to 40% by weight of protein relative to the total wet weight of the first composition. Preferably, the first composition may contain up to 38% by weight of protein relative to the total wet weight of the first composition, more preferably up to 35% by weight, and even more preferably up to 30% by weight of protein relative to the total wet weight of the first composition. Thus, the first composition may contain 10% to 40% by weight of protein relative to the total wet weight of the first composition. Preferably, the first composition may contain 15% to 38% by weight of protein relative to the total wet weight of the first composition, more preferably 20% to 35% by weight, and even more preferably 25% to 30% by weight of protein.

[0085] In particular, first composition 130 includes animal proteins obtained from cultured non-human animal cells.

[0086] Animal protein Unlike conventional protein yarn manufacturing methods, the animal protein used herein is not a protein produced from slaughtered animals, but rather a protein produced by non-human animal cells grown in a cell culture facility, and when animal protein is referred to herein, it is clearly a non-human animal protein.

[0087] As previously explained, the animal protein can be introduced into the first composition in the form of intact or disrupted cultured cells, or in the form of extracted proteins.

[0088] Thus, the step of preparing the first composition 130 can include the addition of a cultured cell extract, disrupted cultured cells, and / or intact cultured cells. More preferably, the first composition comprises food-grade non-human animal cells (intact or disrupted) harvested from a cell culture (suspension or adherent, preferably suspension), or an extract of said cells, preferably a protein extract of said cells.

[0089] Preferably, the first composition comprises cultured non-human animal cells. The cultured non-human animal cells may comprise disrupted cultured cells and / or intact cultured cells.

[0090] In particular, the first composition can comprise at least 1% dry weight of cultured non-human animal cells relative to the total wet weight of the first composition. Preferably, prior to contacting step 160, the first composition can comprise at least 2% dry weight of cultured non-human animal cells relative to the total wet weight of the first composition, more preferably at least 4% dry weight, and even more preferably at least 8% dry weight of cultured non-human animal cells relative to the total wet weight of the first composition.

[0091] In particular, the first composition can include up to 30% by dry weight of cultured non-human animal cells relative to the total wet weight of the first composition. Preferably, prior to contacting step 160, the first composition can include up to 28% by dry weight of cultured non-human animal cells relative to the total wet weight of the first composition, more preferably up to 25% by dry weight, and even more preferably up to 22% by dry weight of cultured non-human animal cells.

[0092] In particular, the first composition may comprise 1% to 30% by dry weight of cultured non-human animal cells relative to the total wet weight of the first composition, preferably 2% to 28% by dry weight, more preferably 4% to 25% by dry weight, and even more preferably 8% to 22% by dry weight of cultured non-human animal cells relative to the total wet weight of the first composition.

[0093] The amount of cultured non-human animal cells can be measured after harvesting and centrifugation and corrected based on the moisture content. The moisture content can be measured according to international standard ISO 1442:1997. When carrying out a method for producing an edible product or raw material, the origin of the raw material is generally known. Therefore, those skilled in the art who practice the present invention can know whether the protein to be added is an animal protein derived from cultured non-human animal cells. Animal proteins derived from cultured non-human animal cells are, for example, proteins produced by non-human animal cells grown in a bioreactor or fermentation reactor.

[0094] In particular, the first composition can comprise at least 1% dry weight of the cultured non-human animal cell extract relative to the total wet weight of the first composition. Preferably, prior to contacting step 160, the first composition can comprise at least 2% dry weight of the cultured non-human animal cell extract relative to the total wet weight of the first composition, more preferably at least 4% dry weight, and even more preferably at least 8% dry weight of the cultured non-human animal cell extract relative to the total wet weight of the first composition.

[0095] In particular, the first composition can comprise up to 30% by dry weight of cultured non-human animal cell extract relative to the total wet weight of the first composition. Preferably, prior to contacting step 160, the first composition can comprise up to 28% by dry weight of cultured non-human animal cell extract relative to the total wet weight of the first composition, more preferably up to 25% by dry weight, and even more preferably up to 22% by dry weight of cultured non-human animal cell extract.

[0096] In particular, the first composition can comprise 1% to 30% by dry weight of the cultured non-human animal cell extract relative to the total wet weight of the first composition. Preferably, prior to contacting step 160, the first composition can comprise 2% to 28% by dry weight of the cultured non-human animal cell extract relative to the total wet weight of the first composition, more preferably 4% to 25% by dry weight, and even more preferably 8% to 22% by dry weight.

[0097] The concentration of protein, particularly animal protein, in the first composition can affect the texture of the protein yarn.

[0098] The first composition may comprise at least 0.5% animal protein derived from cultured non-human animal cells relative to the total wet weight of the first composition, preferably at least 1% by weight, more preferably at least 2% by weight, and even more preferably at least 4% by weight, of animal protein derived from cultured non-human animal cells relative to the total wet weight of the first composition.

[0099] The first composition may comprise up to 30% by weight of animal protein derived from cultured non-human animal cells relative to the total wet weight of the first composition, preferably up to 28% by weight of animal protein derived from cultured non-human animal cells relative to the total wet weight of the first composition, more preferably up to 25% by weight, and even more preferably up to 22% by weight relative to the total wet weight of the first composition.

[0100] Therefore, the first composition can contain 0.5% to 30% by weight of the animal protein derived from cultured non-human animal cells relative to the total wet weight of the first composition. Preferably, the first composition can contain 1% to 28% by weight of the animal protein derived from cultured non-human animal cells relative to the total wet weight of the first composition, more preferably 2% to 25% by weight, and even more preferably 4% to 22% by weight relative to the total wet weight of the first composition.

[0101] The first composition may comprise at least 5% animal protein derived from non-human cultured animal cells relative to the total weight of protein in the first composition. Preferably, the first composition may comprise at least 10% by weight, more preferably at least 20% by weight, even more preferably at least 30% by weight, at least 40% by weight, at least 50% by weight, or at least 60% by weight relative to the total weight of protein in the first composition.

[0102] The first composition may contain up to 100% by weight of animal protein derived from non-human cultured animal cells relative to the total weight of protein in the first composition. Preferably, the first composition may contain up to 90% by weight of animal protein derived from non-human cultured animal cells relative to the total weight of protein in the first composition, and more preferably, may contain up to 80% by weight of animal protein relative to the total weight of protein in the first composition.

[0103] Therefore, the first composition can contain 5% to 100% by weight of the animal protein derived from cultured non-human animal cells relative to the total weight of the protein in the first composition. Preferably, the first composition can contain 10% to 100% by weight of the animal protein derived from cultured non-human animal cells relative to the total weight of the protein in the first composition, more preferably 20% to 100%, and even more preferably 30% to 100% by weight, 40% to 90% by weight, 50% to 100% by weight, or 50% to 90% by weight relative to the total weight of the protein in the first composition.

[0104] Preferably, the amount of animal protein can be measured using Kjeldahl titration in accordance with the international standard ISO-937:1978. When carrying out a method for producing an edible product or ingredient, the origin of the ingredient is generally known. Therefore, a person skilled in the art who practices the present invention can know whether the added protein is an animal protein derived from a non-human cultured animal cell.

[0105] The protein concentration is considered in relation to the polyelectrolyte concentration, and controlling this ratio can optimize the behavior of the protein thread.

[0106] Therefore, the first composition can contain animal protein (derived from cultured cells) and a polyelectrolyte capable of forming a heat-stable gel at a concentration such that the ratio of the weight of the animal protein to the weight of the polyelectrolyte is at least 5, preferably at least 10, more preferably at least 15, and even more preferably at least 20.

[0107] However, the first composition may contain animal protein and a thermostable gel-forming polyelectrolyte at a concentration such that the ratio of the weight of animal protein to the weight of polyelectrolyte is 200 or less, preferably 100 or less, more preferably 80 or less, and even more preferably 60 or less.

[0108] Therefore, the first composition can contain animal protein and polyelectrolyte at concentrations such that the ratio of the weight of the animal protein to the weight of the polyelectrolyte is 5 to 200, preferably 10 to 100, more preferably 15 to 80, and even more preferably 20 to 60.

[0109] In the context of the present invention, the presence of certain molecules, particularly proteins, can be crucial for improving the quality of the final protein yarn. The amount of certain molecules, particularly proteins, can be measured by techniques well known to those skilled in the art, such as enzyme-linked immunosorbent assay or mass spectrometry.

[0110] For example, the animal protein can include heat shock proteins.

[0111] Preferably, the animal protein comprises a myofibrillar protein, for example, the myofibrillar protein may comprise actin, myosin, tropomyosin, troponin, actinin, connectin, titin, nebulin, C protein, M protein, desmin, or a combination thereof.

[0112] Preferably, the myofibrillar protein is selected from actin, desmin, myosin, troponin, titin, nebulin, or a combination thereof. More preferably, the myofibrillar protein is selected from actin, desmin, myosin, troponin, or a combination thereof. Even more preferably, the myofibrillar protein is selected from actin, myosin, or a combination thereof.

[0113] The first composition can comprise at least 0.25% by weight of myofibrillar protein relative to the total weight of protein, preferably at least 0.5% by weight of myofibrillar protein, more preferably at least 1% by weight of myofibrillar protein, and even more preferably at least 2% by weight of myofibrillar protein relative to the total weight of protein.

[0114] The first composition can comprise up to 70% myofibrillar protein, preferably up to 60% myofibrillar protein, more preferably up to 50% myofibrillar protein, and even more preferably up to 40% myofibrillar protein, relative to the total weight of protein.

[0115] Therefore, the first composition contains myofibrillar protein in an amount of 0.25% to 70% by weight relative to the total weight of protein, preferably 0.5% to 60% by weight of myofibrillar protein, more preferably 1% to 50% by weight of myofibrillar protein, and even more preferably 2% to 40% by weight of myofibrillar protein relative to the total weight of protein.

[0116] The concentration of myofibrillar protein in the first composition can also be considered in relation to the concentration of polyelectrolyte. Controlling this ratio can optimize the behavior of protein yarns. Therefore, the first composition can contain myofibrillar protein (derived from cultured cells) and polyelectrolyte at concentrations such that the weight ratio of myofibrillar protein to polyelectrolyte is at least 2.5, preferably 5, more preferably 7.5, and even more preferably 10. However, the first composition can contain myofibrillar protein and polyelectrolyte at concentrations such that the weight ratio of myofibrillar protein to polyelectrolyte is at most 100, preferably at most 50, more preferably at most 40, and even more preferably at most 30. Therefore, the first composition can contain myofibrillar protein and polyelectrolyte at concentrations such that the weight ratio of myofibrillar protein to polyelectrolyte is 2.5 to 100, preferably 5 to 50, more preferably 7.5 to 40, and even more preferably 10 to 30. As described above, myofibrillar protein can be quantified by mass spectrometry, similar to polyelectrolytes.

[0117] In particular, the first composition preferably contains a specific myofibrillar protein, for example, the first composition may contain at least 0.0125% by weight of troponin protein, preferably at least 0.025% by weight, more preferably at least 0.05% by weight, and even more preferably at least 0.1% by weight of troponin protein relative to the total weight of protein in the first composition.

[0118] The first composition may comprise up to 3.5% by weight of troponin protein relative to the total weight of protein in the first composition, preferably up to 3% by weight, more preferably up to 2.5% by weight, and even more preferably up to 2% by weight of troponin protein relative to the total weight of protein in the first composition.

[0119] Therefore, the first composition can contain 0.0125 to 3.5 wt% troponin protein relative to the total weight of protein in the first composition, preferably 0.025 to 3 wt%, more preferably 0.05 to 2.5 wt%, and even more preferably 0.1 to 2 wt% troponin protein relative to the total weight of protein in the first composition.

[0120] Troponin can be produced by cultured animal cells, but it can also be recombinant troponin, which can be produced by cultured cells, be they plant cells, insect cells or microbial cells.

[0121] In the present invention, the presence of certain molecules can improve the protein threads produced. In particular, the presence of extracellular matrix animal molecules (ECM), such as ECM polysaccharides, ECM proteoglycans, or ECM proteins, can improve the protein threads produced. ECM proteins are particularly preferred. For example, the first composition can contain at least 0.1% by weight of extracellular matrix animal molecules relative to the total weight of protein. Preferably, the first composition contains at least 0.5% by weight of extracellular matrix animal molecules relative to the total weight of protein, more preferably at least 1% by weight, and even more preferably at least 1.5% by weight of extracellular matrix animal molecules relative to the total weight of protein. When the first composition contains multiple extracellular matrix animal molecules, the concentrations are accumulated to assess the amount of extracellular matrix animal molecules in the first composition.

[0122] Such molecules can be added to the first composition via cultured cells, preferably cultured animal cells such as fibroblasts, which are known to produce such molecules.

[0123] The first composition may comprise collagen, and may preferably comprise at least 1 wt. % collagen, preferably at least 2 wt. %, more preferably at least 3 wt. %, and even more preferably at least 4 wt. % collagen, relative to the total weight of protein in the first composition.

[0124] The first composition may comprise up to 16% by weight of collagen relative to the total weight of protein in the first composition, preferably up to 14% by weight, more preferably up to 12% by weight, and even more preferably up to 10% by weight of collagen relative to the total weight of protein in the first composition.

[0125] Therefore, the first composition can contain 1 to 16 wt% collagen, preferably 2 to 14 wt%, more preferably 3 to 12 wt%, and even more preferably 4 to 10 wt% collagen, relative to the total weight of protein in the first composition.

[0126] The collagen may be collagen produced by cultured animal cells, or it may be recombinant collagen produced by cultured cells such as plant cells, insect cells, or microbial cells.

[0127] The first composition may comprise elastin and / or tropoelastin, and preferably comprises at least 0.065% by weight of elastin and / or tropoelastin, preferably at least 0.13% by weight, more preferably at least 0.2% by weight, and even more preferably at least 0.25% by weight of elastin and / or tropoelastin relative to the total weight of protein in the first composition.

[0128] The first composition may comprise up to 7.5% by weight of elastin and / or tropoelastin relative to the total weight of protein in the first composition, preferably up to 7% by weight, more preferably up to 6.5% by weight, and even more preferably up to 6% by weight of elastin and / or tropoelastin relative to the total weight of protein in the first composition.

[0129] Therefore, the first composition may contain 0.065 to 7.5 wt% elastin and / or tropoelastin relative to the total weight of protein in the first composition, preferably 0.13 to 7 wt%, more preferably 0.2 to 6.5 wt%, and even more preferably 0.25 to 6 wt% elastin and / or tropoelastin.

[0130] Preferably, the elastin and / or tropoelastin is produced by cultured animal cells, but may also be recombinant elastin and / or tropoelastin, which may be produced by cultured cells, such as plant cells, insect cells, or microbial cells, and the elastin and / or tropoelastin is preferably in a soluble form.

[0131] As previously mentioned, the first composition can include animal proteins derived from cultured non-human animal cells, although the first composition can also include proteins of other origins.

[0132] In particular, the preparation of first composition 130 can include the addition of non-animal proteins, such as plant proteins, microbial proteins, algal proteins, and / or fungal proteins. As described in the Examples, the addition of some non-animal proteins can adjust the viscoelastic or flow properties of the first composition containing cultured non-human animal cells.

[0133] In certain embodiments, the first composition further comprises a vegetable protein, preferably the vegetable protein may be selected from the following: sunflower protein, soy protein, pea protein, canola protein, mung bean protein, chickpea protein, fava bean protein, lentil protein, seaweed protein, potato protein, quinoa protein, nut protein, wheat protein, chickpea protein, bambara bean protein, dulse protein, mesquite bean protein, duckweed protein, dried fava bean protein, dried cowpea protein, lupin protein, jackfruit protein, amaranth protein, millet protein, oat protein, chia protein, hemp seed protein, rice protein, or a combination thereof.

[0134] Preferably, the preparation of the first composition 130 can include the addition of non-animal protein from at least two different sources. For example, the first composition can include plant protein from at least two different plants. Indeed, combining multiple sources can improve the characteristics of edible protein fiber and establish an optimal amino acid profile for human nutrition. More preferably, the preparation of the first composition 130 can include the addition of pulse protein and cereal protein.

[0135] The first composition 130 includes either a multivalent ion or a multivalent electrolyte.

[0136] As described below and shown in the Examples, the method 100 for producing edible protein yarns in accordance with the present invention uses multivalent ions and polyelectrolytes that are complementary and, when combined, produce a heat-resistant gel.

[0137] Typically, if the first composition includes one of a multivalent ion or a polyelectrolyte, the second composition includes the other. The multivalent ion and polyelectrolyte can be combined in step 160, contacting the first composition with the second composition.

[0138] Thus, as shown in FIG. 1, a method 100 for producing edible protein yarn according to the present invention includes preparing a second composition 140.

[0139] This step is specifically designed to prepare a second composition complementary to the first composition, and to form protein threads with the expected texture when the two compositions are brought into contact, thus contributing to the solution of the problem solved by the present invention.

[0140] In particular, the step of preparing the second composition 140 includes adding a polyvalent ion to the second composition if the step of preparing the first composition 130 includes adding a polyelectrolyte, or includes adding a polyelectrolyte if the step of preparing the first composition 130 includes adding a polyvalent ion.

[0141] As mentioned above, in the sense of the present invention, polyelectrolytes and multivalent ions are complementary components that, when combined, form a thermostable gel.

[0142] It should be noted that if the first composition includes a polyelectrolyte, the first composition may still include one or more multivalent ions, but such multivalent ions may not be able to form a thermostable gel with the polyelectrolyte. Their nature and / or concentration should be such that they are unable to form a thermostable gel with the polyelectrolyte.

[0143] Polyelectrolyte As mentioned above, a polyelectrolyte suitable for forming a thermostable gel can be added to the first composition or the second composition. A minimum amount of polyelectrolyte can be important for improving yarn properties.

[0144] Thus, the first or second composition may comprise at least 0.01% by weight of polyelectrolyte relative to the total wet weight of the relevant composition, preferably at least 0.02% by weight of polyelectrolyte relative to the total wet weight of the relevant composition, more preferably at least 0.05% by weight, and even more preferably at least 0.1% by weight of polyelectrolyte relative to the total wet weight of the relevant composition.

[0145] In particular, when the first composition contains a polyelectrolyte, the first composition may contain at least 0.1% polyelectrolyte relative to the total wet weight of the first composition. Preferably, the first composition may contain at least 0.2% by weight of polyelectrolyte relative to the total wet weight of the first composition, more preferably at least 0.3% by weight, and even more preferably at least 0.4% by weight of polyelectrolyte relative to the total wet weight of the first composition. When referring to the concentration of polyelectrolyte herein, it preferably refers to the concentration of the polyelectrolyte salt.

[0146] The first or second composition can contain up to 15% by weight of polyelectrolyte relative to the total wet weight of the relevant composition. Preferably, the first or second composition can contain up to 10% by weight of polyelectrolyte relative to the total wet weight of the relevant composition, more preferably up to 5% by weight, and even more preferably up to 3% by weight of polyelectrolyte relative to the total wet weight of the relevant composition. In particular, as shown in the examples, the first or second composition can contain less than 2% by weight of polyelectrolyte relative to the total wet weight of the relevant composition. More specifically, the first composition contains less than 2% by weight of polyelectrolyte, such as alginate, relative to the total wet weight of the first composition.

[0147] The first or second composition may contain 0.01% to 15% by weight of polyelectrolyte relative to the total wet weight of the relevant composition, preferably 0.02% to 10% by weight of polyelectrolyte relative to the total wet weight of the relevant composition, more preferably 0.05% to 5% by weight of polyelectrolyte relative to the total wet weight of the relevant composition, and even more preferably 0.1% to 3% by weight of polyelectrolyte relative to the total wet weight of the relevant composition.

[0148] As discussed above, when complexed with a polyvalent ion, the polyelectrolyte becomes a heat-stable gel-forming polyelectrolyte. Also, the polyelectrolyte is preferably not a heat-activated gel-forming polyelectrolyte. For example, the polyelectrolyte does not require heat treatment at temperatures above 80° C., preferably above 60° C., for activation.

[0149] Additionally, the polyelectrolyte is intended for human consumption and is therefore digestible by the human digestive system.

[0150] For example, the polyelectrolyte can be either cationic (e.g., chitosan) or anionic (e.g., alginate). Preferably, when the polyelectrolyte is an anionic polyelectrolyte, the polyvalent ion is a polyvalent cation. Also, when the polyelectrolyte is a cationic polyelectrolyte, the polyvalent ion is a polyvalent anion. More preferably, the polyelectrolyte is an anionic polysaccharide.

[0151] Not all of the monomers in a polyelectrolyte need have ionizable groups. For example, a polyelectrolyte is a polymer in which at least 10% of the monomers have ionizable groups, preferably at least 20%, more preferably at least 30%, and even more preferably at least 40% of the monomers have ionizable groups. Preferably, the ionizable groups are selected from amine, carboxyl, sulfate, or phosphate groups.

[0152] For example, a polyelectrolyte is a polymer in which at least 10% of the monomers can be negatively charged, preferably at least 20%, more preferably at least 30%, e.g., all of the monomers can be negatively charged. Preferably, the negatively chargeable monomer contains at least one anionic group. Preferably, the anionic group is selected from a carboxyl group, a sulfate group, or a phosphate group, more preferably, the anionic group is a carboxyl group.

[0153] For example, a polyelectrolyte is a polymer of at least 10% positively chargeable monomers, preferably at least 20%, more preferably at least 30%, even more preferably at least 40%, e.g., all of the monomers are positively chargeable. Preferably, the positively chargeable monomers contain at least one cationic group. Preferably, the cationic group is an amine.

[0154] Preferably, the polyelectrolyte is a high molecular weight polyelectrolyte, e.g., the average molar mass of the polyelectrolyte is at least 2 kg.mol -1 Preferably, the average molar mass of the polyelectrolyte is at least 10 kg mol -1 and more preferably, the average molar mass of the polyelectrolyte is at least 20 kg mol -1 and more preferably, the average molar mass of the polyelectrolyte is at least 30 kg mol -1 is.

[0155] For optimal texture, the molecular weight of the polyelectrolyte should not be too high. For example, the average molar mass of the polyelectrolyte should be 3000 kg.mol -1 Preferably, the average molar mass of the polyelectrolyte is at most 2500 kg mol -1 and more preferably, the average molar mass of the polyelectrolyte is at most 2000 kg mol -1 and more preferably, the average molar mass of the polyelectrolyte is at most 1500 kg mol -1 is.

[0156] Thus, for example, the average molecular weight of a polyelectrolyte is 2 kg.mol -1 to 3000 kg.mol -1 Preferably, the average molar mass of the polyelectrolyte is 10 kg mol -1 ~2500 kg mol -1 and more preferably, the average molar mass of the polyelectrolyte is 20 kg mol -1 ~2000 kg mol -1 and more preferably, the average molar mass of the polyelectrolyte is 30 kg mol -1 ~1500 kg mol -1 is.

[0157] The average molecular weight of the polyelectrolyte can be measured by size exclusion chromatography combined with light scattering, preferably according to the instructions of the international standard ISO 16014-5:2019.

[0158] In the present invention, several polyelectrolytes can be used. However, several polyelectrolytes are preferred. Preferably, the polyelectrolyte is selected from low methoxyl pectin, low methoxyl pectin derivatives, high methoxyl pectin, high methoxyl pectin derivatives, alginate, alginate derivatives, xanthan, xanthan derivatives, chitosan, chitosan derivatives, ionic carboxymethylcellulose derivatives, ionic pullulan derivatives, ionic dextran derivatives, ionic starch derivatives, or combinations thereof.

[0159] More preferably, the polyelectrolyte is selected from alginate, alginate derivatives, low methoxyl pectin, low methoxyl pectin derivatives, chitosan, chitosan derivatives, or combinations thereof.

[0160] More preferably, the polyelectrolyte is selected from alginate, low methoxyl pectin, chitosan, or a combination thereof.

[0161] Multivalent ions As mentioned above, in the method of the present invention, multivalent ions combine with polyelectrolytes to form a heat-resistant gel.

[0162] The multivalent ions can be either multivalent anions or multivalent cations. For example, the multivalent ions can be selected from calcium, magnesium, iron, manganese, copper, zinc, carbonate, oxalate, sulfate, sulfite, phosphate, or a combination thereof. Preferably, the multivalent ions can be selected from calcium, magnesium, iron, manganese, copper, zinc, carbonate, oxalate, sulfate, phosphate, or a combination thereof.

[0163] The polyvalent ions are obtained by dissolving them in the form of salts. For example, the counter ions can be selected from calcium, magnesium, sodium, ammonium, chloride, gluconate, lactate, sulfate, carbonate, phosphate, or a combination thereof. When referring to the concentration of polyvalent ions herein, it preferably refers to the concentration of the polyvalent ions themselves.

[0164] The multivalent ion may be present in either the first composition or the second composition at a concentration of at least 0.001% by weight relative to the wet weight of the composition, preferably at least 0.002%, more preferably at least 0.005%, and even more preferably at least 0.01% by weight relative to the wet weight of the composition.

[0165] The maximum concentration of the multivalent ion is typically determined by the solubility of the multivalent ion, for example, the multivalent ion may be present in either the first or second composition at a concentration of up to 3%, preferably up to 2%, more preferably up to 1%, and even more preferably up to 0.5% by weight relative to the wet weight of the composition.

[0166] Thus, the multivalent ion may be present in either the first or second composition at a concentration of 0.001 to 3% by weight relative to the wet weight of the composition. Preferably, the multivalent ion is present in either the first or second composition at a concentration of 0.002 to 2% by weight relative to the wet weight of the composition. More preferably, the multivalent ion is present at a concentration of 0.005 to 1% by weight relative to the wet weight of the composition, and even more preferably, at a concentration of 0.01 to 0.5% by weight relative to the wet weight of the composition.

[0167] The first and second compositions can undergo various preparations to improve the efficiency of protein thread formation. The drawings and this specification should not be understood as limiting the present invention to embodiments in which the first composition is prepared before the second composition. Those skilled in the art will recognize that within the scope of the present invention, the second composition can also be prepared before or simultaneously with the first composition.

[0168] The step of preparing the first or second composition may include adjusting the pH of either composition. For example, the pH of the first composition is 2 to 12, preferably 3 to 11, more preferably 4 to 10, and even more preferably 5 to 9. For example, the pH of the second composition is 2 to 12, preferably 3 to 11, more preferably 4 to 10, and even more preferably 5 to 9.

[0169] As already mentioned, the temperature at which the compositions are prepared and used in the context of the present invention can have a significant effect on the quality of the protein threads produced.

[0170] Preferably, the first composition is not heated to a temperature of 65°C or above for more than 10 minutes, and preferably, the first composition is not heated to a temperature of 60°C or above, more preferably 55°C or above, and even more preferably 50°C or above for more than 10 minutes.

[0171] For example, the temperature during the step of preparing the first composition is below 65°C, preferably below 60°C, more preferably below 55°C, and even more preferably below 50°C.

[0172] Preferably, the second composition is not heated to a temperature above 85°C for more than 10 minutes, preferably above 75°C, more preferably above 65°C, even more preferably above 55°C for more than 10 minutes.

[0173] For example, the temperature during the step of preparing the second composition is 85°C or less, preferably 75°C or less, more preferably 65°C or less, and even more preferably 55°C or less.

[0174] The first and second compositions may also be subjected to physical treatments such as blending, mixing, homogenization, sieving, or sonication. Preferably, the preparation step for the first or second composition may include a blending, homogenization, emulsification, or stirring step. These steps are specifically designed to homogenize or mix all of the components. These steps may also at least partially disrupt cultured animal cells, if present. The preparation step for the first composition may include disruption of the membranes of non-human animal cells. Thus, the homogenization step may be blending. For example, this may be used when the first or second composition is supplemented with other ingredients, such as plant materials or food additives. Preferably, the homogenization, mixing, emulsification, or stirring step is performed using a homogenizer, such as a high-speed mixer, a rotor-stator homogenizer, a cutter, or a colloid mill.

[0175] Homogenization can be carried out for at least 30 seconds, preferably at least 1 minute, more preferably at least 2 minutes, for example, at least 10 minutes. The homogenization step can be carried out for up to 1 hour, preferably up to 45 minutes, more preferably up to 30 minutes, even more preferably up to 10 minutes, for example, up to 2 minutes. Thus, homogenization can be carried out for 30 seconds to 60 minutes, preferably 1 minute to 45 minutes, more preferably 2 minutes to 30 minutes, for example, about 10 minutes.

[0176] Homogenization can be carried out at a speed of at least 100 rpm, preferably at least 1000 rpm, more preferably at least 2000 rpm, for example at least 5000 rpm. Homogenization can be carried out at a speed of up to 30,000 rpm, preferably up to 25,000 rpm, more preferably up to 20,000 rpm, for example up to 15,000 rpm. Thus, homogenization can be carried out at a speed of 100 rpm to 30,000 rpm, preferably 1000 rpm to 25,000 rpm, more preferably 2000 rpm to 20,000 rpm, and even more preferably 5,000 rpm to 15,000 rpm. Homogenization preferably induces an emulsion, more preferably a microemulsion, in the first composition.

[0177] supplement The first or second composition, preferably the first composition, can further comprise cross-linking molecules such as peptidases, α-galactosidase, alcalase, thermolysin, pepsin, trypsin, chymotrypsin, asparaginase, elastase, subtilisin, glucose oxidase, laccase, transglutaminase, pectinesterase, sortase, tyrosinase, oxidoreductases such as lysyl oxidase and peroxidase, genipin, riboflavin, monoamine oxidase, or combinations thereof, preferably laccase, transglutaminase, or combinations thereof. Such molecules function as natural cross-linking molecules and help to modulate the physical properties of the protein yarn.

[0178] As noted in the Examples, the presence of a supplemental fat, such as a non-animal fat, in the first composition can improve the properties of the resulting protein thread. Advantageously, therefore, the step of preparing the first composition further comprises the addition of a fat, such as a non-animal fat. Preferably, the supplemental fat is a vegetable fat or a fat obtained by fermentation.

[0179] For example, the first composition may contain at least 1% by weight of supplemental fat, preferably vegetable fat or fat obtained by fermentation, relative to the total wet weight of the first composition. Preferably, the supplemental fat is at least 2% by weight, more preferably at least 5% by weight, and even more preferably at least 10% by weight, relative to the total wet weight of the first composition. However, the amount of supplemental fat should not be too large. For example, the first composition may contain at most 50% by weight of supplemental fat, relative to the total wet weight of the first composition. Preferably, the weight of the supplemented fat is at most 40%, more preferably at most 30%, and even more preferably at most 25% relative to the total wet weight of the first composition.

[0180] Thus, the first composition may comprise 1% to 50% by weight of supplemented fat relative to the total wet weight of the first composition. Preferably, the weight of the supplemented fat is 2% to 40% relative to the total wet weight of the first composition, more preferably, 5% to 30% by weight, and even more preferably, 10% to 25% by weight relative to the total wet weight of the first composition.

[0181] For example, the first composition can further comprise a vegetable fat, preferably the vegetable fat comprises a fat or oil extracted from edible plant matter including flowers, fruits, stems, leaves, roots, germs and seeds, more preferably the vegetable fat comprises a fat or oil extracted from oil seeds or fruits, in particular the vegetable fat comprises fat extracted from canola seeds (rapeseed), castor, coconut, flaxseed, allanbrachia, olive, sunflower, soybean, peanut, illipe, cottonseed, shea, palm, avocado, safflower, sesame, lemon, grape seeds, macadamia, almond, sal, kokum, mango, or combinations thereof.

[0182] In particular, the vegetable fat used according to the invention may be chosen from olive oil, palm oil, sunflower oil, avocado oil, almond oil, or combinations thereof.

[0183] Fermented fats can include fats or oils extracted from cells cultured in anaerobic or aerobic fermentation processes, particularly those involving the cultivation of oleaginous microorganisms or animal cells other than human cells. For example, fermented fats can include fats derived from cyanobacteria, microalgae, yeast, fungi, bacteria, or cultured animal cells other than human cells, such as adipocytes. Preferably, fermented fats include fats or oils extracted from oleaginous yeasts, such as Rhodosporidium toruloides, Lipomyces starchii, and Yarrowia lipolytica.

[0184] Preferably, the supplemented fat is composed primarily of triglycerides, with at least 60% by weight of triglycerides compared to the total weight of fat. More preferably, the supplemented fat comprises fatty acids, e.g., fatty acids in the form of triglycerides, selected from butyric acid, isobutyric acid, isovaleric acid, caproic acid, caprylic acid, pelargonic acid, capric acid, lauric acid, myristic acid, palmitic acid, oleic acid, docosahexaenoic acid, stearic acid, arachidic acid, linoleic acid, linolenic acid, arachidonic acid, palmitoleic acid, and eicosapentaenoic acid, or mixtures thereof.

[0185] As shown in FIG. 1, the method of the present invention can include a step of adding a food additive 150. The food additive can be added to the first composition, the second composition, or both. This step is specifically designed to improve the flavor, texture, appearance, or shelf life of the protein yarn. The first or second composition can include 0.01-25% by weight of the food additive relative to the total wet weight of the relevant composition, and preferably 0.01-10% by weight of the food additive relative to the total wet weight of the relevant composition. Meanwhile, the protein yarn may include 0.01-10% by weight of the food additive relative to the total weight of the yarn, preferably 0.01-4% by weight of the food additive relative to the total weight of the yarn.

[0186] The food additive may be selected from a seasoning, a flavor additive, a texture additive, a food coloring, a preservative, or a combination thereof.

[0187] Food additives include, for example, seasonings, mineral salts, flavor additives, moisturizing additives, texture additives, antifoaming agents, emulsifying additives, hardening agents, gelling agents, stabilizing additives, thickeners, food coloring agents, preservative additives, or combinations thereof.

[0188] The seasonings may be selected from, for example, aromatic herbs and / or spices such as salt, pepper, rosemary, sage, mint, oregano, parsley, thyme, bay leaves, cloves, basil, chives, marjoram, nutmeg, cardamom, chili, cinnamon, fennel, fenugreek, ginger, saffron, vanilla, coriander, etc.; alcohols such as wines such as Jurançon, Sauternes, Pacherank, etc.; spirits such as Cognac, Armagnac, etc.; or any combination thereof.

[0189] The flavor additive may be selected from, for example, a flavor enhancer, a sweetener, or any combination thereof.

[0190] The texturing additive can be selected from, for example, a bulking or thickening agent, a drying agent, a hardening agent, or any combination thereof. For example, the first composition can further comprise a texturing additive such as carrageenan, pectin, pullulan, dextran, starch, etc.

[0191] The preservative additive may be selected from, for example, an antimicrobial agent, a pH adjusting agent, or any combination thereof.

[0192] The food coloring agent may be selected from coloring agents extracted from natural sources, such as carotenes, anthocyanins, tomatoes (lycopene), beetroots (betacyanins and betaxanthins), or mixtures thereof. The edible food product may contain 0.01% to 4% of the food coloring agent relative to the total weight of the edible food product.

[0193] In particular, in a first embodiment, as shown in the examples, the first composition can comprise: - at least 10% by weight total protein based on the total wet weight of the first composition, wherein the first composition comprises at least 5% by weight non-human animal protein based on the total protein weight, and the animal protein is derived from non-human cultured animal cells; and - at least 0.1% by weight of polyelectrolyte relative to the total wet weight of the first composition.

[0194] Preferably, as shown in the examples, the first composition may comprise: - at least 10% by weight total protein based on the total wet weight of the first composition, wherein the first composition comprises at least 5% by weight non-human animal protein based on the total protein weight, and the animal protein is derived from non-human cultured animal cells; and - at least 0.1% by weight of a polyelectrolyte relative to the total wet weight of the first composition, and at least 1% of vegetable fat or fat obtained by fermentation, relative to the total wet weight of the first composition;

[0195] Preferably, the first composition may comprise: - at least 10% by weight total protein based on the total wet weight of the first composition, wherein the first composition comprises at least 5% by weight non-human animal protein based on the total protein weight, and the animal protein is derived from non-human cultured animal cells; and - at least 0.1% by weight of a polyelectrolyte relative to the total wet weight of the first composition, and - at least one bridging molecule.

[0196] Preferably, as shown in the examples, the first composition may comprise the following components: - at least 10% by weight total protein based on the total wet weight of the first composition, wherein the first composition comprises at least 5% by weight non-human animal protein based on the total protein weight, and the animal protein is derived from non-human cultured animal cells; and - at least 0.1% by weight of a polyelectrolyte relative to the total wet weight of the first composition, and at least one bridging molecule, and at least 1% of vegetable fat or fat obtained by fermentation, relative to the total wet weight of the first composition;

[0197] More preferably, as shown in the examples, the first composition can comprise: - at least 10% by weight total protein based on the total wet weight of the first composition, wherein the first composition comprises at least 5% by weight non-human animal protein based on the total protein weight, and the animal protein is derived from non-human cultured animal cells; and - at least 0.001% by weight of multivalent ions relative to the total wet weight of the first composition, and at least one bridging molecule, and - at least 1% of vegetable fat or fat obtained by fermentation, based on the total wet weight of the first composition, and at least one extracellular matrix non-human animal molecule, preferably at least 0.1% of extracellular matrix animal molecules compared to the total weight of proteins;

[0198] More preferably, the first composition may comprise: - at least 10% by weight total protein based on the total wet weight of the first composition, wherein the first composition comprises at least 5% by weight non-human animal protein based on the total protein weight, the animal protein being derived from non-human cultured animal cells, and at least 0.25% by weight myofibrillar protein based on the total protein weight; and - at least 0.1% by weight of polyelectrolyte relative to the total wet weight of the first composition.

[0199] More preferably, as shown in the examples, the first composition may comprise: - at least 10% by weight of total protein based on the total wet weight of the first composition, wherein the first composition comprises at least 5% by weight of non-human animal protein based on the total protein weight, the animal protein being derived from non-human cultured animal cells, and wherein the first composition comprises at least 0.25% by weight of myofibrillar protein based on the total protein weight; and - at least 0.1% by weight of a polyelectrolyte relative to the total wet weight of the first composition, and at least 1% of vegetable fat or fat obtained by fermentation relative to the total wet weight of the first composition;

[0200] More preferably, the first composition comprises: - at least 10% by weight total protein based on the total wet weight of the first composition, wherein the first composition comprises at least 5% by weight non-human animal protein based on the total protein weight, and the animal protein is derived from non-human cultured animal cells; and - at least 0.1% by weight of polyelectrolyte relative to the total wet weight of the first composition; wherein the weight ratio of myofibrillar protein to polyelectrolyte is at least 2.5.

[0201] In particular, in a second embodiment, as shown in the examples, the first composition can comprise: - at least 10% by weight total protein based on the total wet weight of the first composition, wherein the first composition comprises at least 5% by weight non-human animal protein based on the total protein weight, and the animal protein is derived from non-human cultured animal cells; and - at least 0.001% by weight of multivalent ions relative to the total wet weight of the first composition.

[0202] Preferably, as shown in the examples, the first composition may comprise: - at least 10% by weight total protein based on the total wet weight of the first composition, wherein the first composition comprises at least 5% by weight non-human animal protein based on the total protein weight, and the animal protein is derived from non-human cultured animal cells; and - at least 0.001% by weight of multivalent ions relative to the total wet weight of the first composition, and - at least 1% of vegetable fat or fat obtained by fermentation relative to the total wet weight of the first composition.

[0203] More preferably, as shown in the examples, the first composition may comprise: - at least 10% by weight total protein based on the total wet weight of the first composition, wherein the first composition comprises at least 5% by weight non-human animal protein based on the total protein weight, the animal protein being derived from non-human cultured animal cells, and at least 0.25% by weight myofibrillar protein based on the total protein weight; and - at least 0.001% by weight of multivalent ions relative to the total wet weight of the first composition.

[0204] More preferably, as shown in the examples, the first composition may comprise: - at least 10% by weight total protein based on the total wet weight of the first composition, wherein the first composition comprises at least 5% by weight non-human animal protein based on the total protein weight, the animal protein being derived from non-human cultured animal cells, and at least 0.25% by weight myofibrillar protein based on the total protein weight; - at least 0.001% by weight of multivalent ions relative to the total wet weight of the first composition, and - at least one bridging molecule.

[0205] Preferably, as shown in the examples, the second composition is an aqueous composition.

[0206] In particular, in a first embodiment, as shown in the examples, the second composition may comprise: - at least 0.001% by weight of multivalent ions relative to the total wet weight of the second composition.

[0207] Preferably, the second composition may comprise: - at least 0.001% by weight of multivalent ions relative to the total wet weight of the second composition, and - at least one bridging molecule.

[0208] In particular, in a second embodiment, as shown in the examples, the second composition can comprise: - at least 0.1% by weight of polyelectrolyte relative to the total wet weight of the first composition.

[0209] Preferably, the second composition may comprise: - at least 0.1% by weight of a polyelectrolyte relative to the total wet weight of the first composition, and - at least one bridging molecule.

[0210] As shown in Figure 1, a method 100 for producing edible protein yarns in accordance with the present invention includes a step 160 of contacting a first composition with a second composition. This step is specifically designed to form edible protein yarns. More specifically, multiple edible protein yarns can be prepared simultaneously.

[0211] Advantageously, as shown in the examples, the temperature of the first composition when contacted with the second composition is less than 65° C. Preferably, the temperature of the first composition when contacted with the second composition is less than 60° C., more preferably less than 55° C., and even more preferably less than 50° C. This can improve the texture, handleability, or appearance of the edible protein yarn.

[0212] Furthermore, once prepared, the first composition should not be subjected to heat treatment at a temperature above 65°C, preferably above 60°C, more preferably above 55°C, and even more preferably above 50°C.

[0213] For example, the temperature of the first composition used in the contacting step 160 is 1°C to 65°C, preferably 3°C to 60°C, more preferably 3°C to 55°C, and even more preferably 3°C to 50°C.

[0214] It has been shown that the second composition can be used at higher temperatures without affecting the quality of the edible protein thread produced. Thus, the temperature of the second composition when contacted with the first composition can be greater than 35°C. However, the temperature of the second composition when contacted with the first composition should preferably be 85°C or less, more preferably 75°C or less, and even more preferably 65°C or less.

[0215] The step 160 of contacting the first composition with the second composition can be performed by known methods such as extrusion spinning, gel spinning, melt spinning, centrifugal spinning, bath-assisted 3D printing, wet spinning, or dry-jet wet spinning. Preferably, the step 160 of contacting the first composition with the second composition is performed by wet spinning, dry-jet wet spinning, or bath-assisted 3D printing. More preferably, the step 160 of contacting the first composition with the second composition is performed by wet spinning or dry-jet wet spinning.

[0216] In particular, the step 160 of contacting the first composition with the second composition may include impregnating a yarn made from the first composition with the second composition, preferably immersing the yarn made from the first composition in a bath made from the second composition. Preferably, a flow can be created in the second composition (e.g., via a vortex collector) and / or the yarn can be wound (which can be advantageously done to straighten the yarn).

[0217] In particular, the contacting step 160 includes immersing the first composition in the second composition and forming edible protein threads in a bath made of the second composition.

[0218] Contacting 160 the first composition with the second composition can occur for at least 10 milliseconds, preferably at least 100 milliseconds, more preferably at least 500 milliseconds, and even more preferably at least 1 second.

[0219] Contacting the first composition with the second composition 160 can occur for up to 20 minutes, preferably up to 10 minutes, more preferably up to 5 minutes, and even more preferably up to 4 minutes.

[0220] Thus, step 160 of contacting the first composition with the second composition can be carried out for a period of time between 10 milliseconds and 20 minutes, preferably between 100 milliseconds and 10 minutes, more preferably between 500 milliseconds and 5 minutes, and even more preferably between 1 second and 4 minutes.

[0221] As shown in FIG. 1, the method of the present invention can include a step 170 of converting the edible protein thread.

[0222] This step is specifically designed to prepare the edible protein yarn suitable for subsequent steps, particularly the production of edible food products, which may be edible meat substitutes. The step of converting the edible protein yarn can also include combining the edible protein yarn with another food matrix.

[0223] For example, the protein threads can be subjected to further processing, such as marinating, crushing, squeezing, braiding, cutting, mincing, grinding, mixing, shredding, squeezing, dosing, molding, pressing, 3D printing, extruding, baking or cooking steps (smoking, roasting, frying, surface treating, coating, etc.), or combining with other ingredients, to produce an edible food product that mimics known conventional meat products. In particular, the edible product can be a meat substitute product intended to mimic known meat products, with or without processing.

[0224] The methods of the present invention can also include combining the protein thread with other food matrices, such as a fat matrix, a protein matrix, a carbohydrate matrix, a plant matrix, and / or a fiber matrix.

[0225] As shown in FIG. 1, the method of the present invention can include a step 180 of preparing an edible food product that includes an edible protein thread.

[0226] This step is specifically designed to obtain an edible food product with meat-like organoleptic properties, in particular meat-like properties such as meat-like texture and flavor.

[0227] According to the present invention, conditioning 180 the edible food product can include steps that affect the moisture content, shape, texture, flavor, and even shelf life of the edible food product.

[0228] For example, according to the present invention, preparing 180 an edible food product can include steps such as drying, dehydrating, freeze-drying, filtering, or combinations thereof.

[0229] For example, according to the present invention, preparing 180 an edible food product can include steps such as sterilization or pasteurization.

[0230] Finally, in accordance with the present invention, preparing 180 an edible food product can include steps such as cooling, refrigerating, deep-freezing, packaging, or a combination thereof.

[0231] In another aspect, the present invention relates to an edible protein yarn obtainable from the method of the present invention. Preferably, the edible protein yarn is obtainable by the method of the present invention.

[0232] In particular, the edible protein yarn comprises a non-human animal protein, said non-human animal protein being a cultured non-human animal cell protein. Further, the edible protein yarn can comprise a heat-stable gel formed by a polyelectrolyte in combination with an appropriate multivalent ion.

[0233] Thus, the present invention preferably relates to an edible protein yarn obtainable from the method of the present invention, said edible protein yarn comprising an animal protein and a thermostable forming polyelectrolyte, said animal protein being a cultured non-human animal cell protein.

[0234] Advantageously, the edible protein yarn has improved meat-like flavor and / or meat-like texture compared to edible protein yarns made solely from plant proteins.

[0235] Several embodiments, whether preferred or not, have been described above in connection with the inventive method for producing the inventive protein yarn. Thus, the inventive protein yarn can include each of the features and any of the steps described above in connection with the inventive method, either alone or in combination.

[0236] General and preferred properties of each component of the protein yarns and edible food products according to the invention have been described in detail above. These embodiments are applicable to both the edible protein yarns and edible food products according to the invention. Some are listed below to highlight their importance.

[0237] Edible protein threads according to the present invention can be made from a first composition comprising either a multivalent ion or a polyelectrolyte, and a second composition comprising the other.

[0238] In particular, the edible protein threads of the present invention can be made from a first composition comprising a polyelectrolyte or from a first composition comprising a multivalent ion.

[0239] Advantageously, the edible protein yarn is produced from cultured non-human animal cells. Thus, as described in more detail below, the edible protein yarn according to the invention can comprise whole cultured cells, disrupted cultured cells, and / or extracts of cultured cells (such as extracts of disrupted cultured cells), said cells being cultured cells from organisms of the animal kingdom excluding humans. However, in view of preferred embodiments of the inventive method for producing an edible protein yarn, the edible protein yarn may not comprise whole cultured cells.

[0240] The protein yarn may contain at least 0.25% by weight of myofibrillar protein relative to the total weight of protein, preferably at least 0.5% by weight, more preferably at least 1% by weight, and even more preferably at least 2% by weight of myofibrillar protein relative to the total weight of protein in the protein yarn. The protein yarn may contain up to 70% by weight of myofibrillar protein relative to the total weight of protein, preferably up to 60% by weight, more preferably up to 50% by weight, and even more preferably up to 40% by weight of myofibrillar protein relative to the total weight of protein in the protein yarn. Thus, the protein yarn may contain 0.25% to 70% by weight of myofibrillar protein relative to the total weight of protein, preferably 0.5% to 60% by weight, more preferably 1% to 50% by weight, and even more preferably 2% to 40% by weight of myofibrillar protein relative to the total weight of protein in the protein yarn.

[0241] The concentration of myofibrillar protein in a protein yarn can also be considered in relation to the concentration of polyelectrolyte. Controlling this ratio can optimize the behavior of the protein yarn. Thus, a protein yarn can contain myofibrillar protein (derived from cultured cells) and polyelectrolyte at concentrations such that the weight ratio of myofibrillar protein to polyelectrolyte is at least 2.5, preferably 5, more preferably 7.5, and even more preferably 10. However, a protein yarn can contain myofibrillar protein and polyelectrolyte at concentrations such that the weight ratio of myofibrillar protein to polyelectrolyte is at most 100, preferably at most 50, more preferably at most 40, and even more preferably at most 30. Thus, a protein yarn can contain myofibrillar protein and polyelectrolyte at concentrations such that the weight ratio of myofibrillar protein to polyelectrolyte is 2.5 to 100, preferably 5 to 50, more preferably 7.5 to 40, and even more preferably 10 to 30. As described above, myofibrillar protein can be quantified by mass spectrometry, similar to polyelectrolytes.

[0242] The protein yarn can include at least 0.1% by weight of polyelectrolyte relative to the total wet weight of the protein yarn. Preferably, the protein yarn can include at least 0.2% by weight of polyelectrolyte relative to the total wet weight of the protein yarn, more preferably at least 0.3% by weight, and even more preferably at least 0.4% by weight of polyelectrolyte relative to the total wet weight of the protein yarn.

[0243] The protein yarn can include up to 15% by weight of polyelectrolyte relative to the total wet weight of the protein yarn. Preferably, the protein yarn can include up to 10% by weight of polyelectrolyte relative to the total wet weight of the protein yarn, more preferably up to 5% by weight, and even more preferably up to 3% by weight of polyelectrolyte relative to the total wet weight of the protein yarn.

[0244] The protein yarn can contain 0.1% to 15% by weight of polyelectrolyte relative to the total wet weight of the protein yarn. Preferably, the protein yarn can contain 0.3% to 10% by weight of polyelectrolyte relative to the total wet weight of the protein yarn, more preferably 0.4% to 5% by weight, and even more preferably 0.5% to 3% by weight of polyelectrolyte relative to the total wet weight of the protein yarn.

[0245] The protein yarn preferably comprises at least 0.0125% by weight of troponin protein relative to the total weight of protein in the protein yarn, and preferably at least 0.025%, more preferably at least 0.05%, and even more preferably at least 0.1% of troponin protein relative to the total weight of protein in the protein yarn.

[0246] The protein yarn can comprise collagen, preferably at least 1%, preferably at least 2%, more preferably at least 3%, and even more preferably at least 4% by weight of collagen compared to the total weight of proteins in the protein yarn.

[0247] The protein yarn can comprise elastin and / or tropoelastin, and preferably the weight of elastin and / or tropoelastin is at least 0.065% compared to the total weight of protein in the protein yarn, preferably at least 0.13%, more preferably at least 0.2%, and even more preferably at least 0.25% compared to the total weight of protein in the protein yarn.

[0248] As explained, the methods of the invention can include adding a supplemental fat, preferably a vegetable fat or a fat obtained by fermentation, to the edible protein yarn. Thus, the edible protein yarn of the invention can include a fat, preferably a vegetable fat or a fat obtained by fermentation.

[0249] For example, the edible protein yarns of the present invention comprise at least 1% by weight of fat relative to the total wet weight of the edible protein yarn. Preferably, the fat is at least 2% by weight relative to the total wet weight of the edible protein yarn, more preferably at least 5% by weight, and even more preferably at least 10% by weight relative to the total wet weight of the edible protein yarn. However, the amount of fat should not be too high. For example, the edible protein yarns can comprise up to 50% by weight of fat relative to the total wet weight of the edible protein yarn. Preferably, the fat is at most 40% by weight, more preferably at most 30% by weight, and even more preferably at most 25% by weight relative to the total wet weight of the protein yarn.

[0250] Thus, the edible protein yarn can contain, for example, 1% to 50% by weight of fat relative to the total wet weight of the edible protein yarn, preferably 2% to 40% by weight of fat relative to the total wet weight of the edible protein yarn, more preferably 5% to 30% by weight, and even more preferably 10% to 25% by weight relative to the total wet weight of the edible protein yarn.

[0251] Edible protein yarns according to the present invention preferably have shape and / or mechanical properties that allow them to mimic the behavior of conventional meat from slaughtered animals when cooked and tasted.

[0252] For example, the edible protein threads of the present invention have a length of at least 2 mm, preferably at least 10 mm, more preferably at least 50 mm, and even more preferably at least 100 mm.

[0253] For example, the diameter of the edible protein threads of the present invention is at most 4 mm, preferably at most 2 mm, more preferably at most 1 mm, and even more preferably at most 0.5 mm.

[0254] Advantageously, the edible protein yarns of the present invention can be considered as a replacement for traditional meat products from slaughtered animals, or as a raw material for replacing traditional meat products from the slaughtered animals themselves.

[0255] Edible protein yarns are, for example, ready-to-eat foods that can be ultimately consumed as is or after processing steps (e.g., grinding, crushing, braiding, cutting, grinding, mixing, shredding, squeezing, dosing, molding, pressing, 3D printing, extruding, baking or cooking procedures such as smoking, roasting, frying, surface treating, coating, etc.) and / or cooking steps. Edible protein yarns can also be intermediate products for combining with other products to produce ready-to-eat foods. In particular, edible protein yarns can also be substitutes for meat from slaughtered animals, intended to mimic traditional meat products (steaks, sausages, pâtés, etc.). As shown in the examples, edible protein yarns according to the present invention can exhibit improved meat-like texture and / or meat-like flavor compared to edible protein yarns made from plant proteins.

[0256] In another aspect, the present invention relates to a method for producing an edible food product. In particular, the method for producing an edible food product comprises the use of an edible protein yarn according to the present invention. As already mentioned, such edible food products can be considered as edible meat substitutes.

[0257] In a preferred embodiment, the method of producing an edible food product comprises assembling one or more of the edible protein threads produced according to the present invention into an edible food product.

[0258] In particular, a method for producing an edible food product can include using a combination of edible protein yarns of the invention made from a first composition comprising a polyelectrolyte and / or edible protein yarns of the invention made from a first composition comprising a polyvalent ion. In a preferred embodiment, the method for producing an edible food product includes using a combination of edible protein yarns according to the invention made from a first composition comprising a polyelectrolyte and edible protein yarns according to the invention made from a first composition comprising a polyvalent ion. The protein yarns in the combination can include protein yarns that differ from each other in their composition (e.g., protein, cell, fat, or other component) or shape or size (e.g., different diameters).

[0259] In a preferred embodiment, a method of producing an edible food product includes a) producing one or more edible protein threads according to the invention, and b) assembling the one or more produced edible protein threads into an edible food product.

[0260] Assembly of the manufactured edible protein threads can include assembling the manufactured edible protein threads into a structure that mimics the texture of meat, a process that involves assembling and aligning the protein threads and further subjecting them to various treatments to create a meat-like texture and consistency.

[0261] In one embodiment of the present invention, assembling protein threads into a meat-like texture comprises one or more of the following steps, which can be performed in any order: · Collecting and stretching protein threads, orienting the proteins longitudinally, similar to the orientation of muscle fibers in meat; · Arranging the protein threads into a parallel or intertwined configuration followed by a lamination process that compresses the threads; · Protein threads are layered on top of each other, arranged so that each layer contributes to the overall meat-like structure; · Chemical and / or enzymatic and / or physical cross-linking of protein threads to enhance the textural properties and integrity of the assembled protein structure; Applying mild heat and pressure to the protein threads to improve protein-protein interactions; and / or · Weaving protein threads into a fabric-like structure using a loom designed to replicate the interwoven nature of meat muscle fibers.

[0262] It is also appropriate to use compositions such as binders, such as fat matrices and / or connective tissue analogs, when assembling edible protein yarns to improve the mechanical and organoleptic properties of the edible product.

[0263] The edible food according to the invention is preferably a processed food, indeed it does not consist of the flesh of a slaughtered animal per se, but is preferably obtained from a combination of edible substances from different biological sources (for example a hybrid product combining a protein from the animal kingdom with a fat from the plant kingdom, or a hybrid product combining a protein from the plant kingdom and / or the animal kingdom and / or fungi and / or bacteria with a fat from the plant kingdom and / or the animal kingdom and / or fungi and / or bacteria).

[0264] Food manufacturing can include several steps such as: blanching, heat sterilization, evaporation and distillation, dehydration, smoking, baking and roasting, frying, high pressure processing, pulsed electric field (PEF) processing, ultrasound, cavitation, shock wave processing, pasteurization, application of cold plasma, dielectric, ohmic and infrared processing, microwave heating, assisted extraction, food irradiation, UV microbial inactivation, pulsed light technology, supercritical extraction, extrusion, freezing, cooling, modified atmosphere, drying techniques (freeze drying, membrane), fermentation, homogenization, mincing, grinding, chopping, salting, tumbling, brine injection.

[0265] In another aspect, the present invention relates to an edible food product obtained from the process of the present invention. Preferably, the edible food product is obtained by the process of the present invention.

[0266] In particular, the edible food product comprises an edible protein thread according to the present invention.

[0267] In particular, an edible food product can comprise a combination of an edible protein yarn of the invention made from a first composition comprising a polyelectrolyte and an additional edible protein yarn of the invention made from a first composition comprising a polyvalent ion. In a preferred embodiment, the edible food product comprises a combination of an edible protein yarn according to the invention made from a first composition comprising a polyelectrolyte and an edible protein yarn according to the invention made from a first composition comprising a polyvalent ion. Such combinations are particularly interesting because they can enrich the mouthfeel, as no two protein yarns have exactly the same mouthfeel.

[0268] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the present products and practice the claimed methods. The following examples therefore specifically point out preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.

[0269] In another aspect, the present invention relates to a system for producing edible protein yarns, particularly edible food products comprising edible protein yarns.

[0270] The system according to the present invention may include a first composition container capable of containing a first composition comprising a protein. The first composition container may be made of food-grade plastic, ceramic, glass, silicon, stainless steel, or a combination thereof. The first composition container may be associated with a temperature controller configured to measure and vary the temperature of the first composition. The first composition container may be associated with a pressure controller configured to measure the pressure within the first composition container.

[0271] The system according to the present invention can include a second composition container capable of containing the second composition. The second composition container can be made from food-grade plastic, ceramic, glass, silicon, stainless steel, or a combination thereof. The second composition container can be associated with a temperature controller configured to measure and vary the temperature of the second composition.

[0272] The system according to the invention comprises a contacting device. Advantageously, the contacting device is arranged to bring the first composition into contact with the second composition, preferably to form edible protein threads from the first composition. Advantageously, the contacting device has one or more holes connected to the first composition container, said one or more holes being arranged to allow the first composition, while still in thread form, to come into contact with the second composition. The area of ​​the hole is 4 mm 2 Less than 3.5mm, preferably 2Less than 2 mm, more preferably 2 Less than 1 mm, more preferably 2 Less than 0.5 mm, more preferably 2 The holes can be irregular or regular shapes, such as rectangular, circular, oval, etc.

[0273] In one embodiment, the contact device includes a plurality of needles connected to a pump device associated with the first composition container. The needles can be connected to a common chamber or each needle can be connected to a separate chamber. The needles can be arranged parallel to one another and can all exhibit the same pattern (circular, rectangular, oval, irregular) or can exhibit different patterns. Preferably, the needles exhibit different patterns to obtain irregular portions of fiber embedded in the fat, thereby giving the food a more natural appearance.

[0274] The system according to the present invention can include a manufacturing vessel capable of housing the protein thread embedded in the second composition. The manufacturing vessel can be made of food-grade plastic, ceramic, glass, silicone, stainless steel, or a combination thereof. The manufacturing vessel can be associated with a temperature controller configured to measure and modify the temperature of the edible food product.

[0275] Preferably, the system of the present invention may further include a cooling device. Preferably, the system of the present invention may further include a roller.

[0276] Preferably, the system according to the present invention may further include a spooler.

[0277] Preferably, the system of the present invention may further include a cutting or slicing device, or a packaging device. [Example]

[0278] The present invention will be described in further detail with reference to the following experimental examples. These examples are provided for illustrative purposes only and, unless otherwise specified, are not limiting. Therefore, the present invention should not be construed as being limited to the following illustrative examples, but rather as encompassing any variations that become evident as a result of the teachings provided herein.

[0279] Materials and Methods chemicals Sodium Alginate Food Grade Powder, E401 LMC Pectin E440 Gelatin nCAS 9000-70-8 Chitosan nCAS 9012-76-4 Calcium chloride nCAS 10043-52-4 NaCl Vegetable fats and / or fats obtained by fermentation are obtained by mechanical or chemical extraction from seeds or other parts of fruits. They are then refined and, if necessary, purified or chemically modified. Many commercially available references can be used, such as a mixture of CremoFLEX® L and CremoFLEX® E. Transglutaminase (TGase) powder - 125U / g Soy protein solution 20% by weight Elastin - Sigma Recombinant Collagen - Sigma

[0280] Cell culture and preparation The bovine cells were obtained from biopsies or cultured cells. The cultured cells are bovine embryonic stem cells that were originally isolated from bovine embryos and adapted to grow in suspension in serum- and growth factor-free medium. These cells are characterized by their ability to grow in suspension on a large scale in bioreactors.

[0281] Bovine cells are cultured in a 30 L stainless steel bioreactor at 37°C with constant agitation at 50 rpm and pH adjusted to 7.1 with CO2 injection. Four days after inoculation, cells are harvested from the bioreactor, subjected to two stages of centrifugation, and the dried pellet is weighed. Protein dosing can be performed on a sample of harvested cells using the Bradford method.

[0282] The moisture content can be measured and adjusted. The cells can be used immediately, or additional steps such as protein extraction can be performed on the harvested culture.

[0283] Preparation of the First and Second Compositions The first and second compositions are prepared by mixing the following mixture compositions:

[0284] The first composition is homogenized using a homogenizer (8000 rpm, 60 seconds).

[0285] Contacting the first composition with the second composition to produce an edible protein thread Protein threads are spun using a syringe pump (Chemyx), a 10 mL syringe with an 18 G (0.8 mm) or 14 G (1.6 mm) needle, or a spinneret with multiple holes. The injection rate is fixed at 5 mL / min.

[0286] Mechanical testing Protein threads are evaluated by tensile testing using a texture meter (Ametek LS1 equipped with a 10 kg load cell). Briefly, the protein thread is stretched until breakage, and the force is recorded as a function of stretch distance. At breakage, the measured force drops sharply.

[0287] The extension rate is 100 mm / min and measurements are carried out on six samples for each diameter.

[0288] Sensory evaluation Food samples are anonymized before tasting, and panelists are provided with water and crackers to rinse their mouths between samples to reset their taste receptors.

[0289] Panelists evaluate the overall integrity and meat-like sensory attributes of edible food samples by tasting. For example, to evaluate beef substitutes, panelists evaluate beef flavor and texture (tenderness, cohesiveness, oiliness, juiciness).

[0290] Evaluating the effect of protein content in the first composition on protein yarn The purpose of this experiment is to determine the effect of protein content on the mechanical properties of the yarn.

[0291] The first composition is prepared by mixing the ingredients shown in Table 1.

[0292] [Table 1]

[0293] The second composition is a coagulation bath containing 0.5% w / w calcium chloride dissolved in distilled water, ie 0.18% multivalent calcium ion.

[0294] A first composition is used in a spinneret to produce multiple streams of the first composition that contact a second composition that is used as a coagulation bath.

[0295] Results and Conclusions Protein yarns made with a first composition having a protein content of 10.56 wt % (A2), 13.20 wt % (A3), or 17.6 wt % (A4) have significantly improved strain at break, load at break, and tensile stress compared to those with a protein content of 7.1 wt % (A1).

[0296] In particular, the protein yarn produced with the first composition (A4) having a protein content of 17.6 wt. % has a strain at break of +6%, a load at break of +37%, and a tensile stress of +36% compared to that with a protein content of 10.56 wt. % (A2).

[0297] Thus, when forming protein yarns according to the present invention, it is preferred to use a first composition having at least 10% by weight protein relative to the total wet weight of the first composition, preferably at least 13% by weight protein relative to the wet weight of the composition, more preferably at least 17% by weight protein.

[0298] Evaluating the effect of the protein source in the first composition on the protein thread The goal of this experiment is to evaluate the effect of protein origin on the mechanical properties of protein yarns using tensile tests.

[0299] The first composition is prepared by mixing the ingredients shown in Table 2.

[0300] [Table 2]

[0301] The vegetable protein used in the first composition is soy protein.

[0302] The second composition is a coagulation bath containing 0.5% w / w calcium chloride dissolved in distilled water.

[0303] A first composition is used in a spinneret to induce a flow of the first composition into contact with a second composition which is used as a coagulation bath.

[0304] After incubation at 37 °C for 1 h, the mechanical properties of the protein threads are evaluated by tensile testing using a texture meter.

[0305] Results and Conclusions The mechanical properties of the protein yarns are shown in Table 3 below.

[0306] [Table 3]

[0307] The tensile test results are normalized to the values ​​obtained for protein yarns made only from vegetable proteins (B0).

[0308] Protein yarns prepared with animal protein from cells or cell extracts (B80, B40) are more resistant to traction (tensile tests) than those prepared with soy protein under the same conditions. When animal protein from cells or cell extracts (B40) is used in combination with plant protein (myofibrillar protein concentration above 4%), the improvement rate increases by two-fold, and when the concentration of animal protein from cells or cell extracts exceeds 15% by weight (B80), the improvement rate increases by at least three-fold.

[0309] Furthermore, in the method of the present invention, the combination of animal protein derived from cultured cells with plant protein improves the tensile properties of the yarn compared to plant protein alone (B40 compared to B0).

[0310] This indicates that protein yarns produced according to the methods of the present invention have desirable mechanical properties that are far superior to those produced from plant proteins alone.

[0311] Furthermore, the addition of some plant proteins can adjust the viscoelastic or flow properties of the first composition containing cultured non-human animal cells.

[0312] Evaluation of the effect of temperature on protein threads The purpose of this experiment was to determine the maximum temperature that the first composition could withstand before spinning and the effect of elevated temperatures on the second composition at atmospheric pressure.

[0313] The first composition is prepared by mixing the ingredients shown in Table 4.

[0314] [Table 4]

[0315] The second composition is a coagulation bath containing 0.5% w / w calcium chloride dissolved in distilled water.

[0316] A first composition is used in a spinneret to direct a flow of the first composition into contact with a second composition used as a coagulation bath.

[0317] To evaluate the effect of temperature of the first composition in the method of the present invention, the first composition is incubated for 10 minutes at different temperatures: room temperature (RT), 55° C., 60° C., or 65° C. The first composition is used in a spinneret to induce flow of the first composition in contact with a second composition used as a coagulation bath, the second composition in the coagulation bath being at room temperature.

[0318] To evaluate the effect of the temperature of the second composition in the coagulation bath in the method of the present invention, the second composition is incubated at 65°C, 75°C, and 85°C for 10 minutes, and protein threads are produced by contacting the first composition (room temperature) with the second composition (the latter in a calcium coagulation bath). The protein threads are incubated in the second composition for 1 minute before harvesting.

[0319] After the fibers are removed from the calcium coagulation bath, they are evaluated for quality and integrity.

[0320] Results and Conclusions Although spinning is possible when the first composition is at 55°C and 60°C, the morphology of the protein yarn is not as uniform as at room temperature, and some lumps and irregularities may be observed on the surface of the protein yarn.

[0321] If the first composition is incubated at 65°C, spinning becomes impossible. The number of clumps increases significantly, the needle becomes clogged, and discontinuous protein threads are formed. Therefore, the first composition should not be subjected to heat treatment above 65°C for 10 minutes.

[0322] For the second composition, threads produced in the 65°C and 75°C coagulation baths exhibit a similar appearance (no lumps) to threads obtained at room temperature, but are slightly lighter in color. Protein threads formed with the second composition at 85°C are acceptable, but begin to develop a clumpy texture on the outside.

[0323] Evaluating the effect of polyelectrolyte properties on protein threads The goal of this experiment is to determine the effect of polyelectrolyte properties on protein threads.

[0324] The first composition is prepared by mixing the ingredients shown in Table 5.

[0325] [Table 5]

[0326] The second composition is a coagulation bath containing 1.5% w / w calcium chloride dissolved in distilled water, whereas for D4 the second composition is a coagulation bath containing disodium phosphate dissolved at 1 mol / L in distilled water.

[0327] A first composition is used in a spinneret to direct a flow of the first composition into contact with a second composition used as a coagulation bath.

[0328] After 1 h of incubation at 37 °C, the mechanical properties of the protein threads are evaluated by tensile testing using a texture meter.

[0329] Results and Conclusions Using alginate (D1), LMC pectin (D2), and chitosan (D4) allows the formation of good, heat-resistant protein threads, whereas using gelatin (D3) results in protein threads that are completely soluble when cooked in a subsequent step.

[0330] The mechanical properties of the protein yarns are shown in Table 6 below. The tensile test results are normalized based on the values ​​obtained for protein yarns made with alginate (D1). For D3, tensile testing was not performed because these protein yarns melted when cooked.

[0331] [Table 6]

[0332] Thus, in the method of the present invention, either the interaction between the cationic polyelectrolyte and the polyanion or the interaction between the anionic polyelectrolyte and the polycation functions. This highlights the importance of using ion-mediated gelation (polymerization occurs immediately upon contact of the two solutions) to obtain threads that can retain their structure when cooked. This is paramount to ensuring good consumer acceptance of the edible food products of the present invention, which can be cooked and eaten, compared to other alternatives to traditional meat products from slaughtered animals.

[0333] Evaluating the influence of extracellular matrix (ECM) molecules on protein fibers The goal of this experiment is to clarify the effect of the presence of extracellular matrix (ECM) molecules on the properties of protein threads using tensile strength measurements.

[0334] A first composition is prepared by mixing the ingredients shown in Table 7.

[0335] [Table 7]

[0336] The second composition is a coagulation bath containing 0.5% w / w calcium chloride dissolved in distilled water.

[0337] A first composition is used in a spinneret to produce multiple streams of the first composition which contact a second composition which is used as a coagulation bath.

[0338] After incubation at 37 °C for 1 h, the mechanical properties of the protein threads are evaluated by tensile testing using a texture meter.

[0339] Results and Conclusions The mechanical properties of the protein yarns are shown below in Table 8. The tensile test results are normalized based on the values ​​(E0) obtained for protein yarns made from animal proteins derived from cultured cells without added ECM proteins.

[0340] [Table 8]

[0341] As shown in the table above, the addition of elastin as a powder to the first composition does not adversely affect strain at break, but may reduce the load and tensile stress at break (E1p).

[0342] However, when elastin is present in the first composition (E1s), the load at break, tensile stress and Young's modulus can be improved by more than 75%.

[0343] The presence of soluble forms of collagen in the first composition also improves the load at break, tensile stress and Young's modulus of the produced thread.

[0344] This suggests that ECM molecules, particularly ECM proteins, when present in the first composition, contribute to the tensile strength of the protein thread.

[0345] Therefore, the use of cultured non-human animal cells with increased concentrations of ECM molecules, particularly proteins, can increase the stiffness and elasticity of the protein threads produced according to the present invention.

[0346] Evaluating the effect of cross-linking molecules on protein fibers The purpose of this experiment was to determine the effect of adding transglutaminase to the first composition using tensile testing.

[0347] A first composition is prepared by mixing the ingredients shown in Table 9.

[0348] [Table 9]

[0349] The second composition is a coagulation bath containing 1.5% w / w calcium chloride dissolved in distilled water.

[0350] A first composition is used in a spinneret to produce multiple streams of the first composition that contact a second composition that is used as a coagulation bath.

[0351] The first half of the sample is incubated at 37°C for 1 hour to allow transglutaminase activity, and the second half is incubated at 4°C for 1 hour to prevent transglutaminase activity.

[0352] After incubation, the mechanical properties of the protein threads are evaluated by tensile testing using a texture meter.

[0353] Results and Conclusions The mechanical properties of the protein threads are shown below in Table 10. The tensile test results are normalized based on the values ​​obtained from incubation at 4°C (F4) and therefore do not benefit from the activity of transglutaminase.

[0354] [Table 10]

[0355] As shown in the table above, adding transglutaminase to the first composition and incubating at 37°C (F37) does not affect the strain at break of the protein yarn of the invention, but significantly improves the load at break, tensile stress and Young's modulus.

[0356] Therefore, the use of an enzyme as a cross-linking agent in the first composition helps to increase the stiffness and elasticity of the protein threads produced.

[0357] Evaluation of the effect of alginate concentration in the second composition on protein threads The purpose of this experiment was to evaluate the effect of alginate concentration in the second composition.

[0358] A first composition is prepared by mixing the ingredients shown in Table 11.

[0359] [Table 11]

[0360] The second composition is a coagulation bath containing 0.5% or 2% w / w sodium alginate dissolved in distilled water.

[0361] Results and Conclusions Regarding the calcium chloride concentration, threads are obtained at both concentrations of 0.03% (G1) and 0.3% (G2).

[0362] Regarding the concentration of alginate in the second composition, threads are obtained with 0.5% or 2% alginate in the second bath, with a lower concentration being recommended as a 2% alginate bath results in irregular thread morphology.

[0363] This method allows for the creation of protein threads with desirable mechanical properties. Furthermore, protein threads produced using polyelectrolytes in the second composition have a jelly-like texture on the outside, which helps mimic the extracellular matrix, improving texture and mouthfeel.

[0364] Evaluation of cross-linking capacity and edible thread properties in relation to protein content and alginate content in the first composition The purpose of this experiment is to use tensile strength measurements to compare protein threads made with different concentrations of protein and alginate, and to determine the ability of increasing alginate content in the first composition to overcome low protein content and produce an edible food product with meat-like properties.

[0365] The first composition is prepared by mixing the ingredients shown in Table 12.

[0366] [Table 12]

[0367] The second composition is a coagulation bath containing 1.5% w / w calcium chloride dissolved in distilled water.

[0368] After incubation at 37 °C for 1 h, the protein threads are incubated at 70 °C for 10 min. The mechanical properties of the protein threads are evaluated by tensile testing using a texture meter.

[0369] Results and Conclusions Protein yarns with good appearance and heat resistance can be formed from first compositions H1, H2, and H4, but the protein yarn formed from first composition H3 is not optimal.

[0370] The mechanical properties of the protein yarns are shown in Table 13 below. The tensile test results are normalized based on the values ​​obtained for samples made with at least 10% protein and less than 2% alginate (H1). The protein yarns formed with composition H3 could not be evaluated by tensile testing because the measured signal was below the detection limit. Surprisingly, the protein yarns obtained from composition H2 have slightly improved mechanical properties compared to the protein yarns obtained from composition H1.

[0371] [Table 13]

[0372] As shown in the table above, the results of the tensile tests indicate that increasing the concentration of alginate in the first composition cannot compensate for the low protein content to produce a meat substitute. The first composition H4 had a detrimental effect on the samples in terms of load at break, tensile stress, and Young's modulus.

[0373] Furthermore, the protein threads produced with first composition H1 (or composition H2) were very smooth and regular, while the protein threads produced with first composition H4 exhibited an irregular, clumpy shape. Furthermore, sample H4 was gel-like and rubbery, exhibiting a high strain at break, making it incomparable to conventional meat fibers. Therefore, it was found that the appearance and texture of the protein threads improved when the protein content was high and the alginate concentration was low.

[0374] Forming an edible product comprising edible protein threads As previously described, the protein yarn shown in FIG. 2 can be used to make the edible food product shown in FIG. 3.

[0375] The present invention is susceptible to numerous modifications and applications other than those described above. In particular, unless otherwise specified, the different structural and functional characteristics of each of the above-described implementations should not be considered as combined with each other and / or intimately and / or inseparably linked, but rather as simple juxtapositions. Furthermore, the structural and / or functional features of the various embodiments described above may be, in whole or in part, subject to any different juxtapositions or any different combinations.

[0376] Evaluating the effect of protein content and origin in the first composition on the sensory properties of the product The objective of this experiment was to evaluate the effect of protein source and content on the sensory properties of the edible food product of the present invention, as well as the overall palatability and similarity compared to the sensory properties of conventional meat.

[0377] A first composition is prepared by mixing the ingredients shown in Table 14.

[0378] [Table 14]

[0379] The second composition is a coagulation bath containing 0.5% w / w calcium chloride dissolved in distilled water.

[0380] Results and Conclusions Similarity of texture, flavor, and overall liking to conventional meat was rated by trained sensory panelists (n = 16) on a 1-9 point scale, with 1 indicating least preferred or least similar to conventional meat and 9 indicating most preferred or most similar to conventional meat.

[0381] The sensory evaluation of the edible food samples according to the present invention is shown in Table 15 below.

[0382] [Table 15]

[0383] The similarity of the samples to conventional meat was rated equally by the panel without significant differences.

[0384] Panelists found that the edible food of the present invention with the highest protein content from cells or cell extracts (Sample J3) provided the best texture and flavor. Furthermore, Sample J3 was said to have texture and excellent flavor comparable to an edible food with a high plant-derived protein content but a low protein content from cells or cell extracts (Sample J4). These results demonstrate that the source of protein has a significant impact on consumer flavor, particularly texture.

[0385] In addition, when comparing two edible foods with the same protein content, one containing a large amount of protein derived from cells or cell extracts (Sample J2) and the other primarily derived from plants (Sample J1), Sample J2 is superior to Sample J1 in terms of flavor and texture. Sample J5, which has a lower overall protein content, shows lower values ​​for similarity to conventional meat texture and flavor.

[0386] Thus, edible foods of the present invention comprising threads made from high animal protein content derived from cells or cell extracts induce the development of meat-like textures and flavors within the edible foods, and further demonstrate that such foods have organoleptic properties similar to conventional meat.

Claims

1. 1. A method for producing an edible protein thread, the method comprising the steps of: - preparing a first composition (130), wherein said first composition comprises: Animal protein, wherein the animal protein is derived from a non-human cultured animal cell, and - either multivalent ions or polyelectrolytes; - preparing a second composition (140), wherein said second composition comprises: the multivalent ions, if the step of preparing the first composition (130) includes the addition of the polyelectrolyte, or the polyelectrolyte, if the step of preparing the first composition (130) includes the addition of the multivalent ion; and - contacting (160) said first composition with said second composition to form edible protein threads, wherein said first composition has a temperature below 65°C.

2. 10. The method of claim 1, wherein the first composition is heated to a temperature of 60°C or greater for no more than 10 minutes.

3. 3. The method of claim 1 or 2, wherein The non-human cultured animal cells are selected from cells of the kingdom Animalia, preferably the animal cells are selected from mammalian cells, avian cells, actinopterygian cells, malacostraca cells, and combinations thereof; for example, mammalian cells can be bovine cells, cervidae cells, leporidae cells, or sardine cells; avian cells can be anseriform cells or pheasantidae cells; actinopterygian cells can be gadoid cells, hake cells, pleuronectid cells, salmonid cells, or mackerel cells; malacostraca cells can be palaemonid cells; molluscan cells can be cephalopod cells or The non-human cultured animal cells may be bivalve cells; the non-human cultured animal cells include cells selected from the following: stem cells such as embryonic stem cells, satellite cells, induced pluripotent stem cells, germ layer cells, fibroadipogenic progenitor cells, muscle cells such as skeletal muscle cells, cardiac cells, smooth muscle cells; myoblasts, muscle cells, hepatocytes, fibrocytes, fibroblasts, adipocytes, chondrocytes, chondroblasts, keratinocytes, melanocytes, osteocytes, osteoblasts, Merkel cells, Langerhans cells, glial cells, Schwann cells, red blood cells and white blood cells, and combinations thereof.

4. The method according to any one of claims 1 to 3, wherein the animal protein comprises myofibrillar protein, preferably representing at least 0.25% by weight compared to the total weight of protein.

5. 5. The method according to claim 1, wherein the first composition comprises extracellular matrix non-human animal molecules, preferably at least 0.1% by weight of extracellular matrix non-human animal molecules compared to the total weight of proteins.

6. 6. The method of any one of claims 1 to 5, wherein the polyelectrolyte is a thermostable gel-forming polyelectrolyte, especially when complexed with multivalent ions, preferably the polyelectrolyte is a polysaccharide.

7. The method of any one of claims 1 to 6, wherein the first composition further comprises a plant protein, an algae protein and / or a fungal protein.

8. The method of any one of claims 1 to 7, wherein the first composition comprises at least 10% total protein weight compared to the total wet weight of the first composition.

9. 9. The method according to any one of claims 1 to 8, wherein the preparation of the first composition further comprises the addition of a fat, preferably a vegetable fat or a fat obtained by fermentation, preferably in an amount of at least 1% by weight of fat compared to the total wet weight of the first composition.

10. The method of any one of claims 1 to 9, wherein the preparation of the first composition comprises homogenization, preferably homogenization is carried out at at least 100 rpm for at least 30 seconds.

11. The method according to any one of claims 1 to 10, wherein the preparation of the first composition comprises the addition of disrupted cultured non-human animal cells and / or intact cultured non-human animal cells.

12. The method of any one of claims 1 to 11, wherein the first composition comprises: - at least 10% by weight of total protein based on the total wet weight of said first composition, wherein said first composition comprises at least 5% by weight of total protein of non-human animal protein, said animal protein being derived from non-human cultured animal cells; and - at least 0.1% by weight of polyelectrolyte relative to the total wet weight of said first composition;

13. 1. A method of producing an edible food product, the method comprising the steps of: - Producing an edible protein thread according to a method according to any one of claims 1 to 12; and - Assembling one or more of the produced edible protein threads into an edible food product.

14. 13. An edible protein yarn obtainable by the method according to any one of claims 1 to 12, wherein the edible protein yarn comprises an animal protein and a polyelectrolyte that forms a heat-stable gel, and the animal protein is a cultured non-human animal cell protein.

15. 15. An edible food product comprising the edible protein thread of claim 14.

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